Proceedings of the Fifty-Second Annual Meeting of the American Society for Clinical Investigation Held in Atlantic City, N

PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING OF THE AMERICAN SOCIETY FOR CLINICAL INVESTIGATION HELD IN ATLANTIC CITY, N. J., MAY 2, 1960

Robert W. Berliner

J Clin Invest. 1960;39(6):966-1042. https://doi.org/10.1172/JCI104118.

Research Article

Find the latest version: https://jci.me/104118/pdf PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING OF THE AMERICAN SOCIETY FOR CLINICAL INVESTIGATION HELD IN ATLANTIC CITY, N. J., MAY 2, 1960

PRESIDENTIAL ADDRESS

PROBLEMS OF THE SOCIETY, 1960

By ROBERT W. BERLINER

It is one of the ancient and, to the victim, barbaric in some subspecialties, Sunday evening. In order to make customs of this rite of spring for the president to ad- this possible, the Federation gave up its Sunday afternoon dress the members of the Society on some aspect of the general meeting. It was also agreed that following a current scene in clinical investigation. The message is reasonable trial of this arrangement there would be a then recorded in the Journal of the Society where it is review to determine whether changes were indicated. available for posterity, or rather for later presidents in A possible expansion of the section meetings was to be search of suitable quotations pertinent to their own ad- subject to such a review. It was conceded, without com- dresses. I had thought that the subjects I plan to dis- mitment, that the Society might entertain the possibility cuss were of only immediate interest and too topical to that such expansion would replace part of its Monday have any significance beyond the next year or so. How- general scientific session. The time for such a review ever, a reading of the recently compiled history of the is at hand and I feel it appropriate to indicate to the Society indicates that these same subj ects have been members of the Society the possible factors upon which the center of discussion almost since the Society was or- our decision should be based. ganized. These two matters-the form of the meeting I believe it safe to say that the section meetings have and the criteria for the election of new members-again been highly successful. They have permitted the presen- demand the attention of the membership. The considera- tation of a considerable number of papers which, because tions that influence our attitudes on these questions to- of the limitations of time, could not have been included day may differ from those which have been debated in in the general programs of the two societies. More im- the past and from those which will determine the posi- portant, they have permitted the inclusion of a number tion of the Society in the future. Nevertheless, we must of excellent papers which, because they deal with tech- make decisions based on the current situation, recog- nical matters of a higlhly specialized nature, would be nizing that they will require continuous reconsideration inappropriate for a general audience, but are received and possible revision in the future. with interest and insight by workers in the same field. Since action on each of these matters is, at this time, Discussion is far less inhibited by the smaller audience required of the Council and the membership, it seems ap- and the much less overwhelming size of the meeting propriate that the presidential address be devoted to an halls and is encouraged by the greater community of attempt to clarify the issues involved. The time avail- interest and understanding. able for the transaction of business at our annual meet- As to the increase in the number of papers presented, ing is sufficiently limited to preclude much adherence to those submitted to the Society have fared somewhat the forms of democratic procedure. Nevertheless, the better both absolutely and relatively than those sub- officers of the Society act in the conviction that they mitted to the Federation. The section meetings have understand and represent the will of the majority of the permitted the presentation of more than twice as many members and, although the formal opportunities for the of the papers submitted to the Society: just under one members to make known their wishes are few, the in- of every three papers offered finds a place on one or an- formal occasions are many. It is in this spirit that I other program. Because of the sacrifice of part of its place before the Society my personal convictions on the general session, there is a smaller increase in the num- problems requiring decision. ber of papers accepted for the Federation program. In As you may know, three years ago the president ap- addition, because of a continuing rapid increase in the pointed a committee of the Council to meet with repre- number of abstracts submitted to the Federation, only sentatives of the Federation for Clinical Research to ar- one in every six or seven is actually presented. range for the conduct of section meetings on various It is always difficult to evaluate, from an abstract, the subspecialties of clinical investigation. The committee work which it represents, but it is my feeling and the was instructed to plan the meetings, in cooperation with considered opinion of the chairmen of the various sec- the Federation if possible but separately if necessary, and tion meetings, both this year and last, that, except for to arrange that the section meetings should not conflict two of the sections, the programs could not be appreciably with the general scientific program of the Society on expanded without reducing their present high level. Monday. Agreement was reached, and jointly spon- Since three of the sections now meet only in the after- sored meetings were set up for Sunday afternoon and, noon, additional programs could be fitted into the even- 966 AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 967 ings by further subdivision of the more prolific special believe that it would adequately meet this problem. areas, but it must be conceded that the evening sessions It is true that there would be a sharp decrease in the are considerably less satisfactory than those in the candidates for election next year if all those over 40 afternoon. were dropped from consideration and their elimination This, then, is the background upon which a decision from consideration were on such an arbitrary basis that concerning the future of the section programs must be it could not be considered a reflection on them personally. based. I, personally, am opposed to their expansion. Of the 193 candidates for election this year, 86 would There is today, as there was when Dr. Meltzer delivered not be eligible for consideration next year solely on the the first presidential address to the Society, cause for basis of age. An additional 34 candidates from the concern about the science of clinical medicine. The group nominated in 1958 would either have been elected basis for this concern is, however, entirely different. The or dropped from consideration, bringing the total num- establishment of the science of clinical medicine as a ber carried over from this year to something under 73 branch distinct from its practice has long since been ac- and most likely around 50. If we could then anticipate complished. Today we find clinical investigation grown next year a number of new proposals not greater than the so massive that it has become subdivided into many average of recent years, the list to be considered would smaller branches, each of which has tended to fuse back be at its lowest level in the last ten years. However, I with the corresponding basic science, thus leading not doubt that this sitaution could last very long. An ex- only to a widening separation between the science and traordinarily large number of candidates was proposed practice of medicine, but also to an increasing indi- in 1958-so many, in fact, that even after reduction of vidualization of the specialized investigative areas. The the list by two previous elections there were still more growing complexity of technology as well as the snow- candidates up for election this year from those nominated balling accretion of information in each field makes it in 1958 than the totals proposed in either 1959 or 1960. virtually impossible for an individual to be expert in Furthermore, of the candidates proposed this year the more than one. Nevertheless, few of us can afford ig- unusually large number of 26, or 39 per cent, would not norance in fields outside our own, and a single day's be eligible next year under the proposed amendment, 18 exposure to a reasonable sampling of the better work in of these 26 being already past the age of 40. Thus the clinical investigation is all too inadequate. number who would be dropped this year is unusually large That only a minority of papers submitted finds a place in several respects. It seems clear that the membership, on the program is, perhaps, unfortunate, but so long as anticipating possible adoption of the amendment, has this fact does not discourage submission of excellent ma- proposed the names of a large number of candidates for terial for the program it should not be an important con- whom it might be the last chance. It seems virtually cer- sideration. There is, these days, no lack of opportunity tain, however, that in the future, in view of the limited for communication. The ease and speed of travel and period of eligibility, candidates will be proposed in equal the availability of funds to support it has led to such numbers but at an earlier age, when it may be even more a massive proliferation of meetings, conferences and difficult to evaluate their independent contributions. symposia that one wonders when the participants will In seeking a satisfactory policy for the election of new find the time to get any new work done. It is, perhaps, members, it is essential that we have a clear idea of what a self-limiting disorder since those most involved must we wish to accomplish by that policy. In other words, eventually run out of things to talk about. In any what is to be the significance of membership in the So- case, we need not feel that limitations on the size of our ciety? It seems to me that election to membership repre- programs significantly diminish the service to our mem- sents primarily recognition of the accomplishment of bers and guests. I believe our aims are best served by significant original and independent research in clinical continuing to hold a program of the highest quality and and related fields. It seems, furthermore, that election limited size and continuing to provide the opportunity for to membership is, more or less, an end in itself. The less formal communication, both technical and otherwise, rights and privileges which pertain to membership are which traditionally goes on outside this hall. otherwise relatively trivial. A regrettably small minority The other problem that the Society must face is one is privileged to hold office or serve on the committees of for which the solution is less apparent and which will the Society and thus to play an active role in the con- not be adequately handled by simple maintenance of the duct of its business. While only members, active or status quo. That problem concerns the election of new emeritus, may introduce papers at the meetings, it is members. The problem has been very clearly outlined unusual for any investigator with worthy material to be by Dr. Dole and his associates in the material circu- unable to find a sponsor. As for the sponsorship of can- lated in support of the constitutional amendment which didates for membership, the problem is only too clear. they have proposed. The amendment would eliminate I therefore find it difficult to believe that most candi- from consideration all candidates for election to the So- dates over the age of 40 have failed of election only be- ciety who have reached the age of 40. If the amendment cause the Councils have preferred persons who could alone or in combination with a moderate increase in the participate as active members for five or more years. rate of election of new members would restore the bal- If election is to be considered recognition of accom- ance between candidates proposed and those elected, it plishment in research, however, it behooves us to make would be a welcome forward step. However, I do not certain that those elected are those whose accomplish- 968 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING ments best warrant the distinction. If we can have com- a good word for a nominee solicited by his sponsors. plete confidence that this is the case, ve can live with any I have painted the worst side of the picture. Despite arbitrary limitation of the number elected and not feel the limitations, I believe the Council makes the best of that any injustice has been done those rejected, although a very difficult situation and, by and large, recommends we should certainly adjust the arbitrary limitation to for election each year those best qualified for membership. the number of really highly qualified candidates. The However, I feel that the process could be considerably question we then must ask ourselves is whether our improved, the burden on the Council somewhat relieved, evaluation of candidates is adequate to provide com- and confidence in the adequacy of the selection process plete confidence that those most qualified have been strengthened. This end might be served by the appoint- elected. I must frankly admit that I do not feel that ment of a qualifications committee, composed of several our present system uniformly warrants such confidence. representatives of each of the various specialized areas Let me hasten to say that I believe the Council makes of investigation. Such a group might review the work every effort to base its decisions solely upon the sig- of each candidate in sufficient detail to be able to provide nificance, originality and independence of an individual's to the Council an evaluation based upon a familiarity contributions. But the ability of the Council to evaluate both with the field and the standing of the candidate these on the basis of the information available to it is within it. The final selection of those to be recommended limited. The high degree of specialization of which we to the Society for election would remain the respDnsi- spoke earlier makes it unlikely that any Council mem- bility of the Council, which would not necessarily be ber will be familiar with the details of the work of many bound by the recommendations of the committee and candidates outside of his own field of interest. The would be expected to maintain a reasonable balance short time available and the large number of candidates among the various fields of investigation. The number to be considered make it difficult for most Council mem- of individuals recommended by the committee as highly bers to read more than a small selection of the papers of qualified might also be of value in providing to the many of the candidates. Consequently they must rely Council some guide as to the desirable annual rate of largely upon the letters of the nominators, which rarely election. Whether or not such a procedure is adopted are restrained in their support of the candidates, upon I feel that it is essential that the nomination date be ad- an attempt to evaluate the bibliographies from the titles vanced so as to provide, to those who are to evaluate the of the papers and the reputation of the journals in which candidate, sufficient time to familiarize themselves more they appear, upon the advice of colleagues who may be thoroughly with his work. With some such assurance familiar with the work of some of the candidates, and of the adequacy of our selection process and with a clear upon the letters received from members. The latter are understanding among the proposers of new members of not too helpful, since they so rarely offer a comparative the criteria for election, we need have little concern about evaluation of various candidates and so often represent the number or age of the candidates for election. PAPERS PRESENTED AT THE FIFTY-SECOND ANNUAL MEETING 1960

1. The Induction of Heterologous Immunity to Influenza 12. Effect of Acute Uremia on Arterial Blood Pressure by Aerosol Administration of Inactivated Virus. in Rabbits. CARMELO GIORDANO, HERMAN WEINSTOK JEROME L. SCHULMAN and EDWIN D. KILBOURNE,* and JOHN P. MERRILL,* Boston, Mass. (990) New York, N. Y. (1026) 13. A "Precursor Solution" of Pancreatic Juice: Evi- 2. On the Role of Virus Sulfhydryl Groups in the At- dence for an Exchange Mechanism. H. D. JANO- tachment of Enteroviruses to Cells. LENNART WITZ * and D. A. DREILING, New York, N. Y. (1000) PHILIPSON and PURNELL W. CHOPPIN, New York, N. Y. (introduced by Igor Tamm). (1017) 14. The Effect of Wheat Instillation into the Proximal Ileum of Patients with Idiopathic Sprue. CYRUS E. 3. The Capacity of Tubercle Bacilli to Assume a Latent RUBIN, LLOYD L. BRANDBORG, ARNOLD L. FLICK, State In Vivo. ROBERT M. MCCUNE, New York, CHERILL PARMENTIER, PATRICIA PHELPS and SALLY N. Y. (introduced by Walsh McDermott). (1009) VAN NEIL, Seattle, Wash. (introduced by Clement A. Finch). (1023) 4. Studies on the Mechanism of Fever Following Intra- venous Inoculation of Staphylococci. ELISHA AT- 15. The Effect of Changes in Atrial Systole on the Rela- KINS * and LAWRENCE R. FREEDMAN, New Haven, tion Between Mean Atrial Pressure and Stroke Conn. (969) Work. STANLEY J. SARNOFF,* JERE H. MITCHELL and JOSEPH P. GILMORE, Bethesda, Md. (1025) 5. Relationship of Phagocytosis to the Fall in Spinal Fluid Glucose in Experimental Meningitis. ROBERT 16. Metabolic Regulation of Cardiac Output. WILLIAM G. PETERSDORF, Baltimore, Md. and Seattle, Wash. E. HUCKABEE,* Boston, Mass. (998) (introduced by W. M. M. Kirby). (1016) 17. On the Mechanism of Carotid Sinus Function. 6. Studies of Granulocyte Kinetics. JOHN W. ATHENS, LYSLE H. PETERSON,* ERIC 0. FEIGL and PETER ALVIN M. MAUER, SPENCER 0. RAAB, OTTO P. HAAB GOURAS, Philadelphia, Pa. (1017) and GEORGE E. CARTWRIGHT,* Salt Lake City, Utah. 18. Effect of Antihypertensive Treatment in the Rat on (969) the Potentiation of Atherogenesis by Experimental 7. Presence of an Altered Amino Acid Sequence in Hgb Hypertension. QUENTIN B. DEMING,* MARIE M. M (Boston Type). PARK S. GERALD, MARY L. EFRON DALY, JAIME BLOOM, LILI BRUN and RUTH KAPLAN, and MARGARET J. PEASE, Boston, Mass. (introduced New York, N. Y. (980) by Louis K. Diamond). (989) 19. Chromosome Constitution in Human Gonadal Dis- 8. Oxidative Hemolysis and Precipitation of Hemo- orders. AVERY A. SANDBERG,* THEODORE S. HAU- globin: Heinz Body Anemias as an Accelerated Form SCHKA, EDWIN GORDY and GEORGE F. KOEPF, Buffalo, of Red Cell Aging. JAMES H. JANDL * and DAVID W. N. Y. (1024) ALLEN, Boston, Mass. (1000) 20. Transmanganin, the Specific Manganese-Carrying 9. The Enzymatic Defect of Orotic Aciduria. LLOYD H. Protein of Human Plasma. GEORGE C. COTZIAS * and SMITH, JR. and CHARLES M. HUGULEY, JR., Boston, ALBERT J. BERTINCHAMPS, UPTON, N. Y. (979) Mass. and Atlanta, Ga. (introduced by Anne P. Forbes). (1029) 21. Inhibition of Aromatic Amino Acid Decarboxylation in Man and Associated Pharmacological Effects. 10. Relation of Hormone-Receptor Bonding and Antidi- JOHN A. OATES, JR., LOUIS GILLESPIE, JR., J. RICHARD uretic Action of Vasopressin. IRVING L. SCHWARTZ,* CROUT and ALBERT SJOERDSMA,* Bethesda, Md. HOWARD RASMUSSEN, MARY ANNE SCHOESSLER, (1015) CONRAD T. 0. FONG and LAWRENCE SILVER, Upton, N. Y. (1026) 22. Immunoassay of Plasma Insulin Concentrations in Normal and Diabetic Man: Insulin Secretory Re- 11. Micropuncture Study of Net Transtubular Movement sponse to Glucose and Other Agents. ROSALYN S. of Water and Urea in the Rat Kidney. WILLIAM E. YALOW and SOLOMON A. BERSON,* Bronx, N. Y. LASSITER, CARL W. GoTTsCHALK * and MARGARET (1041) MYLLE, Chapel Hill, N. C. (1004) 23. The Krebs Cycle and Diabetic Ketosis. DANIEL W. * Member. FOSTER and MARVIN D. SIPERSTEIN,* Dallas, Tex. ( ) Page number of abstract. (986) 968a 968b PAPERS PRESENTED AT THE FIFTY-SECOND ANNUAL MEETING 1960

24. Mannose Metabolism, an Index of Glucose Utiliza- LEONORE M. DECARLI and RUDI SCHMID,* Boston, tion. GEORGE F. CAHILL, JR., FRANCIS C. WOOD, JR., Mass. (1007) BERNARALBsERTLEBOEUF and E. RENOLD,* Boston, 26. The Effect of Cell Structure and Growth Hormone Mass. (976) on Protein Synthesis in Striated Muscle. DAVID M. 25. Stimulation of Hepatic Fatty Acid Synthesis by KIPNIS and ERIC REISS, St. Louis, Mo. (introduced Ethanol in Vivo and in Vitro. CHARLES S. LIEBER, by Carl V. Moore). (1002) ABSTRACTS

Antidiuretic Action of Oxytocin. R. ABDUL-KARIM and day. In 3 patients with chronic myelocytic leukemia in N. S. ASSALI, Los Angeles, Calif. (introduced by remission after treatment with Myleran these values had Joseph F. Ross). returned almost to normal. It is generally believed that oxytocin exerts a minimal In 4 subjects with infection and leukocytosis the values antidiuretic action when compared to vasopressin. Actu- were increased over normal but only moderately so. In ally, oxytocin increases water and electrolyte excretion two subjects with chronic infections and no leukocytosis in the rat and may even antagonize the antidiuretic action the values were within the normal range. of vasopressin. The TBGP has been shown to consist of two compart- ments To test the renal action of oxytocin in man, studies in rapid equilibrium with each other, the circulat- were performed on 12 females (5 pregnant and 7 non- ing granulocyte pool (CGP) and the marginal granu- pregnant) and 5 males. One natural (Pitocin) and 2 locyte pool (MGP). The size of the CGP is calculated synthetic (Syntocinon and DuVigneaud's polypeptide) from the blood volume and the absolute granulocyte count. preparations were infused intravenously in doses varying The MGP is the difference between this value and the from 6.5 to 315 mU per minute. In each instance, TBGP. The distribution of granulocytes in these two oxytocin induced a marked fall in urine flow which lasted compartments of the TBGP has been studied in the sub- for the duration of the infusion. Renal plasma flow jects described above. Thirty-five to 46 per cent of the (PAH), glomerular filtration rate (inulin), electrolyte granulocytes in the TBGP was found to be in the CGP. excretion and osmolal clearance did not change sig- Cells shift from the MGP into the CGP after epinephrine nificantly, whereas free water clearance fell and often injection or exercise. became negative. In the pregnant subjects who received oxytocin for labor induction, the antidiuretic action be- Studies on the Mechanism of Fever Following Intra- came evident before the oxytocic effect. venous Inoculation of Staphylococci. ELISHA ATKINS* To investigate the possibility that oxytocin antidiuresis and LAWRENCE R. FREEDMAN, New Haven, Conn. might be mediated through stimulation of ADH secretion, Although gram-positive microorganisms produce some oxytocin was infused into 2 patients with diabetes in- of the most pyrogenic infections in man, the sequence of sipidus and 3 normal subjects who ingested 180 ml of events leading to fever with these bacteria has remained whiskey. The same fall in urine flow and in the free obscure except where purulent foci have been established. water clearance occurred in these subjects, suggesting a There has been conflicting evidence as to whether staphy- direct action of oxytocin on the kidney. It is concluded lococci are capable of causing fever after that in man intravenous oxytocin has antidiuretic properties closely injection since they do not possess endotoxins which are similar to those of vasopressin. present in gram-negative bacteria. In the experiments to Studies of Granulocyte be described, injection of either live or autoclaved staphy- Kinetics. JOHN W. ATHENS, lococci resulted in fever after a characteristic latent ALVIN M. MAUER, SPENCER 0. RAAB, OTrO P. HAAB period of 1 hour. A similar latent seen and GEORGE E. CARTWRIGHT,* Salt Lake City, Utah. period is with fever produced by inoculation of either influenza virus or In these studies blood was withdrawn from each sub- antigen in specifically sensitized recipients. By studies ject into a plastic bag, the granulocytes labeled with involving passive serum transfer, the fever was shown radioactive diisopropyl fluorophosphate (DFP') and the to be directly caused by a circulating endogenous pyrogen labeled cells returned to the circulation. The size of the (EP). The whole microorganism rather than one of its pool into which the labeled cells are infused, the total products liberated into the culture medium appeared to blood granulocyte pool (TBGP), has been measured with be essential in releasing this endogenous material. No this method. The granulocytes have been shown to leave cross-tolerance to bacterial endotoxin was demonstrable the TBGP in a random manner and the time in which although there was a diminished response to other heter- half of the cells leave (Ti) has been determined. From ologous pyrogens after injection of staphylococci. Toler- the size of the TBGP and the Ti, the granulocyte turn- ance in this situation appears to be due to a temporary over rate (GTR) has been calculated. inability of the recipient given staphylococci to mobilize In 40 normal subjects the mean values for these param- further amounts of EP to another stimulus. eters were: TBGP 0.83 x 109 cells per kg, T1 6.9 hours The experiments suggest that a number of unrelated and GTR 2.1 x 109 cells per kg per day. agents, including bacterial cells and other substances of In 6 patients with chronic myelocytic leukemia in re- large molecular size, produce fever by releasing an EP. lapse the mean values were: TBGP 20 x 109 cells per The interaction of these substances with kg, 49.2 hours and GTR 5.7 x 109 cells per kg per circulating poly- Ti morphonuclear leukocytes is believed to be the initial step * Member. in this process and may therefore be essential to the de- 969 970 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING velopment of fever in a wide variety of different situa- phosphate-loaded dogs results in progressive elevation of tions, both clinical and experimental. urine pH and reduction in titratable acid excretion, while administration of the less mobile anions, sulfate or ferro- Preservation of Platelet Viability at 40 C. MARIO cyanide, is associated with sustained or increased urine BALDINI and SHIRLEY EBBE, Boston, Mass. (introduced acidity and excretion of near theoretical maximum by William Dameshek). amounts of titratable acid. The effectiveness of platelets in promoting hemostasis As a possible mechanism to account for these findings, it is proposed that the various anions affected urine pH depends chiefly upon their viability. In the present study on electrical the viability of platelet concentrates preserved in different and acid excretion through their influence the gradient established by active sodium transport across the media at 40 C was measured. renal tubular cells. According to this hypothesis, the A rabbit model was first adopted, and viability of magnitude of the gradient is determined by the mobility stored rabbit platelets labeled in vitro with Cr5' or in vivo of the available anions, those which penetrate the tubular with P32 was investigated by following platelet radio- activity in the circulation of normal recipients and also epithelium more readily having a greater tendency to by enumerating the circulating platelets after infusion into follow sodium and thus to reduce the potential difference. Changes in urinary pH and titratable acid excretion have thrombocytopenic animals. Two parameters were used to determine viability: 1) the platelet lifespan and 2) the tentatively been attributed to the passive movement of hydrogen ions in response to variations in the transtubular maximum platelet recovery in the recipient's circulation. electrical gradient. Although active transport of hy- From these, the platelet "viability index" was calculated drogen may also be involved in the overall acidifying and was expressed in per cent of the value obtained with fresh platelets. process, the present data suggest that passive diffusion of hydrogen plays an important role in the mechanism gov- Platelet concentrates stored for 24 hours in saline had erning urinary acidification and the renal excretion of a viability index of less than 2 per cent. When stored in acid. gelatin the viability index was less than 1 per cent. A new storage medium containing inosine, adenine, glucose, plasma and phosphate buffer was then investigated. With An Alternate Schedule for Poliomyelitis Immunization. the use of this medium, the viability index of platelets EUGENE V. BARNETT and SAMUEL BARON, Rochester, was 37 per cent after 24 hours and fell to 11 per cent N. Y. and Bethesda, Md. (introduced by Howard B. only after 6 days of storage. Removal of single com- Slavin). ponents from this medium demonstrated that its favorable The current dosage schedule of three 1 ml injections of effect on platelet viability was due mainly to 1) plasma, poliomyelitis vaccine over 8 to 12 months produces an and 2) the purine metabolites. The effect of the latter adequate antibody response in most nonimmunes follow- was more striking after the third day of storage. There ing the second or third dose. The present study was un- was evidence that the purine metabolites delayed the dertaken to determine a dosage schedule which would natural aging process of the stored platelets. provide rapid immunization, maintain detectable antibody Experiments with human platelets preserved as Cr51- levels throughout the immunization period and produce labeled concentrates confirmed the favorable effect of the the high levels of antibody which have been correlated medium containing plasma and the purine metabolites. with long duration of immunity. Such a schedule would After 48 hours of storage, platelet viability was 3.3 times be particularly desirable for those who seek rapid im- higher than in the medium containing only plasma, and munization, such as contacts of paralytic cases. was 15 times higher than in the saline medium. One group of nonimmune children was given vaccine of low antigen content according to the current schedule The Influence of Anion Mobility on Urinary Acidification of three 1 ml doses over 7 months. Another group of and the Excretion of Titratable Acid. NORMAN BANK nonimmune children was given an initial dose of 10 ml and WILLIAM B. SCHWARTZ,* Boston, Mass. of the same vaccine followed by a 1 ml booster at 7 The influence on acid excretion of anions of different months. A group of adults was given an initial 10 ml mobility (membrane-penetrating ability) has been studied dose of vaccine of high antigen content followed by 1 ml in dogs whose sodium and chloride excretion had been doses at 1 and 7 months. The results may be summarized reduced by dietary restriction. It was demonstrated that as follows: 1) Significant quantities of antibody to the 3 administration of the sodium salt of phosphate (pH 7.4), poliovirus types appeared between the second to sixth a buffer anion of low mobility, leads to a marked reduc- day following 10 ml of high antigen-content vaccine. 2) tion in urine pH and the excretion of near theoretical Antibody to Type 1 and 3 polioviruses followed inocula- maximum amounts of titratable acid. These results sug- tion of a single 10 ml dose or two 1 ml doses but soon gest that in the nonacidotic dogs a disproportion between declined to undetectable levels. It was usually boosted by sodium and anion reabsorption was the critical factor in a third inoculation at 7 months. 3) Antibody which re- determining not only the pH gradient between blood and sulted from an initial 10 ml dose followed by a 1 ml dose urine but also the excretion of titratable acid. This hy- at 1 month, remained detectable throughout the immuniza- pothesis is supported by the observation that infusion of tion period and was boosted to higher levels by a 1 ml the more mobile anions, chloride or thiocyanate, into dose at 7 months. AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 971

The Significance of the Instantaneous Aortic Blood Ejec- there was no other evident change or event in the clinical tion Velocity. G. OcTo BARNETT, SAMUEL M. Fox, course. Excepting one subject, who developed ascites 3RD, ALEXANDER J. MALLOS, JOSEPH C. GREENFIELD, JR. terminally 7 years later, edema fluid did not recur in a and DONALD L. FRY, Bethesda, Md. (introduced by subsequent observation period of 3 to 7 years. James H. Baxter). The onset of natriuresis occurred slowly over several with sodium The time relationships between and days urinary increasing from tenths to 1 to pressure blood 5 mEq per day. This early increment a velocity in the aorta can be used to define represented ascending the several-fold increase in sodium excretion but was mechanical function of the myocardial pumping mecha- in- sufficient to influence either urinary volume or the nism. To the extent that mechanical function is altered As the natriuresis early in disease, evaluation of these patient's weight. continued, it followed pressure-velocity re- the same pattern in all 5 patients. The daily urinary lationships has important biophysical and clinical im- sodium excretion doubled 3 plications. Recent evidence indicates that changes of every days (approximately) until normal sodium excretion was achieved. At this blood velocity may be a sensitive index of altered me- chanical rate, on a diet of 50 to 150 mEq of sodium, a month function. It was the purpose of this study to from the of the natriuresis until determine to what extent certain disease elapsed beginning its processes, effect was reflected in a decrease in weight. The con- pharmacological agents, and experimentally produced tinued increase of myocardial ischemia altered the pressure-velocity relation- urinary sodium from tenths of a mEq to over 100 was in with con- ships. These mEq per day keeping the relationships were evaluated by the simul- cept of a first order process. One possible explanation taneous estimate of central aortic pressure and in- for this pattern is an increase in hepatic aldosterone stantaneous blood velocity obtained by the computed clearance. pressure gradient technique using a catheter system de- veloped in this laboratory. These observations were made on the intact unanesthetized man and dog. Changes in Gross Body Composition During Controlled Diffuse myocardial disease in man and experimentally Weight Loss. NATHANIEL I. BERLIN and DONALD M. produced myocardial damage in the dog were associated WATKIN, Bethesda, Md. (introduced by Charles G. with changes in the blood velocity curve, although the Zubrod). blood pressure remained essentially constant. These Three obese patients were placed on an 800 calorie, 8 g changes in blood velocity consisted of a decreased initial N per day metabolic balance regimen for 60 to 120 days. acceleration and a diminished peak velocity. In man Changes in gross body composition [fat, water and dry with myocardial disease, and in dogs, acetyl strophanthi- lean tissue (minerals and proteins) ] were calculated from din, norepinephrine, and isoproterenol caused an increase serial measurements of total body water with tritiated in ejection velocity. In the dog, methoxamine, which has water and body density, determined with a helium dilu- predominantly a peripheral vasoconstrictor action, caused tion device, and from N, Na and K metabolic balance a decrease in ejection velocity. data. This approach, using the computed pressure gradient Following a phase of initial rapid weight loss of 5 to technique, would appear to open new avenues to a more 10 days' duration, due primarily to water loss, all 3 sensitive evaluation of cardiovascular dynamics, such as subjects lost weight and fat at an approximately constant the computation of cardiac power and work, in the intact rate, proportional to initial body fat content. In 2 pa- man. tients fat loss calculated from metabolic balance data and body water and density measurements were in good agree- A Pattern of Sodium Excretion in Patients with Decom- ment; in the third patient the fat loss could be calculated pensated Cirrhosis of the Liver Who Recover from from the N balance and body water data, but not from Their Edematous State. EUGENE Y. BERGER, RENE N, Na and K balances. The measured changes in dry PECIKYAN and GRACE KANZAKI, New York, N. Y. lean tissue constituents varied from negligible to 1.25 kg (introduced by J. Murray Steele). and were in good agreement with those calculated from body water and density data. Daily urinary sodium was measured in 5 patients with The decompensated Laennec's ratios of caloric expenditures, calculated by the cirrhosis of the liver who ex- Newburgh insensible water loss technique, to that calcu- creted less than 2 mEq of sodium per day and who, with- lated from the difference in caloric intake out the use of diuretic agents or a low sodium diet, went and caloric on to exhibit a natriuresis, lose their ascites and recover content of feces and urine plus 9.2 calories per g fat loss from their edematous state. From the onset of ascites per day, were 1.40, 1.32 and 1.07, the last occurring in a which required paracentesis, to the onset of the natri- patient with steroid diabetes. uresis, 3 to 11 months elapsed during which 5 to 14 para- The combination of metabolic balance data with body centeses were performed. Two of the 5 patients had water and density measurements permits a satisfactory febrile episodes at the onset of natriuresis; the third measurement of changes in body composition. An 800 patient received 50 mg of A1-androstene-3,17-dione in- calorie, 8 g N per day intake in obesity results in a rate tramuscularly and developed localized tenderness at the of fat loss proportional to initial body fat content, with a site with associated fever; in the remaining 2 patients small but variable change in lean tissue mass. 972 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING

