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TODAY'S MEDICINE, TOMORROW'S SCIENCE ESSAYS ON PATHS OF DISCOVERY IN THE BIOMEDICAL SCIENCES

JUDITH P. SWAZEY AND KAREN REEDS

Prepared Under Contract NOI-CO-55315 for the National Cancer Institute

DHEW Publication No. (NIH) 78-244 U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE Public Health Service National Institutes of Health

A Note on the Authors

Judith P. Swazey received her Ph.D. in the history of science from Harvard University. She is a Professor in the Department of Socio-Medical Sciences at Boston University School of Medicine and an Associate Professor in the Department of History, Boston University. Her research and writing has dealt with the history of the neuro-sciences, the processes of therapeutic innovation, and the social and ethical implications of contemporary biomedical developments.

Karen Meier Reeds received her Ph.D. in the history of science from Harvard University. She has been a Mellon Postdoctoral Fellow at Bryn Mawr College and currently is a Research Fellow at the University of California, Berkeley.

Preface

Today's Medicine, Tomorrow's Science is a study that grew out of the shared interest of the National Cancer Institute's Program Analysis and Formulation Branch and the authors in examining the ways that categorical or disease-oriented research have contributed to the elucidation of fundamental biological phenomena and processes. For a number of reasons, as we point out in chapter one, little attention has been paid to these paths of inquiry and discovery while, conversely, the flow from basic research to applications in the understanding of disease processes and means of diagnosing, treating, or preventing man's ills has been charted frequently by analysts of science and medicine.

Throughout the course of preparing this volume we have received invaluable help from many sources. The study was made possible by the initiative and support of Dr. Abraham Cantarow, Dr. Robert Love, and Mr. Louis Carrese of the National Cancer Institute.

1 We extend our thanks to the following persons, who shared their expertise with us by critically reviewing various chapters of the book: Dr. Garland Allen, Dr. Merriley Borrel, Dr. Gerald Edelman, Dr. Virginia Fiske, Dr. Joseph Fruton, Dr. Diana Hall, Dr. Vernon Ingram, Sir Rudolph Peters, and Dr. Frank Putnam. We are especially grateful to Dr. Julius Comroe, who undertook the task of reviewing the entire manuscript for us.

For their aid in preparing various portions and stages of the manuscript, our thanks go to Consuelo Alvarez, Chris Suntala, Ruth McNeeley, and Phyllis Schiarizzi.

Preface ...... Chapter 1. On Blind Men, Elephants, and Floppy-eared Rabbits ...... 1 Chapter 2. Louis Pasteur: Science and the Applications of Science ...... 11 Chapter 3. Beriberi and the Coenzyme Function of Vitamin B I ...... 27 Chapter 4. Disease and the Ductless Glands ...... 53 Chapter 5. "rhe Lesson of Rare Maladies:- Sickle Cell Anemia and the Genetic Control of Protein Structure ……….. 73 Chapter 6. A Crucial Experiment of Nature: Multiple Myeloma and the Structure of Antibodies ……… 93 Chapter 7. Disease and Discovery …………………… 115

CHAPTER ONE ON BLIND MEN, ELEPHANTS, AND FLOPPY-EARED RABBITS

The essays in this book revolve around a common theme: many paths of inquiry and discovery about life processes have begun through efforts to understand and to intervene in states of disease. This route of inquiry and discovery is a familiar one to those working in clinical medicine and basic biomedical research, and to historians of medicine and biology. But, at the same time, it is a route that has received little more than anecdotal mention in the literature on the nature and history of scientific research. For that literature, by and large, deals with what has become in many instances a stereotyped view of how research and discovery proceed - by some sort of necessary progession from disinterested "pure" research to the application of the basic knowledge thereby gained for the solution of "practical problems." Such a progression, however, is neither necessary or logical, but instead only one possible path to scien _ tific discovery, new knowledge, or the solution of practical problems. Our purpose in this study, initiated by the National Cancer Institute, thus is to examine one aspect of the complex paths of research, in the area of the biomedical sciences. That aspect is the flow of interest and inquiry that runs from a particular disease problem to major advances in our understanding of fundamental biological phenomena. Or, in abbreviated popular parlance, we are mapping routes that run "from the patient's bedside to the laboratory bench," rather than the more frequently discussed and charted routes from 't e enc to the bedside." After surveying a wide range of nineteenth and twentieth century medical and biological work, we chose a small number of disease-oriented research problems for close historical study, utilizing primarily a detailed tracing and analysis of primary sources. The examples we selected, drawn from bacteriology, nutrition, biochemistry, endocrinology, genetics, and immunology , are presented in the form of case studies. In addition to representing a range of biomedical inquiry that began with a clinical problem, these cases illuminate other aspects of the complex processes of research and discovery. They represent, too, a variety of ways of examining and presenting historical materials in the- form of narrative essays. For, we would argue, just as there is no single way to "do" science, so too there is no single way to describe how it was done. Thus, our first case study deals with a particular scientist, the illustrious Louis Pasteur, and focuses on the early period of his career when he investigated diseases of wine and vinegar. Many lessons emerge from the

2 story of Pasteur's work, among them the variety of interests and influences that shape an investigator's work, the multiple interpretations that can be made about that work, and the consequent difficulties, even in historical retrospect, of neatly categorizing and analyzing research in terms of its "applied" or "basic" nature. Our second case study deals with a particular disease rather than with a particular scientist. It documents how efforts to understand and halt a ravaging disease, beriberi, played a central role in the discovery of a class of substances vital to the animal economy, vitamins. Then, we examine how subsequent research on the "beriberi vitamin," thiamin, helped to elucidate the biochemical roles of vitamins, through a long and arduous series of studies that led to the identification of thiamin as a coenzyme in intermediary metabolism. In the third case, we paint a broader canvas, looking at a group of disease problems that, perhaps preeminently, exemplify a flow of interest and effort "from the clinic to the laboratory." This case study shows, both in broad outline and through particular examples, the interrelations between the medical problems of diseases of the "ductless glands" and the development of a major field of biology and medicine, endocrinology. Our final two case studies, like the beriberi study, treat more specific disease problems, tracing how efforts to understand their causes and nature led, in often unforeseeable ways, to new understanding of fundamental biological phenomena. Thus, in Chapter 5 we examine the role that work on sickle cell anemia played in defining the genetic control of protein structure, while in Chapter 6 we follow a trail of researches that began in 1846 with the discovery of a strange urinary product in a patient with multiple myeloma and ended in the 1960's with the first complete mapping of an antibody's molecular structure. In our work, we have made no attempt to quantify or specifically categorize the various lines of research we examine, as has been done in some recent historical studies. In our essays, indeed, we seldom use terms such as basic or categorical research, for reasons that have to do with two other themes or viewpoints running through our study. The first of these, as we have noted, is that there is no single path, no unique route, by which we arrive at new knowledge of life processes, nor is there a solitary path to knowledge of and means to prevent or treat disease. Rather, as one begins to realize in thinking about the word biomedical, our understanding of life processes, both normal and abnormal or diseased, has come from many intersecting lines of research and discovery. Some of these routes begin with research efforts that are usually labelled as "basic" or "fundamental" -efforts said to be aimed primarily at improving our understanding of a particular phenomenon or area of science, without regard for immediate practical uses of that understanding. Other avenues in the biomedical arena begin with what today is called "categorical" or "applied" research -inquiry that is centered around one or more aspects of.a disease (its etiology, diagnosis, prognosis, or treatment). But whatever the starting point, the end point of a given biomedical research problem is seldom, if ever, reached by a straightline path of inquiry. And often, the end point can be traced only in retrospect, for research frequently leads its practitioners in directions that are hard to foresee and, at the time, seemingly remote from the immediate objectives of their work. This view of biomedical research as a complex network of processes and events, objectives and outcomes, in turn bears upon another theme, the problems of meaningfully labelling or characterizing biomedical research activity with terms such as basic or applied. In this first chapter, as a framework for the case studies themselves, we will deal briefly with the why and what of efforts to label types of biomedical research, and, closely related to these efforts, with the oft misstated role of chance or serendipity in research.

Blind Men and Elephants: Labelling Biomedical Research

In the seventeenth century, historian Herbert Butterfield has observed, the "proper method" of scientific inquiry was "one of the grand preoccupations, not merely of the practicing scientist, but . . . amongst the general thinkers and philosophers" (Butterfield 1958, p. 97). Future historians, looking back upon science in the latter decades of the twentieth century, may judge that one of this period's "grand preoccupations" was the nature of and relationship between "basic" and 11 applied" research, or, in current biomedical parlance, between "basic" or "fundamental" and "categorical" or "mission -oriented" research.

3 This issue is by no means a new one, for, as physicist Alvin Weinberg reminded the participants at a 1966 conference on "the development and use of biomedical knowledge," "the argument about the relation between applied and basic research . . . has been raging since the 1700's" (Weinberg 1967, p. 33). There are many reasons, having to do with the history, sociology, and philosophy of science, and with its practitioners and patrons, for the origins and long persistence of this argument. One set of factors, for example, related both to perceptions about the nature of scientific discovery and to the value system within the scientific community, is a tradition of viewing "pure" research as a "higher form" of intellectual activity than "applied" research. "There exists in some circles," Beveridge observed in his noted text on The Art of Scientific Investigation, 11 a certain amount of intellectual snobbery and tendency to look disdainfully on applied investigation. This attitude is based on the following two false ideas: that new knowledge is only discovered by pure research while applied research merely seeks to apply knowledge already available, and that pure research is a higher intellectual activity because it requires greater scientific ability and is more difficult. Both these ideas are quite wrong" (Beveridge 1957, p. 169), Both in the past and today, arguments about the scientific and social roles of basic and applied research also have involved practical quests for patronage - for the support of researchers by public and private sponsors. In the recent history of biomedical research in the United States, much (but by no means all) of the concern about basic-applied distinctions has been linked with the emergence of the federal government as the major research patron in the years since World War 11. Social and economic policy decisions about how the patron should disperse his finite monies among many competing areas of research, in relation to various desired outcomes of that research, have generated an often heated dialogue about the nature of and "proper

Balance among various lines of biomedical investigation and have tended to foster a strict and competotive division between basic and applied research.

To the extent that it revolves around funding conbasic-applied biomedicine debate has mounted levels of intensity in the past decade. The principal catalyst was a statement made by President Lyndon inscin on June 15, 1966, on the occasion of the 'Jre program's debut. "A great deal of research has ,,One," the President declared, "but ... the time has to zero in on the targets ... to get our knowledge pplied. There are hundreds of millions ... spent on -1 Ory research that may be made useful to human if large-scale trials on patients are initiated in areas. Now Presidents . . . need to show Merest in what the specific results of medical are during their lifetime and during their ,tration . . . And we are determined that the vital tween pure research and practical achievement , be broken" (Quoted in Weinberg 1967, p. 33). Yent Johnson's interest in seeing biomedical 0 "pay off" in terms of "reducing deaths and Aft 10 was scarcely a new concern at the upper 76 government. Rather, it was one in a series of 1ts and reports about the conduct of biomedical to and from the executive and legislative of the federal government, that began in the years with Vannevar Bush's study for President

A, released in 1945 as Science - The Endless Frontier. . Roosevelt had asked Bush for information and . I lenclations on how scientific knowledge devel during the war could be rapidly applied for peace uses, and how a national program of medical research could be organized (Bush 1945). As one com- mentator has remarked, "Science - The Endless Fronfate of many other reports: often cited but seldom read." Readit, one senses again how often history repeats itself, in Johnson's 1966 statement, and one can begin to trace , over three decades, how "the conflicting ideas of n the relation of government to science and of the proper of science in American society became partisan issues in the years after 1945" (England 1976, p. 46) But if Johnson's call to "zero in on the targets" was the A a new one, it was one that nonetheless had profound and receivers of federal funds for As Weinberg commented a few after the event, "the world of biomedical research atleast that portion of it that regards itself as folowing the Newtonian tradition of research for it’s own sake ,was thrown into a mild state of shock by the President's remarks (Weinberg 1967, p. 32). In retrospect, Johnson's remarks, and the responses to them from within and without the biomedical research community, was but one in a series of events and policy decisions at the end of the 1960's that signalled the start of a new fiscal and political era for biomedical research. After two decades of burgeoning growth, with the federal government as its major moral and financial supporter, the research enterprise faced new deliveryoriented competitors such as Medicare for the nation's health dollars (Berliner and Kennedy 1970). A series of critical questions began to be asked, by many sectors of society: about how research priorities were being or should be decided, about the policy, or lack thereof, that had guided federally funded research efforts, and, as epitomized by President Johnson's statement, about what the "health payoffs" had been from the massive amounts of money, equipment, facilities, and manpower devoted to biomedical research in the years since World War 11.

4 These and other questions about biomedical research have continued to be asked, with increasing demands for answers, in the 1970's. Thus, for example, the Department of Health, Education, and Welfare's "Forward Plan for Health," dealing with fiscal years 19761980, noted that:

While there is no serious challenge to the assertion that a major federal role in the health industry is the support of basic biomedical and behavioral research, there are growing concerns as to the size and direction of that investment. For example, there are current questions about how priorities are set for biomedical research programs, why the cost of doing research is climbing so rapidly, what the appropriate relation should be between research and health service needs, what the effect of increasing pressure for targeted programs is, and whether there is sufficient "balance" between and around the various investment targets in the research portfolio. (Quoted in Culliton 1974, p. 617)

Given the long history of concerns about defining the "nature" and "proper methods" of science, and the more recent social and political history of biomedical research, it is easy to understand why there has been of

reiationsniP between "basic and applied" research. And, as one reviews the literature that this preoccupation has generated, one appreciates Alvin Weinberg's cautionary words a decade ago: "that the question about the basic-applied relationship is once more asked in sharp and urgent terms, particularly with respect to biomedical research, by no means implies that new or particularly cogent insights have been attained" (Weinberg 1967, p. 33).

Indeed, especially when viewed in historical compass, much of the discussion about the fundamental-categorical nature of modern biomedical research is reminiscent of the oft-told Hindu fable of the six blind men who examined an elephant. The first man, failing against the elephant's side, bawled that an elephant is very like a wall; a second, seizing a leg, declared the elephant to be a kind of tree; another, grasping a tusk, held it to be a spear; the fourth, feeling the trunk, knew it was like a snake; the fifth man, feeling an ear, said the elephant was like a fan; and the last man, holding the tail, pronounced it just a rope. Each statement about the elephant was a fair inference, but in sum they did not hang together.

As they first approach the "biomedical research elephant," scientists and laymen alike generally feel they will have little trouble in identifying a given part of the elephant as "basic" or "applied." For if one views research in any area of science as involving a spectrum that runs from "very basic" to "very applied," it seems evident that examples at either end of the spectrum should not be hard to come by. But biomedical research, a hybrid word fusing biological and medical, virtually by definition involves human health/disease-related objectives, however distant. It is a research enterprise that fits well a conception of science that philosopher Lewis Feuer has called "predominantly that of the utilitarian hedonist; the pursuit of science, apart from joy in itself, is an instrument for the improvement of the lot of mankind" (Feuer 1963, p. 15).

For such reasons, even those involved in a given piece of biomedical research may find it difficult to apply one or another label to their activity. A simple but striking discussion of this difficulty was given by Beveridge in The Art of Scientific Investigation. Research, he observed, is commonly divided into "applied" and "pure."

This classification is arbitrary and loose, but what is usually meant is that applied research is a delib tance, in contradiction to pure research done to gain knowledge for its own sake ... However, often the distinction between pure and applied research is a superficial one as it may merely depend on whether or not the subject investigated is one of practical importance. For example, the investigation of the life cycle of a protozoon in a pond is pure research, but if the protozoon studied is a parasite of man or domestic animal the research would be termed applied. A more fundamental differentiation, which corresponds only very roughly with the applied and pure classification is (a) that in which the objective is given and the means of obtaining it are sought, and (b) that in which the discovery is first made and then a use for it is sought. (Beveridge 1957, pp. 168-69.)

Implicitly or explicitly, most commentators on the basic-applied biomedicine issue end up, like Beveridge, agreeing that there is at best an elusive dividing line among types of biomedical research, and that many different elements can enter into a given definition of what part of the elephant is being grasped. The definition of a research project as basic or applied, for example, may vary according to whether one's primary

5 rame of reference is the problem being investigated, the objective of the researcher, the locus and organizational form of the research, or the objective of the research funder.

The struggle to carve up the biomedical research elephant according to such variables has produced in the past decade a profusion of studies, hearings, and reports and a sometimes bewildering array of terms. Thus, one can read discussions of basic research, fundamental research, intrinsic basic research, mission-oriented basic research, and non-mission-oriented basic research, and how these endeavors may differ from applied research, mission oriented research, targeted research, programmatic research, systematic research, and categorical research. And, in virtually every such discussion, there are caveats about how difficult it is, in practice, to apply these categories, about the complex feedbacks that occur between the various suggested types, and about t e continuum that is biomedical research (see, for example, Beecher, 1960; Bode 1965; Brooks 1965; Comroe and Dripps 1974, 1976; Frederickson 1977; Horsfall 1965; Kistiakowsky 1965; Shannon 1967; Stewart 1965, 1967). If the many past and present commentators on the of biomedical research could be gathered together reach a consensus about the basic-applied argument, suspect that in the final analysis they would concur h a statement by Paul A. Weiss, whose own investigations into morphogenesis have ranged from submicropic cellular biology to surgical methods for nerve eneration.

Unbroken lines of developmental changes (in science) are apt to go unnoticed by those most closely and continuously involved in them, and it usually is left to the historians later to trace them and package them artificially into separate epochs, stages and phases. A similar artifact is the customary categorical distinction between "basic" and "applied" research. No more realistic is the conceptual separation between "theory" and "practice". . All such distinctions are a matter of degrees of interest and focus and varying proportions in the mixtures of methodologies applied, but certainly are not properties of the subjects under study. As nature knows no pigeonholes, so knowledge, and the research leading up to it, constitute unbroken continua. That i's to say, no borders, fence posts, or other signs of discontinuity are met along the roads from . . . the most elementary discoveries in the cellular and developmental biology of animals to the prevention and cure of human disease. Pigeonholing is plainly a managerial device for the convenience, expedience and efficiency in handling practical affairs; in the infinitely graded diversity of the real world, however, there is no counterpart for the labels that designate the various pigeonholes.

Of course, curious and purposeful investigators and practitioners alike ignore straight jackets to their thoughts and searchings imposed by extraneous formalisms. They shuttle freely between the "basic" and "applied" directions of research continuum. (Weiss 1971, pp. vii-ix)

As Weiss in part suggests, there are a number of pragmatic reasons for making pronouncements about the identity of whatever part of the biomedical research elephant one is grasping. Each utterance may be a fair inference for the time, place, and reasons it is made, but in sum it is hard to make a whole elephant out of them.

The discussion and categorization doubtless will continue, but it would be well to bear in mind their history. That history indicates that the efforts to categorize various types of research often have little relation to the actual doing of the research itself, nor that they will be of great utility in predicting where a given line of research will lead. As illustrated by the following two statements, this has long been realized by many investigators, and, more recently, by many who have sought to determine the shape of a national research policy.

Biochemist Sir Edward Mellanby, in 1935: "It is no more unlikely that discoveries of first-class scientific interest will result from work directed to the solution of practical problems of disease than that discoveries of first-class interest to medicine will result from the study of academic physiological problems." (Quoted in Platt 1956, p. 399.)

Report of the Research Staff on National Goals to the President, 1970: "The issue of the relevance of scientific research to social needs is much more complex than is often assumed. On the one hand, there is no serious research, no matter how theoretical or basic in intention, which does not have some potential for generating knowledge which can ultimately lead to some socially valuable application. On the other hand, the most deliberately utilitarian research, whether basic or applied, can yield results which have theoretical significance. The history of science is one of reciprocity between theory and experiment, between insight and application, and between knowledge and utility. It is misleading to conceive of a one-way relationship, or to speak of research oriented primarily to scientific knowledge in contrast to science undertaken for the sake of

6 its potentially useful applications, as though they could be independent activities. Whatever the primary motivation of the research project the results are likely to include both, in difficult-to-estimate proportions." (Toward Balanced Growth 1970, p. 102)

Floppy-eared Rabbits and the Cult of Serendipity

In his 1945 volume of autobiographical essays, the distinguished physiologist Walter B. Cannon included a chapter entitled "Gains from Serendipity." In it he discussed a long recurrent theme in literature on the nature of science, a theme addressed particularly by scientists themselves: the role of chance or accident in research new term; it had been coined by Horace walpoie neany two hundred years earlier, But, until Cannon's essay, serendipity was an almost unknown word, and the great American physiologist surely had no prevision of how it would become used and misused, and elevated into a quasi-philosophical concept about a, if not the, dominant characteristic of basic research.

In 1754, Cannon wrote, Horace Walpole, in a chatty letter to his friend Horace Mann, proposed adding a new word to our vocabulary, "serendipity." The word looks as if it might be of Latin origin. It is rarely used. It is not found in the abridged dictionaries. When I mentioned serendipity to one of my acquaintances and asked him if he could guess the meaning, he suggested that it probably designated a mental state combining serenity and stupidity an ingenious guess, but erroneous.

Walpole's proposal was based upon his reading of a fairy tale entitled The Three Princes of Serendio. Serendip, I may interject, was the ancient name of Ceylon. "As their highnesses traveled," so Walpole wrote, "they were always making discoveries, by accident or sagacity, of things which they were not in quest of." When the word is mentioned in dictionaries, therefore, it is said to designate the happy faculty, or luck, of finding unforeseen evidence of one's ideas or, with surprise, coming upon new objects or relations which were not being sought. (Cannon 1945, p. 68; italics added)

The trouble with the use of serendipity since 1945 is that few of those who employ it seem to have read Cannon, or his many predecessors who also commented on the role of chance or accident in scientific investigations. Thus, too often, serendipity is described, as in a popular book on the subject, as "the art of happy ceident," leaving one with the impression that scientific dvances often are totally fortuitious, and that scientists, like the three princes of Serendip, "just couldn't miss stumbling onto the most marvelous things even when they weren't searching for them" (Halacy 1967, pp. 9-10).

In more sophisticated statements, scientists may not be portrayed as people who blunder onto monumental discoveries. But, in discussions of the differences be research, with little or no attempt to say what is meant by ascribing a scientific advance as due to chance. Rather, one usually reads simple declarative statements to the effect that serendipity is a major avenue to new knowledge or discovery in basic research, and hence the directions or outcomes of basic research, to a large extent, are uncertain, unpredictable, or uncontrollable (in contrast, that is, to applied research, which is characterized as being a programmatic or systematic progression toward clearly specified objectives). One of innumerable such characterizations of basic research is found in the Department of Health, Education, and Welfare's Forward Plan for Health, FY 1977-1981, in the section on "relative investments in fundamental and applied research." "Effective 'targeting' of resources to particular disease processes requires an adequate fundamental science base to be effective, but articulate exposition of the often serendipitous process of scientific advance is absolutely essential to greater public understanding of this process" (Forward Plan 1975, p. 70).

What such cryptic allusions to serendipity leave out and they are critical omissions when the serendipitous nature of basic research is argued in policy discussions is what it means to say that chance or serendipity plays an important role in research. Yet, what it does mean has been said, many times, by sociologists, historians, and philosophers of science, and, most lucidly, by scientists themselves. The same year that Cannon wrote of serendipity, for example, Robert K. Merton drew the attention of sociologists of science to "the serendipity pattern," describing it as involving the "unexpected, anomalous and strategic datum which exerts pressure upon the investigator for a new direction of inquiry which extends theory." By strategic, Merton emphasized, he was "referring rather to what the observer brings to the datum than to the datum itself" (Merton 1957, p. 104). For sociologists and their readers, Merton's point about the importance of what the observer or investigator brings to his chance encounter was given substance by Barber and Fox's now classic study of "the case of the floppy-eared rabbits." Theirs was an account of "serendipity gained and

7 serendipity lost," exploring the reason why one clinical investigator pursued an unexpected observation: after rabbits received an intravenous injection of the proteolytic enzyme papain, their ears

about the same time, another researcher endently made the same observation, but his an example of serendipity lost. Primarily because his "research preconceptions and the occurence of or serendipitous phenomena in the same experital situation" he did not pursue the case of the py-eared rabbits, which eventually led to new knowlabout cartilaginous tissues (Barber and Fox 1957).

A central point about serendipity that emerges from sociological literature, then, is that while research

s involve the unexpected happening or chance nt, what happens as a result is not fortuitous, but ends, for many reasons, upon what the investigator ings to the occurrence of serendipity.

Scientists themselves have made this point repeatedly they have reflected on the role of chance in their rk. In an 1895 lecture on "accident in invention and iscovery," for example, the great German physicist d philosopher of science Ernst Mach said: But granting that the most important inventions are brought to man's notice accidentally and in ways that are beyond his foresight, yet it does not follow that accident alone is sufficient to produce an invention. The part which man plays i's by no means a passive one . . . In all such cases, the inventor is obliged to take note of the new fact, he must discover and grasp its advantageous feature, and must have the power to turn that feature to account in the realization of his purpose. He must isolate the new feature, impress it upon his memory, unite and interweave it with the rest of his thought; in short, he must possess the capacity to profit by experience...

The disclosure of new provinces of facts before unknown can only be brought about by accidental circumstances, under which are remarked facts that commonly go unnoticed. The achievement of the discoverer here consists in his sharpened attention, which detects the uncommon features of an occurrence and their determining conditions from their most evanescent marks, and discovers means of submitting them to exact and full observation. (Mach 1943, pp. 266, 270)

The same thesis about the investigator's critical role in ensuring gains from serendipity was stated briefly and elegantly by Louis Pasteur: "chance favors the prepared mind." And, as Cannon noted, "Even before Pasteur,

Joseph Henry, the American physicist, enunciated the same truth when he said, "The seeds of great discoveries are constantly floating around us, but they only take root in minds well prepared to receive them" (Cannon 1945, pp. 75-76).

Thus, as Beveridge wrote in describing and discussing a range of unexpected discoveries, "the history of discovery shows that chance plays an important part, but on the other hand it plays only one part even in those discoveries attributed to it. For this reason it is a misleading half-truth to refer to unexpected discoveries as 'chance discoveries' or 'accidental discoveries' (Beveridge 1957, p. 46).

Finally, if serendipity means chance occurrences external to an investigator, which leads to an "unexpected discovery" only if the investigator is prepared to notice, interpret, and act on the chance clue, is there any reason why "serendipity" should be confined to so-called "basic" research? The true meaning of "serendipitous discovery" suggests not. And, indeed, the range of examples of "chance discoveries" cited by various writers on the subject shows that they are not confined to any one area of inquiry (see for example Beveridge 1957; Cannon 1945; Mach 1943; Taton 1962). Thus, to cite a few examples, chance, coupled with keen observation and reason, entered into the discovery of electricity, the development of the wave theory of light, the invention of the battery and the ophthalmoscope, the discovery of sub-clinical conditions, penicillin, the principles of immunization with attenuated pathogens, and the invention of dynamite.

In sum, those who use, or more often misuse the word serendipity, would do well to remember its history and full meaning. If this were done, we might soon disband what Rend Dubos has called "the cult of serendipity." When W. B. Cannon borrowed the word serendipity from Horace Walpole, he used it merely to symbolize the fact that scientific investigators are likely to discover many interesting facts other than the ones they are looking for. Oddly enough, this simple concept has been given so much importance and dignity during the past few decades that it has become a dominant scientific philosophy. If one were to judge from much recent writing, even by some scientists, the justification for doing research on almost any subject is the

8 statistical chance of achieving by accident, useful and practical results. It would be out of place to discuss here the historical tallacies on Which this belief is based. I cannot refrain, however, from stating my view that the cult of serendipity is based on an erroneous interpretation of the history of science, and furthermore amounts to an abdication of intellectual and ethical responsibility. Serendipity is the equivalent of Stephen Vincent Benet's line, 'We don't know where we're going, but we're on our way." (Dubos 1967, p. 128) The Advancement of Knowledge for the Nation's Health: A Report to the President on the Research Programs of the National Institutes of Health. 1967. Prepared Under the Direction of the Office of Program Planning, National Institutes of Health. Washington, DC: Government Printing Office. Basic Research and National Goals. A Report to the Committee on Science and Astronautics, U. S. House of Representatives, by the National Academy of Sciences. 1965. Washington, DC: Government Printing Office.

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Culliton, B. 1974. "NIH: Robert Stone is in Trouble with

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Dubos, R. 1967. "Opportunities and Pitfalls." Research in the

Service of Man: Biomedical Knowledge, Development and Use. Washington, DC: Government Printing Office, pp. 125129.

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10 CHAPTER 2 LOUIS PASTEUR: "SCIENCE AND THE APPLICATIONS OF SCIENCE"

During a long and productive career, Louis Pasteur established himself as one of the most famous figures in the history of science, one whose researches, cutting across many fields of endeavor, had a profound impact upon our health and understanding of a range of fundamental physico-chemical and biological phenomena. Of the many topics which occupied Pasteur's attention during five decades, from the mid-1840's to the late 1880's, he perhaps is best known for his work on the causes and prevention of infectious diseases. In these studies, he succeeded admirably in identifying the microbes responsible for silkworm disease, anthrax, chicken cholera, and swine erysipelas, and in developing means to prevent these and other major agricultural, veterinary, and human diseases. Concomitantly, Pasteur's work on infectious diseases yielded basic techniques for research in microbiology, the establishment of the germ theory of disease, valuable insights into the physiology of microorganisms, and the first workable concepts of virulence and immunity.

An earlier and less familiar period in Pasteur's career, which we will examine in this chapter, was the research on "diseases" of wine and vinegar that occupied his attention from 1855-1865. One familiar result of his work during this time was the invention of "pasteurization," but that was only one of many remarkable accomplishments of this decade of intense work. Pasteur himself saw his fermentation research as the indispensable prelude to his justly famous medical research. But it resulted as well in a revolutionary new theory of fermentation and putrefaction, a solid disproof of spontaneous generation, and a new understanding of the importance and ubiquity of microorganisms in nature.

Our account of Pasteur's 1855-1865 work, however, will deal less with the outcomes of his experiments than with their beginnings. We will consider the debated question of why Pasteur chose this particular line of investigation, given his earlier interests and his ideas about the nature of life. And we will explore how Pasteur's feelings about the "proper conduct" of scientific research, and how tensions about the roles of and relationships between "applied" and "basic" research in the mid-19th century, bore upon Pasteur's own interpretation of his work.

Pasteur's work, as well as analyses of that work by Pasteur and by his chroniclers, epitomize the difficulties that can occur in attempting to unravel the relationships between various lines of research, whether historical or contemporaneous. His many biographers have treated Pasteur as a pure scientist who kept getting "sidetracked" onto applied problems, as a "physician without a degree," as a "free-lance of science," and as an applied scientist who made immensely fruitful fundamental discoveries. These various depictions, however, are no more ambivalent than Pasteur's own views, as reflected in his statements about the importance of his work on industrial problems and disease. At times, he argued that the pursuit of "practical applications of science" could be a snare for the "servant of pure science." Thus, shortly after he had spent a triumphant week at Emperor Napoleon I I I's palace in 1865, demonstrating his discoveries on the microbial causes of wine and silkworm diseases, Pasteur wrote to the Emperor that the scientist who works on practical problems "clutters up his life and thinking with preoccupations which paralyze his faculty for discovery," and he risks both "his peace of mind and the success of his investigations" (Pasteur, Correspondence //, 19 Dec. 1865, p. 237).

While in charge of the science faculties at Lille and at the E~cole Normale in Paris, Pasteur campaigned in favor of a strong emphasis on pure sciences in the curriculum. The student who mastered the theory, principles, and methods of pure science, he insisted on many occasions, would have no trouble turning his knowledge to applications in 5rance's time of need.' Yet, Pasteur himself taught very popular courses in applied chemistry and worked on a long series of practical problems. And, as we read in his letters to Napoleon III, the Emperor's Aide de Camp General Fave, and various ministers of education he often justified his requests for research funds by pointing to the immediate economic or medical significance of his investigations.

Pasteur had a favorite and much-quoted aphorism, that he used to underscore the primacy of pure science, the trees whose fruit were the applications of science.2

There does not exist a category of sciences to which we can give the name of 'applied sciences.' There are science and the applications of science. uy rasteur aoout --applieci science,'' it is important to realize that he

11 had a rather different understanding of the term from our own today, an understanding conditioned by the nature of and relationships between technology and science in the mid-19th century. 3 Pasteur considered the phrase "applied science" to be a "shocking" and "false" combination of words. For, as technology and science were viewed at the time, it suggested that the largely empirical discoveries of industry were more than a mere "collection of recipes" and rules-ofthumb; it suggested that they too could claim to rest on the rigorous methods, theories, and experiments that were seen as the foundations of pure science.

Pasteur's formulation about "science and the applications of science" also had the advantage of making his own work on industrial fermentations and the diseases of men, animals, wines, vinegars, and beers appear an indivisible part of his "purely scientific" work on crystals and spontaneous generation. It saved him from feeling defensive about the years he devoted to practical problems when he wished to regard himself as a "servant of pure science."

Pasteur's earliest research, before his work on fermentation, did not require him to cope with questions about the "applied/pure" nature of his work that would trouble him later in his career. As a student at Paris'

Ecole Normale Superieur from 1843-1846, he was infected by his professors' enthusiasm for the new science of crystallography, which then offered physicists and chemists one of the few ways to recognize and obtain pure laboratory samples of a substance and to gain 4 knowledge of molecular structures (Burke 1966) .

Pasteur's entry into the field of crystallography began at the end of 1846, when the chemist Auguste Laurent joined the laboratory where Pasteur was working as an assistant. Laurent asked Pasteur to assist him with some experiments, and Pasteur recalled that "one day it happened that M. Laurent -studying, if I mistake not, some tungstate of soda, perfectly crystallized and prepared from the directions of another chemist, whose results he was verifying - showed me through the microscope that this salt, apparently very pure, was evidently a mixture of three distinct kinds of crystals, easily recognizable with a little experience of crystalline forms. The lessons of our modest and excellent professor of mineralogy, M. Delafosse, had long since made me love crystallography; so. . .1 began to carefully study the formations of a very fine series of combinations, all very

Fasteur continued: "Another motive urged me to preter the study of those particular forms. M. cle la Provostaye had just published an almost complete work concerning them; this allowed me to compare as I went along my own observations with those, always so precise, of that clever scientist" (Merz 1965, pp. 404-405).

In the course of these researches, Pasteur became fascinated by the problem of explaining the relationship between common tartaric acid and its isomer, racemic acid (also called paratartaric acid), which possessed all the chemical properties of tartaric acid but which exhibited a strikingly different optical behavior in polarized lights Pasteur examined crystals of racemic and tartaric acid under the microscope with great care and discovered in 1848 that racemic acid was in fact a mixture of two crystals which were mirror-images of one another. Moreover, one of these two crystals was absolutely identical - in its crystalline geometry, its chemical properties, and its response to polarized light - to ordinary tartaric acid. From this discovery came the realization that the left- or right-handedness of a dis-symetrical (to use the word Pasteur coined to describe this special kind of asymmetry) crystal and its underlying molecular structure can affect its physical and chemical properties - the basis of stereochemistry (Alworth 1972).

Both the ordinary right-handed crystals of tartaric acid and the mixture of right-handed and left-handed tartaric acid crystals in racemic acid were produced as a by-product of the alcoholic fermentation of grapes. Tartaric acid was commonly found as crystals on barrels of wine, while racemic acid had only been found occasionally and in small quantities. In 1852 Pasteur spent a busy month visiting industrial refineries of tartaric acid in hopes of discovering the conditions under which the rare little tufts of racemic acid would form in the midst of the large tartaric acid crystals. He was able'to establish that only the crude mother-liquors of tartaric acid would produce racemic acid before the industrial processes of refinement took place, but he still could not say why racemic acid arose on the rare occasions that it did. Instead, he went back to his laboratory at Strasbourg, where he was now a professor of chemistry, and there succeeded in turning ordinary tartaric acid crystals into racemic acid by complicated chemical manipulations.

In his work on tartaric acid and many other organic compounds which also showed dissymetry, Pasteur

12 observed that only one kind of crystal was normally found when these compounds occurred as natural products of living things. Thus, the right-handed crystal of tartaric acid was commonplace; its left-handed mirrorimage - either alone or in the racemic acid - was exceptional. And, the right-handed one reacted differently with other optically active compounds from the left-handed counterpart.

Having demonstrated with chemical reactions that his structural principle of molecular dissymmetry influenced chemical affinities, Pasteur began to speculate about the cause of dissymmetry. Sharing the desire of many 19th century scientists to find universal forces or principles, Pasteur imagined that the dissymmetry of organic compounds reflected a cosmic or universal dissymmetry:

The universe is an asymmetric whole. I am inclined to think that life, as manifested to us, is a function of the asymmetry of the universe and of the consequences it produces. The universe is asymmetrical; for, if the whole of the bodies which compose the solar system moving with their individual movements were placed before a glass, the image in the glass could not be superposed upon the reality. Even the movement of solar light is asymmetrical. A luminous ray never strikes in a straight line upon the leaf where plant life creates organic matter. Terrestrial magnetism, the opposition which exists between the north and south poles in a magnet and between positive and negative electricity, are but resultants of asymmetrical actions and movements . . . Life is dominated by asymmetrical actions. I can even imagine that all living species are primordially, in their structure, in their external forms, functions of cosmic asymmetry" (Quoted in Dubos 1950, p. 111).

Viewing the rotation of the earth and its magnetic polarity as two possible dissymmetric influences on living matter, Pasteur began to devise experiments to see what happened to the dissymmetric chemistry of plants when the earth's magnetic forces were reversed by a solenoid or when the sun's rays appeared to move from west to east. Although his friends and mentors begged him not to waste his time on research that probably would lead nowhere, Pasteur had great hopes. His wife wrote to her father-in-law during this period that "Louis is rather too preoccupied with his experi

ments. You know that those he is undertaking this year ought to give us - if they succeed - a Newton or Galileo" (Pasteur, Correspondence /, 10 Nov. 1853, p. 324).

The experiments, however, did not go well, and Pasteur abandoned them for the time being. But he never rejected the idea of a universal dissymmetry manifested in the dissymmetry of living matter. Throughout his career he returned to the subject, discussed it in papers and public lectures, and picked up his abandoned experiments once more to distract himself from the bitterness of France's defeat in the Franco-Prussian War. At the end of his life he regretted that he had not pursued the idea further, that he had not explained the ultimate "cause of one of the greatest mysteries of nature" (Pasteur, Correspondence /, 7 Dec. 1853, p. 325; P. Vallery-Radot, 1957b, P. 185).

In September 1854, Pasteur accepted the position of professor of chemistry and dean of the new Faculty of Sciences at Lille. Lille was the industrial center of northern France, and the town itself had underwritten the new science school in hopes of training its young men in the theory and industrial applications of science. The teaching in the school stressed practical matters and thus "appeared to be the public's taste," Pasteur reported to his superior, the rector of the Academie de Douai, in 1855 (Pasteur, Correspondence /, 26 Jan. 1855, P. 359). Pasteur's course in chemistry attracted the largest number of students and auditors -nearly 250, he boasted to a friend. He was particularly pleased at the young men who had already finished their education and started to work in industry, yet came to the school to profit from studying in subjects most closely connected to their future careers. There was, for example, the son of a distillery owner who would take over his father's business in a few years; since he obviously needed to know more chemistry than physics or natural history, he was allowed to take the chemistry course by itself and to pay reduced fees.

Pasteur's mixed feelings about the relative importance of pure science and applied science are revealed in his correspondence and papers during his three years at Lille. In his inaugural address as dean, for instance, he first spoke eloquently to the citizens about the excitement their sons would feel when they were introduced to the daily utility- of science. "Where in your families will you find a young man whose curiosity and interest will not be immediately awakened when you put into his hands a potato, when with that potato he may Pasteur, "Discours," 7 Dec. 1854, Oeuvres V11, pp. 130131). But, he hastened to add, "God forbid that

13 theory should ever disappear from this teaching. We must not forget that theory is the mother of practice. Without theory, practice is just routine born of habit."

The Minister of Public Instruction, in fact, feared that Pasteur would put too much emphasis on theory at the expense of practical knowledge. Even while he praised Pasteur's teaching, the Minister urged him to keep the industrial needs of the region constantly in mind: "M. Pasteur must always guard against being carried away by his love for science, and he must not forget that the teaching of the Faculties, while keeping up with scientific theories, in order to produce useful results and extend its happy influence, should through the most numerous applications adapt itself to the real needs of the country" (Pasteur, Correspondence p. 374 n. 1).

Pasteur apparently heeded his superior's advice. His activities at Lille included an analysis of fertilizers at the request of the General Council of the Department du Nord, which saw it as an important piece of work in so rich an agricultural region, and also as an opportunity to popularize and increase the influence of the new Faculty. One Easter vacation was spent in Belgium visiting metallurgical factories "since my position as chemistry professor," he wrote his father, "requires I know a good deal about this" (Pasteur, Correspondence 1, 11 Jan. 1856, p. 386; Nov. 1856, p. 406). In 1855 and 1857 he offered a course on "chemistry applied to industries" which was received with great enthusiasm by both his audience and the Minister of Public Instruction. Every lesson was followed by a field trip to a local factory where Pasteur would discuss technical details and clarify the techniques by drawing pictures on the blackboard (Pasteur, "Compte Rendu des travaux cle la Faculte des Sciences cle Lille pendant I'annee scolaire, 1855-1856," Oeuvres V11, P. 144-145).

Pasteur put a special emphasis on teaching about and helping the sugar-beet industry of northern France. A large part of his course on applied chemistry was devoted to the details of the manufacture and refinement of sugar and alcohol from beet juice. He was impressed by the industry's eagerness to keep up with scientific innovations and to adopt new processes. But, he remarked in 1855, "a prejudice which must be a separate body of doctrines, when really the applications of the sciences are nothing but deductions from purely scientific discoveries" (Pasteur, Correspondence /, 15 Nov. 1855, pp. 382-383). Much of Pasteur's later career confirmed his belief that "applications are nothing but deductions from purely scientific discoveries." Yet, there is irony in the fact that this earliest formulation of his aphorism about applied science sh ould concern the industry whose practical problems were soon to stimulate Pasteur into entering the field of biology and there making fundamental new discov eries.

In 1856 Pasteur's interests in molecular dissymetry and his responsibilities as a teacher of the practical applications of science converged in the study of beet juice fermentations. A student of his, Emile Bigo, had tried to make vinegar from sugarbeet alcohol, but relying only on the standard reference works and his notes on Pasteur's lectures, the young man had little success. Pasteur wrote on his behalf to a friend, a physics professor at Nancy, to enquire about a method of preparing vinegar using beechwood shavings that Pasteur had seen at the friend's brother-in-law's house (Pasteur, Correspondence /, July 1856, p. 394-395). We do not know what became of young Bigo's experiments on vinegar in Pasteur's laboratory, but Pasteur's encouragement and interest in his attempts had important consequences. Bigo's father - the owner of a beet juice distillery in Lille - sought Pasteur's help on a problem he was having in his factory. The sugarbeet juice was not fermenting into alcohol, but into lactic acid.

Pasteur soon was so absorbed with the puzzle brought to him by the elder Bigo that he went to the factory every day; Madame Pasteur complained wryly that he was living "neck-deep in beet juice" (Pasteur, Correspondence /, 10 Dec. 1856, p. 412). Pasteur's early experiences with a microscope now proved its value. He spent long hours comparing samples of beet juice and testing one idea after another. On the first day he began studying the fermenting juice, he observed little globules which grew and budded in the liquid. In the samples of juice that were producing the unwanted lactic acid, he soon noticed another kind of globule, smaller and longer than the globule he found in the healthy vats. Grasping the practical implications of his observations, Pasteur told the Bigos to test the healthiness of the fermentation by watching the shape of the microscopic globules; if they were large and round, the fermentation was going well; if they became elongated, lactic acid fermentation was replacing the desired alcoholic fermentation (R. Vallery-Radot 1960, p. 79; P. Vallery-Radot, 1958a, p. 10).6

We have little information about this period in Pasteur's career, and it is not clear just when and how he realized that the two kinds of globules he saw in the fermenting beet juice were in fact living organisms and

14 the cause of the fermentations. His reports on his investigation to the Society of Science in Lille only described his chemical analyses of the fermentations, and not his microscopic studies. He told his students "how the ferment looks under the microscope" but did not suggest that the globules were alive and actively making the juice turn to alcohol or lactic acid (P. ValleryRadot, 1958a, p. 10). Pasteur, however, was predisposed to consider such an explanation, even though it directly opposed the two prevailing theories of fermentation championed by the most influential chemists of the day, Liebig and Berzelius.7 Berzelius regarded the yeast of alcoholic fermentation as simply a kind of chemical catalyst; Liebig believed that yeast caused fermentation by its disintegration and decomposition, which in turn disturbed the sugar molecules so that they broke down into alcohol. Pasteur, though, had long been convinced that the dissymmetry of organic molecules was somehow directly correlated with the fact that they were produced by living - not disintegrating - entities. In 1855, after giving up his romantic experiments on the ultimate cause of molecular dissymmetry, he had gone back to studying examples of organic compounds which exhibited optical activity. Amyl alcohol was a case which especially aroused his curiosity, because it did not behave according to the rule he had formulated for tartaric acid and similar compounds. Amyl alcohol showed optical activity, rotating polarized light like tartaric acid, but its crystals did not show the dissymmetric structures Pasteur expected.

Lille provided Pasteur with ample opportunity to study amyl alcohol, for it was a common by-product of several industrial fermentations, including the beet juice lactic acid fermentation which was giving M. Bigo such trouble. Pasteur did not take long to decide that Liebig's explanation of amyl alcohol's optical activity was untenable. Liebig argued that optically active sugar molecules, excited by the disintegration of the unstable yeast, broke down into amyl alcohol

molecules which preserved the optical activity of the precursor. Pasteur voiced his opposition to Liebig's views in an 1857 "Memoir on Lactic Acid Fermentation, " which he presented to the Society of Sciences, Agriculture, and Arts of Lille. In his Memoir, which Bulloch characterizes as epitomizing "the essential points of all Pasteur's work on fermentation, and indeed of bacteriology," Pasteur argued as follows (Bulloch 1938, p. 60).

The molecular constitution of sugars seems to me to be very different from that of amyl alcohol. If this alcohol, when active, originated from sugar, as all chemists agree, its optical activity would derive from that of sugar. I am loath to believe this, considering the present state of our knowledge, for every time that one tries to find the rotatory property of a substance in its derivatives, it promptly disappears. The fundamental molecular group must remain in some measure intact in the derivative if the latter is to continue optically active, a result that can be foreseen from my investigations, since the property of optical activity is entirely due to a dissymmetric arrangement of elementary atoms. But I think that if the molecular group of amyl alcohol does derive from sugar, it is too distantly connected to retain the dissymmetric arrangement of atoms. (Pasteur, "Memoire sur la Fermentation appele lactique," 1857, Oeuvres, /1, pp. 3-4; Conant 1952, p. 25) What then in the beet juice was responsible for altering the arrangement of the sugar molecule?, Pasteur asked.

Pasteur confessed that his preconceived ideas about the role of molecular dissymmetry in the "organization of living organisms" led him to examine the juice under the microscope for living organisms. In the grey nitrogenous scum on the top of the juice, he discerned "little globules or very short segmented filaments, isolated or in clusters," which he admitted would look very like tiny bits of disaggregated protein to anyone who was not forewarned! But he then invented ingenious techniques for isolating and growing these little globules in a pure state, and demonstrated that lactic acid fermentation took place whenever a trace of the living grey deposits was sown in a suitable mixture of alkaline liquid and sugar.

In his first paper on fermentation in 1857, Pasteur told his audience in Lille how his earlier study of amyl alcohol had made him ask about the process of fermentally prepared to Tind living organisms, how he isolated and grew the lactic acid ferment, and how he could always produce lactic fermentation (and no other kind) whenever he put the globules into the proper medium. To the naked eye, he said, this newly discovered ferment resembled in appearance and activity the well-known product of alcoholic fermentation, brewer's yeast. Each of the two ferments, however, was absolutely specific: pure lactic acid ferment never caused alcholic fermentation, and pure brewer's yeast never produced lactic acid. Moreover, the two throve in different conditions: brewer's yeast grew best and turned sugar to ethyl alcohol most efficiently in a neutral medium,while the globules of lactic acid ferment preferred an alkaline environment. Most important, the yeast or ferment had to be alive. The process of fermentation, Pasteur affirmed, was simply a manifestation of the

15 globules' living organization, development, and physiological activity, and not, as Liebig held, a sign of their death and putrefaction.

Not long after presenting this paper on lactic acid fermentation, Pasteur observed something that clearly demonstrated the intimate connection between fermentation and molecular dissymmetry. He noticed that a solution of racemic ammonia tartrate (ammonia paratartrate) lying about in his laboratory had become moldy and begun to ferment.8 Such accidents were common enough, but at this point any kind of fermentat(on wou/d have attracted Pasteur's eager attention. To his great satisfaction, he found that the mold was choosing between the two forms of ammonia tartrate, fermenting the right-handed one and leaving the lefthanded one alone. It was a far easier way to isolate the left-handed isomer than the elaborate chemical techniques Pasteur had worked out a few years before. He was so pleased with this elegant finding that he submitted a paper about it to the illustrious Academy of Sciences to be considered for the Prize in Experimental Physiology. He received the prize early in 1860 for his work on fermentation in general, but this observation won special mention from Claude Bernard and the other judges. When he reviewed his life work many years later, Pasteur ignored his interest in amyl alcohol and M. Bigo's request for help and made it sound as if his discovery of this signal fact was what had led him from crystallography to the study of fermentations (P. ValleryRadot 1958a, p. 12; Pasteur, "La Dissymetrie Moleculaire," 1883, Oeuvres /, p. 376).

or putretaction required its own peculiar microorgan,-Between 1857 and 1863, he published paper identifying these specific living agents of far,-,tation and describing the conditions they required" survival. One of these papers, published in 186tr particularly notable. In the course of systematk studying the products of lactic acid fermentef' Pasteur noticed that the microorganisms assocL' with the formation of butyric acid from lactic

behaved differently from the infusoria now famc' to him from a variety of fermentations. When he w~ --, ad the infusoria of the lactic acid ferment move a'-' in a drop of liquid under a coverslip, he could see io' cluster about the edges of the coverslip. But the but,, acid infusoria appeared to shun the edges of the cc' -'slip. He followed up this observation with experitpwhich demonstrated that the butyric acid ferment C& live in the absence of free oxygen, and that, in oxygen would kill the tiny microbes. Turning back,-, consider the oxygen needs of other ferments, Patt came to the conclusion that "fermentation was without air." Some microbes, like the butyric ferment, were strictly anaerobic (a word coined, Pasteur): they could live and produce butyric acid el in the absence of oxygen. Most microbes, though, aerobic: they preferred to live in the presence of oxy:` yet they too cou)d survive without oxygen by ferr1_1.:__ ing, by breaking down organic compounds in their environment to obtain the energy they needed to live.

Yeast, Pasteur found, can live either aerobically or anaerobically, and in studying the conversion of sugar into alcohol and yeast protoplasm by yeast grown with and without oxygen, he discovered what later was termed the "Pasteur effect": sugar is converted into yeast protoplasm more efficinetly under aerobic conditions than anaerobically, but, for complex reasons, the actual utilization of sugar is lowered.9 The Pasteur effect subsequently was demonstrated in freshly picked fruit by Pasteur in 1872, and then in frog muscle tissue by Paschutin in 1874. The mechanism of the Pasteur effect continues to intrigue physiologists and biochemists to this day, and it surely counts as one of the most interesting basic discoveries to come out of Pasteur's research on fermentation.

During these years in which Pasteur generalized the phenomenon of fermentation, he also worked hard on two closely related lines of research. In the fall of 1857, the important post of director of scientific research studies at his beloved 9cole Normale Sup6rieure in Paris. His official duties no longer required him to worry about the practical problems of industrial fermentations, but a similar problem of even greater economic importance was soon set before him: what caused the diseases of vinegars and wines?

Pasteur's earliest research on this subject may have been inspired by a kind of local patriotism. For in 1858 he wrote a friend that he was taking a microscope with him on his trip home to Arbois in order to study the grape-must disease, "which requires my presence in Arbois throughout the month of September" (the month of the grape harvest). The first spoiled wines Pasteur looked at were those of his native Jura vineyards, and in them he spied microorgnaisms similar to the lactic acid ferment he had discovered the year before (Pasteur, Correspondence //, 28 August 1858, pp. 35-36; "Introduction," Oeuvres, //1, p. V).

16 He did not, however, spend much time just then on the maladies of wine and vinegar, because the fundamental assumption of his experiments on fermentation was under attack, and Pasteur became embroiled in one of the now classic controversies in the history of science. In maintaining that each kind of fermentation and putrefaction was caused by a specific kind of living microorganism - the germ theory of fermentation Pasteur had implicitly ruled out the possiblility of spontaneous generation of microscopic life. For if microbes could come into life from the random jostling of organic matter in the course of decomposition and fermentation, then there was no sense to the specific actions of the ferments that Pasteur had watched with such care. In 1858, a professor of medicine in Rouen, Felix Pouchet, presented his "proofs" of spontaneous generation before the Academy of Sciences in Paris; a year later he described his experiments and metaphysical ideas about the spontaneous generation of microorganisms in an immense book, Heterogenie. Pasteur's direct involvement in the debate stirred up by Heterogenie began when Pouchet asked him in a private letter whether he thought the lactic acid ferment might not be spontaneously generated. Pasteur replied that there was not as yet enough evidence to say, and that Pouchet might have been too hasty in asserting the reality of spontaneous generation, given the ease with which microbes like the lactic acid ferment could contaminate the air and the broths of organic matter from which the microbes has seemingly been created (Farley and Geison 1974, p. 179).

After this polite response, Pasteur joined the debate in earnest - much against the advice of his friends. In 1860-1861 he published five papers on his spontaneous generation experiments, which have become justly famous for their elegance and meticulous technique. He drew out the necks of glass flasks of boiled broths into long curves which allowed air to flow in easily, but which caught the dust-motes ' germs, and spores in the air on the damp sides of the neck: the broths remained perfectly clear. He carried flasks of sterile broths high into the Alps to prove that, even in the mountain air, germs could contaminate the broths, and also that some samples of mountain air were completely germ-free. (Pasteur, "Memoires sur les corpuscles organisees qui existent clans I'atmosph6re," 1861, Oeuvres, //, pp. 210-294; Conant 1953).

Pasteur's carefully designed and executed experiments dealt several damaging blows to the theory of spontaneous generation, and the observations and experiments on which it rested. Pasteur, for example, demonstrated that micororganisms or their germs floated in the air, whereas Pouchet only claimed that the "eggs" or "germs," rather than the adult microbes, came into being spontaneously from decomposing organic matter. He showed too that air alone could not initiate the generation of living things, as Pouchet and others had argued, and that simple measures of heating and air filtration could prevent "organic broths" like those prepared by Pouchet from showing any signs of life.

The question of spontaneous generation was pit before a commission of the Academy of Sciences for a decisive judgment in 1864. The dramatic clarity of Pasteur's experiments, his scornful condemnations of Pouchet's logic and technique, his self-assured explanations of his own results, and finally Pouchet's affronted withdrawal from the official demonstrations of the major experiments, convinced the commission that spontaneous generation did not occur. Another round of the controversy, with the English physician, Henry Charlton Bastian, defending spontaneous generation, took place in the 1870s, but for the time being Pasteur could consider the question settled triumphantly in his favor and he could give more time to his work on the diseases of vinegars and wines (Duclaux 1920, pp. 109111; Farley and Geison, 1974, p. 161-198).

Pasteur's researches on the diseases of vinegar and wine were a logical but not a necessary sequel to his general studies of fermentation and spontaneous generation. In his Studies on the Mycoderms: The Role of These Plants in Acetic Fermentation, delivered at the Academy of Sciences in February 1862, Pasteur explained that he had long suspected that microscopic fungi were involved in the production of vinegar from wine (i.e., acetic acid from alcohol):

In the researches on fermentations which I have pursued for many years now, various indications led me to think that the mycoderms could not be alien to the formation of acetic acid. These indications multiplied and defined themselves more and more; I applied all my efforts to follow them up with direct experiments. (Oeuvres, ///, pp. 7-12)

Indeed, he had started noticing these "indications" even before he started his work on alcoholic and lactic fermentation. As we have seen, young Emile Bigo had tried to produce vinegar while working in Pasteur's lab at Lille in 1857, and Pasteur had then sought information from his friends about processes for making vinegar. It 'is clear from Pasteur's first report on acetic fermentation, to the Chemical Society of Paris on July 26, 1861, that he had fully expected what he found: a microscopic fungus, mycoderma, covered the wine's surface with a delicate transparent film and turned the alcohol into vinegar. The German process in which the

17 wine was poured over beechwood chips entirely depended on the presence of this fungus on the chips. The beechwood chips had no mysterious catalytic property, as Liebig and many other eminent chemists had maintained in their textbooks." Six months after this first report, Pasteur patented a reliable method for sowing the mycoderm into wine and then put the process into the public domain. No longer would the vinegar-makers of Orleans have to wait anxiously for the thin veil of the fungus to establish itself by chance.