The Demonstration of Three Types of inhibitors and One otide and reveals the fate of a portion of the RNA dur- Type of Stabilizer of the Latex Fixation Test for ing maturation. Rheumatoid Arthritis. GERSON C. BERNHARD and Reticulocytes were obtained from anemic rabbits, DAVID W. TALMAGE,* Denver, Colo. washed, and incubated in serum-bicarbonate buffer (pH under a continuous flow of cent 5 cent The demonstration of three distinct types of inhibition 7.4) 95 per 02, per CO2. Glucose concentration in the medium and pH were and one type of stabilization of the latex fixation system maintained by continuous addition of glucose-bicarbonate has been possible by varying the order of addition of the solution under pH-stat control. and inhibitor and the reagents in the standard test procedure. Adenine-8-C" sodium formate-C14 were added separately to individual The Singer method for latex agglutination with a positive flasks at zero time, and samples were taken up to 24 rheumatoid serum was performed in cuvets. Optical hours. The washed cell mass of each sample was dis- densities were measured photometrically before incuba- solved in sodium and were tion and subsequent to incubation and centrifugation. The dodecyl sulfate, polynucleotides precipitated ethanol or acid per cent optical density (A) remaining in the super- with perchloric (PCA). After hydrolysis with alkali or hot 70 per cent PCA, the natants after centrifugation was plotted against the dilu- nucleotides or bases were separated by paper chroma- tions of rheumatoid serum. Standard curves without in- tography. The extracellular fluid of each was hibitor were compared with those obtained when aliquot dialyzed and desalted on Dowex-1 resin. Purine and conditions were altered by addition of an inhibitor at pyrimidine components were isolated by paper chroma- various steps in the procedure. Incubating bovine serum tography. or heat-inactivated normal human serum albumin (BSA) Three compounds were isolated from the extracellular (NHS) with latex prior to coating the latex with human fluid at 24 hours and tentatively identified as hypo- gamma globulin (HGG) gave curves showing both xanthine, cytidine and uracil. Both adenine and formate stabilization (increased per cent A remaining in super- labeled cell polynucleotide adenine, but formate also natants of controls) and inhibition (reduced agglutina- labeled polynucleotide guanine. The results suggest that tion by rheumatoid factor). Neither BSA nor heated maturing reticulocytes synthesize purine nucleotides and NHS produced inhibition when added after latex was polyribonucleotide de novo during the period of net RNA coated with HGG. However, fresh NHS produced in- loss; and as maturation proceeds, products of acid hibition when added at this stage. The inhibition by nucleic fresh NHS was greatest when it was incubated with breakdown appear outside the cell. HGG-coated latex before the addition of rheumatoid factor. Heating to 560 C at this stage did not destroy Oral Glutamate Therapy in Ammonemia due to Blood in inhibition. However, fresh NHS preincubated with the Gastrointestinal Tract. ALICE N. BESSMAN, Balti- rheumatoid factor and then heated to 560 C before con- more, Md. (introduced by George S. Mirick). tact with HGG-coated latex was not inhibitory. There- It is generally accepted that elevated blood ammonia fore, the inhibitor in fresh NHS appears to act on levels play a significant role in the production of con- HGG-coated latex. Aggregated HGG and certain anti- fusion and coma in many cases of hepatic decompensation. gen-antibody precipitates are known to remove rheuma- Intravenous sodium (or sodium-potassium) glutamate ad- toid factor from serum. Thus inhibitors can cause re- ministration has been shown to lower blood ammonia duced agglutination by reacting with: 1) the uncoated levels, but the amount of glutamate that can be ad- latex; 2) the HGG-coated latex; 3) the rheumatoid fac- ministered is limited by the large quantities of intravenous tor. In addition, stabilizers can be distinguished from fluid and electrolyte that had to be administered. In the inhibitors by their effect on the latex suspension in the recent literature oral sodium glutamate has been reported absence of rheumatoid factor. to be an effective adjunct to low protein diet in lowering elevated ammonia levels in patients with liver disease. Metabolic Turnover of the Ribonucleic Acid of Mam- The greatest unanimity of opinion regarding the effec- malian Reticulocytes During Maturation In Vitro. tiveness of intravenous glutamate therapy has concerned JOHN F. BERTLES, CHARLES J. WILTz and WILLIAM S. those cases precipitated by gastrointestinal bleeding. We BECK,* Boston, Mass. have studied the effectiveness of orally administered sodium glutamate in patients with liver disease who had The development of reticulocytes in mature erythrocytes ingested test meals of whole blood. Six patients with involves the progressive loss of certain biosynthetic capac- Laennec's cirrhosis, each serving as his own control, ities and of ribonucleic acid (RNA). Little experi- ingested 1) whole blood containing a known amount of mental evidence is available on the fate of the RNA and protein nitrogen and 2) the equivalent amount of blood on the question of whether RNA synthesis continues plus 25 g of sodium glutamate. Arterial and venous during the period when net loss of RNA is taking place. blood ammonia levels were followed over a 5 hour period. Both questions bear on the overall problem of cellular All patients showed a rise in blood ammonia levels fol- differentiation; the latter relates to the current contro- lowing ingestion of test loads. In no patient did the versy on the cellular sites of RNA synthesis. The present addition of sodium glutamate diminish this rise and in investigation shows incorporation of certain labeled some patients there was grea'ser ammonemia after blood nucleic acid precursors into reticulocyte polyribonucle- plus glutamate than there was after blood alone. It was AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 973 concluded that oral administration of sodium glutamate patients receiving gold therapy. Administration of 10 in the treatment of ammonia intoxication resulting from mg of gold sodium thiomalate to rats one week prior to blood in the gastrointestinal tract is of no value. assay resulted in a 70 per cent decrease in the specific activity of the glucosamine synthesizing enzyme in con- Variability of Response of Human Tumor Cells to nective tissue, but no change in activity of the enzyme in Chemotherapeutic Agents. H. R. BIERMAN* and G. J. liver. A decrease in the acid mucopolysaccharide content MARSHALL, Los Angeles, Calif. of the connective tissue in the sponge implants also The response of patients with neoplastic diseases to occurred after gold sodium thiomalate administration. chemotherapeutic agents is often highly variable despite Sodium salicylate also inhibited the activity of the the identical histologic pattern of the tumor. Quantita- glucosamine synthesizing enzyme in both liver and con- tive cytometric and cytologic measurements of normal nective tissue homogenates. Twenty per cent inhibition and neoplastic cells in effusions of 15 patients were per- occurred with a concentration of 2.1 x 10' M, but very formed following 40 single or multiple intracavitary ad- high concentrations were needed to cause complete inhibi- ministrations of tracer amounts of 2 classes of alkylating tion. A decrease in the activity of this enzyme occurred agents (amine mustards and Myleran). in both liver and connective tissue following hydro- Prompt decrease in the neoplastic cell population as- cortisone administration to rats, but this steroid had no sociated with cytoplasmic, nuclear and nucleolar changes effect in vitro. without similar damage to normal cells was usually cor- DPNH cytochrome C reductase was inhibited by gold related with beneficial clinical response when full thera- chloride and salicylate in vitro in concentrations similar peutic doses were employed. However, identical tumor to those which inhibited the glucosamine synthesizing cell types in 5 patients failed to exhibit these changes at enzyme in each type of homogenate. Quinacrin also concentrations of the chemotherapeutic agent which inhibited the DPNH cytochrome C reductase in vitro in caused profound changes in the normal cell population. a concentration of 12.5 x 10' M, but this concentration did Intracellular incorporation of H8-thymidine showed varia- not affect the glucosamine synthesizing enzyme. tion in uptake between tumor cell types and a variation in uptake prior to and following the chemotherapeutic agent. The Alveolar Lining: A Method of Extraction; The Sur- Combined chromosome, microsome and membrane changes face Tension Lowering Effect of Cigarette Smokc. in a decreasing tumor cell population with decrease in STUART BONDURANT, Indianapolis, Ind. (introduced by H3-thymidine uptake are indicative of significant altera- John B. Hickam). tions in intracellular metabolism. Nuclear changes alone The stable coexistence of alveoli of varying size in proved inadequate in predicting ultimate chemotherapeutic continuity with the trachea has been attributed to a de- efficacy. Resistant cell populations became evident in 4 crease of surface tension of the alveolar lining layer as of the 15 patients and could not be eliminated at the surface area is decreased. The surface tension of films highest chemotherapeutic concentrations employed. obtained by compressing the lung in saline is 35 to 45 The data suggest that the convenient term "biologic dynes per cm' (T1) decreasing to <20 dynes per cm' variation" as applied to these agents refers in part to (T2) when surface area is decreased by 80 per cent. subtle differences in intracellular biochemical mecha- (Saline T1 = T2= 72 dynes per cm2.) There is strong nisms of neoplastic cells undisclosed by conventional histo- evidence that the alveolar lining layer has similar unique pathologic technics. Assays of chemotherapeutic agents surface properties. Such films are not consistently ob- on neoplastic cells in the human host aids in the selection tained and cannot be easily harvested for further analysis. of effective agents and affords an opportunity to study the Surface tension has been measured (Wilhelmy balance) mechanism of DNA synthesis and duplication. on extracts prepared by 5 methods (5 rats each): com- pression of the lung in saline T1 = 39 -+- 8, T2= 15 + 13; Effects of Antirheumatic Agents on Connective Tissue homogenized lung T1 = 40 -+- 10, T2= 24 +- 4; saline wash- Metabolism. ALFRED JAY BOLLET,* Charlottesville, Va. ing of trachea T1 =46 -+- 4, T2=22 -+- 7; endotracheal The metabolic effects of agents used in therapy of bubbles produced by vascular perfusion with distilled rheumatic diseases are under investigation in an attempt water while the lungs are mechanically ventilated T1 = to elucidate changes which occur during suppression of 40 + 10, T2=20 + 4; same procedure, saline perfusion, inflammation. Thus far, these studies have been concerned T1=34+7, T2=11+4. primarily with two enzymes, the transamidase which The bubbles can be evaporated at room temperature synthesizes glucosamine-6-phosphate from fructose-6-phos- to yield 75 to 100 mg of of amorphous white powder per phate and glutamine and DPNH cytochrome C reductase, rat. Ten to 100 mg of this powder added to a 90 cm' and with two tissues, liver and the connective tissue which saline surface causes T1 = 35 + 7, T2= 15 + 4. Human forms in polyvinyl sponges in rats. lungs similarly treated also yield large quantities of sur- Gold chloride almost completely inhibited the activity face active material. of the enzyme which synthesizes glucosamine-6-phosphate Exposure of the films to cigarette smoke for one minute in a concentration of 50 x 10' M in liver homogenates, causes a decrease in surface tension: T1 from 46 + 7 to and 5 x 10' M in connective tissue homogenates. The 27 ± 7, T2 from 15 ± 4 to 11 + 4 (15 rats, both latter concentration is in the range found in the serum of p < 0.001.) In 6 extracts from 2 normal human lungs, 974 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING

T1 decreased from 36 + 13 to 24 + 3 (p <0.05) and T2 wig has been revived, in which it is conceived that decreased from 11 ± 5 to 9 ± 3 (p > 0.05) with applica- glomerular filtrate is largely reabsorbed across a segment tion of cigarette smoke. of the tubular barrier which is permeable to electrolytes A method is described for extracting surface active as well as water, in answer to osmotic forces in the material, probably the alveolar lining layer, from the peritubular area. If this latter model is correct, then, as lung. Exposure to cigarette smoke causes a decrease pointed out by L. E. Bayliss, active transport of glucose in the surface tension of this material. Such a change should contribute importantly to peritubular osmotic ac- occurring in vivo would alter the stability of alveolar size tivity. Accordingly, 6 dogs were infused with normal favoring the development of overdistended alveoli. saline at the rate of 5 cc per minute. In some instances a priming dose of 150 cc 5 per cent sodium chloride was Observations on the Operation of Starling's Law of the infused over a 30 minute period. After several hours a Heart in Man. EUGENE BRAUNWALD, ROBERT L. FRYE, steady state of urinary output was achieved with output JOSEPH W. GILBERT and MAURICE AYGEN, Bethesda, of salt and water closely approximating input. Glucose Md. (introduced by Robert P. Grant). in a concentration of 2 or 3 per cent was then added to There has been considerable controversy concerning the saline infusion for 30 minutes followed by resumption the applicability of the Frank-Starling mechanism to the of normal saline infusion. Blood glucose was determined human heart. This investigation was designed to deter- as well as urine sodium, chloride, and osmolal concen- mine whether left ventricular end-diastolic fiber length trations. In all cases the output of sodium chloride and (EDFL) and end-diastolic pressure (EDP) are im- water decreased markedly (circa 20 to 40 per cent) dur- portant determinants of ventricular contraction. Systemic ing periods of hyperglycemia (circa 160 mg per 100 ml) arterial pressure and effective EDP were measured at returning to baseline outputs as blood glucose returned operation in 20 adult patients with rheumatic mitral valve to normal values. Inulin and PAH clearances appeared disease and atrial fibrillation. Continuous alterations of to show no significant changes. These data support the the length of a segment of left ventricular muscle were concept that a portion of glomerular filtrate is reabsorbed recorded simultaneously in 7 patients by means of a across some segment of the renal tubule permeable to mercury-filled resistance gauge sutured to the surface of electrolytes as well as water. In this area active reab- the left ventricle. The variability of diastolic length re- sorption of glucose contributes importantly to effective sulted in beat-to-beat alterations of ventricular filling peritubular osmotic activity. which resulted in variations of EDFL and EDP. For each individual beat the peak systolic ventricular pressure Electrical and Osmzotic Characteristics of the Isolated was utilized as an index of maximum tension developed, Turtle Bladder. WILLIAM A. BRODSKY* and THEODORE the area beneath the left ventricular pressure curve (ten- P. SCHILB, Louisville, Ky. sion-time index) was employed as a measure of the total Electrical potential difference (P.D.) and ohmic re- tension developed and the systemic arterial pulse pressure sistance across the isolated turtle bladder were measured. was utilized to indicate relative changes in stroke volume. Mounted in a chamber between two identical oxygenated In every patient alterations in EDFL and EDP corre- Ringer's solutions, the interstitial side was positive in an lated closely with changes in systolic pressure, tension- external circuit with respect to the mucosal side by 100 time index, and arterial pulse pressure. In order to de- to 120 mv, while the D. C. resistance (change of voltage termine whether these relationships also exist in intact for 1 to 10 pa of externally applied current) was 2,500 unanesthetized subjects, effective EDP was measured by ohms per 0.3 cm2 of tissue. Such levels of potential and means of transseptal left heart catheterization in 4 pa- resistance were maintained for 24 to 48 hours with the tients. The correlations of EDP with systolic pressure, tissue in oxygenated media. Nitrogenation of the bathing tension-time index, and pulse pressure were similar to solutions caused a decrease of P.D. to 60 to 65 mv, and those in obtained the patients studied at operation. These an increase of resistance to 4,200 ohms per 0.3 cm2 of results are consistent with the view that when filling is tissue, which levels were maintained for 4 to 6 hours of altered abruptly, Starling's law of the heart operates in nitrogen bubbling. We attempted to relate the electrical man and that EDFL and EDP are important determi- properties of the bladder with its ability to transport nants of the characteristics of the subsequent ventricular water and solute. Whole bladders, filled with 1/3 contraction. Ringer's, were immersed in full-strength Ringer's. Rate of weight loss of such "bladder bags" was 0.20 to 0.25 The Effect of Hyperglycemia Without Glycosuria on Salt g per hour per 10 to 30 g of total weight of bladder plus and Water Excretion. EMANUEL H. BRESLER and content. Osmotic activity of inside solution, initially JERRY N. GILMARTIN, New Orleans, La. (introduced 60 mOsm per kg, decreased to values of 40 to 50 mOsm by Grace Goldsmith). per kg after 4 to 24 hours of incubation. Addition of The forces involved in transtubular movement of vasopressin, 0.1 unit per 1 ml of external solution, ac- glomerular filtrate have not been adequately defined. It celerated the water efflux in one experiment, and induced is widely held that the tubular wall in the main forms an but little change in another. Dilution of the internal impermeable barrier with respect to diffusion of sodium fluid was observed with or without the hormone. This chloride. Recently, an older concept advanced by Lud- demonstrates directly a movement of solute (NaCl) AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 975 against the chemical potential gradient, and probably, sample was drawn. Estrone, estradiol-17,, and estriol the movement of sodium ion against the electrochemical were determined in plasma and urine samples by a chem- potential gradient. ical method which measures conjugated and unconjugated forms of each estrogen together. Serum Factors in Acquired Hemolytic Anemia in Pa- Mean clearance rates for estrone and estradiol-17,j tients with Lymphoma and Leukemia. JEROME I. BRODY were 15.6 ml per minute (SD 5.0) and 12.0 ml per min- and STUART C. FINCH,* New Haven, Conn. ute (SD 0.2), respectively. In contrast, the mean estriol The purpose of this study was to determine whether clearance was 296 ml per minute (SD 46.0). The mean or not the erythrocyte-coating globulin found in the sera U/P ratios of estrone, estradiol-17,8, and estriol were of patients with acquired hemolytic anemia could be char- 3.3, 2.5, and 64.2, respectively. The differences in clear- acterized as erythrocyte antibody, using the technique of ance rates are unlikely to be due to technical factors. immune adherence. This method was chosen because Recoveries of each of the 3 estrogens added to both immune adherence occurs only as a consequence of an urine and plasma were similar (75 to 85 per cent). antigen-antibody reaction and would not occur if the The low clearance rates of estrone and estradiol-17j3 globulin was not erythrocyte antibody. may be due to the physicochemical state of these hor- Sera obtained from lymphoma and leukemia patients mones in plasma, or possibly to glomerular filtration and with an indirect Coombs positive reaction were compared tubular reabsorption. The clearance rate of estriol on immunologically to ABO-specific isologous and heterol- the other hand is approximately twice average published ogous erythrocyte antibody. The technique consisted of figures for glomerular filtration rate in late pregnancy. incubating serial dilutions of test serum with known con- This is supported by simultaneous endogenous creatinine centrations of human red cells, human complement, and clearance rates obtained in some of our subjects. Thus, freshly washed guinea pig platelets. Immune adherence it appears that in late pregnancy some component of the of red cells to the indicator platelets developed in the total estriol of plasma is secreted by the renal tubule. presence of complement and red cell antibody. This pro- This component is likely to be estriol glucosiduronate. duced both macroscopic and microscopic flocculation of the red cells. It was found that either type of group Thyroidal Iodide Secretion. BELTON A. BURROWs,* D. specific erythrocyte antibody reacted strongly in immune WARD SLINGERLAND, ANTHONY Liuzzi, ELIZABETH adherence with normal red cells in this system. Coombs S. DELL and DOROTHY E. GRAHAM, Boston, Mass. positive red cells from patients with lymphoma and Thyroidal secretion of nonthyroxine iodide has been leukemia also reacted strongly in immune adherence in suggested by previous studies in normal subjects, hyper- the presence of rabbit antihuman globulin. These reac- thyroid patients, and experimental animals. In the pres- tions did not take place in the absence of complement. ent study discrepancies in the conventional model of In contrast, Coombs indirect positive sera from non- iodine turnover (iodide to thyroid iodine to circulating transfused patients with acquired hemolytic anemia did hormonal iodine) are demonstrated and the proportion of not react in immune adherence with either their own or nonthyroxine iodine released by the thyroid is estimated. group-compatible homologous erythrocytes. Intravenous tracer doses of radioiodine to These studies given hyper- demonstrate that in these patients with thyroid patients virtually disappeared within one day lymphoma and leukemia the erythrocyte-coating globulin from of the extrathyroidal iodide space, as shown by an acquired hemolytic anemia is not characterized as initial rapid decrease in both the plasma inorganic radio- erythrocyte antibody by the immune adherence technique. iodide concentration and the rate of urine radioiodide It is believed that these abnormal globulins, with affinity excretion. Therefore, after Day 1, radioiodide for the red appearing cell surface, are nonimmunologic and may in plasma or urine had made one or more passages represent a form of dysproteinemia. The data also indi- through the thyroid. For the next several days, plasma cate that heterologous and isologous group-specific red cell inorganic radioiodide concentration and the rate of uri- antibody reactions are complement-dependent in im- nary radioiodide even serum mune adherence. excretion decreased, though protein-bound radioiodine concentration increased two- fold. If the urine radioiodine were derived exclusively Renal Handling of Estrogens in Late Pregnancy. C. from the degradation of labeled thyroid hormone, the HARMON BROWN, BENJAMIN D. SAFFAN and JOHN rate of urinary radioiodine excretion could be expected R. K. PREEDY,* Atlanta, Ga. to increase, rather than decrease, with increased serum Analysis of urinary and plasma estrogen levels in late protein-bound radioiodine levels. Therefore, the con- pregnancy have indicated marked differences between the tribution to urinary radioiodine excretion of thyroidal U/P ratios of estrone and estradiol-17,3 on the one hand, radioiodine in some form other than thyroxine is indi- and of estriol on the other, suggesting differences in the cated. renal handling of these steroids. To investigate this fur- Urine iodine specific activity was as much as 3 times ther, 7 women in the thirty-sixth to fortieth week of greater than serum protein-bound iodine specific activity normal pregnancy were given sufficient water to establish during this interval, and showed considerable day to day a diuresis of 3 to 10 ml per minute. Urine was collected variation. With the administration of blocking doses of for 90-minute periods, in the middle of which a plasma Tapazole, this variation in urine iodine specific activity 976 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING was reduced, and the values were less than serum protein- necrotic cirrhosis, were tested for circulating antinuclear bound iodine specific activity to a degree consistent with globulins by the fluorescent antiglobulin technique. Fixed daily dietary increments of stable iodine. It would ap- homologous leukocytes in normal peripheral blood smears pear that prior to Tapazole, nonthyroxine iodide of vary- were the source of nuclear material. No attempt was ing specific activities, and in amounts at least comparable made to quantitate the titer of antinuclear globulins in to thyroxine iodine, was being released by the thyroid. this study. Of 10 patients with positive fluorescent antinuclear Mannose Mfetabolismn, an Index of Glucose Utilization. tests, 7 had nutritional cirrhosis and 3 had postnecrotic GEORGE F. CAHILL, JR., FRANCIS C. WTOOD, JR., BER- cirrhosis. The latter group included 2 women with NARD LEBOEUF and ALBERT E. RENOLD,* Boston, Mass. transiently positive lupus erythematosus (L.E.) cell prep- Intravenous administration of D-mannose or D-glucose arations who had been clinically designated "lupoid to normal humans (0.5 g per kg given in 3 to 4 minutes) hepatitis." Of particular interest was the finding that resulted in similar disappearance rates. Injection of 7 (50 per cent) of the 14 women in this series had posi- insulin (0.1 unit per kg) with mannose increased equally tive antiglobulin tests as compared to only 3 (17 per the disappearance rates of both endogenous glucose and cent) of the 18 men. infused mannose. Injection of mannose, unlike glucose, As a group, the patients with positive tests did not did not increase serum insulin activity as measured by differ clinically from the others in any respect, including adipose tissue bioassay (in agreement with pancreatic in- history of alcoholism when present, duration and severity fusion data from experimental animals). Thus, mannose of liver disease, age or physical findings. Liver function is "insulin-responsive" similar to glucose, but unlike glu- tests, serum protein levels and total leukocyte counts cose, does not stimulate the fl-cell to secrete insulin. were also comparable in both groups but the latex fixa- Since there is no significant endogenous source of free tion test was positive in every case with a positive anti- mannose, mannose disappearance is an index of removal nuclear test. alone, and being insulin-responsive, reflects insulin These findings indicate that an abnormal circulating activity. Recycling as with C14-labeled substrate is globulin with nuclear affinity may be present in the sera obviated. of certain patients with chronic liver disease. This fac- Prolonged mannose infusions in two normal humans tor was demonstrable in both patients with positive L.E. (0.5 g per kg per hour for 10 hours) resulted in a cell preparations. In the present study antinuclear globu- marked and persistent reduction in blood glucose to 10 lins were encountered more frequently in women. and 23 mg per 100 ml (without hypoglycemic symptoms, since brain presumably utilizes mannose) suggesting a The Effect of Partial and Total Denervation of the Heart marked suppression of hepatic glucose output. Transient on Left Ventricular Function. CARLETON B. CHAP- elevation in serum bilirubin was noted without altera- MAN,* ToM A. BRUCE, ORLAND BAKER and JOSEPH N. tion in other hepatic function tests. In a mild diabetic, FISHER, Dallas, Tex. blood glucose levels were suppressed by a 5 hour mannose The role of intrinsic myocardial reactivity, the central infusion, while in a severe ketotic diabetic off insulin, feature of Starling's Law, is difficult to assess if cardiac there was no in blood change glucose. Disappearance innervation is intact. For this reason cardiac sympa- rates of mannose acutely infused into diabetics were de- thectomy (removal of both chains from middle cervical pressed similar to those of glucose, reflecting a decreased ganglion to T-5) was done in 4 normal dogs. Ten days utilization of both sugars. In two patients with Cush- later, LV volume and pressure curves were recorded ing's at syndrome, mannose levels fell at a considerably rest and during light exercise in intact anesthetized ani- faster rate than administered glucose, suggesting that mals using biplane cinefluorographic methods. Bilateral the level of the latter was sustained by accelerated hepatic vagal section was then done and the studies repeated. gluconeogenesis. Thus mannose is able to serve as a Three dogs were studied before and after vagal section "marker" for glucose "utilization" or "turnover." alone. Additional studies with mannose-C14 in isolated tissues In unoperated anesthetized animals (9 dogs), exercise corroborated the above findings, namely, a parallel de- produced 22 and 6 per cent increase in pulse rate and crease in glucose and mannose metabolism in diabetic stroke volume, respectively. Cardiac output and minute tissues including adipose tissue, kidney and liver. Insulin work rose 33 and 51 per cent, in vitro respectively. With total equally stimulated glucose and mannose metab- cardiac denervation, exercise produced no change in pulse olism in adipose tissue as measured by recovery of label rate, which remained at 125, but stroke volume rose in C02, 21 fatty acids and glyceride-glycerol. per cent. Cardiac output and minute work increased 18 and 26 per cent, respectively. Vagectomy alone caused Circulating Antinuclear Globulins in Patients Iwith marked resting tachycardia; exercise produced modest Chronic Liver Disease. PAUL CALABRESI and MORTI- further increase in rate (180 to 187) and in stroke vol- MER GREENBERG, New Haven, Conn. and Boston, Mass. ume. Similar changes occurred with sympathectomy (introduced by S. R. Lipsky). alone except that rates were lower (108 to 115) and in- The sera of 32 patients with chronic liver disease, 24 crease in stroke volume larger. Decrease in end-diastolic alcoholics with cirrhosis and 8 individuals with post- volume during exercise occurred in normal dogs but AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 977 slight increase was the rule with partial or total denerva- dog brain thromboplastin produces a prompt and severe tion. reduction in fibrinogen, Factor V, prothrombin, anti- Although total denervation depresses cardiac response hemophilic globulin, and platelets. When the same quan- to exercise in absolute terms, considerable increase in tity of thromboplastin was given to heparinized dogs no stroke volume and work is still possible. Increase in significant decrease in these coagulation factors occurred. rate, the dominant mechanism in normal dogs, makes no Histologic sections of lung tissue showed fibrin deposits contribution if the heart is denervated. The behavior of in small vessels of the lung in the dogs receiving only end-diastolic volume during exercise in denervated ani- thromboplastin, while none was observed in dogs which mals opposes the view that residual response is due to were heparinized prior to the thromboplastin infusion. adrenal medullary release. The most likely stimulus to The amount of intravascular fibrin deposition was greatly the left ventricle under these circumstances is increased increased in dogs which were given homologous fibrin- filling. ogen during the thromboplastin infusion. Patients with visceral neoplasms demonstrating ab- Erythropoiesis and Blood Destruction in Congestive normalities of the coagulation mechanism due to in- Heart Failure. ROBERT B. CHODOS, WILLIAM CHAFFEE travascular clotting were studied. One patient with and RAYMOND WELLS, JR., Syracuse, N. Y. (introduced hemorrhagic manifestations and three patients with by B. V. Jager). thromboembolic phenomena had low fibrinogen (110 to 150 mg). Additionally, in these patients, other coagula- The red cell volume, as well as the plasma volume, tion abnormalities of variable degree included platelet may be increased in congestive heart failure. Following counts as low as 90,000 per cu mm, prothrombin levels compensation, red cell volume may decrease in certain to 40 per cent, and Factor V levels to 40 per cent. In patients. The mechanism responsible for this decrease vitro evidence for increased fibrinolysis could not be has not been demonstrated. Studies have therefore been demonstrated in any of the patients. When given undertaken to determine whether this decrease is due to parenteral heparin there was significant improvement or depression of erythropoiesis, an increase in destruction of complete return to normal of these alterations in a period red cells, or a combination of these processes. of 3 to 7 days. Relapse occurred in the two patients in Red cell and plasma volumes were measured inde- whom heparin was discontinued. pendently during failure and after compensation, employ- It is concluded that heparin therapy can control the ing radiochromium (or radiophosphorus) and Evans manifestations of intravascular clotting and that fibrin- blue methods, respectively. Erythropoiesis was esti- ogen administration alone might be hazardous if throm- mated by radioiron turnover techniques. Destruction of boplastic substances are continuing to enter the circula- red cells was evaluated by radiochromium red cell sur- tion. vival and by estimation of fecal urobilinogen excretion. Red cell volume decreased significantly in 5 of 10 pa- Serial Changes in Body Water, Exchangeable Sodium, tients studied, plasma volume in 6 of 10, and total blood Exchangeable Potassium and Serum Electrolyte Concen- volume in 8 of 10. There was no evidence of increased trations During the Treatmient of Ascites. BERNARD blood destruction. cell survival was normal in all Red F. CLOWDUS, JOHN A. HIGGINS, A. L. ORVIS and W. patients and there was no increase in fecal urobilinogen. H. J. SUMMERSKILL, Rochester, Minn. (introduced by During cardiac decompensation, the half-time of plasma H. R. Butt). iron disappearance was rapid (0.33 to 1.15 hours) and plasma and red cell iron turnover were both consistently Measures used in treating ascites have been held re- increased (43 to 182 mg per day). After compensation sponsible for sodium and potassium depletion, with sub- the half-time of plasma iron disappearance increased sequent hyponatremia, uremia and coma, in patients with (1.0 to 2.0 hours) and plasma and red cell iron turnover liver disease. Serial determinations of total body water both decreased strikingly. Control studies in normal (TBW), exchangeable sodium (NaE), and exchangeable subj ects did not show significant changes in iron turn- potassium (KE) in relation to serum electrolyte concen- over. trations were, therefore, made in 10 patients with cir- These data indicate a definite decrease in iron turnover rhosis and ascites during therapy. The latter comprised following compensation from congestive failure. It is a low sodium diet and treatment with diuretics (mercap- suggested that the mechanism responsible for any de- tomerin, chlorothiazide, prednisone and/or Aldactone). crease in red cell volume is a compensatory decrease in External sodium and potassium balances were carried out erythropoiesis. in 9 instances. Before treatment, TBW and NaE were high in every Correction by Heparin of Coagulation Abnormnalities Due patient; in 4 patients judged to be free from excess fluid to Intravascular Clotting. DALLAS V. CLATANOFF, after therapy, mean initial values were 13 and 50 per cent, Madison, Wis. (introduced by Robert F. Schilling). respectively, above the final readings. During treatment, Low fibrinogen may be the result of intravascular weight loss followed water loss, but the latter correlated clotting with or without demonstrable increase in fibrino- poorly with sodium loss; the ratio of sodium to water lytic activity. The intravenous infusion of homologous (NaE/TBW) varied widely, both initially (100 to 72 978 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING mEq per L) and in its change with treatment (+ 1.4 to Conjugation of Sulfobromophthalein Sodium (BSP) -23.0 mEq per L). KE and potassium balance did not with Glutathione by an Enzyme in the Supernatant alter significantly throughout the studies. Fraction of Liver. BURTON COMBES and GENEVA SUE Serum sodium (Nas) and potassium (Ks) concentra- STAKELUM, Dallas, Tex. (introduced by Gladys tions had no direct relationship to NaE and KE, and Fashena). sodium or potassium depletion was not observed. As changes in NaE correlated with the external sodium Conjugation of BSP with glutathione or cysteine, in bal- the liver, is now considered to account for the ance, fluctuations in Nas appeared to reflect ninhydrin- movements reacting BSP compounds excreted in bile of of sodium between intracellular and extracellular com- man, rat and dog. The present studies indicate that partments, rather than its "sequestration" in or "mobiliza- conjugation of BSP is an in the tion" from a catalyzed by enzyme supernatant frac- the skeleton as result of treatment. tion of Hyponatremia uremia in liver. Furthermore, glutathione is by far the and patients with ascites were preferred substrate. associated with high rather than low NaE values. Low- Incubation of BSP with rat liver normal NaE values were found only after effective treat- homogenate (prepared in 0.1 M phosphate at 370 in room ment with an aldosterone antagonist, and in two patients buffer, pH 7.8) C, air, yields a ninhydrin-reacting BSP compound identical slight hyponatremia and uremia developed under these circumstances. chromatographically and electrophoretically with the major BSP compound in rat bile. When S3`-labeled glutathione is added to the homogenate-BSP mixture, The In Yivo Glucuronide Conljutgation of Radioactive radioactivity is incorporated into the synthesized BSP Steroids by the Dog Kidney. GEORGE L. COHN and compound. Hydrolysis of this compound (5.9 N HCl, MICHAEL HUME, New Haven, Conn. (introduced by 1000 C, 16 to 18 hours) yields glycine, glutamic acid David Seligson). and alanine. The latter has been shown to be cysteine It has been established that the liver is the main site minus its sulfhydryl group. It is concluded, therefore, of steroid glucuronide formation, but it is not known that the compound formed is a BSP-glutathione conju- whether or not the kidney can carry out a similar func- gate. Conjugate is not formed by liver homogenates tion. Dutton has observed that kidney tissues contain previously heated at 1000 C for 5 minutes. All of the enzyme systems which can form glucuronides. In order enzyme activity can be accounted for in the supernatant to test the hypothesis that the dog kidney can form fraction of liver; none is demonstrable in microsomes or glucuronides in vivo, the following experiments were mitochondria. carried out. With a Potts clamp on the renal vein at Capacity to synthesize conjugate was restored to super- its junction with the inferior vena cava, radioactive natant fraction previously dialyzed in distilled water or 17-ketosteroids were injected into the renal artery and phosphate buffer, by the addition of glutathione, cysteinyl blood was obtained simultaneously from the catheterized glycine or cysteine. The relative quantity of conjugate renal vein and internal jugular vein. Urine formed dur- formed by rat liver with equimolar amounts of each of ing the course of the perfusion was also collected. these substrates was 15: 2: 1, respectively. BSP conju- Nephrectomy was carried out at the conclusion of the gate was not formed with glycine, glutamic acid or experiment. Steroids were isolated by organic solvent alanine. No requirement for cofactor has been demon- extraction, paper electrophoresis and paper chromatog- strated. raphy. It was found that during the perfusion of the Conjugating enzyme has also been found in homog- radioactive steroid, a small proportion was converted to enates of liver obtained from man, dog, guinea pig, mouse the glucuronide. When a tracer quantity of etiocholano- and cat. The observation that glutathione is the optimal lone-H8 was injected, approximately 8 per cent was found substrate in these species provides strong evidence that in the free fraction, while 16 per cent was converted to conjugation of BSP with glutathione is the major path- the glucuronide. When androsterone-H' of the same way of BSP metabolism. specific activity was injected, 28 per cent was found in the free and 14 per cent in the conjugated form. Some Studies on the Nature of Quinidine-Albuonin Inter- The glucuronides were characterized by their mobility action. HADLEY L. CONN, JR.* and ROBERT J. LUCHI, on paper electrophoretograms and paper chromatograms Philadelphia, Pa. using pure reference standards, and by hydrolysis with The fundamental nature of quinidine action on the 0-glucuronidase and subsequent paper chromatography heart remains unknown. From earlier data, the authors of the free 17-ketosteroids. Radioactivity measurements have postulated that the prime tissue reaction of quinidine were performed throughout the procedures. No sig- is a selective binding to certain protein sites. Some of nificant radioactivity could be found in the urine, internal these contribute to the normal "binding" of ions. If the jugular vein blood, or in the organic solvent extractions binding involves a "critical" cell protein, the result is a of the kidney homogenates. No sulfates or phosphates pharmacologic or toxic effect. If it involves a "non- were detected. It is concluded that the dog kidney can critical" protein such as albumin, drug activity is re- convert 17-ketosteroids in vizo to their respective glu- duced. As a start toward collecting data pertinent to curonides. The implications of these experiments will this hypothesis, the nature of quinidine-albumin interac- be discussed. tion was studied in a dialysis equilibrium system. AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 979

The results showed a binding of 1 mole of quinidine Transmnangaitin, the Specific Manganese-Carrying Protein per mole of albumin at pH 7.4 and an association constant of Human Plasma. GEORGE C. COTZIAS* and ALBERT of 1.3 x 104, indicative of tight and rather specific bind- J. BERTINCHAMPS, Upton, N. Y. ing. With increasing pH, binding increased. Further- We have shown earlier that the body's manganese exists more, at pH 10 the mole ratio became 3 quinidine to 1 largely as readily dissociable complexes. In spite of albumin, suggestive of alteration in albumin structure. loose binding, manganese follows a highly specific path- Ionic effects in addition to that of hydrogen ion were way leading chiefly through the mitochondria. The investigated. Chloride weakly and calcium ions strongly specificity of this element's behavior in vivo was dis- interfered with quinidine-albumin binding. Studies of cordant with the lack of specificity it displayed in vitro. competition for albumin sites between various fragments This discord suggested the existence of a specific manga- of and the total quinidine molecule revealed that the two nese-carrying plasma protein (transmanganin). major quinidine binding sites involve the quinoline ring Bound Mn5 and Fe' localized on the p,-globulins elec- nitrogen and the bridging secondary alcohol. The reac- trophoretically. The metals exchanged with their own tive sites in albumin were investigated by studying bind- isotopes but not with each other, a fact confirmed with ing after preliminary combination of the protein with ultrafiltration. Purified transferrin was avid for iron, various compounds known to react with specific groups. not for manganese. The results suggest that the albumin sites involved are The preponderant valence of transmanganin-bound a lysine e amino group and a spatially adjacent carboxyl manganese was intermediate between valences 2+ and 4+. group. Therefore most probably it is valence 3+, which is un- Conclusions are that 1) albumin-quinidine binding is stable when not bound. This was shown by extensive of a rather specific nature; 2) in so far as this reflects tests employing aerobiasis, anaerobiasis, oxidation and re- interference with ion metabolism, hydrogen and calcium duction for breaking and formation of the complex. Mag- seem most affected. It is of special interest to discover nesium competed only with divalent manganese. whether quinidine-albumin binding is a prototype of all Serial ultrafiltrations of the same sample (fractional quinidine-protein interactions. ultrafiltration) showed a nonartifactual, \striking, pro- gressive fall of the Mn5 concentration in the ultrafiltrates. Stimulation of Steroid Formation of Adrenocortical On the basis of the mass law, this meant the linking of Homogenates by Aledulla and Catecholamines. D. Y. more than one globulin molecule per manganese atom. COOPER, OTrO ROSENTHAL and W. S. BLAKEMORE, Similar behavior was displayed by insulin-bound Zn', Philadelphia, Pa. (introduced by Robert E. Forster). plasma- and transferrin-bound Fe59. Control systems In a study of in vitro corticosteroid synthesis by using some of these same metals showed the classical adrenal slices we noticed that slices containing medulla stability on fractional ultrafiltration. and cortex produced more steroids than slices containing These experiments reconcile the int vitro with the in cortical tissue alone, although medullary slices by them- vivo behavior of manganese: the physiologically trans- selves were inactive. To explore the mechanism of this ported trivalent manganese necessitates stabilization by phenomenon we have studied the effect of medullary binding to transmanganin. This globulin exemplifies a homogenates upon conversion of progesterone to 17,21-di- novel group of metalloproteins in which the metal binds hydroxycorticosteroids (Silber-Porter chromogens) by more than one protein molecule. The ambient redox po- bovine cortical homogenates in the following system: tential controls association and dissociation, thus deter- Krebs Ringer-bicarbonate-2 per cent albumin (7 ml); mining specificity and direction of transport. Such be- progesterone (1 mole) ; glucose-6-phosphate (24 havior should be looked for intracellularly (where moless; TPN (2.4 Iumoles); cortical homogenate (ap- manganese concentrates) in order to define the bio- proximately 100 mg) ; and gas phase 5 per cent C02 in chemical specificity of this important cofactor. 02; 370 C; 4 hours. Addition of 15 mg of medulla in- creased corticoid formation by 30 to 100 per cent. Larger Red Cell Shape and Red Cell Life Span in Hereditary additions (up to 120 mg) produced additional though Spherocytosis. WILLIAM H. CROSBY* and MARCEL E. smaller increments. The supernatant fraction of centri- CONRAD, Washington, D. C. fuged medullary homogenate from 15 mg tissue was as Subj ects of the study were two healthy young men effective as the whole homogenate. However, larger with hereditary spherocytosis (HS). Both had a well amounts did not produce additional increments. Cate- compensated hemolytic disease (short red cell life span cholamines (0.3 molee of epinephrine, norepinephrine or without anemia). They were phlebotomized each week isoproterenol) produced stimulation indistinguishable until they developed iron deficiency anemia and their from that of the medullary supernatants. These results erythrocytes became hypochromic and flattened. The ab- show that acceleration of corticoid formation by medul- normal behavior of the HS red cells in fragility tests was lary tissue is due in part to catecholamines. Paper greatly improved and in one case became normal. How- chromatographic analysis of the hydroxylation products ever, the short life span of the cells was not improved by of progesterone, 17a progesterone, 1 1-desoxycorticoster- the change in shape. Ti of Cr51-labeled red cells before one and 11-desoxycortisol indicate catecholamines enhance and after phlebotomy was 9 days in one patient and 15 hydroxylation at C-21 and perhaps C-17 but not at C-11. days in the other. Later, splenectomy corrected the 980 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING hemolytic disease in both patients. In one of them the Hormonal Control of Collagen Synthesis by Cartilage iron deficiency was not treated, but after the operation the Studied In Vitro. WILLIAM H. DAUGHADAY* and IDA hypochromic cells survived normally in his circulation. KOZAK MARIZ, St. Louis, Mo. In the other one the administration of iron restored the Collagen and chondroitin sulfate, as chondromucopro- red cells' spheroidal shape, and they too survived normally tein, are the principal extracellular products of growing after splenectomy. chondroblasts. Cartilage from hypophysectomized rats The shape of the red cell in HS does not appear to be forms chondroitin sulfate at a reduced rate and this has responsible for its premature destruction by the spleen. been demonstrated in an in vitro system. Moreover, syn- Iron deficiency corrects the spherocytosis but it does not thesis is greatly increased by adding normal serum to the correct the hemolytic disease; splenectomy corrects the medium. To study the parallel process of collagen syn- hemolytic disease but it does not correct the spherocytosis. thesis, the conversion of proline to collagen hydroxy- proline has been measured. No other source of this Physiological Studies in a Family with Nephrogenic unique amino acid is known. (Vasopressin-Resistant) Diabetes Insipidus (N.D.I.). Pools of 15 costal cartilage segments were incubated in RALPH CUTLER, CHARLES R. KLEEMAN,* J. THOMAS enriched medium containing essential amino acids (Eagle) DOWLING and MORTON H. MAXWELL, Los Angeles, and proline-U-C'4. After 24 hours, cartilage hydrox- Calif. yproline was isolated in satisfactory radiopurity by Dowex-50 chromatography. The hydroxyproline from Three adults and one child (age 11) of a family with cartilage of young rats, hypophysectomized 2 to 3 weeks hereditary nephrogenic diabetes insipidus (13 per cent previously, had a specific activity of half that obtained affected members in 7 generations) were studied. from normal rats. In other experiments, costal car- Urinary output averaged 15 to 20 and 3 L per day in the adults and child, respectively. Maximal urinary osmolali- tilages from 6 hypophysectomized rats were distributed randomly into four flasks; the medium for two flasks ties remained markedly hypotonic (60 to 100 mOsm per contained 18 per cent normal rat serum and for the other kg) during vasopressin and 5 per cent saline infusions. rat serum. Hydrox- Dehydration (2 kg weight loss) did not enhance maxi- two, 18 per cent hypophysectomized yproline isolated from cartilage incubated with two mum osmolalities except in the child who attained 442 separate pools of normal serum had a mean specific mOsm per kg. Cortisol administration (180 mg daily activity of 68 per cent (range 25 to 106) greater than x 5) augmented all parameters of maximal water diuresis (M.W.D.); isoncotic expansion of plasma volume did hydroxyproline from incubations with hypophysectomized rat serum. The results have been corrected for slight not augment M.W.D. differences in initial medium proline pools determined by A 20 to 50 per cent reduction in GFR caused marked antidiuresis with minimal increase in urinary osmolality. isotope dilution chromatographic methods. Conversion of medium proline to cartilage hydroxyproline was 1.7 in one adult (upright position) a hexa- During M.W.D. and 1.7 ug per 100 mg of cartilage in two incubations methonium infusion decreased GFR to less than 15 per with hypophysectomized rat serum and 2.6 and 3.0 with cent of and urinary volume to 0.5 ml per minute normal normal rat serum. without producing hypertonic urine (max. osm 270). In These changes in hydroxyproline formation in vitro are adult cases (1.5 g per day) caused a 50 to chlorothiazide evidence of impaired collagen synthesis after hypophys- 75 per cent decrease in daily urinary volume without ectomy and its partial correction by humoral factors in producing hypertonic urine (max. osm 174), a reversal normal serum. It is unknown whether the same factors of the normal diurnal pattern of water execretion, and at stimulate both collagen and chondroitin sulfate synthesis. least a 30 per cent reduction in GFR. When the nega- tive salt balance induced by chlorothiazide was prevented Effect of Antihypertensive Treatment in the Rat on the by a high salt intake daily, urinary volumes did not de- Potentiation of Atherogenesis by Experimental Hyper- crease. Chlorothiazide did not enhance the renal (tubu- tensio~n. QUENTIN B. DEMING,* MARIE M. DALY, lar) response to vasopressin infusions. Conclusions: 1) In the adult subjects, dehydration did JAIME BLOOM, LILI BRUN and RUTH KAPLAN, New not concentrate urine greater than vasopressin. 2) York, N. Y. Chronic cortisol administration augments M.W.D. in In man, dog, rabbit and rat, the hypertensive state is absence of any tubular response to vasopressin, while accompanied by increased severity of atherosclerosis. plasma volume expansion did not augment M.W.D. 3) Whether nonspecific control of blood pressure would In N.D.I. a hypertonic urine could not be produced by a prevent this effect has not been known. marked GFR reduction during M.W.D. This contrasts In the present experiments, atherosclerosis has been with animal results (Berliner et al. J. clin. Invest. 1956, produced in rats by suppression of the thyroid combined 35, 690) and in vasopressin-sensitive diabetes insipidus with addition of cholesterol and cholic acid to the diet. (Kleeman et al. Proc. Soc. exp. Biol (N. Y.) 1957, 96, Hypertension has been produced by constriction of one 189). 4) Chlorothiazide-induced antidiuresis in N.D.I. renal artery. Operated animals whose blood pressures is probably due to altered body sodium and renal hemo- failed to rise have been used as normnotensive controls. dynamics rather than to a direct effect on the concentrat- In the same length of time on diet, hypertensive rats de- ing segment of the nephron. velop more extensive atherosclerosis than normotensives. AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 981

Comparison was made between normotensive animals, scribed. Of two untreated patients with documented hypertensive animals and hypertensive animals rendered postpartum pituitary necrosis, one had two adrenal crises normotensive by adjusted doses of reserpine, hydralazine, two years ago, with hyponatremia and shock precipitated mecamylamine and chlorothiazide given concurrently. by chronic bleeding and water intoxication, respectively. The same dietary program was maintained in the three Urinary aldosterone, determined by double isotope deriva- groups. tive assay (normal range 5 to 20 jug per day), was 0.6 Pharmacological control of established renal hyper- and 2.8 ,ug on normal salt diet prior to therapy; only 5.0 tension resulted in a degree of atherosclerosis significantly and 8.0 lig in two studies on 10 mEq Na diets plus 25 mg less than that occurring in matched, untreated hyperten- cortisone, during which adequate Na retention occurred sive animals and approximating that in untreated normo- without decompensation; and 10.0 and 15.0 /ig on ACTH, tensive animals. which also increased urinary steroids to normal levels. The patient did well on cortisone, thyroid and liberal salt Serum Thrombotic Accelerator (STA) Activity as a intake. Persistent aldosterone defect was highlighted by a Measure of the Antithrombotic Effect of Dicumarol. recent crisis off cortisone during which urinary aldoster- DANIEL DEYKIN, STANFORD WESSLER* and STANLEY one was only 1.0 Ag. Normal salt balance is now main- M. REIMER, Boston, Mass. tained on ACTH alone. The second patient had hypo- natremia and coma with aldosterone value of only 1.2 ,ug Experimentally-induced intravascular thrombosis can per day but now maintains sodium balance on 25 mg be initiated through the "intrinsic" clotting system (in the cortisone alone. A third patient with breast cancer and absence of tissue thromboplastin). The administration of cranial metastases had mild hypopituitarism after an- coumarin-type drugs results in the depression of clotting drogen therapy and achieved aldosterone levels before factors known to be required components of this pathway. hypophysectomy of only 3.5 Atg on low salt diet despite There is no experimental evidence, however, to indicate normal Na retention, and 6.5 Ag on ACTH. Postopera- that these drugs can inhibit in vivo thrombosis mediated tive hyponatremia required both cortisone and mineral- through this intrinsic system. The quantitative bioassay ocorticoid therapy, and withdrawal of either steroid has of the STA activity of thrombin-free mammalian serum, precipitated mild adrenal crises during which aldosterone previously demonstrated in this laboratory, is a measure values were 2.5 and 2.7 ,ug, respectively. These studies of experimental in vivo thrombosis mediated through the show preservation of sodium homeostasis in hypoaldo- intrinsic pathway. This assay permitted exploration of steronism by some protective or potentiating action of the intrinsic system as a possible site for an antithrom- cortisone. Although basal aldosterone secretion may botic action of Dicumarol. Nine dogs received Dicumarol continue in hypophyseal disorders, increased secretion may -in doses adequate to maintain the one-stage prothrombic require aldosterone-stimulating actions of ACTH, the activity at less than 10 per cent of the control values for absence of which may result in relative hypoaldosteronism 2 to 48 days without inducing hemorrhage in any animal. and sodium loss in hypopituitarism during stress. Three dogs served as controls. The following parameters were followed serially in all animals: weight, temperature, hematocrit, in vitro assays of one-stage prothrombic Enzymatic Activity of Isolated Kidney Glomeruli in Two activity, clotting factors II (prothrombin), V (ac- Types of Experimental Nephrosis. ULRICH C. DUBACH globulin), VII (convertin), VIII (antihemophilic globu- and LILLIAN RECANT,* St. Louis, Mo. lin), IX (PTC), X (Stuart), Hageman and PTA; and In nephrosis, attention has been focused upon specific an in vivo assay of STA activity. A statistically sig- alterations in glomerular structure. The foot process nificant antithrombotic effect as measured by the loss of lesion identified by electron microscopy has been found in STA activity was observed in the sera of 8 of the 9 many varieties of this syndrome. Since it seemed pos- treated animals. This was related, in part, to the dose of sible that specific enzymatic alterations might be associ- anticoagulant and was independent of the depression of ated with this lesion, microbiochemical analyses of single any clotting factor, singly or in combination, known to be glomeruli from normal nephrotic animals were made. affected by Dicumarol. This is considered to be the first Previous reports from this laboratory have described experimental demonstration of an in vivo antithrombotic certain enzymatic activities in the normal glomerulus. action of Dicumarol mediated through the intrinsic In fully established aminonucleoside nephrosis, enzymatic clotting system. analyses revealed a decrease in alkaline phosphatase and an increase in glucose-6-phosphate dehydrogenase activity. In the present study, an attempt was made to trace the Hypoaldosteronism in Panhypopituitarism. JOSEPH F. development of the changes in these enzymes in pre- DINGMAN, EDUARDO GAITAN, MAX C. STAUB, AKIRA proteinuric (6 animals) and proteinuric (9 animals) ARIMURA and RALPH E. PETERSON,* New Orleans, La. phases of aminonucleoside disease. Similar studies were and New York, N. Y. made of antikidney serum nephrosis (6 animals). In ad- There are several reports of hypoaldosteronism with dition, enzymatic analyses of liver and pancreas were selective adrenocortical insufficiency but hypoaldosteron- carried out to assess specificity of renal enzyme changes. ism from adenohypophyseal destruction has not been de- During maximal proteinuria in aminonucleoside nephrosis, 982 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING glomerular phosphatase decreased 40 per cent (mean This may mean that the protein moiety and Mg compete normal 3.12 moles per kg dry weight per hour), while for the same binding sites. dehydrogenase increased 40 per cent (mean normal 1.15 moles per kg per hour). On the fourth and seventh day Studies Suggesting the Prescsnce of an Extrahypothalanmic of aminonucleoside injection (prior to proteinuria), ACTH-releasing Center in the Dog. RICHARD H. neither phosphatase nor dehydrogenase activities were EGDAHL, Richmond, Va. (introduced by David M. altered. Nephrosis induced with antikidney serum pro- Hume). duced similar changes. Enzymatic analyses of kidney homogenates showed changes similar to those noted in It is established that properly placed hypothalamic glomeruli in both types of nephrosis. Analyses of liver lesions markedly diminish or prevent the increased adrenal and pancreas homogenates were variably altered in both cortical secretion following operative trauma. The present types. experiments provide evidence that another, lower, CNS These observations indicate that two etiologically differ- area has potential for regulatory function in ASTH re- ent nephroses result in specific, similar enzymatic activity lease, and that this center is normally inhibited in the changes for glomeruli as well as for whole kidney homo- intact animal by nervous impulses from the cerebral genates. These findings appear to correlate with the mag- cortex. Three types of CNS operative procedures were nitude of proteinuria. It is suggested that they represent performed in dogs with chronic adrenal vein cannulas: the "deleterious" effects of continued protein transport total brain removal down to the inferior colliculus with through the glomerular structures. intact pituitary ("isolated pituitary") ; midbrain tran- section; bilateral decortication. Adrenal venous content Bindinq of ilfagnesium to llficrosonmal Nucleoprotein of 17-hydroxycorticosteroids was determined by the (RNAP) and Ribonuclic Acid (RNA). I. S. method of Nelson and Samuels, and adrenal venous out- EDELMAN,* P. 0. P. Ts'o and J. VINOGRAD, San put per minute was calculated. Francisco and Pasadena, Calif. Results were as follows: 1) All dogs with "isolated pituitaries" revealed elevated "resting" corticosteroid out- Thorough understanding of the mechanisms of amino puts 24 to 72 hours after surgery, and in two-thirds of acid assembly in RNAP in the process of protein syn- these animals nerve stimulation resulted in further in- thesis requires detailed knowledge of the structural or- creased adrenal cortical secretion. 2) Decorticate dogs ganization of microsomes. Earlier studies have shown demonstrated "elevated" resting levels of adrenal cortical that the structural integrity of microsomal RNAP is secretion. Nembutal anesthesia prevented the high rest- critically dependent on the attachment of an optimal num- ing levels but left intact the increased adrenal cortical ber of Mg ions in the particle. We therefore undertook secretion following nerve stimulation. 3) The response to study the details of the binding of Mg to RNAP and of dogs with midbrain transaction was similar to that of RNA. the "isolated pituitary" dogs. Microsomal particles were isolated by differential It is concluded: 1) High resting levels and responses ultracentrifugation from reticulocytes obtained from rab- to stimulation in "isolated pituitary" dogs indicate that bits with phenylhydrazine anemia. The RNA was ex- there is a lower CNS ACTH-releasing center, probably tracted from the same lot of microsomal RNAP. Ex- located in the hindbrain. A neurohumor (HBF) which change studies were carried out with Mg28 using dialysis results in ACTH secretion by the pituitary must be chambers in one set of experiments and serial ethanol released from this area, inasmuch as the anterior precipitation in another. Mg content was determined by pituitary is not innervated. 2) Decorticate dogs have eriochrome-EDTA titration and RNA content by ultra- high resting corticosteroid outputs because the cerebral violet absorption and by phosphorus (P) analysis. Par- cortex is a principal inhibitor of the HBF-releasing area. ticle size and homogeneity were evaluated by analytical The latter area can be depressed with Nembutal but a ultracentrifugation. normal response to nerve stimulation is present because Mg bound to RNAP and to RNA exchanged com- of an intact hypothalamic mechanism. 3) Results of pletely with buffer Mg28 in 30 minutes at 30 C. In 0.14 midbrain transaction experiments indicate that cerebral M KCl, 0.0015 M MgC12 buffer the Mg :P molar ratio inhibition of the HBF-releasing area is effected through was 0.18 and 0.23 for RNAP and RNA, respectively. In nervous and not humoral pathways. buffer of low K:Mg ratio, ribosomes consisted mostly of 80S particles which dissociated into subunits in buffer of high K:Mg ratio. Three lines of evidence were obtained Bone Mlagnesiumi, Calcium and Citrate Interrelationships. to indicate that all of the Mg is bound by the RNA LEONARD P. ELIEL,* JOSEPHINE HAWRYLKO and JOHN moiety: 1) the kinetics of Mg exchange were indis- COMSTOCK, Oklahoma City, Okla. tinguishable in RNAP and in RNA, 2) the Mg2" bound Hypoparathyroid patients or intact dogs, given para- to RNAP was quantitatively recovered in the RNA thyroid extract, and patients with malignant osteolytic moiety and, 3) the Mg :P molar ratios of RNAP and disease, display losses of Mg and Ca in a much higher RNA showed a parallel dependence on the buffer Mg Mg/Ca ratio than exists in bone. Blood and urine concentration. Moreover, removal of the protein moiety citrate generally parallel the changes in Mg and Ca uncovered about 30 per cent more binding sites for Mg. balances in these conditions. Tissue analyses have not AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 983 revealed the site of Mg depletion. Dog bone and syn- linoleic and palmitic acid into the cell was largely depend- thetic hydroxyapatite have been incubated with dog ent upon anaerobic glycolysis, since iodoacetic acid and plasma or synthetic plasma ultrafiltrate to clarify the sodium flouride inhibited uptake of labeled fatty acids, relationships between extracellular fluid Mg, bone Mg, whereas dinitrophenol and KCN had little or no effect. bone Ca, and citrate concentration. No transport occurred during incubation at 40 C. Meta- Powdered dog bone (1 g) incubated with plasma or bolic utilization of accumulated linoleic-1-C" and palmi- ultrafiltrate (10 ml) removed Mg, Ca, P, and Na from tic acid-l-C" during incubation at 370 C was evidenced the supernatant. Ultrafiltrate concentrations of these by appearance of increasing amounts of C' activity: 1) ions were reduced 0.6, 0.4, 0.6, and 9 mEq per L, respec- in C02 produced; 2) in water-washes of lipid extracts of tively. Incubation with 0.01 M citrate resulted in con- successive samples of cells and medium; 3) in other tributions of these ions, by bone to ultrafiltrate, of 1.6, fatty acids of lipids of cells and medium. 3.6, 1.1, and 6 mEq per L, respectively; while 6.9 mEq It is concluded that for maintenance of intracellular per L of citrate was removed from the ultrafiltrate to fatty acid composition, and leukocytes rely partly upon bone. The increase in Mg was 10 times that anticipated transport inward and that in the case of linoleic acid, from the normal Mg/Ca ratio in dog bone. Citrate-incu- transport is the sole mechanism. bated bone, washed and re-equilibrated with ultrafiltrate alone, removed more Mg (1.0 mEq per L) than bone Studies on the Equilibration of Thyroxine Between the initially incubated without citrate. Synthetic hydroxya- Intravascular and Extravascular Spaces. NORMAN H. patite (0.5 g) showed great Mg affinity, removing 1.6 ENGBRING, EDWARD J. LENNON and WILLIAM W. ENG- mEq per L from the ultrafiltrate. Removal was pre- STROM,* Milwaukee, Wis. vented by addition of citrate. These studies suggest that: 1) Plasma or ultrafiltrate In an effort to define factors involved in the in vivo is supersaturated with Mg in respect to dog bone or transport of thyroxine, the rate of disappearance of I'"' hydroxyapatite. 2) Citrate can accomplish, by ionic L-thyroxine (T4*) from the intravascular compartment exchange, removal of Mg and Ca from bone in a much of human subjects was acutely determined during the higher Mg/Ca ratio than exists in bone. 3) The location first hour after intravenous injection. A linear regres- of Mg in the hydration shell of bone crystals may make sion, log Y=a+bx, was found in each during the inter- it more available than Ca for ionic exchange. 4) The val from 20 to 50 minutes after injection (p = 0.01). degree of Mg saturation of the hydration shell, local The mean half-time of disappearance for 16 normal, citrate concentrations and parathyroid activity may be euthyroid subjects was very constant, 73.7 + 6.6 minutes. significant factors governing Mg exchange and concen- Covariance analysis indicates parallel regression slopes. trations in extracellular fluid and bone. Studies on patients in other situations revealed: 1) Greatly prolonged survival in spontaneous myxedema (9 cases), 110.7 + 21.9 minutes, and in liver disease (10 Preferential and Active Transport of Linoleic Acid by cases), 163.6 ± 21.5 minutes-undoubtedly due to in- Polymorphonuclear Leukocytes. PETER ELSBACH, New creased avidity of serum proteins for T4* in the former York, N. Y. (introduced by L. W. Eichna). and defective hepatic extraction in the latter. 2) Mark- Previous experiments using acetate-i-C" as an index edly shortened survival in hyperthyroid Graves' disease of synthesis have shown that polymorphonuclear leuko- (8 cases), 54.9 ± 9.0 minutes and in hypoproteinemic cytes, obtained from rabbit peritoneal exudates, synthesize nephrosis, indicating that short T4* survival and thy- most fatty acids in vitro. However, little or no radio- rotoxicosis are not necessarily related. 3) Minimal ef- activity was found associated with linoleic, linolenic and fect on survival in 4 normal subj ects fed 20 grains of arachidonic acid, following recovery of individual fatty dried thyroid, 67 ± 5.6 minutes. 4) Lack of prolonged acids after gas-liquid chromatographic separation. De- survival (68.6, 78.8 and 85.0 minutes) in three profoundly spite this evidence that these three essential fatty acids myxedematous patients after treatment of Graves' dis- were not synthesized, it was found that the composition ease, indicating a persistence of a defect in equilibration. of the fatty acids remained constant in leukocytes actively 5) Estrogen administration to normals quickly (within metabolizing in a medium containing serum lipids 48 hours) prolonged T4* survival, even before changes (ascitic fluid). It was therefore concluded that intra- in protein-bound iodine (PBI), indicating very rapid cellular levels are maintained by preferential transport effects on the serum proteins. and/or relative exclusion from metabolic utilization. While correlation exists (p = 0.01) between level of The results of the present study indicate that prefer- PBI and T4* survival among normals, those with spon- ential transport does take place and that the fatty acids taneous myxedema and untreated Graves' disease, ob- accumulated are also metabolized. Leukocytes were in- servations 4 and 5 indicate T4* survival may change cubated in ascitic fluid containing either linoleic acid- independent of PBI. 1-C" (not synthesized by the leukocytes) or palmitic This method is complementary to in vitro determina- acid-i-C" (actively synthesized). Linoleic acid was re- tions of serum thyroxine-binding capacity and to long- producibly concentrated in the cell-lipids at a rapid rate, term T4* survival, the latter presumably measuring thy- while palmitic acid was incorporated erratically and roxine degradation rate after equilibration among the usually at a much slower rate. The transport of both binding compartments. The reported method is well 984 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING

adapted to study factors involved in rate of egress of ing the potassium supplement period. Urine sodium de- thyroxine from the circulation. creased upon withdrawal of potassium supplements. One of these patients subsequently given a higher sodium in- Inhibition of Incorporation of Orthophosphate into ATP take (76 mEq) showed no significant response to po- by Fatty Acids. A. B. FALCONE, R. L. MAO and E. tassium supplementation. In four patients receiving low SHRAGO, Madison, Wis. (introduced by Ovid 0. sodium diets with spironolactone, reducing daily potas- Meyer). sium intake from 85 to 15 mEq yielded a decrease in urinary sodium of 50 per cent or more. Natriuresis was Numerous studies by others have demonstrated the restored when the low potassium diet was supplemented rapid and considerable oxidation of fatty acids by various with 78 mEq of potassium. In one patient exhibiting tissues. Pressman and Lardy (Biochim. biophys. Acta net sodium loss of 147 mEq daily during spironolactone 1956, 21, 458) reported that various fatty acids bring with a low sodium diet, addition of 68 mEq to the sodium about a marked stimulation of mitochondrial ATPase intake for 6 days reduced net loss to 96 mEq. activity. These studies indicate that high sodium or low po- In rat liver mitochondrial systems we have found that tassium intake decreases and the reverse of these in- various saturated and unsaturated fatty acids inhibit the creases the net sodium loss during siprolactone adminis- incorporation of P32-labeled orthophosphate into ATP tration. Maximum response appears to be obtained by a which occurs in the absence of oxygen uptake. This ex- low ratio of sodium to potassium intake. change reaction is thought to represent a portion of the coupling mechanism of the process of oxidative phos- phorylation (Boyer, Luchsinger and Falcone, J. biol. Evidence that Atheroma Fatty Acids Are in Flux. JOHN Chem. W. FARQUHAR, ROBERT L. HIRSCH and E. H. AHRENS, 1956, 223, 405). Inhibition by fatty acids was New greatly augmented by blocking electron transport in the JR.,* York, N. Y. respiratory chain by inhibitors such as potassium cyanide, Assuming that human atheromata are not synthesized sodium azide, amytal, and antimycin-A. Strict anaerobic and degraded entirely in situ, we must postulate that conditions had a similar effect. Significant inhibition was their various lipid components exist in some sort of observed with concentrations of fatty acids which did not dynamic equilibrium with circulating lipids. In view of stimulate mitochondrial ATPase activity. Surface active recent experiments in man with diets rich in unsaturated agents such as desoxycholic acid and sodium lauryl sul- fats, it was asked whether such diets alter the fatty acid fate were found to be inhibitory at concentrations one composition of atheromata. order of magnitude greater than that found for fatty Tissues were obtained at autopsy or exploration from acids. 5 patients who had received corn oil as sole dietary fat The above findings point to a possible role of fatty (40 per cent of caloric intake) for 4, 5 and 9 weeks and acids as important regulatory agents in the overall 18 and 36 months. "Control" tissues were obtained at energy economy of the cell. autopsy from 5 males of comparable age (31 to 55 years). Aortic atheromata were analyzed separately as small and The Effect of Sodium and Potassium Intake Upon the thin (grade 1) and larger and thicker (grade 2) lesions; Natriuretic Response to Spirolactones. WILLIAM W. also, studies were made of "normal" aortic intima and FALOON, FREDERIC F. TAYLOR, SAMUEL HELLMAN and media. Plasma and liver lipids were studied as examples SIMON OHANESSIAN, Syracuse, N. Y. (introduced by of actively metabolizing fatty acid compartments. Con- Eugene L. Lozner). centrations of lipid classes (triglycerides, cholesterol esters and phospholipids) were measured. After separa- The effect of variations in electrolyte intake upon tion of these classes by silicic acid chromatography, their natriuresis during spirolactone administration has been constituent fatty acids were measured by gas-liquid studied in patients with hepatic cirrhosis and ascites. chromatography. Spirolactone daily dosage was either 1 g of the 19- The two atheroma grades showed similar concentra- noranalog (SC 8109) or 400 mg of thioacetoxy analog tions of lipid classes and fatty acid compositions of (spironolactone) orally. triglycerides and cholesterol esters. All tissues of corn In two patients receiving SC 8109, average net daily oil-fed patients, including atheromata, showed higher sodium loss in comparable 6 day periods was 86.9 mEq contents of linoleic acid (18: 2) in all lipid classes than during 10 mEq sodium intake compared to 8.6 mEq dur- those of the control group; differences were greater the ing 112 mEq sodium intake, the diet being otherwise longer the intake. Most marked changes in fatty acid constant. In four patients receiving SC 8109 with diets composition occurred in triglycerides (i.e., from 10 to containing 10 mEq of sodium, infusion of hypertonic 40 per cent 18: 2). Alterations in fatty acids occurred saline (1.8 mEq per kg of estimated dry weight) de- most rapidly in the plasma and liver, while the rate at creased the net sodium loss by 60 per cent or more on which atheroma lipids acquired the characteristics of the the infusion day. In four patients given spironolactone fed fat was far slower and comparable to adipose tissue. and low sodium diets (10 to 20 mEq) increasing po- The findings constitute strong evidence for the existence tassium intake from 80 mEq daily to 160 mEq for 4 days of a slow flux of atheroma fatty acids and interchange increased sodium excretion by 50 per cent or more dur- with dietary fat. AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 985

The Binding Capacity of Tuberculous and NonTubercu- The Effect of Hypercalcemia on Renal Tubular Function. lous Human Serum for P31-Labeled Extracts from THOMAS F. FERRIS, HOWARD LEVITIN and FRANKLIN Tubercle Bacilli. RICHARD S. FARR and HUBERT H. EPSTEIN,* New Haven, Conn. BLOCH, Pittsburgh, Pa. (introduced by Wallace N. Jensen). A 6 year old boy with congenital hypoparathyroidism had been treated for two years with vitamin D. He had Circulating antibodies against antigens from Myco- several confirmed episodes of hypercalcemia during this bacterium tuberculosis have been detected in many tu- period and finally entered the hospital with a two month berclous patients. A sensitive quantitative method is history of polyuria and anorexia. Laboratory tests on needed to evaluate the clinical significance of these anti- admission showed: C02 7.4 mm per L, K 1.9 mEq per L, bodies and the nature of the antigens which elicit their Cl 111 mEq per L, BUN 10 mg per 100 ml. The urine production. The present study describes a quantitative was alkaline and hypotonic to plasma. Polyuria and system which measures binding between constituents of hyposthenuria were resistant to vasopressin. At autopsy M. tuberculosis and antibodies in sera from tuberculous he was found to have nephrocalcinosis. patients. Prompted by this clinical history, a study was under- A cell-free aqueous extract composed of multiple taken to evaluate the renal effects of hypercalcemia in- antigens of M. tuberculosis was labeled with I181 and duced in rats by vitamin D; 200,000 units of vitamin D incubated with 0.0125 ml human serum at 40 C for 24 were given intraperitoneally for 4 consecutive days. hours. Soluble antigen-globulin complexes were then This dose maintained an elevated serum calcium for at precipitated with rabbit antihuman globulin. The I" least 12 days without an elevation in BUN. Rats so activity of the precipitate was used to measure the bind- treated were found to have a diminished ability to con- ing capacity of patient sera for the bacterial extract. As centrate the urine but minimum urinary osmolality at- a result of specific bonds between antibodies and I18`- tained during water diuresis was unimpaired. Rats put labeled antigens in the extract, the average binding ca- on a low K diet for 6 days and then rendered hypercal- pacity in sera from tuberculous patients was higher than cemic were found to have a diminished ability to conserve in sera of normal individuals or patients with a variety of nontuberculous diseases. In a series of sera from 88 potassium when compared to pair-fed controls. This tuberculous patients, 37 had a higher binding capacity potassium wasting continued up to 7 days after the cessa- than was found in any of the 63 nontuberculous control tion of vitamin D, when the animals were sacrificed. The patients. Enhanced binding capacity was found in a ability of hypercalcemic rats to excrete an acid load was tuberculous patient with negative tuberculin tests (10 mg essentially unchanged when compared to controls, until Old Tuberculin) and was absent in normal subjects with the dosage of 1.75 mmoles NH4CL per 100 g rat was positive tuberculin tests. Specificity was established by attained. At this level the hypercalcemic animals dis- demonstrating that antigenic material from sources other played a diminished ability to excrete acid, accompanied, than tubercle bacilli (analogous extracts from E. coli, however, by an elevated BUN. heterologous red cells and bovine serum albumin) failed It appears that experimentally induced to interfere with the reaction, whereas 1 Ag of the un- hypercalcemia labeled test antigen greatly reduced precipitation of the produces a renal tubular lesion characterized by loss of I"1'-labeled antigen. concentrating ability and impairment of potassium con- These results suggest that the humoral immune re- servation. Repeated episodes of vitamin D intoxication sponse in tuberculosis can be quantitated more precisely may have produced analogous pathological and functional than previous methods have permitted. changes in the case described. 986 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING In Vitro Stimulation of Glucose Oxidation in Thyroid the acids of the citric acid cycle has been developed. By Slices by Thyroid Stimulating Hormone. JAMES B. use of ether extraction of NaCl-saturated homogenates FIELD, IRA PASTAN, PHYLLIS JOHNSON and BETTY and subsequent fractionation on glutarate polyester col- HERRING, Bethesda, Md. (introduced by J. Edward umns, 0.01 Mg of each Krebs intermediate can be re- Rall). covered and quantitated. Livers from normal and se- In vitro effects of thyroid stimulating hormone (TSH) verely ketotic (total blood ketones 25 to 194 mg per on thyroidal iodide and phospholipid metabolism have 100 ml) alloxan diabetic rats were studied. Levels of oxalacetate in the normal rat liver varied from 39 to been previously demonstrated. Since these effects are 151 in from to not felt to the ,g per g and the diabetic, 34 309 ,ug per g. delayed they are represent primary action This does of TSH. slices incubated with radioactive not represent a significant difference. Fuma- Thyroid glu- rate in the from to 6.2 cose showed a ratio greater than when C"02 de- normal liver ranged 2.5 ,g per g unity and in the diabetic, from 7.2 to 19.8 rived from glucose-i-C" is compared to C"02 obtained Ag per g. Succinate from This indicates the existence of the concentration in the normal was 107 to 177 ug per g; in glucose-6-C". the diabetic 120 hexose monophosphate pathway. Addition of TSH to to 179 ,ug per g. Assay of isocitrate, a two- to increase citrate and cis-aconitic acids likewise failed to show any thyroid slices produced eightfold in differences glucose-i-C" oxidation to C"02 while the increase in between the two groups. marked. effect of These studies indicate that intermediates of the tri- glucose-6-C" oxidation was less This carboxylic TSH was manifest within minutes after the start of acid cycle are not decreased in diabetic liver 5 and that contrary the incubation and suggests that the primary action of to present concepts, the ketosis of dia- TSH might be on glucose metabolism in the betes must be due to factors other than deficiencies of the thyroid. Krebs cycle substrates. Adrenocorticotropin, prolactin, growth hormone or fol- licle-stimulating hormone were ineffective. TSH had no effect on glucose oxidation in liver or testis slices. Although TSH increased glucose uptake by thyroid slices this does not appear to be its mode of action, since insulin also increased glucose uptake but did not stimulate glu- cose oxidation. The activity of glucose-6-phosphate Some Properties of the Breakdown Products of Human dehydrogenase and 6-phosphogluconate dehydrogenase in 7S Gamma Globulin and Antibodies. E. C. FRANKLIN, thyroid slices was not increased by TSH. Gluconate- New York, N. Y. (introduced by H. S. Lawrence). 1-C" oxidation was not stimulated by TSH. None of the Treatment of human 7S gamma globulin with cysteine antithyroid drugs interfered with glucose oxidation to and papain by the method of Porter yielded 75 to 90 per C"02 although there was some inhibition of the TSH cent nondialyzable fragments. Ultracentrifugal examina- stimulatory effect when propylthiouracil was present. tion revealed a major peak with an s rate of 3.4 to 3.5S Acetylated TSH itself was inactive and when present and small amounts of more rapidly sedimenting material. in 5 times the concentration of TSH there appeared to The fragments can be fractionated into two major com- be some inhibition of the TSH effect. Serotonin, epine- ponents (I and II) by gradient elution chromatography phrine and norepinephrine also stimulated glucose oxida- on a carboxymethylcellulose column, pH 7.6 (0.01 to tion to C"02 while histamine, acetylcholine, tryptophan 0.4 M). Each can be further separated into two peaks and 5-hydroxyindoleacetic acid were inactive. (IA and B), (IIC and D) on a diethylaminoethylcellu- lose column, pH 8 (0.01 to 0.3 M). Fractions A, B, and C each had s rates of 3.4 to 3.5S and molecular weights of 40,000 to 55,000. Fraction IID comprised less than 5 per cent of the total and was heterogeneous. Each The Krebs Cycle and Diabetic Ketosis. DANIEL W. fraction retained some antigenic properties of FosTR and MARVIN D. Tex. gamma globulin when tested with a rabbit antiserum. SIPERSTEIN,* Dallas, Agar double diffusion studies and immunoelectro- It is generally held that the overproduction of ketone phoresis demonstrated close antigenic relationship of bodies which accompanies diabetes is due primarily to Fractions IA and IIC while IB contained an antigeni- an inability of diabetic liver to maintain normal levels cally distinct component. More than three-fourths of of Krebs cycle carboxylic acids, in particular of oxala- the carbohydrate was associated with Fraction I (hexose cetic acid. With deficiencies of these acids, oxidation of approximately 3 per cent; hexosamine approximately 2 acetyl-CoA to C02 should proceed at a decreased rate, per cent) while Fraction II contained less than 0.4 per resulting in accumulation of acetyl CoA, which must in cent of each. Fragments prepared from antibody to turn condense to form excessive ketone bodies. Despite streptokinase and diphtheria toxin retained the ability wide acceptance of this theory of ketogenesis, there is to neutralize the antigen. More than three-fourths of little evidence that the Krebs cycle in diabetic liver is, this antibody neutralizing activity was associated with in fact, deranged. Fraction IIC. The remainder was present in Fraction As part of an investigation of the problem of ketogene- I. Fragments of precipitating antibody against horse sis, a sensitive gas chromatographic method of measuring serum from a patient with serum sickness no longer AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 987 precipitated. The whole digest and Fraction IIC in- 165 x 1.8 cm, of Amberlite IR-120. Two hundred 3 ml hibited precipitation by the original serum almost com- fractions were collected, and aliquots were analyzed for pletely while Fraction IA was much less active. When ninhydrin color before and after hydrolysis. Three peaks tested by reverse passive cutaneous anaphylaxis in the appeared after hydrolysis where little or no color was guinea pig none of the fractions fixed to the skin, but the detected prior to hydrolysis. These fractions were ex- active fraction, IIC, greatly diminished the passive amined further by paper chromatography, analytical cutaneous anaphylactic reaction of the original serum column chromatography employing an automatic record- with horse serum. ing apparatus, solvent extraction, and other appropriate tests. One peak was shown to contain phenylacetyl- The Effect of Insulin upon the Peripheral Uptake of glutamine, a substance present in normal urine but not Plasma Free Fatty Acids in Man. SAMUEL J. FRIED- previously identified in blood. Another peak, not retained BERG, ROBERT F. KLEIN, MORTON D. BOGDONOFF, E. on the acidic resin, was eluted near column volume with HARVEY ESTES, JR. and DAVID L. TROUT, Durham, the blood glucose. This peak could not be resolved N. C. (introduced by William M. Nicholson). further by chromatography on a basic resin or on paper. In the dog, insulin has been shown to cause a prompt After hydrolysis the amino acids identified were: aspartic reduction in the release of plasma free fatty acids (FFA) acid, glutamic acid, threonine, serine, glycine, alanine. from peripheral tissues into the blood, with no change in The third peak displayed properties similar to those of the rate of disappearance of FFA from the blood. The the second. present study, in man, shows that an increased removal These studies identify some of the increases in previ- of FFA does occur following insulin administration. ously "unidentified nitrogen," or the nonprotein, nonurea, Ten male subj ects were studied in the resting state nitrogenous substances in the plasma of patients with following an overnight (12 to 15 hour) fast. Palmitic- uremia. acid-l-C1' (0.004 to 0.012 Aic) was given by constant in- fusion through a forearm vein. Simultaneous arterio- The Occurrence of Rheumatoid Factor and other Gamma venous samples were drawn at intervals from the oppo- Globulin Abnormalities in the Families of Patients with site brachial artery and antecubital vein or from a femoral Agammaglobulinemia. HUGH FUDENBERG, JAMES L. artery and vein. Following the attainment of a constant GERMAN, III and HENRY G. KUNKEL,* New York, level of radioactive FFA in the arterial blood, 5 sub- N. Y. into jects received 2 units of HGF-free insulin injected The sera of relatives of 13 individuals with congenital the a sampled artery. In all 5 subjects, there was marked agammaglobulinemia and of 10 with idiopathic "acquired fall in a in both arterial and venous FFA, rise specific agammaglobulinemia" have been examined for quantita- activity, and in addition, a consistent fall in the level of tive aberrations in gamma labeled FFA in globulins and for certain the arterial blood. The overall effect qualitative abnormalities, particularly the rheumatoid and was a reduction in FFA turnover. In 5 control sub- lupus factors. The gamma globulin levels in the probands jects there was no change in the levels of FFA or of labeled of the congenital group were all less than 100 mg per FFA. As long as the infusion was maintained 100 ml and in the acquired group the values ranged from there was a positive arteriovenous difference of labeled material. 50 to 250 mg per 100 ml as determined by zone electro- phoresis. Three of the patients in the congenital These data suggest that in man insulin not only inhibits group the had arthritis, and three in the acquired group developed release of FFA into the blood but also increases the various atypical manifestations of rate of disappearance of FFA from the blood. "connective tissue disease." Hypergammaglobulinemia was found frequently in family members of both groups. In addition, marked Conjugated Amino Acids in Deproteinized Plasma of diminution of Patients with Renal the p2A or 82M immunoglobulins was present Failure. GEORGE W. FRIMPTER and in some of the families. In parents and of DAVID D. THOMPSON,* New York, N. siblings pro- Y. bands with "acquired agammaglobulinemia," the incidence Free alpha amino nitrogen in deproteinized plasma of "rheumatoid factor," as evaluated by four different from patients with uremia was found to be approximately serologic parameters, was II of 24, or 46 per cent. This normal. When deproteinized plasma was hydrolyzed with was significantly different from a control group of 23 6 N HCI, a mean increase of alpha amino nitrogen families with an incidence of 4 out of 88 positive reactions amounting to 3.0 mg per 100 ml (a 65 per cent increase) or 5 per cent (p. <0.001). Nine of the ten probands occurred as determined by the manometric ninhydrin with the "acquired" form of the disorder had at least one method. This is contrasted to a mean increase of 0.6 mg parent or sibling with rheumatoid factor. This incidence per 100 ml (11 per cent) in normal subjects. The com- of positive families was also significantly different from pounds giving rise to increased alpha amino nitrogen the incidence in normal families. In addition, antinuclear dialyzed rapidly through cellophane, suggesting that the (lupus) factors were present in relatives in two of the conjugated compounds were of relatively small molecular "acquired agammaglobulinemia" families. In congenital weight. agammaglobulinemia the familial incidence of rheumatoid Chromatographic separation of these compounds was factor was not significantly increased (4 of 38 relatives). performed employing a preparative ion-exchange column, These data suggest a familial predisposition to aber- 988 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING ration in immunologic response, sometimes resulting in proven cases of cholera during the 1959 Thailand epidemic agammaglobulinemia, sometimes in hypergammaglobulin- failed to substantiate this classic concept. emia, and often in serologic abnormalities of the "rheu- Serial biopsy specimens obtained with the "Crosby matoid factor" type. The studies perhaps aid in connect- Capsule" at several intestinal levels during active diarrhea ing on a genetic basis the various diseases associated with showed acute enteritis with intact mucosal epithelium. these gamma globulin aberrations. Cessation of the clinical diarrhea could not be correlated with any specific change in pathology. An additional Effect of In Vitro Sonic Oscillation on Distribution of microscopic finding in all cases consisted of atrophic Serum Lipoproteins in Idiopathic "Essential" Hyper- thinning of the mucosal layer with blunting and fusion of lipemia and Other Hyperlipidemic States. ROBERT H. villi. These chronic changes were comparable to those FURMAN, K. LAKSHMI, LEONARD N. NORCIA and R. seen in dietary deficiency states such as sprue and re- PALMER HOWARD, Oklahoma City, Okla. (introduced mained after the enteritis had cleared. The possibility that by Stewart Wolf). areas of desquamation were missed by the biopsy tech- Ultracentrifugal partition of the serum lipids in a nique seemed unlikely, since intravenously injected inulin variety of hyperlipidemic disorders has been carried out or Evans blue dye failed to leak into the bowel lumen. at the following solvent densities: 1.006, 1.019, 1.063 and Reports that the stool in cholera contains insignificant 1.21 g per ml. In idiopathic hyperlipemia, 40 to 90 per amounts of protein, and demonstrations by Gordon that cent of the serum cholesterol and phospholipid may be intravenous radioiodinated PVP does not appear in found in the chylomicron-rich d < 1.006 g per ml frac- abnormal amounts in the cholera stool lend additional tion, while less than normal amounts are found in the support to the presence of an intact epithelium. Stool d> 1.063 g per ml (a) and d> 1.019 < 1.063 g per ml potassium, pH, and bicarbonate were shown by Watten, (X) fractions. In "normal" sera only 10 to 14 per cent Phillips and colleagues to be higher than simultaneous of the serum cholesterol and phospholipid is found in the plasma values implying that cellular "work" was involved very low density fraction. in the elaboration of the cholera stool. Prompt and com- When normal sera are subjected to sonic oscillation (10 plete absorption of tritiated water and iodine' molecules kc per second, 250 W, 2 hours) the lipoprotein distribu- by the upper intestine was shown in acute cholera, during tion remains essentially unaltered. However, when sera periods when transit time through the bowel was less than from hyperlipemic patients are so treated, ultracentrifugal 2 hours. partition of the lipids reveals marked decrease in the The stimuli initiating the unique clinical and biochemi- amount of cholesterol and phospholipid found in the low cal characteristics of cholera diarrhea remain unknown. density fraction, while there is a quantitatively similar However, it seems possible that atrophic changes in the increase in the lipid content of the a lipoprotein fraction. intestinal mucosa associated with nutritional deficiencies For example, in two hyperlipemic patients with serum may predispose an individual to clinical cholera. cholesterol levels of 458 and 350 mg per 100 ml, respec- tively, only 32 and 11 mg were present as a lipoproteins, An Evaluation of the Urine Hydrolysis Test for Primi- while 390 and 278 mg were present as low density lipo- tive White Blood Cell Differentiation. EDWARD proteins. After sonic oscillation, a cholesterol increased GARDNER, JR., CLAUDE-STARR WRIGHT* and BETTIE Z. to 216 and 101 mg, respectively, with equivalent losses WILLIAMS, Augusta, Ga. from the low density fractions. Accurate identification of primitive leukemic white These and similar data from other patients indicate that blood cells in peripheral blood and bone marrow is de- in "essential" hyperlipemia significant amounts of high sirable for most efficient therapy. Even with a variety of density a lipoproteins are "bound" to low density lipo- technical aids it is often difficult to classify the cell type. proteins and/or chylomicrons. In primary "essential" A means of differentiating cells of the myelocytic series hypercholesterolemia and in hypercholesterolemia second- from other leukocytes by subjecting them to urine was ary to hypothyroidism or nephrosis, no redistribution of first proposed by Brachet. The test involves exposure of ultracentrifugally-defined lipids is noted following sonic bone marrow or peripheral blood films, previously fixed oscillation. In sera from patients with hypercholestero- in methyl alcohol, to the activity of fresh urine. The lemia secondary to biliary obstruction, redistribution of nuclei of myelocytic cells are "lysed" while lymphocytic lipids following sonic oscillation indicates marked binding and monocytic nuclei remain relatively intact. of high density lipoproteins to "beta" lipoproteins. The present study is concerned with three phases of the urine hydrolysis test: 1) conditions influencing the test; Studies of Gastrointestinal Pathophysiology in Acute 2) the nature of the substances in urine responsible for Asiatic Cholera. EUGENE J. GANGAROSA, WILLIAM R. the "lytic" effect; and 3) application of the test to BEISEL, CHANYO BENYAJATI and HELMUTH SPRINZ, clinical material. Washington, D. C. and Bangkok, Thailand (introduced It was found that three factors, time and temperature by John B. Youmans). of incubation and pH of urine, are important. Optimum The diarrhea of Asiatic cholera has been ascribed to conditions for cell differentiation were incubation of blood massive transudation of fluid across a bowel wall denuded films in neutral or slightly alkaline urine at a temperature by epithelial desquamation. Intestinal biopsy specimens of 600 C for 15 minutes. Most urine specimens had to be and physiological studies obtained in bacteriologically adjusted from a slightly acid pH. AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 989