Pasteur's long memoir of 1864 on acetic fermentation, vinegar, and the mycoderma simply elaborated the points he had made in the first paper, for by this time his interest had shifted to the maladies of wine. Pasteur's 1866 book, Studies on Wine, Its Diseases, and Causes Which Provoke Them, with new processes for conserving and aging wine, opened with a dedication to Napoleon I 11:

In the month of July 1863, the Emperor urged Me to turr. my researches toward the understanding of the diseases of wines. Directed by observations of detail which my studies on fermentations had suggested to me, I had already caught sight of the possibility of a worthwhile piece of work on this to which I have dedicated myself ever subject since with the thought of his concern for one of the greatest agricultural products of France and with the desire to respond to the kindness of an August patron. (Pasteur, Ittudes sur le vin, Oeuvres ///, pp. 112-113). Although Occupied With his work on fermentation and his experiments on spontaneous generation, Pasteur had begun thinking about the diseases of wines several years before the Emperor made his suggestion. In 1858. We have noted, he had examined diseased wines from his native countryside, and in 1859 he had justified his fermentation studies to the Minister of Public Instruction by pointing out a practical result of his research: healthy wine contained not only alcohol, but also hitherto unsuspected by-products of fermentation such as glycerine and succinic acid, which helped give wine its "pleasant properties." Then, in 1861, his friend, the chemist Balard, asked Pasteur to look at some diseased wines from the vineyards of Montpellier; Pasteur was pleased to recognize the micro-organisms he had seen in the Jura wines. And, of course, Pasteur's work on acetic fermentation was, in effect, a study of the commonest ailment of wines, their souring and turning to vinegar. But the Emperor's encouragement - and the financial support that it implied - made Pasteur set to work in earnest in.

During the wine harvests of 1863 and 1864, Pasteur and a small band of his favorite former students left Paris for the wine growing regions "to watch the fermentation practices . ., to study diseased wines on the spot, and to collect the observations and views of men competent in these matters" (Pasteur, Correspondence //, 14 July 1863, p. 125). As he had expected, he was soon able to associate every malady and evil taste of wine with its own microorganism. One turned the wine sour, another made it bitter, yet others made it ropy or oily. Given an apparently healthy sample of wine, he could predict from microscopic studies of its alien ferments among the yeast globules just how it would taste in a few days. At the same time, he showed that the only rationale for the traditional fear of air reaching the wine was that the air could carry in the germs of these diseases; otherwise, oxygen actually helped healthy wine to mature in flavor and color. Moreover, just like the yeast, the undesirable ferments were normally present on the grapes themselves. If the diseases were to be prevented, the parasitic microorganisms had to be killed or weakened without destroying the wine at the same time.

Pasteur first experimented with tasteless antiseptic agents (inorganic phosphates and sulphates), but with no great success. He then turned to heat, for his experiments on spontaneous generation had proven that heat could kill microbes. Although centuries of tradition forbade heating wine, Pasteur tried the experiment and announced the happy results to the Academy of Sciences in May 1865, less than two years after acting on the Emperor's suggestion. Wine heated to 50-60*C, well below boiling, did not lose its flavor or color and it did not spoil (Duclaux 1920, pp. 141-144; Pasteur, "Nouvelles observations au sujet de la conservation des vins," 1865, Oeuvres ///, pp. 418-422). He patented the technique of heating the wine and put the process in the public domain, as he had done with his vinegar processes. The technique was quickly dubbed "pasteurization" and used for all kinds of perishable foods and liquids. That fall the Emperor invited Pasteur to spend a week at the palace, where the scientist gave himself the pleasure of showing Napoleon III that the microorganisms of wine diseases could be found even in bottles from the Imperial cellars (Pasteur, Correspondence //, November-December 1865, pp. 216-238).

To us, it sounds odd to call an unwanted change in fermentation a "disease," but the usage goes back to antiquity. Indeed, men had often tried to cure diseased wines with the same remedies they used to cure their own ailments (Majno 1975, pp. 221-224). Linked to this was another traditional analogy: the corruptions of

18 contagious disease were like the "metamorphoses of fermentation" and putrefaction. In ancient medicine a whole class of diseases were labeled "putrid diseases," as Pasteur reminded the Academy of Sciences in 1863, and to him these connotations of "disease" and "fermentation" Were tremendously suggestive. As early as 1859, he was arguing to the Minister of Public Instruction and to the Emperor himself that fermentations, putrefactions, and contagious diseases played similar parts in the "unending circle of life and death" and that they owed their existence to similar causes (Pasteur, "Note remise au Ministre de I'Instruction publique et des cultes," 1859, Oeuvres ///, 1. 481). This became a constant theme in his progress reports to the Minister and Emperor during the early 1860's. However, even though Pasteur recognized the "interest and ability" of research on the general problem of infection by microorganisms, he did not entei the field of his own accord. Until 1865, using a strategy common to scientists seeking funds for their work, he was content to appeal to the possible medical applications of his work to justify to his sponsors the costs of his researches on fermentation and spontaneous generation. The year 1865, however, marked an important turning point in Pasteur's career. Until then he could consider himself a chemist who "happened" to work on problems that involved living matter. But, while Pasteur was still engrossed in the details of pasteurization, he was suddenly asked by his beloved teacher, J. B. Dumas, to investigate a disease of higher organisms. Pasteur's student and assistant, Emile Duclaux, vividly recalled the day when his master was asked to find out what was killing silkworms and ruining the economy of southern France.

. . . Pasteur, returning to the laboratory, said to me with some emotion in his voice: "Do you know what M. Dumas has just asked me to do? He ants me to go into the South and study the disease of silkworms." I do not recall my reply; probably it was that which he had made himself to his illustrious master: "is there then a disease of silkworms? And are there countries ruined by it? " (Duclaux 1920, p. 145)

Pasteur protested his complete ignorance of silkworms to Dumas, expressing astonishment, according to one anecdote , at learning that inside every silk cocoon there is a silkworm turning into a moth! Dumas only replied, "So much the better! For ideas you will have only those which shall come to you as the result of your own observations" (Dubos 1950, pp. 213-214).

From then on, Pasteur worked primarily on problems of infectious disease, first dealing with two epidemics which were simultaneously devastating the silkworm industry, then anthrax in cattle, fowl cholera, swine erysipelas, and finally rabies in dogs and people. The twenty years that Pasteur spent on medical research, years in which he contributed signally to the rise of medical bacteriology and the development of immunology, were a splendidly successful elaboration and application of the ideas and techniques he had worked out during the previous ten years of fermentation research.

The year 1865; then, is an appropriate place to close our account of Pasteur's early career, and to turn to a discussion of the pattern of his discoveries during the years of crystallographic and fermentation research. Pasteur's published scientific papers and lectures offer us many statements of his views on the subject. To the modern reader, Pasteur's papers - written for oral delivery before scientific societies - seem astonishingly informal and conversational in style, including incidental remarks that no scientist today would be able to publish. The reports of the experiments frequently lack the measurements and details of technique that other researchers would need to duplicate them, Pasteur prefaced many papers with comments about the line of thought or preconceived ideas that had led to the experiments at hand, and ended them with speculations about the ultimate significance of the results. Ren6 Dubos has wittily imagined how an editor of a scientific journal today would react if young Pasteur had sent him that first paper on lactic fermentation:

"Dear Dr. Pasteur," the editor would write, "you have observed a few interesting phenomena, but your account of them is almost useless for lack of precise description and of quantitative data. Furthermore, you would do well to dissociate more clearly than is done in your paper wellestablished facts from your personal opinions concerning the nature of life processes. Allow me to tell you, for your own good, that these romantic opinions are entirely out of place in a dignified scientific paper - enjoyable as they may be when heard over several glasses of beer or wine in the twilight of an evening's conversation." (Dubos 1958, p. 16)

Pasteur, however, used his autobiographical and metaphysical digressions to immense rhetorical effect, for such artless candor about the workings of a scientist's mind could not fail to charm his audiences. It is very easy to see from his papers why his lectures were so popular.

19 By continually harking back to his earlier work, Pasteur acted as his own historian. He often claimed that his life's work had been guided by the principle of molecular dissymmetry which he had discovered in his early crystallographic studies of racemic acid. In 1883 he wrote in a memorable passage: "Carried on, enchained should I say, by the almost inflexible logic of my studies, I have gone from investigations of crystallography and molecular chemistry to the study of ferments; I have been engrossed with the thought of admitting dissymmetry into chemical phenomena" (Pasteur, "La dissymetrie moleculaire," 1883, Oeuvres /. p. 736).

Similarly, when he jotted down an outline in 1877 for a possible book on "Studies on contagious or transmissible diseases," he reminded himself to include his 1860 memoir on alcoholic fermentation, his reports on anaerobic fermentation, and his studies of the diseases of wines and silkworms (Gieuvres, VI: intro., p. V). Because the germ theory of contagious disease was so clearly implied by these earlier researches, he believed they would make the best, the most logical introduction to the projected book. In his own reconstruction of his .researches, the immediate reason for Pasteur's actually commencing work on infectious disease - Dumas' urgent request that he study the dying silkworms seemed to be beside the point. Sooner or later, he implies, something would have provided a similar occasion for starting research on diseases of animals and men.

Given Pasteur's training and orientation as a nineteenth century French scientist, his Cartesian insistence that "logic" determined the sequence of his work is not surprising, and his retrospective arguments to that effect are very persuasive. His grandson's seven volume edition of Pasteur's collected works was designed to underscore the rational unity that Pasteur saw in his researches; each volume's introduction comments directly on the formidable logical chain that binds all of Pasteur's work together.

Ren6 Dubos, however, makes two cogent criticisms of Pasteur's belief that he was pushed by "an almost inflexible logic" to make the discoveries he did. First, Dubos points out, the origins of the ideas owe little to logic but a great deal to intuition, keen observation, and bold guessing. "In the work of Pasteur, logic is evident in the demonstration and exploitation of his discoveries rather than in their genius. It is the phase of his work devoted to the development of his ideas which makes the bulk of his long papers, and which gives the impression of orderly logical progression" (Dubos 1950, p. 362). Second, Dubos observes, the logical chain of ideas was far more flexible than Pasteur granted. "His career might have followed many other courses, each one of them as logical, and as compatible with the science of his time and with potentialities of his genius" (Dubos 1950, p. 377).

Dubos argues that Pasteur may have felt apologetic both to himself and to his contemporaries and posterity because he had failed to follow up his earliest ideas on molecular dissymmetry with the vigor and skill he brought to his other work. Perhaps this is why in the 1880's Pasteur implied so strongly that the chance observation of the fermenting tartrate solution, rather than the study of amyl alcohol and M. Bigo's beet juice, had carried him from crystallography to fermentation research. The fermenting tartrate solution was so good an example of the correlation of molecular dissymmetry with the process of life that it should have been the link in the logical chain of discovery. The studies of amyl alcohol, let alone the industrial problems of beet juice fermentation, were less direct, less reasonable, less satisfying intermediate steps and therefore harder to acknowledge.

There is yet another kind of "almost inflexible logic" that governed Pasteur's account of his work. To Pasteur, as we have seen, it was axiomatic that "pure" science preceded its applications. The very word "application" implied the pre-existence of pure science standing ready to be used. Furthermore, within the intellectual and scientific heritage of his time - a heritage that persists today - Pasteur viewed pure science, the establishm nt of theory upon sound experimental studies of natural phenomena, as a superior kind of activity; that was how a scientist ought to spend his limited time and energy. Consequently, Pasteur often ignored in his autobiographical remarks the immediate practical problems or motives that may have provided the occasion for taking up a line of research. If we had to rely entirely on Pasteur's published scientific papers and lectures we would never guess, for example, that the lactic acid ferment research had begun wiih his visits to a beet juice distillery. The logic of the relationship between science and its applications demanded that this commonplace problem could not be the starting point for a major scientific discovery. Instead something else, something that was unquestionably pure science, had to come first. The puzzle of amyl alcohol's peculiar optical activity, even though it meant Pasteur had to admit to working from preconceived ideas, was a more suitable beginning than an industrialist's request for help.

20 Once Pasteur had established the basic concept of the microbial cause of fermentation, it was easier for him to admit to working on practical questions: he was simply using the theory to explain everyday examples of the general phenomenon. Even so, it is interesting to see that he preferred to give only the most impressive reasons for straying from the road of pure science. He studied the diseases of wine, he says, partly because the Emperor suggested it, partly because France needed it. He apologized for undertaking the investigations on silkworm diseases: he was badly prepared for it, he doubted he could carry it "to a logical conclusion," he regretted abandoning the research on ferments and spontaneous generation so dear to his heart. But, again, it was a patriotic duty, and the Minister of Agriculture (he does not mention J. B. Dumas' request) had himself made the request (Lechevalier and Solotorovsky 1965, pp. 39-40). Nationalistic fervor also inspired his last contribution to the problems of fermentation. He had bitterly watched France's defeat in the Franco-Prussian War of 1870 and resolved to prove to the world that France could surpass Germany even at the thing Germany did best: with processes derived from the fecund principles of his research on wines, vinegars, and silkworm disease, he declared, France could produce a "beer of national revenge" superior to Germany's f i nest brew. 12

We must, in short, be wary of accepting Pasteur's version of the origins, motivations, and character of his research. His day-to-day correspondence and his laboratory notebooks provide more reliable evidence than his published papers. In his papers he was all too likely to make progress of his research appear to conform to his notions of the way science ought to proceed: always from theory to practice. No wonder his biographers cannot agree whether he was primarily a pure scientist distracted by practical concerns or a brilliant and lucky applied scientist. Pasteur's own historiography makes it difficult now to say just which of his many contributions to biology arid physiology and to medicine and industry were the result of research directed toward a problem of immediate human importance and which were the result of research inspired by scientific curiosity. Both kinds of research certainly fed into his most general scientific accomplishment, the founding of the discipline of microbiology, although his spectacular later achievements (joined to those of Robert Koch) in research on disease gave the new science a decidedly medical bent.

Nevertheless, the careful study of some of Pasteur's discoveries reveal where new insights into life processes flowed from research on a practical problem. His patriotically inspired studies of beer, for instance, although "based on the same principles" as his studies of wine and silkworm diseases, forced Pasteur to work out the biology of yeasts and to formulate his general theory of fermentation as "life without air." One particularly noteworthy result of these studies, given the contemporary debates on spontaneous generation and Darwin's theory of evolution, was the demonstration that one kind of microscopic mold could not turn spontaneously into another, as Pasteur and many others had once firmly believed (Duclaux 1920, pp. 193-197). Before then, Pasteur's studies of vinegar manufacture -especially his comparisons between the open barrel system of the Orlean industry and the beechwood chip system of the Germans - had yielded the information he needed to disprove part of Liebig's theory of fermentation.

The most important example, of course, is Pasteur's work on lactic and alcoholic fermentation in M. Bigo's beet juice distillery. His 1857 paper stated several fundamental new ideas in biology explicitly, and hinted at others. He asserted that fermentations are caused by microscopic living organisms; that fermentations are correlated with the development and organization of the tiny creatures, not with their death and decomposition; that each kind of fermentation is due to a specific kind of microorganism; and that each kind of microorganism needs a particular set of environmental conditions for its growth and reproduction. He gave ingenious methods for growing pure cultures of microorganisms, the technical prerequisite for any successful microbiological research. He commented on the specific differences in the microbes' vulnerability to changes in their surroundings - the acidity of the medium or the presence of an antiseptic like the oil of onion juice (and how, one wonders, did Pasteur come to think of trying onion juice?). He described the competition between microorganisms for organic nutrients. And, he implied that spontaneous generation was impossible, that even germs had parents like themselves - a conclusion that modern geneticists see as "the axiomatic foundation of molecular biology" (M. R. Pollock, in Monod and Borek 1971, p. 83; Handler 1970, p. 19). But, above all, Pasteur made it plain that microorganisms, until then little more than a curiosity of science, played a literally vital role in the economy of nature not just in the economy of France.

Notes Chapter 2

21 1 . See, for example, Pasteur's "Note sur 1'enseignement professional" (1863) and his "Porquoi la France We pas trouve d'hommes sup6rieurs; au moment du peril?" (1871), in Oeuvres 1111, pp. 187-190, 211-221.

2. Pasteur's meaning comes through most clearly in his earliest and least epigrammatic expression of these views. in 1855 he wrote a letter about the receptivity of the distilleries of Lille to science, and commented that they were handicapped by their belief that there was applied science, that the applications make up a body of doctrine. In truth, of course, Pasteur continued, the applications of science are only deductions from purely scientific discoveries (Correspondence /, p. 382-383, to the Rector of the Academia de Douai, 15 Nov. 1855). His biographers have relied on his later statements made in hi 1863 note on professional education addressed to the Mini:ter of Public Instruction, Victor Duruy (Oeuvres 1/11, p. 188) and his 1871 article "Pourquoi la France We pas trouve d'hommes supdrieurs au moment du peril?" (Oeuvres V11, p. 215). He used the aphorism once again in a speech on the difference between the taste of the grapes and the wine made from them at the Congres viticole et sericole de Lyon in 1872 (Oeuvres I//, p. 464).

3. For materials on the status of and relationships between science and technology in the 19th century see Morz 1965, especially Vols. I and 11; Singer at al. 1958; and Taton 1965.

4. Among the significant early developments in crystallography that formed a background to Pasteur's early researches was Malus' 1808 discovery that all reflected light is polarized: the vibrations of reflected light are not in all directions perpendicular to the light ray, as in ordinary light, but are restricted to one direction. The plane formed by the ray and its perpendicular vibrations was called the plane of polarization. Then ' in 1810, Hauy discovered small facets (or faces) on quartz crystals, facets which could not be predicted from the normal form of the crystal, a regular hexagonal prism bounded by two six-faced pyramids. Hauy hypothesized that crystals are composed of characteristic molecules arrayed in three-dimensional patterns, a conviction shared by Pasteur. To others, the tiny facets in the varied, complex, rough structures of crystals represented random flaws, not characteristic variations of structure.

From observations of polarized light passed through quartz crystals, Argo reached several conclusions in 1811, among them that quartz crystals could split white polarized light into several colored rays, which were deflected at different angles upon leaving the quartz crystal. Then, in 1813 Biot gave lawful form to these and many other physical phenomena of the deviation of polarized light by quartz crystals. One observation, which he studied with precise measurements, was that different quartz crystals deviated polarized light sometimes to the right and sometimes to the left. Finally, and importantly for Pasteur's later work, Blot discovered that certain solutions of organic processes, one such solution being tartaric acid, deviated the plane of polarized light. While inorganic crystals deviated polarized light, inorganic solutions never did. The organic solutions were unique. From these findings Biot concluded, though not stated so precisely, that rotation of the plane of polarization of light depended somehow on the crystal form of quartz, and on the molecular form of organic solutions.

5. In 1819, Mitscherlich discovered that chemical compounds having the same number of atoms, irrespective of the nature of those atoms, took the same crystal form, a phenomenon termed isomorphism. Its definition was later modified to state that compounds having the same number of atoms took roughly similar crystal forms. Then Liebig, in 1824, was one of several chemists who discovered that compounds having the same elements in the same numerical proportions could have entirely different qualities, a phenomenon named isomerism by Barzelius in 1830.

Tartar was a by-product of the fermentation of grapes. In 1770 Scheele, using the double salt of potassium and sodium tartrate, prepared the first organic acid, tartaric acid, which found wide use in the textile indus" and in medicine. Tartaric acid's remarkably similar cousin, racemic or paratartaric acid, was discovered by Gay-Lussac in 1826, in crystalline form mixed with crystals of ordinary tartaric acid during wine fermentation. The study of isomerism began to preoccupy chemists after Berzelius in 1830 found that the salts of tartaric and racemic acid were chemically identical. He then asked Mitscherlich to study the crystals of these salts in hopes that "should the forms be different, then the awkward difficulty of the dual relationship of bodies with the same composition would be solved in a simple and possibly correct manner" (Bernal 1953, PP. 188-190). Biot communicated Mitscherlich's results to the French Academia in a note in 1844. It was Pasteur's reading of this note in 1846, when he was a student at the 6ole Normale, that led him into his later famous studies of tartaric and racernic acid.

22 6. It is not clear from published accounts whether Pasteur (or the Bigos) believed that the round yeast-globules changed shape as the fermentation changed from alcoholic to lactic acid, or whether he realized that there were two kinds of globules corresponding to the two kinds of fermentation and that the longer globules were supplanting the round ones. In Pasteur's later work on silkworm diseases, he made recommendations to the silkworm growers long before he understood the nature of the disease. It is not impossible that he gave the Bigos a convenient rule-of-thumb to follow before he knew that the differentshaped globules were different kinds of living organisms.

7. The controversy over the nature of fermentation that raged during the 19th century, involving such eminent figures as Liebig, Berzolius, and Pasteur, was central to the debates among chemists and biologists about the nature of the differences between inorganic and organic matter, and to the debates between "vitalists" and "reductionists" about the nature of living matter. For accounts of the fermentation controversy see: Bulloch 1938, pp. 41-55; Conant 1952; Merz 1965, 1: pp. 191-218 and II: pp. 106-117, 387-396; Farley and Geison 1974.

8. The date on which Pasteur observed this is in dispute. Bulloch (1938, p. 60) gives 1858, the year Pasteur published his "Memoire sur ia fermentation do I'acide tartarique," Oeuvres //, p. 24-28. Dubos ~1950) says first 1854 (p. 41) and then 1857 (p. 106) without noticing the contradiction. R. Vallery-Radot (1919) gives no date but suggests by his narrative that the incident occurred in 1854. P. Vallery-Radot (0ouvres //, p. vi) gives 27 August 1857 as the date on which "Pasteur set in motion the experiment which showed that, when ammonia racemate started fermenting, the left-handed tartrate appeared and the righthanded tartrate decomposed." However, in his "The Story of Pasteur's Discovery" (1957a), he suggests that Pasteur had made the observation before he published the 1857 lactic acid fermentation paper, but did not Work on it until afterwards. The first mention of the observation in Pasteur's correspondence is in a letter to his mentor, the celebrated old chemist, J. B. Biot, 7 September 1957 (Correspondence 11, pp 427428 and notes), which makes the 27 August 1857 date most plausible.

9. On the discovery of the "Pasteur effect," see Lechevalier and Solotorovsky 1965, pp. 23-29; Pasteur, "Sur la fermentation visqueuse at la fermentation butVrique," 1861, Oeuvres //, pp. 134-136; and "Faits nouveaux pour servir a la connaissance do la th6orie des fermentations proprement dites," 1872, Oeuvres /1, pp. 387-394.

10, In this very brief account of the Pasteur-Pouchet debate, wo have had to leave aside several important scientific and political issues. Emile Duclaux (1920, pp. 109-111), Pasteur's student and collaborator, pointed out that, if Pouchet had not withdrawn, Pasteur would have found it hard to explain away Pouchet's experiments With hay broth: the spores of the hay bacillus readily withstood the preliminary boiling of the broth, and the subsequent growth of the bacillus looked very much like spontaneous generation.

Farley and Geison (1974) have shown that the French scientific establishment found Pasteur's disproof of spontaneous generation congenial on religious and political grounds. The commission itself, they note, was strongly biased in Pasteur's favor from the start; two members, for example, were teachers and close friends of Pasteur and one (Balard) had even suggested the use of swan-neck flasks for the experiments in spontaneous generation!

11. Liebig died in 1873, still believing that fermentation in general was a process of decomposition and that vinegar was produced by a catalysis caused by the beechwood chips.

12. See Oeuvres V, p. 5, and Geison 1974, p. 354. For a critical evaluation of the practical results of Pasteur's research on beer, see K16cher 1903, pp. 4-6, 9, 353.

Chapter 2 Bibliography

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Cuny, H. 1965. Louis Pasteur, The Man and his Theories. Transi. P. Evans. London: The Souvenir Press.

De Bary, A. 1887. Lectures on Bacteria, 2nd improved edit. Transl. H. E. F. Garnsey; rev. 1. B. Balfour. Oxford: Clarendon Press.

Delaunay, A. 1973. LInstitut Pasteur des Origines a Aujourdhui. Paris; Editions France-Empire.

Doetsch, N., Ed. 1960. Microbiology: Historical Contributions from 1776 to 1908. New Brunswick, N. J.: Rutgers University Press.

Dubos, R. 1950. Louis Pasteur, Freelance of Science. Boston: Little, Brown and Company.

Dubos, R. 1958. "Pasteur and Modern Science," Pasteur Fermentation Centennial, 1857-1957. New York: Charles Pfizer& Co., Inc., pp. 17-32.

Dubos, R. 1960. Pasteur and Modern Science. Garden City, New York: Anchor Books, Science Study Series.

Dubos, R. 1962. The Unseen World. New York: The Rockefeller Institute Press.

Duclaux, E. 1 erments et Maladies. (Cours professii a la Sourbonne an 1879-1880) Paris: G. Masson.

Duclaux, E. 1920. Pasteur, The History of a Mind. Transi. E. P. Smith and F. Hedges. Philadelphia: W. B. Saunders Go.

Duclaux, E. 1898. Traitb de Microbiologie: Tome I, Microbialogia Ghn6rale. Paris; Masson et Cie.

Duncan, J. E. 1975. "A Three-Dimensional Consideration of the Liebig-Pasteur Fermentation Controversy," Unpublished paper.

Farley, J., and G. L. Geison. 1974. "Science, Politics and Spontaneous Feneration in Nineteenth Century France: The Pasteur-Pouchet Debate." Bulletin of the History of Medicine 48: 161-198.

Flexner, S., and J. T. Flexner. 1941. William Henry Welch and the Heroic Age of American Medicine. New York: The Viking Press.

Fruton, J. S. 1972. Molecules and Life: Historical Essays on the Interplay of Chemistry and Biology. New York: Wiley-Inter science.

24 Geison, G. 1974. "Pasteur," in Dictionary of Scientific Biography, Ed. Charles C. Gillispie, Vol. X: 350-416. New York: Charles Scribner's Sons.

Handier, P., ed. 1970. Biology and the Future of Man. New York: Oxford University Press.

K16cher, A. 1903. Fermentation Organisms, A Laboratory Handbook. Transl. G. E. Allan, J. H. Miller. London: LongMans, Green, and Co.

Lechevalier, H. A., and M. Solotorovsky. 1965. Three Centuries of Microbiology. New York: McGraw-Hill.

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Merz, J. T. 1965. A History of European Thought in the Nineteenth Century. 4 vols. (1904-1912). New York: Dover Publications.

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Vallery-Radot, P. 1958 Pasteur as I Remember Him," The Pasteur Fermentation Centennial, 1857-1957. New York: Charles Pfizer & Co., Inc., pp. 185-187.

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25 CHAPTER 3 BERIBERI AND THE COENZYME FUNCTION OF VITAMIN B,

The bacteriological triumphs of Louis Pasteur, Rudolph Koch, and their followers so caught the imagination of scientists and doctors in the last two decades of the nineteenth century and early years of the twentieth century that it was almost impossible to imagine a disease caused by anything but microbes or poisons. Medical research was primarily directed at the difficult but immensely satisfying tasks of tracking down the micro-organisms responsible for a variety of infectious diseases, isolating microbial toxins, and preparing vaccines and antitoxins. In 1890, when the Dutch government commissioned a team of physicians to investigate a disease which had begun to devastate the Dutch East Indian colonies about twenty years earlier, everyone fully expected to find a bacterium or parasite or, just possibly, some kind*of poison. The slow realization that the disease, beriberi, was not caused by some tiny organism or a potent toxin, but rather by the lack of minute quantities of an unknown and indispensable nutrient in the diet, required a major shift in medical and biological thinking. Over the first four decades of this century, research on beriberi and on vitamin B, (thiamine)', both exemplified and helped to bring about remarkable changes in our assumptions about disease, nutrition, and the intimate workings of the living cell.

The number of vitamins, the variety of their physiological effects, and their confusing propensity for appearing in combinations make the history of their discovery a long and complicated story. R. R. Williams, who synthesized thiamine in 1936 and who then wrote the first book devoted to a single vitamin, used as a frontispiece to Vitamin 81 (Thiamin) and Its Use in Medicine a thick cable: each separate strand represented one of the various components of the vitamin B, complex as they had been laboriously unraveled by medical, physiological, and chemical research between 1897 and 1938.

In this chapterwe will trace only one of these strands, that involved with the discovery and identification of vitamin B, and then with the elucidation of its biochemical role, as a coenzyme which functions in the metabolic breakdown of sugars.

Disease, Biochemistry, and Nutrition: 1900

Many physicians reacted with scorn when Pasteur promulgated the germ theory of infectious disease in the 1860's and 70's, but by the last decade of the nineteenth century the medical profession had largely adopted the new ideas. By 1900, when microbiology was a wellestablished discipline, biochemistry was just beginning to emerge as a field. The word itself had hardly been invented, and the first journals, societies, university departments, and classes in biological chemistry or biochemistry were founded only in the first decade of the twentieth century.

Biochemistry's most obvious progenitor was physiological chemistry, but the problems that the new science addressed were in some ways more general than those of physiological chemistry - as the bio prefix implied and ir, other ways much more specific. In particular, the mixed crew of physiological and organic chemists, medical researchers, pathologists, bacteriologists, and physiologists who came to call themselves biochemists paid special attention to the chemical processes that occurred within the living cell. The development of the new field was given impetus by several discoveries in the 1890's which suggested that cells produce catalysts which perform the chemical reactions of oxidation, fermentation, respiration, and synthesis inside the cell. Catalysts, or enzymes, which could start or accelerate the breakdown of compounds outside the cell had long been known, but in 1897 scientists first learned that the enzyme of fermentation, zymase, could be extracted from ground-up cells and used to ferment sugars. This discovery, which earned Edward Buchner one of the earliest Nobel Prizes in Chemistry in 1907, showed that the chemical components of the cell could be studied piece-meal, that the cell was not a single structural unit which would lose all its physiological properties the moment its membrane was broken (Kohler 1970, pp. 162-178; Kohler 1971, pp, 35-61; Kohler 1973, pp. 181-196).

The two major problems that the new science of biochemistry set out to solve were the chemical structure and role of enzymes, and the chemical properties colloidal, jelly-like chemical compound of immense size and complexity, and of such fragility that it had to be studied intact. This mysterious substance somehow absorbed and incorporated nutrients, synthesized all manner of products, respired and fermented, grew and

26 reproduced. Biologists and chemists were daunted by protoplasm, and any attempt to analyze it was considered foolhardy by many.

However, Buchner's extraction of zymase as well as the work being done on toxins and antitoxins suggested to biochemists that at least some of the cell's chemical components did have what was then understood to be molecular structure. These compounds and the reactions in which they participated could be studied in cell extracts in the test tube. However, the difficulties that early biochemists faced were tremendous. Their extracts were crude; methods of extracting cell consitutents were harsh; techniques for analyzing the extracts were arduous and time-consuming. Even the basic chemical nature of enzymes was a matter of great debate, for many biologists and chemists did not believe that enzymes were proteins even after James Sumner in 1926 purified and crystallized the enzyme urease, and showed that it was a protein (Fruton 1972, p. 156 ff).

The first generation of biochemists spent little time studying nutrition on the cellular and intracellular level, although for at least a quarter of a century nutrition had been one field where physiology and organic chemistry had come together. In the late nineteenth century, research in nutrition had followed two main lines: determining the energy values of foodstuffs, and trying to devise the physiologically and economically optimal balance of "the nutritional trinity" of protein, carbohydrate, and fat (Armsby 1906, quoted in McCollum 1957, P. 153; lhde and Becker 1971, pp. 20-26). To early ments of human energy needs and of the caloric content of foods were extremely impressive, because they provided strong quantitative data that seemed to demon

Strate a "real measure of nutritional needs, independent of, and apparently superior to, considerations based upon chemical details (Hopkins 1929, pp. 211, 216). In contrast, analysis of the proportions of the elements in different foodstuffs to obtain a "proximate analysis" of their protein, carbohydrate, fat, mineral, and fiber content seemed to be leading nowhere, although over 55; lhde and Becker 1971, pp. 20-26).

Both kinds of nutritional research ignored a point which was becoming more and more obvious in practical animal feeding experiments: the theoretical categories of nutrients were too poorly defined to be useful. Even research on minerals, which were obviously chemically distinct and comparatively easy to study, was hampered by assumptions about the nature of life and protoplasm. Bunge, whose students turned out so many papers on mineral nutrients, firmly believed that animals could only use the more complicated organic mineral compounds produced by plants. Bunge's view followed from the general feeling that animal protoplasm was chemically so complicated and unique a substance that it could only make use of organic substances like itself. This assumption made Bunge ignore the implications of work done by two of his students, Lunin and Socin, who came close to proving the existence of unknown nutrients later named vitamins -.simply because the synthetic diets Lunin and Socin had tried on mice included inorganic salts rather than the vital organic compounds (Hopkins 1929, pp. 213-214).

Beriberi, Chickens, and Rice

The disease beriberi has long been known in the Far East, for there is a Chinese record of a beriberi epidemic dating from 2697 BC (Williams and Spies 1938, p. 17). In the early seventeenth century, a Dutch physician, Jacobus Bontius, described the symptoms of beriberi which he had observed in himself and his patients in Java, and a similar disease named ship beriberi had been known to attack Western sailors on long voyages (Bontius 1642, pp. xxi-xxiii, 106, 111). In the latter half of the nineteenth century, beriberi became a major public health problem in Asia. It became endemic in arkd was, P.%9e.6aU\( a9t ta devastate groups of people who were compelled to live, sleep' and work together, such as prisoners, sailors, hospand lunatic asylum patients, and mothers and tL71 infants. In any of the forms it took, beriberi ravaging disease. Some victims suffered from ar.-~ burning sensations on the skins of their feet and grave weakness or paralysis in the legs as they waa.' away to mere skeletons. For others, the emaciation muscular atrophy was disguised by the swellingsL, edema, Still others, babies especially, died suddenly, acute heart failure. Many patients suffered all the various manifestations in succession, and all too often the disease was fatal.

The military were particularly alarmed by the ravages of beriberi in army camps and on navy vessels. A young medical officer in the Japanese navy, K. Takaki, heard from his father how beriberi had killed many men of the Imperial Palace Guard in 1862, and in his own work at the Naval Hospital, Takaki sometimes found as many as three-quarters of the patients ill with beriberi. "Such conditions," Takaki wrote in 1906 when he explained why he had devoted so much of his life to the study and eradication of beriberi, "used to strike my heart cold when I came to think of the future of our Empire, because if such a state of health went on

27 without discovering the cause and treatment of beriberi, our navy would be of no use in time of need" (Takaki 1906, p. 1369; Williams 1961, pp. 19-25; Harris 1938, pp. 52-56). Takaki's fears were shared by the Dutch colonial authorities in Indonesia - who were losing so many soldiers to beriberi that they could not win a guerilla war against Surnatran rebels - and by the American colonial authorities in the Phillipines - who watched helplessly as a tenth of the native police force came down with beriberi every year (Jansen 1956, p. 260; Williams 1961, p. 4). For such reasons, governments thought it important to find out what caused and what might cure beriberi.

Since the disease obviously seemed to be contagious, its cause was assumed to be a micro-organism. The first task thus was to isolate the bacterium responsible, and Takaki spent five years (1875-1880) in England learning bacteriology for this purpose alone. The starting point for the ultimately successful Dutch investigation also was bacteriological. In 1886 the Dutch government appointed a pathologist, Pekelharing, and a neurologist, Winkler, as a commission to study the destruction of the peripheral nervous system by beriberi and to search for the infectious agent. Before going to Java, Pekelharing and Winkler stopped in Berlin to visit Koch's lab and learn about the latest developments in bacteriology. There they met Christian Eijkman, a young Dutch doctor on leave from the Dutch East Indies colonial army, who asked to join the commission as an assistant. In the colonies, the commission soon believed they had found a coccus in beriberi patients which could not be found in healthy people. Although they published this conclusion, Pekelharing still had doubts as to whether they really had isolated the bacterial cause of beriberi.

A young medical officer, J. van Eecke, who worked closely with the commission and did bacteriological research on his own, put it very bluntly: "Whether the micro-organism we found is the pathogen or not is a question that must be settled unequivocally. The short history of bacteriology is already over-rich in examples of premature conclusions" (van Eecke 1887, p. 85).

To decide the question according to the rigorous canons of bacteriology he had learned from Koch, Eijkman remained in Djarkarta as a civilian medical researcher after Pekelharing and Winkler went home to the Netherlands in 1888. Eijkman began by trying to give animals beriberi, inoculating them with infectious matter from beriberi patients in the hospital. For his experimental animals, he made a somewhat unorthodox but fortunate choice. He used chickens, probably because they were cheap, easy to obtain, and easy to care for, but also perhaps because he had heard that, during an outbreak of beriberi in the Moluccas, large numbers of the birds and hens had also suffered from the disease (Jansen 1959, pp. 70, 74). The bewildering variety of forms that beriberi took presented Eijkman with a problem: how would he know if his hens had caught beriberi? He decided to look for evidence of degeneration of the peripheral nerves (polyneuritis), a prominent and painful symptom of human beriberi. But for several months the hens showed no signs of anything resembling beriberi (Jansen 1956, pp. 260-65).

Like Pasteur, however, Eijkman was prepared to take advantage of accidents and "chance" observations. One day in 1889 he found his chickens sick with something that closely resembled beriberi. The signs of polyneuritis were especially plain: the unhappy birds staggered and collapsed, their wings drooped in partial paralysis, their heads and necks were pulled sharply backwards by peculiar muscular spasms. But it seemed unlikely that the inoculations were responsible, for the inoculated chickens and the uninoculated controls were equally sick and nearly three-fourths of each flock died. When Eijkman examined the dead birds, he found that both groups showed the distinctive signs of nerve degeneration that he knew so well from his microscopic studies of nerves from beriberi victims. Six months later, the few birds who survived what Eijkman cautiously called polyneuritis gallinarum (polyneuritis of chickens) recovered quite suddenly and did not relapse.

On investigating the sudden outbreak of polyneuritis and its equally sudden and mysterious disappearance, Eijkman discovered that the chickens' diet had been changed twice. When he began his unsuccubtions, the birds were being fed crude unmilled rice. Then the servant in charge of feeding them begged table scraps of polished white rice from the hospital; after a few weeks of eating this rice, the birds fell ill. Later, when a new hospital cook "refused to allow military rice to be taken for civilian chickens," their feed again consisted of the crude, unpolished rice (Eijkman 1929, p. 203). Soon afterwards the chickens got well and stayed healthy. Moreover, sick birds recovered when they were fed the rice polishings and rice germ. Clearly, something about the white rice caused polyneuritis gallinarum, and something in the red skin and germ of the unmilled rice prevented and cured the disease.

28 Eljkman's "lucky accident" and his simple test of his observation gave him a new freedom in his work on beriberi, for he could now produce the disease at will in his experimental animals. This still left him open, however, to the criticism that chicken polyneuritis was not the same disease as human beriberi, a problem that was met when Eijkman took advantage of a "natural experiment" with Javanese prisoners. He asked the medical inspector of the prison system to collect information about the incidence of beriberi in the island's jails and about the kinds of rice their 300,000 prisoners ate. The inquiry revealed that in jails where prisoners milled their own rice by hand according to their local custom, the inmates came down with beriberi at only 1/300th the rate found in jails where prisoners were given industrially milled polished white rice. Indeed, all but one of the 37 jails where prisoners prepared their own rice were completely free from beriberi (Eijkman 1929, pp. 205-206). To Eijkman, although not to the prison authorities or other experts in tropical medicine, the results seemed unequivocal.

When Eijkman tried to explain why polished rice should be so dangerous and unmilled rice with its germ and skin intact should be so beneficial, he fell back on the prevailing assumptions of bacteriology. If the cause of a disease was not a micro-organism, he reasoned, then there must be a toxin which was neutralized by something in the red skin and germ. Eijkman continued to believe for many years that the starchy white grain contained an active toxic substance, a belief strengthened by his showing in 1896 that beriberi was not caused by a dietary protein deficiency. 2

In 1896 Gerrit Grijns, a young surgeon who had served periodically as Eijkman's assistant since 1893, tory. The Dutch East Indies government asmuu to "investigate the physiological and pharmacological properties of the tannin contained in red rice, and possibly other constituents of this kind of rice which might require consideration in relation particularly to beriberi" (Grijns 1935, p. 1). He was assured that he should not restrict his research to this problem, but instead should extend Eijkman's results to discover more about the connection between diet and polyneuritis and about the anti-beriberi properties of red rice.

Grijins continued the work on chicken polyneuritis, first by finishing Eijkman's incomplete experiments on "salt-starvation" as a possible explanation for the harmful white rice diet. Over the next four years, he improved Eijkman's experimental techniques, and he explored many ideas. Did the origin of the rice make a difference? Was the loss of fats contained in the rice skin significant? Was the starch of rice, sago, and tapioca really positively harmful, as Eijkman had concluded in his 1895 and 1896 reports? What was the active substance in rice skin? Could other foods cure beriberi as effectively as rice skin and rice germ?

In casting about for other foods that prevented or cured beriberi, Grijns happened on a kind of pea, Phaseolus radiatus (Katjang idjoe in Malayan), which was often used in Java to supplement chicken-feed. The results of tests with this kind of pea in birds who were so ill with beriberi that they had to be fed by hand were striking; within three or four days they could feed themselves, and within ten days they could walk easily again. Both the skin and kernel of the pea were equally efficacious, but the peas lost their curative and preventive powers when cooked in steam at 120'C. The discovery of the peas' effectiveness led to a series of experiments which neatly disproved Eijkman's theory of toxins produced by starches, by showing that polyneuritis could be produced in pigeons and fowl with a diet that contained no starch or carbohydrate at all.

Grijns' report in 1901 on his five years of work with nearly two hundred fifty birds began by recapitulating and arguing against theories of a bacterial or toxic cause of beriberi. At the same time, he stressed that his work strengthened Eijkman's important finding that chicken polyneuritis and human beriberi were the same. He concluded with the first clear statement of the existenc: of previously unknown "protective substances" whose absence from the diet led to "partial starvation."

There occur in various natural foods, substances, which cannot be absent without serious injury to the peripheral nervous system. The distribution of these substances in the different food-stuffs is very unequal. Of those examined, phaseolus radiatus and cajanus indicus were the richest, and polished rice the poorest, in these substances. The separation of these substances meets with the difficulty that they are so easily disintegrated. This disintegration, which takes place in a damp, warm place, shows that they are very complex substances. They cannot be replaced by simple chemical compounds (Grijns 1935, p. 38).

To judge by the dates of Grijns' various experiments, ee had had serious doubts about Eijkman's toxin theory Within a year of starting his research, and he probably hypothesized the existence and distribution of the iprotective substance early in 1898. His experiments between 1898 and 1901, designed to clear up

29 details about the chemical constituents of substitues for rice skin and the rice skin itself, were founded on Grijns' belief that a new vital dietary substance was involved.

Griins' first paper had little effect when it was pub'i lished in 1901. Both Eijkman and he published their results in the medical journal of the Dutch East Indies and summarized them in the leading medical journal of the Netherlands; but their articles were in Dutch, which severely limited their audience. Moreover, as late as 1906 Eijkman was still hesitant about accepting Grijns' "partial starvation" theory, for he would not discard his own toxin hypothesis until he had repeated all the experiments on potato starch and peas (Eijkman 1906, pp. 156-58). (it thus is interesting to see how carefully Eijkman avoided discussing his long-held toxin and antidote theory in his 1929 Nobel Prize lecture; he only spoke about the "anti-neuritic principle" of the rice skin, not at all about the poisons in the starchy grain! ) Only two Norwegian researchers, Holst and Frolich, who were studying ship beriberi, tried to follow up Eijkman's and Grijns' research immediately. Believing that tests on mammals would be more instructive than tests on birds for explaining human pathology, they chose the guinea pig as their experimental animal. Their results were unexpected; they were able to produce scurvy, but not beriberi, and switched their research to take advantage of this finding (Holst 1907, pp. 619-633).

Although little attention was paid to Eijkman's and Grijns' work at the time, the problem of beriberi was not being ignored. During the first decade of the 20th century, several other physicians in Asia arrived more or less independently at the connection between a diet of polished rice and beriberi, and provided valuable newevidence forwhat would become the vitamin theory. Braddon, an English doctor, observed striking differences among four cultures living in Malaya in regard to their use of rice and their incidence of beriberi. The Malays who milled their rice at home by hand, the Tamils who parboiled their rice before removing the rice skin, and the Europeans who did not eat rice were al I f ree f rom beriberi, but the Ch inese who ate imported rice suffered severely from the disease. Braddon believed that a toxin, perhaps from a saprophyte, formed in white rice as it grew stale. His theory was tested by Fletcher on patients in a lunatic asylum, where nearly half the patients had beriberi and a quarter of those died of it. He confirmed the relationship between the rice diet and the disease, but left the explanation open.

In 1907, Fraser and Stanton tried different kinds of rice diets on Javanese laborers who were building roads in isolated areas of Malaya. After finding that a white rice diet did indeed produce beriberi, they tried something that, rather surprisingly, Eijkman and Grijns had not done: they tried to extract the hypothetical toxins from white rice. In addition, they showed that if an alcoholic extract were made of parboiled rice, the rice after extraction was no longer any better than milled white rice. But the extract could be fed along with polished rice and prevent the beriberi that the polished rice diet would usually cause. Fraser and Stanton concluded that the polished white rice lacked "some substance or substances essential for the normal metabolism of nerve tissues" - a clear statement of the deficiency theory (Williams 1961, pp. 43-49). Fraser and Stanton presented their work at the first meeting of the Far Eastern Association of Tropical Medicine in 1910, at which time the association resolved that: "sufficient evidence has been produced in support of the view that beriberi is associated with the continuous consumption of white (polished) rice as the staple article of diet, and the Association accordingly desires to bring this matter to the notice of the various governments concerned" (Williams 1961, pp. 48-49).

Fraser and Stanton's report was heard by an American, Vedder, who also knew of Eijkman's and Grijns' work. Vedder enthusiastically adopted Fraser and Stanton's deficiency theory because he had already seen that Griins' results implied a kind of nutritional deficiency that was simpler and more fundamental than that implied by Griins' phrase "protective substances." To help him find out exactly what the essential substances in extracts of rice skin or parboiled rice were, Vedder recruited a young chemist at the Manila Bureau of Science, R. R. Williams. Vedder's enthusiasm was contagious, and for years Williams worked in his spare time until he at last succeeded in isolating and synthesizing the active anti-beriberi principle (Williams 196 1, pp. 1, 48-50, 95 f f).

Rats, Milk, and Minimal Diets

While physicians in Asia were proving a Strong connection between disease and diet and formulating the new theory of nutritional deficiencies of unknown substances, a different kind of research in the United States and England yielded other important evidence of the need for accessory food factors. For these workers, the impetus for undertaking such research did not Come out Of a desire to understancl a particuiar disease,

30 although several of them did have practical applications in view. They hoped to determine the simplest diet for farm animats anci experimental animals and to use that knowledge as the basis for further nutritional experimentation. By 1906, there were already about a dozen papers scattered throughout the literature and published in at least four different languages, which described chemically simplified or synthetic diets. As a rule, the authors of these papers knew little, if anything, about one another's work, and very often they did not follow up on their own observations. Looking back, we can see that they were on the right track, but they had no way of being sure at the time.

The great French chemist, Dumas, for example, attempted to invent a substitute for mother's milk during the siege of Paris in 1870. The disastrous effects of his mixtures of emulsified fat and sweetened protein - in theory a complete food - led him to conclude that a vitally important ingredient was lacking, but Dumas did not try to identify the differences between real milk and his artificial concoction. The work by Lunin in 1880 and Socin in 1891 on inorganic minerals and salts in the diet also suggested that milk and egg yolks contain more than just the basic protein, fat, carbohydrate, and minerals. They used very small numbers of experimental animals, so their results were not conclusive; and their professor, Bunge, as we have mentioned, discouraged them on theoretical grounds from continuing (Ihde and Becker 1971, pp. 11-12). Ironically, the one scientist who did take Lunin and Socin's work seriously was C. A. Pekelharing, the Dutch pathologist who had sought a bacterial cause of beriberi in 1888. In 1905 he published the results of several years of research which demonstrated the existence of -an unknown substance in milk which even in very small quantities is of paramount importance to nourishment. If this substance is absent, the organism loses its power to assimilate the well-known principal parts of food. . Undoubtedly this substance not only occurs in milk, but in all sorts of food stuffs both of vegetable and animal origin" (Pekelharing 1905, p. 122).

In Great Britain the most important work on simplified diets at the beginning of the twentieth century was done by Frederick Gowland Hopkins, one of the most influential figures in the history of vitamin research and biochemistry. As a young man, Hopkins trained for a career as an "analyst," which then meant a professional analytic chemist. At the age of 28, he decided to study medicine and as a medical student began doing valuable research in physiological chemistry and pathology. As he recalled in his 1929 Nobel Prize address, however, his clinical work excited an interest in nutrition:

Early in my career I became convinced that current teaching concerning nutrition was inadequate, and while still a student in hospital in the earlier eighteen nineties I made up my mind that the part played by nutritional errors in the causation of disease was underrated. The current treatment of scurvy and rickets seemed to me to ignore the significance of the old recorded observations. I had then a great ambition to study those diseases from a nutritional standpoint (Hopkins 1929, p. 217).

Rather than going into clinical medicine, Hopkins took up an unexpected offer in 1898 to "develop ... teaching and research in the chemical side of physiology" at Cambridge (Needham and Baldwin 1949, p. 20). Sixteen years later, the university founded a chair of biochemistry, appointed Hopkins the first professor, and put him in charge of the new department of biochemistry. Hopkins' research in his first years at Cambridge concentrated on proteins and amino acids. "I realized," he said in 1929, "as did many others at the last century's close, that for a full understanding of so many other ,,Xcts of biochemistry, further knowledge of proteins then a prerequisite; and . . . I did my best to contribute AS to that knowledge" (Hopkins 1929, p. 217). By Pitalizing on his discovery of an impurity in acetic "-7id while teaching a laboratory session on proteins, .4opkins was able to isolate and identify trVptophane, amino acid whose existence had only been postuAtted before then. Hopkins followed this achievement ~y showing that the protein of maize, zein, lacked this ismino acid. More important still, he found that an Artificial diet which contained adequate amounts of fats, carbohVd rates, and minerals with zein as the only protein, could not keep mice alive; as soon as tryptophane was added to supplement the zein, the mice survived much longer. Hopkins realized clearly and quite early in his work on proteins that a variety of nutrients were required for health and life. In an interesting speech to the Society of Public Analysts in 1906, "The Analyst and the Medical Man," Hopkins stressed that:

No animal can live upon a mixture of pure protein, fat, and carbohydrate, and even when the necessary inorganic material is carefully supplied the animal still cannot flourish. The animal body s adjusted to live either upon plant tissues or on the tissues of other animals, and these contain countless substances other than the proteins, carbohydrates, and fats. Physiological evolution, I believe has made some of these well-nigh as essential a; are the basal constituents of diet. He cited lecithin as one such substance that was already known, and pointed to rickets and scurvy as diseases in which empirical dietary cures had been

31 found long ago. These illnesses and other less obvious or severe 11 nutritive errors" were, Hopkins insisted, certainly due to "minimal quantitative factors" in the diet. He asked the analytic chemists to help the doctors by identifying and isolating these "unknown substances with unknown properties, present in complex mixtures." His audience's response to this appeal was disappointing; only one chemist addressed himself to the question of diet and disease, and then simply to point how impossible difficult, expensive, and time-consuming such analysis of foodstuffs would be (Hopkins 1906, pp. 394-96, 401).

At the time of this speech, Hopkins had been using simplified diets to test the qualitative effects of different proteins and amino acids on the growth of young rats. Although the addition of tryptophane to zein length ened survival time, the rats still did not grow properly. By substituting casein (milk protein) for zein, by adding a small quantity of milk to the simplified diet, or by using yeast extracts to make the dull, tasteless diet more appetizing, he could bring the young rats' growth patterns back to normal. From 1906 to 1912 Hopkins tried to understand how these small changes in the diet could make so great a difference in the rats' growth. For a number of reasons, however, he published nothing and only gave a couple of talks on his experiments before 1912 (Hopkins 1929, p. 218; Dale 1948, pp. 130-131 ).3

While Hopkins moved from studies of the nutritive values of different proteins to the inadequacies of synthetic diets, American researchers at the Wisconsin and Connecticut agricultural stations undertook similar research. Like Hopkins, they began with the assumption that all proteins do not possess equal food value. At the Connecticut Agricultural Experiment Station in New Haven, Thomas Osborne began preparing pure vegetable proteins and analyzying their amino acid content from the 1890's on. A young chemist from Kansas, Elmer V. McCollum, came to Yale in 1904 to do graduate work in organic chemistry. He specialized in physiological chemistry, took courses on nutrition from Lafayette Mendel and Russell Chittenden, the leading American authorities, and worked on amino acid determinations at Osborne's laboratory. McCollum recalled hearing Mendel lecture on Hopkins' work on supplementing zein with tryptophane in 1906-07; he also remembered that none of his teachers ever mentioned beriberi, scurvy, rickets, or pellagra (McCollum 1953, p. 301).

In 1907, while Osborne and Mendel proceeded with tests of the nutritive values of their purified proteins, McCollum went to the University of Wisconsin's College of Agriculture and Research Station to perform chemical analysis of cattle feeds. McCollum did a thorough search of the literature on animal nutrition and collec-A accounts of other attempts to restrict diets in order t, determine the requirements for particular substances. He was "astonished to find that every effort which had been made to feed animals on such mixtures (of isolated proteins, carbohydrates, fats, and mineral-salt mixtures) had resulted in prompt failure of their health," and he realized then "that the most important discovery to be made in nutrition would be the elucidation of the cause or causes of these failures" (McCollum 1953, pp. 304-05).

For a number of reasons, ..ie cattle-feeding experi cal. And so, in 1907, he set up the first rat colony in America for nutritional studies. McCollum kept in touch with his Yale professors and in 1909 Mendel and Osborne also began using rats as experimental animals for their protein tests.

Like Hopkins, McCollum worried about the palatability of the insipid simplified diets he used on his rats, and tried to add flavor and variety to their diet. In so doing, he unwittingly saved his rats from vitamin deficiencies by allowing them to eat whey -contam i nated lactose and their own feces - both sources of vitamins not otherwise provided by the simplified diet. In a 1913 paper, McCollum and his co-worker, Margaret Davis, showed that fats, or something associated with fats, extracted by ether from butter or egg yolks, contained something necessary to the growth of young rats. As long as the diet consisted of casein, carbohydrates, salt, and lard, they were able to keep the rats alive and growing for a few weeks, after which the rats did not gain any more weight. But the addition of ether-extracts of butter or yolk to the diet at this point would set the rats growing rapidly (McCollum and Davis 1913).

Meanwhile, at the Connecticut research station, Osborne and Mendel had been working along similar lines; establishing a basic diet of "protein-free (skim~ milk," carbohydrates, salts, and lard, and then testing the effects of different proteins. But until 1912 they were unable to get rats to grow full size on their basic diet (Osborne and Mendel 1911, pp. 618-698). Then, in desperation, they reconsidered every constituent of milk, realized that all their milk substitutes and diets lacked the cream of milk, and tried replacing lard with butterfat. The results were so immediately successful that they began to write up the new experiments for publication (Osborne and Mendel 1913a, b). To their chagrin, however, their rivals McCollum and Davis won what had been a conscious race between the Connecticut and Wisconsin researchers. In the summer of 1913 Osborne wrote to Mendel, "I have just received the July issue of the Journal of Biological Chemistry and

32 notice McCollum's article in it, which I assume you have seen. If not, I might say that he could just as well have taken his data from our notebooks as from his" (Becker 1970, p. 158-59).