The effect of purified ribonuclease and desoxyribo- transmitted cyanosis which simulates congenital methemo- nuclease and various concentrations of sodium chloride globinemia. Both the Hgb M and normal adult hemo- solutions on blood films was determined. Only a solu- globin may be demonstrated by electrophoresis of tion of desoxyribonuclease in buffered saline produced hemolysates from cyanotic members of such families. In results comparable to the effect of urine. addition to being electrophoretically abnormal, the Hgb Application of the urine test to blood and/or bone M pigments are spectroscopically anomalous when in the marrow films from 18 cases of leukemia substantiates the methemoglobin state. This latter unusual property has validity of the test in 17 cases. been thought to be due to the formation of an internal complex between the ferric iron and a new active group Pregnanetriol Excretion and Urinary Pregnanetriol/Al- in the globin portion of the molecule. dosterone Ratio in Normal Subjects and Patients with Chemical studies of the protein portion of Hbg M Arterial Hypertension. JACQUES GENEST, WOJCIECH (Boston type) have now been accomplished by the NOWACZYNSKI, ERICH Koiw and THOMAS SANDOR, "fingerprint" technique developed by Ingram. Comparison Montreal, Canada (introduced by J. S. L. Browne). of the tryptic digest fragments from Hgb M (Boston Extension of our study on the role of adrenal cortical type) with those from the Hgb A1 fraction of the same hormones in human hypertension has shown a significant patient, as well as from normal adults, has revealed the decrease in mean urinary pregnane-3a, 17a, 20a-triol presence of an amino acid alteration in at least one such excretion in groups of patients with essential, renal and fragment. This is evidenced by the absence from the malignant hypertension as compared to that of normal Hgb M (Boston type) fingerprint of a spot constantly subjects. Sixty-eight determinations in 56 normal sub- present in the Hgb A1 fingerprint, and in addition, the j ects and patients with essential, renal and malignant appearance in the former of a "new" spot which reacts hypertension were made. Normal females used for the with staining reagents for tyrosine. From these data, it study collected their urine only during the first part of seems reasonable to postulate that the mutation repre- the menstrual cycle in order to avoid the rise in urinary sented by Hgb M (Boston type) results in the substitu- pregnanetriol during midcycle. Urine collections from tion of a tyrosyl residue for the amino acid which occurs women with hypertension were made without regard to at this point in the sequence of Hgb A1. the menstrual cycle. All patients and normal subj ects This is the first demonstration that abnormal spectro- were on unrestricted diets. scopic properties of the hemoglobin molecule need not Mean urinary pregnanetriol excretion determined ac- result solely from changes in the heme group, but may cording to Bongiovanni's procedure is significantly de- be attributable to alterations in the amino acid sequence creased in groups of patients with essential, renal and of the globin. malignant hypertension as compared with that of normal subjects (p <0.01). During this study, Bongiovanni's Family Studies on the Chromosomal Complement of procedure was found to be inaccurate in many cases be- Mongols. JAMES L. GERMAN, III and ALEXANDER G. cause of the presence of two substances interfering with BEARN,* New York, N. Y. pregnanetriol determination. One of these has already The karyotypes of two female mongols and their been identified as Compound III, previously described, parents were studied by means of short-term bone mar- and the second was identified by various procedures, in- row cultures. The first patient, aged 18 months and the cluding infrared spectrometry, as the 5-pregnene-3p8, 17a, third of three siblings, showed the classical mongoloid 20a-triol. But the validity of results obtained with appearance, congenital cardiopathy, and mental deficiency. Bongiovanni's procedure was confirmed by the use of a The mother and father, aged 36 and 35 years, respectively, specific method recently developed in which pregnanetriol when the child was born, showed no mongoloid character- is determined after isolation in a high degree of purity. istics. The mongol showed 47 chromosomes, the small The ratio of two urinary steroids varying in opposite acrocentric number 21 being trisomic. Sex chromosomes directions, pregnanetriol/aldosterone, is significantly de- present were (XX). The mother had 46 chromosomes creased in all three groups of hypertensive patients and is (XX), the father 46 (XY). In the second patient, aged below lower limits of normal range in 92 per cent of two months and the fourth of four siblings, pathogno- patients with various types of arterial hypertension. monic clinical features of mongolism were notably absent, These findings have great interest because of recent although the facies was suggestive of mongolism. The observation of the hypotensive action of progesterone in mother was 26 when the child was born, the father 30. experimental and human arterial hypertension by Arm- Despite the difference in the clinical findings in the two strong. This adds further evidence for a disturbance in cases, the karyotypes were grossly identical. Abnormal- the adrenocortical function of hypertensive patients. ities of chromosome number 21 were not detected in the parents. Presence of an Altered Amino Acid Sequence in Hgb M Disturbances in chromosomal mechanics giving rise to a (Boston Type). PARK S. GERALD, MARY L. EFRON trisomic complement could arise in several ways, includ- and MARGARET J. PEASE, Boston, Mass. (introduced by ing nondisjunction during miosis, somatic nondisjunction Louis K. Diamond). during early embryonic cell division, or the presence of an The group of abnormal hemoglobins categorically additional chromosome in the parental gonial cells. H~ow- known as the Hgb M's are associated with dominantly- ever, in view of the well recognized maternal age effect 990 PROCEEIDINGS OF THE FIFTY-SECOND ANNUAL MEETING it seems probable that the majority of mongols are the has permitted the use of random urine specimens col- result of maternal nondisj unction. No evidence for lected from patients following no specific diet. As with somatic mosaicism was found in either parent examined the original test, no false-negative results were obtained. in the study. However, the possibility that parental A series of 31 cases of pheochromocytoma gave uni- germinal mosaicism might account for a proportion of formly positive test results. Ten urine samples, which cases, particularly in those born to young mothers in had previously given false-positive results due to in- whom there is more than one affected member in the adequate dietary control, were negative when tested in sibship, is not excluded by the present findings. the following manner. A urine aliquot equivalent to 1.0 mg creatinine was hydrolyzed at pH 2 and 1000 C Effect of Acute Uremia on Arterial Blood Pressure in for 10 minutes, after which the pH was buffered at 4.0 Rabbits. CARMELO GIORDANO, HERMAN WEINSTOK and and the urine extracted with ethyl acetate. The aqueous JOHN P. MERRILL,* Boston, Mass. layer was acidified with HC1 to a pH of 0.5 to 1.0, ex- Neither ureteral ligation nor bilateral nephrectomy tracted with ethyl acetate, and the organic layer blown to causes hypertension in the rabbit. Present experiments dryness and dissolved in dilute K2CO3. Diazotized p- were designed to study the effect of the addition of urea nitroaniline was added and the purple azo-VMA com- loading to ureteral ligation on arterial blood pressure. pound extracted into n-amyl alcohol: ethanolamine In previous investigations we have shown both in vitro (100: 1) for reading on a Beckman DU spectrophotom- and in vivo that elevated concentrations of urea (0.05 M) eter at 450 and 550 mAl (vs. H20 blank). The ratio will inhibit monoamine oxidase activity of mytochondria (R)-density 450 m1 normally exceeded 1.25 (mean = from rabbit kidney or liver. Twenty animals were density 550 mAJ divided into 3 groups. In Group 1 bilateral ureteral 1.63), whereas no test of urine from a patient with a ligation was done and 10 to 20 minutes afterward 2 g of pheochromocytoma exceeded 1.20 (mean = 0.70). This urea per kg body weight was administered by stomach simple and rapid test should enable the physician to tube. Such a load increased the blood urea nitrogen screen large numbers of hypertensive patients for the concentration (BUN) to 150 ± 10 mg per 100 ml. presence of pheochromocytoma. Group 2 served as control, having only ureteral ligation. Group 3 received only the urea load. Blood pressures Blocking Antibodies after Brucella Hyperimmunization were taken at 5-minute intervals for 20 minutes before and Infection in Rabbits and Mice. HARRY GLENCHUR, and 4 hours following the urea load using an ear mano- HORACE H. ZINNEMAN and WENDELL H. HALL,* metric capsule. A significant rise in blood pressure ap- Minneapolis, Minn. peared in Groups 1 and 3 during the first 2 hours after Continued injection of heat-killed Br. abortus resulted urea loading. No significant change in blood pressure in persisting agglutinating antibody in rabbits. Weeks occurred in Group 2. Death occurred in Group 2 in 3 to of massive injections were required to produce blocking 4 days, whereas death occurred in Group 1 in 10 to 12 antibody (slow-moving gamma globulins). With fur- hours following the urea load. The curves of serum ther injections, the blocking activity moved into the osmolality, hematocrit and BUN indicated that urea had ,8-globulins. This migratory phenomenon was also ob- completely equilibrated in total body water in 10 to 15 served in a man with acute brucellosis. Hyperim- minutes and could not therefore account for blood pres- munized rabbits also had a marked rise in serum gamma sure rise at 2 hours on the basis of expansion of extra- globulins, the majority of which were absorbable by cellular fluid. homologous antigen. Brucella-infected rabbits, too, The possibility that the relation of urea loading to demonstrated increased serum gamma globulins which, increased arterial pressure may be related to inhibition of however, were mostly not absorbable. Hyperimmuniza- monoamine oxidase activity is considered. tion did not impair the rate at which the animal con- trolled a brucella bacteremia. A Rapid Urine Test for Pheachromocytoma. STANLEY Serum precipitins appeared in hyperimmunized rabbits E. GITLow and ELIZABETH KRUK, New York, N. Y. after 2 to 15 weeks. The time of appearance of precip- (introduced by Milton Mendlowitz). itins was inversely correlated with the weekly dosage of Analysis of urine for vanillylmandelic acid (VMA) has antigen. As immunization continued, more precipitin proven to be a reliable diagnostic technique for pheo- lines appeared, until there were 3 to 4. Absorption of chromocytoma, but the procedures heretofore used for the hyperimmune sera with whole brucella cells removed VMA measurement have been either complex or pro- some but not all the precipitins. The precipitins dis- longed and therefore unsuitable for use by a routine appeared 14 weeks after cessation of immunization (the clinical laboratory. absorbable one first) although the agglutinins persisted A rapid urine test for the presence of excessive quan- in low titers. In brucella-infected mice, the blocking tities of VMA was reported in 1959, but it required that antibody appeared early, associated with an increase of the patient avoid certain foods and drugs prior to sample both j8- and gamma globulins. Mice infected with collection. Failure to do so resulted in a false-positive brucella showed only a single serum precipitin after 80 test, necessitating the use of the time-consuming confirma- days of infection. tory tests. A modification of this simple screening test Since the antigen for the precipitin test was from AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 991 disrupted brucella, this work suggests that antibodies averaging 4 and 1.5 cc per minute, respectively. Within form to internal antigens as degradation of the bacterial one hour these values had returned to control levels. cell occurs in vivo. An outpouring of nonspecific globu- Thereafter a more sustained and progressive increase in lin also occurs in active infection. Immunization with Coom developed which reached an average peak increment intact, killed brucella cells produces antibody globulin but of 12 cc per minute two hours after the administration no nonspecific gamma globulins. In response to infec- of meralluride. Coincidentally, urine flow (V) progres- tion, the mouse produces more 8-globulins and early sively increased to the same extent so that CH2O re- blocking antibodies. Thus species differences in the mained constant throughout this phase of the solute serological response to brucella infection are emphasized. diuresis. At the time of maximum mercurial response an infusion of calcium or sodium sulfate caused a prompt Electron Microscopy of the Human Glonterulus in Early further increase in Comm and CH20, averaging 6 and 3 cc Diabetes. F. C. GOETZ, J. F. HARTMANN and A. LAZ- per minute. AROW, Minneapolis, Minn. (introduced by C. J. Wat- In 4 comparably hydrated subjects infused with a son). hypotonic mannitol load, meralluride likewise produced Needle or surgical biopsies of the kidney have been a two-phase response, the first phase corresponding to obtained in 23 patients with diabetes mellitus, the dura- that described above. The second more sustained re- tion of the disease varying from 5 months to 28 years. sponse produced maximum increases in Cosm and V, They have been studied by accepted light and electron averaging 24 cc per minute or twice that observed with- microscopic techniques, with special attention to the width out the solute load. As above, this solute diuresis oc- and appearance of the glomerular basement membrane. curred without a measurable change in CH20. The ad- Control material was provided by nondiabetic human ministration of dimercaprol during the second phase of biopsy and autopsy material (Bloom, Vernier and Hart- meralluride diuresis in hydrated subjects eliminated the mann). similar increments in Cosm and V so that CH2O remained The diabetic glomerulus characteristically showed ir- unchanged. regular thickening of basement membrane. In two 19 The transient initial phase of mercurial diuresis seems year old patients with duration of diabetes well docu- explicable by the inhibition of proximal salt absorption. mented at 6 and 22 months, respectively, basement mem- The persistently isosmotic character of the tubular re- brane measurements showed that significant thickening jectate during the sustained phase of diuresis, despite the was already present (2,385 to 5,565 and 2,772 to 5,568 capacity for CH20 to be independently increased before A units, in comparison with the normal value of 3,000 + and during this phase, is difficult to explain by either a 550 A, [SD]). Neither of these patients showed clini- proximal or early distal effect. Unless meralluride in- cal or laboratory evidence of renal or other vascular dis- hibits both proximal and distal salt absorption with a ease, and by light microscopy the glomeruli appeared unique balance of differential effects so as to maintain normal. Nearly all patients with 5 years or more of CH20 constant, these data are consistent with the hy- diabetes showed some degree of change, whether or not pothesis that mercurials prevent a late distal isosmotic clinical evidence of renal disease was present. Where salt reabsorption in hydrated man. changes were extreme, basement membrane reached 13,000 A in thickness, with complex folding and forma- The "Diabetic" Pituitary: Observations on the Carbohy- tion of anastomosing trabeculae of basement-membrane- drate Metabolism of Pituitary Tissue. CHARLES J. like material in nodular masses. These severe changes GOODNER and NORBERT FREINKEL,* Boston, Mass. were accompanied by marked broadening of some epithe- Despite the classical evidence that hypophyseal hor- lial foot-processes and by thickening and vacuolation of mones may influence diabetes mellitus, the responsiveness endothelial cytoplasm. of normal pituitary tissue to insulin and the carbohydrate It is concluded that a characteristic glomerular change metabolism of the pituitary in diabetes mellitus have never may begin within one to two years of the apparent on- been examined. Since these factors may condition pitui- set of diabetes, and that this change progresses in a more tary function, the following studies were undertaken. or less smooth continuum as the duration of diabetes in- Isolated portions of anterior (AP) and posterior (PP) creases, finally leading to extreme changes with the for- pituitary from normal rats and calves were incubated mation of nodular masses. Involvement of glomerular separately with differentially-labeled glucose-C". Ap- capillaries in diabetes may occur much earlier than had preciable hexose-monophosphate shunt activity was dem- heretofore been suspected. This finding has implications onstrated in AP and PP. In addition, both structures important to understanding of the pathogenesis of dia- exhibited rapid glycogen turnover, net glycogenesis in betic kidney disease. vitro, and appreciable levels of phosphorylase activity. AP and PP of both species were responsive to insulin Meralluride-Induced Solute and Water Diuresis in Hy- in vitro. Addition of insulin to the suspending medium drated Man. MARVIN GOLDSTEIN, A. DANIEL HAUSER significantly (p < 0.01) enhanced glucose assimilation and MARVIN F. LEVIrr,* New York, N. Y. and the disposition of radioactivity into glycogen-C', In 7 maximally hydrated subjects meralluride pro- lipid-C" and C1"02. To assess the effects of acute in- duced a prompt but transient increase in CH2o and Cosm, sulin deprivation, pituitaries were excised from rats 20 992 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING hours following total pancreatectomy. During incubation concentration. Increased absorption of iron in rats, as with glucose-U-C" in vitro, AP from these animals measured by total body counting, was found to begin formed 40 per cent less C1402 (p < 0.001) and 10 per after 6 to 8 hours of hypoxia and was preceded by an ele- cent less lipid-C" (p < 0.05) than AP from sham-op- vation rather than a fall in SI. erated controls. Administration of insulin during the The temporal relationship of SI turnover to absorp- postoperative period prevented this deterioration of AP tion was studied in 48 rats whose iron stores had been carbohydrate metabolism. No abnormalities in the dis- labeled by the intravenous injection of 20 uc of Fe"CI3 position of labeled glucose were demonstrable during in- one week previously. In a 3 x 2 factorial design, 3 groups cubation of PP from depancreatized rats. of 16 rats each received hypoxia (Po2 = 76 mm Hg) for Conclusions: The presence within the pituitary of 0, 2 and 8 hours, and one-half of each group received 1 pathways for 1) the storage of glucose, 2) the rapid mg of ferrous sulfate intragastrically 2 hours before mobilization of glycogen, and 3) the generation of sacrifice for determination of SI and serum Fe'9. The reduced pyridine nucleotides during oxidation of glucose- fasted rats had SI concentrations of 170, 211 and 155 6-phosphate suggests mechanisms whereby glandular ug per 100 ml for the 0, 2 and 8 hour periods. The cor- carbohydrate economy may influence hormonogenesis. responding values for the iron-fed rats were 234, 299 and Potential derangements of pituitary glucose metabolism 310 /Lg per 100 ml. Serum Fe'9 levels, corrected for in diabetes mellitus are suggested by the responsiveness hemoglobin radioactivity, were 346, 982 and 727 cpm per of hypophyseal structures to insulin in vitro. That this 2 ml for the fasted rats and 374, 721 and 958 counts for is indeed the case, at least for AP, is suggested by the the others. By analysis of variance the elevations in SI finding in rats rendered acutely aninsulinemic by total for hypoxia and for iron feeding are significant at the pancreatectomy. 0.01 and 0.001 levels, respectively, and the elevation of Fe'9 with hypoxia is also highly significant (p <0.001). Studies on the Origin of the Fecal Fat. HYMIE GORDON, Thus mobilization of storage iron, apparently not Cape Town, South Africa (introduced by Victor A. blocked by an oral iron load, is responsible for the rise McKusick). in serum iron after 2 hours of hypoxia. After 8 hours, Fifteen normal subj ects were maintained on constant when enhanced absorption can be demonstrated, the SI very low fat diets (6.1 to 8.6 g of fat daily) for periods in the fasted rats is falling, suggesting utilization of iron varying from 6 to 24 days. While on this diet, each sub- by the bone marrow. In the iron-fed rats the SI and ject's stools were collected in 3 or 5 day batches and Fe"9 remain high, reflecting both movement out of storage their fat content was determined. The mean daily fecal and enhanced absorption. These data suggest a primary fat content was 2.4 g (SD 1.2). effect of hypoxia on serum iron turnover; the latter Various dietary manipulations were then carried out may be the factor directly related to enhanced absorption. on groups of these subjects. WVhen the dietary calories were progressively increased without altering the fat in- Jfechanismii of Neutromuiscular Block in Mllvasthcenia take, there was no significant change in the fecal fat Gravis. DAVID GROB* and RICHARD J. JOHNs, Brook- content. Increasing the dietary crude fiber intake in- lyn, N. Y. and Baltimore, Md. creased the fecal fat only when very large artificial fiber Essential differences have been found in the effect of loads were given. transmitter substance (acetylcholine, ACh) on neuro- The addition of either saturated or unsaturated fat to muscular transmission of normal subjects and myasthenic the diet increased the fecal fat content; in general, the patients. Intra-arterial administration of ACh produces, greater the dietary load of fat, the greater was the fecal in both, block of neuromuscular transmission, which is fat content. To determine whether this increase in fecal more marked and prolonged in myasthenic patients. fat was due to unabsorbed dietary fat or to the increased The nature of the block may be characterized by its effect excretion of fat from the body, a series of experiments on the subsequent injection of ACh. Only in myasthenic was carried out in 5 subjects using intravenous cotton- patients does this ACh-induced block inhibit the de- seed oil ("Lipomul I.V."; Upjohn). When the intravenous polarizing action of subsequently injected ACh. The fat was administered instead of dietary fat, the fecal fat block may be further characterized by observing whether content fell toward the basal (very low fat diet) level; or not it is reversed by ACh. In normal subjects it is when it was administered in addition to dietary fat, there not. In myasthenic patients who respond well to anti- was no further increase in the fecal fat content. These cholinesterase medication, the ACh-induced block is re- observations support the hypothesis that in subjects con- versible by ACh, but in the minority who respond poorly suming diets containing ordinary amounts of fat, much of it is not. The latter ACh-inhibitory, non-ACh-reversible the fecal fat is derived from unabsorbed dietary fat. block may be termed "ACh-insensitive." Patients may become ACh-insensitive during exacerbation of their dis- Iron Absorption and Turnover in Hypoxia. MORTIMER ease, or rendered locally ACh-insensitive by repeated S. GREENBERG, HELENA WONG, STEPHEN A. MILLER, intra-arterial administration of ACh or choline, or by ROBERT W. SCARLATA and THOMAS C. CHALMERS,* prolonged repetitive nerve stimulation, which causes local Boston, Mass. accumulation of endogenous transmitter. Patients in the Enhancement of iron absorption during experimental ACh-insensitive state are often treated with excessive hypoxia has been attributed to a fall in serum iron (SI) doses of anticholinesterase compounds which render them AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 993 even more insensitive. Cessation of anticholinesterase facilitate cardiac output response to volume loads in less medication, or potassium administration or hypothermia severely ill patients. usually increases responsiveness to ACh and may be fol- lowed by clinical improvement. Amino Acid and a-Keto Acid-Induced Hyperinsulinism These observations indicate that myasthenia gravis is in the Leucine-Sensitive Type of Idiopathic Hypogly- due to an ACh-inhibitory neuromuscular block produced cemia. MELVIN M. GRUMBACH * and SELNA L. KAP- by endogenous transmitter. It is likely that ACh com- LAN, New York, N. Y. bines with several forms of receptor substance in the motor end-plate to produce different types of block: non- A group of infants and children with "idiopathic" ACh-inhibitory in normal subjects and ACh-inhibitory hypoglycemia has been shown to exhibit a sharp fall in in myasthenic patients, in most of whom the block is the concentration of blood sugar following the adminis- ACh-reversible, but in some is non-ACh-reversible. In tration of L-leucine. In the present studies, data obtained myasthenic patients there may be one or more abnormal in two children with leucine-induced hypoglycemia forms of receptor substance, and the predominant form (L.-I.H.) indicate that certain amino acids and keto acids may determine the type of block, clinical state, and re- promote an increase in circulating insulin. sponse to anticholinesterase medication. When L- or DL-leucine, or L-isoleucine was fed (150 mg per kg), a rapid and striking fall in blood glucose Cardiac and Renal Responses, to Acute Volume Loading concentration without glycosuria was obtained. D-leucine, in Patients with Heart Disease. JACOB GROSSMAN, L-alanine, L-valine, DL-threonine, and L-glycine did not ROBERT ROSENBLUM, SUN HING LAU and MoRRs have a significant effect. The a-keto acid analog of WOLFMAN, New York, N. Y. (introduced by Louis leucine, a-ketoisocaproic acid (a-KIC) induced hypo- Leiter). glycemia when administered intravenously, but its keto acid metabolites did not. Examination of the plasma In normal subjects and 14 patients with mitral stenosis amino acids and urinary amino and a-keto acids before of minimal to moderate severity, the effects of acute in- and after the administration of leucine indicated 1) a crease in body fluid volume on cardiac and renal functions normal pattern and 2) a rise in serum leucine consistent were determined. Cardiac catheterization was performed with that obtained in normal subjects. Thus, evidence with control measurements of pressures in the pulmonary of a defect in amino acid or keto metabolism was not circulation, cardiac output, renal hemodynamics and found. In incubation experiments a direct effect of electrolyte excretion. Two L of a hypotonic solution of leucine on the rate of oxidation of labeled glucose by glucose in water containing sufficient vasopressin (0.25 whole blood was not demonstrated. Epinephrine masked mU per kg per hour) to maintain low urine flow (1 ml the hypoglycemic effect of leucine. It was shown that per minute) were infused in 2 hours. Renal hemody- leucine caused a rise in plasma lactate and a fall in namics and electrolyte excretion were measured continu- plasma phosphate and potassium, findings consistent with ously. Repeat determinations were performed twice at an insulin-like action, although a significant increase in the end of volume loading, and again 1 hour later. peripheral glucose uptake was not detected. Plasma in- Changes in hematocrit and plasma sodium concentra- sulin determined directly by immunoassay by Yalow and tion reflected rapid equilibration. In normal subjects, Berson after feeding leucine rose sharply to levels 14 to this relatively small (5 per cent) expansion in fluid vol- 19 times the fasting concentration in 15 to 45 minutes. ume was associated with increase in cardiac output, renal The mother and sister of one patient showed a moderate hemodynamics and sodium excretion. A delayed rise in response to leucine, whereas none was found in normal urine flow despite vasopressin resulted from mild osmotic subjects nor in one patient with an islet-cell carcinoma. diuresis. Of 5 patients with normal responses, 1 with It is concluded that leucine, isoleucine, and their keto normal cardiac output and 2 of 4 with slightly reduced acid analog induce an increase in the concentration of outputs (cardiac index 2.4 to 3.0) presented no history plasma insulin in L.-I.H. irrespective of the blood glucose of failure. In 5 additional patients (cardiac index 1.9 level, an effect which may be attributed to their action to 2.7), renal hemodynamics rose significantly in the ab- on the insulin-secreting mechanism. sence of or preceding the increase in cardiac output. Fluid loading failed to increase pulmonary pressures. Correlation between normal pulmonary pressure and car- Intensification of Rabbit Atherosclerosis by Chronic diac response to volume was not observed. Five of 7 Hypothalamic Stimulation. C. G. GUNN, Oklahoma patients whose cardiac output rose had elevated pul- City, Okla. (introduced by Meyer Friedman). monary pressures; conversely, of 5 patients whose car- The following experiments were performed to test the diac output remained unchanged, 4 exhibited normal hypothesis that central nervous system mechanisms may initial pressures. Electrolyte and water excretion gen- influence experimental atherosclerosis. Chronic stimu- erally paralleled renal hemodynamics rather than cardiac lating or mock electrodes were implanted in diencephalic output. areas of male rabbits of comparable age, size and serum Under volume loading, cardiac patients, prior to fail- lipid level; 14 experimental and 15 control rabbits were ure, behave as normal subj ects. After failure, renal then fed a cholesterol-added diet for 3 months. The ex- response may vary independently of cardiac output. perimental group differed by receiving electrical stimu- Moderate elevation of pulmonary arterial pressure may lation via hypothalamic electrodes intermittently 5 days 994 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING each week at parameters slightly above behavioral and this hormone also enhances proliferation of erythroblasts autonomic thresholds. A third group of 4 stimulated or activates reticulum so successive periods of stimulation animals received a noncholesterol diet. Blood pressure, induce progressively the differentiation of unexpectedly cholesterol, phospholipid, total lipid and 17-OH-cortico- large numbers of stem cells. A single erythropoietin is steroid concentrations were measured each month. sufficient to induce both erythroblast proliferation, as After 3 months, postmortem examination included gross measured by reticulocyte production, and hemoglobin and microscopic grading of aortic and coronary athero- formation, as measured by Fe59 incorporation. Finally, sclerosis and actual analysis of aortic cholesterol content. these simple experiments, in which consistent results are All determinations were performed and recorded without obtained following administration of erythropoietin to knowledge of the source of the material. All groups ate animals whose erythropoiesis is suppressed without ap- a comparable amount and showed similar weight gains. parent damage, suggest that this system provides a useful The stimulated animals on a noncholesterol diet showed model for the investigation of many facets of cell growth. neither artherosclerosis nor lipidemia. On the other hand, the stimulated cholesterol-fed rabbits demonstrated Significance of the Distribution and Cytology of Iron in more than a twofold increase over the control rabbits' Primary Hemochromatosis During Treatment with aortic and coronary atherosclerosis and aortic cholesterol Phlebotomies. GABRIEL HAIBY, PRAWASE WAsI an1d content (4.63 vs. 2.0 g per 100 g dry weight; t = 3.5, MATTHEW H. BLOCK,* Denver, Colo. p <0.01). These stimulated animals also showed sig- nificantly higher serum cholesterols (1,587 vs. 1,215 mg), The distribution and cytologic characteristics of storage phospholipids (609 vs. 435 mg) and total lipids (2,785 vs. iron in patients with primary hemochromatosis were 2,117 mg) (t = 3.4, 3.6, and 3.4, respectively, p < 0.01 studied before, during and while completing treatment throughout). The concentration of corticosteroids was with phlebotomies. Before treatment there was moderate not significantly different among the three groups. cirrhosis with iron present in Kupffer cells, hepatic cells, Hypothalamic stimulation thus significantly intensified and portal connective tissue. Most of the iron, seen as the atherosclerosis and increased the circulating blood coarse, brownish crystals measuring 2 to 5 ,^, was in the lipids in cholesterol-fed rabbits. This response seemed hepatic cells at the periphery of the lobule and in the most marked when electrodes were in or near the ventro- portal connective tissue. Less iron, in amorphous form medial nucleus and less pronounced when anterior hypo- or in small yellowish-green crystals measuring 0.5 to 2 ts, thalamic areas were stimulated. was demonstrable in the hepatic cells at the center of the lobule and in Kupffer cells. The Kinetics of Erythropoiesis. CLIFFORD W. GURNEY After 60 to 80 phlebotomies the iron in the central and EDWARD FILMANOWICZ, Chicago, Ill. (introduced hepatic cells and in Kupffer cells was markedly decreased. by Leon 0. Jacobson). There was little change in the amount of iron in other areas. In contrast, after about 100 phlebotomies, the Erythropoiesis was investigated in the hypertransfused patients with continued phlebotomies developed an anemia mouse in which erythrocyte production is eliminated with- with cytologic and biochemical characteristics of iron out known alteration of metabolism or marrow damage. deficiency. There was little iron left in the peripheral Following one injection of erythropoietin, a highly puri- hepatic cells. Practically all the iron, in the form of fied a2-glycoprotein extracted from plasma of anemic large yellow crystals, was in the portal tissue. sheep, a wave of erythropoiesis swept through the mar- The iron of the central hepatic cells and Kupffer cells row, culminating in a reticulocytosis in the peripheral was probably the most recently deposited because, as de- blood, maximal at 72 hours and returning to zero by 144 scribed elsewhere, the most recently deposited iron is the hours. first used for erythropoiesis and is present in amorphous the With increasing doses of erythropoietin, peak form or as small green crystals. We postulate that the until a maximum of 1.4 reticulocytosis increased response residual large brown crystals of iron, found in the portal per cent was reached ml (12 sheep plasma equivalent). tissue after Since mice have the capacity to produce erythrocytes far treatment, were originally laid dowin in in excess of this rate, the effect of multiple doses was peripheral hepatic cells and later incorporated into the investigated. The reticulocytosis obtained clearly ex- proliferating connective tissue which had replaced these ceeded that predicted from the proj ected summation of cells after they had degenerated. This iron is mobilized individual doses. For example, from the time and mag- for erythropoiesis with difficulty, thus explaining the nitude of response to a single submaximal dose, a peak paradox of an iron deficiency anemia in patients with reticulocyte response of 2.1 + 0.6 per cent was expected considerable tissue iron. following 5 daily injections; however an observed re- sponse of 5.2 + 1.3 per cent was found on the sixth day. Maximum Rates of Excretion of Titratable Acid. JOSEPH A similar result was obtained when Fe"9 uptake by newly S. HANDLER, ANDRZEJ WOJTCZAK and MARTIN GOLD- formed erythrocytes was employed as a measure of BERG, Philadelphia, Pa. (introduced by J. Russell erythropoiesis. Elkinton). These studies suggest a more complex mechanism of action of erythropoiesis than simply the initiation of Under pentobarbital anesthesia, dogs were given 0.1 N differentiation from stem cells. It would appear that hydrochloric acid and sodium phosphate (pH 7.3) at AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 995 varying rates intravenously. When phosphate was ad- The characteristics of penicillin antibody were ex- ministered so that serum phosphate concentration rose amined. Enhancement of agglutination of penicillinized slowly to a level of 10 to 20 mmoles per L, the excretion cells coated with antibody by an anti--y2-globulin rabbit of titratable acid reached and remained at an apparent serum was taken to indicate in three instances that the maximal rate (range 20 to 30 AEq per minute per kg patient's antibody was a -y-globulin. Enhancement by an body weight). During this plateau in titratable acid anti-non--y2-globulin rabbit serum in three instances, and excretion the rate of phosphate excretion continued to dependency of agglutination upon heat-labile normal increase, urinary hydrogen ion concentration was con- serum component (s), was considered to be consistent siderably lower than it was earlier in the same animal, with complement fixation. Zone electrophoresis in one in- and in some dogs the rate of potassium excretion also stance resulted in the separation of the hemagglutinating rose. The plateau was unaffected by increasing serum antibody among the fast moving gamma globulins. Pco2. Whenever serum phosphate rose rapidly due to Hemagglutinating antibody to penicillin develops in- accelerated administration of phosphate, urinary concen- frequently, except in patients developing allergic reac- tration of hydrogen ion rose and the rate of excretion tions to penicillin, and in systemic lupus erythematosus of titratable acid increased considerably (to more than and acquired hemolytic disease. Its development in the 40 AEq per minute per kg body weight) even in situa- latter diseases may be a reflection of an enhanced capacity tions where previously a plateau had been obtained in for patients with these two diseases to make antibody. titratable acid excretion. The highest rates of titratable acid excretion did not necessarily occur in those dogs Inadequacy of the "Pore Theory" to Account for the who manifested the highest rate of phosphate excretion Action of Neurohvpophyseal Hormones on Water and or the most severe acidosis as measured by whole blood Solute Movement Across a Living Membrane. RICH- pH. Lower rates of titratable acid excretion were ob- ARD M. HAYS and ALEXANDER LEAF,* Boston, Mass. tained in dogs that did not receive an in addi- acid load It is customary to regard movement of water across tion to the phosphate load. Occasionally, but not re- living membranes as occurring by diffusion or by bulk the producibly, clearance of phosphate exceeded the flow (Poiseuille flow). In vitro measurements of net simultaneous clearance of creatinine. (Aw) and unidirectional (,O) fluxes of water across the The data suggest two mechanisms for titratable acid toad bladder were made gravimetrically and isotopically, excretion under these circumstances. The first is hy- respectively. Without vasopressin, A, is very small drogen ion secretion into tubular fluid rich in phosphate (2.1 4l per cm2 per hour) even with large osmotic buffer wherein the rate of titratable acid excretion is gradients across the membrane and large 0/w (334 4ul per limited by a maximum rate of hydrogen ion secretion. cm2 per hour). These findings define an average pore The second mechanism, apparently related to serum phos- radius of some 6 A and are consistent with transport by phate concentration and its rate of increase, may involve diffusion. With vasopressin added to serosal medium tubular secretion of an acid phosphate ion as postulated without an osmotic gradient, A, remains essentially zero in "phosphate aciduria." (no active transport of water) but 0w nearly doubles (584 4l per cm2 per hour). However, in presence of Penicillin Antibody in Disease. JEAN HARRIS and JOHN osmotic gradients, A, increases strikingly with hormone VAUGHAN,* Rochester, N. Y. without further change in o,. For gradient of 163 mOsm per kg water A, averages 4l per The hemagglutination test for the detection of penicillin 225 cm' per hour, a very large value comparable to the hydraulic flow through antibody (Ley) has been applied to a study of patients porous artificial membranes. This response to with 1) recent penicillin reactions, 2) recent penicillin hormone therapy without reactions, 3) autoimmune hemolytic dis- is independent of sodium or potassium in the bathing medium and is shown to result, largely if not entirely, ease, 4) systemic lupus erythematosus, and 5) miscel- laneous medical illnesses. from an action of the hormone on the mucosal surface of the membrane. Diffusion could account Six of 16 patients diagnosed clinically to have de- for less than 1 per cent of such net movement water veloped allergic reactions to penicillin had positive of and conven- tional calculations of mean pore radius, assuming Poi- hemagglutination reactions. Thirteen patients were also skin tested; 6 had positive immediate reactions and 4 had seuille flow, yield values as high as 40 A. Such large delayed reactions. Only 3 patients had both hemag- pores are inconsistent with the low permeability of the membrane to most small glutinating antibody and positive skin tests. molecules and ions, i.e., re- flection coefficients for Cl and Sera from 167 patients, obtained at random from the thiourea (radii < 3 A) of 0.9995 and 1.00, respectively, in spite of very large net hospital chemistry laboratory, were all negative for transfers of water. Movement of water through "pores" hemagglutination; 31 sera selectively obtained from the likewise would fail to explain the very striking and medical ward services from patients 3 to 7 days after spe- cific increases in permeability of the membrane following completing a course of penicillin therapy also were nega- vasopressin to urea, acetamide, proprionamide, butyra- tive. Three of 307 sera from patients receiving oral mide, cyanamide and dimethylformamide while permeabil- prophylaxis for rheumatic fever were positive. Four of ity to many molecules and ions of similar and smaller 12 patients with lupus erythematosus and 4 of 21 patients radii is unaffected by hormone. with acquired hemolytic disease were positive. An alternative hypothesis will be presented. 996 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING

Supportive Effect of Adrenal Steroids on Acetate tional status of man. These results suggest the serumii Metabolismn. ALLEN R. HENNES and TOMMY W. folic acid activity may be due largely to one or more REDDING, Oklahoma City, Okla. (introduced by R. H. polyglutamates (possibly diglutamyl derivatives) of one Bayley). or more of the folic acid coenzymes. Should such prove acid may represent We have studied the effect of maintenance doses of to be the case, the serum folic activity primarily folic acid converted in the liver to metabolically cortisone on utilization of acetate-l-C'4 in two patients active triglutamyl pteridine forms, rather than unchanged with adrenal insufficiency. Each patient served as his ingested folic acid-active materials. Parenthetically, it own control. Prior to one of their two studies, each was observed that the L. casei assay medium used sufficed patient was taken off maintenance cortisone for two days. as a basal medium for S. faecalis and L. citrovorunii, At the start of all experiments, patients were normo- for different media for the tensive, and glucose, sodium, potassium, and chloride thereby eliminating the need three microorganisms. concentrations were normal. However, both subjects developed definite symptoms of adrenal insufficiency 6 to Fluorescent when cortisone had Reaction of Rheumatoid Leukocytes zith 8 hours after start of experiment Aggregated Gamma Globulini. EVELYN HEss and been omitted. Plasma lipids were fractionated on silicic MORRIS ZIFF,* Dallas, Tex. acid columns. C"402 was determined by the method of Frederickson and Ono. In a study of the binding of rheumatoid factor to cellu- When cortisone was omitted, the following defects in lar elements, it was observed that washed leukocytes ob- synthesis of plasma lipids were found in both patients. tained from the blood of rheumatoid arthritis patients 1) Peak specific activity in plasma triglycerides was one- with high titers of rheumatoid factor were agglutinated eighth to one-tenth of control. 2) Disappearance of by aggregated human gamma globulin (Cohn Fraction triglycerides from plasma was definitely slower than in II). Leukocytes from nonrheumatoid individuals were the control state. 3) Appearance of C"4 in plasma free not agglutinated under the same circumstances. They cholesterol was one-half and three-fourths of control. 4) were rendered agglutinable, however, by incubation with Esterification of cholesterol was significantly slower than rheumatoid serum. These observations suggested that the in the control state. C`402 specific activity curves were leukocytes of rheumatoid blood are coated with rheuma- different in adrenal insufficiency from control curves, in toid factor. one case strikingly so, indicating an effect of adrenal In order to further establish this fact, buffy coats were steroids on this extremely stable parameter of metabolism. separated by a phytohemagglutin sedimentation method It is concluded that a maintenance level of 17-hydroxy- and stained with a fluorescein isothiocyanate labeled and corticoids is required for normal synthesis and disap- heated sample of FII. The presence or absence of fluores- pearance of plasma triglycerides. Synthesis of free and cent staining of the leukocytes was observed under a long esterified cholesterol is definitely increased by maintenance wave ultraviolet lamp. Fluorescence, which could be cortisone, but effects are less striking than onl triglyceride blocked by prior treatment with unlabeled FII, was ob- synthesis. Defects in synthesis and removal of triglyc- served in the case of the rheumatoid leukocytes even when erides and abnormal utilization of acetate appear to be the titer of rheumatoid factor in the serum was low or major and early abnormalities in adrenal insufficiency. negative. This occurred with 39 of 42 rheumatoid prepa- rations and 8 of 13 from children with rheumatoid Serumn Folic Acid Activity: Assay and Natntre. VICTOR arthritis. Sera from 7 of the 8 positively reacting juvenile HERBERT, Boston, Mass. (introduced by ". B. Castle). preparations gave negative serological tests for the rheu- matoid factor. Among 37 nonrheumatoid buffy coats, one Prior attempts to assess folic acid deficiency by micro- from a patient with erythema nodosum and a second assay of human serum have met with scant biologic from a patient with lues fluoresced. Both of these also success. The present studies were undertaken in part to gave positive serological tests by the latex fixation determine why a more recent attempt was successful, method. Thus, fluorescent staining of buffy coat appears in part to the nature of the folic acid and determine to provide a sensitive test for the "rheumatoid factors." activity in serum. It was found that most of the serum In addition to providing the basis for a test, the folic acid activity is highly labile unless protected (pre- indicates that the rheumatoid fac- sumably against oxidative destruction) by ascorbic acid phenomenon described tors are adherent to cells which are in contact with or d-iso-ascorbic acid. When adequately protected, serum serum. Fluorescent staining of blood smears has shown may be assayed directly, without autoclaving to pre- that both granulocytes and nongranulocytes are coated. or folic acid activity. Activity cipitate protein "release" The mechanism of the binding of the rheumatoid factor by may be converted from labile to stable form by auto- leukocytes and its possible significance will he discussed. claving in the presence of adequate ascorbic acid. Serum folic acid activity, either protected by ascorbic acid or Factors Deoxyribonucleic Acid III converted to stable form, allows only slight growth of Influencintg Syntthesis Vivo. HOWARD H. HIATT- an1d TADEt-SZ B. BOJARSKI, S. faecalis and L. citrovorumn. The lability of this ma- Boston, Mass. terial, as well as its relative unavailability for S. faecalis and L. citrovorum, may explain the failure of earlier at- Thymidylate (TMP) kinase is an essential enzyme ir tempts to assess microbiologically the folic acid nutri- the reactions leading to deoxyribonucleic acid (DNA') AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 997 synthesis. We have found kinase specific activity to be much greater than that in untrained animals fasted for 24 low in adult human and rat tissues known to synthesize hours. little DNA (liver, kidney, lung), and high in proliferat- These studies demonstrate the remarkable capacity of ing tissues (fetal and neonatal liver and kidney, gastro- adipose tissue to adapt to manipulation in the time of intestinal mucosa, bone marrow, tumors). Activity in feeding and have interesting implications in the overall human liver is threefold and in dog liver eightfold that scheme of intermediary metabolism. present in rat liver. Orotic acid-C1' incorporation studies indicate a correlation between the level of kinase activity The Effects of Mephentermine on Cardiac Output in and the amount of DNA synthesis. Kinase activity in Normotensive and Hypotensive Dogs. A. W. HORSLEY regenerating rat liver increases up to 30-fold beginning and JOHN W. ECKSTEIN, Iowa City, Ia. (introduced by 18 hours following partial hepatectomy, and returns to E. L. DeGowin). resting levels within 120 hours. A less marked increase There are conflicting reports concerning the hemody- follows CC14 administration. The increase in regenerating namic responses to mephentermine (Wyamine) in intact liver can be prevented by 5-fluorouracil (FU), which is dogs. These studies were done to determine the effects known to interfere with TMP synthesis. Up to a ten- of the drug on cardiac output. Right atrial and femoral fold increase in activity in several tissues is observed arterial pressures and cardiac output were measured in following prolonged administration to normal rats of Nembutal-anesthetized and also in gallamine-paralyzed thymidine, which is presumably converted to TMP within dogs before and after intravenous administration of the cell. The effects of FU and thymidine are probably mephentermine sulfate (0.3 to 0.6 mg per kg). Observa- explained by our in vitro observations that kinase activity tions were made in normotensive dogs and in those made is labile in the absence of TMP, but stable in its presence. hypotensive by hexamethonium (100 mg i.v.). Indo- Thus, enzyme activity in vivo probably changes with cyanine green dye was injected into the right atrium and variations in the level of substrate, which acts as protec- continuously recorded time-concentration curves were ob- tor. Our data also suggest a regulatory mechanism for tained from the carotid artery. Control values were DNA synthesis in regenerating liver at a reaction earlier obtained during a 5 minute period before mephentermine than that involving TMP kinase. injection. Experimental observations were made when arterial pressure became stable after having risen to a Hyperlipogenesis in Adipose Tissue of Rats Limited to a maximum level. Daily Two Hour Feeding Period. Guy HOLLIFIELD, Cardiac output increased in each of 22 experiments Charlottesville, Va. (introduced by William Parson). when mephentermine was given. It increased from an average of 2,421 ml per minute during control periods to Glucose loads, fed to rats after a fast, cause a prompt an average of 2898 ml after the drug in 6 paralyzed nor- increase in the amount of C"-labeled acetate incorporated motensive dogs (p < 0.01); from 1,799 to 2,465 in 6 into lipids by liver slices. This increase in lipogenesis is paralyzed hypotensive dogs (p < 0.01); from 2,457 to accompanied by a rise in the activity of pentose phosphate 3,280 in 5 anesthetized normotensive animals (p < 0.01): enzymes. Liver slices from rats trained to eat their day's and from 2,754 to 3,394 in 5 anesthetized hypotensive dogs ration in one hour also show increased lipogenesis. (p < 0.02). Significant increases in stroke volume were While adipose tissue is known to be the major site of noted only in the normotensive dogs. Stroke volume did lipid synthesis and has high pentose phosphate enzyme not increase in the dogs with ganglionic blockade because activity, little is known of its adaptive response to of marked increases in heart rate with mephentermine. manipulation of food intake. Heart rate tended to decrease in the paralyzed normo- In this study rats were allowed ground chow only 2 tensive group and increase in the anesthetized normoten- out of each 24 hours; 5 to 6 rats were sacrificed at the sive group. Atrial pressure tended to increase in each end of the feeding period after 1 to 7 days on this group but the changes were small a id insignificant. Cal- program. The in vitro incorporation of C`-labeled acetate culated "central blood volume" increased in 21 of the 22 into lipids by epididymal fat, liver glycogen, and free experiments. fatty acid content (FFA) of adipose tissue was measured. The increased cardiac output and central blood volume The incorporation of C`-labeled acetate into lipids by may be associated with systemic venous constriction. adipose tissue increased over 30 times by the fifth day of Venous constriction has been observed in man following this regimen. This remarkable increase in the rate of mephentermine administration. lipogenesis was accompanied by a gradual fall in the FFA content of adipose tissue while liver glycogen rose Lipid, Carbohydrate anid Purine Abnormalities in Von for the first 3 days. Adipose tissue from animals trained Gierke's Disease. R. RODNEY HOWELL, DORIS ASHTON for 5 days had 300 to 400 per cent more glucose-6-phos- and JAMES B. WYNGAARDEN,* Durham, N. C. phate and 6 phosphogluconate dehydrogenase activity than Three siblings with glycogen 1 storage disease of liver did animals fed in this way for day. The activity of showed fasting abnormalities of serum FFA (1,100 to these enzymes in livers from these animals was un- 2,500 ,umoles per L), triglycerides (570 to 1,145 mg per changed. The glycogen content of livers from animals 100 ml), phospholipids (370 to 500 mg per 100 ml), fasted for 24 hours after 4 or 5 days of training was very cholesterol (287 to 485 mg per 100 ml), total lipids 998 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING

(1,535 to 2,735 mg per 100 ml), ketones (12 to 29 mg per higher rates of antibodies to the DA virus than were 100 ml), lactate (47 to 98 mg per 100 ml), pyruvate (1.8 found in sera from persons in the same area but without to 1.9 mg per 100 ml), glucose (G) (18 to 59 mg per overt evidence of hepatitis. Nine of 11 serial serum series 100 ml), and urate (9.5 to 11.7 mg per 100 ml). Glu- from experimentally induced hepatitis in humans showed cagon infusionis led to gradual rise of blood glucose (max. fourfold or greater rises in the DA-treated cell ag- 100 to 140 mg per 100 ml) and fall of FFA (1,569 to 360 glutinin. No rises of this agglutinin were noted in serial umoles per L), prompt rise of lactate (198 mg per 100 sera from cases of measles or poliomyelitis, but elevated ml), and pyruvate (3.8 mg per 100 ml), and fall of titers were noted with frequency in certain types of serum C02 (11 mEq per L). Liver obtained at operation hepatic cirrhosis. from one sibling (7 year old male) showed < 10 per cent The data indicate an association between positive sero- of normal G-6-phosphatase activity, but normal phos- logical tests involving the DA virus and certain forms of phorylase, G-6-P dehydrogenase, 6-P-gluconate dehydro- liver disease, but do not warrant conclusions as to any genase, phosphoglucomutase and fructose diphosphatase specific etiologic role of this virus. activities. Liver fat was 15.3 per cent, glycogen 9.3 per cent, lactate 438 mg per 100 ml, nitrogen 1.4 per cent (of .M etabolic Regulation of Cardiac Output. WILLIAMI E. wet weight of liver). Glucose-1-C'4 and -6-C'4 were HuCKABEE,* Boston, Mass. utilized by liver slices in normal proportions (C'402 from In mild exercise, no correlation was found between C-1/C-6 = 2.3 at 45 minutes). However, total utilization change in 02 consumption (AV02) and change in cardiac of glucose per gram of liver N was decreased, possibly output (A C.O.) in normal or heart failure patients, nor explaining diabetic type glucose tolerance observed. Liver consistent differences between the groups in the slope of slices incubated with or epinephrine glucagon showed this "response curve." However, the response of C.O. to appreciable glycogen breakdown and lactate production. an 02-sensing mechanism has not been clearly established; A postulated is: dynamic sequence phosphatase defi- biochemical changes which are only irregularly related to ciency -* increased G-6-P disposition into glycogen antd Po2 might be responsible. Therefore, the tissue metabolic via oxidative and -* increased glycolytic pathways produc- changes of hypoxia were induced in dogs in such a way tion of TPNH, DPNH, acetyl CoA, and lactate -> in- that blood and cellular 02 levels would remain high, creased of i.e., synthesis cholesterol, fatty acid, triglyceride by inhibition of cytochrome oxidase with cyanide, 0.02 to -> fatty liver and hyperlipidemias. The hyperlactic acid- 0.04 mmoles per kg, intra-arterially. C.O. increased emia -> increased muscle glycogen (1.7 per cent; forearm markedly, reaching sustained levels of 700 per cent in- A/V lactate = 47.7/38.3 mg per 100 ml, decreased urate crease. Despite continuing increase in venous blood clearance (2.8 ml minute and and per per m2) hyperuricemia. tissue Po2 (80 to 90 mm Hg), the C.O. increase was Hypoglycemia -> epinephrine and/or glucagon release progressive for 15 to 20 minutes. Vo, and Vco2 were glycogen breakdown and accentuation of all of above. reduced 20 to 50 per cent; blood pH was constant. The cardiac response, of a magnitude previously produced only Serological Reactions zwith a Newly Isolated Myxovirus. by muscular exercise, was associated with reduced ven- G. D. HSIUNG, PETER ISACSON and ROBERT W. tricular diastolic pressure and was not prevented by MCCOLLUM, New Haven, Conn. (introduced by John vagotomy, adrenalectomy (plus phentolamine), ganglionic R. Paul). blocking agents, antihistaminics, or clamping of the carotid Using human or rhesus monkey kidney cell cultures, a arteries. Several metabolic inhibitors blocking virus was isolated from the blood of a fatal case of in- at levels lower than cytochrome oxidase had no stimulat- fectious hepatitis which occurred during an epidemic. ing effect; the highest block was effected at pyruvic de- This agent, designated as the DA virus, grew in primate hydrogenase by oxythiamine. C.O. was diminished by kidney cells but without cytopathic effect, its presence this procedure in dogs and pigs; it remained normally being detected by the formation of plaques under agar responsive to infusions, epinephrine and cyanide, and overlay. A hemagglutinin was present, the characteris- always returned to the reduced level again after these tics of which served to classify this agent as a myxovirus stimuli. It was concluded that: 1) Blood flow is not closely related to mumps-Newcastle disease viruses. Sero- responsive to changes in molecular 02, but to metabolic logical studies have also indicated antigenic crossing with processes in the tissues without regard to Po2 or V02. mumps and certain simian myxoviruses. Human sera 2) The effective metabolic change is not all-inclusive, but have been tested for antibodies to this virus using tech- lies between cytochrome oxidase and pyruvic dehydro- niques of plaque neutralization, hemagglutination-inhibi- genase. 3) The metabolic change induces a cardiac re- tion, complement-fixation, and agglutination of chick red sponse by unknown mechanisms, not involving filling cells treated with the DA virus, a phenomenon originally pressure, catecholamines or sympathetic nerves. demonstrated by Burnet and Anderson in 1946, in which another myxovirus had been shown to have cell sensitiz- High Altitude Putlmonary Edemza. HERBERT N. HULT- ing properties which give rise to hemagglutination with GREN,* WARREN B. SPICKARD and KURT 0. HELLRIEGEL, sera from cases of infectious mononucleosis and hepatitis Palto Alto, Calif. and some other diseases. Sera from jaundiced cases of Eighteen patients with a unique variety of acute infectious hepatitis from the same epidemic showed pulmonary edema, occurring upon exposure to an altitude AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 999 of 12,200 to 15,300 feet, were observed at the Chulec In earlier studies designed to elucidate the physiologic General Hospital in La Oroya, Peru from 1950 to .1959. function of TBG, estrogens increased TBG and con- Initial manifestations appeared 12 to 36 hours after arrival comitantly decreased clearance of thyroxine from the from a lower elevation. Symptoms consisted of cough, plasma by peripheral tissues. However, changes in hemoptyses, dyspnea, and weakness. hormonal metabolism secondary to alterations in TBG Physical examination revealed tachycardia, hypotension, could not be distinguished from possible direct effects of cyanosis, and pulmonary rales. Signs of heart failure estrogen on hormonal degradation. In the present pa- were absent. Temperature elevations exceeding 1000 tient, control studies of thyroxine metabolism revealed were seen in only 3 patients and leukocytosis greater than that, while PBI was low and total hormonal turnover 12,500 cells was seen in only 5 patients. X-ray study normal, the rate of clearance of thyroxine was abnormally revealed bilateral pulmonary densities which were usually rapid. Although a six week course of estrogen induced patchy. In 6 patients diffuse pulmonary exudates filled fluid retention and pronounced gynecomastia, it did not both lung fields. No enlargement of the left ventricle or increase TBG or change the kinetics of thyroxine metab- left atrium was noted, but transient prominence of the olism. Direct estrogenic effects on hormonal degrada- pulmonary artery occurred in 7 patients. Electrocardio- tion were thereby excluded. grams revealed right ventricular "strain" patterns and These findings indicate that TBG regulates the rate of peaking of the P waves which became more normal on removal of thyroxine from plasma, presumably by limit- recovery. ing its availability to the cell. They further suggest that Bed rest and oxygen administration resulted in com- in man, thyroid activity is regulated by the delivery of plete clinical recovery and clearing of the pulmonary hormone to peripheral tissues, rather than by the concen- exudate in 24 to 48 hours. Digitalis was ineffective. No tration of hormone in the circulation. deaths occurred. Fifteen of the 18 patients had been thoroughly ac- Effects of Alcohol on the Liver: Mechanism of the Im- climatized, and developed pulmonary edema upon return- paired Galactose Utilization. KURT J. ISSELBACHER* ing to the mountains after a 1 to 3 week stay at sea and ELIZABETH A. MCCARTHY, Boston, Mass. level; 3 patients had not previously been exposed to high altitude. None gave a history of a preceding respiratory Previous investigators have observed that small tract infection. Eleven patients were less than 18 years amounts of alcohol ingested by normal individuals pro- of age and 14 were males. Recurrent episodes in 4 duce immediate and striking inhibition of galactose toler- patients and 2 occurrences in siblings suggest an in- ance, reflecting an impairment of hepatic galactose utiliza- dividual and a familial susceptibility. There was no racial zation. Since the galactose tolerance test is a very predilection. sensitive index of liver function, we have investigated the High altitude pulmonary edema thus appears to be a mechanism whereby alcohol can produce such pronounced unique effect of anoxia upon the circulation. The data alterations in carbohydrate metabolism. suggest that factors other than left ventricular failure Observations that the administration of alcohol to rats may be important in its pathogenesis. results in a doubling of the hepatic levels of reduced diphosphopyridine nucleotide (DPNH) suggested that the inhibition of galactose metabolism by alcohol might be Thyroxine Metabolism in the Absence of Thyroxine- secondary due to the increased DPNH levels-especially binding Globulin and its Implications with Regard to since a key enzyme in galactose metabolism (uridine di- the Physiological Role of Hormonal Binding. SIDNEY phosphogalactose-4-epimerase) is inhibited by DPNH. H. INGBAR,* Boston, Mass. Consistent with this hypothesis were the findings that the A patient with a newly-recognized syndrome, charac- alcohol inhibition of galactose oxidation in rat liver terized by absence from the plasma of thyroxine-binding homogenates was enhanced by factors increasing the globulin (TBG) has afforded a unique opportunity to metabolism of alcohol (resulting in concomitant increases evaluate the hypothesis that TBG is a regulator of the in DPNH) and completely reversed by metabolites re- peripheral turnover of thyroxine. Despite a protein- ducing the DPNH concentration. In erythrocytes, which bound iodine of only 2 ,ug per 100 ml, *there were no readily metabolize galactose but have no alcohol dehy- clinical stigmata of myxedema; BMR, cholesterol, and drogenase (ADH), there was no inhibition of galactose thyroid uptake of PIl" and I' were normal. These dis- oxidation by alcohol. However, when exogenous ADH crepant findings prompted electrophoretic studies of the was added, alcohol produced a marked inhibition of patient's serum, which revealed normal binding of galactose oxidation by red blood cells. Examination of all thyroxine by pre-albumin, but no demonstrable binding the soluble enzymes involved in the conversion of ga- by TBG. Associated abnormalities included atrial septal lactose to C02 revealed no inhibition of enzyme activity defect and testicular atrophy. Androgenic hormones de- by alcohol. In the presence of ADH however, even very crease thyroxine-binding by TBG. However, the pa- low concentrations of alcohol produced an 80 to 90 per tient's manifest hypogonadism and failure of prolonged cent inhibition of one enzyme, namely UDP-galactose-4- administration of prednisone to restore TBG in the serum epimerase. excluded gonadal and adrenal androgens as the cause of These studies indicate that alcohol inhibits galactose his binding abnormality. utilization in the liver by producing increased cellular 1000 PROCEEDITGS OF THE FIFTY-SECOND ANNUAL MEETING