The "Vitarnine" Theory: 1911-1920

Hopkins, McCollum and Davis, and Osborne and Mendel gave a new direction to nutritional studies by factor in milk, by their demonstration of the value of rats as experimental animals, and by their use of growth rate as the measure of dietary deficiency. But much of the force of their papers came from their explicit recognition that their work and the research on beriberi and scurvy were closely allied. For this idea, they were indebted in some measure to Casimir Funk, a young Polish chemist working in England, who had begun to study the beriberi-preventing substance in rice skin and yeast in 1911. Funk's review of the literature, his bold speculations about the nature of deficiency diseases, and his invention of the word "vitamine" as a name for the mysterious food factors implicated in deficiency diseases, all argued that there was an underlying unity to the diverse studies of beriberi, scurvy, and the growthpromoting factor in milk. In effect, Funk did what Hopkins had failed to do with the insights so clearly expressed in the 1906 speech to the analysts: he provided a theory of nutrition and disease that was as dramatic and as amenable to scientific verification as Pasteur's germ theory of infectious disease. From 1912, when the new word first appeared in print, to the 1940's, vitamin research was as exciting a field as bacteriology had been in the late nineteenth and early twentieth century.

After receiving his Ph.D. in organic chemistry in 1904, and then studying under the noted biochemist, Gabriel Bertrand, at the Institut Pasteur in Paris, Funk did research on amino acids and proteins in synthetic diets for dogs with the physiological chemist, Abderhalden. In 1910, an English friend in Abderhalden's laboratory found Funk a job at the Lister Institute in London, where his attention was soon directed to the problem of beriberi (Harrow 1955, pp. 35-39).

C. J. Martin, the first director of the Lister Institute of Preventive Medicine, was interested in tropical diseases and encouraged his friend Braddon's work on beriberi in Malaya. Martin also was a close friend of Hopkins, and knowing Hopkins' research on tryptophane deficiency in zein, Martin guessed that the cause of beriberi might be some other sort of amino acid deficiency in polished rice (Chick 1956, pp. 134-39, 197-98). Since Funk had experience with amino acid determinations, Martin suggested that he analyze the amino acid content of the rice and rice skins Braddon had sent to the Institute (Harrow 1955, p. 39). At the time, Martin's amino acid hypothesis was as plausible as any other theory about beriberi - and there were a good many. Although there was considerable agreement by 1911 that eating too much polished rice caused beriberi (witness the resolution of the Far East Association of Tropical Medicine), the reason for the ill effects of polished rice and the good effects of the rice skin was by no means 'settled. In addition to the toxinantidote theory held by Eijkman, Braddon, and others, and the protective substance-deficiency theory held by Grijns and Fraser and Stanton, there were other reasonable suggestions: a deficiency of fat, a deficiency of phosphorus, a deficiency of nitrogen, an excess of carbohydrate, a lack of a particular phosphorus-containing substance called phytin (Williams 1961, pp. 14-15).

When Funk and the first of several collaborators, E. A. Cooper, set to work analyzing extracts from rice skins in 1911, they had very little idea what to look for. The extant literature, they remarked, gave very few clues to the chemical nature of the protective substance from rice polishings, except that it seemed to be neither a salt or a protein.

Cooper and Funk's first experiments sought to learn whether the substance could still cure beriberi after it was subjected to extensive chemical manipulations, whether it had a simple or complicated chemical structure, and whether it belonged to a known class of chemical compounds. They quickly showed that "it is very improbable that polyneuritis is the result of a deficiency in phosphorus compounds." The active extract they obtained also lacked carbohydrate or protein groups, although it did include a significant amount of nitrogen. And, as others had found, very small amounts of the extract sufficed to cure beriberi in pigeons (Cooper and Funk 1911, pp. 1266-67; Funk 1911, p. 400; Funk 1912a, p. 149).

Funk coined the word "vitamine"4 for his 1911 paper in the Journal of Physiology to describe the nitrogen-containing substance he and Cooper had extracted from yeast and rice skin. But the staff at the Lister Institute, to whom he was required to submit drafts of his research papers, and the editors of the Journal of Physiology disliked the new word. Their opposition was quite sensible: the substance had not been proved to be an amine or amino acid - as the word implied - and, in general, the indiscriminate coining of words in science led to confusion. Funk was not obliged, however, to show drafts of review articles to his

33 colleagues at the Institute, and at the suggestion of a fellow Polish scientist, he wrote an article on "The Etiology of Deficiency Disease" for the widely read Journal of State Medicine in 1912 (Harrow 1955, pp.

41-43). Here he was free to introduce and explain what he meant by his catchword, "vitamine." It is now known that all of these diseases (beriberi, polyneuritis, epidemic dropsy, scurvy, experimental scurvy in animals, infantile scurvy, ship beriberi, pellagra), with the exception of pellagra, can be prevented and cured by the addition of certain preventive substances; the deficient substances, which are of the nature of organic bases, we will call "vitamines;" and we will speak of a beriberi or scurvy vitamine, which means a substance preventing the particular disease (Funk 1912a, p. 164).

Although Funk did not give the etymology of "vitamine" in this paper, it clearly was a compound of vita, "life" in Latin, and amine, a nitrogenous base. Thus, a "vitamine" meant a nitrogenous amine which is necessary to life: a much larger claim than "a substance preventing the particular disease," and a claim that made Funk's colleagues at the Institute and on the Journal of Physiology uneasy. In later years Funk protested that he did not mean that all vitamines had to be amines, but this point was not clear in his early papers and caused considerable confusion (Funk 1922, p. 169, n. 1).

In his essay of 1912, Funk also surveyed the evidence for grouping human and experimental beriberi, scurvy, and ship beriberi under the general category of "deficiency diseases," and perspicaciously argued that pellagra and possibly rickets also belonged to this class although the vitamines involved were probably different from those of beriberi and scurvy. He told of the attempts by himself and other investigators to extract the active substance, the beriberi vitamine, from rice skins, yeast, and Katjang idjoe beans. Speculating on the possible chemical and metabolic relationships between the beriberi vitamine and the scurvy vitamine, it seemed likely to him that the animal body could transform the scurvy vitamine into the beriberi vitamine, but not vice-versa. And, based on what he knew of the experiments by Osborne and Mendel and by Hopkins with simplified diets supplemented by milk, Funk supposed "that the substance facilitating growth found in milk is similar, if not identical, with the vitamines described by me" (Funk 1912a, p. 169).

The reactions to Funk's work were mixed. In general, his attempts to isolate the active substance in rice skin were greeted with enthusiasm. Only a few weeks after t,ooper ana runK S preiiminary iii i i communication on rice-skin extracts in Lancet, a column in Lancet described how a German explorer in New Guinea had successfully prevented beriberi among the members of his expedition by eating a pottage of red rice and Katiang idjoe beans cooked together every day. The anonymous Lancet writer remarked on the new progress of knowledge about the disease:

In future we are to expect that, thanks to the work of Mr. E. A. Cooper and Dr. Casimir Funk (The Lancet, Nov. 4, p. 1266), the leader of an expedition will be able to take all the special substance required to keep beriberi away from his men in a one-ounce bottle in his pocket. Of such are the triumphs of medicine. (Anonymous 191 1c)

But the proposal of the new word, vitamine, and its theoretical implications aroused much controversy. During the next ten years Funk campaigned vigorously on behalf of his idea. In 1914 he published a long German monograph, Die Vitamine, which was revised and translated into English in 1922. Although he moved several times - from England to Canada, from Canada to the United States, then to Poland, to Paris, and back to the United States in 1939 - Funk continued to do research on vitamines and to publish papers on his work in English, German, and Polish (Harrow 1955, pp. 5598). Less than five years after Funk coined the word, "vitamine" became a widely used and accepted term in scientific and popular writing. In September 1915, for example, Scientific Amerkan published a condensation of an article on "Vitamines and Their Importance for the Maintenance of Health." A year later an editorial in Science, "A New Phase in the Science of Nutrition," described the progress of vitamine research and showed how well Funk's term caught on:

The word "vitamine" has come into our vocabulary since the latest dictionaries were published. Etymologically it means an amine that is essential to life, and it was coined by C. Funk as a generic name for a group of substances, of unknown chemical composition, small quantities of which appear to be a necessary constituent of a wholesome human diet . . . An absence or insufficiency of vitamines in the diet brings on diseases now known as "avitaminoses" or "deficiency diseases," of which scurvy and beriberi are the principal representatives. Science already recognizes two vitamines - viz., antiscorbutic vitamine, which

34 prevents beriberi in man and polyneuritis in b There may be others. The investigation of the ' vitamines has m great strides in the past two years. The sub is beginning to crop up in the newspapers ant general literature, not to mention the small of the dinner table, where everything on menu invites classification from the point view of the "vitaminologist." (Science 19 p. 453)

By the early 1920's writers were using the t metaphorically: "A book . . . . so full of the vitami of literature," "The vitamines of the spirit and. . . true religion" (Oxford English Dictionary Suppleme, 1933). But, although widely used, the word and t concepts it stood for still upset many scientists. late as 1948, the eminent physiologist and pharmac ogist, Sir Henry Dale, regretted that Hopkins had n used "the right, which would have been generall accorded to him (in view of his contribution to vitami research)" to suppress so inappropriate a term (Dal. 1948, p. 131). Osborne and Mendel, in their second 1913 paper, '7he Influence of Butter-fat on Growth," wrote that butterfat probably did contain "something analogous to the so-called vitamines which Funk considers to be necessary for life," but they saw some important objections to Funk's generalization. Without minimizing the importance of the new field of research and the new viewpoints in nutrition which are presented by these recent findings, we may nevertheless hesitate to accept the extreme generalizations which have already been proposed on the basis of the evidence obtained largely from the investigation of pathological conditions. . . It is still rather early to generalize on the role of accessory "vitamines" when the ideal conditions in respect to the familiar fundamental nutrients and inorganic salts adequate for prolonged maintenance are not completely solved (Osborne and Mendel 1913b, pp. 429-30).

They argued further that a substance which maintained health, like Funk's beriberi and scurvy vitamines, might be a very different kind of thing from their butterfat accessory factor which promoted normal growth. In any case, their butterfat growth -promot i ng substance was not nitrogenous - the one chemical characteristic Funk seemed to insist upon by his use of the suffix amine. Collum made similar criticisms in his popular ares orv nutrition in 1918. He disliked the vita 44ix because it implied that vitamines were more initial to life than, for example, the indispensable .no acids. And, McCollum felt, Funk had exaggerated number of deficiency diseases, erred in his chemical ription of the curative substances, and foolishly led the significance of the fat-soluble substance. Ae popularity of Funk's word, McCollum said bluntiy, s deplorable: "There has become fixed in the minds students of nutrition and of the reading public an together xtravagant idea regarding the importance the substances to which Funk gave the name 'vita ordnes' " (McCollum 1918, pp. 84, 113-14). Hopkins and Abderhalden preferred other terms to express the ,'lea of indispensable dietary constituents of undeter , 'pined chemical composition: "accessory food factors," food hormones," "nutramines" (Needham and Baldwin i1949, p. 166; Abderhalden 1919, p. 39).

Despite (or perhaps because of) these disagreements *bout words and the things they stood for, more and "ore scientists became interested in the nature of vitamins and in the practical applications of what McCollum in 1918 called "the newer knowledge of Onutrition." Funk's former colleagues at the Lister Institute (he had taken an appointment at the London Cancer Hospital in 1913) made vitamin research their main contribution to the war effort: they studied the quantitative distribution of vitamins in different foods; and, in response to an epidemic of beriberi among Australian soldiers stationed on Lemnos, they prepared vitamin-concentrates from yeast and eggs to supplement army rations. Funk's assistant, Jack C. Drummond, performed some early experiments on the effects of vitamins on tumor growth rates and worked with Hopkins on the wartime Food Committee of the Royal Society, making recommendations about food rationing and vitamin enrichment of margarine. In the United States, McCollum, with various collaborators, continued his research on rat diets, trying to distinguish among the various indispensable food complexes by their different physiological effects. R. R. Williams was given unofficial permission by his biochemist-chief at the Food and Drug Service to work part-time with Atherton Seidell on the isolation and synthesis of the beriberi vitamin, even though "at that time, of course, vitamins were not recognized as having a legitimate place in food chemistry" (McCollum 1918, passim; Williams 196 1, p. 107).

In the Netherlands and Java, Eijkman and his coworkers continued their studies of beriberi and the active substance in rice skins; similar research was underway in Japan. Pellagra and rickets, which Funk and Hopkins had guessed might be due to vitamin deficiencies, received new attention from physicians, scientists, and public health services in American and Britain.

In 1920 Jack Drummond helped lessen the remaining disagreement in the new field by proposing a reform of nomenclature. He acknowledged the great convenience of having a single word to name all of the "so-called accessory food factors" and noted the confusion that accompanied the proliferation of synonyms

35 for Funk's vitamines. But the word vitamine, despite its wide adoption by 1920, was still unfortunate because "the termination '-ine' is one strictly employed in chemical nomenclature to denote substances of a basic character, whereas there is no evidence which supports his (Funk's) idea that these indispensable dietary constituents are amines" (Drummond 1920, p. 660). Drummond suggested that the final e in "vitamine" should be dropped. The result, "vitamin," would fit under the Chemical Society's nomenclature rule, which allowed "a neutral substance of undefined composition to bear a name ending in '-in'." The various individual vitamins could then be called by the letters of the alphabet which had already been used to differentiate among them: vitamin A, vitamin B, vitamin C. Funk opposed the change because, he said in 1922, "1 still believe in the nitrogenous nature of these substances," and he continued to call them by the name he had invented until 1937 (Funk 1922, p. 39 n. 2; Harrow 1955, p. 200). Otherwise, the new spelling was adopted rapidly, although, as we shall see, the designations of individual vitamins presented other difficulties.

If the "young science of vitamins" needed further legitimation it was provided in 1929 by the award of the Nobel Prize in Medicine and Physiology to Christian Eijkman and Sir Frederick Gowland Hopkins for their "discoveries of the antineuritic and the growth-promoting vitamins .... which .... are foundation stones of the science of vitamins" (LiIjestrand 1929, p. 198). Hopkins used the occasion to recount the early history of vitamin studies, paying tribute to the foreshadowings of Lunin, Socin, and Pekelharing, noting Grijns' correct interpretation of Eijkman,s experiments on chicken beriberi, and assessing the work and writings of Casimir Funk.

The award to Hopkins must have made Funk bitterly angry. Three years before, Funk had protested in a letter " the discoverer of vitamines," that Hopkins' 1912 paper came "so late that it exerted a relatively small influence on the development of the whole subject," and that Hopkins' earlier work, described in the 1906 speech, had been unknown to other workers until 1912 (Funk 1926, pp. 455-56). Hopkins' conclusion in the Nobel Prize lecture that Funk had "not received too much, but too little credit for his vitamin research as a whole" was hardly consoling, since Hopkins still claimed priority for his own experiments and ideas on the physiological functions of vitamins (Hopkins 1929). As in all priority disputes, both sides had justification for their rival claims to recognition. It is idle for us to try to apportion credit or judge the relative significance of their various contributions, not to mention those of their predecessors like Pekelharing and Eijkman or their contemporaries like McCollum and Osborne. But we should underscore one point: the rapid acceptance of the idea of vitamins was probably as much due to Hopkins' and Funk's enthusiastic, skillful evangelizing as to the two biochemists' actual scientific work on the chemistry and physiology of accessory food factors (Becker 1970, pp. 159-161).

The Race for Vitamin B I

No doubt scientists would have tried to isolate, analyze, and synthesize the anti-beriberi factor in rice skins and the growth-facilitating factor in butterfat even if there had been no theory linking the two, for it was clear that each of these substances was of physiological and medical importance. But the vitamin hypothesis made it all the more imperative to obtain the substances in pure form. Only then could one know whether the crude extracts contained more than one active substance, whether vitamins did constitute a new class of chemical compounds (as Funk seemed to assert), and whether the various vitamins all performed similar physiological functions. At least four different research teams were engaged in the effort to isolate the beriberi vitamin, a task of very great difficulty. Then, once the pure vitamin had been isolated, it was possible to work out its correct structure. Once the structure was known, the synthesis of vitamin B I in turn was a comparatively simple albeit also lengthy task.

R. R. Williams, who was deeply involved with every stage of this endeavor, estimated in 1938 that no other substance in the history of biochemistry had cost so much to isolate and identify as vitamin B, : "The first several hundred thousand dollars" (Williams and Spies 1938, P. 138). That cost is only one measure of the importance, both theoretical and practical, that scientists attached to gaining knowledge of the anti-beriberi substance. The quest for vitamin B, served as a school for biochemists from 1912 to 1940. "To mention all the names of those who have participated in some phase of the project," Williams wrote, "is to call the roll of half the mature biochemists in England and the United States. The project bulked equally large upon the horizon of Dutch, Japanese . . . French (and, somewhat later, German) biochemistry. (Williams and Spies 1938, p. 138).

The high cost in labor, time, and materials was largely due to the nature of the vitamin itself. To those who worked on its isolation, the vitamin's ability to act effectively in very small doses raised two immediate

36 problems: the anti-beriberi factor was to be found in nature only in very small quantities, and its presence in crude extracts was hard to assay.5 The enormous quantities of rice skin or yeast that it took to yield even a tiny bit of the vitamin astonished everyone who worked on the problem. For example, Jansen and Donath, who carried on the long tradition of Dutch beriberi research in Eijkman's laboratory in Djarkarta, began with 100 kilograms of rice bran to obtain 100 milligrams of pure vitamin (Jansen 1956, pp. 274-277).

The problems involved both in extracting a tiny bit of vitamin from tons of rice bran or from yeast and in measuring its ability to cure beriberi were complicated by a confusion that was as much a dispute over theory as it was a problem with chemical techniques. During the first years of vitamin research, the leading researchers could not agree how many vitamins there were. Funk, for instance, believed for several years that only the beriberi vitamin and the scurvy vitamin fit his criteria for a vitamin. Accordingly, he at first rejected McCollum's fat-soluble growth factor because it did not cure beriberi (or any other known deficiency disease) and because it was not an amine (Funk 1922, p. 117; Funk 1925, pp. 157-58). McCollum, in turn, insisted that only his fat-soluble growth factor, "fat-soluble A", which did cure an eye disease, and Funk's anti-beriberi factor, "water-soluble B", were true vitamins. His early experiments led him to assert that scurvy, rickets, and pellagra were not vitamin deficiency diseases (McCollum 1918, pp. 30 ff). But the nature of vitamin B - as Drummond had renamed the beriberi vitamin and $1 water-soluble B" - was itself in question. McCollum showed in 1918 that "water-soluble B" was as necessary to the growth of rats as "fat-soluble A". Was vitamin B then a single substance with several distinct physiological functions, or was vitamin B a mixture of different active substances?

Early in the 1920's Eijkman and Jansen, among others, declared their belief that the water-soluble growth factor which could be extracted from yeast was not identical to the beriberi-preventing factor (Williams and Spies 1939, p. 130). Jansen and Donath clearly made this a working assumption in their choice of an assay for vitamin B - only the cure or prevention of beriberi could serve as a valid test for the presence of the beriberi vitamin. Their decision was vindicated in 1926 by Smith and Hendrick, who demonstrated that the anti-beriberi factor in yeast could not survive pro longed heat, while the rat-growth factor in yeast did retain its activity after heating (Williams and Spies 1938, pp. 130-31). Thus, vitamin B was not a single substance but rather a complex of at least two vitamins: the thermolabile, anti -neuritic factor (that would be called vitamin 131, aneurin, or Thiamine), and the thermostabile, growth-promoting factor (Dutcher 1928, 6 pp. 206-209).

Despite the many problems of technique and theory, Jansen and Donath succeeded in isolating aim ost pure crystals of the beriberi vitamin in 1926. Williams, who also was working intensively to isolate the vitamin, regarded this feat as a "landmark . . . . example of a systematically planned and executed pursuit of a chemical objective" (Williams and Spies 1938, p. 139). After eight years of labor, the-Dutch chemists obtained 100 milligrams (1/280 of an ounce) of the almost pure substance. They sent "some scores of milligrams" to Eijkman, who had the pleasure of confirming their claim that this substance was the vitamin and that astonishingly small amounts of it could cure or prevent beriberi in birds (Jansen 1956, p. 274-77).

Jansen and Donath's long and tedious work had shown that the anti-beriberi vitamin could be isolated from natural sources, and, they believed, they also had determined its correct empirical formula. Other groups immediately set out to repeat and modify the isolation process, and to check Jansen and Donath's analysis of thiamine's chemical components. In 1931, a team of chemists at Gottingen led by A. Windaus found that Jansen and Donath had made a critical error in their analysis: they had overlooked the presence of sulphur in the thiamine molecule. As Williams commented, "What a shock it must have been to Jansen and Donath to learn of this mistake after all the years of grueling work they had expended on the isolation! (Williams 1961, p. 117).

Two years later, in 1933, successful isolation was also reported by R. A. Peters and his colleagues in England, and by Williams' group in the United States. Williams' group confirmed Winclaus' empirical formula, and, by several innovations in the isolation process, succeeded in improving the yield of pure crystalline th iam ine by f our or f ive f old.

Now an intense race began to determine the structure of thiamine and then to synthesize the vitamin (see Williams 1961; Wuest 1962). The race was triggered not only by the desire of chemists to understand how thiamine was made and to synthesize it, gaining undeniable prestige in the process. The pharmaceutical industry knew that the commercial rewards, too, would be high for the company that acquired patent rights to manufacture the anti-beriberi vitamin. Thus, impelled by a variety of motives, three major groups of researchers and pharmaceutical companies pursued the structure and synthesis of thiamine: R. R. Williams

37 et al. in the U. S., at first independently, but later supported by Merck and Co.; A. R. Todd and F. Berjel in England, sponsored by the Swiss company, Hoffman-La Roche, Ltd; and, in Germany, H. Andersag and K. Westphal in the Elderfeld Laboratories of the giant chemical firm, 1. G. Farben.

Scientific priority in the race fell to R. R. Williams and his colleagues in the U. S., who first published the complete synthesis procedure in August 1936. Not having realized that his work on thiamine had significant commercial interest, Williams was surprised to find that he was engaged in a highly competitive race, and even more surprised to find himself embroiled in a legal dispute over patent rights with 1. G. Farben. Williams and his colleagues had assigned the patent to a nonprofit Research Corporation, to insure that the profits from the manufacture of vitamins would be used to support scientific research and to eradicate dietary diseases. The courts ultimately awarded the patent rights in the United States and Canada - which soon became the largest market for vitamin B I in the world to Williams et al., Research Corporati6n, and its two licensees, Merck and Co. and Hoffman-La Roche. After eight years of effort, we recall, Jansen and Donath in 1926 had finally extracted 100 milligrams of crystalline iniammu tivii, have envisaged that by 1950 the U. S. manufacturers of synthetic thiamine would be producing 100 metric tons a year!

Nomenclature continued to be a problem for several more years after thiamine had been analyzed and synthesized. Vitamin B, was a widely accepted term, but seemed to belong too much to the era of confusion over the heterogeneity of vitamin B. Jansen proposed "aneurin" as an abbreviation for "anti-neuritic vitamin," a word still used in the British Pharmacopeia. But the American Council of Chemistry and Pharmacy of the American Medical Association rejected this name because it made a therapeutic claim for the substance, and because it could be confused with "aneurism." The Japanese vitamin researchers tended to use the name

oryzanin," while R. A. Peters in England preferred torulin" - words that referred to the source of the vitamin in rice and yeast. In 1937 R. R. Williams suggested that, since "aneurin" had met with such objections in the United States, it might be convenient and acceptable to use a name which reflected the vitamin's chemical peculiarities: "thiamin," where the thiaprefix referred to the sulphur atom and thiazole ring (Williams and Spies 1938, pp. 134-35). Later, the American Chemical Society noted the presence of an amino group in the molecule and changed the name to "thiamine" - a small indirect victory for Casimir Funk's original nomenclature.

Thiamine, Coenzymes, and Carbohydrate Metabolism

While organic chemists were hard at work isolating, analyzing, and synthesizing pure vitamin B1, physiologists and biochemists were busy trying to understand the vitamin's role in metabolism. Unfortunately, the organic chemists and the biochemists and physiologists of the 1920s and 30s could not help each other with their tasks, for the chemical structure of the vitamin offered no clues to its functions, and vice versa. There was no chemical test for the presence of vitamin B I, for instance, that would determine its distribution in the tissues, and the low yields of the early isolation procedures forestalled any systematic physiological experimentation with the pure crystals. As a result, the concurrent investigations of the chemistry and physiology of vitamin B I did'not converge until 1937, when the newly synthesized vitamin was used to confirm the identity of thiamine with cocarboxylase, a coenzyme which had just been shown to play a major role in carbohydrate metabolism. postulated as the cause of berioen, scientists suggesting a variety of ways that such a factor might act in metabolism. Eijkman's original hypothesis was that it neutralized toxins produced by starches. The edema suffered by many beriberi patients made some observers think that the vitamin was involved in water metabolism, while others felt that the vitamin was involved in the body's ability to use phosphorus. Funk's discovery that the vitamin contained a pyrimicline ring suggested to him that the vitamin might have something to do with nucleic acids, although the functions of the latter were completely obscure at the time. The rapidly accumulating knowledge of hormones and their importance in metabolism made other early researchers, such as Hopkins, Schaumann, and Funk think that vitamins might work as "exogenous hormones," that is, hormones that had to be supplied by nutriments rather than by biosynthesis within the body. Somewhat akin to this idea was Hopkins' early view of "nutritive errors": the effects of the missing food factors were comparable to the "inborn errors of metabolism" described by his close friend, Sir Archibald Garrod (see Chapter 5). Thus, the accessory food factors might be necessary at particular stages of metabolic pathways, although Hopkins could not specify whether the factors served as catalysts or substrates (Hopkins 1906, p. 396). In 1911 Funk suggested that vitamins might serve as "mother-substances," precursors to other essential metabolites (Funk 1914, p. 6). Because such small amounts of vitamins had

38 such striking effects, Seidell among others speculated that they might be related to enzymes (Seidell 1924, p. 440).

Funk and others considered yet another possibility, which proved to be the best guess about the function of the beriberi vitamin. In 1914 Funk observed that if pigeons on a vitamin-free diet were fed extra carbohydrates, they developed polyneuritis more rapidly. Experiments on pigeons fed with various combinations of carbohydrates and vitamin extracts suggested that vitamin-free artificial diets with a high proportion of carbohydrates cause a "marked disturbance of the carbohydrate metabolism" which could be quickly reversed by the vitamin. Braddon and Cooper came to similar conclusions at the same time (Funk and Schbnborn 1914, pp. 328-33 1).

Although the idea that vitamin B, was somehow involved in carbohydrate metabolism gave a focus to research, it was not as helpful as the vitamin researchers must have hoped, for carbohydrate metabolism was Itself one of the central problems of biochemistry. common refrain in historical accounts of develop .ts in biochemistry is, "the process was turning to be much more complex than had been imagined years before" (Fruton 1972, p. 343; Peters 1939, 1071, note). In the case of carbohydrate metabolism thiamine, it took several decades before the varied rimental observations of fermentation, glycolysis, iration, and vitamin activity could be sorted into a the breakdown of sugar and the two quite different lines of biochemical work led to conclusion in 1936-37 that vitamin B, acted as a nzyme in the reactions of pyruvic acid, an important termediate in both the anaerobic fermentation of sugar alcohol and carbon dioxide, and the aerobic break own of sugar in respiration to carbon dioxide and ter. The research by Karl Lohmann, which culminated in the isolation of cocarboxylase and the recognition of this coenzyme as the phosphoric ester of thiamine, was part of a long, intensive investigation into what is now called the Embden-Meyerhof pathway: the anaerobic degradation of glucose to pyruvic acid, and thence to ethanol and carbon dioxide in yeast (fermentation), or to lactic acid in muscle (glycolysis). The second line of research, that by R. A. Peters and his collaborators, was specifically aimed at understanding the cell's need for thiamine: they wanted to pinpoint the lesion caused by the vitamin deficiency, but they also realized that their work ought to cast some light on the details of carbohydrate metabolism.

Karl Lohmann worked as a senior staff member in Otto Meyerhof's research laboratory from the mid-1920s to 1936, where his ability as a superb organic chemist beautifully complemented Meyerhof's keen scientific imagination in their studies of glycolysis, muscle contraction, and fermentation (Proc. Conf. Hist. Devel. ftenerg. 1973, pp. 70-73, 169). Among his many accomplishments, the achievement for which Lohmann is best known was his isolation of adenine triphosphate (ATP) in 1929. Later, ATP was shown to be the compound which trapped the free energy to drive other reactions in the cell. Although Lohmann recognized some of the implications for energy transport by ATP, he was chiefly interested in the compound as a coenzyme required for the transfer of phosphate to and from intermediates in the pathway of glycolysis and fermentation (Cori 1973, pp. 163, 166; Fruton 1972, pp. 366-69).

The question Lohmann wanted to answer once h had isolated ATP was: what does the coenzyme AT have to do with the coferment of zymase discovered b) Harden and Young in 1906 ?7 It was through the pursui of this question, in a series of complex biochemica studies, that Lohmann and Schuster in 1937 announced the purification of a new coenzyme, cocarboxylase, and proved that "the organic 'ground substance' of cocar boxylase, is diphosphorylated aneurin (vitamin Bj;' that is, "cocarboxylase is diphosphorylated aneurin (vitamin B0- (Lohmann and Schuster 1937, p. 300)

Unfortunately, it is not clear from Lohmann's ac count of his investigations when or how he first sus pected the identity of the coenzyme as thiamine diphos phate. The publicity that had been given to R. R Williams' recent analysis of thiamine's structure, and the subsequent race to synthesize the vitamin may well have alerted Lohmann to compare the two compounds. Obviously, only the availability of pure crystals of thiamine from natural sources and from Williams' synthesis made a definitive identification possible.

There was another reason, though, for Lohmann to think of his coenzyme in terms of vitamins. Between 1932 and 1935 Warburg, Kuhn, and Theorell had shown that riboflavin (vitamin B2 in one nomenclature) was the coenzyme for the so-called "yellow enzyme" which, according to Warburg, shuttled hydrogen from the oxidation of sugar to the respiratory chain to react with oxygen (Ball 1973, pp. 98-99). Lohmann mentioned the work on riboflavin in the discussion part of his cocarboxylase paper and drew attention to another similarity between the two vitamins: not only were they both the "ground-substances" of coenzymes, but they

39 also needed phosphate groups to be added onto the vitamin molecule before they could act as coenzymes. It was a point that the discoverer of ATP was bound to notice. It is also proof that Lohmann did not yet know that the nicotinic acid amide in Warburg's other coenzyme, DPN, was the anti-pellagra vitamin although Warburg himself had suggested this identity When he first crystallized nicotinic acid amide crystals from DPN in 1934, Warburg declared, "I am quite sure this will turn out to be a vitamin." But despite his research on the coenzyme function of riboflavin, Warburg was not interested enough in vitamins to follow up his hunch about nicotinic acid amide (Theorell 1962, p. 2). Thus, it was not until late 1937, after Lohmann and Schuster's paper on cocarboxylase, that Conrad Elvehiern published his identification of niacin as yet within so short a span of time of three members of the vitamin B complex as parts of coenzymes involved in major metabolic pathways suggested that all vitamins have coenzyme functions, setting in motion a great deal of new biochemical research.

The second major line of research leading to the identification of vitamin 131's coenzyme function was that pursued by R. A. Peters and his colleagues at Oxford. While Peters and Lohmann arrived, independently, at the same conclusions about thiamine's role in intermediary metabolism, their work was strikingly different in both techniques and goals. Karl Lohmann, as we have noted, was an organic chemist, whose work relating to vitamin B, was part of a complex series of basic biochemical analysis being conducted by Meyerhof's laboratory group. R. A. Peters, on the other hand, had been trained as a physician, and for personal and professional reasons "could never forget the hospital and the wish to improve care for people" (Peters, personal communication). Peters saw his research on thiamine as an attempt to explain the pathology of vitamin deficiency in biochemical rather than histological or anatomical terms. He introduced the striking phrase "biochemical lesion," to "crystallize the idea that pathological disturbances in tissues were initiated by changes in their biochemistry" (Peters 1963, p. 1). Although the ultimate aim of Peters' research was to pinpoint a derangement in a biochemical pathway, his use of pigeons as an assay forced him to keep in mind the gross consequences of the biochemical lesion upon the whole organism.

After completing his clinical studies, Peters served as a medical officer in France until he was recalled to England at the request of the eminent physiologist, Sir Joseph Bancroft, "to work on antidotes to gas poisoning" (Peters, personal communication). It was in this particular context of war-impelled research that Peters' attention was first directed to vitamin B1. In 1920, working in Hopkins' laboratory at Cambridge on the effects of poisons on protozoa, Peters found that his protozoa would not grow on their artificial medium unless an alcoholic extract of yeast was added, and thus his interest was directed to vitamin B. Beginning in 1922, and continuing after his appointment as professor of biochemistry at Oxford in 1923, Peters began the effort to isolate vitamin B, from bakers' yeast. to continue our work with yeast, because it would become necessary ultimately to be sure that the vitamin B, in yeast was the same compound as that in rice polishings. And, we realized early that the Jansen and Donath crystals were not quite pure" (Peters, personal communication). Thus, along with the Winclaus group in Germany and Williams and Seidell in the United States, Peters' group pursued and eventually accomplished the goal of obtaining pure vitamin B, extract from yeast.

From 1922/23 on, Peters states, his work on vitamin B 1 "was part of a larger plan," one inspired both by his clinical training and his work as a biochemist. "I thought that by isolating one factor dealing with a specific clinical condition, I could begin to clear up the muddle as to how many B factors existed" (Peters, personal communication). Within this framework, the problem of testing the vitamin activity of the yeast extract fractionations led Peters to the question: how did the vitamin deficiency produce the dramatic symptoms of beriberi? He and his colleagues felt that the cure of polyneuritic symptoms in pigeons was a more certain sign of vitamin activity than either the prevention of symptoms or the progress of normal growth. But, as Williams and others had objected, it was all too easy to produce false cures or remissions by injecting the sick birds with glucose and water. So, during the mid-1920s, Peters sought a reliable test of a cure. Opisthotonus, the convulsive neck spasm which pulls the head of a pigeon sharply back, seemed to be the most obvious and important clinical sign of a genuine vitamin deficiency. "Hence," Peters recalls, "I sat down to make the pigeon opisthotonus test quantitative and reliable. This I succeeded in doing by standardizing conditions of feeding, and by never using birds which took over a month to develop the head retraction, when other deficiencies seemed to intervene" (Peters, personal communication) ' If the opisthotonus appeared after a pigeon had lived for a couple of weeks on a polised rice diet, Peters found, it could often be relieved for a day or two by glucose and water. But, if the neck spasm re-appeared within a month or less on the deficient diet and could no longer be relieved by glucose, then the only cure was the vitamin (Peters 1963, pp. 6-8).

Study of the head retraction symptom, and their observations of pseudo-cures with glucose, led Peters

40 the next stage of research: the convulsive state induced by the deficiency drew my attention to the central nervous system and provided the stimulus to examine this for possibly enzymic changes; until then attention had been focused upon the peripheral nervous system, which had become emphasized wrongly through the extensive use of the word "Polyneuritic." (Peters 1963, pp. 8-9). he temporary "cures" produced by glucose suggested at the glucose might counteract a low blood sugar el in the sick birds. Comparison of the blood sugar level in sick and normal pigeons did not give any real answer, so Peters tried to lower the blood sugar in healthy birds by insulin injections. As others had also discovered, Peters reported in 1929, he found that ordinary doses of insulin had no effect upon the birds. Large doses generally gave convulsions .... to my surprise the convulsions exactly resembled those of avitaminosis . . . . So far as I know this has not been previously pointed out. In view of this fact, Mr. Kinnersley and I were led to examine systematically the various features of the carbohydrate "cycle" in the brains of avitaminous birds. (Peters 1929, p. 272).

By 1936, the study of the "carbohydrate cycle" undertaken by Peters and his associates had yielded important findings about the function of thiamine. He and his co-workers had shown as well that biochemistry made a "new approach to pathological analysis" possible. Working with rather large amounts of wet tissue rather than with the cell-thick sections used by histologists, they could detect "changes too subtle to be revealed upon the histological specimen, changes in the behavior of essential enzyme systems present." They had, by this approach, first located the site of the biochemical lesion of vitamin B, deficiency in the lower parts of the brain, and then pinpointed the lesion in the oxidation system of the 3-carbon stage (lactic and pyruvic acid) of sugar metabolism. While Embden and Meyerhof's experiment had left open the possibility that the presence of pyruvic acid was an artifact of their techniques, Peters' techniques proved that pyruvic acid actually was a normal intermediary in carbohydrate metabolism. They also had shown that the acute symptom of the neck spasm in thiamine-deficient pigeons was probably not due to a toxic build-up of pyruvic

43 acid, but to the deficiency of the energy which the oxidation of pyruvic acid would normally produce. In short, Peters and his colleagues had shown "that an in-vitro research. . . which takes advantage of the in-vitro labours of biochemists can be applied to in-vivo events" (Peters 1936). Even in its unfinished state, Peters' work was a convincing demonstration of the power of biochemical research to explain normal and pathological phenomena at a new level of detail.

Lohmann's isolation of cocarboxylase in 1937 was a triumphant confirmation of Peters' conclusions that vitamin B, was the coenzyme directly concerned with the metabolism of pyruvic acid. But Lohmann's results raised new questions. The first and easiest to answer was: was there any important difference in the activity of the free vitamin B, which Peters had used and the phosphorylated vitamin B, which Lohmann had found? Between 1937 and 1939 Peters' group included the Spanish biochemist, Severo Ochoa, who had just come from Meyerhof's laboratory at Heidelberg and thus knew about Lohmann's work first hand (Proc. Conf. Hist. Deveh Biberierg., 1973 pp. 169-170, 184). Ochoa showed that both in brain and yeast, the free vitamin had to be phosphorylated to become cocarboxylase before it could act on pyruvic acid; the in vitro brain tissue preparations Peters had been using luckily contained the enzymes and phosphates necessary for this step (Peters 1963, pp. 18-19).

A much more puzzling problem remained: what was the aerobic oxidative reaction of pyruvic acid and cocarboxylase in carbohydrate metabolism in the brain? Lohmann had shown how pyruvic acid was broken down to acetaldehyde by a simple removal of carbon dioxide (i. e. decarboxylation) in the anaerobic fermentation reaction of yeast. But Peters was dealing with a system which included oxygen and in which pyruvic acid was somehow completely broken down by both oxidation (i.e. dehyd roge nation) and decarboxylation to yield carbon dioxide, water, and energy. The most satisfactory path for the oxidation of pyruvic acid would have been the citric acid cycle which Hans Krebs and W. A. Johnson proposed in 1937. Unfortunately, when Banga, Ochoa, and Peters tried to test this pathway, they found that only a few of the intermediates in the citric acid cycle would help speed the oxidation of pyruvic acid (in the presence of cocarboxylase). So in 1939 Peter's group was forced to conclude that, in the brain at least, cocarboxylase set pyruvic acid on some other important pathway which also required some of the compounds found in the Krebs cycle served to transport hydrogen from pyruvic acid to the respiratory chain and ultimately to oxygen (Fruton 1972, pp. 380-381; Banga, Ochoa, and Peters 1939a, b). With this proposal, they claimed that "we

41 may . . . . consider that we know now the main facts about the biochemistry of vitamin 131" (Banga, Ochoa, and Peters 1939a, P. 1109).

Epilogue

Time would prove that Banga, Ochoa, and Peters were in fact premature with their claim to know the "main facts of the biochemistry of vitamin B1.- Over the next decade, Fritz Lipmann - with important contributions from Ochoa and Feoclor Lynen - clarified the role of pyruvic acid as the "crossroads" or "hub" of carbohydrate metabolism. In working out the steps which link pyruvic acid to Kreb's citric acid cycle, Lipmann showed that cocarboxylase did indeed mediate both the simple decarboxylation of pyruvic acid in alcoholic fermentation and the oxidative decarboxylation of pyruvic acid in the first step of the aerobic pathway (Lipmann 1971, pp. 27-54, 119-127).

Lipmann's work marked the end of an era of vitamin B, research. For those who had participated in the discovery of vitamin-deficiency diseases, the identification of vitamins, their chemical isolation and synthesis, and finally the elucidation of their function in intermediary metabolism, the pioneering excitement was over and it was time to turn to other fields. But there were still many questions about vitamin B, which needed answers, many practical implications to work out, and many new avenues of research that would be opened up.

One major line of research, the practical application of knowledge about the vitamin's chemistry and function has, in effect, continued the original thrust of Eijkman's beriberi research. His original hope in studying beriberi was to find the cause of the disease and then to find a cure, but, as we have seen, Eijkman was more successful at finding a cure for beriberi than at finding out the primary cause.8 It is impressive to see how, ever since E ijkman, many of the scientists who worked hardest at basic research arising out of his observations of beriberi's cure also became leaders in the application of their results to medicine and human nutrition. After isolating vitamin B1, for example, Jansen immediately turn, assigned the profits from his patent of thiamine synthesis to the nonprofit Research Corporation, the American Friends Service Committee, and WilliamsWaterman Fund for the Combat of Dietary Diseases, in order to sponsor research in human nutrition and dietary diseases (Williams 1961, pp. 168-189; Williams 1956). And, during World War 11, R. A. Peters turned his knowledge of the pyruvate oxiclase system back to the problem of poison gases, which had indirectly led him to the study of thiamine. He and his laboratory were able to show how the arsenical gas, levvisite, interfered with pyruvic acid metabolism, and they subsequently developed an effective antidote.

Medical researchers and physiologists also have investigated a variety of factors which affect the thiamine requirements of micro-organisms, experimental animals, and man: the proportions of fat, protein, and total calories in the diet, the use of antibiotics or sulfa drugs, the intestinal bacterial synthesis of thiamine or its inhibitors, and interactions with hormones, minerals, and other vitamins. Such work has suggested other metabolic pathways in which thiamine could be involved, and it has become clear that thiamine in its phosphorylated form takes part in at least two dozen biochemical reactions - more than any other coenzyme known (Bhuvaneswaran and Screenivasan 1962, pp. 580585; Williams 1961, pp. 140 ff; Brin 1962; Breslow 1962; Gunsalus 1956).

From the vantage point of historical retrospect, we can see that the scientific research inspired by the study of beriberi was clond at two different levels of complexity and detail, which correspond roughly to the concerns of the science of nutrition and of the science of biochemistry. For both sciences, the beriberi and vitamin B, research posed important problems and contributed to the theoretical foundations of the field. When Eijkman began his investigation of the relationship between diet and beriberi, nutritional science was at a standstill. The results of the beriberi research, coupled with the work on simplified diets, gave the study of nutrition an entirely new entity to deal with. What were these mysterious 'vitamines'? How many were there? How did their absence cause disease? What was their chemical nature? The glory and intellectual satisfaction that shortly before had gone to the discoverer of a new microbial pathogen now went to the scientist who discerned the deficiency of a

vitamin in a disease or who isolated or synthesized a vitamin. The attempt to understand the function of vitamins at a deeper level paralleled the development of biochemistry as a discipline, and a remarkable

42 number of eminent biochemists cut their scientific teeth on the problem of vitamin B1. The successful elucidation of thiamine's precise role in carbohydrate metabolism was seen as a triumphant vindication of the primary program of biochemical research, the dissection of intermediary metabolism.

At the first level, it is easy to see how closely basic nutritional research was tied to the clinical study of disease. This connection is somewhat less direct, less obvious in the biochemical stage of thiamine research, yet even here the disease of beriberi continued to serve as the inspiration for much of the work. It is probably true that Lohmann's work on cocarboxylase was aimed at understanding one more piece of a complex metabolic process, and as far as we know, he and Meyerhof had no special medical question in mind. For them, the basic research puzzle of carbohydrate metabolism was quite difficult and interesting enough in itself. But for R. R. Williams (and probably B. C. P. Jansen) the motives for studying vitamin B, were certainly mixed. Williams has testified how strongly his first-hand experience of the ravages of beriberi in the Phillipines and the dramatic cures with Vedder's rice-polish moved him to pursue the problem to the end, despite the many -obstacles he faced. Part of the urgency he felt in the race to synthesize thiamine arose from his certainty that his competitors, all organic chemists in pharmaceutical firms, would claim exclusive rights over thiamine manufacture for their own firms and thus raise the price of thiamine beyond anything that beriberi victims in Asia could afford. And, Williams also found thiamine an absorbing problem in organic chemistry.

When I began my work with Vedder in Manila, and for twenty years thereafter, I never thought of the antineuritic vitamin as something having monetary value. It was merely a baffling scientific problem, the solution of which would interest the rice eaters of Asia. For several years I did not visualize any probability that the knowledge of its structure would be highly pertinent to the basic science of nutrition, nor that its availability in pure form would become a significant factor in the economy or welfare of any people in the West. I worked at the job of isolating it, partly to justify my curiosity as to why or how it

worked, and partly as a humanitarian contribution to the very poor and ignorant of Asia. (Williams 1961,p.164)

For R. A. Peters, the motives are still harder to untangle. In additionto hisclinical interests, he gladly acknowledged thegreat influence that Hopkins and Hopkins' outlook had on his choice of problems and methods. His success in f inding the biochemical lesion of vitamin 8 1 deficiency proved, Peters has often said, how right Hopkins had been to urge the unraveling of metabolic pathways as the chief problem of "the dynamic side of biochemistry" (Peters 1963, p. 15; Peters 1929, p. 216; Peters 1957, pp. 371 ff). But Peters' initial decision to study vitamin B I was as a side issue which needed to be settled before the research on the pharmacological effects of poison gases could proceed. Then, we have seen, he became absorbed by the challenge of isolating vitamin B 1 as part of the larger puzzle of how many vitamin B factors there were. Through this research, in turn, Peters was led to search for the primary "lesion" of thiamine deficiency, a biochemical quest in part founded on the bizarre character of the neck spasm in vitamin-deficient pigeons and the astonishing speed of the cure. These two phenomena caught his imagination in a way that the tediously slow rat-growth test simply could not match:

I always felt that at least for myself a very important aspect of vitamin B studies was the dramatic change in the polyneuritic pigeon, so-called, on dosing with thiamine, i.e., the change from convulsive opisthotonus to normality. The impact which these facts made upon myself was no more remarkable than that upon students .... it was easy to realize that something fundamental would be found out if these events could be understood and this is what induced me to study much further this and the biochemical conditions of the brain. (Peters 1963, pp. 8, 13; 1957, p. 373)

Although the biochemical puzzle quickly became the major focus of Peters' research, his use of the polyneuritic pigeons kept him from ever losing sight of the in vivo effects of the biochemical phenomena he saw in vitro. And it was this clearcut connection between in vitro and in vivo events that made Peters' explanation of thiamine's role in pyruvic acid oxidation in the brain so convincing to both physicians and biochemists.

In the course of half a century, research on beriberi and thiamine has directly contributed to two major ence of vitamins, and then the discovery of the role of vitamins as coenzymes which catalyze crucial steps in intermediary metabolism. Is there any prospect that the study of beriberi can still lead to new fundamental

43 ideas in biology? Or has the study of vitamins at all levels been so successful that, as some vitamin researchers have mournfully argued in recent years, there is nothing left to do, that the field is complete and therefore dead? (Wuest 1962, p. 400; Schneider 1963, p, 157; Zbinden 1962, p. 550.

History has often showed that it is unwise to speak eulogies over a dead science, for too often the corpse has proved to be a phoenix. At least two major questions about beriberi and vitamin B 1 were not answered during the golden age of vitamin research. How exactly do all the terrible symptoms of beriberi -the burning of the nerves, paralysis, edema, emaciation, heart failure, convulsive spasms - follow from the initial biochemical lesion, whether that lesion be the cocarboxylase deticiency in pyruvic acid metabolism or the lack of thiamine in yet another process? And why is there such variation among individuals and among species in their susceptibility to thiamine deficiency? The first question hints at the possibilities of new levels of complexity in neurophysiology. The second suggests a new approach to the interaction of environment and heredity (Schneider 1963, pp. 162-169. It may still be premature to claim that we know the main facts about beriberi and vitamin B 1.

Notes Chapter 3

1 . "Thiamin" was later changed to "Thiamine" by the American Chemical Society to reflect the presence of an amino group in the molecule.

2. Between 1882 and 1906 Takaki succeeded in virtually eliminating beriberi from the Japanese navy by adding European foods to the Japanese rice-based diet; his theory was that the ratio of nitrogen to carbon (i.e., protein to carbohydrate) in tne Japanese sailors' diet was too small. A Dutchman, Van Leent, had developed a similar theory in 1879 after observing the differences in diet and the rate of beriberi between the Europeans and the East Indians in the Dutch East Indian navy. Eijkman, who knew Van Leent's work, but not Takaki's, was able to show in 1896 that the amount of nitrogen in the rice skin and germ was negligible, and that replacing such a small amount of protein with protein from other foods would not cure beriberi. Therefore, he concluded, beriberi was not caused by a dietary deficiency of protein. He also initiated research into the possibility that the rice skin might contain necessary mineral salts. (Eijkman 1929, pp. 200-201).

3. Because Hopkins did not publish, it is hard to trace the lines of his research except through his recollections and those of his friends. He apparently began by performing experiments like those of Lunin and Socin (although he was ignorant of their work and Pekelharing's) to convince himself that the diet of purified basic constituents really was inadequate for survival, let alone growth. He noticed that the mice grew fairly well when fed some batches of commercial casein, while on others they died quickly. The mysterious growth factor could be extracted by alcohol from the growth-supporting casein, leaving that casein incapable of helping the mice grow. Later, Hopkins discovered that the yeast extract he had been using to make the purified diets more tasty also contained this factor and indeed was more effective than the casain extracts. (it was at this point that he made his comments to the public analysts in 1906.) Most of his research up to 1912 dealt with yeast extracts and fractionations - time-consuming work because each test had to follow the growth of rats over 4 to 9 weeks (Dale 1948, p. 131; Hopkins 1912, pp. 454-460).

4. "Vitamine" was the accepted spelling until 1920. Because Funk's spelling had definite chemical and theoretical implications, we will use this spelling in discussing this early phase of vitamin research.

5. The work that went into the development of reliable, sensitive biological or chemical tests of thiamine's presence could be the subject of a separate chapter, one that would illustrate well the importance of technique in both clinical and basic research. Significant assays developed for thiamine after the time period covered in this chapter include that discovered in 1936-37 by Bergel and Todd: an oxidation reaction of thiamine to thiochrome, a compound which emits a strong blue fluorescence that can be easily and accurately measured. Then, in 1938, M. Lwoff devised a sensitive microbiological assay for thiamine which used protozoa to test for the release of thiamine by stimulated nerves (Sebrell and Harris 1972, p. 147). These and other assays have been widely used to find out how much thiamine is needed for the normal growth of microorganisms, plants, animals, and people.

44 6. Over the next few years, the heat-stable factor was itself shown to be a mixture of several physiologically and chemically distinct substances, Ironically, Funk and a team of researchers had identified one of these substances, nicotinic acid (niacin) as early as 1911-12 in their attempts to isolate the beriberi vitamin (McCollum 1957, pp. 310-311). But it was not until 1937 that niacin was proven to be the vitamin lacking in the diets of pellagra victims, a deficiency disease fully as devastating as beriberi (Etheridge 1972, p. 205 ff). While the intricate details of the unraveling of the vitamin B complex do not concern us here, it is important to realize how perplexing the multiple properties of vitamin B were to the people who had to use these properties as their only means of determining the presence of the vitamin they hoped to isolate.

7. The discovery of coenzymes began in 1906, when Arthur Harden and William John Young at the Lister Institute showed that zymase from yeast could not ferment glucose unless two other factors were present: phosphate and "a dialysable substance which is not destroyed by heat" (Harden and Young 1906, p. 25; Fruton 1972, pp. 297, 344). In 1918 this second factor - called coferment by Harden and Young, and cozymase by Euler and Myrback in the 1920's - was found in animal tissues by Otto Meyerhof. Then, in 1921, he and Gustav Embden showed that coferment was as necessary to glycolysis in muscle tissue as it was to alcoholic fermentation in yeast. Meyerhof, Embden, and several other biochemists saw that this common requirement for coferment implied a fundamental similarity between the pathways of alcoholic fermentation and glycolysis and, even more important, it implied a "unity of biochemistry" among all kinds of living organisms (Fruton 1972, p. 346). However, little was done to determine the chemical identity of this necessary companion to zymase until the 1930's.

8. Beriberi is still the most important manifestation of vitamin B, deficiency, and although much research has been done on its pathology, cure, and prevention, it has remained a significant public health problem in southeast Asia. Its modern victims, though, are now more likely to be rural women and babies than the soldiers, sailors, prisoners, and hospital patients who suffered so often from beriberi at the turn of the century. In the course of this century, more and more people have taken to eating factory-polished white rice rather than milling their own rice. the polished tastes and cooks better, it keeps better, and it has a certain 'social prestige.' To enrich the ric)f-adds to its cost; laws requiring enrichment are often hard to enforce; and so beriberi persists (Salcedo 1962, p. 573; Sebrell 1962, pp. 566567). In the U.S. beriberi has never been a great problem, save in conjunction with other dietary deficiencies like pellagra (now rare, thanks to the general enrichment of staple cereal products) or the general malnutrition of acute alcoholism.

Abderhalden, E. 1919. Diu Grundlagen Unserer Erna Hrung Und Unseres Staffwechsels. 3rd rev. ed. Berlin: Verlag von Julius Springer.

Adams, R., and F. Murray. 1972. Body, Mind, and the 8 Vitamins. NY: Larchmont Books.

Addinall, C. R., compiler. 1939. The Story of Vitamin 81, 1st ad. Rahway, N. J,: Merck & Co., Inc.

Addinall, C. R., compiler. 1940. The Story of Vitamin 81 (Thiamine Hydrochloride U. S. P.). rev. ed. Rahway, N. J.: Merck& Co., Inc.

Anonymous: 1911a. Book review of The Etiology of Beri-beri (1911), by Henry Fraser and A. T. Stanton. Lancet (Sept. 16): pp. 828-829.

Anonymous: 1911b. "The Etiology of Beri-beri," The Lancet (Sept. 16); 842.

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51 CHAPTER 4 DISEASE AND THE DUCTLESS GLANDS

"By their diseases ye shall know them - the endocrines." The title of this essay by J. H. Means aptly characterizes the development of endocrinology, a field to which his own clinically -oriented researches ' on the thyroid contributed so greatly (Means 1960). For, endocrinology is a science which grew out of the interest of physicians in the malfunctioning of glands whose functions were not yet well understood, and it has continued to draw many of its problems from the study of endocrine disorders.

In the pages that follow, we will look at the ways in which research on endocrine disorders gradually revealed the nature and functions of the hormones - the chemical messengers produced by the endocrine glands - and the complexity and integration of the endocrine system's interactions. Our account of endocrinology's development defines four phases of research which, though often overlapping in time and effort, are each marked by the appreciation of a new level of complexity in the functions of the endocrine glands. Phases one and two, in which physicians and physiologists recognized the effects of individual endocrine glands on other parts of the body, and then discovered how glands communicate with one another, will receive the most detailed treatment in this chapter. For it was in these phases of inquiry that problems of medicine raised a fundamental biological problem, that of the nature of the chemical regulation of physiological processes: a problem around which endocrinology emerged as a new field of research in the early years of the twentieth century. To provide some understanding of the problems that endocrinologists subsequently pursued we will sketch, more briefly, the content of endocrinology's third and fourth phases. Phase three was marked by discoveries about how the endocrine glands and the nervous system influence each other's activities, while in the fourth and current phase, endocrinologists and molecular biologists seek to understand the elaborate mechanisms of communication between the endocrine system and the organelles of individual target cells.

The broad approach to the development of endocrinology taken in this chapter in part reflects the nature of the endocrine system itself. The system's most strik

ing feature is its ability to coordinate extremely complex interactions among its own disparate elements, the rest of the body, and the external environment. Each gland's activity impinges on the others and on the body as a whole. As textbooks of endocrinology despairingly point out, it is artificial and misleading to depict the development and nature of endocrinology in terms of a particular disease, gland, hormone, or even a set of interacting parts. The fact that often the peculiar manifestations of endocrine disorders initially drew attention to the interrelationships of glands, hormones, nerves, environment, and target cells underscores the importance of treating the history of research on the endocrine system in as holistic a manner as possible.1

The Enigmatic Glands

Like most other branches of science, endocrinology has no obvious birthday to celebrate. Most of the endocrine glands were discovered by the "kitchen anatomy" of hunters and cooks in prehistory or by the medical anatomists of classical antiquity and the Renaissance; a few - like the tiny parathyroids embedded in the thyroid - were not found until the last century. Similarly, ancient, medieval, and Renaissance physicians noted several disorders diabetes, goiter, cretinism, dwarfism, and gigantism which we now know are caused by endocrine malfunction; but other equally striking endocrine diseases were not described until this century. And, whether by superstition or empirical trials, physicians in many cultures and periods have employed animals' glands as drugs -precursors of such modern glandular products as insulin and cortisone. These early observations of anatomy, disease, and therapy were not linked together, however, until the latter half of the nineteenth century and the beginning of the twentieth. Only then did a combination of clinical studies and the physiological experiments they generated suggest that these mysterious glands had an important role in the coordination of growth and metabolism.