levels of DPNH. The increased DPNH effectively in- Cl and HCO3 in an isosmotic fluid; and 3) dependence hibits a key enzyme in galactose metabolism. The results of secretion on carbonic anhydrase activity. serve to emphasize the importance of cellular concentra- Pancreatic juice in man was obtained by duodenal tions of pyridine nucleotides, as well as their oxidation- intubation with a collecting system which obviated gastric reduction state, in the overall regulation of carbohydrate contamination and minimized intestinal loss. The rate metabolism. of flow and composition of secretin stimulated secretion (1.0 a per kg) was studied in 27 subjects before and Oxidative Henmolysis and Precipitation of Hemoglobin: after cholinergic blockade (Lakeside, compound No. JB Heinza Body Anemiiias as an Accelerated Form of Red 323, 340). Although the rate of flow was markedly re- Cell Aging. JAMES H. JANDL* and DAVID W. ALLEN, duced (mean depression 65 per cent, range 38 to 89 per Boston, Mass. cent) there was no significant alteration in HCO3 con- centration (mean change + 3 per cent, range 0 to 8 per Although the basis for hypersusceptibility to the hemo- cent). Similar dissociation of HCO3 and flow rates has lytic effect of certain aromatic amines has been well been reported by us after carbonic anhydrase inhibition characterized recently, the actual mechanism of the with Diamox (J. clin. Invest. 1957, 36, 904). This is in hemolytic process is poorly understood. Comparisons marked contrast to the reciprocal rise in Cl and fall in were made of the effects in vitro of drugs such as HCO3 concentrations w-hen rate of flow is reduced by phenylhydrazine upoIn red cells and upon solutions of adrenal cortical hormones (cortisol, prednisone) in nor- crystallized hemoglobin. In either state hemoglobin was mal subjects or that which occurs spontaneously in pani- initially transformed reversibly to methemoglobin and creatitis. Na and K concentrations, which approximate then irreversibly to "sulfhemoglobin" and a fast moving plasma levels, remain independent of rate of flow in all component on electrophoresis and column chromatogra- circumstances. phy. Eventually hemoglobin in solution was precipitated These results suggest that the pancreas elaborates a into small, blue-staining coccoid bodies identical in ap- "precursor solution" of isotonic Na and K HCO3 which pearance and manner of evolution to Heinz bodies. These is then modified by a process in which Cl is exchanged denaturative changes in hemoglobin apparently involved for HCO3 within the gland, and requires carbonic anhy- oxidation of the thiol groups of hemoglobin, since they drase activity. It is presumed that this exchange occurs were slowed by reduced glutathione (GSH) and partially in the intercalated duct recently shown by electron- reversed by various thiols. GSH in the presence of microscopy to bear microvilli. phenylhydrazine and hemoglobin became bound to hemo- globin by forming mixed disulfides; in so doing GSH "Trachonia Viruses" Isolated in the Untited States. increased the size, while diminishing the number, of ERNEST JAWETZ,* PHILLIPS THYGESON, LAVELLE "Heinz bodies" formed. These interactions may explain HANNA, CHANDLER DAWSON and YUKIHIKO MITSUI, the disappearance of red cell glutathione and certain San Francisco, Calif. and Tokushima, Japan differences in Heinz body size previously observed by Trachoma is an eye disease affecting 400 million others during drug-induced anemias. per- sons in the world, often causing blindness. It is very Since these drugs appeared to catalyze the oxidative prevalent in Asia and Africa but rare in the United denaturation of hemoglobin by oxygen, the effects of States. Recently strains of "trachoma virus" have been prolonged exposure of red cells and hemoglobin alone to isolated in Africa and Asia. We have succeeded in grow- atmospheric oxygen in vitro were investigated. Under ing for the first time "trachoma viruses" from patients in 02, but not under CO, the same sequence of oxidative the United States. injury slowly evolved: methemoglobin, sulfhemoglobin, a The viruses were isolated by injecting conjunctival fast moving hemoglobin on electrophoresis, and, finally, scrapings in broth-saline containing 4 the mg per ml strepto- formation of typical Heinz bodies. mycin into the yolk sacs of 7-day embryonated eggs, in- It is concluded that: 1) Hemolytic aromatic amines cubated at 350 C. After 1 to 4 blind passages cause embryos oxidative denaturation of hemoglobin and its pre- died and elementary bodies were seen in stained yolk sac cipitation as Heinz bodies. 2) These drugs are redox smears. After additional passages egg LD50 titers reached intermediates between oxygen and hemoglobin because of 107 per ml. The agents fixed complement with specific their capacity to react well either with paired or with antisera to psittacosis virus, but were not infectious for single electrons. 3) Heinz body anemias represent an small laboratory animals or tissue cultures by usual tech- acceleration of normal mechanisms of red cell aging. nics. One strain (Bour) was isolated from acute trachoma A "Precursor Solution" of Pancreatic Juice: Evidence for in a white California resident, with a serum complement- an Exchange Mechanism. H. D. JANOWITZ* and D. A. fixing (CF) titer of 1: 64 to psittacosis-LGV group DREILING, New York, N. Y. antigens. This virus diluted 10' produces intense fol- licular conjunctivitis with abundant inclusions in M. Any hypothesis of pancreatic electrolyte secretion must cynoniolgn1s. Instilled into the eyes of human volunteers at least account for 1) the apparent direct relationship it produces typical acute trachoma with inclusions. A between HCOs concentration (range 25 to 150 mEq per second strain (Asgh) was isolated from a trachomatous L) and rate of flow; 2) the reciprocal relation between relapse in a Pakistani who had acquired the infection AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 1001 more than 20 years ago. During the past 5 years, living serum was studied. The thoracic duct was cannulated in Britain and the United States, he had been free from in the neck in 2 subj ects and samples of lymph were col- ocular symptoms. His serum CF titer to the group lected in the fasting state and after feedings of 100 g of antigen was 1: 4. That virus diluted 101 produces only corn or coconut oil. Serum samples were obtained after minimal follicular conjunctivitis with rare inclusions in comparable corn and coconut oil feedings. monkeys. Thus it appears that either the geographic Chylomicrons were isolated from all samples of serum origin or the persistence in an infected host may influence and lymph by centrifugation at 26,000 x G (sp. gr. 1.005) pathogenetic properties of "trachoma viruses." Three for 30 minutes and then purified by saline washes and re- additional strains were isolated from active trachoma in peated centrifugations in the same gravitational field. Apache Indian children at the San Carlos Reservation. After removing the chylomicrons, lipoproteins of the The pathogenetic position of these strains is under study. various density classes were separated from the infra- nates by successive ultracentrifugations at specific gravi- Characterization and Significance of Inhibitors Control- ties of 1.019, 1.063 and 1.21. The fatty acid composition ling the Activation of Fibrinolysis in Alan. ALAN J. of each fraction was determined by gas chromatography. JOHNSON,* New York, N. Y. The fatty acid composition of the lymph chylomicrons The level of circulating inhibitors in the plasma of 8 hours after ingestion of fat became virtually identical normal individuals is immediately concerned with the with that of the corn oil or the coconut oil in the test control and regulation of fibrinolysis in man. The mech- meal. The fatty acid composition of the serum chylo- anism of fibrinolysis by streptokinase (SK) may be divided microns showed similar changes to a lesser degree. into 3 stages: 1) activator formation by streptokinase and Comparisons were made of the changes in the fatty acid proactivator; 2) plasmin formation by activator and composition of the other lipoprotein fractions of the plasminogen; and 3) fibrinolysis production by plasmin serum and lymph. Little change was produced in the and fibrin. Urokinase, an activator from human urine, low density fraction 1.019 to 1.063 by either the corn or reacts with plasminogen directly as in Stage 2 above. cocoInut oil. The density classes 1.005 to 1.019 and An inhibitor of streptokinase, Stage 1, and an in- 1.053 to 1.21, however, showed definite enrichment with hibitor of activator, Stage 2, will be described in the the predominant components of the dietary fat. present studies. Both occur in the a-globulin fraction The prompt appearance of the fed fatty acids in the and appear to be nonenzymatic proteins since they are high density lipoprotein fraction is in accord with the nondialyzable, digested by trypsin and pepsin, fast-acting hypothesis, based on analysis of the peptide composition, and are relatively temperature independent. They are of a close association between chylomicrons and high labile at 40 C and are readily denatured by organic density lipoproteins. solvents. The streptokinase inhibitor is measured spe- cifically by SK inhibition and is not affected by urokinase A Comparative Study of Pulmonary Circulatory Effects in vitro or by spontaneous activator in vivo. It resembles of Intravenous Isoproterenol in Pulmonary Emphysema, SK antibody which also acts at Stage 1, neutralizing SK, Heart Failure and Mitral Stentosis. KAYE H. KILBURN because it usually increases in vivo 5 to 10 days after and HERBERT 0. SIEKER,* Durham, N. C. parenteral SK injection or following natural infection The cardiopulmonary circulatory effects of intravenous with the streptococcus. The increase was not propor- isoproterenol were determined in various types of cor tionate, however, with respect to SK antibody. The pulmonale in order to separate causes of pulmonary hy- streptokinase inhibitor is different from SK antibody pertension which often coexist, e.g., chronic pulmonary since it is: 1) ether labile, 2) forms no precipitate with insufficiency and heart failure. The results were com- SK in gel-diffusion studies, and 3) shows no reaction on pared with those obtained during exercise, oxygen breath- passive cutaneous anaphylaxis. The activator inhibitor is ing, acetylcholine infusion, acetyl strophanthidin ad- measured specifically, by urokinase inhibition. It differs ministration, and unilateral occlusion of the pulmonary from the streptokinase inhibitor in its pH stability and artery. Studies were done on 37 patients with pulmonary does not give a delayed response to the parenteral in- emphysema with and without heart failure, 8 with heart jection of SK. failure alone, 8 with mitral stenosis, and 7 normal con- These (two) inhibitors have been found to determine, trols. Cardiac output and pressures in the right heart, in part, the thrombolytic effect of intravenously ad- pulmonary circulation and arterial system were measured. ministered SK, to control physiological spontaneous Pulmonary vascular resistance was calculated. fibrinolysis, and to be deficient, if not pathogenetic, in Exercise increased cardiac output and pulmonary artery pathological spontaneous fibrinolysis. pressure in the 3 groups of patients. In heart failure, isoproterenol increased the cardiac index (1.9 to 3.0 L Fatty Acid Patterns of Human Lymph and Serum after per minute per m2), lowered pulmonary artery pressure Corn Oil or Coconut Oil Feeding. HERBERT J. KAY- (41 to 35 mm Hg), and lowered pulmonary vascular re- DEN, ARTHUR KARMEN, ALLAN E. DUMONT and JOSEPH sistance (466 to 297 dynes-sec-cm'). In pulmonary BRAGDON, New York, N. Y. and Bethesda, Md. (intro- emphysema, it increased the cardiac index and pulmonary duced by Charles E. Kossmann). artery pressure (2.9 to 3.9 L per minute per m2; 30 to 35 The effect of different dietary fats upon the fatty acid mm Hg). Patients with mitral stenosis showed a simi- composition of the lipoproteins of human lymph and lar response. In emphysema, oxygen breathing and 1002 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING acetylcholine lowered pulmonary artery pressure without intracellular amino acid pool does not appear to be of altering cardiac output, and acetyl strophanthidin nim- functional significance in protein synthesis. icked the effect of isoproterenol. Unilateral pulmonary artery obstruction increased pulmonary artery pressure Observations on1 the Mechanism of Tolerance to the Local in emphysema but not in heart failure. Patients with Shwartzman Phenomenon Using Isotopically Labeled emphysema and heart failure combined showed the heart Endotoxin. DIETER KOCH-WESER* and JOI-IN M. failure type of response to isoproterenol. MOSES, Cleveland, Ohio. It is suggested that isoproterenol is useful in separating Tolerance to endotoxins has been studied using the local pulmonary hypertension related to left ventricular failure Shwartzman phenomenon as a measure of host reactiv- from that associated with structural changes in the lungs ity. When appropriate doses of lipo-polysaccharide from or mitral valve. These studies also imply that left ven- E. coli (LPS-C) are used, this is a reliable index of tricular failure in patients with pulmonary emphysema tolerance. Tolerance was induced in two different ways, will be manifest primarily as right ventricular failure. either by 7 or more daily injections of the same LPS-C, Finally, it is suggested that isoproterenol may be useful or by 7 daily injections of a sonic cell wall preparation in treatment of heart failure either with or without pul- of Paracolon, containing another lipo-polysaccharide monary emphysema. (LPS-P). The mechanism of tolerance was investigated by using LPS-C labeled either with carbon"4 or with The Effect of Cell Structure and Growth Hormone on chromium51. Continuous blood clearance, the role of Protein Synthesis in Striated Muscle. DAVID M. Kip- "trapping" in the lung and of removal by the RES were NIS and ERIc REISS, St. Louis, Mo. (introduced by determined by cannulating one carotid artery and leading Carl V. Moore). the cannula through a scintillation counter into the op- The effects of cell structure and growth hormone on posite jugular vein, as well as by assaying the radio- the metabolic events regulating protein synthesis in activity in several organs after an intravenous injection muscle form the basis of the present report. Use of the of labeled LPS-C. "intact" in vitro rat diaphragm permitted investigation, Approximately 96 hours after the first injection, the under steady state conditions, of 1) intracellular trans- serum of rabbits made tolerant with LPS-C contains port of amino acids as measured with the nonutilizable precipitins which combine in vitro with added labeled amino acid a-aminoisobutyric acid-l-C" (AIB); 2) intra- LPS-C. The precipitate, which by counting can be cellular amino acid concentration (pool size) as reflected quantitated in terms of LPS-C content, has no endo- by lysine and proline determinations; and 3) protein toxin activity as measured by its ability to prepare for synthesis as determined by incorporation of labeled amino the local Shwartzman phenomenon. When serum from acids into tissue protein. an animal made tolerant by injections of LPS-C is given Amino acid transport decreased 50 per cent following to a normal rabbit, the recipient becomes tolerant to hypophysectomy and was restored to normal by in vivo LPS-C for at least 48 hours. If labeled LPS-C is given administration of growth hormone. This effect appeared intravenously to rabbits made tolerant by repeated in- to be partially mediated by insulin since it was greatly jections of LPS-C, the radioactive material is rapidly reduced in alloxan-diabetic hypophysectomized animals. trapped in the lungs, presumably as an endotoxin-precip- Furthermore, in vitro addition of bovine or human growth itin complex. The serum of rabbits made tolerant to hormone produced small and, at times, equivocal results. LPS-C by repeated injections of LPS-P does not con- Intracellular pools of lysine and proline were not ap- tain precipitins to LPS-C and, injected into normal ani- preciably changed by hypophysectomy or growth hormone mals, does not produce tolerance to LPS-C. administration; similar results were obtained with AIB It would appear, therefore, that there are at least two in vivo. Amino acid incorporation into protein decreased mechanisms involved in tolerance, one associated with 50 per cent following hypophysectomy and was restored antibody formation, which can be passively transferred, to normal by bovine growth hormone administered in the other nonspecific, which cannot be passively trans- vivo but not in vitro. Despite this hormonal effect, the ferred and seems to be related to an activated RE sys- specific activity of the intracellular amino acid pool was tem. similar under all conditions, suggesting direct shunting by Diet Amino Acid Pattern in of amino acids entering cells to sites of protein synthesis Modification of' Urinary Cystinuria. FELIX 0. KOLB and HAROLD A. HARPER, or a very small "effective" pool. This conclusion was San Francisco, Calif. (introduced by Peter H. For- strengthened by the observation that label appeared in sham). protein as a linear function of time whereas specific ac- The amino acid of with of urinary pattern patients tivity the amino acid pool approached equilibrium cystinuria and cystine stones shows a specific hyperexcre- exponentially. tion of lysine, cystine, arginine, and ornithine in the Growth hormone stimulates both amino acid transport presence of normal plasma concentrations on the basis of and protein synthesis. These processes are apparently a renal tubular reabsorptive defect, virtually complete so intimately coordinated that changes in one are accom- for lysine and cystine and partial for arginine and orni- panied by similar changes in the other. The "total" thine. The oral administration of methionine and cys- AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 1003 teine markedly enhanced the cystine excretion in cys- period. Doses in the 30 mg per kg range do not produce tinurics, while minor increases were observed in normals. this effect. Three patients with multiple myeloma im- In contrast, oral administration of cystine produced no proved; two symptomatically without alteration in ab- increase in urinary cystine even in cystinurics. This is normal proteins, and a third patient has had two remis- readily explained by differences in the solubility of indi- sions with positive decrease in abnormal protein only vidual amino acids affecting absorbability from the in- after the initial large dose of the drug. testine. After methionine and cysteine feeding, the plasma Cyclophosphamide induces the same bone marrow de- levels increased significantly while no appreciable in- pressant effects in vivo as other alkylating agents. It is crease was noted after cystine. Drastic restriction of not active in bone marrow in vitro. It may be useful in dietary methionine- and cysteine-containing proteins has treatment of multiple myeloma. been studied over a period of 4 years in 14 human sub- jects and in 1 cystinuric dog. By dietary means urinary Acute Adult Glomerulonephritis: Studies of Healing and cystine was reduced without consistently altering urinary Progression to Chronicity. DANIEL S. KUSHNER, lysine or arginine. Under different dietary conditions PAUL B. SZANTO and ALVIN DUBIN, Chicago, Ill. various excretion patterns of the 4 affected amino acids (introduced by S. Howard Armstrong, Jr.). have been produced without any fixed ratios. On only intravenous glucose as a source of calories a human Most adults (analogously to children); have been cystinuric showed the same urinary excretion of cystine presumed to recover completely from j8-streptococcal and lysine as when on marked dietary restriction, sug- acute glomerulonephritis; some hold nonstreptococcal gesting that this is a minimum of cystine derived from pathogenesis for ominous adult chronic glomerulonephri- amino acids of endogenous protein. Dietary restriction tis. Correlation of serial renal biopsies with recurrent has been of little value in reducing cystinuria in 3 cys- streptococcal infections, quantitated incidental pyelone- tinuric children whose greater need for protein anabolic phritis, electrophoretically quantitated proteinuria, stepto- processes might not have been met by the diet. Two of coccal antibodies, and crude glomerular filtration rates, the three children continued to form stones. In contrast, in all adults since 1956 at Cook County Hospital with the reduction of urinary cystine by diet in 11 adults and presenting diagnosis of acute glomerulonephritis was a 1 dog correlated with decreased stone formation and first step in evaluation of these presumptions. Analysis even resolution of stones. of 45 patients leads us to this breakdown: Histologic healing: 10; streptococcal relationship ini- tially: all; subsequently: 2; associated nephritic ex- Studies With a Cyclic Phosphamide Ester of Nitrogen Mustard: Cyclophosphamide. D. R. KORST, F. D. acerbations: 1; incidental pyelonephritis: 1; average JOHNSON, C. D. COBAU, M. H. RENNIE and E. P. follow-up only 8 months. FRENKEL, Ann Arbor, Mich. (introduced by Paul S. Histologic chronicity, clinical healing: 7; streptococcal Barker). relationship initially: 6; subsequently: 4; associated nephritic exacerbations: 3; incidental pyelonephritis: 3; Cyclophosphamide (Cytoxan), an ester of nitrogen average follow-up 19 months. mustard, is activated by intracellular enzymes to the Histologic and clinical chronicity: 4; streptococcal re- active bis, 8-chlorethyl group. We have performed lationship initially: all; subsequently: 1; no nephritic studies in animal and man to determine tolerance, bone exacerbation or pyelonephritis; average follow-up 21 marrow effect, and plasma protein changes. months. The turnover of radioiron (Fe') and tritiated thymi- Uncertain initial classification: acute vs. chronic with dine (H'T) has been used in rats and in rat bone mar- hematuric exacerbation: 16; streptococcal relationship row 24 hour culture; 22 mg per kg of drug in 6 rats initially: only 8; subsequently: 1 (no nephritic exacer- results in a 24 hour RBC uptake of 5.3 + 2.3 per cent. bation); incidental pyelonephritis: 7; average follow-up Normal rat Fe59 turnover is 18.2 + 2.5 per cent and after 16 months. 0.25 mg per kg of HN2 is 10.3 + 3.7 per cent. Mice with Declined biopsy: 8; streptococcal relationship initially: myeloma tumor (X5563) and gamma globulin peaks all; subsequently: 1; no nephritic exacerbation or pyelo- show the same degree of suppression of iron turnover nephritis; GFR's <70 ml per minute: 4; average fol- after the drug without significant alteration in protein low-up 18 months. pattern. P32 localizes in highest concentration in marrow, Carbohydrate-rich a,-globulin was prominent in initial intestine and tumor. The change in H8T turnover is not proteinuria (maximum 10 g per 24 hours) of which as significant as Fe". The in vivo changes are not seen gamma globulin never exceeded 12 per cent. in the marrow culture experiments where cyclophos- When present, histopathologic stigmata of chronicity phamide is added, while depression is obtained with comprised: 1) Residuals of glomerular inflammation agents like NaF. This indicates lack of cyclophos- qualitatively approaching, while quantitatively not equal- phamide activation in vitro. Erythropoietic stimulation ing, those considered characteristic of chronic glomer- is possible in vitro in presence of the drug. ulonephritis. 2) Persistent focal interstitial lymphocytic Patients receiving up to a total of 100 mg per kg total infiltrates. 3) Focal tubular atrophy; interstitial fibrosis. dose show almost complete failure of plasma clearance The persistence of these changes in patients apparently and turnover of Fe'9 in the immediate post-treatment fully recovered raises the question as to whether existing 1004 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING presumption of the long-term innocuous character of vidual samples, the average fluid/plasma inulin ratio in- streptococcal acute glomerulonephritis in the adult may creased progressively along the proximal convolution and require revision. averaged 2.8 in the final 30 per cent of the convolution. The fluid/plasma urea ratio, on the other hand, quickly Rapid Induction of Isolated Riboflavin Deficiency in Man. reached a value of approximately 1.5, but showed little MONTAGUE LANE, CHARLES E. MENGEL and DOROTHY further increase along the length of the convolution. J. DOHERTY, Bethesda, Md. (introduced by Stanley M. Hypo-osmotic fluid from the first portion of the distal Levenson). convolution had an average fluid/plasma inulin ratio of 7.9. The urea ratio in early distal samples was similar Evidences of a deficiency state have been observed to that of inulin. Urea concentration decreased along occasionally in human riboflavin deprivation studies, but the distal convolution, while the inulin concentration only after months of dietary restriction. Previous efforts tended to rise. Ureteral urine/plasma urea ratios in to induce clinically recognizable deficiency rapidly with these animals were low, averaging one-tenth that of in- riboflavin antagonists have been unsuccessful. Pharma- ulin. cological studies have shown that galactoflavin is a ribo- These results suggest that, in the presence of anti- flavin antagonist and antitumor agent in rodents. It is diuretic hormone, the entire cortical nephron is permea- absorbed following ingestion by man and is not degraded ble to urea, except for the ascending limb of the loop of to inactive metabolites. This agent was therefore se- Henle, which is also believed to be relatively impermeable lected, in conjunction with a riboflavin restricted diet, to water. Concentration gradients presumably favor for trial in the present investigations. diffusion of urea out of all segments of the nephron ex- Three adult males with metastatic neoplasms were placed cept the loop of Henle, where net diffusion appears to on a semisynthetic diet restricted to a maximum of 0.5 occur into the thin descending limb from the medullary mg of riboflavin per day, adequate in calories, protein, interstitium. The high medullary interstitial concentra- fat, and minerals, and supplemented with other vitamins. tion of urea is thought to result from diffusion of urea Galactoflavin was given orally in doses of 1.5 to 3.0 g out of the collecting duct and the action of the counter- per day. Urinary riboflavin excretion exceeded intake current system. following the administration of galactoflavin, indicating that it displaced body riboflavin. Signs of riboflavin de- ficiency were evident 21 to 30 days after the start Renal Tubular Secretion of Uric Acid in the Mongrel Dog. WILLOUGHBY of galactoflavin administration. All patients developed LATHEM,* BERNARD B. DAVIS anId seborrheic dermatitis of the face and ears, angular GERALD P. RODNAN, Pittsburgh, Pa. stomatitis, and cheilosis. One patient demonstrated ex- The prevailing view of the mechanism of uric acid tensive loss of filiform papillae and "mushrooming" of excretion in the mongrel dog is that a filtration-reabsorp- fungiform papillae of the tongue, burning, and loss of tion mechanism is involved. This hypothesis was exam- taste. Seborrheic dermatitis of the scrotum was also ined in the present study by increasing the rate of urate noted. The described signs were rapidly and completely excretion by two means: mannitol-induced osmotic reversed with riboflavin therapy. A striking finding, not diuresis and intravenous sodium urate loading. Anes- previously reported in human deprivation studies, was thetized dogs were used and urine was collected from the the development of reticulocytopenia and severe anemia ureter or the bladder. Sodium urate was given intra- in the three patients. Blood loss or hemolysis was not venously at a rate of 30 mg per minute and maniiitol evident. The anemia did not respond to parenteral ad- (20 per cent) was administered simultaneously at a rate ministration of iron and vitamin B12. Reticulocytosis of 9 ml per minute. Under these conditions the plasma occurred promptly upon supplementation with riboflavin. urate concentration increased from an average of 0.30 In the patients studied clinical riboflavin deficiency was mg per 100 ml to levels of 5 to 25 mg per 100 ml and induced rapidly and riboflavin appeared to be essential the rate of urate excretion increased strikingly. In 11 for normal erythropoiesis. of 18 animals the rate of urate excretion exceeded the rate of filtration (P urate x creatinine clearance) by 5 Micropunlctutre Study of Net Transtubular Movement of to 35 per cent. This high rate of excretion could not be Water and Urea in the Rat Kidney. WILLIAM E. accounted for by non-ionic diffusion; there was no sig- LASSITER, CARL W. GOTTSCHALK* and MARGARET nificant relationship demonstrable between urinary pH MYLLE, Chapel Hill, N. C. and urate excretion. These results are therefore indicative of tubular urate Anesthetized, nondiuretic male rats were infused with secretion. An attempt to demonstrate the site of secre- C`-labeled inulin carboxylic acid or urea, and fluid was tion by stop flow analysis was not successful. Reab- subsequently collected by micropuncture from proximal sorption in a proximal site could be demonstrated, but se- and distal convolutions on the surface of the exposed cretion could not. kidney. Radioactivity of tubular fluid, ureteral urine, The demonstration of tubular urate secretion in the and plasma was determined in a windowless flow counter. mongrel and Dalmatian dogs and in man and the rabbit Puncture sites were localized by microdissection. suggests that this is a general or common mammalian Although there was considerable scatter among indi- transport mechanism. AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 1005 Antibody Production in Systemic Lupus Erythematosus vitamin therapy produced unsustained alterations in (SLE) and Rheumatoid Arthritis (RA). S. L. LEE, circulating levels in this group. Infusion of 15 per cent L. E. MEISELAS, S. B. ZINGALE and R. RICHMAN, ethanol for 60 minutes (maximum blood levels 100 to Brooklyn, N. Y. (introduced by Robert Austrian). 125 mg per 100 ml) under conditions of hepatic vein To determine whether or not hetero-, iso-, and auto- catheterization produced an increased hepatic output antibodies vary in similar fashion in patients with SLE of pantothenic acid. and RA, antibodies in each category were followed in a Circulating pantothenic acid appears to provide a highly series of patients after their exposure to bacterial and sensitive index to liver injury. Decreased ability of the rickettsial antigens. Five patients with clinical and liver to bind pantothenic acid causes increased circulating laboratory evidence of SLE and 15 patients with typical levels and depletes stores of this vitamin. This results abnormal fat RA were inoculated with vaccines of brucellae and in decreased coenzyme A activity and rickettsiae. Antibodies to Br. abortus, Br. melitensis and metabolism. R. ricketsii were assayed by appropriate serologic reac- tions. The titers of isoagglutinins and of the "abnormal The Relationship Between Cationic Amino Acids antd antibodies" of SLE and RA were measured simultane- Potassium in Rat Muscle In Vitro. NORMAN G. ously in all patients. LEVINSKY, IAN TYSON and ARNOLD S. RELMAN,* Bos- Antibacterial and antirickettsial antibodies appeared in ton, Mass. the patients with SLE at comparable times and in titers An intact rat diaphragm technique which permits pro- similar to those observed in a control group of patients; longed observation of steady state relationships has been in the patients with RA the titers were somewhat higher. used to study cation equilibrium. Unlike the in vivo When titers of antibacterial antibodies rose, correspond- situation, intracellular K in vitro is unchanged (about ing increases in isoagglutinins were observed regularly. 150 mEq per L) as external K is reduced from 5 to 1 Antinuclear "antibodies" of SLE were observed, how- mEq per L. Since it has been reported that cationic ever, to vary independently of heterologous and isologous amino acids (CAA), primarily lysine, accumulate in antibodies. On the other hand, variations in the titer of K-depleted muscle, we have studied the in vitro interac- the latex fixation reaction in patients with RA corre- tions between CAA and K. sponded with the observed changes in heterologous and At bath K's from 5 to 1 mEq per L, addition of lysine isologous antibodies. The lack of correlation between approximately at in vivo CAA plasma concentration (0.8 variations in the titer of "auto-antibody" and of other mEq per L) lowered muscle K by 5 per cent or less. antibodies in patients with SLE suggests that, in this Further increases in bath lysine to 4 mEq per L progres- disease, auto-antibodies may be relatively uninfluenced by sively reduced muscle K, but there was no more change factors which affect the behavior of other antibodies. at 8 or 16 mEq per L of lysine; up to 30 to 40 per cent of muscle K was lost. This loss was always approxi- Pantothenic Acid, Fatty Liver and Alcoholism. CARROLL mately equal to the lysine accumulated, and Na did not M. LEEVY, WILLIAM S. GEORGE, HERMAN ZIFFER and increase. At any external lysine concentration, replace- HERMAN BAKER, Jersey City, N. J. and New York, ment of muscle K by lysine was usually increased by a N. Y. (introduced by Harold Jeghers). decrease in bath K. Other CAA, including nonmetabo- Circulating levels of pantothenic acid, thiamine, nico- lizable 2,4-diaminobutyric acid, also produced significant tinamide, folic acid, pyridoxine and cyanocobalamin were losses of muscle K. By contrast, neutral amino acids determined by microbiological techniques in 20 normal caused little or no loss of K, and anionic amino acids subjects and 36 patients hospitalized with acute alcoholism caused none. A high external lysine concentration did without clinical stigmata of vitamin deficiency. Liver not slow the rapid accumulation of K when diaphragms biopsy in the latter group revealed normal liver in 12, previously leached of K were incubated in a bath con- fatty liver in 10 and cirrhosis in 14. Results were corre- taining K. lated with histopathology and tissue vitamin levels. A It is concluded that CAA compete with K for intra- consistent finding was a two- to threefold increase in cellular position as cations in muscle, but probably not circulating pantothenic acid with a decrease in hepatic by competing for transport at the cell surface. At the tissue levels in patients with fatty liver and cirrhosis expected plasma levels in K-depletion in vivo, this com- with fatty metamorphosis and necrosis. This was ac- petition is apparently not sufficient to produce the ob- companied by a decrease in circulating thiamine, nico- served muscle K loss or Na accumulation. Factors other tinamide and folic acid; pyridoxine levels were un- than CAA and lowered external K must play a role. changed. Cyanocobalamin, unlike pantothenic acid, was normal in patients with fatty metamorphosis and in- A Study of Tuberculin Reactions in an Indian Hospital creased with liver cell necrosis. Vitamin levels were Population. RICHARD A. LEVINSON and MARTIN M. within normal range in both alcoholic subjects without CUMMINGS,* Oklahoma City, Okla. hepatic lesions and in patients with inactive cirrhosis. In order to determine the prevalence of infections due Mobilization of liver fat by diet was associated with to human, avian and atypical mycobacteria, tuberculin a concomitant decrease in pantothenic acid levels and skin tests were performed on 179 patients and employees an increase in thiamine, nicotinamide and folic acid; of the U.S.P.H.S. Indian Hospital (Shawnee, Okla.). 1006 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING

This Indian group was chosen for study because of their munication. Other factors, the etiology, extent and loca- well defined demographic characteristics and their un- tion of the cysts, are probably more important. Pulmo- usually high degree of tuberculin reactivity. In addition nary air cysts uniformly displace or destroy normal pul- 19 Caucasian employees of the hospital were tested. The monary tissue as shown by the lowered diffusing capacity, subjects were tested intradermally with 0.1 ml (0.0001 but this decrease in diffusing capacity, also, could not be mg) PPD-S, PPD-Avian and PPD-Battey. All reac- related to the patency of bronchial communication. tions were observed and measured at 48 hours. A reac- tion was considered "positive" when more than 5 mm of The Relation of Erythropoiesis to Iron Absorption. induration was observed. Seventy-four per cent of the ALLYN B. LEY, New York, N. Y. (introduced by Indians reacted to PPD-S, 34 per cent to PPD-B and 46 David A. Karnofsky). per cent to PPD-Avian. The relationships between the number and size of reactions to the different tuberculins The endogenous factors which have principally been were analyzed using the Chi-square test. Thirty per related to the control of absorption of iron from the gut cent reacted both to PPD-S and PPD-B, 43 per cent to have been 1) the amount of the body stores of iron, 2) PPD-S and PPD-Avian and 26 per cent to PPD-B and the degree of anemia, and 3) the rate of erythropoiesis. PPD-Avian. The degree of cross-reactivity between We have observed the development of hemochroma- avian, Battey and human tuberculins was significantly tosis in 3 patients with refractory anemia but with high. None of the Indians reacted to PPD-Avian alone erythroid hyperplasia of the marrow. In none could the and only 2 reacted to PPD-B alone. However, 4 of 19 anemia be ascribed to hemolysis. In none could the white employees reacted to avian tuberculin alone and 2 iron overload reasonably be attributed to transfusion. In others reacted to avian and Battey tuberculin. These the one patient so studied, plasma iron turnover was findings suggest that infections with avian tubercle bacilli markedly increased. Hence, in this patient, and by in- as well as atypical mycobacteria may be responsible for ference in the other two, while "effective" erythropoiesis the unusual tuberculin hypersensitivity patterns being was markedly diminished, "ineffective" erythropoiesis was observed in certain geographic areas. markedly increased. Measurements of the gastrointestinal absorption of a The Effect of Pulmonary Air Cysts on Respiratory 1 mg dose of radioiron showed values of 55 to 86 per Mechanics and Pulmonary Diffusing Capacity. BEN- cent while the patients were anemic. When the same test JAMIN M. LEWIS, L. C. REED, JR., AKIo FURUSHO and dose was used after the anemias had been corrected by THOMAS H. SNIDER, Detroit, Mich. (introduced by transfusion, the per cent absorption fell to 11 to 25 per Gordon B. Myers). cent. Similar studies were done in 3 patients with thymomas Resistance to airflow, compliance, diffusing capacity who were equally anemic, but whose marrows showed and residual volume was measured in 9 patients with practically no erythroid cells and whose plasma iron large pulmonary air cysts. All 9 had decreased diffusing turnover rates were very low. In this group, the ab- capacity. Four patients had normal respiratory me- sorption, while the patients were anemic, varied from chanics, and abnormalities in the other 5 ranged from a 25 to 33 per cent. After transfusion to normal or near marked elevation of inspiratory resistance to moderate normal levels of hematocrit, the per cent absorption fell increases of expiratory resistance without relation to the to 10 to 21 per cent. helium dilution residual volume. Specifically, the resid- These results are interpreted to indicate that iron ab- ual volume was low in 4 patients, indicating poor sorption is predominantly controlled by the rate of bronchial communication of their cysts. One patient had erythropoiesis, whether erythropoiesis is effective or in- a marked elevation of inspiratory resistance, probably due effective. It appears likely, however, that anemia itself, to expansion of the cysts as intrathoracic pressure fell, regardless of erythropoiesis, also promotes the absorption together with moderate elevation of expiratory resistance of iron. and low compliance. Two, one with histologically proved sarcoid, had moderate elevations of expiratory and in- Human Hor- spiratory resistances and low compliance. The final pa- The Cytological Localization of Grozwth tient had normal respiratory mechanics. Three patients mone with Fluorescent Antibody. A. LEZNOFF, J. had normal residual volumes, indicating that the cysts FISHMAN, L. GOODFRIEND, E. E. McGARRY, B. RoSE communicated with the bronchi. Two of these had normal and J. C. BECK,* Montreal, Canada. values for compliance and resistance, while the third had Previous attempts to demonstrate the cytological locali- moderate increases of expiratory and inspiratory resist- zation of anterior pituitary hormones with fluorescent ance and a lowered compliance. The last 2 patients had antibody have been inconclusive, probably because of the an elevated residual volume, supposedly indicating a com- lack of purity of the hormones used as antigens. Using municating cyst plus emphysema of the surrounding lung, gel diffusion and hemagglutination techniques, our lab- yet resistance and compliance were normal in one and oratory and others have shown that antisera to Raben's inspiratory and expiratory resistance moderately increased more highly purified preparation of human growth in the other. Thus, the mechanical effects of large air hormone are species specific and hormone specific. cysts can not be related to the patency of bronchial com- Antisera to human growth hormone were conjugated to AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 1007 fluorescein isothiocyanate and applied to sections of increased formation of DPNH produced on ethanol normal human pituitaries, eosinophilic adenomata from oxidation. patients with acromegaly, chromophobe adenomata, and an unclassified pituitary adenoma from a patient with Stability of Protein in Intestinal Epithelial Cells. severe Cushing's disease. The fluorescent antisera could MARTIN LIPKIN, THOMAS P. ALMY* and HENRY be seen to localize almost exclusively in pituitary QUASTLER, New York, N. Y. and Upton, N. Y. eosinophilic cells and in eosinophilic adenomata. There was no staining of other organs or adenomata. The Previous studies by others and ourselves have demon- fluorescent staining could be inhibited with nonfluores- strated rapid incorporation of amino acids into intestinal cent antihuman growth hormone and absorbed out by mucosal proteins, and rapid removal or "turnover" of prior incubation with human growth hormone. Unstained protein in the entire intestinal mucosa. The stability of sections and sections stained with heterologous fluorescent synthesized protein within individual cells, however, has conjugates showed no fluorescent localization. Fluores- not been studied. For this purpose, rates of leucine cent antihuman gamma globulin localized in blood vessels incorporation into various cell types and intracellular and fibrous septa of the pituitary. locations in j ej unal mucosa of mice were measured by These studies contribute additional evidence for the quantitative microautoradiography, following injection of specificity of our antisera to human growth hormone, and leucine-H8. constitute direct evidence that the pituitary eosinophil is Leucine incorporation is most rapid in epithelial cell the cellular site of production or storage of growth cytoplasm adjacent to growing microvilli. Incorporation hormone in the human. Using similar techniques further is successively decreased in epithelial cell cytoplasm studies on the cellular site of corticotropin are in adjacent to nuclei, in nuclei, and in connective tissue. progress. Incorporation is rapid in crypt epithelial cells which are undergoing division and differentiation, and is succes- Stimulation of Hepatic Fatty Acid Synthesis by Ethanol sively decreased in cells at the base, center and tip of the in Vivo and in Vitro. CHARLES S. LIEBER, LEONORE villi. As epithelial cells migrate to tip of villus, label M. DECARLI, and RUDI SCHMID,* Boston, Mass. is retained at original sites within individual cells. In cortisone-treated animals, identical results are seen. At To investigate the mechanism by which ethanol pro- comparable intervals after leucine injection, label is found duces a fatty liver, 14 male rats were given a tracer dose to be rapidly removed from Paneth's cells and pancre- of acetate-i-C14 intraperitoneally, and 8 g per kg ethanol atic acinar cells. or isocaloric amounts of glucose by gastric tube. They The results indicate a high degree of stability of syn- were sacrificed 16 hours later. In all animals, specific thesized protein in intestinal epithelial cells, rather than activity of fatty acids in the liver was much greater than rapid intracellular protein turnover. The stability of in adipose tissue. Compared to litter mates fed glucose, synthesized protein in these mammalian cells in vivo is in ethanol-treated rats, the concentration of total fatty sifnilar to that found in growing cultures of Escherichia acids in the liver was increased; C" incorporation into coli, and recently in mammalian cells in tissue culture. fatty acids was enhanced by 40 per cent in the liver and decreased by 50 per cent in adipose tissue, suggesting that Bleeding Diathesis in Children with Liver Glycogen Dis- ethanol stimulates hepatic fatty acid synthesis. This was ease and in Their Parents. CHARLES U. LOWE,* confirmed in 14 other rats given labeled acetate 5 days JULIAN L. AMBRUS, CLARA M. AMBRUS, Luis L. prior to a 4 day course of or ethanol glucose administra- MOSOVICH, IRVING B. MINK and JOSEPH E. tion. In contrast SOKAL, to the previous experiment, the specific Buffalo, N. Y. activity of fatty acids in liver was less than in adipose tissue. In the ethanol-treated animals, hepatic fatty acid Detailed examination of hemostatic factors revealed concentration was increased and specific activity reduced, abnormalities in each of 6 children with liver glycogen as compared to animals pair-fed with glucose. disease (LGD) and also in at least one parent of each. In comparison to glucose or acetate, incubation of rat Hemorrhagic episodes were encountered in 4 of the pa- liver slices with ethanol demonstrated both a three- to tients. Both the hepatic enzyme abnormalities and the eight fold increase in incorporation of acetate-i-C1' into hemostatic defects varied. Children with different hepatic fatty acids (p < 0.001) and increased net synthesis of defects had different clotting defects. Hemostatic ab- fatty acids (p < 0.02). In adipose tissue, which lacks normalities could not always be demonstrated, but when alcohol dehydrogenase, ethanol had no effect on fatty present, the pattern was consistent for a given patient. acid synthesis. Two of the subjects were siblings; their abnormalities In the liver, oxidation of ethanol is coupled with re- were similar: prolonged clotting and bleeding time, de- duction of disphosphopyridine nucleotide (DPN) ; in vitro, fects in PTA, PTC, and possibly in Stuart-Prower Fac- another DPNH generating system (sorbitol-fructose) tor, and increased Factor V. In one family in which both reproduces the effect of ethanol on fatty acid synthesis. parents had abnormalities, they resembled those of the Furthermore, a hydrogen acceptor such as methylene blue child-definitely prolonged recalcified plasma clotting abolished this effect. This indicates that the stimulation time, slight decrease in prothrombin, and increase in of hepatic fatty acid synthesis by ethanol results from Factor V. All of the children and some of the parents 1008 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING had thrombocytosis oni one or more occasions, reaching Formiiminioglutamic Acid versus Serun "Folic Acid" as values as high as 1,200,000 per mm3. No defects were ait Index of Folic Acid Deficientcy. A. LEONARD found in members of the fibrinolysin and retractozyme LUHBY and JACK M. COOPERMAN, New York, N. Y. systems, in platelet factors, or in capillary morphology. (introduced by Alvin F. Coburn). Fresh serum was more effective than whole blood in con- We have shown that measurement of urinary formi- trolling bleeding. In one instance, blood from a patient's minoglutamic acid (FIGlu) after a standard histidine mother failed to control hemorrhage; however, blood metabolic load is an early and sensitive index of folic from a normal donor proved effective. The mother was acid deficiency. This has been confirmed by Spray and found to have a clotting deficiency. Witts. Recently, Baker and Herbert and others have This occurrence of hemostatic defects seems too regu- proposed a test for folic acid deficiency based on de- lar to be coincidental and suggests that such abnormali- termination of serum "folic acid" activity by Lactobacilluts ties may constitute a basic feature of LGD. Studies of casei. carbohydrate metabolism have proved unsuccessful in Urinary excretion of FIGlu after a 15 g L-histidine identifying the possible heterozygous state of this familial monohydrochloride metabolic load for two to three days disease; it is intriguing that a study of clotting factors was compared with serum "folic acid" by L. casei assay has revealed abnormalities among parents without his- in patients with megaloblastic anemias of various origins. tory of bleeding. It would appear that the manifestations A new, modified L. casei assay for serum "folic acid" of LGD, as well as the genetic mechanisms involved, are activity was employed which increases the accuracy and more complex than has been generally appreciated. sensitivity of previous procedures. Urinary FIGlu of over 30 ,ug per ml or over 35 mg Inhibitiont of Serotonin Production by Isonticotinic Acid per 24 hours by the end of the loading period was found Hydrazide with Conttrol of Symiptoms in the Malignant indicative of folic acid deficiency as established by in- Carcintoid Syntdromiie. GEORGE D. LUDWIG, Philadelphia, variable, dramatic response to minute doses of folic acid Pa. (introduced by Francis C. Wood). but not to vitamin Bm The author has previously shown that isonicotinic acid Serum "folic acid" was found in normal subjects to hydrazide (INH) inhibits growth of higher plants by vary between 2 and 30 m,ug per ml. Initial serum "folic blocking conversion of tryptophan to indole-3-acetic acid, acid" in patients found folic acid-deficient by urinary the main plant growth hormone. It was shown that INH FIGlu after standard histidine loading and by therapeutic forms a Schiff-base complex with pyridoxal phosphate, trial with very small doses of folic acid ranged from 2 thus depriving a decarboxylase or transaminase of re- to 15 m,ug per ml. quired cofactor. Anticipating that INH might inhibit Thus, serum L. casei "folic acid" activity, known not 5-hydroxytryptophan decarboxylase, and thus 5-hydroxy- to be specific for folic acid, was not a good measure of tryptamine (serotonin) production, 2 patients with proven folic acid deficiency since values found in folic acid-de- metastatic carcinoid were given 400 mg of the drpg ficient patients fell within range of normals. Urinary daily. Serotonin concentrations in whole blood and FIGlu determination after histidine loading, however, platelet fractions were measured spectrofluorometrically based upon a specific biochemical defect in folic acid before and during INH administration. Daily urinary deficiency, was invariably found an early and accurate excretion of 5-hydroxyindoleacetic acid (5-HIAA) was index of folic acid deficiency. measured with the nitroso-naphthol and Ehrlich reagents, and individual urinary indoles by two-dimensional paper In Vitro Stimultlation of Hexokinase by Insulin. ROBERT chromatography, for 5 control days and 10 days during M. MACLEOD, ROSE BROWN and WILLIAM S. LYNN,* INH administration. In both patients general clinical Durham, N. C. improvement and decrease of diarrhea occurred. In one, roentgenograms showed reversal to normal of a previ- Previous studies performed in this laboratory indicate ously disordered motor pattern of the small bowel. Co- that incubation of rat epididymal adipose tissue with in- incident with the symptomatic improvement, concentra- sulin stimulates the conversion of glucose-l-C" and -6-C'4 tions of serotonin in the whole blood and the platelet to glycogen, C02, glyceride-fatty acid, lactate and the fraction were decreased 30 and 50 per cent, respectively; oxidation of glucose via the hexosemonophosphate shunt, average daily excretion of 5-HIAA, as measured with when incubated in Krebs phosphate buffer with air as Ehrlich reagent, was decreased 30 to 40 per cent, and the gas phase. Since insulin stimulates glycolysis, gly- paper chromatograms showed a marked decrease in cogenesis and the hexosemonophosphate shunt, it was 5-HIAA, 5-HIAA-sulfate, and serotonin. concluded that the locus of action of insulin must be In contrast, iproniazid (Marsilid), which differs from either to increase tissue permeability to glucose, or to INH in being a potent monamine oxidase inhibitor, failed stimulate hexokinase. Measurements of intracellular to inhibit plant growth or conversion of tryptophan to glucose and glucose-6-phosphate concentrations have indi- indole-3-acetic acid, and other authors have shown that cated that insulin caused a marked decrease in intracellu- iproniazid aggravates symptoms of metastatic carcinoid, lar glucose, with a concomitant increase in glucose-6- presumably by increasing the circulating serotonin con- phosphate concentration. However, total water, urea, centration as a result of inhibiting its oxidation to raffinose, inulin, and sodium spaces remained unchanged 5-HIAA by monamine oxidase. in the hormone-treated tissue as compared to controls. AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 1009

The intracellular glucose concentration in control is 45 of intravenously administered glucose on hepatic glucose per cent lower than extracellular glucose; however, this balance before and after insulin administration in normal, limitation of transport can be overcome by addition of mildly diabetic and severely diabetic dogs. Hepatic digitonin, i.e., intracellular glucose equals extracellular glucose balance was determined in 24 studies from meas- glucose. Under these conditions the addition of insulin is urements of hepatic blood flow (Bradley's technic) and still capable of lowering intracellular glucose and increas- arteriohepatic venous glucose concentration difference ing glucose-6-phosphate concentrations, as measured by (Somogyi iodometric method) in dogs with portacaval enzymatic and radioisotopic methods. Therefore, under shunts, a preparation permitting measurement of hepatic these conditions, the action of insulin must be to stimu- rather than splanchnic glucose balance. late the phosphorylation of glucose rather than the trans- In normal dogs after 60 minutes of intravenous glucose, port of glucose into adipose tissue. the liver, which initially was contributing 46 mg per minute was now storing (extracting) 24 mg per minute, The Capacity of Tubercle Bacilli to Assume a Latent a net positive balance of 70 mg per minute. Hepatic State In Vivo. ROBERT M. MCCUNE, New York, N. Y. glucose storage started at a mean arterial glucose con- (introduced by Walsh McDermott). centration of 116 mg per 100 ml. Insulin pretreatment An increasing proportion of the disease load in hos- prior to glucose loading lowered this storage threshold pitals today is being produced by microbial infections to 92 mg per 100 ml. Although glucose infusions in that have been resurrected from a dormant or latent mildly diabetic dogs (mean fasting blood sugar 145 mg state. The term latent is used in the strict sense to per 100 ml) also resulted in hepatic glucose storage, this describe an infection whose presence in the tissues cannot occurred only when mean arterial glucose concentration be demonstrated by any known technique, yet the fact reached 200 mg per 100 ml. In contrast, hepatic glucose that infection is present is established by its subsequent storage never occurred in the more severely diabetic dogs reappearance. The capability of viruses to assume the (mean fasting blood sugar 240 mg per 100 ml) despite latent state has long been recognized, but the extent to elevation of arterial glucose to levels as high as 490 mg which bacteria possess this same capability has only re- per 100 ml. When these more severely diabetic dogs cently been extensively studied. Thus far, the experi- were pretreated with insulin prior to glucose infusion, mental induction of a latent bacterial infection has been hepatic glucose storage occurred and at a mean arterial accomplished only in the case of tubercle bacilli as pre- glucose concentration of only 164 mg per 100 ml. viously reported from this laboratory. In the experi- These data indicate that insulin controls the magnitude mental model, large populations of tubercle bacilli com- of the role of the liver in the disposition of a carbohy- pletely vanish from the tissues of mice treated with a drate load. Not only is the arterial glucose level at which nicotinamide derivative, pyrazinamide, and a companion the liver starts storing glucose progressively elevated drug according to certain established time-dose relation- with increasing severity of diabetes but this level is ships. promptly lowered toward normal by insulin administra- In the present studies it has been found that: 1) tion. latency has been induced in at least four-fifths of the animals from which the tubercle bacilli have vanished; The Cleavage of Disulfide Bonds in Thyroid Tissue by 2) time is an important factor-at least 9 weeks are Thiourea. F. MALOOF and M. SOODAK, Waltham and necessary for induction of latency and 6 weeks to many Boston, Mass. (introduced by J. Lerman). months for the resurrection of the bacilli; 3) once the We have previously described a thyroidal cytoplasmic latent state is assumed it is remarkably stable and resur- particulate system which desulfurates S5-thiourea. Thio- rection during the first month after induction cannot be cyanate ion and reduced diphosphopyridine nucleotide are produced even by large doses of hydrocortisone; 4) the required. Liver and kidney tissue are inactive. nature of the tissue environment is an essential factor in The major sulfur product is protein-bound sulfur the assumption of the latent state; 5) the concurrent ad- (PB-S5; 50 per cent). The S5 is firmly bound to pro- verse influence on the tubercle bacilli, that is a necessary tein, but about 80 per cent of it can be displaced by prerequisite for experimentally induced latency, appar- nucleophilic anions (0.01 M), as sulfite or cyanide, by ently must be specific. Three drugs with widely varied 0.1 N NaOH or by thiols (0.01 M) as cysteine. This antituberculous effectiveness can exert such an influence, is evidence that the S5 is bound to protein in disulfide but penicillin cannot. Experiments to determine the linkage (PB-S-S35). role of vaccine-induced immunity as an "adverse in- Potent inhibitors of this system are sulfite and thiols fluence" are virtually completed. which effectively split disulfide (S-S) bonds: RSSR' + SO3& >± RSH + R'SSO9 (idem thiols). Preincubation Insulin's Control of the Role of the Liver in the Disposi- of the particulate fraction with sulfite (0.01 M), eysteine tion of a Glucose Load in Diabetic and Nondiabetic (0.01 M) or thioglycollic acid (0.01 M) leads to about Dogs. LEONARD L. MADISON, BURTON COMBES and 80 per cent inhibition of the subsequent desulfuration of REUBEN ADAMS, Dallas, Tex. (introduced by Elias thiourea. Studies with sulfhydryl (SH) group inhibitors Strauss). reveal that a free SH group is not involved. Incubation The physiological role of the liver in disposing of a with labeled sulfite (Na2SWOs) yields protein-bound carbohydrate load was examined by measuring the effects radioactivity (PB-S-S503). The S'Os is firmly bound 1010 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING to protein but can be displaced by precisely the same re- phrine administration, have been documented in 3 types agents that remove the S35 from the PB-S-S3. Hence of endocrinopathy. 1) Of 8 hyperthyroid patients, mean sulfite and the sulfur of thiourea seem to be attached to fasting serum FFA was 1,183 ,uEq per L; of 6 controls, thyroid protein in a similar manner. 731 AEq per L; A = 452, p < 0.01. After ingestion of 75 g These data suggest that a disulfide bond in thyroid of glucose the decrease in FFA concentration was greater tissue is cleaved by thiourea to form a new-S-S-bond. but briefer than normal, and the rise 2 to 4 hours later This formulation is supported by chemical studies in was excessive (p <0.05). 2) In uncontrolled mild dia- which thiourea was found to cleave the disulfide bond betes mellitus (9 patients) mean fasting serum FFA was of cystine. Of interest is that this thyroid particulate elevated (1,179 AEq per L, p < 0.05) and FFA response preparation has 3 and 7 times the cystine content of to glucose ingestion was impaired; on chlorpropamide similar preparations of kidney and liver. Parallel ob- therapy mean fasting FFA was lowered (to 812 ,Eq servations on the in vitro iodination reaction suggest a per L, A = - 367, p <0.01); the mean FFA values after sulfenyl iodide (SI+) intermediate resulting from cleav- glucose ingestion were also lower in the treated patients, age of a disulfide bond. Competition for this site may although the decrease from fasting values was less. 3) explain the inhibitory action of thiourea in the thyroid. In primary and secondary hypoadrenalism the rise of serum FFA which normally is present one-half hour after Thrombolytic Activity of Mold Fibrinolysin (Aspergillini administration of epinephrine (0.3 mg s.c.) was impaired 0) in Vivo. HECTOR M. MARIN, MARIO STEFANINI,* (p < 0.05; mean A = 173 /iEq per L for 8 patients versus FRANCO SOARDI and LISELOTTE MUELLER, Boston, Mass. 410 ,uEq per L for 6 controls). Aspergillin 0, an agent with strong fibrinolytic activity The data are all compatible with the thesis that serum isolated from filtrates of cultures of Aspergillus oryzae FFA reflects directly the rate of utilization and mobiliza- B-1273, was injected into 70 dogs at average dose of 10 tion of fat, and indirectly and inversely the rate of glucose mg per kg body weight. A venous thrombus was pro- utilization. They support also the following interpreta- duced in 15 dogs with sodium morrhuate technic pre- tions. 1) In hyperthyroidism the normal metabolic re- liminary to the injection. Animals showed no changes sponses to fasting and food-taking occur in accelerated in temperature or pressure. There was oozing only at and exaggerated form; the demonstrable abnormalities site of surgical incisions for about one-half hour after are presumably largely a consequence of the raised injection of Aspergillin 0. Coagulation studies indicated metabolic rate produced by thyroid hormone. 2) The increased fibrinolytic activity without simultaneous con- blood-sugar-lowering effect of chlorpropamide in dia- sumption of profibrinolysin, depletion of clotting ac- betics reflects amelioration of their impaired rate of celerators (labile factor V) and some degree of hypo- utilization of glucose. fibrinogenemia for 1 to 4 hours. Generation of thrombin was slow and incomplete; activity of prothrombin and Steroid Hormone Inhibition of Mammalian Glutcose-6- stable factors was slightly affected; the generation of Phosphate Dehydrogenase. PAUL A. MARKS * and thromboplastin was minimally delayed. Direct inspec- JULIA BANKS, New York, N. Y. tion and venographic studies indicated recanalization of This study demonstrates a highly specific-steroid hor- occluded veins within 3 days of production of thrombi. mone inhibition of glucose-6-phosphate dehydrogenase A strong inhibitor developed temporarily following ad- (G-6-PD), an enzyme catalyzing reduced triphosphopy- ministration of Aspergillin 0. Addition of 2 mg of ridine nucleotide (TPNH) formation. Dehydroiso- heparin sodium to Aspergillin 0 inactivated the inhibitor androsterone (DHA) and pregnenolone (PE) and cer- and enhanced the fibrinolytic and clot-delaying activity tain other C19 and C-n steroids with a ketone group at of the drug. This occurred occasionally, however, when C-17 or C-20 inhibit G-6-PD. At 10-' M, DHA and doses of 5 mg per kg or less were administered; it was PE inhibit G-6-PD activity 20 to 30 per cent, and at prevented by the simultaneous administration of small 10' M, 65 to 85 per cent. These steroids inhibit purified doses of heparin. G-6-PD from human erythrocytes and G-6-PD in crude Studies have been conducted in 55 humans, examples preparations of various human and rat tissues including of which will be given in detail. They indicated that liver and adrenal, but not G-6-PD of yeast or spinach. Aspergillin O is not toxic or antigenic and exhibits lytic Isocitric and 6-phosphogluconic dehydrogenases, which effect on venous and arterial thrombi, while favoring also catalyze TPNH formation, are not inhibited by establishment of collateral circulation following vascular these steroids. Estrogens, testosterone, corticosteroids, occlusion. Coagulation studies gave results similar to progesterone and cholesterol have little, and generally those obtained in animal experiments. Ino inhibitory effect on G-6-PD at concentrations as high as 10' M. The steroids are noncompetitive inhibitors Endocrine Influences ont Serum Free Fatty Acid (FFA) of G-6-PD with regard either to glucose-6-phosphate in Man. BERNARD H. MARKS and A. GORMAN HILLS,* or TPN. This G-6-PD inhibition does not involve a Miami, Fla. TPN-linked reduction or oxidation of the steroids. Significant deviations from normal serum FFA con- Steroid inhibition of G-6-PD might be a regulatory centration measured after a 14 hour fast, or of the re- mechanism of significance in glucose metabolism. TPNH sponse of serum FFA to glucose ingestion or to epine- is required for steroid biosynthesis. DHA and PE are AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 1011 important intermediates in steroid synthesis. Inhibition circulations, 13 had left ventricular failure (CHF), and of TPNH generation by DHA or PE may function as 4 had coronary sclerosis (CAD). a control mechanism in the formation of these key inter- Normal oxygen supply pattern was: resting CBF= mediates. 80 cc per 100 g LV per minute increasing 25 per cent during exercise, CVR = 69,400 dynes-sec-cm', decreasing In Vitro Transfer of P" Anti-Rh0 (D) from "Sensi- 26 per cent during exercise, coronary venous oxygen tized" Red Cells. S. P. MASOUREDIS, San Francisco, content (CV 02) =4.6 vol per cent increasing 3 per cent Calif. (introduced by R. J. Havel). during exercise, A-VO2/AO2= 71 per cent remaining con- stant or decreasing during exercise, and ME = 44 per cent The in vitro "transfer" of I"81 anti-Rh0 (D) from increasing 32 per cent during effort. "sensitized" red cells to "unsensitized" Rho(D) positive In resting CHF patients with a CI above 3.0, CBF= red cells has been demonstrated and the conditions which 111, CVR = 46,000, CVO2= 5.8, A-VO2/AO2 =69 per cent, affect this process have been studied. and ME = 25 per cent; with a CI below 3.0, CBF = 77, Rho (D) positive red cells "sensitized" with I"' anti- CVR = 61,500, CVO2 = 3.6, A-VO2/AO2 = 79 per cent, and Rho(D) were incubated with "unsensitized" Rh0(D) ME = 22 per cent. In all pure CHF patients, A-VO2/AO2 positive red cells of a different ABO phenotype. The and CVO2, whether normal or abnormal at rest, remained recipient cell was isolated from the "sensitized" donor constant during exercise, indicating increased CBF ade- cell after reaction by differential agglutination of the quate to M Q02; CBF increased 44 per cent and CVR donor cell with the appropriate ABO isoantibody. The decreased 32 per cent with exercise. effectiveness of this procedure in separating the two cell Coronary artery disease patients had normal resting populations was evaluated for each experiment by using oxygen supply patterns. During exercise, however, CBF an Rh0(D) negative cell of the same ABO phenotype as increased only 16 per cent, CVR fell only 13 per cent, the recipient cell. Under these conditions from 30 to 75 CV02 fell 19 per cent, and A-VO2/AO2 increased 8 per per cent of the "sensitized" donor cells were carried cent, while ME increased normally. over into the recipient cell suspension as a result of in- In normals, increased M Q02 is supplied through in- complete agglutination of the donor cells. creased CBF secondary to coronary vasodilatation, with- Both A and B cells participate equally with A, B or 0 out A-VO2/AO2 alterations. In low CI, CHF patients, recipient cells in the transfer of I' anti-Rho(D). The increased resting A-VO2/AO2 together with a normal maximum quantity of antibody was transferred between ability for coronary vasodilatation on effort, suggests a 370 and 450 C. About 15 per cent of the antibody con- coronary vasoconstrictor reflex acting in the resting state. tent of the "sensitized" donor cells was found in the The persistence of, but not increase in, a widened recipient cells after one hour's incubation at 370 C. No A-VO2/AO2 despite greatly increased CBF on effort "transfer" of antibody occurred at 40 or at 560 C. About suggests incomplete abolition of this constrictor tone by 9 per cent of the donor cell antibody content was trans- the competing influence of increased M Q02. In con- ferred within 30 minutes and a maximum of about 15 trast, the CAD group normal resting oxygen supply per cent was found between 120 and 180 minutes. No pattern is replaced on effort by abnormal oxygen ex- increase in quantity of antibody transferred could be traction. demonstrated by prolonging the time of incubation. Re- cipient cells containing the rh'(C) antigen accepted less Erythrocyte Glutathione Synthesis. AARON MILLER and I'13 anti-Rh0(D) than did rh'(C) negative cells. The MARTHA HORIUCHI, Boston, Mass. (introduced by antibody transferred in these experiments was sufficient Charles P. Emerson). to saturate from 5 to 20 per cent of the Rho(D) sites on the recipient cell. Glutathione in erythrocytes has an important role in their function and survival. Synthesis of erythrocyte The Influence of Peripheral and Coronary Hemodynamics glutathione (glutamylcysteinylglycine) was studied in Upon Patterns of Human Myocardial Oxygen Supply. vitro with labeled precursors. A method for isolation of J. V. MESSER, R. J. WAGMAN, H. J. LEVINE, W. A. radiochemically pure glutathione was developed. Protein- NEILL and R. GORLIN,* Boston, Mass. free filtrates of erythrocytes which had been incubated up to 5 hours in media containing S5-cystine, C1-glycine, Patterns of human myocardial oxygen supply may C14-glutamic acid or C14-glutamine were chromatographed vary dependent upon the influence of coronary artery after elution from an anion-exchange resin (IR45) and disease, extracardiac moderator reflexes, and the appli- specific activity of the separated glutathione determined. cation of acute stress. Thirty-two patients were studied Erythrocytes incorporated labeled cystine and glycine by coronary sinus (CS) and pulmonary artery catheteri- into glutathione progressively without change in the zation with measurements of coronary flow (CBF), A-V glutathione concentration. Erythrocyte glutathione labeled 02 differences, and systemic hemodynamics at rest and with C14-glycine did not exchange with added unlabeled during exercise. Myocardial oxygen consumption (M glycine, indicating that labeled glycine was incorporated Q02), A-VO2/AO2, coronary diastolic vascular resistance into glutathione by true synthesis. No incorporation of (CVR), myocardial efficiency (ME), and cardiac index labeled glutamic acid into glutathione was found. Intra- (CI) were calculated. Fifteen patients had near normal cellular ratios of 1 molecule of labeled glutamic acid to 1012 PROCEEDINGS OF THE FIFTY-SECOND ANNUTAL MEETING

115 molecules of glycine were ouoserved usinIg paired Thus, the edematous state per se is unrelated causally samples of blood. Similarly, 1 molecule of labeled glu- to ventilatory and gas exchange disturbances of primary tamic acid to 7 molecules of glutamine (the monoamide of cor pulmonale. glutamic acid) was found within the cells; however, in- corporation of labeled glutamine into the glutamic acid Abnorimal Insulin-Binding Fractiont Demlonistrated by moiety of glutathione was not observed. The Electrophoresis on1 Ion Exchange Paper of Scra In summary, labeled cystine and labeled glycine were From Diabetic Patientts. MARVIN L. MITCHELL, Boston, used to demonstrate true synthesis of glutathione in red Mass. (introduced by J. M. Hayman). terminal glycine with unlabeled cells. Exchange of the Quantitative differences in I131-insulin-binding by sera glycine was not noted. Labeled glutamic acid could not diabetic pa- of erythrocyte from insulin-resistant and insulin-responsive be incorporated into glutathione because been demonstrated by interaction Labeled glutamine entered the red cells tients have previously impermeability. analysis using anion exchange resins. To determine more glutamic acid. However, incorpora- readily than insulin was bound by different protein labeled glutamine into the glutamic acid moiety of whether or not the tion of fractions, an ion exchange paper was introduced as the could not be demonstrated because of either glutathione supporting medium for the electrophoresis of serum con- limited cell entry relative to that of labeled glycine, or the intracellular pool of unlabeled glutamine, taining P`1-insulin. dilution by Sera from 12 normal subjects, 35 insulin-responisive or conversion to glutamic acid, or any combination limited and 6 insulin-resistant diabetic patients (300 to 5,000 of these factors. units daily) were equilibrated for approximately 30 minutes with IP1`-insulin. Following equilibration, electrophoresis The Role of the Edemnatous State to Respiratory Func- (veronal buffer, pH 8.6) of the sera was carried out on tion in Patiecnts with Primzary Cor Pulmonale. paper which had been prepared from a mixture of a- WILLIAM F. MILLER, IVAN E. CUSHING and NANCY cellulose pulp and a finely ground cation exchange resin, W\TU, Dallas, Tex. (introduced by E. E. MIuirhead). Amberlite IR-120. The protein patterns were similar to the observed after electrophoresis of serum on Digitalis and diuretics often are vigorously employed patterns filter paper. in treatment of primary cor pulmonale on the assumption applied directly to the resin paper remained that the edematous state plays some specific role in IP1`-insulin sites of the resin respiratory symptoms and altered pulmonary function. at the origin; when the insulin binding paper had been saturated with stable insulin, the radio- Our own observaticns led us to believe that changes in insulin migrated with a mobility slightly less than that of pulmonary function are related to alterations within the albumin. IP1`-insulin in botlh normal sera and in sera lung per se and bear no direct relationship to the edem- diabetic patients migrated with atous state. The formation of edema is merely a mani- from insulin-responsive the a- or In contrast, P`31-insulin migrated festation of disturbed venous hemodynamic function and a-globulins. in sera from the insulin-re- is secondary to pulmonary parenchymal disease. solely with the y-globulins sistant diabetic patients. In order to examine this contention, patients were care- Serum from the same diabetic patient was studied dur- fully selected as examples of chronic cor pulmonale, per- of insulin resistance and insulini sensitivity. sistently edematous but without evidence of acute infection ing periods The radioinsulin moved with the gamma globulin frac- or other acute disturbance which would account for the insulin-resistant phase and with the a- patients were tion during changes in respiratory function. Fifteen Thus, Except for reduced physical activity globulins during the insulin-responsive phase. selected for study. P`1-insulin serum protein com- and improved dietary intake (regular diet), hospital normal distribution of the plex is associated with insulin responsiveness as compared treatment was presumably unchanged from outpatient the pattern seen with true insulin re- treatment. Lung volume, compliance, resistance and blood with abnormal sistance. gas measurements were made every other day for a 10 day period then, biweekly thereafter. Nine patients ex- hibited acute changes in respiratory function, demon- The Myocardial Exchange of Potassium71e and Sodium strated to be due, either to clearing of retained bronchial During Respiratory Acidosis. JOHN C. MITHOEFER secretions or to clearing of acute infection. The remain- and FRED D. HOLFORD, Cooperstown, N. Y. (intro- ing 6 patients suitable for study failed to exhibit sig- duced by James Bordley, III). nificant change in ventilatory function as a result of Concentrations of K+ and Na+ were measured in blood spontaneous diuresis, presumably due to decreased physi- samples from the aorta and coronary sinus of anesthe- cal work, or induced mercurial diuresis (average weight tized dogs during oxygen breathing and respiratory loss 6.8 kg). There was no significant change in gas acidosis. Acidosis was produced after a control period of exchange functions except in those patients in whom a oxygen breathing by inspired mixtures of 8 or 19 per reduction of physical activity resulted in decreased C02 cent C02 in oxygen or by the technique of diffusion production. In two patients the edematous state was respiration. K+ and Na+ concentrations were measured reinduced by large salt and water loads and in two pa- by flame photometer. tients by increased physical exertion. In Ino instance was During control periods arterial serum K+ concentrationi functional change apparent. slightly exceeded that of the coronary sinus blood (mean AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 1013

0.1 mEq per L). During diffusion respiration as the bility of diabetics to staphylococcal infection cannot be PACo2 rose a progressive arterial-coronary venous K+ related to deficient serum bactericidal power. difference (arterial higher than venous) developed, reach- ing a mean value of 0.7 mEq per L when PAco2 was The Biliary Dynamics of the Metabolites of Sulfobromo- 100 mm Hg then decreasing to 0.3 mEq per L when phthalein (BSP) in Man. L. S. MONROE and A. L. PAco2 was 140 mm. After two minutes' recovery when KITInNGER, La Jolla, Calif. (introduced by E. L. PAco2 was 60 mm the relationship was reversed, venous Keeney). concentration slightly exceeding arterial. Similar results When sulfobromophthalein (BSP) is excreted into the were produced by respiratory acidosis from C02 breath- bile following intravenous injection, the dye maintains ing. the same absorption spectrum and indicator characteristics During acidosis the venous Na+ concentrations were as the pure compound. For this reason it had been as- higher than the arterial when the reverse relationship sumed that the dye was unchanged. However, in recent existed for K+. However, concomitant reciprocal changes years there has been increasing evidence that BSP in the arteriovenous (A-V) difference of these ions undergoes molecular changes in the process of excretion. could not be demonstrated but may have been masked by Column chromatography utilizing cationotrophic alumina the small percentage changes which occurred in Na+ and descending paper chromatography with a t-butanol: concentration as compared to K+. formic acid: water system demonstrates 5 fractions of Respiratory acidosis was associated with progressive BSP which appear in the bile following the intravenous rise in arterial K+ concentration but this was shown not injection of the dye. Four of these fractions chromato- to be the cause of the A-V K+ difference which developed, graph differently from the pure dye. nor was the latter the result of shift of K+ from plasma During an attempt to correlate BSP metabolites with to RBC. The A-V K+ difference is interpreted as evi- various liver diseases, marked variation in the biliary ex- dence of a transfer of this ion from blood to myocardium cretion rates of the metabolites has been found. A series in response to acidosis, a shift opposite to the known loss of 18 postoperative patients recovering from choledochot- of K+ from cells of skeletal muscle. omy were used to study this variation. After intravenous injection of BSP (5 mg per kg), timed bile specimens Destruction of Pathogenic Staphylococci by Human were subjected to column chromatography and the ex- Serum. LEWIS A. MOLOGNE and ABRAHAM I. BRAUDE,* cretion rates of the metabolites were studied for a Pittsburgh, Pa. minimum of 2 hours. The most marked changes were noted in the percentage excretion rates of Fractions I Pathogenic staphylococci, unlike many pathogenic gram- and IV. Following an average level of 54 per cent at negative bacteria, are reputedly resistant to the bacteri- 20 minutes after injection, Fraction I (chromatographi- cidal power of normal human serum. In order to ac- cally similar to pure BSP) decreased progressively count for the susceptibility of diabetics to staphylococcal throughout the period of observation. Fraction IV hav- infections, their sera were compared with nondiabetic sera ing an average level of 10 per cent at the 20 minute for killing of staphylococci. period increased progressively at nearly the same rate. Bactericidal activity was measured by inoculating ap- The percentage excretion rates of Fractions III and V proximately 20,000 to 30,000 coagulase-positive staphy- increased gradually, while that of Fraction II gradually lococci per ml of serum and performing serial plate decreased following injection of the dye. Improvement counts for 24 hours. Sera from 18 nondiabetics and 22 in hepatic function was accompanied by an increase in the diabetics were examined for their ability to kill various slope of the plotted curves of the percentage excretion coagulase-positive staphylococci isolated from 55 infected rates of Fractions I and IV. patients and nasal carriers. Plasma from all subj ects, The slope of the curves of the excretion rates, plotted whose sera were examined for bactericidal power, pos- during the first hour period after injection, probably sessed coagulase factor as measured both by clumping mirrors the functional status of the liver and may repre- and clotting. sent the maximum possible transfer of metabolites by a Diabetic and nondiabetic sera killed coagulase-positive rate-limited transfer mechanism. Any attempt to cor- staphylococci with equal frequency. Marked killing and relate liver disease to BSP metabolites should take into sometimes sterilization of staphylococci was noted in 118 consideration the time elapsed after injection. Pre- of 161 tests, while heavy growth occurred in only 43. liminary studies indicate that the BSP metabolites are Staphylococci isolated from infected materials were as not all ninhydrin-reactive, and the nonreactive fractions susceptible to killing by serum as those recovered from may be excreted by a mechanism different from the cur- nasal carriers. Bactericidal activity was resistant to heat rently proposed conjugation reactions. and independent of the phage type and antibiotic sensi- tivity of the staphylococci. Different strains of staphy- An Evaluation of the Acidifying Capacity of the lococci of the same phage type varied greatly in suscepti- Chronically Diseased Kidney in the Experimental bility to the bactericidal power of a given serum. Animal. PETER A. F. MORRIN, NEAL S. BRICKER* and These results demonstrate that human sera kill S. WESLEY KIME, JR., St. Louis, Mo. pathogenic staphylococci as well as pathogenic gram- Metabolic acidosis is a characteristic feature of chronic negative bacteria, and suggest that the alleged suscepti- renal insufficiency. Whether the acidosis develops solely 1014 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING

because of a loss of functioning nephrons, or whether taining less than 8 mEq of potassium per kg for 4 specific tubular defects in hydrogen ion secretion also months; they became potassium-deficient as measured by exist in ordinary forms of chronic renal disease is un- serum and muscle potassium levels, and the TmPO4 was known. measured as before. During the last 12 days of the Hydrogen ion secretion by diseased kidneys has been potassium-deficient diet, 25 mg of desoxycorticosterone investigated in acidotic (NH4Cl-loaded) dogs. Per- acetate (DCA) in oil was injected daily into each animal manent hemibladders permitted separate urine collections and the TmPO4 measured again. from both kidneys. Data include: 6 studies on 5 dogs The mean tubular maximum for phosphate reabsorp- with unilateral pyelonephritis or aminonucleoside-nephri- tion in each animal during each of the periods of the tis, and 4 studies on 1 dog with bilateral pyelonephritis. experiment, respectively, was as follows in mmoles per Urinary pH, tritratable acid (TA), ammonia and bicar- minute: in the control period 0.092 (SE + 0.008) and bonate excretion were measured bilaterally during infu- 0.084 (SE + 0.005); in the period of potassium deficiency sion of mannitol and phosphate. Urine and arterial blood 0.081 (SE + 0.007) and 0.093 (SE ± 0.006); in the samples were collected anaerobically. period of potassium deficiency and DCA administration The absolute excretion rates (UV) of ammonia and 0.082 (SE + 0.005) and 0.085 (SE ± 0.006). No sig- TA were less for diseased (experimental) than for con- nificant difference was observed between the values for tralateral control kidneys; and in the animal with bi- either animal under the different conditions. lateral disease, excretion rates were lower for the more It was concluded that the renal tubular lesions of severely diseased (i.e., experimental) organ. However potassium deficiency did not interfere significantly with glomerular filtration rates (GFR) for the experimental phosphate reabsorption by the kidney. kidneys were decreased proportionately and when ex- cretion rates were calculated per unit of GFR, no differ- Ethyl Ether Elimination by the Lungs, a New Measure ences were observed between experimental and contra- of Effective Alveolar Ventilation. THEODORE H. lateral kidneys. UVNHIS/GFR for experimental kidneys NOEHREN and DOUGLAS S. RIGGS, Buffalo, N. Y. (in- averaged 95 per cent of values for contralateral kidneys troduced by John H. Talbott). [in 9 experiments ratios averaged 0.99 (range, 0.84 to 1.14); and in 1 experiment, 0.55]. UVTA/GFR for ex- Theoretical considerations of the exchange of inert perimental kidneys averaged 99 per cent of contralateral gases in the lungs have indicated that pulmonary ventila- values (range, 0.93 to 1.08). Essentially no bicarbonate tion, cardiac output, and the solubility of the individual was excreted and bicarbonate reabsorption/GFR there- gas in blood are major factors controlling the pulmonary fore was always equal bilaterally. If total hydrogen ion elimination of the gas. The relative importance of excretion was expressed as UVNH,+ UVTA+ bicarbonate cardiac output and pulmonary ventilation is determined reabsorption, values per unit of GFR remained equal by the solubility. The highly soluble gases, such as bilaterally. ethyl ether, should be eliminated at a rate directly pro- These results suggest that residual nephrons of the portional to the volume of alveolar ventilation, with diseased kidneys secreted hydrogen ion in an orderly and very little influence by alterations in blood flow unless efficient manner. The data provide confirmatory evidence alveolar ventilation is considerably larger than blood that the surviving nephrons of diseased kidneys in the flow. dog retain a high degree of functional integrity. The present study was designed to test the accuracy of this hypothesis. Ethyl ether solution was administered The Reabsorption of Phosphate the in Potas- intravenously at a constant rate to dogs anesthetized with by Kidney pentobarbital. Ventilation was altered by electrical sium-Deficient Dogs. ASHTON B. MORRISON, VARDAMAN stimulation of the phrenic nerves M. BUCKALEW, RAY MILLER D. through implanted JR., and JOHN LEWIS, electrodes. Intravenous isoproterenol (Isuprel) was used Philadelphia, Pa. (introduced by Archer P. Crosley, to vary cardiac output. The procedure was safe and did Jr.). not necessitate achieving anesthetic concentrations of Low serum phosphate levels are often found in patients ether in these animals. with potassium deficiency and have also been reported in The results indicate a linear relationship between ether potassium-deficient rats. To find whether or not the clearance by the lungs and alveolar ventilation within the hypophosphatemia results from interference with phos- ranges studied. Alterations in cardiac output had very phate reabsorption in the kidney by the lesions caused by little influence on the amount of ether excreted. These potassium deficiency in the tubular cells, the renal tubular results are in good accord with theory. maximum for phosphate reabsorption (TmPO4) was This technique offers a new way of measuring effec- measured in 2 dogs before and during potassium defi- tive alveolar ventilation without significant disturbance ciency. by circulatory changes. The one-way pulmonary gas ex- Two trained female dogs were fed a control diet ade- change achieved by administering the gas intravenously quate in all nutrients. On 7 separate occasions, over a offers distinct advantages in the study of the cardio- period of 3 months, 4 determinations of TmPO& were pulmonary system. It is applicable in a variety of made in each animal by standard methods during phos- physiological and pathophysiological problems in experi- phate loading. The animals were then fed a diet con- mental animals, and should be safe for clinical trial. AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 1015