It was well before the latter half of the nineteenth century, however, that physicians and biologists began to formulate the concept of animal hormones or internal secretions. In 1766, for example, the celebrated Swiss

52 anatomical peculiarities - the lack of a secretory cluct and a plentiful supply of blood vessels - and suggested that these "vascular glands" poured special substances directly into the veins and thus into the general circulation. A few years later, in Recherches sur les maladies chroniques, French physician Theophile de Bordeu formulated a concept akin to what we now think of as a hormone. De Bordeu speculated that "each organ of the body gives off emanations which are necessary and useful to the body as a whole," and he particularly stressed "the tonic effects of testicular and ovarian emanations, and their influence on the secondary sex characteristics as demonstrated by the obvious alterations in the body in general which follow castration" (Young 1970, p. 129, 130).

Thus, although the great French physiologist, Claude Bernard, has often been credited with first formulating the concept of internal secretion in the 1850s, the idea that what we now call endocrine glands secrete substances into the circulation was current well before Bernard's time. It is to Bernard, however, that we owe the term "internal secretion," and the first direct demonstration of one internal secretion (the liberation of glucose into the bloodstream by the liver) (Houssay 1967; Young 1970). Although Bernard's conception of internal secretions, which included the possibility that these substances might function to maintain the composition of the blood, was far broader than that later formulated by endocrinologists, his preeminence in nineteenth century physiology helped to focus attention on the ductless glands.

It was in 1855, in a lecture at the Coll6ge de France, that Bernard discussed the liver's internal secretion. The same year, a book appeared that, perhaps more than any other single work, deserves to be called the foundation of modern endocrinology: Dr. Thomas Addison's On the Constitutional and Local Effects of Disease of the Supra-renal Capsules. Addison's work epitomizes a sequence of observation and experiment that textbooks often call the "classic method" of endocrine research, in which the clinical features of a disease were compared and correlated with pathological changes in a gland. Along with, or usually following, this study, went observations on the effects of removing a gland thought to be overactive, or, conversely, the effects of administering an extract of a gland that was atrophied or damaged. his monograph is such a descriptive masterpiece that modern works on Addison's disease have not been able to improve greatly upon its accuracy of detail (Hartman and Brownell 1949, p. 343). From the study of only eleven cases, Addison was able to pick out the "leading and characteristic features of the morbid state. . . anemia, general languor and debility, remarkable feebleness of the heart's action, irritability of the stomach, and a peculiar change of the color in the skin (Addison 1885, p. 214). Post-mortem examinations revealed a diseased condition of the "supra-renal capsules," as Addison and earlier anatomists called the small pyramidshaped glands which sit on top of the kidneys. In some of his cases the adrenals had been damaged by tuberculosis or cancer, but Addison could point to at least one case in which the adrenals were the only abnormal organs in an otherwise healthy body. This was good evidence that the symptoms and condition he described were, in fact, due to a malfunction of this gland alone (Addison 1855, p. 210).

The monograph did more than describe a new disease, however, for Addison saw clearly that his study of the adrenals was a "first and feeble step towards inquiring into the functions and influence of these organs" (Addison 1855, p. 210). It seemed probable to him that the general state of ignorance about the adrenals, as well as the other "gland-like" organs, the spleen, thymus, and thyroid, would be overcome by pathological studies where physiological research had previously failed. "Although pathology . . . is necessarily founded on physiology," he wrote, "questions may, nevertheless, arise regarding the true character of a structure or organ, to which occasionally the pathologist may be able to return a more satisfactory and decisive reply than the physiologist" (Addison 1855, p. 209). Indeed, Addison himself had "stumbled upon" these "curious facts" about the disease of the adrenals while searching for the organic lesion of another mysterious disease, a fatal anemia of unknown etiology. (His description in 1849 of this anemia, now known as pernicious anemia and known to be caused by a vitamin deficiency, also is classic.) Among his anemic patients, Addison had noticed a few with a distinctive darkening or bronzing of the skin, who at autopsy were found to have abnormal adrenals. Addison was, of course, unable to say why damage to the adrenal glands should alter the complex ion or cause a gradual, fatal debility. But, by directing attention to the disease and the site of its lesion and by emphasizing the utility of studying a diseased organ to understand the functions of a healthy one, Addison invited systematic research into the role of the ductless glands in the general economy of the body.

Thirty years after Addison's monograph, a French physician, Pierre Marie, described and named another remarkable malady, again exemplifying the early classic method of endocrine research. In his 1866 paper, "Two Cases of Acromegaly: An Unusual Hypertrophy of the Head and Upper and Lower Extremities," Marie discussed two patients whom he had observed and another seven cases he had found described in the

53 medical literature. He pointed out the excessive growth of the viscera and the bones of the face, hands, and feet, and the thickening of soft tissues like the tongue, lips, and nose, which made the features of acromegalic patients gradually become strikingly coarse and elongated. Other signs and symptoms that Marie noted as characteristic of the chronic condition included severe headaches, intense thirst and appetite, cessation of menstruation, changes in the thyroid, and damaged vision. Later papers by Marie, his student Souza-Leite, and other physicians added to the number of cases and confirmed Marie's orginal description of the disease.

Marie hesitated in his first paper to offer an explanation of the disease he had defined, because only one autopsy had been performed. In that one case, published a few years earlier by another physician, an egg-sized tumor had been found in the brain "in the position of the pituitary body" (Marie 1886, p. 20). Marie considered this a lesion of the nervous system and thought that the hypertrophy or enlargement of the pituitary gland was suggestive of the hypertrophy of the bones of the face and extremities. But, he carefully added, the two might have had no cause and effect relationship this was only one of several plausible hypotheses about the cause of acromegaly. Marie's reluctance to seize upon the abnormality of the pituitary gland as the primary cause of acromegaly may also have been influenced by the prevalent belief among physiologists that this gland had "little, or perhaps no, use in the organisms of the higher vertebrates" (Rolleston 1936, p. 55). But Marie's clear account of the syndrome, even without speculations about its cause, encouraged others to search the literature, clinics, and anatomical museums for further examples. Two years after Marie's first paper, Otto Minkowski - more famous for his work on the pancreas and diabetes - explicitly made the connection between acromegaly and a disease of the pituitary gland. Then, in 1890 and 1891, Marie reported that enlarged pituitaries always were found in post-mortern examinations of persons with acromegaly, and he hypothesized that the abnormal growth of the pituitary caused a glandular deficiency and hence toxemia (Rolleston 1936, p. 86).

By implicating a particular gland in a specific disease, Addison and Marie gave physicians and physiologists both a reason and a method for studying the ductless glands. Over the centuries, as we have indicated, there had been occasional attempts to see what happened when a gland was removed or transplanted, but no one had followed up on them seriously. The well-known effects of castration on men and animals, for example, had led the eighteenth century surgeon, John Hunter, to try transplantation experiments with the gonads and accessory sex organs of hens and roosters. In 1849 similar experiments were carried out by the German physician, A. A. Berthold, out of a particular interest in the structural changes in the transplanted gonads and a general interest in the nature of sex, heredity, and the "sympathies" between different parts of the body (Jdrgensen 1971, pp. 27-30). Astley Cooper and T. W. King tried removing the thyroids of dogs in the 1820s and 30s and suggested that the thyroid might form some "particular material" which exerted an influence "upon the circulating fluid [which] may be more or less needful for the healthy subsistence of the entire animal" (Rolleston 1936, pp. 18, 50). Addison's monograph immediately inspired the French physiologist, BrownSdquard, and others to do systematic extirpation experiments on the adrenals, with contradictory results. Brown-S6quard believed that the adrenals were necessary to life because he found that the removal of both adrenals from various animals was always fatal; Phillipeaux, on the other hand, was able to excise the adrenals and other glands from white rats without killing them. The different results were due partly to different surgical techniques and precautions against infection and partly to the existence of unnoticed accessory adrenals in white rats which enabled them to survive the loss of the main pair, as well as differences in resistance to stress and infection among different strains of animals. Similar debates over the indispensability of the pituitary and the thyroid glands took place in the 1880s.

The variable results of extirpation experiments underscores the importance of surgical and aseptic Medicine in 1909 for his work on thyroid diseases, he began his lecture by exalting Pasteur's discoveries in bacteriology and Lister's application of them to surigical practice. Thanks to aseptic procedures, Kocher emphasized, it at last was possible for surgeons and physiologists to "make all the organs accessible to direct observation, and to alter the conditions in which they exercise their functions (Kocher 1909, pp. 330-33). Surgical technique, too, often meant the difference between a clearcut and an inconclusive experiment. The results of thyroid extirpation had been complicated by the failure to recognize and leave intact the tiny parathyroid glands in the midst of the thyroid tissue; the effects of removing the pituitary gland were confused by the effects of surgical damage to the hypothalamic region of the brain.2

Perhaps the most celebrated example of the importance of surgical skill for the development of endocrinology is Minkowski's discovery of the role of the pancreas in diabetes in 1889. The physician von

54 Mering was studying the problem of fat absorption in diseases of the pancreas and wanted to know how the decreased secretion of pancreatic juices into the intestine affected the breakdown of fats. When Minkowski asked why he did not remove the pancreas, von Mering answered that this was impossible. Minkowski, proud of his surgical skill, replied brashly, "Why should it be impossible? Bring me a dog and I will remove the pancreas." The operation succeeded, but to Minkowski's annoyance the supposedly well-trained dog began urinating frequently and copiously. On an impulse, or at the suggestion of his chief, von Naunyn - stories differ - Minkowski collected some of the urine from the floor and tested it for sugar. The sugar content was surprisingly high, a familiar sign of diabetes mellitus. After a week of further operations on other dogs, Minkowski was able to tell von Mering that total removal of the pancreas resulted in diabetes mellitus (Nothman 1954, pp. 272274; Allen et al. 1919, p. 38; Young 1970, pp. 137-140).

Surgical skill and asepsis also helped to decide between two competing theories of the function of the ductless glands. The first of these theories, widely held in the late nineteenth century and well into the first two decades of the twentieth, was that these glands somehow neutralized or removed poisons in the blood; consequently, disease or removal of these organs caused toxemia. It is easy to see in this theory the strong explanation, triai ine yidfiub stances into the bloodstream, had a long history of illustrious proponents such as von Haller. Cooper and King's experiments in thyroid removal in the 1820 and 30s, as we have seen, led them to this thesis: that glands such as the thyroid released a special substance directly into the blood. In the 1840s George Gulliver, surgeon and Fellow of the Royal Society, reported that under the microscope spheroidal bodies could be seen in both the adrenals and the veins leading from them; he argued that the veins served as the duct for some "peculiar matter which doubtless had some special use" and which was produced by the adrenals. Gulliver considered this "an interesting and important subject for further inquiry," but neither he nor anyone else pursued it at the time. Like many other early examples of the internal secretion hypothesis, this work was ignored until the hypothesis was firmly established and its history began to be written (Rolleston 1936, pp. 18-20).

The first good evidence for the internal secretion explanation of the glands' function came through research on thyroid diseases and the unhappy results of thyroid surgery on humans in the 1870s and 80s. In England W. W. Gull and W. M. Ord described a condition which Ord called myxedema: a swelling of the face and body from an excess of a gelatinous substance, mucin, accompanied by impairment of the mental faculties, lethargy, and extreme sensitivity to cold. In its advanced stages, myxedema strongly resembled cretinism. Cretin ism, in turn, was associated with thyroid abnormalities but in very confusing ways; many victims of "endemic cretinism" had immense goiters, while cases of "sporadic cretinism" generally had tiny atrophied thyroids. Ord noted that in all five of his cases of myxedema the thyroid was small, but he thought it had simply been squeezed by the excess mucin. In Switzerland, meanwhile, where goiter was especially common, removal of the grossly enlarged thyroids became popular once the antiseptic method made such surgery a less dangerous affair. Unfortunately, some patients whose goiters had been removed began to suffer a new malady which was variously ascribed to injury of the nerves or trachea or to loss of some "blood-making" function of the gland.

In both England and Switzerland a few bold physicians speculated that all three conditions -sporadic cretinism, myxedema, and the illness that followed thyroid operations - resulted from the absence or degeneration of the thyroid. Schiff, who had done experimental thyroidectornies on animals thirty years earlier and thought then that the thyroid produced an internal secretion, now saw the clinical significance of his earlier research; he tried implanting thyroids into the abdomens of thyroidectornized animals and succeeded in preventing myxedema. At this point it was clear that the thyroid was necessary for health, but the blood cletoxification hypothesis was still tenable. Then, in 1891, George Murray described his successful attempt to treat human myxedema with extracts of sheep thyroid glands; and three years later, Magnus-Levy showed that such extracts speeded up metabolism. Thus, the thyroid and its secretion gradually was recognized as having an active function unrelated to detoxifying the blood but indispensable to health (Paget 1919, pp. 54-67; Rolleston 1936, pp. 29-30, 151-153).

The discovery that thyroid extracts or grafts could restore normal intelligence and vigor to the myxedematous patient attracted a great deal of both scientific and popular attention,3 for it showed that a disease could be caused by an insufficiency of the secretion of a ductless gland and, in turn, that the symptoms of such a disease could be cured by what was termed replacement or substitution therapy. The interest caused by clinical studies of the thyroid gland, however, was overshadowed by more sensational claims for the actions of internal secretions, associated with the theories and activities of Charles-Edouard Brown-S6quard.

55 In retrospect, Brown-S6quard has been recognized as "a remarkable pioneer in endocrinology," whose therapeutic studies, "although they later fell into disrepute, sparked the imagination of many investigators [and) led to an active search for 'internal secretions' in animal tissues" (Rolleston 1937, p. 261; Borell 1975, p. 1). Elected to England's Royal Society and France's Academie des Sciences, Brown-S6quard's peripatetic career saw him practicing medicine, teaching, and doing research in locales such as Paris, Boston, New York, Philadelphia, London, and Dublin.

Addison's book on the symptoms of adrenal insufficiency, as we noted, sparked Brown-Sdquard's interest in the ductless glands, and in 1856 he concluded that the adrenals are essential to lite after performing total adrenalectomies on dogs. In his 1869 course of lectures at the Paris Faculty of Medicine he set aside his earlier views about the detoxifying function of the adrenals in favor of the theory that the, "glands have internal secretions and furnish to the blood useful if not essential principles" (Major 1943, p. 375). Then, in 1889, after he had succeeded Claude Bernard as professor of medicine at the Colldge cle France, Brown-Sdquard reported to the Society of Biology that, in experiments performed on himself, he had shown the rejuvenating effects of testicular extracts from healthy young guinea pigs. Between 1889 and his death in 1894, BrownS6quard and his assistant, d'Arsonval, extended their investigations to include the therapeutic effects of extracts from other animal tissues, based on BrownS6quard's expanded concept of internal secretions. Every tissue of the organism, he postulated, secretes its own special product into the blood and this influences "all other cells which in this way are dependent on each other, by a mechanism different from the nervous system" (Houssay 1967, p. 165; Brown-S6quard 1889, pp. 415-422).

To the world at large, including many physicians, the details of Brown-Sdquard's concept were far less exciting than the therapeutic possibilities it implied; by 1890, an estimated 12,000 physicians were giving testicular extracts to their patients, despite the skepticism and embarrassment expressed by many medical journals (Olmsted 1964, pp. 209-211).

Brown-S6quard, too, was concerned about the sensational publicity that his experiments received, and angered at the lucrative business of glandular extracts engaged in by some physicians. Nonetheless, he believed strongly that his experiments with testicular and other extracts were premised on sound physiological thinking, and would open up a new realm of therapeutics.

. . now we believe that all the tissues, glandular or not, give something special to the blood, that every act of nutrition is accompanied by an internal secretion. We believe, in consequence, that all the tissues will be able to be and ought to be employed in special cases as a mode of treatment; that there is, in short, a new therapeutics to create, in which the medicaments will be products produced by the different tissues of the organism.

The bacterial products taught us how active the chemical compounds created by the infinitely small were: the living cell, of each tissue that belongs to the organism, must, by analogy, secrete some products, of which the efficacy is no less. (Brown-Sdquard and d'Arsonval 1891; trans. Borell 1975, pp. 3-4)

endocrinology, one that many later endocrinologists would have preferred to ignore because of the cloud that it cast over clinical endocrinology. The editor of the British Medical Journal in 1937, for example, wrote of the organotherapy movement as follows:

After the discovery that preparations of thyroid gland taken by mouth were of benefit in myxoedema and cretinism, thyroid extract began to be given for many and varied conditions, an extracts of other ductless glands were prescribed with an enthusiasm that outran knowledge. Indiscriminate endocrine therapy brought about the inevitable reaction, and clinical endocrino ogy came to be looked upon with suspicion. The intensive research work carried out over the past decade has gone far to remove the disfavour into which endocrinology had fallen, and it is now becoming possible to put the matter in some kind of perspective. Solid achievements have been made. Possibilities are daily becoming probabilities. But the

56 exploitation of these probabilities in the interest of the patient must be carried out with due caution and a healthy skepticism if endocrinoloby is not to be done at the disservice of again becoming a fashion. (The Endocrines in Theory and Practice 1937, Preface)

Despite the negative effects of the excesses committed in the name of organotherapy, however, the movement was a positive force for the development of endocrinology in other respects. Fo: , as historian of endocrinology Merriley Borell points out, "much was learned about the action of internal secretions by means of these therapeutic efforts. The organotherapy effort was fundamental in attracting the attention of scientists to the field, who then sought to set up standards for the proper investigation of these therapeutic effects. Here, clearly, clinical concerns pointed the way to new scientific problems" (Borell, personal communication).

One such route was followed by the British physician George Oliver, who experimented with extracts of various tissues, including the adrenal and thyroid glands and the brain. Oliver had been administering extracts to his son, and then using an instrument he had developed to measure their effects on the diamEter of his son's radial artery. In 1893, he observed that adrenal extracts professor of physiology at University College, London, and caught him - so tradition has it - at a moment when he was measuring the blood pressure of an anaesthetized dog. Schafer was dubious about Oliver's claim, but agreed to inject some of the extract into the animal's veins. To Schafer's astonishment, the mercury in the blood-pressure manometer shot up so quickly and so high that the recording float was nearly lifted out of the tube (Dale 1938, pp. 461-463; Barcroft and Talbot 1968, pp. 6-8). Oliver and Schafer's studies of this pressor effect of adrenal extract not only led to the first isolation of a hormone (epinephrine), but also to a better understanding of the physiological role of the glands of internal secretion.

The arguments over the possible blood detoxifying function of the adrenals subsided once the definite, measurable effect on blood pressure was demonstrated. Oliver and Schafer also correlated function with anatomy; they showed that extracts made from the inner part of the adrenals, the adrenal medulla, would raise blood pressure, while extracts from the outer layer, the cortex, were ineffective (Rolleston 1936, pp. 29, 331). Research into the physiological implications of anatomical distinctions between parts of other glands, such as the anterior and posterior lobes of the pituitary, and the islets of Langerhans in the pancreas, quickly followed and proved fruitful. The fact that the internal secretion of a gland might actually contain several different active substances, each produced by a particular kind of tissue or cell, was at first confusing but later became axiomatic, and helped to direct attention to the complicated interactions of hormones and glands. Finally, the isolation of the pressor substance from the adrenal medulla at the end of the nineteenth century and its crystallization as a pure compound by Jokichi Takamine and T. B. Aldrich (working independently) in 1901 demonstrated concretely the existence of an internal secretion, and enabled scientists to study the pressor effect and other characteristic actions of epinephrine in much more carefully controlled experimental situations (MartiIbanez 1952, p. 233).

When Oliver and Schafer first reported their study of the pressor effects of adrenal extract to the Physiological Society in March 1894, their audience included two young investigators in Schafer's laboratory, William Bayliss and Ernest Starling. In 1902, investigating the control of pancreatic secretions, Bayliss and Starling identified a new internal secretion that they named secretin. Their discovery was made when they attempted to pinpoint the pathway of the neural reflex that the Russian physiologist, Ivan Pavlov, had declared must be responsible for the flow of pancreatic secretions into the intestine to neutralize digestive acids. Bayliss and Starling, however, found that even when all the nerves between the small intestine and the pancreas were severed, acid in the duodenum would still provoke the flow of pancreatic juices. According to C. J. Martin, who was present when the experiment was performed, Starling concluded at once, "Then it must be a chemical reflex." He promptly made a crude extract from a piece of duodenum, injected it into the jugular vein of the dog, and within a few moments had the pleasure of seeing the pancreas respond with a heavy secretion (Barrington 1975, p. 11).

Starling used the occasion of the Croonian Lectures to the Royal College of Physicians of London in 1905 to bring together what was known about "the chemical correlation of the functions of the body." In these lectures he introduced the word "hormone" (from the Greek, hormao, to excite or arouse to action) for the general class of substances "which, speeding from cell to cell along the blood stream,. . coordinate the activities and growth of different parts of the body ' (Starling 1905, p. 340). As examples of these chemical messengers, he named epinephrine, secretin and a number of similar gastric hormones which had been discovered in the meantime, extracts from the thyroid and from the interstitial cells of the testes and ovary, and - more tentatively - the supposed internal secretion of the pancreas. The simplicity and convenience of

57 the word "hormone" soon made it popular even though, as etymological and physiological purists pointed out, it applied to substances which could inhibit activity as well as excite it. The words "endocrine" and "endocrinology," (from the Greek, I separate within) came into use about half a dozen years later (Rolleston 1936, pp. 24).

Interrelationships Among the Endocrine Glands

Building upon the observations and clinical studies of physicians, the discovery of secretin in 1902 and Starling's elaboration of the hormone concept in 1905 represent a watershed in the emergence of endocrinology as a field of basic as well as clinical research. In the case of the thyroid, adrenals, and the duodenal extract named secretin, physicians and physiologists had succeeded in their effort to confirm the long-presumed existence of potent secretions in animal tissues. That proof came both from the ability of glandular extracts to cure specific diseases such as myxedema and from the analysis of specific physiological responses to the chemical substances in adrenal and duodenal extracts.

When secretin was discovered, as Borell observes, the term "internal secretion" "no longer provided an adequate description of the phenomenon under study. It did not specify the messenger role, the activity potential of chemicals elaborated by certain tissues," as did the word "hormone" (Borell 1975, p. 8). And, as Starling and other investigators recognized at the turn of the century, the chemical coordination of physiological processes by hormones was now a major subject for biological research, one that seemed of equal import to understanding the integrative action of the nervous system. For, as Starling wrote in Lancet in August 1905:

If control of the different functions of the body be largely determined by the production of definite chemical substances within the body, the discovery of the nature of these substances will enable us to interpose at any desired phase in these functions and so to acquire an absolute control over the workings of the human body. Such a control is the goal of medical science. (Maisel 1965, p. ix)

Building upon the idea of a human control of physiological processes, the next phase of research in endocrinology began to delineate and emphasize the intricate functional interactions among the glands. The early endocrinologists had tacitly assumed that each gland produced its own special hormone and had its own particular effect on the body, and endocrine diseases thus were viewed as the consequence of an increase or decrease in the secretion of a hormone. By the 1920s, however, closer study of endocrine diseases coupled with animal experimentation made endocrinologists see that this picture of the endocrine system was greatly oversimplified. The central fact -and the central problem of endocrinology between the World Wars was that activities of one gland could control and be controlled by the activities of others.

Research now concentrated on pairs or groups of glands, and the choice of which constellation of glands to study was still largely determined by clinical data and interests. The sexual abnormalities found in many different illnesses, the apparenx Ene"pluriglandular" preparations, the ability of epinephrine to alleviate but not to cure Addison's disease - each of these clinical observations led to new insights into the complexities of metabolism and to new approaches to therapy.

Three areas of research, moving back and forth from the clinic to the laboratory, proved especially successf ul and fruitful in this period. First, in the lineage begun by Thomas Addison, there was a body of work on the adrenals by Cannon, Selye, von Euler, Kendall, Moore, and many others. Their researches, in brief synopsis, helped to elucidate the roles of the two layers of the adrenal - the medulla and the cortex - in maintaining homeostasis and coordinating the body's response to emergencies and stress; established the nature of the medulla's close connections to the sympathetic nervous system; provided a better understanding of the relationships between the pituitary gland and the adrenal cortex; and identified, isolated, purified, and ultimately synthesized ACTH, the pituitary gland hormone which stimulates the adrenal cortex, and the several hormones secreted by the adrenal's two layers.

Secondly, there was a focus of research on the endocrinology of sex and reproduction. Work in this area received a powerful stimulus from the joint decision of the Bureau of Social Hygiene, the National Research Council, and the Rockefeller Foundation in 1921 to support "a program of research on fundamental problems of sex," and in 1931 to sponsor a major survey of sexual endocrinology: Sex and Internal Secretions. This

58 review of the recent progress in the biology of sex and the sex glands, first published in 1932, attracted many new workers into the field (Young and Corner 1961, 1, ix-xii; Hall, work in progress).

A third major area of research, dealing with the functions of the pituitary gland and, in particular, the relations between pituitary disorders and diabetes, culminated in establishing for a time a view of the anterior pituitary as "the master gland" or "conductor of the glandular orchestra." The methods shared by all three areas of research included not only the familiar techniques of extirpation and replacement by transplants and extracts, but also the use of isolated and purified hormones like thyroxin and insulin as these the nature of this second phase in the development of endocrinology by highlighting research on the pituitary gland or hypophysis, physiologically the most complex and important of the ductless glands. About 1890, as we have seen, Marie, Minkowski, and others explained the condition of acromegaly in terms of a lesion of the hypophysis which decreased the gland's internal secretion. Five years later, in the course of their experiments on the dramatic power of adrenal extracts to raise blood pressure, Oliver and Schafer discovered that hypophyseal extracts also had a pressor effect. The hypophysis, like the adrenals, has two anatomically distinct parts: the anterior lobe or adenohypophysis and the posterior lobe or neurohypophysis. By 1898, Howell and Schafer and Vincent had shown that only extracts of the posterior lobe exerted a pressor effect, and so research centered on this lobe of the gland for many years. During the first decade of the twentieth century, a bewildering array of specific effects were attributed to the posterior lobe. While an extract of the posterior lobe as a whole increased blood pressure, one fraction of it had the opposite effect of lowering blood pressure. The extract produced in tense contractions of the uterus and weaker contractions in some other plain muscles. It provoked the secretion of urine almost immediately, but this diuretic effect soon gave way to an even stronger anticliuretic effect.4 In some mammals posterior lobe extracts made milk flow more easily and in greater amounts than usual. In frogs the posterior lobe seemed to control the contraction and expansion of the pigments cells and thus the color of the frog's skin (Sharpey-Schafer 1924-26, 11, pp. 252-257).

These varied effects of posterior lobe extracts were both interesting and useful. The uterine contraction response provided a quantitative bioassay for the potency of posterior lobe extracts and found an application in obstetrical practice. The anticliuretic effect was first discovered when patients with diabetes insipidius - a form of diabetes characterized by a great flow of dilute urine and a correspondingly great thirst - were relieved by experimental injections of pituitary extract; this quickly became a standard treatment of the disease (Sharpey-Shafer 1924-26, 11, p. 244). But none of the actions of the posterior lobe of the pituitary could explain acromegaly.,

As the American neurosurgeon Harvey Cushing wrote uently, the great difficult for all pituitary gland searchers was getting at the gland in the first place.

Nature saw fit to enclose the central nervous system in a bony case lined by a tough protecting membrane, and within this case she concealed a tiny organ which lies enveloped by an additional bony capsule and membrane like the nugget in the innermost of a series of Chinese boxes. No other single structure is so doubly protected, so centrally placed, so well-hidden. (Cushing 1930, p. 5)

In 1904 Cushing forecast that techniques of brain surgery were improving so much that "it is not impossible that a diseased pituitary may someday be successfully attacked" (Fulton 1946, p. 267). Only two years later the British surgeon, Victor Horsley, successfully operated on several cases of pituitary tumors (Paget 1919, p. 180). Soon Cushing too was actively engaged in pituitary gland surgery and research.

Cushing had been interested in the hypophysis ever since 1901 when he had failed to recognize that a case of sexual infantilism accompanied by obesity, headache, retarded growth, and impaired vision was a case of pituitary tumor. His pride was nettled because, soon after finding the tumor at autopsy, he learned that A. Frohlich in Vienna had seen a similar case, made the correct diagnosis, and had the tumor removed successfully (Fulton 1946, pp. 271-272). In 1907-1908 Cushing's interest was renewed by the announcement of a new technique for removing the gland from dogs and by a series of lectures on the pituitary that Schafer delivered at John Hopkins. Cushing and his students at Johns Hopkins then began a long series of pituitary gland extirpation experiments on dogs, correlating the results with the conditions he found in his patients.

Cushing's chagrin over his misdiagnosis of what came to be called "Frohlich's syndome" was redeemed by the first major result of these experiments. Earlier reports on removal of the gland said that the animals

59 always died after the operation. Some of Cushing's dogs, however, survived the surgery and lived many months although they became "extraordinarily fat, logy, sexless creature(s)." The general opinion among Cushing's students was that somehow the operation had not been complete, but they drew no further conclusions from the peculiar state of the animals. One day in the winter of 1908-1909 Cushing "caught sight of [one of] these animals while it was still alive and said at once, 'Here is Frohlich's asexual adiposity.' " Because the animals lack all or nearly all of the pituitary, Cushing deduced that Frohlich's syndrome must be caused by a lack of the pituitary secretion. Moreover, if this state of asexual adiposity was the result of too little pituitary secretion, then acromegaly must be the result of an excessive secretion - exactly the reverse of Marie's original explanation of acromegaly (Fulton 1946, pp. 280-282).

Over the next four years, Cushing confirmed his deductions in further experiments and studies of patients with a variety of pituitary gland disorders. He demonstrated that under- or over-secretion by the anterior lobe alone was responsible for Frohlich's syndrome and acromegaly, as well as for gigantism and a form of dwarfism. He also stressed the effects that pituitary lesions had on the rest of the endocrine system:

In view of the apparent interrelation of many of the glands of internal secretion it is quite probable that certain of the symptoms known to accompany hypophyseal disease may be consequent upon a secondary change in other glands which follow the primary change of the hypophysis. These changes are seemingly more outspoken and more widespread after a lesion of the pituitary body than after a corresponding lesion of any other member of the group of ductless glands, and in view of its unusually well-protected position one might have conjectured that it must represent a vitally important organ, (Fulton 1946, p. 301)

Cushing's strong awareness of the secondary endocrine effects of hypophyseal disorders led twenty years later to his elucidation of what had been vaguely known as "polyglandular syndrome" and usually ascribed to a malfunction of the adrenal cortex. In acromegaly he had noted in the anterior lobe a preponderance of cells which take up eosin dyes, and he concluded that acromegaly was due to oversecretion by these eosinophilic cells. At the same time he argued that oversecretion by the other distinctively staining cells of the anterior lobe, the basophiles, must also be responsible for some kind of syndrome, even though he could not say what that syndrome was. In 1930 he read of a case of "adipositalgenital dystrophy" which at autopsy revealed a tiny adenoma (tumor) of the basophilic cells. The case resembleil in every detail cases of "polyglandular syndrome" he had seen over the years, and further post acromegaiy ana poiygianuuid ,Yllurulllu, U-Illu Ilong ago concluded that the eosinophilic cells secreted a hormone which regulated growth. Now he could add that the basophilic cells elaborated "a sex-maturing principle" or 11 a gonad -stimulating factor" (Cushing 1932, pp. 114-119, 155-161; Fulton 1946, pp. 614616).

Inevitably, the situation turned out to be more complex than Cushing's account in 1932 made it appear. More sophisticated cell staining techniques revealed many specialized cell types within the general categories of eosinophile and basophile. Moreover, the original explanation of the polyglandular syndrome (since renamed "Cushing's syndrome") in terms of an adrenal cortex disorder was vindicated: excessive production of the steroids of the adrenal cortex was the immediate cause of the symptoms of the disease, although such hypersecretion of steroids was often in turn the result of oversecretion of ACTH by the anterior pituitary. However, the main outlines of Cushing's explanation of the syndrome and his characterization of a growth factor and a gonad- stimulating factor proved to be sound.

Concurrent research by Bernardo Houssay in Argentina and by Herbert McLean Evans at Berkeley did much to confirm and extend Cushing's clinical insights into the workings of the anterior lobe and its ties to other endocrine glands. In 1907, the year that Cushing began his extensive work on the hypophysis, a precocious young medical student in Buenos Aires saw a patient with acromegaly and immediately became interested in the gland. Forty years later, in his Nobel Prize lecture, Bernardo A. Houssay commented that he had been attracted to the hypophysis by both its medical and physiological aspects: "the microscopic picture showed glandular activity and its lesions were accompanied by serious organic disturbances, such as acromegaly, dwarf ism, etc." (Houssay 1947, p. 211; Young and Foglia 1974, p. 254). Beginning with his medical thesis in 1911, and continuing through a long series of researches, Houssay developed techniques for removing the hypophysis from frogs, toads, and dogs, and examined the physiological effects of posterior lobe extracts and implants on muscles, the uterus, carbohydrate metabolism, the thyroid, and the adrenals.

Houssay's most important research in endocrinology began after Banting and Best announced the discovery of insulin in 1921, a year that Houssay later saw as a

60 in extracting it from the pancreas, but with insulin's existence proven in 1921, no one could doubt any longer the existence of hormones or their ability to regulate metabolism. Moreover, as the association between acromegaly and diabetes had been noted often by clinicians, Houssay saw that insulin could serve as a new tool for investigating the suspected physiological reactions between the hypophysis and the pancreas (Houssay 1956, p. 213; 1967, pp. 167-168).

Houssay had to devote several years to the preparation and standardization of insulin for his experiments, for large-scale extraction of insulin for treatment began only in 1923-1924, and pure crystalline insulin was not prepared until 1926. He and Magenta demonstrated in 1924 that a dog without its hypophysis became remarkably sensitive to insulin's ability to lower blood sugar levels. It was not clear from their experiments, however, whether it was the loss of the anterior lobe or of the posterior lobe that was responsible for this hypoglycemic effect. Until 1929 the general opinion was that "if the hypophysis had any action on carbohydrate metabolism and on the diabetes of acromegaly it was due to its posterior lobe" (Houssay 1943, p. 247). The anterior lobe was ignored by researchers because the actions of its extract seemed to be transient and because it was hard to extirpate the lobe surgically (Houssay 1956, p. 213).

Houssay's early studies of the hypophysis in amphibians, dogs, and man proved their value in 1929 when he realized that the anatomy of the toad's pituitary made it the ideal experimental animal for testing these widely-held views of the pituitary's role in carbohydrate metabolism. Houssay found that removing the lobe which corresponds to the anterior lobe in man produced great sensitivity to insulin, while removal of the posterior lobe had no such effect. Without the anterior lobe, the toad rapidly used up glucose, and the injection of insulin exacerbated this high rate of sugar utilization. Thus the anterior lobe clearly was implicated in the regulation of carbohydrate metabolism (Houssay 1936 a, b). The diabetic's problem, though, was that his body used sugar too slowly. Houssay and Biasotti proved in 1929-1930 that the effect of the anterior pituitary on glucose metabolism was antagonistic to the effect of the pancreas. If a toad or dog was made diabetic by the removal of its pancreas, the removal of its anterior lobe would then counteract the diabetes. If, on the other and, the animal was made mildly diabetic by the artial removal of its pancreas, an injection of anterior Vituitary extract would drastically increase the severity ,of the diabetes. While the pancreas secreted insulin to increase the utilization rate of sugar, the anterior pituitary secreted something with the opposite effect, something which inhibited the supply of sugar.

These experiments and results were not well understood or accepted in the early 1930s, both because :Houssay published them in Spanish-lanquage journals, and because they contradicted the current theories of hypophyseal function so completely that journals in the U. S. would not publish his English summaries. One distinguished physiologist declared that Houssay had to be wrong because it was well-known that the posterior lobe's primary function was metabolic control (Barrington 1975, p. 54; Young and Foglia 1974, p. 256). In addition, others who tried to reproduce the experiments failed, chiefly because they did not follow Houssay's techniques for preserving the activity of pituitary extracts (Houssay 1943, p. 252; Wrenshall et al. 1962, p. 65). Eventually, however, the "diabetogenic" effect of the anterior lobe in mammals was confirmed in 1931-1932 through the researches of Herbert McLean Evans and Miriam Simpson, who had been working with anterior lobe extracts from a very different point of view.

Evans and Simpson observed the diabetogenic effect in the course of a wide-ranging program of basic research on the effects of the anterior pituitary extracts on growth and reproduction, work which in turn fed back into clinical aspects of endocrine disorders through the type of feedback cycle so characteristic of the development of endocrinology (Evans 1923-24). In the midst of their efforts to isolate and purify the growth hormone (a task achieved' in 1944-45),. Evans and Simpson noticed in 1931 that one of their dogs in which gigantism had been induced with the growth hormone ate and drank excessively, yet became thin and weak. The dog also "excreted great amounts of urine to which flies were attracted." Like Minkowski in 1889 (and Evans, who was very interested in history of science, surely knew the anecdotes about Minkowski's discovery), Evans and Simpson tested the urine and blood for sugar and found that the dog was suffering from diabetes. The experiments proceeding from that observation gave the first clear evidence that the anterior lobe produced an antagonist to insulin and reassured Houssay that his remarkable results with amphibians were correct (Amoroso and Corner 1972, pp. 128-129).5

By the mid-1930s, then, there were strong medical and experimental reasons for believing that the anterior lobe was, as Houssay put it, "the central and directing organ in the endocrine constellation" (Houssay 1936 b, p. 961). Little over a decade later, concentrated research in biochemistry had proven that the gland's control of growth, reproduction, metabolism, and the activities of other endocrine organs was carried out through the

61 secretion of six different hormones, and all six hormones were purified by 1950. However, immed iately after proclaiming the central importance of the anterior lobe in his Dunham Lectures at Harvard in 1935, Houssay was forced to add that the other endocrine organs "in some . . . cases ... also have an influence on the pituitary" (Houssay 1936 b, p. 961). Intensive research into the reciprocal influences of the endocrine glands and the hypophysis on rates of internal secretion kept pace with identification and purification of the anterior lobe's trophic hormones through the 1930s and 40s. The general conviction of the early 1920s that the endocrine organs did not act independently - an opinion based primarily on clinical data - now rested on the solid ground of physiological and biochemical experimentation, and the availability of newly purified hormones gave hope that many more of the complex interactions would be successfully untangled in the future.

The Rise of Neuroendocrinology

Between the World Wars endocrinology became firmly established as a scientific discipline with a distinct perspective of its own towards physiological problems. Endocrinologists regarded the endocrine glands, hormones, and their interactions under the pituitary gland's direction as the body's long-term system for coordinating and regulating its activities, a system that neatly complemented the more rapid integrative functions of the nervous system. Most endocrinologists would have described the two systems as separate but equal, although some enthusiasts claimed that the glands controlled the brain and nerves (Vincent 1925, pp. 11, 18; Rolleston 1936, pp. 57-9; LiIjestrand 1936, p. 398; Berman 1930, pp. 200-218). For some time, though, evidence had been building up in a variety of fields human pathology, psychology and psychiatry, comparative anatomy, experimental physiology and endocrinology, and animal behavior - to suggest that, once again, the picture was more complicated than either the independent modes of internal communication after all, but interconnected in very interesting ways.

Out of the study of these interconnections a new hybrid discipline, neuroenclocrinology, emerged in the 1940s and 50s. Early clues to a relationship between the endocrine and nervous systems came from observations of the emotional disturbances associated with abnormal endocrine activity in the thyroid and adrenal medulla. Graves' disease (thyrotoxicosis, toxic goiter), for instance, seemed to be triggered by severe psychic distress, such as a sudden shock or long, continuing anxiety, and nervous irritability was among its most common symptoms. Because clinical manifestations of nervous system involvement in the thyroid matched anatomical observations of sympathetic nerves in the thyroid, it seemed possible that the thyroid's secretions were directly controlled by the sympathetic branch of the autonomic nervous system. Although research in the 1930s concentrated instead on the humoral control of the thyroid by the anterior pituitary's thyrotropic hormone, the earlier notions of neural control continued to interest physicians, psychiatrists, and physiologists (Rolleston 1936, pp. 222-224, 240: Reichlin 1966, pp. 445-446, 502-506).

The isolation and synthesis of epinephrine in the early years of the twentieth century made it comparatively easy to study the relations between the sympathetic nervous system and the adrenal medulla. Almost immediately after Oliver and Schafer's discovery of the pressor effect of adrenal medulla extracts, neurophysiologists noted how closely the actions of the extract (and later, of pure epinephrine) mimicked the effects of the sympathetic nerves. As early as 1904, T. R. Elliot hypothesized that the sympathetic nerves acted by releasing epinephrine at the ends of the nerve fibers. The histology and embryology of the adrenal medulla hinted at this possibility too, for the medulla was laced with sympathetic nerve fibers which ended on the medullary cells, which had themselves developed from embryonic nerve cells. And, electric stimulation of the nerves leading to the medulla were found to induce the medullary cells to secrete epinephrine (Rolleston 1936, pp. 321-323; Dale 1962, p. 72).

From 1909 on, the American physiologist, Walter Cannon, emphasized the psychosomatic importance of the neural connections to the adrenal medulla. In the course of studying the mechanics of digestion in movements of its stomacn Hnu VVW--- -equally suddenly. Over the next five years Cannon. investigated the effects of emotional stimuli on epinephrine secretion, which led to his famous theory of the emergency function of the adrenal medulla. In his 1915 monograph, Bodily Changes in Pain, Hunger, Fear, and Rage, he argued that strong emotions stimulated the sympathetic nerves and thence the secretion of ep inephrine; all the varied effects of the hormone on the body could be seen as quick preparations for "fight or flight" (Brooks and Koizumi 1975, p. 52).

Although neural control over the adrenal medulla and the thyroid seemed probable, the physiological

62 mechanisms were obscure. How did the nerves provoke secretion? In 1921 Otto Loevvi in Austria showed that the nerve cells themselves secrete neurohumors, tiny amounts of chemical substances which travel the short distance from the secreting end of the neuron to the membrane of the cell being excited. In one of the most elegant biological experiments ever devised, Loevvi proved that, when the parasympathetic vagus nerve to the heart is electrically stimulated, it releases a neurohumor which will slow the heart.

H. H. Dale and Loevvi subsequently identified this Vagus-stoff as acetylcholine, a compound which was already known to mimic the actions of the parasympathetic nerves as closely as epinephrine mimicked the sympathetic nerves. (Later, acetylcholine proved to be the neurohumor released by the voluntary nerves as well.) Meanwhile, Cannon and his co-workers believed that the sympathetic nerves also liberated chemical transmitters. After much controversy and reinterpretation of experimental data and theories, Cannon's younger colleague, Bacq, and Ulf von Euler, proved that the sympathetic nerves do indeed secrete epinephrine in order to inhibit activity of the target cell, and that they also release norepinephrine to excite the target cell (Bacq 1975, pp. 68-76).

The most logical place to look for neurcienclocrine interactions, however, was in the connections between the brain and the pituitary gland. The posterior lobe attracted attention first, for anatomically it possesses a rich supply of nerve fibers which join the lobe to a tract of the hypothalamus by way of the pituitary gland's stalk. Oliver and Schafer's discovery of internal secretions from the posterior lobe raised a difficulty, however: histologically the posterior lobe had many e fibers but no obvious secretory cells. How then Id it produce its blood-borne effect on blood pressure, ter metabolism, and uterine contraction? Studies of diabetes insipidus further complicated the roblem. In 1913 two clinical investigators noted the ility of posterior lobe extracts to relieve the excessive rine flow of patients with diabetes insipidus. Presumbly, in these patients a damaged posterior lobe failed to secrete enough antidiuretic hormone, but there was conflicting evidence. Emotional and physiological stress also seemed to have an antidiuretic effect, which implied that the nervous system exerted some control over water metabolism - the parallel to Cannon's emergency theory of adrenal medulla function was easily drawn (Pickford 1975, pp. 209-210). In 1912 and 1913 Aschner and Roussy and Camus argued that diabetes insipidus (not to mention obesity, sexual dysfunction, genital atrophy, and increased sugar in the blood - many of the symptoms, in short, associated with pituitary gland disorders) were caused by damage to the brain in the hypothalamic region. In 1920 they produced experimental diabetes insipidus in dogs by making lesions only in the hypothalamus, leaving the pituitary gland untouched, a feat that led them to deny any function at all to the hypophysis (Rolleston 1936, pp. 56-56; Abel 1923-24, pp. 202-207; Harris 1955, pp. 200-204).

These rival explanations of diabetes insipidus and of the roles of the posterior lobe and the hypothalamus were gradually reconciled through new studies of the nerves uniting the brain and hypophysis. 6 13Y 1940 it was reasonably clear that the hypothalamus exerted direct control over the antidiuretic and oxytocin secretions of the posterior lobe, and that this control was somehow mediated by the nerves leading from a welldefined tract of the hypothalamus to the posterior lobe. The source of the secretions themselves remained a question that was not fully answered for another decade, although the most important clues had already been discovered. Ernst Scharrer had shown in his doctoral thesis in zoology in 1928 that certain hypothalamic neurons in fish functioned like glandular secretory cells, and he and his wife Berta subsequently found neurosecretory cells in many vertebrate and invertebrate species. But for many years, in a pattern typical of resistence to scientific discoveries, they could not persuade either the endocrinological or the neurological communities that their cytological evidence would explain the confusing relationship between the hypothalamus and posterior pituitary. For, as Berta Scharrer later wrote, the proposition that there were special hypothalamic neurons with an endocrine function "was a bold concept that did not fit into any existing mold, and it is not surprising that it was received with skepticism. Why should members of a class of cells as readily defined as neurons be capable of functioning as glands of internal secretion? " As Scharrer goes on to note, however, "What is less understandable . . . is the almost universal rejection by the scientific community of the validity of cytological evidence for the existence of a secretory process" (Scharrer 1975 b, pp. 225-260).

After World War 11, an old friend of the Scharrers, Wolfgang Bargmann, became irritated by the negative attitude of his fellow scientists toward the Scharrers' conception of neurosecretion. Bargmann decided to try a new approach based on the well-known usefulness of cell staining techniques in endocrinology. His first trial in 1948-1949 of a recently developed staining technique succeeded splendidly, for he could see individual

63 neurons of the hypothalamic tract extending themselves without interruption down into the posterior lobe. By 1957 Bargmann and the Scharrers had established the fact that the posterior lobe was little more than a storage depot for the hormones produced by the hypothalamus (Bargmann 1975, pp. 38-43; Scharrer 1975 b).

The connections between the anterior pituitary and the nervous system were equally hard to understand, although many observations, especially about the reproductive functions of the anterior lobe, implied the existence of connections. For instance, women had long known that emotional distress and other psychological factors can affect their menstrual cycles. Farmers, hunters, and naturalists had recognized for centuries that many environmental factors like temperature, food supply, light, and the sensation of coitus can alter an animal's ability to ovulate.

Further evidence came from the clinic: tumors of the hypothalamus disturbed many endocrine functions usually associated with the anterior lobe (Fulton, Ranson, and Frantz 1940, pp. xiii-xxx, 864-874). Following this lead, Camus and Roussy produced Frohlich's syndrome of asexual adiposity by making minor lesions in certain tracts of the hypothalamus, just as they had caused diabetes insipidus with lesions in the tract connected to the posterior lobe. Joseph Hinsey and Joseph Markee at Stanford brought together in 1933 Markee's observations on the nervous control of ovulation and Hinsey's experience with the chemical transmitters of sympathetic and parasympathetic nerves. In a the anterior 1oUe, in corrEra5i: TV Lilt: 11L.11 1111lulvateof the posterior lobe, implied a humoral mechanism for the brain's control over the anterior lobe's civulatory function (Hinsey 1975, pp. 135, 139). But these various observations, experiments, and speculations did not excite much interest until the 1940s. The only mention of "neuro humoral mechanisms" in the authoritative and lengthy 1939 survey, Sex and Internal Secretions, for example, was to be found in a psychologist's review of research an the sex drive (Allen, Danforth, and Doisy 1939, pp. 1213-1220).

Before anyone could make sense of the miscellaneous assortment of observed nervous system-anterior lobe interactions, much more detailed, direct experimentation on the anatomy and physiology of the hypothalamus, pituitary stalk, and anterior lobe was necessary. It was the English anatomist, G. W. Harris, who "more than anyone else brought the new endocrinology into focus in the late 1930s" and 1940s (Price 1975, p. 229). Although Harris' early experiments suggested that nerves led from the hypothalamus to the anterior lobe, his examination of the lobe's anatomy raised doubts in his mind, for the anterior lobe abounded in secretory cells and blood vessels but almost completely lacked neurons.

The other possible path for messages between the central nervous system and the anterior lobe was through the rich network of blood vessel that surrounded the pituitary stalk. A Roumanian pathologist, Rainer, had noticed in 1927 that these blood vessels were especially prominent in people who had died suddenly and violently; he persuaded a young medical student, G. R. Pope, to study these vessels and their connections to the capillaries of the hypothalamus and the hypophysis. Popa and Fielding published an account of this work in 1930, in which they said that the blood in these short portal vessels flowed upwards from the hypophysis to the hypothalamus. Six years later Wislocki and his colleagues in Boston disputed this assertion and argued from histology that the blood flowed down from the brain to the anterior lobe. Wislocki, Popa, and Harris debated ("at times quite vehemently," Harris recalled much later) the question of the direction of blood flow over the next ten years (Harris 1955, p. 30).

In 1947, Harris and Green tried a new approach: they now watched the flow of blood directly in living, anesthetized animals, and in so doing confirmed Wis had first been proposed in 1933 by Hinsey and Markee. Harris devoted the next half dozen years to proving the theory that the hypothalamus sends blood-borne chemical messengers down the pituitary stalk's portal vessels into the anterior lobe, where they stimulate the release of the anterior lobe's pituitary hormones (Harris 1955, pp. 20-31; Tepperman 1973, p. 36; Vogt 1972, pp. 310-313; Jacobson 1975).

Once the neural control over the anterior lobe's secretions were widely accepted, research centered on isolating the "releasing factors." Early trials with known nerve transmitter substances showed that these compounds were only indirectly concerned with the hypothalamus/anterior lobe system. In the mid-1950s and the 60s neurciendocrinologists and biochemists succeeded in assaying, isolating, and purifying a series of small polypeptides from hypothalamic tissue and blood. By 1973 eleven different releasing or inhibiting factors had been found, eight of them purified, and the structure of three worked out in detail (McCann and Dhariwal 1966, pp. 261-296; Williams 1974, p. 5; Maugh 1975, p. 921).

64 As endocrinologists learned to see the anterior lobe as the "concert master" rather than the "conductor" of the "endocrine orchestra," they reconsidered the interactions among the glands that had been so arduously worked out before. In the 1940s, Norbert Wiener, the inventor of cybernetics, had popularized the concept of feedback control in engineering and in neurophysiology (Dempsey 1975, pp. 85-86; Wiener 1948, pp. 7-33, 113-114). Walter Cannon's concept of homeostasis agreed well with Wiener's ideas, and endocrinologists gradually realized that the language of feedback control provided an effective way of describing and thinking about endocrine interactions. They saw that the presence of the hypothalamus in the endocrine constellation required more elaborate feedback systems than the simple see-saw reciprocity envisaged by earlier investigators.

Following the publication of Harris' monograph, Neural Control of the Pituitary Gland, in 1955, with its diagrams of feedback loops involving the higher brain, the hypothalamus, the two pituitary lobes, the gonads, adrenals, thyroid, and sympathetic nervous system, neurclendocrinologists started intensive research into the mechanisms by which hormones act on the brain and hypophysis. The principle of feedback loops has become so fundamental a part of endocrinological thinking in recent years that textbooks now commonly discuss the feedback integration of the endocrine system before they even name and describe the components of the system, the glands and hormones (Williams 1974; Tepperman 1968, 1973; Barrington 1975).

Reading the Message: Hormone Receptors and Molecular Biology

There was a continuity of outlook through the three phases of endocrine research discussed so far, as researchers concentrated on seeking the source of control over the actions and interactions of glands and their secretions. In the earliest phase, control seemed to come from the gland itself, in the second, from the hypophysis, and in the third, from the nervous system as modified by feedback loops. The response of target organs was used to assay hormones, but otherwise that response was simply taken for granted. The interactions of the endocrine system were studied on the tissue or organ level; and, while biochemists played an important role in purifying and characterizing hormones, they could not contribute much to the conceptual foundations of the discipline.

The question, "How do organs and cells receive and read the hormone message?" could not be answered, or even asked, effectively in this context. It made sense only at the cellular level, and this was - until the late 1950s - the separate province of molecular biologists and biochemists. For among biologists "there was a widespread feeling . . . that hormone action could not be studied meaningfully in the absence of organized cell structure" (Sutherland 1972, p. 401). Between 1955 and 1962, however, Earl Sutherland and his co-workers, proceeding on the conviction that "there was a real possibility that hormones might act at the molecular level," proved that breaking up cells did not destroy their sensitivity to some hormones (Sutherland 1972, p. 401). In studying the influence of epinephrine and the pancreatic hormone, glucagon, on the liver's ability to break glycogen down to glucose, Sutherland and his associates found in liver cell fragments a molecule called cyclic AMP which carried the hormone message to the enzymes that governed the rate of glycogen - glucose reactions. Since then, cyclic AMP has also been implicated in the cellular reception of information from the luteinizing hormone, the adrenocorticotropic hormone, thyroid-stimulating hormone, parathyroid hormone, and many others. Its ubiquity in the animal kingdom at all levels of organization suggests that, in evolutionary terms, it may be as significant a compound as DNA, RNA, and ATP (Robison, Butcher, Sutherland 1971, p. 74 and chap. 12). Research on the biochemical and structural details of the hormone-membrane receptorcyclic AMP-enzyme activation pathways in the cell has become one of the fastest growing fields in contemporary endocrinology and molecular biology.

Sutherland's discovery of cyclic AMP came directly out of research on the enzymes and biochemical pathways of carbohydrate metabolism - a tradition very different from endocrinological research in the first half of this century. Unlike endocrinologists, for example, researchers in intermediary metabolism had long been accustomed to experimenting with cellfragments and with enzyme-substrate reactions in cellfree systems. Another difference lay in the primary research orientation of investigators such as Sutherland. His mentors Carl and Gerty Cori, and others who elucidated metabolic pathways in the 1920s, 30s, and 40s, had medical training and clinical experience, and their work was supported largely by medical schools and medical research institutes. But they conducted their research on the hormonal control of carbohydrate metabolism in the tradition of basic research, with questions about the cause or cure of disease playing little part in their discoveries (Sutherland 1972, pp. 401-408; Cori 1969, pp. 1-20).

65 Virtually all areas and phases of endocrinology, however, are marked by recurring interactions between basic and clinical pursuits, and contemporary molecular endocrinology is no exception. Thus, cyclic AMP workers have begun looking into the relationship between defects in the formation or action of this nucleotide and a variety of human diseases, with the ultimate hope that "The results of this research can eventually be applied to increase the quality of human life... [since] the chief end of all scientific research either is or ought to be the promotion of human happiness" (Robison, Butcher, and Sutherland 1971, pp. 454-455). Simultaneously, the reverse application of knowledge from the study of disease to fundamental questions so common in the earlier history of endocrinology is also taking place in the new molecular endocrinology. For example, physicians have known for some time that the lethal effect of the cholera bacteria's enterotoxin is a devastating diarrheal loss of fluids and salts from the body; the most effective therapy is the immediate replacement

67

toxin mimics the ability of cyclic AMP in intestinal cells to provoke cell membranes into actively transporting fluid and salts out of the cells. Carpenter theorizes that the toxin behaves like a hormone by binding itself to hormone receptors on the cell membrane; the receptors stimulate production of cyclic AMP, which in turn stimulates the membrane's active transport system. Unlike ordinary hormones, though, cholera enterotoxin is not particular about the kinds of cells or the membrane binding sites it will act upon. This makes it a useful, if potentially fearsome tool for investigating basic questions about the structure of hormone receptors and about the biochemical role of cyclic AMP within the cell (Carpenter 1972; McCann 1974, pp. 323-325). Thus, the discovery and study of diseases stemming from cyclic AMP malfunction promises to yield both the objective of treating diseases more effectively, and also that intense intellectual satisfaction which comes to the scientist when his puzzle-solving researches fit together to produce a new understanding of life processes.

"A New Physiology"

As we have explored some of the ways in which a"cl-i-l~gy has ev~;-np f~~m at7cient "k;tchen anatomy" to present molecular researches, we see clearly the interplay between the clinic and the research laboratory in the generation of problems, concepts, and facts concerning endocrine function. What we have called phases three and four of endocrine research - the development of neuroenclocrinology and current molecular studies of hormone action - belong primarily to that portion of endocrinology's history where research had moved from a disease-initiated focus into the province of basic research. We have, accordingly, treated these phases only briefly, to suggest where that flow led and is leading investigators of the ductless glands.

It is in looking at the emergence of endocrinology as a field of biology in the latter decades of the nineteenth century and early years of the twentieth century that we see, most preeminently, the imprint and influences of medical concerns. For, particularly in France and then Britain, countries in which clinical medicine more generally shaped the problems pursued by physiologists, attention by physicians to disorders of the ductless glands provided a new range of phenomena for scientific investigation. At the turn of the century, as represented most significantly by Bayliss and Starling's new anG Tunuamentai uiiuui4tai--j endocrine gland's chemical mediators in physiological and biochemical functioning.

Reflecting in 1933 on the impact of endocrinology upon not only medicine but upon physiology as well, R. G. Hoskins chose to underscore the words of the great British physiologist and pioneer endocrinologist, Edward Sharpey-Schafer:

The changes in physiology which have resulted from this [new endocrine] knowledge constitute not merely an advance in degree but an alteration in character. The doctrine of internal secretions forms a new departure. We must in the future explain physiological changes in terms of chemical regulation as well as of nervous regulation. It is therefore justifiable to speak of the doctrine as a New Physiology, seeing that it has completely altered our outlook on many of the problems with which physiology deals and consequently on those met with in medicine and surgery. (Hoskins 1933, pp. 347-348)

68

66 Notes

1 . Within the confines of a single chapter, we have had to omit some of the most interesting fundamental consequences of disease-related research in endocrinology. Some examples drawn from the history of diabetes and insulin research will give some sense of the range of problems suggested by clinical endocrinology and some sense of our ornissions,

Clinical endocrinologists have imposed great demands on the ingenuity of biochemists, to the benefit of both. For example, the first protein to have its amino acid sequence worked out completely was insulin. Sanger demonstrated conclusively by his analysis of the hormone that "proteins are well-defined molecules in which the amino acid units are linked by pe,)tide bonds to form long polypepticle chains," a definition that was oy no means certain when he began his decade of work in 1945. In the process of analyzing the structure of the insulin molecule, Sanger developed an armory of biochemical techniques for attacking the structure of other proteins and macrornoleculesThese techniques soon revealed subtle differences among the insulins of different species and forced evolutionary biologists to ask why, for example, pig insulin resembles human insulin more than cow insulin does.

Through both human and animal research on the nature of endocrine disorders, investigators have discovered both how universal endocrine mechanisms are in the animal kingdom and how idiosyncratic each group can be. A 1972 review of research on diabetes, insulin, and carbohydrate metabolism, for example, points out that insulin is an especially important hormone in carnivores who cannot count on a steady supply of food and whose metabolism must cope with sudden gluts and long-term shortages. In such creatures - man included -diabetes will be a serious disease; Jn herbivores and especially the ruminants who nibble all day long, diabetes is much less dangerous (Fritz 1972, pp. 166-180).

Another area we do not treat is that of comparative endocrinology. We deal only with research on the endocrinology of vertebrates; and within the vertebrates, almost solely with laboratory animals - dogs, cats, rats, rabbits - and man. We bypass entirely the fascinating work on invertebrates, plants, fungi, algae, and bacteria. Moreover, we have ignored the contributions of clinical endocrinology to our understanding of evolution, ecology, social and sexual behavior, psychology , embryology, and development. Finally, we have played favorite. within the endocrine system too. Thus, for example, the story of parathyroid and calcium metabolism receives no mention. The elaborate relationships of the sex hormones and glands with reproduction, growth, development, and behavior are only touched upon, chiefly because the history of sex endocrinology currently is being dealt with extensively by historian Diana Long Hall.

2. The importance of special instruments for the advancement of both neuroenclocrinoiogy and neurosurgery is illustrated by the sterecitaxic instrument developed in 1908 by British neurosurgeon and neurophysiologist Victor Horsley and his colleague R. H. Clarke. The Horsley-Clarke device, a special kind of head-clamp combined with three-dimensional micrometer measuring devices, enabled physicians and laboratory researchers to make surgical or electrolytic lesions in precisely determined spots in the brain.

3. For instance, the discovery was the basis for Dorothy L. Sayer's 1928 short story, "The Incredible Elopement of Lord Peter Wimsey."

4. The diuretic response later was shown to be an artifact due to the experimental conditions, in contrast to the antidiuretic response which is in endocrine function of the posterior pituitary (Barrington 1975, p. 93).

5. Despite their obvious importance, these discoveries of the growth hormone and the cliabetogenic effect of the anterior pituitary were something of a side issue for Evans and his colleagues- So was the laboratory group's participation in the discovery of the thyroid-stimulating hormone (TSH) and the adrenocorticotropic hormone (ACTH), both hinted at in Evan's analysis of Smith's early research on tadpoles (Evans 1923-24, pp.

67 216-218; Evans, Sparks, and Dixon 1966, pp. 319-320; Amoroso and Corner 1972, pp. 129-131). The interrelationships between the hypophysis and the ovaries remained the central problem for Evans and his co-workers. In seeking the reasons why their gigantic rats who had been treated with growth hormone failed to ovulate, Evans, Simpson, and Smith uncovered the existencd of still another pair of hormones, the gonadotropic luteinizing hormone (LH) and foil icle-stimu lating hormone (FSH). Their method of preparing crude extracts of growth hormone had, they gradually realized, retained both the growth hormone and the luteinizing hormone but destroyed the follicle-stimulating hormone. For successful ovulation both gonadotropic hormones had to be present in the right proportions. Once distinctions among the effects of the luteinizing, foil icle-stimu lating and growth hormones were drawn and analogous effects of these hormones in male animals were recognized, the research effort turned to the isolation and purification of the three hormones; in 1949, Li, Simpson, and Evans finally obtained homogenous FSH.

6. In the 1930s at Chicago, Fisher, Ingram, and Ranson used the Horsley-Clarke sterecitaxic instrument to make small, accurately placed lesions in different areas of the hypothalamus in cats (see note 2). Lesions in the tract originally described by Ramon y Cajal had two related effects: atrophy of the posterior pituitary, and a polyuria very much like that in clinical cases of diabetes insipiclus. Ranson and his colleagues also noticed that these lesions interfered drastically with labor in pregnant cats a result which correlated well with the strong uterine contractions invoked by the oxytocin ("quick childbirth") fraction of posterior lobe extracts. Injections of posterior pituitary extract counteracted the polyuria and difficult labor. Lesions in other tracts of the hypothalamus did not incur any of these disorders although they did have other effects resembling pituitary malfunction (Harris 1955, pp. 200-204, 224-225).

E,

Studies of Pituitary Principles." The Harvey Lectures, Series XIX, pp. 154-211 ~

Addison, T. 1855. "On the Constitutional and Local Effects of Disease of the Supra-renal Capsules." In A Collection of the Published Writings of the late 'Thomas Addison, M. D., eds. Dr. Wilks and Dr. Dalcly. London: The New Sydenham Society, 1968, pp. 209-239.

Allen, F. M., C. H. Danforth, and E. A. Doisy, ads. 1939. Sex and Internal Secretions: A Survey of Recent Research. 2nd ad. Baltimore: The Williams & Wilkins Co.

Allen, F. M., E. Stillman, and R. Fitz. 1919. Total Dietary Regulation in the Regulation of Diabetes. Monographs of the Rockefeller Institute for Medical Research (New York), No. 11.

Amoroso, C., and G. W. Corner. 1972. "Herbert McLean Evans, 1882-1971." Biog. Mem. Fellows Roy. Soc. 18:83186.

Bacq, Z. M. 1975. "Walter B. Cannon's Contribution to the Theory of Chemical Mediation of the Nerve Impulse." In The Life and Contributions of Walter Bradford Cannon, 1871-1945: His Influence on the Development of Physiology in the Twentieth Century. Eds C. McC. Brooks, K. Koizimu, and J. 0. Pinkston. State Univ. of New York, Downstate Medical Center, Brooklyn, pp. 68-83.

Barcroft, H. and J. F. Talbot. 1968. "Oliver and Schafer's Discovery of the Cardiovascular Action of Supernal Extract." Postgraduate Medical Journal (London) 44:6-8.

Bargmann, W. 1975. "A Marvelous Region." In Pioneers in Neuroendocrinalogy. Eds. J. Meites, B. T. Donovan, and S. C. McCann. New York: Plenum Press, pp. 37-43.

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73 CHAPTER 4 DISEASE AND THE DUCTLESS GLANDS

"By their diseases ye shall know them - the endocrines." The title of this essay by J. H. Means aptly characterizes the development of endocrinology, a field to which his own clinically -oriented researches on the thyroid contributed so greatly (Means 1960). For, endocrinology is a science which grew out of the interest of physicians in the malfunctioning of glands whose functions were not yet well understood, and it has continued to draw many of its problems from the study of endocrine disorders.

In the pages that follow, we will look at the ways in which research on endocrine disorders gradually revealed the nature and functions of the hormones - the chemical messengers produced by the endocrine glands - and the complexity and integration of the endocrine system's interactions. Our account of endocrinology's development defines four phases of research which, though often overlapping in time and effort, are each marked by the appreciation of a new level of complexity in the functions of the endocrine glands. Phases one and two, in which physicians and physiologists recognized the effects of individual endocrine glands on other parts of the body, and then discovered how glands communicate with one another, will receive the most detailed treatment in this chapter. For it was in these phases of inquiry that problems of medicine raised a fundamental biological problem, that of the nature of the chemical regulation of physiological processes: a problem around which endocrinology emerged as a new field of research in the early years of the twentieth century. To provide some understanding of the problems that endocrinologists subsequently pursued we will sketch, more briefly, the content of endocrinology's third and fourth phases. Phase three was marked by discoveries about how the endocrine glands and the nervous system influence each other's activities, while in the fourth and current phase, endocrinologists and molecular biologists seek to understand the elaborate mechanisms of communication between the endocrine system and the organelles of individual target cells.

The broad approach to the development of endocrinology taken in this chapter in part reflects the nature of the endocrine system itself. The system's most strik ing feature is its ability to coordinate extremely complex interactions among its own disparate elements, the rest of the body, and the external environment. Each gland's activity impinges on the others and on the body as a whole. As textbooks of endocrinology despairingly point out, it is artificial and misleading to depict the development and nature of endocrinology in terms of a particular disease, gland, hormone, or even a set of interacting parts. The fact that often the peculiar manifestations of endocrine disorders initially drew attention to the interrelationships of glands, hormones, nerves, environment, and target cells underscores the importance of treating the history of research on the endocrine system in as holistic a manner as possible.1

The Enigmatic Glands

Like most other branches of science, endocrinology has no obvious birthday to celebrate. Most of the endocrine glands were discovered by the "kitchen anatomy" of hunters and cooks in prehistory or by the medical anatomists of classical antiquity and the Renaissance; a few - like the tiny parathyroids embedded in the thyroid - were not found until the last century. Similarly, ancient, medieval, and Renaissance physicians noted several disorders diabetes, goiter, cretinism, dwarfism, and gigantism which we now know are caused by endocrine malfunction; but other equally striking endocrine diseases were not described until this century. And, whether by superstition or empirical trials, physicians in many cultures and periods have employed animals' glands as drugs -precursors of such modern glandular products as insulin and cortisone. These early observations of anatomy, disease, and therapy were not linked together, however, until the latter half of the nineteenth century and the beginning of the twentieth. Only then did a combination of clinical studies and the physiological experiments they generated suggest that these mysterious glands had an important role in the coordination of growth and metabolism.

It was well before the latter half of the nineteenth century, however, that physicians and biologists began to formulate the concept of animal hormones or internal secretions. In 1766, for example, the celebrated Swiss

anatomical peculiarities the lack of a secretary duct and a plentiful supply of blood vessels - and suggested that these "vascular glands" poured special substances directly into the veins and thus into the general circulation. A few years later, in Recherches sur les maladies chroniques, French physician Theophile de

74 Bordeu formulated a concept akin to what we now think of as a hormone. De Bordeu speculated that "each organ of the body gives off emanations which are necessary and useful to the body as a whole," and he particularly stressed "the tonic effects of testicular and ovarian emanations, and their influence on the secondary sex characteristics as demonstrated by the obvious alterations in the body in general which follow castration" (Young 1970, p. 129, 130).

Thus, although the great French physiologist, Claude Bernard, has often been credited with first formulating the concept of internal secretion in the 1850s, the idea that what we now call endocrine glands secrete substances into the circulation was current well before Bernard's time. It is to Bernard, however, that we owe the term "internal secretion," and the first direct demonstration of one internal secretion (the liberation of glucose into the bloodstream by the liver) (Houssay 1967; Young 1970). Although Bernard's conception of internal secretions, which included the possibility that these substances might function to maintain the composition of the blood, was far broader than that later formulated by endocrinologists, his preeminence in nineteenth century physiology helped to focus attention on the ductless glands.

It was in 1855, in a lecture at the Coll&ge de France, that Bernard discussed the liver's internal secretion. The same year, a book appeared that, perhaps more than any other single work, deserves to be called the foundation of modern endocrinology: Dr. Thomas Addison's On the Constitutional and Local Effects of Disease of the Supra-renal Capsules. Addison's work epitomizes a sequence of observation and experiment that textbooks often call the "classic method" of endocrine research, in which the clinical features of a disease were compared and correlated with pathological changes in a gland. Along with, or usually following, this study, went observations on the effects of removing a gland thought to be overactive, or, conversely, the effects of administering an extract of a gland that was atrophied or damaged. tions of his patients and his diagnostic skill. Comparably, his monograph is such a descriptive masterpiece that modern works on Addison's disease have not been ablu` to improve greatly upon its accuracy of detail (Hartman,, and Brownell 1949, p. 343). From the study of only 't eleven cases, Addison was able to pick out the "leading and characteristic features of the morbid state. . . anemia, general languor and debility, remarkable feeble- j ness of the heart's action, irritability of the stomach, and a peculiar change of the color in the skin (Addison 1885, p. 214). Post-mortem examinations revealed a diseased condition of the "supra-renal capsules," as Addison and earlier anatomists called the small pyramidshaped glands which sit on top of the kidneys. In some of his cases the adrenals had been damaged by tuberculosis or cancer, but Addison could point to at least one case in which the adrenals were the only abnormal organs in an otherwise healthy body. This was good evidence that the symptoms and condition he described were, in fact, due to a malfunction of this gland alone (Addison 1855, p. 210).

The monograph did more than describe a new disease, however, for Addison saw clearly that his study of the adrenals was a "first and feeble step towards inquiring into the functions and influence of these organs" (Addison 1855, p. 210). It seemed probable to him that the general state of ignorance about the adrenals, as well as the other "gland-like" organs, the spleen, thymus,. and thyroid, would be overcome by pathological studies where physiological research had previously failed. "Although pathology . . . is necessarily founded on physiology," he wrote, "questions may, nevertheless, arise regarding the true character of a structure or organ, to which occasionally the pathologist may be able to return a more satisfactory and decisive reply than the physiologist" (Addison 1855, p. 209). Indeed, Addison himself had "stumbled upon" these "curious facts" about the disease of the adrenals while searching for the organic lesion of another mysterious disease, a fatal anemia of unknown etiology. (His description in 1849 of this anemia, now known as pernicious anemia and known to be caused by a vitamin deficiency, also is classic.) Among his anemic patients, Addison had noticed a few with a distinctive darkening or bronzing of the skin, who at autopsy were found to have abnormal adrenals. Addison was, of course, unable to say why damage to the adrenal glands should alter the complex or cause a gradual, fatal debility. But, by directing ntion to the disease and the site of its lesion and emphasizing the utility of studying a diseased organ understand the functions of a healthy one, Addison ited systematic research into the role of the ductless rids in the general economy of the body.

Thirty years after Addison's monograph, a French ysician, Pierre Marie, described and named another -narkable malady, again exemplifying the early classic tthiod of endocrine research. In his 1866 paper, "Two ._s of Acromegaly: An Unusual Hypertrophy of the ad and Upper and Lower Extremities," Marie disssed two patients whom he had observed and another ven cases he had found described in the medical erature. He pointed out the excessive growth of the era and the bones of the face, hands, and feet, and e thickening of soft tissues like the tongue, lips, id nose, which made the features of acromegalic tients gradually become

75 strikingly coarse and elonted. Other signs and symptoms that Marie noted as aracteristic of the chronic condition included severe aclaches, intense thirst and appetite, cessation of enstruation, changes in the thyroid, and damaged sion. Later papers by Marie, his student Souza-Leite, d other physicians added to the number of cases d confirmed Marie's orginal description of the disease. Marie hesitated in his first paper to offer an explana on of the disease he had defined, because only one topsy had been performed. In that one case, published few years earlier by another physician, an egg-sized mor had been found in the brain "in the position of e pituitary body" (Marie 1886, p. 20). Marie consided this a lesion of the nervous system and thought at the hypertrophy or enlargement of the pituitary and was suggestive of the hypertrophy of the bones he face and extremities. But, he carefully added, the might have had no cause and effect relationship was only one of several plausible hypotheses about e cause of acromegaly. Marie's reluctance to seize Pon the abnormality of the pituitary gland as the priary Cause of,:cromegaly may also have been influenced I the prevant belief among physiologists that this and had "little, or perhaps no, use in the organisms of e higher vertebrates" (Rolleston 1936, p. 55). But arie's clear account of the syndrome, even without eculations about its cause, encouraged others to search e literature, clinics, and anatomical museums for rther examples. Two years after Marie's first paper, tto Minkowski - more famous for his work on the pancreas and diabetes - explicitly made the connection between acromegaly and a disease of the pituitary gland. Then, in 1890 and 1891, Marie reported that enlarged pituitaries always were found in post-mortern examinations of persons with acromegaly, and he hypothesized that the abnormal growth of the pituitary caused a glandular deficiency and hence toxemia (Rolleston 1936, p. 86).

By implicating a particular gland in a specific disease, Addison and Marie gave physicians and physiologists both a reason and a method for studying the ductless glands. Over the centuries, as we have indicated, there had been occasional attempts to see what happened when a gland was removed or transplanted, but no one had followed up on them seriously. The well-known effects of castration on men and animals, for example, had led the eighteenth century surgeon, John Hunter, to try transplantation experiments with the gonads and accessory sex organs of hens and roosters. In 1849 similar experiments were carried out by the German physician, A. A. Berthold, out of a particular interest in the structural changes in the transplanted gonads and a general interest in the nature of sex, heredity, and the "sympathies" between different parts of the body (Jdrgensen 1971, pp. 27-30). Astley Cooper and T. W. King tried removing the thyroids of dogs in the 1820s and 30s and suggested that the thyroid might form some 11 particular material" which exerted an influence "upon the circulating fluid [which] may be more or less needful for the healthy subsistence of the entire animal" (Rolleston 1936, pp. 18, 50). Addison's monograph immediately inspired the French physiologist, BrownS6quard, and others to do systematic extirpation experiments on the adrenals, with contradictory results. Brown-Sdquard believed that the adrenals were necessary to life because he found that the removal of both adrenals from various animals was always fatal; Phillipeaux, on the other hand, was able to excise the adrenals and other glands from white rats without killing them. The different results were due partly to different surgical techniques and precautions against infection and partly to the existence of unnoticed accessory adrenals in white rats which enabled them to survive the loss of the main pair, as well as differences in resistance to stress and infection among different strains of animals. Similar debates over the indispensability of the pituitary and the thyroid glands took place in the 1880s.

The variable results of extirpation experiments underscores the importance of surgical and aseptic

Medicine in 19U9 Tor his worK on inyrom oiseaseti, ne began his lecture by exalting Pasteur's discoveries in bacteriology and Lister's application of them to surigical practice. Thanks to aseptic procedures, Kocher emphasized, it at last was possible for surgeons and physiologists to "make all the organs accessible to direct observation, and to alter the conditions in which they exercise their functions (Kocher 1909, pp. 330-33). Surgical technique, too, often meant the difference between a clearcut and an inconclusive experiment. The results of thyroid extirpation had been complicated by the failure to recognize and leave intact the tiny parathyroid glands in the midst of the thyroid tissue; the effects of removing the pituitary gland were confused by the effects of surgical damage to the hypothalamic region of the brain.2

Perhaps the most celebrated example of the importance of surgical skill for the development of endocrinology is Minkowski's discovery of the role of the pancreas in diabetes in 1889. The physician von Mering was studying the problem of fat absorption in diseases of the pancreas and wanted to know how the decreased secretion of pancreatic juices into the intestine affected the breakdown of fats. When Minkowski asked why he did not remove the pancreas, von Mering answered that this was impossible. Minkowski, proud of his surgical skill, replied brashly, "Why should it be impossible? Bring me a dog and I will remove

76 the pancreas." The operation succeeded, but to Minkowski's annoyance the supposedly well-trained dog began urinating frequently and copiously. On an impulse, or at the suggestion of hischief,von Naunyn -storiesdiffer - Minkowski collected some of the urine from the floor and tested it for sugar. The sugar content was surprisingly high, a familiar sign of diabetes mellitus. After a week of further operations on other dogs, Minkowski was able to tell von Mering that total removal of the pancreas resulted in diabetes mellitus (Nothman 1954, pp. 272274; Allen et al. 1919, p. 38; Young 1970, pp. 137-140).

Surgical skill and asepsis also helped to decide between two competing theories of the function of the ductless glands. The first of these theories, widely held in the late nineteenth century and well into the first two decades of the twentieth, was that these glands somehow neutralized or removed poisons in the blood; consequently, disease or removal of these organs caused toxemia. It is easy to see in this theory the strong stances into the bloodstream, had a long history of illustrious proponents such as von Haller. Cooper and King's experiments in thyroid removal in the 1820 and 30s, as we have seen, led them to this thesis: that glands such as the thyroid released a special substance directly into the blood. In the 1840s George Gulliver, surgeon and Fellow of the Royal Society, reported that under the microscope spheroidal bodies could be seen in both the adrenals and the veins leading from them; he argued that the veins served as the duct for some "peculiar matter which doubtless had some special use" and which was produced by the adrenals. Gulliver considered this "an interesting and important subject for further inquiry," but neither he nor anyone else pursued it at the time. Like many other early examples of the internal secretion hypothesis, this work was ignored until the hypothesis was firmly established and its history began to be written (Rolleston 1936, pp. 18-20).

The first good evidence for the internal secretion explanation of the glands' function came through research on thyroid diseases and the unhappy results of thyroid surgery on humans in the 1870s and 80s. In England W. W. Gull and W. M. Ord described a condition which Ord called myxedema: a swelling of the face and body from an excess of a gelatinous substance, mucin, accompanied by impairment of the mental faculties, lethargy, and extreme sensitivity to cold. In its advanced stages, myxedema strongly resembled cretinism. Cretinism, in turn, was associated with thyroid abnormalities but in very confusing ways; many victims of "endemic cretinism" had immense goiters, while cases of "sporadic cretinism" generally had tiny atrophied thyroids. Ord noted that in all five of his cases of myxedema the thyroid was small, but he thought it had simply been squeezed by the excess mucin. In Switzerland, meanwhile, where goiter was especially common, removal of the grossly enlarged thyroids became popular once the antiseptic method made such surgery a less dangerous affair. Unfortunately, some patients whose goiters had been removed began to suffer a new malady which was variously ascribed to injury of the nerves or trachea or to loss of some "blood-making" function of the gland.

In both England and Switzerland a few bold physicians speculated that all three conditions - sporadic cretinism, myxedema, and the illness that followed thyroid operations - resulted from the absence or degeneration of the thyroid. Schiff, who had done experimental thyroidectornies on animals thirty years earlier and thought then that the thyroid produced an internal secretion, now saw the clinical significance of his earlier research; he tried implanting thyroids into the abdomens of thyroidectornized animals and succeeded in preventing myxedema. At this point it was clear that the thyroid was necessary for health, but the blood detoxitication hypothesis was still tenable. Then, in 1891, George Murray described his successful attempt to treat human myxedema with extracts of sheep thyroid glands; and three years later, Magnus-Levy showed that such extracts speeded up metabolism. Thus, the thyroid and its secretion gradually was recognized as having an active function unrelated to detoxifying the blood but indispensable to health (Paget 1919, pp. 54-67; Rolleston 1936, pp. 29-30, 151-153),

The discovery that thyroid extracts or grafts could restore normal intelligence and vigor to the myxedematous patient attracted a great deal of both scientific and popular attention, 3 for it showed that a disease could be caused by an insufficiency of the secretion of a ductless gland and, in turn, that the symptoms of such a disease could be cured by what was termed replacement or substitution therapy. The interest caused bV clinical studies of the thyroid gland, however, was overshadowed by more sensational claims for the actions of internal secretions, associated with the theories and activities of Charles-Edouard Brown-S6quard.

In retrospect, Brown-Sdquard has been recognized as a remarkable pioneer in endocrinology," whose therapeutic studies, "although they later fell into disrepute, sparked the imagination of many investigators [and] led to an active search for 'internal secretions' in animal tissues" (Rolleston 1937, p. 261; Borell 1975, p. 1). Elected to England's Royal Society and France's Academie des Sciences, Brown S6quard's peripatetic

77 career saw him practicing medicine, teaching, and doing research in locales such as Paris, Boston, New York, Philadelphia, London, and Dublin.

Addison's book on the symptoms of adrenal insufficiency, as we noted, sparked BrownSdquard's interest in the ductless glands, and in 1856 he concluded that the adrenals are essential to life after performing total adrenalectornies on dogs. In his 1869 course of lectures at the Paris Faculty of Medicine he set aside his earlier views about the detoxifying function of the adrenals in favor of the theory that the "glands have internal secretions and furnish to the blood useful if not essential principles" (Major 1943, p. 375). Then, in 1889, after he had succeeded Claude Bernard as professor of medicine at the Coll6ge de France, Brown-S6quard reported to the Society of Biology that, in experiments performed on himself, he had shown the rejuvenating effects of testicular extracts from healthy young guinea pigs. Between 1889 and his death in 1894, BrownS6quard and his assistant, d'Arsonval, extendeci their investigations to include the therapeutic effects of extracts from other animal tissues, based on BrownS6quard's expanded concept of internal secretions. Every tissue of the organism, he postulated, secretes its own special product into the blood and this influences "all other cells which in this way are dependent on each other, by a mechanism different from the nervous system" (Houssay 1967, p. 165; Brown-S6quard 1889, pp. 415-422).

To the world at large, including many physicians, the details of Brown-S6quard's concept were far less exciting than the therapeutic possibilities it implied; by 1890, an estimated 12,000 physicians were giving testicular extracts to their patients, despite the skepticism and embarrassment expressed by many medical journals (Olmsted 1964, pp. 209-211).

Brown-S6quard, too, was concerned about the sensational publicity that his experiments received, and angered at the lucrative business of glandular extracts engaged in by some physicians. Nonetheless, he believed strongly that his experiments with testicular and other extracts were premised on sound physiological thinking, and would open up a new realm of therapeutics.

. . . now we believe that all the tissues, glandular or not, give something special to the blood, that every act of nutrition is accompanied by an internal secretion. We believe, in consequence, that all the tissues will be able to be and ought to be employed in special cases as a mode of treatment; that there is, in short, a new therapeutics to create, in which the medicaments will be products produced by the different tissues of the organism.

The bacterial products taught us how active the chemical compounds created by the infinitely small were: the living cell, of each tissue that belongs to the organism, must, by analogy, secrete some products, of which the efficacy is no less. (Brown-S6quard and d'Arsonval 1891; trans. Borell 1975, pp. 3-4 endocrinology, one that many later endocrinologists would have preferred to ignore because of the cloud that it cast over clinical endocrinology. The editor of the British Medical Journal in 1937, for example, wrote of the organotherapy movement as follows:

After the discovery that preparations of thyroid gland taken by mouth were of benefit in MYXoedema and cretinism, thyroid extract began to be given for many and varied conditions, and extracts of other ductless glands were prescribed with an enthusiasm that outran knowledge. Indiscriminate endocrine therapy brought about the inevitable reaction, and clinical endocrinology came to be looked upon with suspicion. The intensive research work carried out over the past decade has gone far to remove the disfavour into which endocrinology had fallen, and it is now becoming possible to put the matter in some kind of perspective. Solid achievements have been made. Possibilities are daily becoming probabilities. But the exploitation of these probabilities in the interest of the patient must be carried out with due caution and a healthy skepticism if enclocrinoloby is not to be done at the disservice of again becoming a fashion. (The Endocrines In Theory and Practice 1937, Preface)

Despite the negative effects of the excesses committed in the name of organotherapy, however, the movement was a positive force for the development of endocrinology in other respects. Fo: , as historian of endocrinology Merriley Borell points out, "much was learned about the action of internal secretions by means of these therapeutic efforts. The organotherapy effort was fundamental in attracting the attention of scientists to the field, who then sought to set up standards for the proper investigation of these therapeutic effects. Here, clearly, clinical concerns pointed the way to new scientific problems" (Borell, personal communication).

78 One such route was followed by the British physician George Oliver, who experimented with extracts of various tissues, including the adrenal and thyroid glands and the brain. Oliver had been administering extracts to his son, and then using an instrument he had developed to measure their effects on the diameter of his son's radial artery. In 1893, he observed that adrenal extracts professor of physiology at University College, London and caught him - so tradition has it - at a moment when he was measuring the blood pressure of an anaes thetized dog. Schafer was dubious about Oliver's claim but agreed to inject some of the extract into the animal' veins. To Schafer's astonishment, the mercury in the blood-pressure manometer shot up so quickly and so high that the recording float was nearly lifted out o the tube (Dale 1938, pp. 461-463; Barcroft and Talbo 1968, pp. 6-8). Oliver and Schafer's studies of thi pressor effect of adrenal extract not only led to th first isolation of a hormone (epinephrine), but also t a better understanding of the physiological role of th glands of internal secretion.

The arguments over the possible blood cletoxifying function of the adrenals subsided once the definite, measurable effect on blood pressure was demonstrated. Oliver and Schafer also correlated function with anatomy; they showed that extracts made from the inner part of the adrenals, the adrenal medulla, would raise blood pressure, while extracts from the outer layer, the cortex, were ineffective (Rolleston 1936, pp. 29, 331). Research into the physiological implications of anatomical distinctions between parts of other glands, such as the anterior and posterior lobes of the pituitary, and the islets of Langerhans in the pancreas, quickly followed and proved fruitful. The fact that the internal secretion of a gland might actually contain several different active substances, each produced by a particular kind of tissue or cell, was at first confusing but later became axio matic, and helped to direct attention to the complicated interactions of hormones and glands. Finally, the isola tion of the pressor substance from the adrenal medull at the end of the nineteenth century and its crystalliza tion as a pure compound by Jokichi Takamine and T. B Aldrich (working independently) in 1901 demonstratec concretely the existence of an internal secretion, ant enabled scientists to study the pressor effect and othe characteristic actions of epinephrine in much mor carefully controlled experimental situations (Marti Ibanez 1952, p. 233).

When Oliver and Schafer first reported their study o the pressor effects of adrenal extract to the Physiologica Society in March 1894, their audience included twi young investigators in Schafer's laboratory, Wilhart Bayliss and Ernest Starling. In 1902, investigating the ntrol of pancreatic secretions, Bayliss and Starling 1dentified a new internal secretion that they named 'secretin. Their discovery was made when they attempted ,,to pinpoint the pathway of the neural reflex that the Russian physiologist, Ivan Pavlov, had declared must 'be responsible for the flow of pancreatic secretions into the intestine to neutralize digestive acids. Bayliss and Starling, however, found that even when all the nerves between the small intestine and the pancreas were severed, acid in the duodenum would still provoke the flow of pancreatic juices. According to C. J. Martin, who was present when the experiment was performed, Starling concluded at once, "Then it must be a chemical reflex." He promptly made a crude extract from a piece of duodenum, injected it into the jugular vein of the dog, and within a few moments had the pleasure of seeing the pancreas respond with a heavy secretion (Barrington 1975, p. 11).

Starling used the occasion of the Croonian Lectures to the Royal College of Physicians of London in 1905 to bring together what was known about "the chemical correlation of the functions of the body," In these lectures he introduced the word "hormone" (from the Greek, hormao, to excite or arouse to action) for the general class of substances "which, speeding from cell to cell along the blood stream,. . coordinate the activities and growth of different parts of the body" (Starling 1905, p. 340). As examples of these chemical messengers, he named epinephrine, secretin and a number of similar gastric hormones which had been discovered in the meantime, extracts from the thyroid and from the interstitial cells of the testes and ovary, and - more tentatively - the supposed internal secretion of the pancreas. The simplicity and convenience of the word "hormone" soon made it popular even though, as etymological and physiological purists pointed out, it applied to substances which could inhibit activity as well as excite it. The words "endocrine" and "endocrinology," (from the Greek, I separate within) came into use about half a dozen years later (Rolleston 1936, pp. 2-4).

Interrelationships Among the Endocrine Glands

Building upon the observations and clinical studies of physicians, the discovery of secretin in 1902 and Starling's elaboration of the hormone concept in 1905 represent a watershed in the emergence of endocrinology as a field of basic as well as clinical research. In the case of the thyroid, adrenals, and the duodenal extract named secretin, physicians and physiologists had suc ceeded in their effort to confirm the

79 long-presumed existence of potent secretions in animal tissues. That proof came both from the ability of glandular extracts to cure specific diseases such as myxedema and from the analysis of specific physiological responses to the chemical substances in adrenal and duodenal extracts.

When secretin was discovered, as Borell observes, the term "internal secretion" "no longer provided an adequate description of the phenomenon under study. It did not specify the messenger role, the activity potential of chemicals elaborated by certain tissues," as did the word "hormone" (Borell 1975, p. 8). And, as Starling and other investigators recognized at the turn of the century, the chemical coordination of physiological processes by hormones was now a major subject for biological research, one that seemed of equal import to understanding the integrative action of the nervous system. For, as Starling wrote in Lancet in August 1905:

If control of the different functions of the body be largely determined by the production of definite chemical substances within the body, the discovery of the nature of these substances will enable us to interpose at any desired phase in these functions and so to acquire an absolute control over the workings of the human body. Such a control is the goal of medical science. (Maisel 1965, p. ix)

Building upon the idea of a human control of physiological processes, the next phase of research in endocrinology began to delineate and emphasize the intricate functional interactions among the glands. The early endocrinologists had tacitly assumed that each gland produced its own special hormone and had its own particular effect on the body, and endocrine diseases thus were viewed as the consequence of an increase or decrease in the secretion of a hormone. By the 1920s, however, closer study of endocrine diseases coupled with animal experimentation made endocrinologists see that this picture of the endocrine system was greatly oversimplified. The central fact -and the central problem of endocrinology between the World Wars was that activities of one gland could control and be controlled by the activities of others.

Research now concentrated on pairs or groups of glands, and the choice of which constellation of glands to study was still largely determined by clinical data and interests. The sexual abnormalities found in many

ciaterent mnesses, PP

"plurigiandular" preparations, the ability of epinephrine to alleviate but not to cure Addison's disease - each of th.ese clinical observations led to new insights into the complexities of metabolism and to new approaches to therapy.

Three areas of research, moving back and forth from the clinic to the laboratory, proved especially

uccessful and fruitful in this period. First, in the lineage begun by Thomas Addison, there was a body of work on the adrenals by Cannon, Selye, von Euler, Kendall, Moore, and many others. Their researches, in brief synopsis, helped to elucidate the roles of the two layers of the adrenal - the medulla and the cortex - in maintaining homeostasis and coordinating the body's response to emergencies and stress; established the nature of the medulla's close connections to the sympathetic nervous system; provided a better understanding of the relationships between the pituitary gland and the adrenal cortex; and identified, isolated, purified, and ultimately synthesized ACTH, the pituitary gland hormone which stimulates the adrenal cortex, and the several hormones secreted by the adrenal's two layers.

Secondly, there was a focus of research on the endocrinology of sex and reproduction. Work in this area received a powerful stimulus from the joint decision of the Bureau of Social Hygiene, the National Research Council, and the Rockefeller Foundation in 1921 to support "a program of research on fundamental problems of sex," and in 1931 to sponsor a major survey of sexual endocrinology: Sex and Internal Secretions. This review of the recent progress in the biology of sex and the sex glands, first published in 1932, attracted many new workers into the field (Young and Corner 1961, 1, ix-xii; Hall, work in progress).

80 A third major area of research, dealing with the functions of the pituitary gland and, in particular, the relations between pituitary disorders and diabetes, culminated in establishing for a time a view of the anterior pituitary as "the master gland" or "conductor of the glandular orchestra." The methods shared by all three areas of research included not only the familiar techniques of extirpation and replacement by transplants and extracts, but also the use of isolated and purified hormones like thyroxin and insulin as these the nature of this second phase in the development of endocrinology by highlighting research on the pituitary gland or hypophysis, physiologically the most complex and important of the ductless glands. About 1890, as we have seen, Marie, Minkowski, and others explained the condition of acromegaly in terms of a lesion of the hypophysis which decreased the gland's internal secretion. Five years later, in the course of their experiments on the dramatic power of adrenal extracts to raise blood pressure, Oliver and Schafer discovered that hypophyseal extracts also had a pressor effect. The hypophysis, like the adrenals, has two anatomically distinct parts: the anterior lobe or adenobypophysis and the posterior lobe or neurohypophysis. By 1898, Howell and Schafer and Vincent had shown that only extracts of the posterior lobe exerted a pressor effect, and so research centered on this lobe of the gland for many years. During the first decade of the twentieth century, a bewildering array of specific effects were attributed to the posterior lobe. While an extract of the posterior lobe as a whole increased blood pressure, one fraction of it had the opposite effect of lowering blood pressure. The extract produced intense contractions of the uterus and weaker contractions in some other plain muscles. It provoked the secretion of urine almost immediately, but this diuretic effect soon gave way to an even stronger antidiuretic effect.4 In some mammals posterior lobe extracts made milk flow more easily and in greater amounts than usual. In frogs the posterior lobe seemed to control the contraction and expansion of the pigments calls and thus the color of the frog's skin (Sharpey-Schafer 1924-26, 11, pp. 252-257).

These varied effects of posterior lobe extracts were both interesting and useful. The uterine contraction response provided a quantitative bioassay for the potency of posterior lobe extracts and found an application in obstetrical practice. The antidiuretic effect was first discovered when patients with diabetes insipidius - a form of diabetes characterized by a great flow of dilute urine and a correspondingly great thirst - were relieved by experimental injections of pituitary extract; this quickly be Came a standard treatment of the disease (Sharpey-Shafer 1924-26, 11, p. 244). But none of the actions of the posterior lobe of the pituitary could explain acromegaly. As the American neurosurgeon Harvey Cushing wrote ,Iquently, the great difficult for all pituitary gland I .,earchers was getting at the gland in the first place.

Nature saw fit to enclose the central nervous system in a bony case lined by a tough protecting membrane, and within this case she concealed a tiny organ which lies enveloped by an additional bony capsule and membrane like the nugget in the innermost of a series of Chinese boxes. No other single structure is so doubly protected, so centrally placed, so well-hidden. (Cushing 1930, p. 5) in 1904 Cushing forecast that techniques of brain surgery were improving so much that "it is not impossible that a diseased pituitary may someday be successfully attacked" (Fulton 1946, p. 267). Only two years later the British surgeon, Victor Horsley, successfully operated on several cases of pituitary tumors (Paget 1919, p. 180). Soon Cushing too was actively engaged in pituitary gland surgery and research.

Cushing had been interested in the hypophysis ever since 1901 when he had failed to recognize that a case of sexual infantilism accompanied by obesity, headache, retarded growth, and impaired vision was a case of pituitary tumor. His pride was nettled because, soon after finding the tumor at autopsy, he learned that A. ~.Frohlich in Vienna had seen a similar case, made the 'correct diagnosis, and had the tumor removed successJulIV (Fulton 1946, pp. 271-272). In 1907-1908 CushOg's interest was renewed by the announcement of a 4W technique for removing the gland from dogs and by series of lectures on the pituitary that Schafer deliv ad at John Hopkins. Cushing and his students at Johns 1:opkins then began a long series of pituitary gland xtirpation experiments on dogs, correlating the results 'Ith the conditions he found in his patients.

Cushing's chagrin over his misdiagnosis of what ~zsme to be called "Frohlich's syndome" was redeemed b the first major result of these experiments. Earlier .reports on removal of the gland said that the animals 41ways died after the operation. Some of Cushing's Oogs, however, survived the surgery and lived many '01nonths although they became "extraordinarily fat, . 'Y' sexless creature(s)." The general opinion among iushing's students was that somehow the operation had siot been complete, but they drew no further concluOons from the peculiar state of the animals. One day 4n the winter of 1908-1909 Cushing "caught sight of Aone of] these animals while it was still alive and said at once, 'Here is Frohlich's asexual adiposity.' " Because the animals lack all or nearly all of the pituitary, Cushing deduced that Frohlich's syndrome must

81 be caused by a lack of the pituitary secretion. Moreover, if this state of asexual adiposity was the result of too little pituitary secretion, then acromegaly must be the result of an excessive secretion - exactly the reverse of Marie's original explanation of acromegaly (Fulton 1946, pp. 280-282).

Over the next four years, Cushing confirmed his deductions in further experiments and studies of patients with a variety of pituitary gland disorders. He demonstrated that under- or over-secretion by the anterior lobe alone was responsible for Frohlich's syndrome and acromegaly, as well as for gigantism and a form of dwarfism. He also stressed the effects that pituitary lesions had on the rest of the endocrine system:

In view of the apparent interrelation of many of the glands of internal secretion it is quite probable that certain of the symptoms known to accompany hypophyseal disease may be consequent upon a secondary change in other glands which follow the primary change of the hypophysis. These changes are seemingly more outspoken and more widespread after a lesion of the pituitary body than after a corresponding lesion of any other member of the group of ductless glands, and in view of its unusually well-protected position one might have conjectured that it must represent a vitally important organ. (Fulton 1946, p. 301)

Cushing's strong awareness of the secondary endocrine effects of hypophyseal disorders led twenty years later to his elucidation of what had been vaguely known as "polyglandular syndrome" and usually ascribed to a malfunction of the adrenal cortex. In acromegaly he had noted in the anterior lobe a preponderance of cells which take up eosin dyes, and he concluded that acromegaly was due to oversecretion by these eosinophilic cells. At the same time he argued that oversecretion by the other distinctively staining cells of the anterior lobe, the basophiles, must also be responsible for some kind of syndrome, even though he could not say what that syndrome was. In 1930 he read of a case of "adipositalgenital dystrophy" which at autopsy revealed a tiny adenoma (tumor) of the basophilic cells. The case resembleil in every detail cases of "polyglandular syndrome" he had seen over the years, and further post long ago concluded that the eosinophilic cells secreted a hormone which regulated growth. Now he could add that the basophilic cells elaborated "a sex-maturing principle" or "a gonad -stimulating factor" (Cushing 1932, pp. 114-119, 155-161; Fulton 1946, rp. 614616).

Inevitably, the situation turned out to be more complex than Cushing's account in 1932 made it appear. More sophisticated cell staining techniques revealed many specialized cell types within the general categories of eosinophile and basophile. Moreover, the original explanation of the polyglandular syndrome (since renamed "Cushing's syndrome") in terms of an adrenal cortex disorder was vindicated: excessive production of the steroids of the adrenal cortex was the immediate cause of the symptoms of the disease, although such hypersecretion of steroids was often in turn the result of oversecretion of ACTH by the anterior pituitary. However, the main outlines of Cushing's explanation of the syndrome and his characterization of a growth factor and a gonad- stimulating factor proved to be sound.

Concurrent research by Bernardo Houssay in Argentina and by Herbert McLean Evans at Berkeley did much to confirm and extend Cushing's clinical insights into the workings of the anterior lobe and its ties to other endocrine glands. In 1907, the year that Cushing began his extensive work on the hypophysis, a precocious young medical student in Buenos Aires saw a patient with acromegaly and immediately became interested in the gland. Forty years later, in his Nobel Prize lecture, Bernardo A. Houssay commented that he had been attracted to the hypophysis by both its medical and physiological aspects: "the microscopic picture showed glandular activity and its lesions were accompanied by serious organic disturbances, such as acromegaly, dwarfism, etc." (Houssay 1947, p. 211; Young and Foglia 1974, p. 254). Beginning with his medical thesis in 1911, and continuing through a long series of researches, Houssay developed techniques for removing the hypophysis from frogs, toads, and dogs, and examined the physiological effects of posterior lobe extracts and implants on muscles, the uterus, carbohydrate metabolism, the thyroid, and the adrenals.

Houssay's most important research in endocrinology began after Banting and Best announced the discovery of insulin in 1921 a year that Houssay later saw as a in extracting it from the pancreas, but with insulin's existence proven in 1921, no one could doubt any longer the existence of hormones or their ability to regulate metabolism. Moreover, as the association between acromegaly and diabetes had been noted often by clinicians, Houssay saw that insulin could serve as a new tool for investigating the suspected physiological reactions between the hypophysis and the pancreas (Houssay 1956, p. 213; 1967, pp. 167-168).

Houssay had to devote several years to the preparation and standardization of insulin for his experiments, for large-scale extraction of insulin for treatment began only in 1923-1924, and pure crystalline insulin was not prepared until 1926. He and Magenta demonstrated in 1924 that a dog without its hypophysis became

82 remarkably sensitive to insulin's ability to lower blood sugar levels. It was not clear from their experiments, however, whether it was the loss of the anterior lobe or of the posterior lobe that was responsible for this hypoglycemic effect. Until 1929 the general opinion was that "if the hypophysis had any action on carbohydrate metabolism and on the diabetes of acromegaly it was due to its posterior lobe" (Houssay 1943, p. 247). The anterior lobe was ignored by researchers because the actions of its extract seemed to be transient and because it was hard to extirpate the lobe surgically (Houssay 1956, p. 213).

Houssay's early studies of the hypophysis in amphibians, dogs, and man proved their value in 1929 when he realized that the anatomy of the toad's pituitary made it the ideal experimental animal for testing these widely-held views of the pituitary's role in carbohydrate metabolism. Houssay found that removing the lobe which corresponds to the anterior lobe in man produced great sensitivity to insulin, while removal of the posterior lobe had no such effect. Without the anterior lobe, the toad rapidly used up glucose, and the injection of insulin exacerbated this high rate of sugar utilization. Thus the anterior lobe clearly was implicated in the regulation of carbohydrate metabolism (Houssay 1936 a, b). The diabetic's problem, though, was that his body used sugar too slowly. Houssay and Biasotti proved in 1929-1930 that the effect of the anterior pituitary on glucose metabolism was antagonistic to the effect of the pancreas. If a toad or dog was made diabetic by the removal of its pancreas, the removal of its anterior lobe would then counteract the diabetes. If, on the other and, the animal was made mildly diabetic by the rtial removal of its pancreas, an injection of anterior ituitary extract would drastically increase the severity f the diabetes. While the pancreas secreted insulin to ncrease the utilization rate of sugar, the anterior pituiry secreted something with the opposite effect, something which inhibited the supply of sugar.

11 These experiments and results were not well under'stood or accepted in the early 1930s, both because Houssay published them in Spanish -lanquage journals, and because they contradicted the current theories of hypophyseal function so completely that journals in the U. S. would not publish his English summaries. One distinguished physiologist declared that Houssay had to be wrong because it was well-known that the posterior lobe's primary function was metabolic control (Barrington 1975, p. 54; Young and Foglia 1974, p. 256). In addition, others who tried to reproduce the experiments failed, chiefly because they did not follow Houssay's techniques for preserving the activity of pituitary extracts (Houssay 1943, p. 252; Wrenshall et al. 1962, p. 65). Eventually, however, the "diabetogenic" effect of the anterior lobe in mammals was confirmed in 1931-1932 through the researches of Herbert McLean Evans and Miriam Simpson, who had been working with anterior lobe extracts from a very different point of view.

Evans and Simpson observed the diabetogenic effect in the course of a wide-ranging program of basic research on the effects of the anterior pituitary extracts on growth and reproduction, work which in turn fed back into clinical aspects of endocrine disorders through the type of feedback cycle so characteristic of the development of endocrinology (Evans 1923-24). in the midst of their efforts to isolate and purify the growth hormone (a task achieved' in 1944-45), Evans and Simpson noticed in 1931 that one of their dogs in which gigantism had been induced with the growth hormone ate and drank excessively, yet became thin and weak. The dog also "excreted great amounts of urine to which flies were attracted." Like Minkowski in 1889 (and Evans, who was very interested in history of science, surely knew the anecdotes about Minkowski's discovery), Evans and Simpson tested the urine and blood for sugar and found that the dog was suffering from diabetes. The experiments proceeding from that observation gave the first clear evidence that the anterior lobe produced an antagonist to insulin and reassured Houssay that his remarkable results with amphibians were correct (Amoroso and Corner 1972, pp. 128-129).5

By the mid-1930s, then, there were strong medical and experimental reasons for believing that the anterior lobe was, as Houssay put it, "the central and directing organ in the endocrine constellation" (Houssay 1936 b, p. 961). Little over a decade later, concentrated research in biochemistry had proven that the gland's control of growth, reproduction, metabolism, and the activities of other endocrine organs was carried out through the secretion of six different hormones, and all six hormones were purified by 1950. However, immediately after proclaiming the central importance of the anterior lobe in his Dunham Lectures at Harvard in 1935, Houssay was forced to add that the other endocrine organs "in some . . . cases . . . also have an influence on the pituitary" (Houssay 1936 b, p. 961). Intensive research into the reciprocal influences of the endocrine glands and the hypophysis on rates of internal secretion kept pace with identification and purification of the anterior lobe's trophic hormones through the 1930s and 40s. The general conviction of the early 1920s that the endocrine organs did not act independently - an opinion based primarily on clinical data - now rested on the solid ground of physiological and biochemical experimentation, and the availability of newly purified hormones gave hope that many more of the complex interactions would be successfully untangled in the future.

83 The Rise of Neuroendocrinology

Between the World Wars endocrinology became firmly established as a scientific discipline with a distinct perspective of its own towards physiological problems. Endocrinologists regarded the endocrine glands, hormones, and their interactions under the pituitary gland's direction as the body's long-term system for coordinating and regulating its activities, a system that neatly complemented the more rapid integrative functions of the nervous system. Most endocrinologists would have described the two systems as separate but equal, although some enthusiasts claimed that the glands controlled the brain and nerves (Vincent 1925, pp. 11, 18; Rolleston 1936, pp. 57-9; LiIjestrand 1936, p. 398; Berman 1930, pp. 200-218). For some time, though, evidence had been building up in a variety of fields human pathology, psychology and psychiatry, comparative anatomy, experimental physiology and endocrinology, and animal behavior - to suggest that, once again, the picture was more complicated than either the all, but interconnected in very interesting ways.

Out of the study of these interconnections a new hybrid discipline, neuroenclocrinology, emerged in the 1940s and 50s. Early clues to a relationship between the endocrine and nervous systems came from observations of the emotional disturbances associated with abnormal endocrine activity in the thyroid and adrenal medulla. Graves' disease (thyrotoxicosis, toxic goiter), for instance, seemed to be triggered by severe psychic distress, such as a sudden shock or long, continuing anxiety, and nervous irritability was among its most common symptoms. Because clinical manifestations of nervous system involvement in the thyroid matched anatomical observations of sympathetic nerves in the thyroid, it seemed possible that the thyroid's secretions were directly controlled by the sympathetic branch of the autonomic nervous system. Although research in the 1930s concentrated instead on the humoral control of the thyroid by the anterior pituitary's thyrotropic hormone, the earlier notions of neural control continued to interest physicians, psychiatrists, and physiologists (Rolleston 1936, pp. 222-224, 240: Reichlin 1966, pp. 445-446, 502-506).

The isolation and synthesis of epinephrine in the early years of the twentieth century made it comparatively easy to study the relations between the sympathetic nervous system and the adrenal medulla. Almost immediately after Oliver and Schafer's discovery of the pressor effect of adrenal medulla extracts, neurophysiologists noted how closely the actions of the extract (and later, of pure epinephrine) mimicked the effects of the sympathetic nerves. As early as 1904, T. R. Elliot hypothesized that the sympathetic nerves acted by releasing epinephrine at the ends of the nerve fibers. The histology and embryology of the adrenal medulla hinted at this possibility too, for the medulla was laced with sympathetic nerve fibers which ended on the medullary cells, which had themselves developed from embryonic nerve cells. And, electric stimulation of the nerves leading to the medulla were found to induce the medullary cells to secrete epinephrine (Rolleston 1936, pp. 321-323; Dale 1962, p. 72).

From 1909 on, the American physiologist, Walter Cannon, emphasized the psychosomatic importance of the neural connections to the adrenal medulla. In the course of studying the mechanics of digestion in equally suddenly. Over the next five years Cannon investigated the effects of emotional stimuli on epinephrine secretion, which led to his famous theory of the emergency function of the adrenal medulla. In his 1915 monograph, Bodily Changes in Pain, Hunger, Fear, and Rage, he argued that strong emotions stimulated the sympathetic nerves and thence the secretion of epinephrine; all the varied effects of the hormone on the body could be seen as quick preparations for "fight or flight" (Brooks and Koizumi 1975, p. 52).

Although neural control over the adrenal medulla and the thyroid seemed probable, the physiological mechanisms were obscure. How did the nerves provoke secretion? In 1921 Otto Loevvi in Austria showed that the nerve cells themselves secrete neurohumors, tiny amounts of chemical substances which travel the short distance from the secreting end of the neuron to the membrane of the cell being excited. In one of the most elegant biological experiments ever devised, Loevvi proved that, when the parasympathetic vagus nerve to the heart is electrically stimulated, it releases a neurohumor which will slow the heart.

H, H. Dale and Loevvi subsequently identified this Vagus-stoff as acetylcholine, a compound which was already known to mimic the actions of the parasympathetic nerves as closely as epinephrine mimicked the sympathetic nerves. (Later, acetylcholine proved to be the neurohumor released by the voluntary nerves as well.) Meanwhile, Cannon and his co-workers believed that the sympathetic nerves also liberated chemical transmitters. After much controversy and reinterpretation of experimental data and theories, Cannon's younger colleague, Bacq, and Ulf von Euler, proved that the sympathetic nerves do indeed secrete epinephrine in order to inhibit activity of the target cell, and that they also release norepinephrine to excite the target cell (Bacq 1975, pp. 68-76).

84 The most logical place to look for neuroenclocrine interactions, however, was in the connections between the brain and the pituitary gland. The posterior lobe attracted attention first, for anatomically it possesses a rich supply of nerve fibers which join the lobe to a tract of the hypothalamus by way of the pituitary gland's stalk. Oliver and Schafer's discovery of internal secretions from the posterior lobe raised a difficulty, however: histologically the posterior lobe had many e fibers but no obvious secretory cells. How then it produce its blood-borne effect on blood pressure, er metabolism, and uterine contraction? Studies of diabetes insipiclus further complicated the oblem. In 1913 two clinical investigators noted the M11' ty f sterior lobe extracts to relieve the excessive inle floo:oof patients with diabetes insipidus. Presumly, in these patients a damaged posterior lobe failed secrete enough antidiuretic hormone, but there was nflicting evidence. Emotional and physiological stress Iso seemed to have an antidiuretic effect, which implied at the nervous system exerted some control over water tabolism - the parallel to Cannon's emergency theory adrenal medulla function was easily drawn (Pickford f,," ' , pp* 209-210). In 1912 and 1913 Aschner and ussy and Camus argued that diabetes insipidus (not ,to mention obesity, sexual dysfunction, genital atrophy, and increased sugar in the blood - many of the symp~toms, in short, associated with pituitary gland disorders) caused by damage to the brain in the hypothalamic ,region. In 1920 they produced experimental diabetes ~insipidus in dogs by making lesions only in the hypothalamus, leaving the pituitary gland untouched, a feat that led them to deny any function at all to the hypophysis (Rolleston 1936, pp. 56-56; Abel 1923-24, pp. 202-207; Harris 1955, pp. 200-204).

These rival explanations of diabetes insipidus and of the roles of the posterior lobe and the hypothalamus were gradually reconciled through new studies of the nerves uniting the brain and hypophysis.6 By 1940 it was reasonably clear that the hypothalamus exerted direct control over the antidiuretic and oxytocin secretions of the posterior lobe, and that this control was somehow mediated by the nerves leading from a welldefined tract of the hypothalamus to the posterior lobe. The source of the secretions themselves remained a question that was not fully answered for another decade, although the most important clues had already been discovered. Ernst Scharrer had shown in his doctoral thesis in zoology in 1928 that certain hypothalamic neurons in fish functioned like glandular secratory cells, and he and his wife Berta subsequently found neurosecretory cells in many vertebrate and invertebrate species. But for many years, in a pattern typical of resistence to scientific discoveries, they could not persuade either the endocrinological or the neurological communities that their cytological evidence would explain the confusing relationship between the hypothalamus and posterior pituitary. For, as Berta Scharrer later wrote, the proposition that there were special hypothalamic neurons with an endocrine function "was a bold concept that did not fit into any existing mold, and it is not surprising that it was received with skepticism. Why should members of a class of cells as readily defined as neurons be capable of functioning as glands of internal secretion? " As Scharrer goes on to note, however, "What is less understandable . . . is the almost universal rejection by the scientific community of the validity of cytological evidence for the existence of a secretory process" (Scharrer 1975 b, pp. 225-260).

After World War 11, an old friend of the Scharrers, Wolfgang Bargmann, became irritated by the negative attitude of his fellow scientists toward the Scharrers' conception of neurosecretion. Bargmann decided to try a new approach based on the well-known usefulness of cell staining techniques in endocrinology. His first trial in 1948-1949 of a recently developed staining technique succeeded splendidly, for he could see individual neurons of the hypothalamic tract extending themselves without interruption down into the posterior lobe. By 1957 Bargmann and the Scharrers had established the fact that the posterior lobe was little more than a storage depot for the hormones produced by the hypothalamus (Bargmann 1975, pp. 38-43; Scharrer 1975 b). The connections between the anterior pituitary and the nervous system were equally hard to understand, although many observations, especially about the reproductive functions of the anterior lobe, implied the existence of connections. For instance, women had long known that emotional distress and other psychological factors can affect their menstrual cycles. Farmers, hunters, and naturalists had recognized for centuries that many environmental factors like temperature, food supply, light, and the sensation of coitus can alter an animal's ability to ovulate.

Further evidence came from the clinic: tumors of the hypothalamus disturbed many endocrine functions usually associated with the anterior lobe (Fulton, Ranson, and Frantz 1940, pp. xiii-xxx, 864-874). Following this lead, Camus and Roussy produced Frohlich's syndrome of asexual adiposity by making minor lesions in certain tracts of the hypothalamus, just as they had caused diabetes insipidus with lesions in the tract connected to the posterior lobe. Joseph Hinsey and Joseph Markee at Stanford brought together in 1933 Markee's observations on the nervous control of ovulation and Hinsey's experience with the chemical transmitters of sympathetic and parasympathetic nerves. In a of the posterior lobe, implied a humoral

85 mechanism for the brain's control over the anterior lobe's ovulatory function (Hinsey 1975, pp. 135, 139). But these various observations, experiments, and speculations did not excite much interest until the 1940s. The only mention of "neuro humoral mechanisms" in the authoritative and lengthy 1939 survey, Sex and Internal Secretions, for example, was to be found in a psychologist's review of research on the sex drive (Allen, Danforth, and Doisy 1939, pp. 1213-1220).

Before anyone could make sense of the miscellaneous assortment of observed nervous system-anterior lobe interactions, much more detailed, direct experimentation on the anatomy and physiology of the hypothalamus, pituitary stalk, and anterior lobe was necessary. It was the English anatomist, G. W. Harris, who "more than anyone else brought the new endocrinology into focus in the late 1930s" and 1940s (Price 1975, p. 229). Although Harris' early experiments suggested that nerves led from the hypothalamus to the anterior lobe, his examination of the lobe's anatomy raised doubts in his mind, for the anterior lobe abounded in secretory cells and blood vessels but almost completely lacked neurons.

The other possible path for messages between the central nervous system and the anterior lobe was through the rich network of blood vessel that surrounded the pituitary stalk. A Roumanian pathologist, Rainer, had noticed in 1927 that these blood vessels were especially prominent in people who had died suddenly and violently; he persuaded a young medical student, G. R. Popa, to study these vessels and their connections to the capillaries of the hypothalamus and the hypophysis. Popa and Fielding published an account of this work in 1930, in which they said that the blood in these short portal vessels flowed upwards from the hypophysis to the hypothalamus. Six years later Wislocki and his colleagues in Boston disputed this assertion and argued from histology that the blood flowed down from the brain to the anterior lobe. Wislocki, Popa, and Harris debated ("at times quite vehemently," Harris recalled much later) the question of the direction of blood flow over the next ten years (Harris 1955, p. 30).

In 1947, Harris and Green tried a new approach: they now watched the flow of blood directly in living, anesthetized animals, and in so doing confirmed Wis had first been proposed in 1933 by Hinsey and Markee. Harris devoted the next half dozen years to proving the theory that the hypothalamus sands blood-borne chemical messengers down the pituitary stalk's portal vessels into the anterior lobe, where they stimulate the release of the anterior lobe's pituitary hormones (Harris 1955, pp. 20-31; Tepperman 1973, p. 36; Vogt 1972, pp. 310-313; Jacobson 1975).

Once the neural control over the anterior lobe's secretions were widely accepted, research centered on isolating the "releasing factors." Early trials with known nerve transmitter substances showed that these compounds were only indirectly concerned with the hypothalamus/anterior lobe system. In the mid-1950s and the 60s neuroendocrinologists and biochemists succeeded in assaying, isolating, and purifying a series of small polypepticles from hypothalamic tissue and blood. By 1973 eleven different releasing or inhibiting factors had been found, eight of them purified, and the structure of three worked out in detail (McCann and Dhariwal 1966, pp. 261-296; Williams 1974, p. 5; Maugh 1975, p. 921).

As endocrinologists learned to see the anterior lobe as the "concert master" rather than the "conductor" of the "endocrine orchestra," they reconsidered the interactions among the glands that had been so arduously worked out before. In the 1940s, Norbert Wiener, the inventor of cybernetics, had popularized the concept of feedback control in engineering and in neurophysiology (Dempsey 1975, pp. 85-86; Wiener 1948, pp. 7-33, 113-114). Walter Cannon's concept of homeostasis agreed well with Wiener's ideas, and endocrinologists gradually realized that the language of feedback control provided an effective way of describing and thinking about endocrine interactions. They saw that the presence of the hypothalamus in the endocrine constellation required more elaborate feedback systems than the simple see-saw reciprocity envisaged by earlier investigators.

Following the publication of Harris' monograph, Neural Control of the Pituitary Gland, in 1955, with its diagrams of feedback loops involving the higher brain, the hypothalamus, the two pituitary lobes, the gonads, adrenals, thyroid, and sympathetic nervous system, neuroendocrinologists started intensive research into the mechanisms by which hormones act on the brain and hypophysis. The principle of feedback loops has become so fundamental a part of endocrinological thinking in recent years that textbooks now commonly discuss the feedback integration of the endocrine system before they even name and describe the components of the system, the glands and hormones (Williams 1974; Tepperman 1968, 1973; Barrington 1975).

Reading the Message: Hormone

86 Receptors and Molecular Biology

There was a continuity of outlook through the three phases of endocrine research discussed so far, as researchers concentrated on seeking the source of control over the actions and interactions of glands and their secretions. In the earliest phase, control seemed to come from the gland itself, in the second, from the hypophysis, and in the third, from the nervous system as modified by feedback loops. The response of target organs was used to assay hormones, but otherwise that response was simply taken for granted. The interactions of the endocrine system were studied on the tissue or organ level; and, while biochemists played an important role in purifying and characterizing hormones, they could not contribute much to the conceptual foundations of the discipline.

The question, "How do organs and cells receive and read the hormone message?" could not be answered, or even asked, effectively in this context. It made sense only at the cellular level, and this was - until the late 1950s - the separate province of molecular biologists and biochemists. For among biologists "there was a widespread feeling . . . that hormone action could not be studied meaningfully in the absence of organized cell structure" (Sutherland 1972, p. 401). Between 1955 and 1962, however, Earl Sutherland and his co-workers, proceeding on the conviction that "there was a real possibility that hormones might act at the molecular level," proved that breaking up cells did not destroy their sensitivity to some hormones (Sutherland 1972, p. 401). In studying the influence of epinephrine and the pancreatic hormone, glucagon, on the liver's ability to break glycogen down to glucose, Sutherland and his associates found in liver cell fragments a molecule called cyclic AMP which carried the hormone message to the enzymes that governed the rate of glycogen - glucose reactions. Since then, cyclic AMP has also been implicated in the cellular reception of information from the uteinizing hormone, the adrenocorticotropic hormone, thyroid-stimulating hormone, parathyroid hormone, and many others, Its ubiquity in the animal kingdom at all levels of organization suggests that, in evolutionary terms, it may be as significant a compound as DNA, RNA, and ATP (Robison, Butcher, Sutherland 1971, p. 74 and chap. 12). Research on the biochemical and structural details of the hormone-membrane receptorcyclic AMP-enzyme activation pathways in the cell has become one of the fastest growing fields in contemporary endocrinology and molecular biology.

Sutherland's discovery of cyclic AMP came directly out of research on the enzymes and biochemical pathways of carbohydrate metabolism - a tradition very different from endocrinological research in the first half of this century. Unlike endocrinologists, for example, researchers in intermediary metabolism had long been accustomed to experimenting with cellfragments and with enzyme-substrate reactions in cellfree systems. Another difference lay in the primary research orientation of investigators such as Sutherland. His mentors Carl and Gerty Cori, and others who elucidated metabolic pathways in the 1920s, 30s, and 40s, had medical training and clinical experience, and their work was supported largely by medical schools and medical research institutes. But they conducted their research on the hormonal control of carbohydrate metabolism in the tradition of basic research, with questions about the cause or cure of disease playing little part in their discoveries (Sutherland 1972, pp. 401-408; Cori 1969, pp. 1-20).

Virtually all areas and phases of endocrinology, however, are marked by recurring interactions between basic and clinical pursuits, and contemporary molecular endocrinology is no exception. Thus, cyclic AMP workers have begun looking into the relationship between defects in the formation or action of this nucleotide and a variety of human diseases, with the ultimate hope that "The results of this research can eventually be applied to increase the quality of human life... [since] the chief end of all scientific research either is or ought to be the promotion of human happiness" (Robison, Butcher, and Sutherland 1971, pp. 454-455). Simultaneously, the reverse application of knowledge from the study of disease to fundamental questions so common in the earlier history of endocrinology is also taking place in the new molecular endocrinology. For example, physicians have known for some time that the lethal effect of the cholera bacteria's enterotoxin is a devastating diarrheal loss of fluids and salts from the body; the most effective therapy is the immediate replacement toxin mimics the ability of cyclic AMP in intestinal cells to provoke cell membranes into actively transporting fluid and salts out of the cells. Carpenter theorizes that the toxin behaves like a hormone by binding itself to hormone receptors on the cell membrane; the receptors stimulate production of cyclic AMP, which in turn stimulates the membrane's active transport system. Unlike ordinary hormones, though, cholera enterotoxin is not particular about the kinds of cells or the membrane binding sites it will act upon. This makes it a useful, if potentially fearsome tool for investigating basic questions about the structure of hormone receptors and about the biochemical role of cyclic AMP within the cell (Carpenter 1972; McCann 1974, pp. 323-325). Thus, the discovery and study of diseases stemming from cyclic AMP malfunction promises to yield both the objective of treating diseases more effectively, and also that intense intellectual satisfaction

87 which comes to the scientist when his puzzle-solving researches fit together to produce a new understanding of life processes.

"A New Physiology"

As we have explored some of the ways in which endocrinology has evolved from ancient "kitchen anatomy" to present molecular researches, we see clearly the interplay between the clinic and the research laboratory in the generation of problems, concepts, and facts concerning endocrine function. What we have called phases three and four of endocrine research -the development of neuroendocrinology and current molecular studies of hormone action - belong primarily to that portion of endocrinology's history where research had moved from a disease-initiated focus into the province of basic research. We have, accordingly, treated these phases only briefly, to suggest where that flow led and is leading investigators of the ductless glands.

It is in looking at the emergence of endocrinology as a field of biology in the latter decades of the nineteenth century and early years of the twentieth century that we see, most preeminently, the imprint and influences of medical concerns. For, particularly in France and then Britain, countries in which clinical medicine more generally shaped the problems pursued by physiologists, attention by physicians to disorders of the ductless glands provided a new range of phenomena for scientific investigation. At the turn of the century, as represented most significantly by Bayliss and Starling's new and fundamentai unaerstwiumy . I... endocrine gland's chemical mediators in physiological and biochemical functioning.

Reflecting in 1933 on the impact of endocrinology upon not only medicine but upon physiology as well, R. G. Hoskins chose to underscore the words of the great British physiologist and pioneer endocrinologist, Edward Sharpey-Schafer:

The changes in physiology which have resulted from this (new endocrine] knowledge constitute not merely an advance in degree but an alteration J . n character. The doctrine of internal secretions forms a new departure. We must in the future explain physiological changes in terms of chemical regulation as well as of nervous regulation. It is therefore justifiable to speak of the doctrine as a New Physiology, seeing that it has completely altered our outlook on many of the problems with which physiology deals and consequently on those met with in medicine and surgery. (Hoskins 1933, pp. 347-348)

Notes

Within the confines of a single chapter, we have had to some of the most interesting fundamental consequences isease-related research in endocrinology. Some examples n from the history of diabetes and insulin research will some sense of the range of problems suggested by clinical crinology and some sense of our omissions.

Clinical endocrinologists have imposed great demands on the ingenuity of biochemists, to the benefit of both, For example, first protein to have its amino acid sequence worked out completely was insulin. Sanger demonstrated conclusively by analysis of the hormone that "proteins are well-defined molecules in which the amino acid units are linked by pe,)tide cls to form long polypepticle chains," a definition that was cy means certain when he began his decade of work in 1945. the process of analyzing the structure of the insulin molecule, at developed an armory of biochemical techniques for proteins and macromolecules. subtle differences among the sulins of different species and forced evolutionary biologists ask why, for example, pig insulin resembles human insulin does.

Through both human and animal research on the nature of 'endocrine disorders, investigators have discovered both how universal endocrine mechanisms are in the animal kingdom and how idiosyncratic each group can be. A 1972 review of research on diabetes, insulin, and carbohydrate metabolism, for example, points out that insulin is an especially important hormone in carnivores who cannot count on a steady supply

88 of food and whose metabolism must cope with sudden gluts and long-term shortages. In such creatures - man included -diabetes will be a serious disease; in herbivores and especially the ruminants who nibble all day long, diabetes is much less dangerous (Fritz 1972, pp. 166-180).

Another area we do not treat is that of comparative endocrinology. We deal only with research on the endocrinology of vertebrates; and within the vertebrates, almost solely with laboratory animals - dogs, cats, rats, rabbits - and man. We bypass entirely the fascinating work on invertebrates, plants, fungi, algae, and bacteria. Moreover, we have ignored the contributions of clinical endocrinology to our understanding of evolution, ecology, social and sexual behavior, psychology, embryology, and development. Finally, we have played favorites within the endocrine system too. Thus, for example, the story of parathyroid and calcium metabolism receives no mention. The elaborate relationships of the sex hormones and glands with reproduction, growth, development, and behavior are only touched upon, chiefly because the history of sex endocrinology currently is being dealt with extensively by historian Diana Long Hall.

2. The importance of special instruments for the advancement of both neurcienclocrinology and neurosurgery is illustrated by the sterecitaxic instrument developed in 1908 by British neurosurgeon and neurophysiologist Victor Horsley and his colleague R. H. Clarke. The Horsley-Clarke device, a special kind of head-clamp combined with three-dimensional micrometer measuring devices, enabled physicians and laboratory researchers to make surgical or electrolytic lesions in precisely determined spots in the brain.

3. For instance, the discovery was the basis for Dorothy L. Sayer's 1928 short story, "The Incredible Elopement of Lord Peter Wimsey."

4. The diuretic response later was shown to be an artifact due to the experimental conditions, in contrast to the antidiuretic response which is an endocrine function of the Posterior pituitary (Barrington 1975, p. 93).

5. Despite their obvious importance, these discoveries of the growth hormone and the diabetogenic effect of the anterior pituitary were something of a side issue for Evans and his colleagues. So was the laboratory group's participation in the discovery of the thyroid-stimulating hormone (TSH) and the adrenocorticotropic hormone (ACTH), both hinted at in Evan's analysis of Smith's early research on tadpoles (Evans 1923-24, pp. 216-219; Evans, Sparks, and Dixon 1966, pp. 319-320; Amoroso and Corner 1972, pp. 129-131). The interrelationships between the hypophysis and the ovaries remained the central problem for Evans and his co-workers. In seeking the reasons why their gigantic rats who had been treated with growth hormone failed to ovulate, Evans, Simpson, and Smith uncovered the existence of still another pair of hormones, the gonadotropic luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Their method of preparing crude extracts of growth hormone had, they gradually realized, retained both the growth hormone and the luteinizing hormone but destroyed the follicle-stimulating hormone. For successful ovulation both gonadotropic hormones had to be present in the right proportions. Once distinctions among the effects of the luteinizing, follicle-stimulating and growth hormones were drawn and analogous effects of these hormones in male animals were recognized, the research effort turned to the isolation and purification of the three hormones; in 1949, Li, Simpson, and Evans finally obtained homogenous FSH.

6. 1 n the 1930s at Chicago, Fisher, Ingram, and Ranson used the Horsley-Clarke stereotaxic instrument to make small, accurately placed lesions in different areas of the hypothalamus in cats (see note 2). Lesions in the tract originally described by Ramon y Cajal had two related effectsz atrophy of the posterior pituitary, and a polyuria very much like that in clinical cases of diabetes insipidus. Ranson and his colleagues also noticed that these lesions interfered drastically with labor in pregnant cats a result which correlated well with the strong uterine contractions invoked by the oxytocin ("quick childbirth") fraction of posterior lobe extracts. Injections of posterior pituitary extract counteracted the polyuria and difficult labor. Lesions in other tracts of the hypothalamus did not incur any of these disorders although they did have other effects resembling pituitary malfunction (Harris 1955, pp. 200-204, 224-225).

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CHAPTER 5 "THE LESSON OF RARE MALADIES" SICKLED CELLS, MOLECULAR DISEASE, AND THE GENETIC CONTROL OF PROTEIN STRUCTURE

Much of our present understanding of the composition, structure, and function of genes has come from the joining of previously disparate work in microbiology, biochemistry, and genetics, a fusion that marked the emergence of molecular biology in the 1940s.1 Since the 1940s, investigators seeking to elucidate gene function have turned by-and-large from the morphological and biochemical complexities of the fruit fly, mice, and maize, materials they had used to unravel problems of gene transmission, to simpler microbial and viral materials. To contemporary geneticists and molecular biologists, aware of the infinitely greater complexities of the human organism, it thus seems "remarkable" and "amazing" that, stemming from work on a human hereditary disorder, sickle cell anemia, "the genetically controlled protein system about which most is known is human hemoglobin" (Sutton 1961, p. 50).

In this chapter we will see how "the existence of mutant proteins in humans afflicted with sickle cell anemia proved to be the critical key in the elucidation of the nature of the genetic apparatus" (Handier 1970, p. 664). As in our other case studies, it is a story in which a clinical problem feeds into and affects the course of a stream of biological research, a stream which is fed by many sources and which in turn has numerous branches. It also is a story that relates how much we have learned from what the British physician, Sir Archibald Garrod, called "the lesson of rare maladies," the inherited abnormalities that provide researchers with a "natural experiment." In this case, the natural experiment, sickle cell anemia, led to the concept of "molecular disease" and thence to a frontal assault on the question of how genes control protein structure.

95 And, in delineating how gene mutations alter the structure of the human hemoglobin molecule, researchers have made major strides in resolving one of the oldest and most difficult problems in genetics, the problem of allelism. (Alleles are alternative or variant forms of genes which occur at the same locus or place on two members of a pair of homologous chromosomes.) "The problem of allelism," as Levitan and Montagu explain,

"arises whenever two traits are found that affect the same cell, tissue, or organ system. . . [For example] every time a new red blood cell antigen, or blood type is discovered, the question arises as to whether it represents a further complexity of a known system (and thus probably conditioned by a new allele at a previously discovered locus) or whether it is a new system conditioned by still another independent locus. This problem goes to the very core of genetics. We are in effect looking for evidence to delineate the unit factor of genetics, the gene" (Levitan and Montagu 1971, p. 549).

Inborn Errors of Metabolism: Garrod's Unlearned Lesson

Conceptually, the bearing of sickle cell research on our understanding of the function of genes is indirectly but inextricably linked with the ideas of Sir Archibald Garrod (1857-1910) concerning the biochemical nature of gene action. Garrod, who died the same year that the first clinical report of sickle cell anemia was published, worked in that post-Mendelian era when genetics and biochemistry were pursued largely as separate endeavors. When those fields were joined, his clinically-based concept of inborn errors of metabolism would be rediscovered and reenunciated as the one gene-one enzyme concept, on the basis of research with the bread mold, Neurospora. Then, in the 1950's, once again growing out of a clinical framework, this time provided by sickle cell anemia, GarrGd's ideas would find their modern echo in the one gone-one polypeptide concept of the genetic control of protein structure.

In 1902 Garrod, an Oxtord-trained pathologist, published the first of a series of papers on alkaptonuria, a disorder manifested clinically by a blackening of the urine on exposuro to air. Six years later, in the Royal Society's Croonian Lectures, Garrod summarized his first decade of researches on alkaptonuria and three other human disorders, albinism, cystinuria, and pentosuria, four 'rare maladies" that he tern-wd "irA)orn errors of metabolism." In his lectures, published in 1909 with a second edition in 1923, Garrod "clearly and in the normal pattern of intermediary metabolism, where some specific enzyme, normally present, was absent because of alteration of the controlling gene" (Glass 1965, p. 231).

Garrod, a physician thoroughly trained in the biological and chemical sciences of his time, drew upon and uniquely joined contemporary researches in protein biochemistry and in the newly rediscovered science of Mendelian genetics. As he pointed out in the first of his 1908 Croonian lectures:

The great strides which recent years have witnessed in the sciences of chemical physiology and pathology, the newly-acquired knowledge of the constitution of proteins and of the part played by enzymes in connexion with the chemical changes brought about within the organism, have profoundly modified our conceptions of the nature of the metabolic processes . . . The conception of metabolism in block is giving place to that of metabolism in compartments. The view is daily gaining ground that each successive step in the building up and breaking down, not merely of proteins, carbohydrates, and fats in general, but even of individual fractions of proteins and of individual sugars, is the work of special enzymes set apart for each particular purpose. (Garrod 1909, pp. 4-6)

In introducing his audience to the anomalies "which may be classed together as inborn errors of metabolism," Garrod first distinguished them from "diseases of metabolism" such as diabetes, gout, and obesity. "The liability to develop diabetes or gout is often inherited, but the diseases themselves are not inherited for they are never congenital. Developing at any period of life, the mischief, once begun, tends to become aggravated as time goes on, but the rate of aggravation differs widely in individual cases and is often conspicuously controlled by appropriate treatment" (Garrod 1909, p. 131). In contrast to these diseases, Garrod explaine

some of the inborn errors of metabolism "are certainly and all of them are probably, present from birth.' Clinically, he stated, The chemical error pursues an even course and shows no tendency to become aggravated as time goes on. With one exception they bring influenced by any therapeutic measures at our disposal. Yet they are characterized by wide departures from the normal of the species far more

96 conspicuous than any ordinary individual variations, and one is tempted to regard them as metabolic sports, the chemical analogues of structural malformations. (Garrod 1909, pp, 13-14)

Today's thick textbooks devoted to inborn errors of metabolism bear witness to Garrod's insight in 1908 that "among the complex metabolic processes of which the human body is the seat there is room for an almost countless variety of such [metabolic] sports." But he acknowledged that in his own day there was adequate evidence for classifying only four anomalies as inborn errors of metabolism.

Of the four, alkaptonuria was Garrod's most comprehensively analyzed inborn error of metabolism. For a substantial body of clinical, biochemical, and hereditary information about alkaptonuria existed by 1908, including Garrod's own investigations, which he uniquely synthesized and interpreted. The blackened urine of alkaptonuria was known to be caused by the excretion of homogentisic or "alkapton" acid, a substance formed in the body from tyrosine and phenylalanine. Garrod recognized that "two explanations are possible of the fact that alkaptonurics excrete homogentisic acid, whereas normal persons do not. Either the alkapton acid is a strictly abnormal product formed by a perverted metabolism of tyrosin and phenyl-alanine . . . or it is an intermediate product of normal metabolism which is usually completely destroyed and does not come to excretion, but which in alkaptonuria escapes destruction" (Garrod 1909, p. 66). Garrod then documented his reasons for concluding that the second of these hypotheses was the correct one. Homogentisic acid, he argued, is a product of normal metabolism, but the alkaptonuric, unlike the normal individual, lacks the "power of destroying homogentisic acid when formed - in other words of breaking up the benzene ring of that compound" (Garrod 1909, p. 79). This inability, Garrod correctly speculated, was due to the absence or inactivity of an enzyme that normally catalyzes the breakdown of homogentisic acid.2 "We may further conceive," he wrote, "that the splitting of the benzene ring in normal metabolism is the work of

a special enzyme, that in congenital alkaptonuria this enzyme is wanting, whilst in disease its working may be partially or even completely inhibited" (Garrod 1909, p. 80).

Garrod's recognition that the metabolic defect in alkaptonuria represented an inborn or inherited condition, in turn, was based on familial patterns of the disease's occurrence. From familial studies, Garrod found that the four metabolic disorders he was analyzing had "the liability for each of them to occur in several members of a family, most often in collaterals of the same generation, born of normal parents" Garrod 1909, p. 22). An explanation for the inborn error's mode of inheritance within such families was provided by William Bateson, who was responsible for having Mendel's paper translated into English, was the first to demonstrate Mendelian inheritance in animals, and who introduced such terms as "genetics" and "allelomorph." Drawing upon Bateson's 1902 report to the Evolution Committee of the Royal Society on "The Facts of Heredity in The Light of Mendel's Discovery," Garrod concluded that "the mode of incidence of alkaptonuria finds a ready explanation if the anomaly in question be regarded as a rare recessive character in the Mendelian sense" (Garrod 1909, p. 26). In Inborn Errors, he examined at length the Mendelian nature of alkaptonuria as a recessive trait, and also presented evidence for pentosuria, cystinuria, and albinism as metabolic defects due to the recessively inherited absence of a single enzyme. "To the students of heredity," Garrod pointed out a scant eight years after the rediscovery of Mendel's work, "the inborn errors of metabolism offer a promising field of investigation." However, he wisely cautioned those who would enter the arena of medical genetics, "their adequate study from this [genetic] point of view is beset with many difficulties" (Garrod 1909, p. 22).

One Gene-One Enzyme. The Rediscovery of Garrod's Concept

When Garrod delivered his Croonian Lectures in 1908, the prevailing concept of gene action offered by the nascent science of genetics was "one gene-one character." As Bentley Glass observes, Garrod's pioneering foray into biochemical genetics represented two major steps forward from this early understanding of gene action: "from recognition of the relation of the specific altered gene to the particular blocked step in the metabolic pattern, and thence to the lack of the specific enzyme governing that pattern" (Glass 1965, p. 233).

But Garrod might well have echoed Gregor Mendel's bitter words, "Meine Zeit wird schon kommen" [my time will surely come] , for the import of the British physician's work, like that of the Austrian monk's, would be unrecognized by geneticists, and by biochemists, for several decades.

97 For those interested in understanding the processes of biomedical discovery, innovation, and diffusion, the question of why work such as Garrod's was "profoundly neglected" during his lifetime is as interesting and significant as the content of the work itself. As with other instances of scientific and clinical discoveries that underwent a long latency period before they were "rediscovered," there are multiple explanations of why Garrod's biochemical -genetic concept of gene action lay fallow for many years. One partial explanation, advanced by Garrod's principal rediscoverer, George Beadle, invokes the familiar argument that "the time was not ripe." Because Garrod was ahead of his time, Beadle argued, neither biochemists nor geneticists would take his concept seriously. Further, Beadle wrote, "I strongly suspect that an important common component of the unfavorable climate for receptiveness in these two instances [Mendel and Garrod] is the persistent feeling that any simple concept in biology must be wrong" (Beadle 1966, p. 3 1).

Other, interrelated explanations have been presented by geneticist and historian Bentley Glass, who also has studied the reception of Mendel's work. Glass argues that Garrod's lectures on human heredity must have been known to geneticists, as was Mendel's 1865 paper in its time. But Garrod, a physician, was presenting studies in biochemical genetics in an era when genetics and biochemistry were separate fields of inquiry. As late as 1946, Beadle still felt the need to urge the fusion that Garrod had personified.

If the maximum possible understanding of what the organism is and what it does is to be obtained, it is clear that our approach must be made from many sides. . the chemist cannot understand what the organism does chemically without considering genes. Therefore, the methods of genetics, which are biological - not chemical, must supplement those of chemistry. In the same way, a biologist would be blind if he were to ignore chemistry in his attempts to understand how the organism is built and how it functions. Biochemical genetics represents an approach in which the cooperation of two disciplines is essential. (Beadle 1946, p. 53) inborn metabolic disorders presented many promem!, for the geneticist, Thus, "there was a common attitude among geneticists who worked with Drosophila or maize or other experimental organisms that human heredity was refractory to analysis and that little basic insight could be gained from studies of hereditary abnormalities in an organism that could not be bred at will" (Glass 1965, P. 232).

Compounding the problems of receptivity created by a physician working in human biochemical genetics, Glass continues, was the relative lack of direct interest in enzymes among geneticists. There were relevant Lines of research pursued by a small group of geneticists, particularly on the topics of flower pigmentation and the fermentation of sugars by yeasts, and by 1940 these researches had "established that many biochemical reactions are, in fact, controlled in specific ways by genes" (Beadle and Tatum 1941, p. 499). But how this genetic control is exerted was unclear, and there was debate as to whether enzymes were genes themselves or merely products of genes, An answer had been proffered by Garrod in 1908, but most of those working through the early 1940s were either unaware of Inborn Errors or, if they had read it, failed to see the signifi cance of Garrod's concept.

For multiple reasons, then, it was not until the mid1940's that Garrod's concept of gene action - one gene/one metabolic block/one enzyme deficiency would be "rediscovered" and clearly enunciated as the one gene-one enzyme hypothesis. The name linked most closely with this rediscovery is George W. Beadle, who in the 1930s and 1940s conducted Nobel Prize winning researches on how genes act on development. Beadle's first researches, conducted with Boris Ephrussi, were carried out on eye pigments of Drosophila. The hypothesis that he and Ephrussi formulated to explain their initially unexpected experimental results, Beadle later reflected, "was a scheme closely similar to that proposed by Garrod for alkaptonuria . . . But at the time we were oblivious of Garrod's work, partly because geneticists were not in the habit of referring to it, and partly through failure of ourselves to explore the literature. Garrod's book was available in many libraries" (Beadle 1958, p. 592).

In 1937, Beadle, now working at Stanford, was joined by a young biochemist, Edward L. Tatum. They found that "isolating the eye-pigment precursors of Drosophila was a slow and discouraging job," and "realized this was likely to be so in most cases of attempting to identify the chemical disturbances underlying inherited abnor to be simple chemically" (Beadle 1958, p. 594).

Thus, Beadle and Tatum realized, the biochemical study of gene function required a new approach and new biological material, something less morphologically complex than the fruit fly or other diploid organisms that had been suitable for defining the laws of genetic transmission. The new approach they selected, Beadle feels, was an "obvious one," but at the time it represented a "bold step" of "inverting the experimental attack on the problem [of gene action] . Instead of waiting for the accumulation of information necessary to

98 understand the biochemical basis of the complex morphological mutants of the higher forms, attention was turned to simpler microbial material" (Handler 1970, p. 25).'

The organism chosen by Beadle and Tatum was the now famous bread mold, Neurospora crassa (see Beadle 1964; Tatum 1964; and Beadle and Tatum 1941). "it is sometimes thought," Beadle said in his 1958 Nobel Prize address, "that the Neurospora work was responsible for the 'one gene-one enzyme' hypothesis - the concept that genes in general have single primary functions, aside from serving an essential role in their own replication, and that in many cases this function is to direct specificities of enzymatically active proteins. The fact is that it was the other way around - the hypothesis was clearly responsible for the new approach" (Beadle 1958, pp. 596-597). For, as Beadle and others understood in retrospect, the one gene-one enzyme concept had been gradually albeit implicitly envolving in the work of many investigators since it was formulated by Garrod in 1908. Once he was aware of Garrod's work, Beadle realized that:

In this long and roundabout way, first in Drosophila and then in Neurospora, we had rediscovered what Gerrod had seen so clearly so many years before. By now we knew of his work and we were aware that we had added little if anything new in principle. We were working with a more favorable organism and were able to produce, almost at will, inborn errors of metabolism for almost any chemical reaction whose product we could supply through the medium. Thuswewereable to demonstrate that what Garrod had shown for a few genes and a few chemical reactions in man, was true for many genes and many reactions in Neurospora. (Beadle 1958, p. 526)

The Discovery of Sickled Cells

At the time, no one could have foreseen the connection between Garrod's work, or the later researches of Beadle and Tatum, and the publication of a paper by Chicago physician James B. Herrick on "Peculiar and elongated sickle-shaped red blood corpuscles in a case of severe anemia." "This case is reported because of the unusual blood findings, no duplicate of which I have ever seen described," Herrick wrote in 1910. He had first seen his patient, a 20 year old black who had been raised in the West Indies, when he came to Herrick's hospital in 1904 with a cough, fever, weakness, dizziness, a headache and nasal discharge, a yellow tinge to the whites of his eyes, and a history of skin ulcerations. The physician's examination revealed one striking finding: a large number of red blood cells which, in contrast to normal red cells, had a nucleus and were oddly shaped.

Nucleated reds were numerous, 74 being seen in a count of 200 leukocytes, there being about 5,000 to the c.mm. The shape of the reds was very irregular, but what especially attracted attention was the large number of thin, elongated, sickle-shaped and crescent-shaped forms. . They were not seen in specimens of blood taken at the same time from other individuals and prepared under exactly similar conditions. They were surely not artifacts, nor were they any form of parasite . . . in the fresh specimen where there was a slight current in the blood before it had become entirely quiet, all of the red corpuscles, the elongated forms as well as those of ordinary forms, seemed to be unusually pliable and flexible, bending and twisting in a remarkable manner as they bumped against each other or crowded through a narrow space and seeming almost rubber-like in their elastic resumption of the former shape. One received the impression that the flattened red discs might by reason of unusual pliability be rolled up as it were into a long narrow bundle. Once or twice I saw a corpuscle of ordinary form turn in such a way as to be seen on edge, when its appearance was suggestive of these peculiar forms. (Herrick 1910, pp. 518-519)

After a month of treatment (rest, nourishment, and iron for his anemia) the patient was discharged. His condition was improved but the odd-shaped and nucleated red cells were still present. Herrick kept track of the patient until early 1907, recording his hospitalizations for problems such as "muscular rheumatism" and "bilious attacks." Clinically, Herrick acknowledged, "no conclusion can be drawn from this case." He and a colleague had unsuccessfully attempted to reproduce the types of red cells seen in the patient, which suggested to Herrick "that some unrecognized change in the composition of the corpuscle itself may be the determining factor." However, Herrick concluded, "the question of diagnosis must remain an open one unless reports of other similar cases with the same peculiar blood-picture shall clear up this feature" (Herrick 1910, P. 521).

Other reports were forthcoming, which confirmed and elaborated upon Herrick's clinical findings and, began to explore the possible etiology of the newly recognized disorder. A second puzzling case of severe anemia with sickle-shaped cells was reported in 1911 by Washburn, and a third in 1915 by two physicians

99 from Washington University Medical School in St. Louis. "It will be seen that there is a striking similarity in both the blood-picture and the clinical history of our case with those of Herrick and Washburn," wrote Drs. Jerome Cook and Jerome Meyer. The three patients "were of Negro blood" and had "peculiar. . recurring leg ulcer," severe anemia, and a "peculiar discoloration of the sclerae" (the coating of the eyeball). "A glance at the blood slides leaves no doubt as to the identical character of the elongated and sickle-shaped red cells" (Cook and Meyer 1915, pp. 650, 651).

As had Herrick and Washburn, Cook and Meyer ruled out syphilis or a parasitic infection as causative agents of the strange disorder. They were able to suggest another possible explanation for the disease, however, based on investigations they had conducted with Dr. Victor Emmel, a colleague "who has made extensive studies of blood development and morphology."

The possibility of an inherited anomaly suggested itself. The fact that the three other children of the family had suffered from severe anemia encouraged investigation in this direction. Examination of the father's blood showed 4,500,000 red cells, 11,100 white cells, and 86 per cent hemoglobin... the stained smear showed none of the peculiarities of the daughter's blood. Dr. Emmel, however, found some resemblance in the behavior of the blood. When preparations of the fresh blood were temperature, the microscope revealed long, snarp projections and elongations from many of the red cells in the blood of both the father and the daughter. Dr. Emmel has not found similar appearances in the blood of any other person. (Cook and Meyer 1915, p. 649)

In his observations of the father's blood, Emmel had seen the phenomenon of sickling in the otherwise norMal-looking red blood cells of carriers, a phenomenon that the physicians correctly interpreted as suggesting A hereditary disorder. Emmel's primary interest in this case was to explain the sickling process, and in a 1917 paper he reported in more detail on his studies of blood samples from Cook and Meyer's patient and her father. Based on these studies, and his knowledge of the development of the blood cells, he proposed a logically consistent hypothesis about the mechanism of red cell sickling. Knowing that undeveloped red blood cells within the bone marrow are spherical in shape, Emmel proposed that sicklinq is "in part due to an accentuated or abnormal activity of the same factor which in normal hernatogenesis are involved in the transformation of the original spherical erythrocyte [red blood cell] into a biconcave disc-shaped form" (Emmel 1917, p. 598).

While Emmel's hypothesis would be proved incorrect, it directed other investigators toward a fruitful line of research into the nature of the sickling process. In a major paper of 1927, E. V. Hahn and E. B. Gillispie reviewed the growing body of clinical literature on 11 sickle cell anemia" - a term introduced by Mason in 1922 - and reported an their own experimental study of sickle cell formation. Hahn and Gillispie set up a gas chamber apparatus under the microscope, along with red blood cell preparations from a patient with sickle cell anemia, and treated the cells with different gases. When oxygen or carbon monoxide was added to the preparation, the sickle cells immediately returned to the normal discoid shape. When carbon dioxide was added, it took only four minutes for all the normal discoid cells to sickle. In addition to this experiment, they also observed the red blood cells at various partial pressures of oxygen, and found that the normally shaped cells of a person with sickle cell trait sickled when oxygen tension fell below 45 millimeters, less than a third of normal oxygen.

The gas chamber experiment simulated different conditions of hemoglobin, the substance in red blood

Yelid their study, Hahn and Gillispie were the first to suggest the role of hemoglobin in sickle cell anemia, which they too viewed as a hereditary disorder.

The red corpuscles of persons with the "sickle cell trait" are transformed into sickle cells in vitro as a result of asphyxia. The transformation takes place when the oxygen tension falls below a partial pressure of 45 mm. of mercury. Oxygen and carbon monoxide induce restoration of the discoid form. All of the facts relating to sickle cells are consistent with a hypothesis that the sickle form is stable when the hemoglobin is dissociated, and that the discoid form is stable when the hemoglobin is combined... Reasons exist for believing that the only specific cause for active sickle cell anemia is the unique hereditary anomaly of the red corpuscles which predisposes to it. (Hahn and Gillispie 1927, p. 254) 5

While Cook, Meyer, and Emmel had proposed in 1915 that sickle cell anemia was hereditary, it would be more than 30 years before the correct mode of inheritance was worked out. In the interim, investigators often grouped together persons with sickle cell anemia and those who were only carriers of the disease, and the

100 literature presented an understandably confusing array of terminology. Thus, for example, Hahn and Gillispie attempted to replace the "awkward" terms "sickle cell," "latent sickle cell anemia" (clinically healthy but with the inherited trait) and "active sickle cell anemia," with terms derived from the Greek word for "sickle": "drepanocyte," "drepanocytemia," and "drepanocytic-anemia" (Hahn and Gillispie 1927, p. 254).

A first attempt to explain the inheritance of sickle cell anemia in Mendelian terms was made in 1923 by J. G. Huck and W. H. Taliaferro. Based on their study of two families, and in the absence of differentiation between the trait and the disease, Huck and Taliaferro concluded that "sickle cell anemia in man is an inherited condition and behaves as a single Mendelian character which is dominant over the normal condition and which is not sex-linked" (Taliaferro and Huck 1923, p. 597; see also Huck 1923).

Taliaferro and Huck's explanation of sickle cell anemia as due to the inheritance of a single dominant "character" or gene was widely accepted until the late 1940's, when it was disproven in the light of a more sophisticated understanding of homozygous and heterozygous modes of inheritance. 6 In the interim, researchers also sought to resolve the confusion between sickle cell anemia and sickle cell trait and to determine whether the trait and the disease were limited to the black population. A trio of investigators from University of Tennessee's Department of Clinical Pathology and the Nutritional Division of the University of Florida's Experimental Station clarified many of these questions about sickled cells through their survey of "normal and hospital negroes and white people" in the early 1930s. Based on their studies of over 8,000 individuals, and on other surveys reported in the literature, Diggs, Ahmann, and Bibb found that the incidence of the sickle cell trait among negroes was 7.3 percent, and that 1 in 40 of those with the trait also would exhibit sickle cell anemia. "The sickle cell trait," they reported, "has not been demonstrated in recorded surveys of white people, and the only reasonably proved instances of the sickle cell trait in families with unmixed blood have been limited to those of the Mediterranean stock" (Diggs et al. 1933, p. 777). Those exhibiting the sickle cell trait without severe anemia, Diggs and his colleagues emphasized, should not be considered as ill, for their observation showed that "the trait is compatible with long life" and "the incidence [of the trait] in hospital cases has not been proved to be higher than in healthy individuals." Thus, they concluded, "the importance of the sickle cell trait appears to be limited to the relatively small group who in addition to the trait have sickle cell anemia" (Diggs et al. 1933, p. 777). But why some of those who inherit the trait, "thought to be transmitted as a dominant Mendelian characteristic," also develop severe anemia remained a mystery "due to factors unknown" (Diggs et al. 1933, p. 769).

Why some persons had sickled cells but not severe anemia became clearer genetically in the late 1940's, when James V. Neel from the University of Michigan's Heredity Clinic demonstrated that sickled cells were not inherited as a dominant Mendelian characteristic. On the basis of Taliaferro and Huck's hypothesis, Neel pointed out in a 1949 paper, "the inference was that this [dominant] gene was more strongly expressed in some individuals (sickle cell anemia) than in others (sicklemia [or sickle cell traitl )" (Neel 1949, p. 64). In a 1947 paper reviewing the chemical detection of genetic carriers, however, Neel had suggested another mode of inheritance: "There is present in the colored population a certain factor which when heterzygous may have no discernible effects but usually results in sickling, and when homozygous tends to result in sickle cell anemia" (Neel 1947,p. 129).

Neel's interpretation of the inheritance of sickle cell anemia was generated by his study of another hereditary blood disorder, thalassemia or Cooley's anemia, a condition which would become significantly linked with sickle cell research in the 1950s. By the mid-1940s, clinicians and geneticists knew that thalassemia was found relatively frequently among persons living in, or descended from residents of, the Mediterranean countries, and that the condition could occur in either a severe or relatively mild form. In 1944, Neel and W. W. Valentine reported on their investigation of 34 parents, siblings, or immediate collaterals of three patients with thalassemia, and one with mild hereditary anemia. From their genetic and hematologic study, they suggested that the mild familial anemia was due to a heterozygous or carrier state, while severe thalassemia resulted from a homozygous inheritance from two carriers. The mild and severe forms of the disorder, they proposed, could be designated as thalassemia minor and thalassemia major (Valentine and Neel 1944)."

Reasoning from his analysis of thalassemia, Neel recognized that there was a relatively simple way to determine whether his hypothesis about the inheritance of sickle cell anemia was correct. "it the homozygousheterozygous hypothesis is correct, then both the parents of any patient with sickle cell anemia should always sickle . . . If, on the other hand, the disease is due to a dominant gene with variable expression, only one parent need sickle, although occasionally, due to the chance marriage of two sicklers, both parents may sickle." (Neel 1949, p. 64) Thus far, Neel reported, he had tested 42 parents of 29 patients

101 with sickle cell anemia for the occurrence of sickling. Although only 13 couples were tested, the results were convincing: the blood of all 42 parents sickled, "This is the result expected from the homozgous-heterozygous hypothesis. On the other hand, the probability of the occurrence of such a number of positive parents under the variable dominant hypothesis is (0.76542 )42 or 0.000013" (Neel 1949, p. 650

Reviewing survey data from the U. S. and Africa on the incidence of sickling, Neel briefly discussed the sub new understanding of the genetic transmission Or SICKiea cells, one could both accurately predict the disease's occurrence and, if so desired, greatly reduce its incidence. In a genetic situation such as appears to obtain here, where the heterozygote, who may be termed the genetic carrier of the disease, may be readily distinguished from normal and from the homozygote, it is possible to predict with a high degree of accuracy which marriages should result in homozygous individuals - in this case, children with sickle cell anemia. Since (homozygous) individuals with sickle cell anemia either die young or, if they reach maturity, have a greatly lowered fertility, the vast majority of cases of the disease are the issue of marriages between two (heterozygous) persons with the sickle cell trait. In the absence of marriage between individuals whose erythrocytes exhibit the sickling phenomenon, the frequency of the homozygote would greatly decrease, and sickle cell anemia would tend to disappear, with only a very rare case arising as a result of mutation in a normal individual married to a person homozygous or heterozygous for the sickling gene. (Neel 1949, pp. 65-66)

How Cells Sickle: Hemoglobin and the Concept of Molecular Disease

The chain of researches that would unravel the puzzling question of how red blood cells sickle, and in so doing greatly clarity our understanding of how genes control protein structure, was set in motion on a spring night in 1945. At the time, chemist Linus Pauling was serving as a member of the Medical Advisory Committee that was assisting Vannevar Bush in the preparation of a report to the President on the state and direction of science at the close of World War 11. "One evening," Pauling later recalled, "Dr. William B. Castle, Professor of Medicine in Harvard University, mentioned to the other members of the committee the disease sicklecell anemia, with which he had had some experience. He told about the discovery of the disease by Dr. J. B. Herrick in 19 10, and described the characteristic change in shape of the red corpuscles and the effect of oxygen in preventing the sickling and of carbon dioxide in accelerating it" (Pauling 1955, p. 216).

Castle's remarks immediately caught Pauling's attention, for in 1935, after studying the structure of relatively simple inorganic molecules for a decade, Pauling had become interested in the homoblobin molecule . beginning wirth an anaalysis of the structural origin of hemoglobin's oxygen equilibrium curve, Pauling and his colleagues at the California Institute of Technology went on to examine the denaturation of hemoglobin and other proteins, and the magnetic properties of hemoglobin and its derivatives. For Pauling, long interested in understanding how molecular structure determines chemical properties, the study of magnetic properties was "especially fruitful in providing information about the nature of the bonds formed by the iron atoms in hemoglobin with the neighboring atoms of . . . the globin, and attached molecules such as the oxygen molecule" (Pauling 1955, p. 216).

Pauling thus was well prepared to respond to Castle's discussion of sickle-cell anemia, particularly his description of Hahn and Gillispie's 1927 experiments on the effects of oxygen and carbon dioxide on sickling. Pauling suggested to Castle that "the action of carbon dioxide was to accelerate the dissociation of oxygen from oxyhemoglobin. . ., and I pointed out that the relation of sickling to the presence of oxygen clearly indicated that the hemoglobin molecules in the red cell are involved in the phenomenon of sickling." (Pauling 1955, pp. 216-217)

In 1927 Hahn and Gillispie had recognized that hemoglobin somehow is involved in sickling. Now, in 1945, Pauling's agile mind quickly formulated an idea about the nature of that involvement, an idea triggered in part by his work on the nature of antigen-antibody relationships.' 0 "1 thought that it was possible that the patients with sickle-cell anemia manufacture an abnormal sort of hemoglobin molecule, such that the molecules are self -complementary, and stick to one another, forming long rods, which then line up side by side to produce a needle-like crystal, which, as it continues to grow, becomes longer than the diameter of the red cell, and thus deforms the red cell into an elongated shape. It was necessary to assume that molecules of sickle-cell oxyhemoglobin, as well as those of normal adult human hemoglobin and oxyhemoglobin, do not have this property of self -complementariness" (Pauling 1960, pp. 2-3).

102 Pauling, a chemist, recognized the wisdom of collaborating with a physician to explore his new interest in sickle cell anemia. That collaborator, Dr. Harvey Itano, arrived at Cal Tech in the fall of 1946 on a three year American Chemical Society Precloctoral Fellowship in chernistry. Itano had received his M. D. from St. Louis niversity School of Medicine in 1945, where one of his professors, Dr. Edward Doisy, suggested that he pursue s interest in chemistry by working with Pauling. Paulg agreed to Doisy's plan, and in correspondence with tano Pauling "suggested that he investigate the hemolobin from the red cells of sickle-cell anemia patients, order to see whether it was different from normal ult human hemoglobin" (Pauling 1955, p. 217).

Itano began his hemoglobin research in the fall of 1946 by replicating Hahn and Gillispie's finding that , 18ickling is prevented by both oxygen and carbon mon.oxide. He then found that a series of other hemoglobin derivatives similarly prevent sickling and developed a diagnostic test enabling physicians to rapidly differentiate between sickle cell trait and sickle cell anemia Otano and Pauling, 1949).

Then, with Pauling's guidance, Itano joined forces with two postdoctoral fellows, S. J. Singer and A. C. Wells, to determine whether there were significant differences in the physical and chemical properties of hemoglobins from normal adults and from persons with sicklemia (sickle cell trait) and sickle cell anemia.

Four months after J. V. Neel's paper in Science, documenting his hypothesis about the homozygous inheritance of sickle cell anemia Pauling, Itano, Singer, and Wells reported the results o; their physical -chemical researches in the same major journal. They found, initially, that most of the properties of hemoglobin from the blood of normal individuals and sickle cell anemia patients were identical. But then a striking and fundamental difference was found, by the painstaking measurement of the different hemoglobins' electrophoretic mobility.1 I

At the time of their November 1949 report, Pauling's group had done eiectrophoresis studies, each taking 6 to 20 hours, on hemoglobins from 15 persons with sickle cell anemia, 8 with sickle call trait, and 7 with normal blood. "The results," they announced, "indicate that a significant difference exists between the electrophoretic mobilities of hemoglobin derived from erythrocytes of normal individuals and from those of sickle cell anemic individuals" (Pauling et al. 1949, p. 544), Electrophoresis of hemoglobin from sickle cell trait carriers also was found to have a characteristic pattern, one that looked like the pattern produced by a roughly equal mixture of normal hemoglobin and sickle cell anemia hemoglobin. At least electrophoretically, therefore, "the two components in sicklemia hemoglobin are identifiable with sickle cell anemia hemoglobin and with normal hemoglobin" (Pauling et al. 1949, p. 546).

Based on a further series of ongoing physical-chemical tests, the Cal Tech researchers reported it seemed probable that the difference between normal and sickle cell hemoglobins resided in the globin portion of the molecule. Their experimental observations, Pauling and his colleagues judged, thus supported the picture of the sickling process that Pauling had first envisaged while talking to William Castle in 1945.

We can picture the mechanism of the sickling process in the following way [the investigators wrote in 19491. It is likely that it is the globins rather than the hemes of the two hemoglobins that are different. Let us propose that there is a surface region on the globin of the sickle cell anemia hemoglobin molecule which is absent in the normal molecule and which has a configuration complementary to a different region of the surface of the hemoglobin molecule. This situation would be somewhat analogous to that which very probably exists in antigen-antibody reactions. The fact that sickling occurs only when the partial pressures of oxygen and carbon monoxide are low suggests that one of these sites is very near to the iron atom of one or more of the hemes, and that when the iron atom is combined with either one of these gases, the complementariness of the two structures is considerably diminished. Under the appropriate conditions, then, the sickle cell anemia hemoglobin molecules might be capable of interacting with one another at these sites sufficiently to cause at least a partial alignment of the molecules within the cell, resulting in the erythrocyte's becoming birefringent, and the cell membrane's being distorted to accommodate the now relatively rigid structures within its confines. The addition of oxygen or carbon monoxide to the cell might reverse these effects by disrupting some of the weak bonds between the hemoglobin molecules in favor of the bonds formed between gas molecules and iron atoms of the hemes. (Pauling et al. 1949, pp. 546-547)

At the time their Science paper was published, Pauling, Itano, and their associates recognized that "some of the details of this picture of the sickling process are as yet conjectural," awaiting future experimental confirmation or disproval. But if the proposal was correct, they

103 ence of 'defective' hemoglobin moiecuies anU Lilt: pathological consequences of sickle cell disease" (Pauling at al. 1949, p. 547).

With this statement, Pauling introduced his fundamentally important concept of sickle cell anemia as a genetically transmitted molecular disease. Discussing the genetics Of sickle cell disease, Pauling and his co-inveStigators briefly reviewed Taliaferro and Huck's dominant gene theory, and Neel's recent report of his study supporting a heterozygous-homozygous mode of inheritance. Then, as investigators are prone to do in the competitive world of research, Pauling's group added a priority claim.12 "Our results had caused us to draw this inference before Neel's paper was published. The existence of normal hemoglobin and sickle cell anemia hemoglobin in roughly equal proportions in sicklemia hemoglobin preparations is obviously in complete accord "pith this hypothesis" (Pauling et al. 1949, p. 547).

Pauling, Itano, Singer, and Wells then reached the core of their paper: an explanation of the probable relationship between the genetics of sickle cell anemia and the mechanism of sickling.

In fact, if the mechanism proposed above to ac count for the sickling process is correct, we can identify the gene responsible for the sickling process with one of an alternative pair of alleles capable through some seri . es of reactions of intro ducing the modification into the hemoglobin molecule that distinguishes sickle cell anemia hemo globin from the normal protein.

The results of our investigation are compatible with a direct quantitative effect of this gene pair; in the chromosomes of a single nucleus of a normal adult somatic cell there is a complete absence of the sickle cell gene, while two doses of its allele are present; in the sickiemia somatic cell there exists one dose of each allele; and in the sickle cell anemia somatic cell there are two doses of the sickle cell gene, and a complete absence of its normal allele. Correspondingly, the erythrocytes of these individuals contain 100 percent normal hemoglobin, 40 percent sickle cell anemia hemoglobin and 60 percent normal hemoglobin, and 100 percent sickle cell anemia hemoglobin, respectively. This investigation reveals, therefore, a clear case of a change produced in a protein italics added)

With this statement, Pauling's group affirmed the soundness of Dr. James Herrick's first hypothesis about the cause of the "peculiar" sickle-shaped red blood cells he had first observed under his microscope in 1904: "that some unrecognized change in the composition of the corpuscle itself may be the determining factor. 11 Pauling and Itano's investigations, part of a sequence of inquiries into the nature of sickle cell anemia that began with Herrick's first case report in 1910, also forged new links between physical chemistry, human and basic genetics, and clinical medicine. That fusion would profoundly alter our understanding of the nature of gene action, and resolve many of the puzzling questions about allelism that we noted at the beginning of this chapter. For, in a line of inquiry that at the time seemed far removed from Beadle, Ephrussi, and Tatum's basic genetic researches with Drosophila and Neurospora, the work of Pauling and his colleagues soon would intersect with and significantly modify the one gene-one enzyme hypothesis.

The Molecular Detectives:

Fingerprinting Hemoglobin

While Pauling et al.'s 1949 report provided strong empirical evidence for the concept of a molecular disease, it left many questions unresolved and, not surprisingly, provided a major catalyst for further study. The torrent of researches on normal and abnormal hemoglobins since 1949 is suggested by the fact that as of 1976 some 300 abnormal hemoglobins, in addition to hemoglobin S (sickle-cell hemoglobin), had been discovered, and the precise chemical nature of 259 of these variants had been established (Neel 1976, p. 57).

In the remainder of this chapter, our focus will be on three interrelated lines of work through the early 1960s that marked the coming of age of "molecular pathology" and that, joining with other areas of genetic research, produced a new understanding of how genes control the structure of proteins.1 3 At the center of these investigations were two critical questions posed by the work of Pauling's group: what is the exact biochemical nature of the difference between normal and sickle cell hemoglobin and where in the hemoglobin molecule is this difference localized?

One of the first areas of study to bear fruit after 1949 was the mechanism of the sickling process. In his first conversation about sickle cell anemia with William Castle in 1945, and again in his 1949 Science paper,

104 Pauling had suggested a mechanism for the sickling process involving the combination of complementary sites on adjacent hemoglobin molecules, analogous to the presumed interactions between antigens and antibodies. With this hypothesis, the known effects of oxygen and carbon monoxide in reversing sickling were explained by a disruption of the weak bonds between the hemoglobin molecules by the bonds formed between the gas molecules and iron atoms of heme.

The essentials of this picture of the sickling process were substantiated by 1951 through several lines of investigation, including further experiments by Pauling (St. George and Pauling 1951). The principal confirmation of Pauling's view of the sickling mechanism came from studies conducted in Boston, Massachusetts and Cambridge, England. (Fittingly, Dr. William Castle directed the work of Dr. John Harris in Boston, in the Thorriclike Memorial Laboratories of Boston City Hospital.) In a 1950 paper, Harris reported on his study with the polarizing microscope of various concentrations of sickle-cell hemoglobin in oxygenated and deoxygenated states. As Pauling had hypothesized, Harris found that the cleoxygenated hemoglobin formed spindle-shaped bodies arranged as tactoids. This formation, Harris explained, "is evidence of a specific arrangement or linkage of the individual molecules with the formation of long chains of hemoglobin elements. (Harris 1950, p. 199).

At first independent of Harris' work, although the two groups later corresponded about their findings, comparable studies were initiated at the University of Cambridge's Cavendish Laboratory by noted protein crystallographer F. M. Perutz. Perutz had spent some 10 years collecting x-ray diffraction data from hemoglobin crystals in an effort to map the protein's threedimensional structure, when in 1950 his attention was directed to sickle cell anemia by Dr, F. Eirich, from the Polytechnic Institute in Brooklyn, New York. Then, following up on a suggestion by Dr. C. A. Stetson of the Rockefeller Institute, Perutz and Mitchison reported in 1950 that their solubility studies of normal and sickle cell hemoglobin, using a polarizing microscope, indicated that "the reduced [cleoxygenated] hemoglobin in sickle cells is in a crystalline state" (Perutz and Mitchison 1950, p. 678). Several months later Perutz reported on further studies, using x-ray diffraction to see whether there were any obvious structural differences between sickle-cell anemia and normal oxyhemoglobin molecules. But the nature of the molecular differences between normal and sickle cell hemoglobin was not to be revealed by x-ray diffraction. "As to the structural differences between normal and sickle-cell anemia haemoglobin, our crystallographic results provide no clue, but merely serve to emphasize the close similarity between the two proteins." To Perutz and his collaborators, "this was a most surprising result, considering that normally two related but slightly different proteins - for example the haemoglobins of closely related animal species - have totally different crystal structures" (Perutz, Liquori, and Eirich 1951, pp. 931, 929). 14

Another line of attack on the problem of localizing the structural basis for sickled cells was undertaken by the eminent hemoglobin researcher W. A. Schroeder. Working with A. M. Kay and A. C. Wells, Schroeder's approach to the molecular disease puzzle was to see whether there were differences in the amino acid content of hemoglobins from blacks with and without sickle cell anemia. Their results, like Perutz's x-ray diffraction studies, were largely negative, for comparative analysis indicated no significant differences in the basic and acidic amino acid content of normal adult and sickle cell hemoglobin. Slight differences, however, were detected in the two hemoglobins' content of four uncharged amino acids. Perhaps, Schroeder suggested, these differences might affect the folding or coiling of the polypeptide chains so as to change the acid or base content of other amino acid groups, and thus indirectly alter the electrophoretic properties of the hemoglobins (Schroeder, Kay, and Wells 1950).

Schroeder's work, like other researches at the time, proceeded on the basis of Pauling's suggestion that the structural differences between hemoglobins A (normal) and S (sickle-cell) lay in the globin portion of the molecule. The correctness of this assumption was demonstrated in 1952 by Havinga and Itano in Pauling's laboratory. By separating the heme from the globin and subjecting the globin portions to electrophoresis, they found that the globins had the same differences in electrophoretic mobility as did the whole hemoglobin A and S molecules (Havinga and Itano 1952). In a series of concurrent experiments, Havinga ruled out a variety of possible reasons for differences between hemoglobins S and A, including optical rotation, phosphorus accounted for normal and abnormal hemoglobin molacules was further complicated between 1950 and 1953 as clinical geneticists, led by James V. Neel and Harvey Itano, discovered three more abnormal varieties of adult human hemoglobin, named in alphabetical order hemoglobins C, D, and E. These newly recognized abnormal hemoglobins, the researchers quickly found, could manifest themselves alone, or in synergistic interaction with sickle cell anemia, or with thalassernia (see note 7). Still another disease, they found, could result if an individual simultaneously had the sickle cell and the thalassernia minor traits. Thus, by 1953, Itano was able to review ten distinct hereditary clinical states associated with abnormal hemoglobin metabolism

105 (Itano 1953; see also reviews by Itano 1955, Pauling 1955). For both clinical and basic researchers in genetics, biochemistry, and physical chemistry, there was now an almost bewildering array of molecular hemoglobin disorders with which to work. In terms of the central questions raised by the molecular disease concept, that of where and how the hemoglobin molecules differed structurally, researchers focused principally on hemoglobins A, S, and C. It structural differences could be pinpointed researchers knew they would gain a critical insight into "a fundamental question in biochemical genetics:" "whether or not the abnormal hemoglobin genes are alleles, that is, whether mutations resulting in the alteration of hemoglobin metabolism takes place at one or more than one chromosomal locus" (Itano 1955, p. 293). In 1949, we recall, Pauling et al. had postulated that the genes for hemoglobins A and S were allelic, and, with the discovery of hemoglobin C in 1950 by Itano and Neel, a series of studies soon "furnished strong biochemical and genetic evidence that the genes for hemoglobins S and C are allelic or closely linked" (Itano 1955, p. 293).

Through the early 1950s, the varied attempts to locate the structural difference between normal and sickle cell hemoglobin, beyond implicating the globin portion of the molecules, had served primarily to rule out a series of possibilities. The one viable hypothesis seemed to be Schroeder et al.'s suggestion that the molecules might be composed of the same polypeptide chains, but folded in different ways. "The interesting possibility exists," said Pauling in his 1955 Harvey lecture, "that the gene responsible for the sickle-cell abnormality is one that determines the nature of the the baffling problem of the specific chemical difference between hemoglobins A and S began to be resolved by the researches of a young protein chemist, Vernon M. Ingram, working in Perutz's laboratory. Although only two decades have elapsed since Ingram's first paper, the strides that have been made in analyzing the sequential and spatial structure of proteins make it difficult to appreciate today the problems facing protein chemists in the mid-1950's and the corresponding elegance of Ingram's work. Proteins are extremely complex macromolecules, and in the early 1950s little was known about the exact sequences in which given amino acids form given proteins, much less about the relationships between sequencing and the three-dimensional shapes of a protein's polypepticle chains (see note 14). After years of arduous work, biochemist Frederick Sanger finished thefirst complete sequence analysisof a protein in 1953, an achievement that "contributed decisively to the proof of the peptide theory" - the long debated theory that protein molecules are built of chains of amino acids bound together by pepticle bonds (Fruton 1972, p. 148). The protein he studied was the hormone insulin, a relatively small protein whose longest polypeptide chain contained only 51 amino acids. Complex as the sequence analysis of insulin's structure proved to be, hemoglobin presented a far more formidable task, for, with a molecular weight of about 65,000, researchers knew it would probably contain about 600 amino acid residues.

Vernon Ingram arrived at the Cavendish Laboratory in 1952, to join in Perutz's x-ray crystallography studies of hemoglobin crystals. Then, Ingram recalls, "about the same time as the Watson-Crick breakthrough in the same lab, I went off in a slightly different direction from Perutz and began to study the relationship between the heme group and its adjacent polypepticle chains" (Ingram, personal communication). Ingram's interest in sickle cell hemoglobin was aroused in 1955 by the arrival at Cavendish of an Oxford physician, A. C. Allison. Allison was one of a number of clinicians then investigating both the incidence of sickle cell trait and disease in Africa, and the hypothesis proposed by Brain in 1952 that the evolutionary significance of hemoglobin S might lie in its conferring protection against malarial parasites (Brain 1952; Allison 1954; Pauling 1955). Allison also became interested in the formation of hemoglobin crys als in sickle cells, and came to the Cavendish to do 4iber x-ray crystallography studies. Given the state of liber x-ray techniques at the time, Ingram remembers, Allison "didn't get any significant x-ray pictures," and ~departed leaving behind some of his sickle cell hemo samples. But Allison's visit to the Cavendish would prove highly significant in other respects, for it "was the quite fortuitous appearance of those sickle cell samples" which triggered Ingram's idea of looking at their protein chemistry (Ingram, personal communica tion). It was not, of course, completely fortuitous. For Ingram,working "in a lab which was passionately inter ested in anything to do with hemoglobin," was familiar with the research of Pauling's group and with the sub sequent attempts to determine the difference between emoglobins A and S.

When Ingram decided that he, too, would seek to uncover how the protein globins of normal and sickle cell hemoglobins differed, he knew from previous investigations that the difference, whatever it was, must be a small one. He knew, too, that the hemoglobin molecule was too large for the type of complete sequence analysis of insulin that Sanger had performed. Faced with this reality, Ingram devised a short cut, utilizing a combination of methods that enabled him to obtain and analyze relatively small pepticle fragments from the whole molecule. In the mid-1950s, one of the newest and most reliable ways of separating pepticles and

106 amino acids was by means of the enzyme trVpsin, which split polypeptide chains by specifically degrading the chemical bonds formed by the carboxyl groups of two amino acids, lysine and arginine. Other studies of hemoglobin's amino acid composition had indicated that there were some 60 of these particular carboxyl groups in hemoglobin A and S molecules. But, because Perutz's x-ray diffraction analysis had indicated that each hemoglobin molecule was composed of two identical "half molecules," Ingram anticipated that "the number of pepticles obtained by the action of the trypsin should be about thirty, an average chain-length of ten amino-acids. Small differences in the two proteins [hemoglobin A and S1 , he reasoned, will result in small changes in one or more of these peptides" (Ingram 1956, p. 793).

To try to detect these small differences, Ingram ingeniously combined paper electrophoresis and the paper chromotagraphy methods that he had watched Sanger use. By this combination he created a twodimensional method that enabled him to comparatively "fingerprint" the hemoglobin S and A fragments he obtained from the tryspin digest. The fingerprints revealed approximately 30 pepticle spots, confirming Perutz's view that the human hemoglobin molecule consists of two identical half molecules. As he also had expected, Ingram saw that the normal and sickle cell globins contained the same number of peptides. But the fingerprints did what other attempts to locate a difference between the S and A globins had been unable to do - they revealed a small difference in the largely identical molecules. As Ingram reported in 1956, ". . . there is one peptide spot clearly visible in the digest of haernoglobin S which is not obvious in the haemoglobin A 'finger print" (Ingram 1956, p. 793).

Prior to the development of Ingram's fingerprinting method, it had been impossible to decide whether the small but critical difference between hemoglobins A and S resided in the amino acid sequences of the polypepticle chains, or in the ways those chains were folded. But by means of the trypsin digest and fingerprinting, as Ingram concluded in his short paper:

One can now answer at least partly the question put earlier, and say that there is a difference in the amino-acid sequence in one small part of one of the polypepticle chains. This is particularly interesting in view of the genetic evidence that the formation of haernoglobin S is due to a mutation in a single gene. It remains to be seen exactly how large a portion of the chains is affected and how the sequences differ. (Ingram 1956, p. 294)

Ingram's experiment thus had opened a new vista in biochemical genetics and protein chemistry, providing the way to show with detailed precision the relationship between a gene and the protein under its control. Less than a year after his first paper, Ingram published a second short report in which he answered his 1956 question of "how large a portion of the chains is affected and how the sequences differ."

I have now found that out of nearly 300 aminoacids in the two proteins, only one is different; one of the glutamic acid residues of normal haemoglobin is replaced by a valine residue in sickle cell anaernia haernoglobin. The latter is an abnormal protein which is inherited in a strictly Mendelian manner; it is now possible to show, for the first time, the effect of a single gene mutation as a change in one amino -acid of the haemoglobin polypeptide chain for the manufacture of which that gene is responsible. (Ingram 1957, p. 326) shown, caused by the smallest possible alteration in the large hemoglobin molecule: the substitution of a single amino acid, glutarnine, by another, valine. The technique of protein fingerprinting, as Ingram realized, had simply and decisively answered one of the most intriguing questions in the explosive new field of molecular genetics, that of precisely how a gene mutation affects the specific structure of a protein.

What, then, was the nature of the mutation within the hemoglobin gene that produced this small but signif icant alteration in the structure of the hemoglobin molecule? Presumably, Ingram wrote in 1957, it is an equally small change in the hemoglobin gene. "it is not known, but it may well be that this involves a replacement of no more than a single base-pair in the chain of the deoxyribonucleic acid of the gene" (Ingram 1957, p. 327). This hypothesis about a human gene and its protein, Ingram pointed out, accorded well with the new knowledge of genetic subunits being gained from the standard organisms of molecular geneticists, NeuroVora, Aspergillus, and bacteriophage.

The results presented in this communication are certainly what one would expect on the basis of the widely accepted hypothesis of gene action; the sequence of base-pairs along the chain of nucieic acid provides the information which determines the sequence of amino-acids in the polypeptide chain for which the particular gene, or length of nucleic acid, is responsible. A substitution in nucleic acids leads to a substitution in the polypeptide. (Ingram 1957, pp. 327-328)

107 Less than a month after Ingram I s 1957 paper, another important report issued from PaUling's laboratory. Perutz's x-ray crystallography studies and Ingram's fingerprint had led them to conclude that the hemoglobin molecule consisted of two identical "half molecules.,, Now, on the basis of complex quantitative chemical analysis, H. L. Rhinesmith, W. A. Schroeder, and Pauling had succeeded in identifying the number and kind of polypeptide chains in normal adult hemoglobin. The globin, they determined, consists of two sets of polypeptide chains, or a total of four chains forming two identical half molecules (Rhinesmith, Schroeder, and Pauling 1957). With this report, there was now a solid line of evidence, derived from a complex human protein, that confirmed and greatly refined the concept

That concept, as we have seen, had been redisco and reenunciated as the one gene-one enzyme hypoth'by George Beadle and his associates in the 1930's early 1940's, through their biochemical genetic 2' searches with Drosophilia and Neurospora. Ingr ecalls that while he was at the Cavendish laboratc;

from 1952-1958 the one gene-one enzyme hyp was "well accepted" by himself and his colleagues,,Yl~ that he promptly saw the "sickle cell hemoglobin bu." ness" as providing a "direct illustration" of the corr . ness of Beadle et al.'s formulation (Ingram, persor- communication). But acceptance of the one gene-O(, enzyme hypothesis was far from universal among ger- icists from the early 1940's through the mid-1950's part because, like Garrod's explanation, it seemed V-1. simple an account of gene action.16 There were, of course, other reasons besides 1' simplicity for the slow acceptance of the one gene-orenzyme concept and all that it implied. It would take some years to understand t', relationship between mutations and enzynit, primarily because so little was known concerni, the structure of proteins at the time there was much discussion of the one gene-one enzyM concept. We may have difficulty in appreciatir 1. the problem unless we keep in mind that U';1 concepts were formulated without any cli, idea of the properties of proteins which conferm specif icity . . . Although proteins were known to Ll composed of polypeptide chains which wei assumed to be folded in specific ways, there w no clear concept of what determined the sequens of amino acids or whether the sequence w-related to the three-dimensional shape. Did ger--, control the specificity of enzymes and if so howu If a mutation changed an enzyme activity was because the protein was changed or produced r smaller amounts? Would the mutation of one gec, change more than one protein? Could genmutate in a variety of ways?

Without a clear concept of the nature of the gar., or of its supposed product, a specific proteIF-' little progress could be made. Geneticists cou.". study the phenotypes produced by mutation had been done for years. The improvement pro vided by Neurospora, which could be grown in a simple, completely defined medium, was that the change in phenotype could be defined much more precisely in chemical terms. In an attempt to simplify the problem, the question was put in the following form. Do any genes have a single primary function and if so what proportion of mutations produces changes of this nature? (Taylor 1965, pp. 4-5)

With this precis of the questions rampant in genetics through the early 1950s, the impact of the researches on sickle cell hemoglobin is apparent. By 1957, principally through the researches of Ingram and of Paul ing's group, geneticists knew that a mutant hemoglobin gene produced a disease by altering the presence of a single amino acid in the polypepticle chain of the hemoglobin molecule. The one gene-one enzyme concept of gene action had been essentially correct. But it had been too simple, because enzymes are proteins, but not all proteins are enzymes. Principally through research from many quarters on the nature of hemoglobin, it was gradually recognized that structural proteins, too, might be the direct products of gene action, and the one gene-one enzyme hypothesis was recast in terms of one gene-one protein. Then, the trail of researches into the nature of sickle cells, that began in 1910 with Herrick's first clinical report, revealed the specificity of gene action within a protein, and one gene-one protein was recast as one gene-one polypepticle. "Thus," wrote Bentley Glass on the centennial of Gregor Mendel's publication, "geneticists were led to the conclusion that, as predicted from the Watson-Crick model of the DNA molecule, the sequence of nucleotides in the DNA molecule specifies the sequence of amino acids in the polypeptides" (Glass 1965, p. 234).

Another core aspect of gene action elucidated by the study of abnormal hemoglobins was allelism. As we have seen, Ingram's fingerprints of hemoglobins A and S provided strong indirect support for the supposition advanced initially by Pauling at al. in 1949: that the mutant gene responsible for sickle cell hemoglobin is an allele or variant of the normal hemoglobin gene, occupying the same chromosomal locus. As we also mentioned, the rapid discovery of other abnormal hemoglobins, beginning with the identification of hemoglobin C in 1950, raised new questions about allelism: were hemoglobins C, D, E, and so on, like hemoglobin S, due to another allele at the same locus, or did it represent a new mutational system at an inde

108 pendent locus? The first answers to this fundamentally important question, once again, were provided by Ingram's fingerprints.

In 1951, shortly before Ingram moved from Cambridge, England to MIT in Cambridge, Massachusetts, he and J. A. Hunt published a short note in Nature: "Allelomorphism and the Chemical Differences of the Human Hemoglobins A, S and C." By preparing fingerprints of hemoglobin C and comparing them with their analysis of the A and S fingerprints, Hunt and Ingram had found that C was caused by a different amino acid substitution at the same site in the polypeptide chain as the A and S variation. While hemoglobin S was produced by the substitution of valine for glutamine, hemoglobin C occurred when the same glutamic acid residue was replaced by lysine. "These results," wrote Hunt and Ingram, "have interesting genetic implications."

They are the first steps in a search for a correlation between the linear fine structure of a gene such as that determined recently by Benzer and Pontecorvo, and the linear structure of the polypeptide chain of the protein the synthesis of which that gene controls...

Our results also shed light on the position of these two mutations on the haemoglobin gene or genes. Genetic evidence shows that the haemoglobin S and C mutations are allelic or occur at linked sites; but the statistical evidence is not sufficiently strong to distinguish between these two possibilities. If we assume a direct relationship between the internal (linear) structure of the gene, and the linear arrangement of the amino-acid residues in the haemoglobin molecule, then the finding that the same amino-acid residue, gfutamic acid, is altered in both mutations implies that they are indeed allelic mutations and occupy the same site on the gene. Thus if these ideas are correct the chemical investigation of proteins such as the human haemoglobins can provide a powerful, though indirect, tool to help the geneticist in mapping the positions on the gene where certain mutations occur.

In order to obtain sufficient statistical data and a sufficient number of mutants, such a programme must eventually turn to proteins from microorganisms. However, it is hoped that the abnormal human haemoglobins will provide a few more

Hunt and Ingram's modest hope that abnormal human hemoglobins would provide "a few more useful examples" of the effects of gene mutations on protein structure has been amply realized in the years since 1958. The study of inherited hemoglobin abnormalities has provided the clearest evidence that similar traits are often determined by multiple alleles of a single locus. A 1971 text, for example, noted that over 175 "hereditary abnormal hemoglobin" diagnoses had been made, and had been traced to variations or alleles in only five loci. "Remarkably," the authors wrote, "one of the clearest demonstrations" of the existence and effects of multiple alleles "has come from a human character: the variations in the polypeptide chains that make up the globin portion of the hemoglobin molecule." (Levitan and Montagu 1971, p. 551)

The Lesson of Rare Maladies

The study of abnormal hemoglobins, which now number some 300, is a continuously unfolding one, involving interactions between many areas of clinical and basic research. We have looked at one portion of that study, the trails of inquiry that led from the discovery of strangely elongated and sickle-shaped red blood cells to new knowledge of how normal and mutant genes govern the structure of proteins. This particular path from clinical to basic knowledge, of course, was neither straight not simple. The definition and elucidation of sickle cell anemia as a molecular disease built upon decades' of prior researches into the nature of hemoglobin, and, as we have seen, human and basic geneticists, crystallographers, physical chemists and biochemists, were among those whose often collaborative researches converged to unravel the genetic principles contained in normal and abnormal hemoglobin molecules, and to link their findings with those from other areas of molecular genetics.

To Dr. Vernon Ingram, reflecting in 1976 on the history of hemoglobin research in which he has played such a central role, it is not surprising that "the study of human or animal diseases will frequently and unpredectably lead to fundamental insights into basic mechanisms in biology." But to Ingram, what happened in the arena of molecular genetics was and is surprising. "The coming field in the molecular biology of genetics in the middle 1950s was bacterial and viral genetics ingram said. So if somebody say in 1955 had predicted from which field would come the first demonstration that one gene difference is related to results in ' specific localized difference in a protein, they wouink have said from bacterial or viral materials. We were

109 at surprised as anyone else that it in fact happened to com_v, from a human protein" (Ingram, personal communication).

Today, two decades after that "surprising" event,,l I the study of hemoglobin abnormalities continues to weave between and impact upon clinical and basic.' research. "The study of the abnormal hemoglobins,"~ James V. Neel wrote in a volume of bicentennial essaysl on advances in American medicine, "has not only prc)7 duced a paradigm for 'molecular' diseases in general,,, but has yielded insights into the fine details of chromo-k some structure in mammals which would not otherwise be available" (Neel 1976, p. 60).

Chapter 5 NO TES

1 . The explosive growth of knowledge generated by molecular biology has revealed Mendel's "characters" or units of heredity to be segments of DNA molecules. Through the intermediary activities of RNA, these DNA segments or genes serve as indirect patterns for the reproduction of polypepticles, chains of amino acids which are, or combine to form, proteins. 'Thus, overall, the gene (DNA) clearly specifies the protein. But without proteins, DNA cannot be made; without RNA, proteins cannot be made. Molecular biology is a drama with these as the three major actors, each owes its existence to the others" (Handler 1970, p. 15).

2. In the second edition of Inborn Errors (1923), Garrod reported that evidence for the correctness of his hypothesis had been provided in 1914 by Gross, who reported finding in normal blood plasma but not in the plasma of alkaptonurics an enzyme that oxidized homogentisic acid. Gross' finding, however, was never replicated, and it was not until 1958 that this hypothesis was confirmed by LaDu and his associates, who demonstrated the absence of the enzyme in liver biopsy specimens from an alkaptonuric patient (Glass 1965, p. 231).

3. This failure is amply illustrated by the 1942 book, New Paths in Genetics, authored by the eminent J. B. S. Haldane. For, as Glass points out:

. . . Haldane, of all geneticists of his time, was best trained in biochemistry and most open to see its genetic significance. Yet the book is singularly lacking in analyses of possible gene-enzyme relationships. Garrod's work of 1923 is cited for its example of human metabolic abnormalities inherited as simple recessives; the relationship of phenylketonuria and alkaptonuria to blockage of specific steps in amino acid metabolism is implicitly, but not explicitly, recognized, but the enzymatic relationship is scarcely hinted at. (Glass 1965, p. 232)

Unawareness of Garrod's work, in turn, is exemplified by Beadle and Tatum's opening sentence in their now classic 1941 paper, "Genetic Control of Biochemical Reactions in Neurospora."

From the standpoint of physiological genetics the development and functioning of an organism consist essentially of an integrated system of chemical reactions controlled in some manner by genes. It is entirely tenable to suppose that these genes which are themselves a part of the system, control or regulate specific reactions in the system either by acting directly as enzymes or by determining the specificities of enzymes. I (Beadle and Tatum 1941, p. 499)

In their first reference Beadle and Tatum then note that "the ,,,possibility that genes may act through the mediation of enzymes ~has been suggested by several authors," and they refer the reader ~to publications by Troland (1917), Wright (1927), and Haldane '(1937) for discussion and further references.

4. "The idea was simple," George Beadle has stated.

"Select an organism like a fungus that has simple nutritional requirements. This will mean it can carry out many reactions by which amino acids and vitamins are made. Induce mutations by radiation or other mutagenic agents. Allow meiosis to take place so as to produce spores that are genetically homogeneous. Grow these on a medium supplemented with an array of vitamins and amino acids. Test them by vegetative transfer to a medium with no supplement. Those that have lost the ability to grow on the minimal medium will have lost the ability to synthesize one or more of the substances present in the supplemented medium. The

110 growth requirements of the deficient strain would then be readily ascertained by a systematic series of tests on partially supplemented media.

"in addition to the above specifications we wanted an organism well-suited to genetic studies, preferably one on which the basic genetic work had already been done." (Beadle 1964, p. 594)

5. Hahn and Gillispie's gas chamber experiments fell into disrepute during the 1930's because other investigators had trouble replicating their findings. In 1940, however, 1. S. Sherman reported that, using similar methods, he could distinguish experimentally between sickle cell trait and sickle cell anemia, based on the amount of oxygen tension needed to produce sickling (Sherman 1940).

6. The term "homozygous" means that an organism has inherited identical genes at a given locus, while "heterozygous" refers to the possession of different alleles at a given locus. Thus, in the case of sickle cell hemoglobin, a homozygous person, who has sickle cell disease, has inherited the sickle cell hemoglobin gene (hemoglobin c), from both parents; the person with sickle cell trait, who is only a carrier of the disease, is heterozygous, having a normal or hemoglobin A gene and its alleiie hemoglobin S gene at the same locus.

7. The thalassemias, too, provide an excellent illustration of the ways that a clinical disorder can generate and interact with fundamental research. Much like sickle cell anemia, study of the thalassernias has produced more detailed knowledge both of 11 molecular diseases," and of the mechanisms involved in the genetic control of protein structure. Thus, as a 1976 review article noted, "the thalassernia literature abounds with syndromes that mix classical and molecular genetics with clinical hematology" (Orkin and Nathan 1976, p. 710). From this sometimes confusing mix of inquiries, researchers have found that thalassernia syndromes are caused by deficient production of specific polypeptide chains. Unlike sickle cell anemia, the globin chain in thalassernias, when it is not absent, is structurally normal. Genetically, therefore, the abnormality in the thalassemias must involve either the globin genes themselves or regulatory elements closely linked to them. Newer technics in cellular and utilizing globin mRNA's as templates to define the molecular defects in the thalassemia syndromes. In one sense the thalassemias are all quite similar in that the defect in globin-chain synthesis can always be traced at least to the level of the globin MRNA's. (Orkin and Nathan 1976, p. 711)

8. As occurs frequently in science, recognition of the correct mode of inheritance of sickle cell anemia was a case of "simultaneous discovery," for which history has largely credited only one discoverer. In 1949, independent of Neel's work, a British physician named E. A. Beet, who had been studying the incidence of sickle cell trait and disease in East Africa also recognized the heterozygous-homozygous mode of inheritance (Beet 1949).

9. The human red blood cell or erythrocyte is nearly all hemoglobin, 95% of the dry weight of the cell. Due to the high concentration of hemoglobin in the cell, and the corresponding lack of such usual cellular components as a nucleus, hemoglobin is easily isolated and had been a favorite subject for protein research since the second half of the 19th century. When Pauling turned his attention to the hemoglobin molecule in 1935, chemists knew that the molecule consisted of four heme or ironcontaining groups with which oxygen combines to form oxyhemoglobin, and globin, a bulky protein in which the heme is "imbedded." At the time, little was known about the detailed structure of the globin portion of the molecule.

10. For a discussion of antigen -antibody theory and research, including Pauling's own work on this topic, see Chapter 6.

11. Pauling and his colleagues, in 1949, were able to utilize a highly sophisticated form of electrophoresis, a technique which permits the measurement of the physical properties of molecules such as hemoglobin, recording characteristic patterns that enable one to identify different molecules or chemical substances. Electrophoresis, for example, can distinguish two different proteins as follows: the proteins are placed in an electric field, with a positive and negative side; proteins have positive and negative surface charges, which causes positively charged proteins to migrate to the negative pole of the electrophoretic field, at a rate depending on the strength of the positive charge. Conversely, a negatively charged protein moves to the positive pole of the field. Hemoglobin electrophoresis had first been performed in 1939, by B. D. Davis and E. J. Cohn.

111 12. As documented by historians and sociologists of science, priority disputes are a frequent and characteristic accompaniment of research, for reasons that are understandable in terms of the values and the rewards of scientific research. For the classic sociologic study of priority disputes, see Merton 1957.

13. Among the many and significant areas of hemoglobin research that we will not touch upon are work on the evolution of proteins, the differences between fetal and adult hemoglobins, and chromosomal relationships of hemoglobin loci. For a succint but comprehensive review and bibliography of these and other aspects of hemoglobin research, see Neel 1976. amino acid s). i nus ine organic how the amino acids are linked together and what their order is within a given linear polypeptide chain. Likewise, the biochemist wishes to know both how the backbone linkages are connected and what trick is used to order the amino acids during synthesis

Aside from the question of sequence, there is also the problem of how polypepticle chains assume their final 3-D configuretions. The correct functioning of almost all proteins depends not only upon possession of the correct amino acid sequence but also upon their exact arrangement in space" (Watson 1965, pp. 169-170). 15. One such line of biochemical evidence was offered in 1954 by clinical geneticist and biochemist 1. Herbert Scheinberg and his associates, from the New York State Psychiatric Institute and Columbia University's College of Physicians and Surgeons.,! Using a new experimental method, paper electrophoresis, Scheinberg's group sought to identify the chemical basis of the known differences in the positive electrical charges between herno-, globins A, S, and C. Their experiments provided soon widely~',, cited evidence "that the difference in charges between herric-l", globins a, [s] , and c are due to differences in their content of free carboxyl groups." But, they acknowledged, "the structordli` basis for the apparent difference in the content of free carboxyj~ groups of these hemoglobins remains obscure" (Schelnbern-~ Harris, and Spitzer 1954, pp. 779, 782). 16. George Beadle remembers that:

"In 1945 1 held a Sigma Xi National Lectureship under which I gave lecures on biochemical genetics and thi one gene-one enzyme interpretation. I was much impressed with the resistance to this notion, especially agricultural colleges where workers were familiar with 4 genetics of such characteristics as egg production, milk production in dairy cattle. They were sure 96 action could not be generally described in the sirni:!, Way we had postulated. It seems to me the status of concept dropped to an all-time low at the Cold Sp(w" Harbor Symposium of 1951. In rereading the volume on those meetings, I have the impression that the numtk_ whose faith in one gene-one enzyme remained stee could be counted on the fingers of one hand – wltW couple of fingers left over" (Beadle 1966, p. 30).

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Tatum, E. L. 1964. "A Case History in Biological Research, Nobel Lectures, Physiology or Medicine, 1946-1962. Amste dam: Elsevier Publishing Co., 602-612.

Taylor, J. H., ed. 1965. Selected Papers on Molecular Genetic, New York: Academic Press.

Valentine, W. N., and J. V. Neel. 1944. "Hematologic an Genetic Study of the Transmission of Thalassemia," Archivd of Internal Medicine, 74: 185-196.

Washburn, R. E. 1911. "Peculiar Elongated and Sickle-Shape Red Blood Corpuscles in a Case of Severe Anemia," Virgin Medical Semi-Monthly, 15: 490.

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115 Overthe past century, beginning with efforts of bacteriologists to find ways of protecting man d his domestic animals from the ravages of infectious iseases, the field of immunobiology has involved a eadily broadening scope of inquiry. From many isciplines, approaches, and interests, clinical and boratory investigators have gradually defined and are 1eeking to understand the three key features of the phenomenon called immunity: recognition, specificity, and memory. That is, first, how does the immune system #,recognize" potentially hazardous foreign antigens, and, of equal import, recognize the body's own constituents as harmless? Or, in the words of Burnet, how does the immune system distinguish between "not self" and 11 self?" The immune system, secondly, operates with remarkable specificity against millions of chemically different antigenic structures. Thus, closely related to understanding how recognition occurs, immunobio ogists want to know how the immune system pro uces a specific antibody against any one of millions-of antigens. The third key characteristic of the immune system is "memory:" how, days or years after its first exposure to an antigen, is an organism able to "remember" that exposure and react more rapidly and strongly to a second encounter?

Finding answers to these questions, investigators have long realized, has obvious medical significance. And , as has become increasingly evident in recent years, the quest to understand how immune defense mechanisms operate also has led us into the "central regions" of biology. "The current explosion in immunological research," as Lewin pointed out in 1974,

CHAPTER 6 A CRUCIAL EXPERIMENT OF NATURE: MULTIPLE MYELOMA AND THE STRUCTURE OF ANTIBODIES throws out waves of discoveries that are giving insights into the science undreamed of a few years ago. Immunobiology is now one of the most rewarding and promising areas of biological research. It is rewarding because it spreads into many areas of acute social misery giving prospects of long-hoped-for cures: the successes of vaccinations against many bacterial and viral infections may soon be followed by assaults on cancer and diseases such as rheumatoid arthritis and the presently unassailable parasitic infections; immunology also holds the key to safe organ transplantations. And it is promising because the drive toward understanding the molecular mechanisms behind the immune responses is certain to reveal features fundamental to the whole of biology. (Lewin 1974, p. 2)

During the relatively brief span of its existence, the field of immunobiology has become so vast, and the lines of disease-oriented and basic research pursued within its compass so complex and intertwined, that no one book could pretend to be a definitive history. Within the even narrower scope of a single essay, we will look at only a single but critical strand in the history of immunology, a strand that exemplifies how "major thrusts toward fundamental progress in immunobiology have been guided by questions formulated from experiences in the clinic" (Bach and Good 1972, p. xiii). These clinical problems, in the main, have centered around what immunologist Robert Good has termed "the crucial experiments of nature," a series of diseases which have "contributed maximally to the development of our modern concepts of immunology" (Good 1972, p. 23).

The particular disease problem that we will examine is multiple myeloma, a relatively rare form of cancer that accounts for only some 0.5 percent of all malignancies and about two deaths per 100,000 persons per year. But although a rare form of cancer, multiple myeloma "probably represents the most important of all experiments of nature" for immunobiologists (Good 1972, p. 25). For, as a trail of medical and scientific research has revealed since the first case of multiple myeloma was documented in 1846, this cancer is a tumor of the plasma cells which are responsible for the production of antibodies. In patients with multiple myeloma, as we shall learn, the immune system's protein metabolism is so altered that the excretion of certain abnormal proteins is a hallmark of the disease. The study of these abnormal proteins, in turn, has generated extensive and significant knowledge not only about multiple eases, and the structure, biosynthesis, and genetic control of antibodies.

The particular strand among this network of research and discovery that we will follow is that which runs from the first clinical descriptions and study of multiple myeloma in the mid-19th century to the analysis of

116 antibody structure in the 1950s and 1960s, an analysis that rested on defining the nature of one of the abnormal proteins of multiple myeloma patients, known as Bence Jones protein. The elucidation of an antibody's structure marked the birth of molecular immunology, an event that future historians may well judge to have been a "revolution" in the science of immunology. One of the first results of that revolution, as we shall see, has been major strides in understanding how an antibody's molecular structure specifies two of its functions: to recognize a specific type of antigen, and to then perform a particular type of defense function, termed the antibody's effector function.

In part because both the history of immunology and our present knowledge of immune phenomena are so complex, we are going to reverse our historical narrative and begin with the end of the story: a brief account of knowledge about the immune system, particularly the structure and function of antibodies, as of the early 1970s. Then we will go back to the mid-19th century, and learn why multiple myeloma has proved to be the critical experiment of nature for unraveling the molecular structure of antibodies and, in turn, for beginning to understand how that structure specifies antibody functions. Separate paths of inquiry about the nature of myeloma proteins and about the nature of antibodies were pursued until the late 1950s, and, mirroring this historical disjunction, we will look separately at attempts to answer two questions: what is Bence Jones protein? , and what is an antibody? Finally, we will see how these two questions converged during the 1960s, when the study of myeloma proteins entered and profoundly affected the mainstream of immunological research.

The Immune System's Architecture

At any given moment, there are some trillion cells in the human body called lymphocytes, a type of white cell which exercises the recognition, specificity, and memory functions of the immune system. In the late phocytes are composed of two cell types: b ceiis ano i cells. The identification of B and T cells resolved earlier decades of often intensive debate about whether immunity is primarily a humoral or cellular phenomenon, for these cells represent a dual humoral and cellular system of immunity. In this dual system, the T lymphocytes are responsible for the cellular immune response, which functions against certain microorganisms and parasites, viruses, cancer cells, and organ transplants. 2 The B cells, in turn are responsible for the production of antibodies, and thus mediate the humoral response of the immune system. In the past few years, researchers have found that this duality is not a complete or simple one. There is an overlap in the responses of B and T cells to common antigens, and the production of antibodies involves not just the response of B cells to certain antigens, but a complex cooperative response involving

T and B cells and another type of white cell, the macrophage. Antibodies have been identified as large proteins, containing about 1300 amino acids in their chains, that are associated with the globulin class of proteins and thus also are termed immunoglobulins. The antibody or immunoglobulin molecules are arranged on the surface of B cells, with any given B cell having only one type of antibody molecule. We have noted that there are millions of possible antigens, and, correspondingly, there are millions of different antibodies, each structured to fit or bind to an antigen like the matching of a lock and key.

The huge variety of antibodies have been grouped into five classes, according to their molecular structure. It is IgG, the major mammalian antibody class, that we shall refer to most often as we trace the role of multiple myeloma research in elucidating the structure of antibodies.

The first major, fundamental information revealed by the molecular analysis of antibody structure, as we have noted briefly, is how the antibody I s two principal functions are determined by its structure. The antibody's recognition function - its ability to "identify" a specific antigen - is governed by the shape of the antigenbinding site in the molecule, and is unique to a given antibody. The antibody's effector function - its ability to execute a given type of defensive action against an antigen - is determined by another part of the antibody molecule, and is common to an antibody class.3

Is of antibody structure that emerged in and 1960s from the union of immunology r biology have shown us that an antibody obulin molecule is composed of four

in chains, linked together with special ical bonds. The arrangement of the four a antibody molecule as a whole the shape

117 y's four protein chains are composed of shorter pair called the light chains, and a called the heavy chains. The light chains chains, in turn, have two common structhat determine the antibody's recognition functions: one part of each chain is strucnt," while the rest of the chain is "variregion" designates that area of the heavy in chain in which the amino acid sequence within a given immunoglobulin class. That is, , the constant regions of heavy and light I IgG antibodies have identical amino acid hus, molecular studies have shown that it t region of the chains which determines an

'glass. And, as we have said, the antibody class by the effector or defense function that class. implies, the variable region is that relaarea of the protein chain in which the amino differs from antibody to antibody. It is region that provides the antibody moleition function, or, stated another way, it the antibody's antigen binding capability. mber of possible amino acid sequences both the heavy and light chains' variable each antibody its unique ability to "recogular type of antigen, forming a specif icaliy

or binding site that matches the antigen mmation, we know today that B lymphovn their surfaces a particular type of large cule, called antibody or immunoglobulin. le consists of two heavy and two light s arranged in a Y-shape. Each end of the 'antigen-binding site, in the variable regions and light chains. These two sites are the given antibody molecule, and they enable to "recognize" and bind with a particular ked to the two light chains in each antibody chains, and it is the long constant regions ins that primarily determine an antibody's its effector function.

The knowledge of the structure of an immunoglobulin molecule that we have summarized is a very recent achievement in the history of immunology. It is knowledge, as we have suggested, that has opened a new, sophisticated, and conceptually powerful era of molecular immunology. And, at the same time that it has opened a new era of research, the recent detailing of an antibody's molecular structure is the end of a trail of investigation that began more than a century ago, in 1845, when Dr. Thomas Watson asked Dr. Bence Jones, "What is it? "

'What is It?" Multiple Myeloma and Bence Jones'Proteins

The Puzzle Begins: The First Case of Multiple Myeloma. Dr. Watson's query to Dr. Bence Jones concerned a urine sample from a seriously ill patient, who died two months after certain peculiar characteristics of his urine had first been noticed by another physician involved in his care, Dr. William Macintyre. The sample, Dr. Watson wrote in his brief note, "contains urine of very high specific gravity. When boiled it becomes slightly opaque. On the addition of nitric acid, it effervesces, assumes a reddish hue, and becomes quite clear; but as it cools, assumes the consistency and appearance which you see. Heat relinquishes it. What is it? " (Bence Jones 1847, p. 52).

This question, about the urinary substance that later became known as Bence Jones protein, would occupy the attention of clinical and basic researchers for more than 100 years. The patient who prompted the inquiry had the first recorded case of multiple myeloma, which his physicians recognized as a malignant bone disease and named "mollities ossium."

Three astute physicians became involved in exploring the nature of this previously unrecognized disease, and, in a series of papers published between 1846 and 1850, they described their findings concerning the clinical features of the disease, the gross and microscopic postmortem findings, and the characteristics of the strange urinary protein. Dr. William Macintyre, a London physician, entered the case in October 1845, when he was asked by Dr. Watson to see a patient whom the latter had been treating for several months. From his careful history and physical examination, Maclntyre discovered several important clinical features of multiple myeloma, including severe bone pains and an unusual protein in the urine. Although history has linked Bence Jones' name with the characteristic proteins of multiple myeloma patients, it was Macintyre who first thought to examine the patient's urine. In so doing, he observed as the urine's temperature was raised, and then reprecipitate as the urine cooled.

Drs. Macintyre and Watson independently sent samples of their patient's urine to another physician for further study. They both chose Dr. Henry Bence Jones, a physician to St. George's Hospital who was noted as a "chemical pathologist." Bence Jones confirmed Macintyre's findings concerning the urinary protein, and went on to study it in greater detail. Macintyre's case report paper in 1850 dealt mainly with the clinical features of the disease, which is doubtless why he failed to receive historical credit for discovering the protein (MacIntyre 1850; Clamp 1967).

118 The third member of this physician trio became involved in the case after the patient's death. John Dalrymple, a surgeon to the Royal Ophthalmic Hospital and a member of the Microscopical Society, was asked to do a histological study of material from the patient's lumbar vertebrae and a rib. His paper, accompanied by two woodcut drawings of the cells he had examined , today would be read as a description of malignant plasma cells (Dalrymple 1846).

The immediate cause of death listed for this first recorded victim of multiple myeloma was -[kidney] atrophy from albuminuria." At the time, "albuminuria" was a term used non-specifically to designate proteinuria - the excretion of large amounts of prtein in the urine. Dr. Macintyre's and Bence Jones' recognition and analysis of their patient's abnormal urinary protein was, in the context of their day, a significant achievement, for the terms "protein" and "albumin" had just entered the lexicon of chemistry in 1838, originally with a meaning different from its modern usage. 5

Bence Jones discussed his study of the protein that would bear his name in 1847 and 1848 papers published in three of England's most prestigious medical and scientific journals: Lancet, the Proceedings of the Royal Society, and the Philosophical Transactions of the Royal Society.

The first paper, in Lancet, was the text of a lecture to the Royal College of Physicians - testimony to the young physician's eminence as a chemical pathologist. The theme of Bence Jones' lecture was the chemical effects of oxygen in the human body, a topic he addressed principally with reference to his own experiments on the urinary products of normal subjects and patients in effect, working as a clinical researcher in the 1840s, seeking to define metabolic processes in normal and diseased human subjects.

It was in this context that Bence Jones introduced the case he had worked on with Drs. Watson and MacIntyre, stating that "I have found another oxide of albumen in the urine, in a case of mollities ossium" (Bence Jones 1847, pp. 91-92). Bence Jones then described the clinical symptoms and read the note from Dr. Watson that accompanied the urine sample, before reporting on his physical and chemical analysis of the "enormous quantities" of the substance in the urine. Reflecting the newness of protein chemistry in 1845, Bence Jones argued that his analysis showed the substance to be an "oxide of albumen," not an "oxide of protein" (see note 5).

"it will immediately be asked," Bence Jones correctly realized, "what is the connection between Mollities Ossium and this state of the urine? " "To such a question," he acknowledged, "I am as yet unable to give a positive answer" (Bence Jones 1847, p. 92). He did, however, have an hypothesis to advance: chlorine, formed by the decomposition of sodium chloride in cells of the bones and kidney, "may have been the cause of the solution of the earthy matter of the bone"~ (i.e., of the pathological fractures of the patient's bones-i . another characteristic of multiple myeloma). This

hypothesis, in turn, generated the question of "why do,,,! the cells take on this peculiar action? " Bence Jones had, no answer, but could only note that "on this question,"' the whole of secretion and nutrition are involved" (Bence Jones 1847, p. 92).

To Dr. Watson's query, "Waht is it? ", Bence Jone-,,,,~ thus could give only a partial answer, albeit as good characterization of the urinary substance's propert!W, as the analytic techniques of his day permitted. He coullc not, he realized, do more than guess at the exact origb~ and nature of the substance, and at its relationship t;--' the disease, multiple myeloma. For he, together wl-' Drs. Watson, Macintyre, and Dalrymple, had uncoverzt, a medical puzzle that would intrigue scores of disee detectives for generations to come.

A Hundred Years Later.- "Much Remains to Learned." As one scans the literature on multi myeloma that developed from the late 1840s to va~ early 1950s, one initially striking impression is hc".

119 !on was devoted to this relatively rare physicians and their research colleagues in chemistry" or biochemistry. "Mollities named myeloma by Rustizky in 1873, be rally recognized disease entity when a Geran alerted physicians in 1889 to the fact in constellation of symptoms usually meant nt had multiple myeloma. Those symptoms, Maclntyre and his colleagues had first noted included deformity and abnormal fragility of ,bone pain, cachexia (a general wasting that chronic diseases), and the presence of Bence in in the urine (Kahler 1889). ians encountered and defined the characterof multiple myeloma, they discovered that the ,of large amounts of Bence Jones protein in the not the only protein abnormality associated isease. Two other changes in protein metabo refound: an increased level of certain abnorIns in the serum, and the deposition of protein body tissues. Of these three protein abnorence Jones proteinuria received particular -because, as physicians searched for its preser disease states, they found that it rarely except in muitiple myeloma. Thus, the presence Jones protein in a patient's urine, as asized, was an almost sure sign of multiple ns became interested in Bence Jones protein, the abnormal serum protein and amyloiclosis, ause they were characteristic indicators of yeloma. From the time it was first observed e etiology or cause of multiple myeloma was ,And, as had Dr. Bence Jones, physicians hoped of the characteristic changes in their patients' tabolism might reveal what caused the disease. the early 1950s, the study of Bence Jones s pursued predominantly as a problem in icine. Physicians and other clinically-oriarchers (mainly physiological chemists) were to solve questions about the abnormal origin and its chemical constitution primarily I to questions about multiple myeloma. reports and discussions of various hypotheses Jones protein appeared as separate articles, ponents of extensive clinical review articles iled hundreds of case reports of multiple

The state of knowledge about multiple myeloma and Bence Jones protein a century after the first case report is exemplified by a 1953 clinical monograph by Snapper, Turner, and Moscovitz, physicians at Mt. Sinai Hospital in New York. Having extensively reviewed the literature on multiple myeloma, and having personally dealt with ninety-seven cases over a seven-year period, the physicians wrote at the beginning of their book: "it is remarkable that today, after more than a century of study, much remains to be learned about the disease in general, and the source, chemistry, and constitution of Bence Jones protein in particular" (Snapper et al, 1953, p. 1).

Over the course of a century, Snapper and his colleagues went on to note, new ideas about the disease's etiology gradually had developed. Most authorities no longer regarded multiple myeloma as "merely" a tumor derived from bone marrow. But having decided what it was merely not, the question of what multiple myeloma was remained a matter of several unproven hypotheses, generated by clinical studies and, after 1935, by experiments with mice in which myeloma-like tumors could be produced. "For the time being," Snapper and his colleagues had to conclude in 1953, "the nature and pathogenesis of multiple myeloma remains completely unknown. In the absence of a proven etiologic agent, our knowledge can only be furthered by repeated and careful clinical observation and study" (Snapper et al. 1953, p. 4).

The status of knowledge about Bence Jones proteinuria in the early 1950s was much akin to that of the etiology of multiple myeloma: many questions, a variety of competing hypotheses, and little definitive knowledge. To the extent permitted by physicochernical analytic techniques as they developed over a century, investigators had pursued three major questions about this abnormal urinary product: what is the metabolic origin of Bence Jones protein, what is its chemical nature, and what is its relationship to normal serum proteins anrl i the abnormal serum proteins found in multiple myelor, patients?

Not surprisingly, ideas about the origin of Bence Jones protein tended to be linked with the development of myeloma itself. Thus, many favored a hypothesis put forward in the 1930s, that Bence Jones protein is formed by the malignant myeloma cells in the bone marrow (Magnus-Levy 1938). One extension of this idea, cited often in the literature after it was proposed by Dent and Rose in 1949, also sought to account for the

Jones protein was one of a small group of proteins, including the tobacco mosaic virus, that lacked the amino acid methionine in its composition. This finding, in light of the current knowledge about the structure and function of viruses, suggested that multiple myeloma "is due to invasion of the body by a virus which lives and multiplies in the plasma cells of the bone marrow . . . It is further suggested that the Bence Jones protein, when combined with nucleic acid in the plasma cells, is the virus itself" (Dent and Rose 1949, pp. 616617).

120 Another view was that Bence Jones protein was more indirectly the product of the bone marrow's malignant myeloma cells. In part reasoning from the fact that the protein had been found to have a relatively low molecular weight, Rundles and his colleagues cautiously proposed that "the serum components. . are produced in all likelihood by the abnormal plasma cells. . Bence Jones proteins could possibly be derived from the abnormal serum constituents, since the latter have a molecular weight about three to six times as great, if the latter were to disintegrate or be split into protein moities filterable through the glomeruli [of the kidneys] " (R undles, Cooper and Willett, p. 1125).

Yet another school of thought held that Bence Jones protein is not an abnormal product somehow triggered by myelorna. Rather, as Meyler suggested, Bence Jones protein is produced by normal bone marrow, but in quantities too small to be detected in a normal person's urine or blood serum. When myeloma develops, however, the protein's production increases so greatly that it is excreted in readily detectable amounts in the urine (Meyler 1936).

These and other ideas about the source of Bence Jones protein were, by and large, as speculative as Dr. Bence Jones' original idea. Lack of knowledge about the etiology of myeloma itself, the fact that a given investigator usually had only one or at best a few patients' samples to study, and technical difficulties in isolating and analyzing Bence Jones protein, all contributed to the high degree of uncertainty about where this urinary protein came from. These same factors also bore upon the decades of conflicting ideas and data about the protein's chemical nature. Up to this point in our narrative we have used the singular, Bence Jones protein, to reflect the historical fact that for many decades it was generally regarded and discussed as a

Derrieu, had suggested in 19UI triat line prutum imire, not be the same in all cases, but the early literatur1li generally cited a 1911 paper by Hopkins and Savoy,,, The latter, after analyzing the amino acid compositiolt' of Bence Jones protein obtained from two patients,' concluded that the chemical composition was identiv' (Hopkins and Savoy 1911).

The plural term, Bence Jones proteins, reflectir; knowledge that the substance was not chernicall identical in all patients, did not enter the literature unt! the 1920s, and one finds even today that the singult~ term continues to be used. Given the ultimate signifl., cance of Bence Jones proteins vis-a-vis our understandir' of antibody structure, it is interesting that attempts ti identify and characterize the abnormal proteins

myeloma patients relied heavily on immunologic mett ods. But, and this is an important historical "but, these immunological methods were used for decadsimply as useful analytic techniques, quite independec, of the work on antibodies by immunologists and W chemists that eventually would involve Bence Jor" proteins.

By the early 1900s, knowledge of antigenic reactioi' gained by the medical bacteriologists who pioneered# i early development of immunology had begun to indic-1, that proteins have individual antigenic specificity (,-,f Bulloch 1938, Lechevalier and Solotorovsky 11974 Thus, immunological reactions were recognized lr physiologists and chemists as a useful way of detect

differences in proteins that, by other criteria, appear.' alike. Two chronologically scattered reports on i;` study of Bence Jones proteins by immunologic methc,' appeared in 1911 and 1921; both showed, contrary

earlier studies, that the protein was not the samee I normal serum protein (Massini 1911; Hektoen 19Z.,

Then, in 1922, these reports were confirmed extended by Bayne-Jones and Wilson from Johns H kins, who used immunologic tests to study nom," serum proteins and twelve samples of Bence,Je4' proteins obtained from five patients. They found, f', J that "the Bence Jones proteins are immunologic different from the proteins of normal human senk' and these results, they noted, supported a newly errv, ing major concept in protein research - "that the sr' ficity of proteins is not dependent upon their biolc-r' origin, but due to their chemical composition" Br', Jones and Wilson 1922a, p. 43). Bayne-Jones

Wilson's work also challenged the "tendency to assume that all preparations of Bence-Jones protein are identical in structure and composition." For they had found that, immunologically, their preparations could be categorized into "two and possibly three groups." Thus, they concluded, Bence Jones "protein" actually is a group of similar but not identical protein substances (Bayne-Jones and Wilson 1922b).

121 Bayne-Jones' and Wilson's work, as well as earlier studies of the constitution and characteristics of BenceJones proteins, was subject to a major criticism. Most preparations of the protein were impure, containing other urinary or serum substances, and thus the results of a given experiment could not unequivocally be attributed to Bence Jones protein. By the 1940s, however, researchers were able to obtain crystallized proteinore readily and reliably. Repeating earlier ad the existence of two (immuno(og(i;a((y of Bence Jones proteins, and found, a given patient may excrete both types also began to use other s such as u I trace ntrif ugation and electrodition to immunological tests, that perprecise qualitative and quantitative characthe metabolic abnormalities in multiple a "broad and flexible analytic and his colleagues noted in 1943, oped to sort out "the multiplicity of oteins and ... their correspondingly varied Jand] the varied serum protein patterns of

Bence Jones proteinuria" (Moore, Kabat ,1943, p. 74). By the early 1950s, the use of of techniques had produced a mass of ation about the properties of Bence and the abnormal serum proteins of nts. But the findings of different researchways accord, and so the exact nature of ip between the various protein abnormaliwith myeloma remained "still a matter (Snapper et al. 1953, p. 58). Siochemical and Immunologic Studies hen Snapper and his colleagues observed "the exact nature of Bence Jones protein n determined," a series of studies had uld yield the f irst tentative answers to originally posed by Dr. Watson in 1845. ions of Bence Jones and other myeloma

proteins in the 1950s fall into two major groups in terms of methods. First, as represented by the work of Frank W. Putnam and his associates at the University of Chicago, new knowledge about the structure and origin of Bence Jones proteins began to emerge from quantitative and qualitative biochemical analyses of multiple myeloma proteins. Secondly, investigators continued to examine the immunological relationships among the various abnormal proteins found in myeloma patients, and in so doing also uncovered new information about the nature of Bence Jones proteins. Representative of this work in the 1950s was the research by Korngold and Lipari at New York's Sloan Kettering Institute for Cancer Research and Cornell Medical College's Department of Biochemistry, and that by Slater and his associates at the Hospital of the Rockefeller Institute for Medical Research.

M exalnining the work on myeloma oroteins in the r96YY, one 6~66' f

the researches. Through the early 1950s the abnormal proteins of multiple myeloma had been investigated primarily -although not exclusively - in relation to the light they might shed on the disease itself. Some investigators, particularly those like F. G. Hopkins who were based in university laboratories rather than hospitals, also studied Bence Jones protein because "a proper understanding of the disturbances involved could hardly fail to throw light on normal protein metabolism" (Hopkins and Savoy 1911, p. 190). But, in the main, the literature on abnormal myeloma proteins through the early 1950s indicates that these proteins were objects of clinical research, studied in reference to questions about multiple myeloma.

From the early 1950s on, however, one finds an increasing focus of interest in the myeloma proteins as 11 experiments of nature," as abnormal substanceswhose study might yield knowledge about the normal synthesis and structure of proteins in general, and about the globulin class of proteins in particular. In the remainder of this chapter, the trails of investigation that we will follow are those that led from Bence Jones proteins to the birth of molecular immunology, Once again, of course, this has not been the exclusive focus of research since the 1950s, for, as in earlier decades, the abnormal proteins of multiple myeloma have continued to be studied in reference to this and other diseases. And, as one looks at sources of support since the early 1950s for the increasingly basic lines of research that utilized Bence Jones proteins, one finds that it has come principal work. Thus, tor example, l1utnam-s worK in Tne izjz)us was supported in part by the National Cancer Institute (NCI) and a private University of Chicago Fund, Korngold's researches by the Atomic Energy Commission and the NCI, and Slater's work by the Rockefeller Institute for Medical Research. Finally, in noting a shift of emphasis in the study of Bence Jones protein in the early 1950s, it is worth re-emphasizing that the work hinged on the existence of patients who, whatever the objectives of the various researchers, served as research subjects because they were afflicted with a critical experiment of nature. 6

One of the major figures in the recent intertwined history of research on Bence Jones proteins and the structure of antibodies is biochemist Frank Putnam. The first of his many papers on multiple myeloma proteins appeared in 1953 when Putnam was at the University of Chicago. In his first foray into myeloma proteins, Putnam used sera from twenty-five patients to do quantitative physicochernical studies of the abnormal serum globulins, seeking, as had others before him, to determine their relationship to normal globulins (Putnam and Udin 1953). He had also launched studies of Bence Jones proteins, seeking to define

122 their chemical structure and their metabolic origin. These serum and urinary proteins, Putnam explained, were of great interest to him as a protein chemist because "the profuse synthesis and diverse nature of the proteins elaborated in multiple myeloma constitute the most profound alteration in protein metabolism in any disease. It may be hoped that the biochemical study of this striking phenomenon may have import in the analysis of the mechanism of protein synthesis" (Putnam and Stelos 1953,p.357).

In his first paper on Bence Jones proteins, Putnam dealt with the still-debated question of whether they were chemically identical substances. His comparative physical and chemical analysis of Bence Jones protein from eighteen myeloma patients, Putnam reported, "has shown that in no instance are the proteins identical in physicochernical properties" (Putnam and Stelos 1953, p. 356; see also Putnam and Miyake 1954). Physicochernical evidence for the conclusion that different patients excrete different Bence Jones proteins, Putnam noted, had been repeatedly supported by immunological studies showing that "there are at least their metabolic origin.

To tackle the question of the origin of Bence Jonw proteins, Putnam and Sarah Hardy conducted isotopkV- studies on two patients, to trace the rate of synthe&W-,, and possible chemical precursor relationships of bo"U11, myeloma globulins and Bence Jones proteins (Hardl and Putnam 1953; Putnam and Hardy 1955; Har*,' and Putnam 1955). Several important findings, beariro, on many debated questions about the nature of and' relationship between serum and urinary proteins, emerge&--' from these technically complex studies. Putnam and Hardy found, first, that the seruf-k globulin and Bence Jones proteins appeared to '~, synthesized independently in the patient's body. Thl, J and other data overturned a long-held hypothesis abou- the origin of Bence Jones proteins: that they wer~ breakdown or degradative products of normal tissc~ proteins or of serum proteins. Rather, Putnam arW Hardy found Bence Jones proteins to be synthesiz~_, "de novo- by the myeloma patient, and to be derive directly from the body's pool of metabolic nitrogq-"- rather than from any tissue or plasma protein precursor :t

Up to this point in their researches, Putnam and h' colleagues had established two important "nots" abolif Bence Jones proteins. They are not physicochemicall, identical, and they are not formed as breakdown prc' ducts of protein metabolism. What the proteins are terms of their structure and the site of their synthepir remained uncertain; but the boundaries of the PUZZ!,' had been greatly clarified by these "nots."

We will resume our account of Putnam's work in~ Bence Jones proteins' structure and origin in a lat_,, section, when we move to the studies in the 1960s th.elucidated antibody structure. Clues to this latter lir-' of research, that would link together the efforts determine the nature of Bence Jones proteins iw-the attempts of immunologists to decipher the structu' of antibodies, emerged in the 1950s, through fur*.'~ immunological studies of myeloma proteins. The woof two research groups, in particular, pointed to V` identification of Bence Jones proteins, and how t~' ; identification would lead to the molecular analysislt antibody structure.

At the Rockefeller Institute for Medical Researckgroup of investigators in 1950 were seeking to define immunological properties of normal human gamma globulin (protein antibodies), which had just been successfully purified. Their work, they found, was hampered because the various gamma globulin fractions were not single proteins. To overcome this problem, they seized upon "the special readily purified" serum proteins found in "tremendous quantity" in myeloma patients, hoping that study of these abnormal serum proteins would help to "clarify the relationship between the fractions of (normal) gamma globulin" (Kunkel, Slater, and Good 1951, p. 190).

The physicians who undertook this projectin 1950 included Dr. H. G. Kunkel, Dr. A. J. Slater, and a young fellow at the Institute, Dr. Robert A. Good. After demonstrating an immunological relationship between myeloma serum globulin and fractions of normal gamma globulin, Kunkel and Slater went on to study the immunological relationships between the myeloma serum globulins from a series of twenty-one patients. Three British investigators had shown in 1950 that myeloma serum globulin has two immunologically distinct components, gamm and beta (Wuhrmann, Wunderly, and Hassig 1950). As had this and previous studies, including Putnam's, the Rockefeller group's work indicated that every one of the myeloma proteins was different and individually specific to a given patient. But there also were commonalities, as the British study had found: the myeloma gamma globulin proteins were related immunologically to each other and to normal

123 gamma globulin; the myeloma beta globulins similarly were interrelated, but were mostly distinct from the gamma globulins.

These findings on the immunological relationships among normal and myeloma globulins, Slater and Kunkel realized, had important implications for understanding the origin of myeloma proteins.

Insufficient knowledge of the properties of normal serum proteins had made it difficult to determine conclusively whether multiple myeloma proteins are truly abnormal entities or simply represent a great elevation of a single one of the many normal globulin components. In general the data obtained in the present study favor the hypothesis that these proteins are not normal but related to normal constituents.

It is tempting to consider the myeloma proteins as intermediates in the synthesis of yglobulin and antibodies with the more closely related myeloma proteins having most of the determinant groups of ,y-globulin. However, definite evidence on this point is lacking. (Slater, Ward, and Kunkel 1955, p. 106)

During the same time period, a more specific focus on the immunologic relationships between Bence Jones protein and normal and myeloma serum proteins was being pursued at Sloan Kettering Institute by Korngold and Lipari.8 Citing both the Rockerfeller group's work and Putnam's researches, and thanking Putnam for his "valuable suggestions and criticisms," Korngold and Lipari reported on their own immunological studies in two papers in Cancer during 1956. In their first paper, on an antigenic analysis of myeloma serum globulins, Korngold and Lipari reported that their data were "compatible with the theory that MM (Multiple Myeloma) globulins are altered gamma-globulins (Korngold and Lipari 1956a, p. 191).

Then, in their second paper, Korngold and Lipari addressed the relationship between Bence Jones (BJ) proteins and serum proteins (globulins). Experiments from the 1920s on, they observed, "have created the impression that BJ proteins are antigenetically distinct and unrelated to eith MM globulin or normal gammaglobulin." But newer techniques had "made a properly controlled study of such antigenic relationships possible." In doing such a controlled antigenic study, Korngold and Lipari demonstrated, in contrast to previously accepted views, that Bence Jones proteins 11 are related to both normal gamma-globulins and the MM globulins" (Korngold and Lipari 1956b, p. 268). They also found, as had many prior invesitgators, that there are antigenic differences among Bence Jones proteins. And, going a step further, they showed that the urine of a given myeloma patient contains two major antigenic types of Bence Jones proteins (subsequently designated as K and L).

As had other investigators for many decades, Korngold and Lipari brought their findings to bear on the question of the origin of Bence Jones proteins. "The immunologic data presented here," they wrote in 1956, show that BJ protein is antigenically related to norma gamma-globulin and the patient's abnormal MM globulin. Since BJ protein contains determinants present in MM globulin but not in normal gamma-globulin, it must be assumed that it is more closely related to the former . . . In the light of the globulin. The incompletely synthesized proteins, which are smaller and more deficient in antigenic groupings than the serum proteins, are excreted into the urine as BJ proteins. (Korngold and Lipari 1956b, p. 217)

In this "speculation," as the next decade of research would show, lay both the answer to Dr. Watson's question about Bence Jones protein, and the means for immunologists to uncover the structure of the antibody molecule.

What is it?: Ideas About Antibody and Immunity

To appreciate the impact that the identification and structural analysis of Bence Jones proteins had upon immunology in the 1960s, we need to look briefly at another "What is it? " question, one asked quite independently of work on myeloma proteins, by those who sought to determine the nature of antibodies and how they interacted with antigens. This path of inquiry, long and complex, can be dealt with here only briefly and selectively, to suggest the nature of theories about antibodies through the 1950s and thus set the stage for seeing how the study of Bence Jones proteins entered into immunology.

The science of immunology began to develop in the last decades of the 19th century as one of the major yields of the new field of bacteriology. "The whole initial concept of immunity," as Burnet has pointed out, "was in relation to infectious disease in man or his domestic animals. . once [Pasteur] had shown the

124 potentiality of immunization with attenuated pathogens (1880) the central objectives of immunological research were defined for the next sixty or seventy years. Immunology was one of the practically important aspects of medical bacteriology and almost all those concerned with its advance were medically trained" (Burnet 1969, p. 5).

By the late 1880s, building on Pasteur's epochal achievements between 1880-1885 with attenuated vaccines against diseases such as fowl cholera, anthrax, and rabies, the new field of immunology began developing in two major, often intersecting directions (Pasteur 1880a,b, 1884, 1885; Pasteur, Chamberland, Roux 1885). The enormous practical implications of vaccines, first, generated continuing efforts to extend the range gists began attempting to explain the mechanisms or processes responsible for immunity. The identification of specific pathogenic bacteria focused attention on the question of why animals, including man, are normally resistant to most bacteria. At the same time, the development of vaccines framed questions about how immunity is produced and about the process of recovery from an infectious disease.

From the mid-1880s through the early years of the 20th century, much of the effort to explain the nature of immunity revolved around what were seen as two opposing explanatory models. The cellular theory held that immunity depends upon the ability of certain white cells in the body, phagocytes, to engulf infective materials and destroy them by a process of intracellular digestion (Metchnikoff 1888, 1901, 1908). The other major contender was the humoral theory, which proposed that certain substances or properties in the body fluids, principally the blood, were responsible for immunity (Nuttall 1888; Buchner 1890, 1900). The debate that flourished between proponents of cellular and humoral theories of immunity was one of the classic controversies that dot the history of science, generating fruitful research and ideas as well as polemical attacks and counterattacks. As often happens, this controversy abated when researchers recognized that their work pointed to the involvement of both humoral and cellular factors in immunity, a view that eventually would be confirmed in the 1960s with the discovery of the dual system of immunity residing in the T and B lymphocytes.

In the midst of the cellular vs. humoral theory debate, a major landmark in the history of immunology was established on December 4, 1890, when the discovery of antitoxin was announced by Emil von Behring and Shibasaburo Kitasato, researchers in Robert Koch's Berlin laboratory. Their discovery posed a major challenge both to the cellular theory and to relatively simple humoral explanations of immunity, opened up the field of serology, and, most importantly for our purposes, focused attention on what later would be termed the antigen -antibody relationship (von Behring and Kitasato 1890, transl. in Brock 1962; von Behring 1901).

Von Behring and Kitasato's experiments provided the first evidence that substances which are able to neutralize foreign materials are formed in blood serum globulin. The incompletely synthesized proteins, which are smaller and more deficient in antigenic groupings than the serum proteins, are excreted into the urine as BJ proteins. (Korngold and Lipari 1956b, p. 217)

In this "speculation," as the next decade of research would show, lay both the answer to Dr. Watson's question about Bence Jones protein, and the means for immunologists to uncover the structure of the antibody molecule.

What is it?: Ideas About Antibody and Immunity

To appreciate the impact that the identification and structural analysis of Bence Jones proteins had upon immunology in the 1960s, we need to look briefly at another "What is it? " question, one asked quite independently of work on myeloma proteins, by those who sought to determine the nature of antibodies and how they interacted with antigens. This path of inquiry, long and complex, can be dealt with here only briefly and selectively, to suggest the nature of theories about antibodies through the 1950s and thus set the stage for seeing how the study of Bence Jones proteins entered into immunology.

The science of immunology began to develop in the last decades of the 19th century as one of the major yields of the new field of bacteriology. "The whole initial concept of immunity," as Burnet has pointed out, "was in relation to infectious disease in man or his domestic animals. . once [Pasteur] had shown the potentiality of immunization with attenuated pathogens (1880) the central objectives of immunological research were defined for the next sixty or seventy years. Immunology was one of the practically important

125 aspects of medical bacteriology and almost all those concerned with its advance were medically trained" (Burnet 1969, p. 5).

By the late 1880s, building on Pasteur's epochal achievements between 1880-1885 with attenuated vaccines against diseases such as fowl cholera, anthrax, and rabies, the new field of immunology began developing in two major, often intersecting directions (Pasteur 1880a,b, 1884, 1885; Pasteur, Chamberland, Roux 1885). The enormous practical implications of vaccines, first, generated continuing efforts to extend the range gists began attempting to explain the mechanisms or processes responsible for immunity. The identification of specific pathogenic bacteria focused attention on the question of why animals, including man, are normally resistant to most bacteria. At the same time, the development of vaccines framed questions about how immunity is produced and about the process of recovery f rom an i nf ectious d isease.

From the mid-1880s through the early years of the 20th century, much of the effort to explain the nature of immunity revolved around what were seen as two opposing explanatory models. The cellular theory held that immunity depends upon the ability of certain white cells in the body, phagocytes, to engulf infective materials and destroy them by a process of intracellular digestion (Metchnikoff 1888, 1901, 1908). The other major contender was the humoral theory, which proposed that certain substances or properties in the body fluids, principally the blood, were responsible for immunity (Nuttall 1888; Buchner 1890, 1900). The debate that flourished between proponents of cellular and humoral theories of immunity was one of the classic controversies that dot the history of science, generating fruitful research and ideas as well as polemical attacks and counterattacks. As often happens, this controversy abated when researchers recognized that their work pointed to the involvement of both humoral and cellular factors in immunity, a view that eventually would be confirmed in the 1960s with the discovery of the dual system of immunity residing in the T and B lymphocytes.

In the midst of the cellular vs. humoral theory debate, a major landmark in the history of immunology was established on December 4, 1890, when the discovery of antitoxin was announced by Emil von Behring and Shibasaburo Kitasato, researchers in Robert Koch's Berlin laboratory. Their discovery posed a major challenge both to the cellular theory and to relatively simple humoral explanations of immunity, opened up the field of serology, and, most importantly for our purposes, focused attention on what later would be termed the antigen -antibody relationship (von Behring and Kitasato 1890, transL in Brock 1962; von Behring 1901).

Von Behring and Kitasato's experiments provided the first evidence that substances which are able to neutralize foreign materials are formed in blood serum infection. And, as they showed in the case xin, these antitoxic substances are highly wake of this discovery, researchers soon hat animals can produce antitoxins range of poisonous substances. The riments and theories of Paul Ehrlich early important, for his work both broad of immunology beyond its early focus diseases, and began to direct the attention ists to what they later would call anti 1891, 1897, 1900, 1908; Marquardt of the century, a series of crucial ques framed about the nature of d how they interact, questions that only be answered in fine detail since the 1960s. quickly revealed one of the properties of antibodies, their specificity, the question of the source or mecha ocificity. Initial ideas centered on the rce of specificity, but by 1900 a variety had shown that antibodies were some in modified toxins, some sort of special acted in specific response theories of immunity to account for the origin and specificity as the "side chain" theory developed by 1897. Ehrlich's theory, a blend of late lological and chemical fact and fancy, certain cells in the body have special hains or groups of atoms that perfectly atoms in a toxin - like a match between . Once a side chain or antitoxin has into a toxin, and its parent cell thus Ehrlich supposed that the body began to a of the side chains, thus accounting uction (Ehrlich 1900). ry generated vast amounts of research by both its supporters and critics. A to his theory, and another significant munology's scope of inquiry, was set 1898 when Pasteur's proteg6, Jules ated the immune lysis (destruction) of I(Bordet 1898, 1899, 1903). Bordet's i,an increasing interest in the immunolo both cells and body fluids. Then, Karl d that any molecule, either natural Id stimulate an immune response under certain conditions, a finding that argued against the validity of Ehrlich's theory (Landsteiner 1930, 1946). For it was hard to conceive, as the side-chain theory demanded, that the body had an infinite number of preformed side-chains or receptors on its cells, capable of fitting specifically to a structurally infinite variety of antigens.

Rather, Landsteiner's work with artificial antigens seemed to indicate that antigens govern the specificity of antibodies by somehow directing or "instructing" the cell's activities. Instructive theories of antibody formation gained in sophistication and status in the 1940s with the entry of Linus Pauling into immunology, for his work

126 began an "effective association of immunology with the developing principles of biochemistry (Burnet 1969, p. 5). Stimulated by conversations with Karl Landsteiner, Pauling had begun to work on the molecular bases of serological reactions in the mid-1930s. From this work, in turn, he developed, in as precise form as the time permitted, what is now recognized as the classical instructive theory of antibody formation (Pauling 1940).

Between 1940 and the mid-1950s, modifications of Pauling's instructive theory were proposed by several researchers, consonant with new methods. and knowledge in protein chemistry and immunology (see Burnet 1963). At the same time, it also became evident that a number of important immunological phenomena could not be explained by instructive theories. In particular, these theories could not account for the persistence of modified immunological reactivity, or for the fact that antibodies usually are not produced against chemical configurations normally present in the body (the phenomenon of immunological "recognition") (Burnet and Fenner 1949). Such considerations led to the development of various "selective" theories of antibody formation, a development linked most closely with the work of Australian virologist and immunologist F. MacFarlane Burnet.

Beginning with his 1941 book, The Production of Antibodies, Burnet had carefully studied and critiqued instructive theories, and sought to interpret antibody formation primarily in biological rather than in chemical terms.9 But, as Burnet himself recognized, the first major alternative to the generally accepted instructive theory came in 1955, when Danish immunologist Niels Jerne proposed a "natural selection theory" (Jerne 1955, 1967). Jerne's theory served to explain why the body does not make antibodies against its own constit deficiencies in Jerne's theory which were met in I vo/ uy new forms of a selection theory, developed independently by David Talmage at the University of Colorado and by Burnet (Talmage 1957, Burnet 195)).

Both Talmage and Burnet recognized the variety of globulins that can be present in the blood, and felt that a satisfactory theory of antibody production should assume that the antibody -repI icati ng elements were cells (like various globulin cells or their precursors), rather than, as in Jerne's view, extracelfular protein able to replicate only if incorporated into a particular cell type like the phagocyte. Burnet developed this conviction in terms of a "clonal" selection theory (the word "clone" designates a group of cells that originate from the same parent cell). "In its simplest form," Burnet has explained:

Looming iarge among Ene clonal selection as the central dogma of modern immunology was the molecular analysis of antibody structure. And, as we resume our chronicle of Dr. Bence Jones' strange protein, we will learn how it served in the 1960s as the crucial experiment of nature for that analysis.

Bence Jones Pro teins, L Igh t Chains, the "clonal selection" theory postulates that, I n the course of embryonic differentiation, a very large number of clones of mesenchymal cells arise, each carrying a specific immunological potentiality to react with a single antigenic determinant. Depending on various circumstances, the reaction following contact with antigen may be manifested in one of three different forms: (a) the cell may be damaged so that its capacity to multiply is lost, or it is actually destroyed; (b) it may be stimulated to proliferate; or (c) it may undergo conversion to plasma cell character and produce and liberate antibody. Antibody production follows the normal rules of protein synthesis, the information needed for its specificity being stored in the cell genome. (Burnet 1963, pp. 91-92)

Through the late 1950s and into the 1960s, Jerne, Talmage, Burnet and other proponents of a selective theory of antibody formation and action argued lengthily and persuasively for the logical correctness of their views. But, as they knew, instructive theories had long held sway, and at the time there was no direct experimental evidence to confirm one or the other school of thought. By the early 1970s, however, new lines of evidence pointed to the essential validity of selective theories and, in the words of Gerald Edelman, "the fundamental idea of these theories is now the central dogma of modern immunology: molecular recognition of antigens occurs by selection among clones of cells already committed to producing the appropriate anti

By the late 1950s, the study of myeloma proteins had generated a number of provocative clues to the identity of Bence Jones Droteins. These myeloma proteins now were known to be antigenically related to both normal and myeloma immunoglobulins, and hence it seemed reasonable to speculate that BJ proteins might represent intermediary or incompletely synthesized immunoglobulins.

During this same period, in the late 1950s, immunologists and molecular biologists began an intensive attack on the structure of antibodies. As we saw in the preced. ing section, there was a substantial body of information and theories about the functions and origins of these proteins, but little was known about their detailed chemical structure. Determining the structure of antibodies, researchers knew, should provide

127 understanding of their unique specificity for antigens, and perhaps a basis for choosing between instructive and selective theories of antibody production. But researchers had been confounded by the experimental problems created by two aspects of antibodies: they are very large proteins (with a molecular weight of 150,000 or greater), and they are very heterogeneous chemically (a problem, too, for those who had been working on Bence Jones proteins).

The ultimately successful quest to define the mole-,; cular structure of an antibody was launched during,.' 1958, in researches centered at the Rockefeller Institute,' in New York, and in the Department of Immunolo9v" at St. Mary's Hospital Medical School in London. Thi leaders of the two research teams were physician-irM munologist Gerald Edelman and biochemist-immunol. gist Rodney Porter, who in 1972 would be honored wiLl the Nobel Prize in Physiology or Medicine for the.. structural studies of immunoglobulines.

Rodney Porter, who had trained as a biocheml' under Frederick Sanger, developed a method of breakif' an immunoglobulin molecule into analyzable fragmer, rotein-dissolving enzymes. One of his major rn this work, which occupied years of effort, immunoglobulin G (IgG) molecule conglobular fragments: an -Fc- fragment all molecules, and two identical "Fab" each of which carried a specific antigen-binder 1959, 1973).

York, Edelman developed another approach IgG molecules that involved breaking their tonds and then exposing the molecule to solvents. By these methods, Edelman found

IgG molecule was not, as had been thought, of a single polypeptide chain, but rather was of several discrete chains linked by disulficle s first phase of his research also indicated gG molecule had two kinds of polypeptide r named heavy and light chains, and that s were not the same as the fragments Porter with his enzyme cleavage method (EdleEdelman and Poulik 1961). Understanding -structure of antibodies, then, would involve the relationship between the polypepticle ad by Edelman's methods and the fragments major obstacle to a structural analysis, Itill confronted researchers in 1960: the chemical diversity of antibodies. As Edelman his Nobel address, this chemical diversity challenging questions." did the observed heterogeneity of antibodies only in the conformation of their polypepchains, as was then widely assumed, or did heterogeneity reflect differences in the pristructures of these chains, as required citly by the clonal selection theory? Second, heterogeneity did imply a large population olecules with different primary structures, could one obtain the homogeneous material for carrying out a detailed analysis? nature rather than direct physicoult" proved to be the means by which his colleagues were able to deal simultaneboth of these challenges. Drawing upon the by Putnam's group and by Slater and Kunkel f-afler. Edelman realized that Bence Jones pro el relatively homogeneous and low molecular weight substance, known to be antigenically related to immunoglobins.

By 1961, Edelman had begun to formulate "a unifying hypothesis . . . for the structure of proteins in the [immunoglobulin] family." IgG molecules, he and Poulik stated, "appear to consist of several polypepticle chains linked by disulfide bonds," and bivalent antibodies "may contain two chains that are similar or identical in structure." Thus, the heterogeneity and antigenic specificity of antibodies "may arise from various combinations of different chains as well as from differences in the sequence of amino acids within each type of chain" (Edelman and Poulik 1961, p. 880).

The discovery that immunoglobulin molecules consist of several polypepticle units, Edelman recognized, also had "a possible bearing upon the pathenogenesis of diseases of gammaglobulin production." "A primary defect in. . multiple myeloma," he suggested, "may be a failure of specificity and control in production and linkage of various subunits to form larger molecules." Thus, he and Poulik were able to propose a structural basis for previous speculations that Bence Jones proteins were incompletely synthesized myeloma globulins. "Bence Jones proteins may be polypeptide chains that have not been incorporated into the myeloma globulins because of a failure in the linkage process" (Edelman and Poulik 1961, p. 881; see also Poufik and Edelman 1961).

Edelman's hypothesis about the nature of Bence Jones proteins was confirmed "one exciting afternoon" when he and a graduate student, Joseph Gaily, "heated solutions of light chains isolated from our own serum immunoglobulins in the classical test for Bence Jones proteinuria": the test used by Dr. Watson in 1845, that had generated a century of efforts to identify Bence Jones proteins. Dr. Watson's question, "What is it? ", at last was answered. Edelman and Gaily, aware of the history underlying their own work, noted that "Dr. Jones concluded that it was the 'hydrated cleutoxide of albumin.' [Our] studies would supply the question with another qualified answer: Bence Jones proteins appear to be polypepticle chains of the L [light chain] type that have not been incorporated into myeloma proteins" (Edelman and Gaily 1962, p. 225). What the

128 Rockefeller investigators had observed was that their serum immunoglobulin light chains "behaved as Bence Jones proteins, the hypothesis (Edelman 1973, p. 831; Edelman and Gally 1962, 1968; Mannik and Kunkel 1962).

The Rockefeller group's experiments, by 1961, had three major implications for a molecular structureactivity analysis of antibodies. First, "because different Bence Jones proteins had different amino acid compositions [as determined by Putnam and others in the 1950s] , it was clear that immunoglobulins must vary in their primary structures." Second, this deduction in turn "lent strong support to selective theories of antibody formation." Third, because Bence Jones proteins now were known to be identical or analogous to light chains, with a relatively low molecular weight, it had become possible to begin "a direct analysis of the primary structure of an immunoglobulin moelcule" (Edelman 1973, p. 832).'('

The first laborious analyses of the amino acid sequences in Bence Jones proteins were undertaken by Hi1schmann and Craig at the Rockefeller and by Frank Putnam, then heading a laboratory group at the University of Florida College of Medicine. By spring 1965, partial sequence analyses of several different Bence Jones proteins had revealed another key feature of antibody structure: the structural diversity between light chains was limited to what became known as the variable region of the chain (Hilschmann and Craig 1965; Titani and Putnam 1965; Titani, Whitley et al. 1965; Putnam and Easley 1965). ' '

Through the mid-1960s structural analysis was focused on the antibody molecule's light chain, because of the natural experiment afforded by Bence Jones proteins. Less work was being done on the other type of polypeptide chain identified by Edelmann's experiments, the heavy or H chain. But by 1964, even in the absence of much detailed knowledge, comparisons between heavy and light chain structure had shed light on another reason for the diversity of antibodies: the existence of antibody classes. Comparisons of chain structure in the first three classes to be identified showed that classes have similar kinds of light chains, and that it is structural differences in their heavy chains which give them their distinctive class characteristics or effector functions (Bull. WHO 1964).

From the mid-1960s on, the analysis of both the heavy and light chains of antibodies moved forward at an accelerating pace, generating an increasingly more an antibody's functions. In lubz, Kooney rut wi wim mcolleagues in London had begun to link their analysis of the molecule's Fc and Fab fragments with the Edelman group's study of polypeptide chains. From this work Porter hazarded what proved to be a brilliant guess: the IgG molecule is composed of two large (heavy) polypeptide chains and two smaller (light) chains, making in total a four polypeptide chain structure (Fleischman, Pain, Porter 1962; Fleischman, Porter, and Press 1963).

Aided by this new understanding of the relationship between chain structure and Porter's enzyme fragments, plus the growing knowledge about the structure of Bence Jones proteins, Edelman and Gally in 1964 developed a topological model of the IgG molecule (Edelman and Gally 1964). Then, in 1965, Edelman judged that it was feasible to begin working out the complete molecular structure of an immunoglobulin molecule, and set to work on a sample of human myeloma IgG.

While Edelman's group was laboring in New York, Frank Putnam and his associates, now at the University of Indiana, were pursuing their painstaking mapping of the amino acid sequences in type K and L Bence Jones proteins (the two major antigenic types identified by Korngold and Lipari in 1955). In 1966, Putnam's group published a "tentatively" complete amino acid sequence for type K, and in 1967 reported a complete sequence analysis for type L Bence Jones protein (Put nam, Titani, Whitley 1966; Wikler, Titani et al. 1967; Putnam 1969). Thus, for the first time, researchers had accomplished the task of determining the structure of a Bence Jones protein, and hence of a light chain in the antibody molecule.12

Solving the complete primary structure of an antibody molecule, the task undertaken with myeloma igG by Edelman , s group in 1965, received a strong stimulus from Putnam I s determination of the L-type light chain structure. Another major input came from Porter's group in London, when they reported on the heavy chain structure of IgG from a myeloma patient and from normal rabbit serum (Porter 1967). Porter and his

129 colleagues focused their efforts on that half of the heavy chain where they believed the antibody's antigen com-, bining site was located. The partial analysis he had com-.' pleted by 1967 encouraged Porter to "claim that, if the~ k . . . on the sequence of the heavy chains . . . can be ried to completion, it appears to offer a feasible perimental approach to obtaining an answer to the stion: does the amino acid sequence alone control ibody specificity, and, if so, how is it achieved? orter 1967, p. 425).

The achievements of groups such as Putnam's and rter's represented important pieces of the puzzle of ibody structure, but its total solution required more. as researchers now suspected, each immunoglobulin olecule consisted of two identical light chains and two

entical heavy chains, a complete map of its primary ructure would be obtained by a sequence analysis of ne light and one heavy chain, and by locating all the !sulfide bonds and the various carbohydrate prosthetic roups attached to the chains.

This was the task that Edelman and five colleagues at Rockefeller Institute had undertaken in 1965, a task that would occupy "a good portion of [their] waking hours" for four years (Edelman 1973, p. 883). Then, at a scientific meeting in 1969 Edelman announced the completion of their work. "We now report the amino acid sequence fo an entire human (IgG1 immunoglobulin (molecular weight 150,000), the location of all disulficle bonds, the arrangement of light and heavy chains, and the length of the heavy chain [variable] region" (Edelman, Cunningham et al. 1969, p. 78).

Within the scope of this essay we have come full circle, for now, in 1969, immunologists had gained an understanding of the primary structure of an antibody molecule, and of how the variable and constant regions in its heavy and light chains make possible an antibody's extraordinary specificity and its class or effector functions. With this understanding the science of immunology, that had begun in the 19th century with the work of medical bacteriologists such as Louis Pasteur and Robert Koch, underwent a major revolution. And, as we have seen, a "crucial experiment of nature," the long puzzling proteins of multiple myeloma patients, occupied center stage in the revolution. The quest to identify the nature and origin of these proteins, begun by Dr. Watson's query in 1845, had ended more than a century later in "the first of the projects of molecular immunology, the task of which is to interpret the properties of the immune system in terms of molecular structures" (Edelman 1973, P. 830).

Those involved in the many facets of immunobiology recognize that the molecular analysis of antibody structure is only a beginning, albeit a profoundly important one, in understanding the nature of the immune system. The structural basis of antibody diversity has been revealed, but, as Gerald Edelman observed in his 1972 Nobel Prize address, "two great problems of molecular and cellular immunology remain to be solved." The first of these problems is "the origin of intrasubgroup diversity:" what are the genetic mechanisms that lie behind the enormous variability of amino acid sequences within portions of the heavy and light chains? (see note 11). The second problem, recognized since Burnet proposed his clonal selection theory, is to explain the induction of antibody synthesis: how is the clonal expansion of lymphocytes triggered after their receptor antibodies combine with antigens? Eventually, immunologists are confident, these major questions too will be answered, and immunology again will be transformed "both as a discipline and as an increasingly important branch of medicine" (Edelman 1973, p. 839).

1 . Immunodeficiency diseases reter to Tnose conumum, frequently inherited, that are caused by various types of defects in immunological function. Because a majority of patients with inherited forms of immunodeficiency have been found to have quantitative deficiencies of T cells, B calls, and their products, the study of these diseases has had a major import for the development of modern immunological concepts (see Bach and ood 1972).

2. Like the B cell, the T cell has receptors on its surface that can specifically recognize antigens. But, whereas the B cell's response to an antigen is to produce antibody, the T cell releases substances called lymphokines. The lymphokines, in turn, attract monocytes (a type of white call) which engulf and digest invaders such as bacteria. The T cells also directly attack foreign proteins.

3. The principal defensive or effector functions of each of the five antibody classes are, briefly, as follows. IgM, the first antibody to be produced in response to the presence of an antigen, is evolutionarily the most primitive of the five types, which perhaps accounts for its relatively weak binding ability. IgM is involved in

130 certain autoimmune diseases, such as rheumatoid arthritis. The major antibody type in mammals is IgG. Both IgG and IgM act against foreign organisms and toxins, but IgG has a more specific antigen binding ability and remains in the blood much longer than IgM. IgG also is the only antibody type able to cross the placenta, to confer passive immunity from mother to fetus. IgA is present in large quantities in the intestine, where it acts as a barrier to prevent the escape of pathogenic organisms from the gut into the blood stream. The known functions of IgE are primarily negative ones, from man's perspective, as this antibody is responsible for triggering allergic and asthmatic responses. The functions of the fifth type of antibody, IgD, presently are unknown (Lewin 1974, pp. 28-31).

4. An antibody's structurally specific antigen binding site results from how the amino acids are arranged in the variable regions of its heavy and light chains, and the vast number of i3ossible binding site shapes are generated by permutations in the possible sequences of the 108 amino acids that make up the variable regions of both the heavy and light chains. Investigators also have found that some areas of the variable regions are more variable in amino acid sequences than others; these are termed hypervariable regions.

5. The views of organic chemists in the 11840s on the nature of albumin and protein were developed, most influentially, by the work of Berzelius, Mulder, and Liebig on the elementary analysis of alburninoid substances. Berzelius proposed the word "protein" in 1838 to designate the "organic oxide of fibrin and albumin," and he felt that this "protein" was the "primitive or principal stubstance of animal nutrition that plants prepare for the herbivores, and which the latter then furnish to the carnivores." Fruton notes that Berzelius' and Mulders' conclusions about the identity of fundamental protein units were adopted by the influential Liebig in 1841 "on the basis of analyses performed by his associates Johann Joseph Scherer and Henry Banco Jones" (Fruton 1972, pp. 96-97). clinical research, we note that multiple myeloma patients participated in two different types of experimental work. First, some patients had blood and urine samples collected for laboratory analysis, or in vitro research. Secondly, some patients were given substances, such as radioactively labelled glycine, and than had blood and urine samples collected for in vitro study.

7. Good was at the Rockefeller Institute on a one-year fellowship, having become interested in immunology through his work as a pediatrician at the University of Minnesota. When Good returned to Minnesota in 1950, he brought with him a number of intriguing and puzzling clinical observations from his year in New York. Among these was his learning from myeloma patients that they had serious problems with infection. Why was this the case, Good wondered, since these patients had such high levels of plasma cells or antibodies in their blood, whose function is to combat infection. It was the pursuit of such clinical puzzles that, among other accomplishments, would lead Good and his colleagues at Minnesota in the 1950s and 1960s to decipher the dual humoral and cellular system of immunity (sea Good 1972; Lewin 1974, ch. 1).

8. Other research groups besides those discussed in the chapter also were conducting important research with Bence Jones proteins, myeloma proteins and normal globulins in the 1950s, that would feed into the molecular analysis of antibody structure. Among these groups were H. F. Deutsch and his colleagues in the Department of Physiological Chemistry at the University of Wisconsin, who worked on physicochernical and immunochemical properties of myeloma and Bence Jones proteins and of the macroglobulins found in patients with Waldenstrom's macroglobulinemia (see Deutsch 1955; Deutsch et al. 1955, 1956; Morton and Deutsch 1958).

9. As part of their indirect template theory, Burnet and Fenner in 1949 predicted the existence of immunological tolerance, a phenomena seen but not understood in 1946 when Owen observed that most twin cattle are born with and retain a mixture of each other's red blood cells. As part of their theory that the body's recognition of self develops slowly during fetal life, rather than being inherited, Burnet and Fenner predicted that an antigen experimentally administered to an embryo would not generate antibodies, but would be accepted as a normal part of the embryo's body. This state of "acquired tolerance" was demonstrated in 1953, in experiments on tissue grafting by Medawar and his colleagues. Medawar's immunological research, in turn, had developed out of his work for England's Medical Research Council in the early 1940s on the problem of skin grafts for W. E. 11 burn victims. In 1960, Burnet and Medawar received the Nobel Prize for their work on acquired immunity (Burnet and Fenner 1949; Medawar 1944, 1945, 1957).

10. Edelman and Gaily recognized that their use of myeloma globulins to analyze immunoglobulin structure confronted them 11 with a peculiar dilemma in structure-activity determination. Either we commit a

131 fundamental heresy in analyzing a myeloma globulin the activity of which is unknown, or a structural heresy in studying the amino acid sequences of an indeterminately

ge mixture Of specific antibodies." Despite this dilemma, they It that the evidence for myeloma globulins being typical munoglobins permitted them to proceed with a "confident alysis of the over-all structure of immunoglobins" (Edelman id Gally 1968, pp. 329-330).

1 The discovery that the variable region of the antibody ecu le is responsible for the enormous range of structurally

antigen binding sites, as Porter observed in his Nobel 1. raised very difficult problems as to the genetic origin many different amino acid sequences" (Porter 1973, 716). A now widely accepted explanation for the sequence

riability seemed at first a heresy, for it contradicted the one gone-one polypepticle dogma established, as we saw in chapter 5, largely through the analysis of abnormal hemoglobins. What geneticists and immunologists accept as the most likely explanation for the structure of antibody chains is a "two genes-one polypepticle chain" control mechanism, in which one gene codes for the constant region and one for the variable region. Two major explanations for a two-gene mechanism have been advanced, the germ-line and the somatic mutation theories. According to the latter theory, the multiple genes that code for the variable regions are the product of somatic mutation of a relatively small number of germ-line genes. (see Porter 1973; Putnam 1969, 1972).

12. Following his.work on the structure of light chains, Putnam and his colleagues went on to "tackle the largest immua noglobulin of all, the IgM macroblobulin produced in large amounts by patients with Waldenstrom's macroglobulinemia" (Putnam 1972, p. 370). The task was a formidable one, for the IgM is many times larger than IgG, with a molecular weight of about 1,000,000, and has several other properties that also complicated the task of analyzing its primary structure (see Porter 1972, pp. 371-378).

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CHAPTER 7 DISEASE AND DISCOVERY

The five case studies presented in this volume, as we pointed out in chapter one, revolve around a common theme that has been largely neglected in analyses of past and present biomedical research: many paths of inquiry and discovery about life processes have begun through efforts to understand the nature of and to intervene in states of disease. Although they focus, in current parlance, on the flow from categorical or disease-oriented research to basic research and consequent advancement of fundamental knowledge, rather than the more frequently charted flow from basic research to clinical application, these case studies belong to a larger body of historical and sociological studies, and less directly, policy studies, that have sought to articulate the processes involved in various areas of research.

In the realm of policy-related studies, a number of reports over the past decade have analyzed selected examples of research-to-application developments in biomedicine and other fields. Either explicitly or implicitly, these reports have been concerned with bolstering a given position on the "proper" allocation of federal funds for "applied" or "basic" research, in response to the debates engendered by President Johnson's call in 1966 to "zero in on the targets by trying to get our knowledge fully applied" (see chapter one).

The first of these studies, commissioned by the Department of Defense, was Project Hindsight, in which a group of engineers and scientists retrospectively analyzed the development of 20 major military weapons (Sherwin and Isenson 1966).

138 Although it was issued only as a "first interim report," and despite methodological criticism of the short historical time-span of the analyses and the validity of transferring findings from the development of military weapons to biomedical research, Project Hindsight's arguments for "targeted" research buttressed the new policy climate in Washington vis a vis how federal support for research ought to be apportioned.

Given the strongly and widely held counter-ethos among those engaged in biomedical research, affirming the importance of "basic, undirected" research for medical progress, it is scarcely surprising that Project Hindsight triggered a series of rebuttals, in the form of case studies documenting the role of basic research in various medical innovations and other areas of technological advances. These included Shannon's account of the development of the polio vaccine and Visscher's study of the rubella vaccine, and the studies of various technological innovations commissioned by the National Science Foundation (Shannon 1967; Visscher 1967; Illinois institute of Technology 1968/69; Batelle/Columbus Laboratories 1973). More recently, critiquing studies such as the above for being either "anecdotal" single case reports or for being biased because the examples were selected by those who did the analyses, Comroe and Dripps used panels of consultants to help them select and analyze the development of ten major advances in cardiovascular and pulmonary medicine and surgery since the early 1940s (Comroe and Dripps 1974, 1976; see also Comroe 1977).

All of these post-Hindsight studies, particularly the work by Comroe and Dripps by virtue of its methodology, argue persuasively for the importance of basic research in the pursuit of greater knowledge about disease states and diagnostic, preventive, and treatment capabilities.

As historians of science and medicine, familiar with a range of developments in many fields over many centuries, we could not but argue that basic research plays an essential role in shaping medical progress. Nor, as we stated in chapter one, has this study sought to argue otherwise. But, accepting the fact that research proceeds in complex ways and by many routes, it would be equally absurd to argue, as did an eminent biomedical researcher in 1977, that "it has yet to be shown that basic science progress can be a byproduct of applied research" (Visscher 1977). Such completely unidirectional statements, while rare, reflect in part both stereotypic views about the relationship between basic and applied research, and the dearth of well-documented studies of how "important elements of the fundamental biomedical sciences of the past have emerged from the study of human beings and their diseases" (Report of the President's Biomedical Retearch Panel, App. A 1976, p. 16).

The present study has been directed primarily toward beginning to fill in this gap in the literature on biomedical research. As such, whatever policy implications may be read into it should be of a different order from the Project Hindsight genre. For, we make no arguments about, and draw no conclusions concerning, the relative importance of basic or categorical research and in turn the relative balance between the two broad categories that should obtain in federal research funding. Indeed, for reasons that we also explored in chapter one, we have by and large avoided labelling the lines of research that we examine in the case studies with terms such as applied, categorical, mission-oriented, fundamental, basic, etc.

From our perspective, if this study has any latent policy "message" it is to underscore the fact that scientif ic knowledge and discoveries come from many sources, by often unforeseen routes, and that we still know far too little about the course and determinants of those research processes that may variously result in therapeutic innovation or in fundamental knowledge. Here, we concur with the views of a Rand Corporation report for the President's Biomedical Research Panel concerning the need for "improved conceptual frameworks of scientif ic progress." The report, characteristically, focuses on the flow from basic to applied biomedicine and notes only parenthetically that "questions arising in an area of clinical medical science may become important research questions for several areas of basic medical science." But, more generally, the report observes that "as a practical matter, no analytical models adequately capture the processes by which biomedical R & D results flow into medical practice [or, we would add, the converse processes] . The search for a single comprehensive model that simultaneously has great generality and great capacity to reflect the detail of these processes is futile. Rather, the search should be for a number of models of intermediate levels of generality, able to handle a wide variety of cases with adequate respect for their detail and complexity" (Report of the President's Biomedical Research Panel, App. 8 1976, pp. 77, 78).

Within the scope of this study, however, we have not attempted to construct an analytical model for the flow from categorical to basic research, nor to test existing models of the flow from basic research to medical practice to see how well they can incorporate the ,*reverse lines" that we have traced in our case studies.

139 Neither, and this would be perhaps the more intellectu ally examined fully the types of researen processes that we have analyzed in relation to various philosophical and sociological theories about the nature of science, as one of us has attempted to for neuroscience research (Swazey and Worden 1975).

All three types of model building and testing are needed to help us better understand how categorical research can contribute to basic science research, and to better understand the nature of scientific research and discovery more generally. Such efforts, however, will be sounder when there is a larger body of detailed case studies, whether of the sort in this volume or the types sponsored by the NSF or conducted by Comroe and Dripps. The type of historical analyses we have done, as we have noted, is only one of several possible approaches, one we used because of our particular shared training in the history of science, and because one of us has found the detailed case study format a particularly congenial and fruitful mode of historical and sociological analysis (Swazey 1969, 1974; Fox and Swazey 1974; Worden, Swazey, and Adelman 1975).

A particular merit that we find in the case study for mat is that it can reveal and illustrate many processes and phenomena involved in research. Thus, each of our cases does more than document the fact that categorical research can contribute in significant ways to the advancement of basic knowledge. The other phenomena and patterns that the cases illustrate include, first, the nature of serendipity, a phenomenon we discussed at some length in chapter one in relation to its frequent misinterpretation as a discovery resulting from a totally fortuitous event, and its invocation as the distinguishing characteristic of basic research. As we noted, more careful historical and sociological analyses show that serendipity involves far more than chance - that, in Pasteur's famous dictum, chance favors the prepared mind - and that there is no inherent reason why serendipity should occur only in' basic rather than applied or categorical research. Both of these points are borne out, once again, in the instances of serendipity recorded in our case studies: for example, Pasteur's observation of fermentation in the solution of racemic ammonium tartrate lying in his laboratory;

Eijkman's pursuit of the sudden outbreak and equally sudden disappearance of polyneuritis in his chickm;4 f Minkowski's discovery that the removal of a dog's parpi creas caused diabetes melfitus; and Ingram's clecis to study the protein chemistry of sickled cells.

A second point about research that we addressed in chapter one was the problems that commonly ensue when, for heuristic, policy, or other purposes, one tries to neatly label a given research activity as basic, applied, mission-oriented, fundamental, etc. These problems are seen in this volume as one thinks about the work on thiamine's structure and functions by researchers such as Williams and Peters, the study of minimal diets by Hopkins, McCollum, and others, the many reasons that the strange proteins of multiple myeloma were studied in the 1950s, and, perhaps preeminently, in the work of Louis Pasteur on the diseases of wine and vinegar.

Another frequently discussed factor in scientific and technological advances is the role of technique, embracing both methods and instrumentation. Technique, not surprisingly, played an important role at many junctures in our cases. To recall but a few examples: technique helped to resolve competing theories, as was the case in surgical and aseptic techniques in endocrinology and with Pasteur's experimental denunciation of the spontaneous generation theory; permitted the identification of a new substance, the beriberi "vitamine," by means of improved assay, extraction, and purification; and detected the molecular basis for sickle cell anemia by "fingerprinting" the hemoglobin molecule.

The case studies also illustrate the important role that interdisciplinary fusions played in many of-the advances we charted. As exemplified by the development of endocrinology as a field of research and the subsequent emergence of neuroendocrinology, and by the enormous yield of the mergers of genetics, microbiology, and biochemistry that engendered molecular biology, the coming together of different disciplines, with a new sharing of perspectives, interests, and methods, is an important pattern in the course of research and discovery.

Another common and important pattern in research seen in our cases, a pattern that has been analyzed in detail by Comroe, is the occurrence of lags, which may be of various types and occur for various reasons (Comroe 1976). In the delays that ensued in the acceptance of the concepts of gene action put forward by

140 Mendel, Garrod, and Beadle and Tatum, for example, we see instances of lags that occurred in part because of the persistent feeling, in Beadle's words, "that any simple concept in biology must be wrong." The conflict of a new finding or theory with prevailing views also can cause lags in acceptance and utilization, as was partly the case in biochemical genetics and as occurred in the resistance that the Scharrer's concept of neurosecretion encountered. Here, we see elements of the pattern of resistance by scientists to scientific discovery, a pattern that has been examined in detail by sociologists, historians, and philosophers of science (see Barber 1961; Frank 1961; Kuhn 1970). Depending on their nature and consequence, as Comroe points out, lag times in research between discovery and rediscovery, discovery and acceptance, discovery and application, etc., may be too long, too brief, or of an appropriate duration. Thus, for example, what may seem at first to be an unduly long period of time between a discovery and the understanding of what that discovery means, as in the over 100 years between the discovery of Bence Jones proteins and their identification, may turn out to be an appropriate "latency" period when one examines the knowledge and technique prerequisite to the proteins' characterization.

The case studies further bear upon several questions about the development of theories that have long drawn the attention of those interested in the nature of science, and engaged them in often intense debates. In traditional formulations, briefly, science is viewed as an "objective" endeavor that uses the cutting "edge of objectivity" to separate truth from error. Traditional accounts of the nature of science, such as those perpetuated in textbooks, also place strong emphasis upon the value of hypothetico-deductive thought, the use of experimentation, and the scientist as one who "does not blindly accept established dogma" (Brush 1974; Gillispie 1960).

Another major component of traditional views of the nature of science is that science progresses incrementally, adding each discovery, like a new brick, to previously laid bricks in the slowly rising edifice of "truth." This image of science, as Ragnar Granit has noted, is reflected in one of the most prestigious symbols of scientific achievement, the Nobel Prize. "The young scientist often seems to share with the layman the view that scientific progress can be looked upon as one long string of pearls made up of bright discoveries. This standpoint is reflected in the will of Alfred Nobel, whose mind was that of an inventor, always loaded with good ideas for application. His great Awards in science presuppose definable discoveries" (Granit 1972, p. 3).

Without asserting that science never operates in the ways outlined above, views contradicting traditionally accepted accounts have long been offered by scientists themselves, as well as by philosophers, historians, and sociologists; perhaps the most imiuc,111.1 _ sial "unorthodox" account in recent years has been Thomas Kuhn's analysis of the structure of scientific revolutions (Kuhn 1970; Lakatos and Musgrave 1970; Scheff ler 1967; Swazey and Worden 1975).

One recurring pattern seen in our case studies that is particularly relevant to these varying interpretations of the nature of science, and on balance favors views such as Kuhn's, concerns the ways that scientific theories develop. Scientific theories, according to the canons of experimental science, arise out of data, and stand or fall solely on the basis of objective scientific evidence. To this thesis our case studies, as have many others, say "not so." We have, for example, already noted the types of resistance by scientists to new discoveries or theories, a recurring pattern that argues against the "edge of objectivity." Prevailing theories, secondly, may determine, at least for a time, the type of evidence that researchers seek, or the explanation they offer for a particular phenomenon. Thus, in two of our cases, we saw how the infectious disease model was applied to both the explanation of beriberi and the search for its cure, and to the view that the ductless glands function to neutralize or remove toxins in the blood.

Correlatively, we have encountered a number of the classic theoretical controversies that run throughout the history of science and medicine. These include Pasteur's engagement in the long debate over spontaneous generation; the competing theories of endocrine function in the late nineteenth and early twentieth century; debates over the nature of what came to be known as vitamins; the long and often heated battle between proponents of humoral and cellular theories of immunity; and the play between selective and instructive theories of antibody formation. The resolution of such controversies involves more than just the accumulation of new scientific evidence or what Kuhn calls "the methodological stereotype of falsification by direct comparison with nature" (Kuhn 1970, p. 77). Rather, as Kuhn argues, historical studies of scientific development indicate that "the act of judgment that leads a scientist to reject a previously accepted theory is always based upon more than a comparison of that theory with the world. The decision to reject one paradigm is always simultaneously the decision to accept another, and the judgment leading to that decision

141 involves the comparison of both paradigms with nature and with each other" (Kuhn 1970, p, 77). between scmin.selves have often made an "unorthodox" acimis5wii. their decisions as to what and how they will observe, as well as how they may subsequently interpret those observations, are predicated by their theoretical constructs, rather than vice versa. One of the best expressions of this way of "doing science" was made by Albert Einstein in 1926, when he responded to Heisenberg'~ statement that only observable magnitudes must be used in formulating a theory like that of relativity.

Possibly I did use this kind of reasoning, but it is nonsense all the same. Perhaps I could put it more diplomatically by saying that it may be heuristically useful to keep in mind what one has observed. But on principle, it is quite wrong to try founding a theory on observable magnitudes alone. In reality the very opposite happens. It is the theory which decides what we can observe. (quoted in Brush 1974, p. 1167)

Three comparable counter-instances to traditional views of doing science come at once to mind from our cases. Pasteur's preconceived and strongly held ideas about the role of molecular dissymmetry in the "organization of living organisms" both helped lead him to search under his microscope for living organisms in ferments, and structured many of his interpretations of his observations. Linus Pauling and his colleagues came to their analysis of sickled cells with Pauling's concept of "molecular disease" already in his mind, a concept triggered when he first discussed sickle cell anemia with William Castle in 1945 and related what he learned to his prior work on the nature of antibodies. Thirdly, reflecting in his Nobel Prize address on his and Tatum's experiment, with Neurospora, George Beadle said, much as hac Einstein: "it is sometimes thought that the Neurospon work was responsible for the 'one gene-one enzyme hypothesis . . . The fact is that it was the other wa, around - the hypothesis was clearly responsible for th new approach."

For us, in sum, Today's Medicine, Tornorrovy Science does more than chart the ways that the study, disease problems can lead to new vistas in our knowled of basic biological phenomena. We do not claim to hz made "discoveries" in this study, as that term is co monly understood to mean new and unexpected ft ings. But if, as Ragnar Granit suggests, the real goal c esearcher's labors is understanding or insight, rather than the "eureka" of discovery, it is hoped that these essays have contributed to our understanding of the complex enterprise that is biomedical research.

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Biomedical Research Programs." Report of the President's Biomedical Research Panel. Appendix B. Wahington, D. C.: DHEW Publication No. (OS) 76-502.

Brush, S.G. 1974. "Should the History of Science be Rated X?,Science 183:1164-1172.

Comroe, J. H. 1976. "Lags Between Initial Discovery and Clinical Application to Cardiovascular Pulmonary Medicine and Surgery." Report of the President's Biomedical Research Panel. Appendix B. Washington, D. C.:DHEW Publication No. (OS) 76-502.

Comroe, J. H. 1977. Retrospectroscope. Insights into Medical Discovery. Menlo Park, Cal. ' Von Gehr Press.

Comroe. J. H., and R. D. Dripps. 1974. "Ban Franklin and Open-Heart Surgery." Circulation Research 35 (Nov.): 661-669.

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142 Fox, R. C., and J. P. Swazey. 1974. The Courage to Fail. A Social View of Organ Transplants and Dialysis. Chicago: University of Chicago Press.

Frank, P. G., ad. 1961. The Validation of Scientific Theories. New York: Collier Books.

Gillispie, C. C. 1960. The Edge of Objectivity. Princeton, New Jersey: Princeton University Press.

Granit, R. 1972. "Discovery and Understanding,.' Ann. Rev. Physiol. 34;-12.

Illinois Institute of Technology Research Institute. 1968/1969. Technology in Retrospect and Critical Events in Science. Prepared for the National Science Foundation under Contract NSF-C535.

Kuhn, T. S. 1970. The Structure of Scientific Revolutions. 2nd ad. Chicago: University of Chicago Press.

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Bobbs-Merrill. otential Contribution

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Sherwin, C. W., and R. S. Isenson, June 30, 1966. First Interim Report on Project Hindsight. Washington, D. C.: Office of Director of Defense Research and Engineering. (Revised October 13, 1966)

Swazey, J. P. 1969, Reflexes and Motor Integration. The Development of Sherrington's Integrative Action Concept. Carnbridge, MA: Harvard University Press.

Swazey, J. P. 1974. Chlorpromazine in Psychiatry. A Study of Therapeutic Innovation. Cambridge, M A: MIT Press.

Swazey, J. P., and F. G. Worden. 1975. "On the Nature Of Research in Neuroscience." In F. Worden, J. Swazey, G. Adelman, ads. The Neurosciences: Paths of Discovery. Cambridge, MA: MIT Press, ch. 31.

Visscher, M. B. 1967. "Applied Science and Medical Progress." in Applied Science and Technological Progress. Washington, D. C.: National Academy of Sciences, pp. 185-206.

Visscher, M. B, 1977. Interview in Medical News, April 11, P. 16.

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