Inihibition of Aromatic Amino Acid Decarboxylation in 0.10, corresponding to a tubular potassium concentration Man and Associated Pharmacological Effects. JOHN of 5.4 + 0.1 mEq per L. In 5 of these experiments, the A. OATES, JR., Louis GILLESPIE, JR., J. RICHARD CROUT potassium concentration in glomerular fluid was com- and ALBERT SJOERDSMA,* Bethesda, Md. pared with that in serum, giving a mean ratio of 1.05 + 0.04. Water absorption, measured with C14-inulin in a Decarboxylation is a requisite reaction in the bio- second series of 12 animals was 33 per cent, a value cor- synthesis of aromatic amines. The present studies demon- responding to a C1'-inulin urine/serum ratio of 1.48 + strate inhibition of this process by a-methyl-3,4-dihydroxy- 0.08. DL-phenylalanine (a-methyl-dopa). Decarboxylation of In these experiments, the mean concentration ratio of 5-hydroxytryptophan (5HTP) was measured in four hy- potassium in fluid collected from the distal end of the pertensives by determining urinary serotonin following proximal tubule is essentially equal to that of inulin. infusion of 30 mg DL-5HTP. Pretreatment with 2.0 g Consequently, no potassium absorption from the proximal a-methyl-dopa orally produced 60 per cent inhibition of tubule of the Necturus kidney was observed under our of serotonin formation. A similar decrease in production experimental conditions. of dopamine from 3,4-dihydroxy-L-phenylalanine was demonstrated as well as 80 per cent inhibition of forma- Elevated Factors VII and X in Pregnancy, A "Hyper- tion of tyramine from tyrosine (125 mg per kg orally) coagulable" State. LIBERTO Mass. and 50 per cent inhibition of tryptamine formation from PECHET, Boston, (introduced by Benjamin tryptophan (50 mg per kg orally). In collaboration with Alexander). S. Udenfriend, inhibition of 5HTP decarboxylation was Factor VII has been found elevated in pregnancy. confirmed in two carcinoid patients in whom daily ad- When first observed, Factor X (Stuart) was unknown. ministration of 5.0 to 6.0 g a-methyl-dopa decreased Owren's assay, used then, is now known to measure both urinary 5-hydroxyindole acetic acid 70 per cent and in- VII (proconvertin) and X. It remained to be deter- creased 5HTP excretion from < 1 and 10 mg to 60 mg mined whether the observed elevation was in VII, X, or per day. in both. During these experiments a hypotensive effect was Factor VII was specifically determined by the clot noted, and observations were extended to 10 hypertensive corrective effects on congenital VII-deficient plasma; X, patients. Treatment with 0.75 to 4.0 g of a-methyl-dopa by the "Stypven" method. Also assayed were "Factor daily for 7 to 28 days reduced average standing blood VII" by the Owren method, and two-stage prothrombin. pressures in all cases (range, -22/-16 to -53/-36 mm Hg) Normal values are 70 to 120 per cent for VII, 70 to 110 and lowered recumbent pressures in 5 patients (-24/-12 per cent for X, and 220 to 300 units prothrombin per ml. to -37/-29 mm Hg). A central effect, manifested as In 51 pregnant subjects Factor X varied from 72 to drowsiness, was limited to the first 3 days of treatment. 200 per cent, average 130 per cent. In 33 it exceeded In addition to offering a basic approach to investigation 110 per cent; in 14, 150 per cent. VII varied between of blood pressure regulation in man, a-methyl-dopa war- 70 and 250 per cent, mean 162. Thirty-six individuals rants therapeutic evaluation in diseases characterized by a exceeded 120 per cent; 31, over 150 per cent, and 8 ex- relative or absolute excess of amines. ceeded 200 per cent. Owren "Factor VII" varied be- tween 86 and 248 per cent, average 170 per cent. Fifty Potassium Concentration in the Proximal Tubule of exceeded 110 per cent; 39, 150 per cent or more; 8 ex- Necturus Kidney. DONALD E. OKEN and A. K. ceeded 200 per cent. Prothrombin was essentially normal. SOLOMON, Boston, Mass. (introduced by Kendall Emer- In some subjects VII was elevated and X normal; in son, Jr.). others X was increased with VII only slightly. In most, both were elevated, with Owren values falling in between. A method has been developed to measure the potassium In some, however, Owren "VII" was much higher than concentration in fluid collected from the proximal tubule both VII and X. of the Necturus kidney. Samples were obtained by a The occasional increased Owren "VII" where VII and modification of the micropuncture technique of Richards X were normal, suggests that the assay may reflect and Walker. Fluid was collected only from the most entity(s) besides VII and X. The disparity in VII and distal segments which had been identified visually and X, and prothrombin, indicates their distinctness as clot- blocked with injection of Sudan black-stained mineral ting entities, and makes it doubtful that VII and/or X are oil. Since tubular fluid was collected at such a rate that prothrombin derivatives. More significant, these high the position of the distal oil droplet remained almost levels in the gravid reflect a truly "hypercoagulable" static, the rate of collection approximated normal tubular state, ready to be triggered by the intrinsic or extrinsic flow. After each experiment, the distal location of the thromboplastin mechanisms, in a condition so frequently collection site was checked by further oil injection. Po- complicated by thromboembolism. tassium concentrations in tubular fluid, glomerular fluid and serum were determined in the flame photometer of Studies of the Metabolism of Calcium, Phosphorus and Solomon and Caton on samples measuring 0.3 to 0.4 ,lI. Magnesium in Subjects with Pseudo-Hypoparathyroid- In 12 experiments, the ratio of tubular potassium con- ism. MAURICE M. PECHET* and EVELYN L. CARROLL, centration to serum potassium concentration was 1.51 Boston, Mass. 1016 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING

In the preparation of vitamin D2 by the ultraviolet fibrinogen levels and thromboplastin generation were irradiation of ergosterol a number of isomeric compounds affected to a minimal degree. With prior heparinization, are formed. Complete metabolic studies were carried the effect of protamine on most of the tests corresponded out in 3 subjects with pseudo-hypoparathyroidism, with only to the portion unneutralized by heparin. Interaction 2 of these isomeric compounds, carefully purified, so as to between heparin and protamine was completed within one represent by chemical determinations single entities. minute. Platelets dropped whether or not heparin had Crystalline dihydrotachysterol II, when administered been given. Results with polybrene were identical ex- orally in doses of 6 mg daily, produced a marked de- cept that larger amounts of heparin were neutralized and crease in fecal calcium, a modest increase in urinary coagulation abnormalities were more severe and per- calcium and a pronounced increase in serum calcium. sistent with the same doses. Fecal phosphorus decreased and urinary phosphorus in- It is concluded that excessive amounts of protamine or creased; these changes occurred without changes in serum polybrene can produce a coagulation defect in vivo. Sig- phosphorus levels. Fecal magnesium decreased and nificantly increased blood loss following open heart urinary magnesium increased while the serum values re- surgery may thus occur, particularly if repeated injections mained unaltered. Changes in fecal and urinary nitrogen are given at intervals of less, than 20 to 30 minutes. In were minimal. The effect of administration of dihydro- no case was the disappearance of protamine or polybrene tachysterol II on urinary phosphorus excretion manifested followed by the reappearance of heparin in the circulating itself promptly and prior to the effect on urinary calcium blood. excretion; in addition, the magnitude of the changes was greater for phosphorus than for calcium. The effects on Relationship of Phagocytosis to the Fall in Spinal Fluid phosphorus excretion are probably not secondary to the Glucose in Experimental Meningitis. ROBERT G. changes in calcium excretion. PETERSDORF, Baltimore, Md. and Seattle, Wash. (in- The daily oral administration of 100 mg of suprasterol troduced by W. M. M. Kirby). I produced a marked increase in fecal calcium and no change in urinary or serum calcium. Fecal phosphorus The mechanism of hypoglycorrhachia in bacterial men- increased to a lesser extent than did the fecal calcium, ingitis is poorly understood, and the fall in GSF glucose and the effect wore off much more rapidly for phos- observed in these infections has been attributed to cells, phorus following the withdrawal of the suprasterol. bacteria, consumption of glucose by neural tissue and Urinary phosphorus increased slightly, and serum phos- changes in the blood-CSF barrier. Previous experiments phorus remained unaltered. Fecal and urinary excretion have demonstrated that a mixture of leukocytes and of magnesium increased and then decreased promptly pneumococci incubated in CSF in vitro act synergistically following the withdrawal of the compound. Whereas to depress CSF glucose, suggesting that a combination of vitamin D2 is known to induce a decrease in fecal ex- cells and bacteria is necessary for hypoglycorrhachia. cretion of calcium, the isomeric suprasterol induced an In the present experiments, aseptic meningitis was increase in fecal excretion of calcium. produced in dogs by the administration of saline into the cisterna magna. Four hours later these animals regularly had an exudate containing 3 to 8,000 WBC per mm8 of Duration of Coagulation Abnormalities Following In CSF, predominantly polymorphonuclear leukocytes. At Vivo Neutralization of Heparin by Protamine and this time pneumococci were instilled intrathecally and Polybrene. HERBERT A. PERKINS, D. J. AcRA and cisternal puncture was performed 3 hours later. In all MARY R. ROLFS, San Francisco, Calif. (introduced by animals with bacterial meningitis superimposed upon Stacy R. Mettier). aseptic meningitis, a profound drop in CSF glucose oc- Following open heart surgery, complete neutralization curred. Control animals given pneumococci without ante- of heparin is necessary to avoid excessive blood loss. cedent production of aseptic meningitis, or animals with The neutralizing agents by themselves have an anti- aseptic meningitis without superimposed bacterial infec- coagulant effect in vitro, but there has been disagreement tion, did not experience a fall in glucose. These results as to whether excessive amounts in vivo are likely to provide evidence for synergistic action between bacteria induce a hemorrhagic state. and leukocytes in depressing CSF glucose in vivo. In Protamine was injected intravenously into dogs, and order to clarify the mechanism of the synergistic effect, blood levels were estimated by the ability of the blood to heat-killed pneumococci were injected intrathecally into neutralize the anticoagulant effect of various concentra- animals with aseptic meningitis. Hypoglycorrhachia oc- tions of heparin. Protamine was detectable for periods curred promptly indicating that bacterial multiplication up to 10 minutes with doses of 5 mg per kg; with 3 mg was not responsible for the fall in glucose. Intrathecal per kg for less 5 administration of India ink particles to dogs with aseptic than minutes. However, coagulation meningitis was also associated with a decrease in CSF abnormalities were evident considerably longer than glucose and a large number of the particles were visible protamine could be detected by our relatively crude tech- within leukocytes. These findings suggest that leukocytes nique. Platelets dropped sharply, the Lee-White clotting in the CSF engaged in active phagocytosis rapidly con- time and cephalin time increased, and prothrombin con- sume glucose, while resting white cells exhibit little sumption was greatly impaired. Prothrombin times, glycolytic activity. AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 1017

On the Mechanzism)i of Carotid Sinus Function. LYSLE H. curic chloride, thimerosal, and iodine also inactivated PETERSON,* ERIC 0. FEIGL and PETER GoURAS, Phila- the hemagglutinating activity of the ECHO and Cox- delphia, Pa. sackie viruses mentioned. The effect of mercuric chloride but not of thimerosal or iodine was reversed by reduced A classic principle of cardiovascular function holds that glutathione. However, thimerosal apparently acts at the arterial blood pressure is regulated via "baroreceptors" same site on the virus protein as PCMB because the such as those in the walls of the carotid sinus. Actually latter was capable of blocking the action of thimerosal. these receptors respond to stretch of the vessel wall rather ECHO 11 and 22 were inactivated also by iodoacetamide, than to pressure per se. The magnitude of vessel wall an alkylating reagent which reacts with sulfhydryl groups. results from intravascular stretch (strain) which pressure Hemagglutinating activity of arbor and myxoviruses was depends upon the mechanical properties of the vessel wall unaffected by PCMB. (elasticity and viscosity). Thus, the relationship between PCMB and thimerosal inactivated the infectivity of arterial blood pressure and the nervous activity of the the following viruses: ECHO 7, 11, 12, and 22 which receptors depends upon these mechanical properties. Tech- hemagglutinate; ECHO 6 and 9 which do not hemag- niques have been developed and used for the accurate, glutinate; two strains of Coxsackie B3, one which hemag- simultaneous measurement of the intracarotid sinus pres- glutinates and another which does not; and poliovirus 2, sure, carotid sinus strain (change in circumference per MEF1 strain. Preliminary evidence indicates that PCMB unit circumference), the electrical of sinus re- activity or thimerosal-treated virus fails to adsorb to monkey ceptors and the mechanical properties of the sinus wall. kidney cells. The results obtained suggest that sulfhydryl Experiments on and anesthetized (pentobarbital Dial) groups of the virus proteins are necessary for attachment dogs have demonstrated that: 1) The mechanical prop- of enteroviruses to erythrocytes and host cells. erties of the sinus wall can change by 100 per cent or more following the application of vasoactive materials to the sinus wall; there are, therefore, contractile elements Electron Microscopic Study of Tissue Mast Cells and in the wall. 2) The mechanical properties of the wall can Chromaflin Cells in Man. JOHN H. PHILLIPS and change reflexly as a result of efferent nerve supply to the RICHARD G. HIBBs, New Orleans, La. (introduced by sinus wall. 3) Receptor activity changes, with respect to George E. Burch). pressure as the wall properties change. From these find- In various species the typical type of mast cell has been ings it can be concluded that the nerve impulse traffic shown to contain serotonin, histamine and heparin. Pre- arising from these receptors is a function of two inde- vious studies in this laboratory revealed chromaffin gran- pendent variables, wall properties and pressure. Thus, the ules in elongated eel-like cells in human skin which classic concept of blood pressure regulation is an over- superficially are quite similar to tissue mast cell and -simplification which may be misleading. In addition to have probably been so called in the past. Available in- the acute "physiological" changes in vessel wall proper- direct physiological and pharmacological evidence sug- ties it is likely that chronic changes due to aging and gests that these granules are a local tissue source of disease may also lead to alterations in the cardiovascular epinephrine or norepinephrine or both. As so-called tis- regulatory mechanisms. sue mast cells may in reality represent several cellular types, electron microscopic studies were undertaken to On the Role of Virus Sulfhydryl Groups in the Attach- clarify possible anatomical differences. Tissues utilized ment of Enteroviruses to Cells. LENNART PHILIPSON in these studies were obtained from human digital and and PURNELL W. CHOPPIN, New York, N. Y. (in- abdominal skin (20 specimens) by punch biopsy and troduced by Igor Tamm). from human gastric mucosa (8 specimens) by suction biopsy. At least two distinct types of "mast cells" were The precise mechanism whereby animal viruses attach identified. One is an elongated cell with granules con- to cells is poorly understood. In the experiments to be taining a homogenous material enclosed by a membrane, described, interaction of entero, myxo, and arbor viruses and the other, an oval circumscribed cell with larger with erythrocytes and host cells was studied. Hemag- granules composed of peculiar subgranular structures in glutinating activity of ECHO 7, 11, 12, and 22, and Cox- the form of clusters of laminated rolled scrolls, all en- sackie B3 virus was eliminated by p-chloromercuri- closed by a distinct membrane. Filling the spaces be- benzoate (PCMB), a compound which combines with tween these scrolls was a finely particulate matter. It is sulfhydryl groups forming mercaptides. PCMB-treated felt that the oval-type cell with its complex granules is virus failed to adsorb to erythrocytes. At 10- M the more classic form of tissue mast cell while the elon- PCMB eliminated the hemagglutinating activity of gated type with its rather simple granule is the chro- ECHO 7 virus; 1045 M PCMB was sufficient to cause maffin cell identified by light microscopy. 50 per cent inactivation. The reaction was rapid in The intimate relationship of mast cells to the dynamics that 101 M PCMB caused 75 per cent inactivation in 2 of the ubiquitous connective tissue is well known. Nota- minutes at 370 C. The effect of PCMB was reversible ble in its effects on the connective tissue is the adreno- by sulfhydryl-containing compounds. Cysteine, reduced corticosteroid. Preliminary electron microscopic observa- glutathione, and mercaptoethanol completely restored the tions following steroid administration have shown quite hemagglutinating activity of PCMB-treated virus. Mer- different effects on the two cellular types described above, 10181PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MIEETING

Thus evidence is presented in which "tissue mast cells" moreover, glomerular involvement was always more se- represent at least two distinct cellular types with many vere than tubular damage. This unusual relative glo- differences in structure and function. merulo-tubular imbalance led us to study quantitatively enzyme activity in the nephron in lupus nephritis, in order Bladder Smooth Muscle Contributions to Micturition. to assess the relationship between enzyme activity in in- FRED PLUM, Seattle, Wash. (introduced by R. H. Wil- dividual anatomical subunits of the nephron and glo- liams). merulo-tubular imbalance. Alkaline phosphatase activity Two types of contractions occur when fluid fills the (APP) was assayed because qualitative data were avail- normal urinary bladder: low amplitude (2 to 5 cm H20), able from histochemical staining techniques; lactic de- unsustained, rhythmically recurring waves, and high in- hydrogenase activity (LDH) was assayed because LDH tensity (75 to 150 cm H20), intermittent micturition is a key enzyme in cellular energy metabolism. contractions. Previous studies leave unclarified the inter- Seven healthy kidneys and 19 renal biopsy specimens relationships of these two responses. This investigation from 13 patients with lupus nephritis were assayed quan- demonstrates the genesis of the bladder rhythm, indicates titatively for enzyme activity by ultramicrochemical tech- its role in stimulating micturition contractions, and shows niques. Activity was measured on individual glomeruli, how purely local bladder changes alter the rhythm to and dissected portions of proximal and distal convolu- produce abnormal cystometric changes traditionally at- tions, medullary rays, medulla and papilla containing 20 tributed to neurological injury. to 200 cells; results were expressed as moles of substrate To separate neurogenic from vesicogenic micturitional split per kilogram dry weight per hour at 370 C. changes, isometric cystometrograms were recorded in APP activity was decreased in lupus nephritis in proxi- cats before and after spinal cordectomy, sacral rhizotomy mal (p=<0.001) and distal convolutions (p=<0.001). and vesical autonomic ganglionectomy. Bladder wall APP activity was similar in lupus glomerulitis (mild damage was induced by overdistention, which, in in- glomerular lesions, normal tubules) and lupus glomerulo- nervated bladders, required either obstructing the urethra nephritis (severe glomerular lesions, abnormal tubules or temporarily suppressing micturition by ether anes- and interstitial tissue). thesia. Afferent stimuli from the bladder were recorded LDH activity was higher in lupus glomerulonephritis in single pelvic nerve or sacral dorsal root fibers. To than in healthy kidneys in proximal (p= < 0.001) and rule out species differences, isometric cystometrograms distal convolutions (p=<0.05). In all structures ana- were recorded from healthy humans before and after lyzed LDH activity was significantly higher in lupus spinal anesthesia plus ganglionic blockade. glomerulonephritis than in lupus glomerulitis. This dif- Rhythmic contractions originated directly in bladder ference was not related to renal functional impairment or smooth muscle and persisted after all denervation pro- to the dosage of prednisone used therapeutically (con- cedures. Rhythmic contractions appeared to provide the firmed by experiments in rats). Increased LDH activity necessary stimulus to micturition, since bladder filling in convoluted tubules in lupus glomerulonephritis would invariably evoked rhythmic waves preceding micturition appear to be an enzymatic expression of the ability of contractions, and maximal afferent nerve activity coin- slightly damaged tubules to respond metabolically to cided with each rhythmic wave's rising phase. Denervat- increased metabolic demands imposed by severely dam- ing the bladder abolished micturition contractions but aged glomeruli. By contrast, in Fanconi syndrome with produced little other cystometric change-additional over- tubulo-glomerular imbalance and severe tubular damage, distention was required to supress bladder rhythm and LDH activity in the convoluted tubules was significantly produce a flat, atonic cystometrogram. Identical cysto- decreased. metric atony was induced by overdistending neurologi- cally intact bladders: rhythmicity was suppressed, afferent Recall of Type Specific Imimnunity by Vaccination of nerve potentials were markedly reduced, and micturition Hum1lant Beinigs with Groutp A Streptococcal Cell Wall was abolished until vesical decompression restored in- Vaccinies. ELIZABETH V. POTTER, GENE H. STOLLER- trinsic bladder rhythmicity. MAN * and ALAN C. SIEGEL, Chicago, Ill. Once distention has suppressed intrinsic bladder Immunity to Group A streptococci appears to be type rhythmicity, obstructive and neurogenic bladder dysfunc- specific and is related primarily to the development of tion cannot be differentiated by cystometrogram. Effec- antibody against the M protein surface antigen. Human tive treatment in either case must be directed toward vaccination with Group A streptococci has been dis- restoring smooth muscle function. couraged by the toxicity of streptococcal products, the relatively low antigenicity of type specific M protein in Quianttitative Assay of Alkaline Phosphatase and Lactic purified form, and the multiplicity of streptococcal sero- Dehydrogenase Activities in the Nephron in Glomerulo- logical types. Primary immunization in man has been Tubular Imbalance. VICTOR E. POLLAK, SJOERD L. unsuccessful so far. The present studies were under- BONTING and ROBERT C. MUEHRCKE, Chicago, Ill. (in- taken to determine whether or not vaccines prepared from troduced by Robert M. Kark). cell walls of streptococci (rich in M protein) are at all Previous studies showed that in lupus nephritis glo- antigenic in the human being. meruli were abnormal before tubules were damaged; Cell wall vaccines of Type 12 streptococci were pre- AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 1019

pared by Mickle disintegration according to the method These studies, then, support the view that Hagernan of Barkulis. Type 12 anti-M antibodies were produced factor is an enzyme which initiates coagulation by its in rabbits by primary immunization with whole cells and effect on plasma thromboplastin antecedent. small doses of the cell wall vaccine were shown to recall these antibodies after their disappearance. Role of Plasma Pco2 and Carbonic Anhydrase Actizity Of 57 children with untreated Type 12 streptococcal in HCO3- Reabsorption. FLOYD C. RECTOR, JR., ALBERT pharyngitis (who were part of a controlled study in prog- D. ROBERTS, JR., JERRY S. SMITH and DONALD W. ress), 41 developed type specific antibody following the SELDIN,* Dallas, Tex. natural infection. Ten of these patients no longer had anti-M antibody demonstrable by bactericidal and long Renal HC08- reabsorption in dogs was examined using chain tests after 2 years of follow-up. These children three types of experiments. In Group I, the effect of received 3 to 4 small booster subcutaneous or intradermal plasma Pco2 on maximum HC0- reabsorptive capacity injections of Type 12 cell wall vaccine (1.7 to 170 ,ug (HCOs- Tm) was assessed before and after carbonic of protein per dose) which were well tolerated. Recall anhydrase (CA) inhibition (acetazolamide 50 mg per of anti-M antibody was successful in all 10 patients. The kg). HCOs- reabsorption increased curvilinearly as levels of antibody recalled, though low, were at least as Pco2 was elevated. CA inhibition depressed HCOa7 high as those observed following the antecedent natural reabsorption by a constant amount at all Pco2's. In infection. Group II, the effects of Pco2 and CA inhibition on the The results indicate that M protein in cell wall vaccines relationship of HCOs- excretion to HCO3- Tm were is antigenic in man in small, tolerated doses and suggest studied. As plasma HCOs- concentration was progres- that primary immunization might be achieved if a suffi- sively increased in the normal state, HC03- excretion cient amount of vaccine can be administered in multiple began before the Tm was reached. A similar HCOi- small doses. leak was noted in respiratory acidosis. CA inhibition, however, caused a HCO8- leak which was of far greater magnitude and occurred even at very low levels of serum An Action of Hageman Factor: Evidence that Plasma Thromboplastin Antecedent is Activated by Hagenman [HCO3-j. Increasing H+ production by markedly ele- Factor. OSCAR D. RATNOFF,* DAVID L. MALLETT and vating plasma Pco2 failed to obliterate the HCO3- leak, EARL W. DAVIE, Cleveland, Ohio. whereas administration of Na2SO4, a salt capable of in- tensely acidifying the urine in the normal state, partially The earliest steps in the process of blood coagulation corrected it. In Group III, CA inhibition during metabo- are obscure. Previous studies have indicated that contact lic acidosis resulted in an inability to acidify urine below of plasma with glass converts Hageman factor from an the pH of blood, despite elevation of plasma Pco2 to levels inactive to an active form. Several authors have sug- as high as 200 mm Hg. gested that this activated Hageman factor then reacts It was concluded that HC03- reabsorption is accom- with plasma thromboplastin antecedent to form a clot- plished by two distinct processes. One process is inde- promoting agent. pendent of CA, has a HCOS- Tm which is dependent on Partially purified preparations of activated Hageman Pco2, and is incapable of establishing pH gradients be- factor, deficient in other known clotting factors, were t-ween blood and urine. The other process is dependent prepared from plasmas obtained from patients with de- on CA, independent of Pco2, and necessary for the es- ficiencies of plasma thromboplastin antecedent. Similarly, tablishment of pH gradients between blood and urine. partially purified preparations of plasma thromboplastin antecedent were prepared from plasmas lacking Hageman Abnormalities of In Vitro Behavior of Structural Lipids factor-, obtained from patients with Hageman trait. When of Red Blood Cells from Patients with Hereditary a mixture of Hageman factor and plasma thromboplastin Spherocytosis. CLAUDE F. REED and Scorr N. antecedent was incubated in silicone-coated tubes at 370 SWISHER,* Rochester, N. Y. C, a clot-promoting agent gradually evolved. The evolu- tion of the clot-promoting agent was temperature- and To explain increased osmotic fragility, increased per- time-dependent. Suggestive evidence was obtained that meability to K+, and premature hemolysis of erythrocytes the formation of the clot-promoting agent was enzymatic. from patients with hereditary spherocytosis (HS), pro- Partially purified, activated Hageman factor was inhibited duced by in vitro incubation at 370 C, several investi- by diisopropylfluorophosphate, as shown by Becker, but gators have postulated an early onset of "degeneration of partially purified plasma thromboplastin antecedent was the membrane," preceding these changes. not affected by this substance. Experiments in which In the present study, serial measurements of the rate the concentrations of plasma thromboplastin antecedent of exchange of plasma and erythrocyte phospholipids, and and of Hageman factor were varied independently sug- of net changes in lipid composition of these erythrocytes, gest that the effect of Hageman factor is to transform were made during 8 to 24 hour periods of in vitro incu- plasma thromboplastin antecedent into a clot-promoting bation. These parameters were correlated with simul- substance. This substance is neither thrombic nor throm- taneous measurements of glucose disappearance, lactic boplastic in activity and appears to act early in the clot- acid production, cellular K+ content and changes in os- ting process. motic fragility. All studies were made before the oc- 1020 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING currence of detectable hemolysis, in an attempt to de- gen requirements of exercise depend upon the posture fine the prehemolytic sequence of changes. of the individual. Under all circumstances studied, the rate of exchange between plasma and erythrocyte phospholipids was sig- The Henmodynamiiic Determinants of the Rate of Pressure nificantly less in HS erythrocytes than in normal erythro- Change of the Left Ventricle During Isometric Con- cytes. A progressive net loss of red cell lipid, signifi- traction. T. J. REEVES, LLOYD L. HEFNER, W. B. cantly greater than that observed in normal erythrocytes, JONES, CECIL COGHLAN, GUSTAVO PRIETO and JOHN occurred during the first 24 hours of in vitro incubation. CARROLL, Birmingham, Ala. (introduced by Tinsley R. The predominant loss was from those cellular lipids Harrison). known to exchange with the corresponding plasma com- The pressure, time derivative of pressure, circumference pounds, i.e. sphingomyelin, lecithin, and cholesterol. and contractility (isometric contractile force-Walton- Lipid loss preceded detectable changes in cellular K+ Brodie strain gage arch) of the left ventricle have been content, gross changes in osmotic fragility or measurable simultaneously recorded in a series of thoracotomized hemolysis. Supplemental glucose retarded, but did not dogs. These parameters were recorded at multiple levels prevent the progressive loss of cellular lipid, as it does of filling pressure, while variations in myocardial function with normal erythrocytes under similar experimental and systemic resistance were induced by the administra- circumstances. tion of epinephrine and methoxamine. These observations illustrate a relationship between The maximum rate of pressure rise (MRPR) of the erythrocyte energy production, maintenance of integrity isometric contraction phase of the ventricular pressure of the structural lipids, and "membrane function." pulse ranged from 456 to 6,610 mm Hg per second. The HS erythrocyte appears to utilize energy ineffi- MRPR was shown to have a significant correlation with ciently in maintaining integrity of its structural lipids; peak ventricular pressure (R = 0.624; p <0.001) with the observed initial loss of cellular lipid may well repre- ventricular pressure area (R=0.287; p<0.01) ventricu- sent the postulated "membrane degeneration." lar end diastolic stretch (R=0.484; p<0.001) and with ventricular end diastolic pressure (R=0.468; p<0.001). The Adaptation of the Circulation to Supine Exercise and When graphically displayed, all of these relationships Treadmill Walking. JOHN T. REEVES, ROBERT F. showed a strong tendency to form families of curves, GROVER, GILES F. FILLEY and S. GILBERT BLOUNT, JR.,* with MRPR per unit systolic pressure, stretch, or end Denver, Colo. diastolic pressure being greater while epinephrine was Walking is the usual form of exercise for man, yet being administered than in the control period. Conversely, cardiac catheterization of normal man during treadmill MRPR was less per unit pressure, circumference or end walking has not been reported. Although the circulatory diastolic pressure when methoxamine was administered. behavior during standing differs greatly from that of Since epinephrine resulted in increased myocardial con- supine rest, the effect of posture on the circulatory re- tractility and methoxamine resulted in a decreased myo- sponse to exercise has not been documented previously. cardial contractility, it was not surprising that a highly To investigate these areas of circulatory physiology, 10 significant correlation between MRPR and contractility normal men each performed several grades of treadmill was found (R = 0.451; p <0.001). A linear relationship exercise with a catheter in the pulmonary artery, per- was found between MRPR and the product of contractil- mitting repeated measurements of cardiac output by the ity and the end diastolic stretch of the ventricle (R = classical Fick method. These and 12 other normal sub- 0.790; p < 0.001). This product may be considered as jects were also evaluated by this technique during supine' an index to the maximum tension that would be gen- exercise and during standing. The behavior of the circu- erated during a completely isometric contraction of the lation of the leg was investigated for the various condi- entire ventricle. tions of the experiment by sampling blood through a The quotient MRPR/end diastolic pressure was found polyethylene catheter introduced into the femoral vein. to have an excellent correlation with contractility (R = The results showed that during exercise a given quan- 0.655; p < 0.001), indicating the possibility of obtain- tity of oxygen was consistently transported by a smaller ing a quantitative index to myocardial contractility from cardiac output (10 to 20 per cent smaller) and a greater the ventricular pressure alone. arteriovenous (A-V) oxygen difference for the upright Influtentce of Extracellular Calcium Concentration Upont than for the supine posture. Further, in the supine Mvocardial Potassium Transfer. TIMOTHY J. REGAN, posture, the A-V oxygen difference for the leg increased GERALD C. TIMMIs, MARTIN J. FRANK, JOHN D. markedly for mild exercise, but with heavier exertion McGINTY and HARPER K. HELLEMS,* Detroit, Mich. increase. However, the A-V there was little further Since calcium and the monovalent cations frequently difference for the leg increased nearly to maximum as have opposing effects upon a variety of physiological the subject assumed the standing posture, and severe phenomena, the influence of varied plasma Ca concentra- treadmill exercise was not accompanied by a further in- tions on myocardial transfer of potassium and sodium has crease. These data indicate that the particular central been studied. and peripheral mechanisms called upon to meet the oxy- The effects of doubling the normal plasma Ca concen- AMERICAN SOCIETY FOR CILINICAL INVESTIGATION 1021 trations upon net myocardial transfer of K and Na have correlation between the magnitude of the fall in WHVP been assessed in 7 intact vagotomized dogs by serial and HBF and, in those patients who failed to show any paired sampling of arterial and coronary sinus blood. drop in HBF, WHVP either did not decrease or fell Rapid infusion of calcium induced a net myocardial up- minimally. Hemodynamic changes appeared a few min- take of potassium over a 4 minute period, paralleling utes after vasopressin was started, fluctuated irregularly the time course of hemodynamic effects. Planimetry of throughout the infusion (15 to 80 minutes) and returned the arteriovenous difference area was + 4.1 cm2 com- toward baseline values soon after the infusion was ter- pared to control period of + 0.5 cm' (p = <0.001). No minated. Systemic symptoms included nausea, pallor, sodium changes were noted. Sustained infusions pro- abdominal cramps and urgency and, in most cases, could duced a similar effect. Increase in heart rate, arterial be controlled by decreasing the rate of infusion. There pressure, stroke work and myocardial oxygen consump- was marked individual variation both in hemodynamic tion resulted from hypercalcemia. Prior studies indicate and symptomatic response to similar doses of vasopressin. that these changes, per se, do not affect ion arteriovenous The best explanation for these effects appears to be differences. splanchnic vasoconstriction with reduced portal inflow. As these hemodynamic and ion alterations are similar Although the hemodynamic changes with vasopressin are to sympathomimetic effects, another series of animals transitory, irregular, and not completely predictable, they was studied. Neither dibenzyline blockade of adrenergic may prove to be of clinical value in patients with bleeding influence nor depletion of myocardial norepinephrine esophageal varices. stores with reserpine modified the response to calcium. Preliminary ultracentrifugation studies of heart muscle The Determination of Pulmonary Blood Flow in Man. indicate that hypercalcemia increases mitochondrial cal- MARIO RIGATTO, GERARD M. TURINO and ALFRED P. cium concentrations while other fractions are unaltered. FISHMAN,* New York, N. Y. Such accumulation implies that calcium's activity is not exclusively dependent upon an extracellular location. In subjects with normal lungs, pulmonary blood flow The consequences of reduced plasma concentrations of may be calculated from the rate of absorption of an in- Ca upon the known K egress induced by strophanthidin spired soluble gas, its mean alveolar concentration, and was evaluated in 5 dogs by pretreatment with Sodium its coefficient of absorption in blood. This approach has Versenate. A 20 per cent plasma calcium reduction in- two advantages over indicator-dilution and direct Fick duced during strophanthidin administration was attended techniques: 1) simplicity and 2) validity in the face of by a significantly greater K loss (planimetered area left-to-right shunts. The main deficiencies of previous - 16.1 cm2) than in controls (-2.19 cm2) without hypo- methods using such gases have been: 1) overestimation calcemia after Calcium Disodium Versenate pretreatment of pulmonary recirculation time, 2) measurement of flow only during (p = < 0.05). expiration, and 3) failure to collect simul- These studies indicate that extracellular calcium ex- taneously all data used for the calculation. hibits a control over myocardial potassium transfer that The present study was designed to circumvent these is a direct function of its concentration. Thus, mainte- difficulties. The uptake of nitrous oxide was measured nance of the transcellular potassium gradient has a close during a vital capacity maneuver of less than 10 seconds. dependence upon the quantitative activity of this divalent The mean alveolar N20 concentration during inspiration cation. was calculated from the volume of inspired N20 and the residual volume; the corresponding concentration during expiration was measured by a The Effect of on Hepatic continuous infrared Vasopressin Hemodynamics in analyzer. The optimal time for the vital capacity ma- Patients with Portal Hypertension. TELFER B. REY- neuver was established by 24 separate determinations of NOLDS,* HERMAN M. GELLER and ALLAN G. REDEKER, the pulmonary recirculation time which involved: Los Angeles, Calif. 1) bronchospirometry, for sampling expired gas from one Infusion of vasopressin (20 to 40 IU per hour) resulted lung after administering N20 to the other; and 2) car- in a fall in wedged hepatic vein pressure (WHVP) in diac catheterization, for sampling mixed venous blood 11 of 13 patients with cirrhosis and portal hypertension. for N20 after a single breath of N20. Pulmonary re- The maximum fall in WHVP varied from 19 to 59 per circulation occurred within 11 + 3 seconds. cent of the original value. Although, in general, the de- In 11 subjects, with cardiac indices ranging from 1.7 cline in WHVP was greater in those with initially higher to 5.4 L per minute per m2, 19 comparisons were made of pressures, the degree of response could not be predicted. the pulmonary blood flow by the N20 and by the Fick In 2 patients there was no change in WHVP despite methods. The average difference between the two groups systemic symptoms indicating activity of the vasopressin. of values was 3 per cent. Individual differences ranged Hepatic blood flow (HBF) was estimated in 15 pa- from + 34 to - 13 per cent, with a mean of ± 12 per cent. tients and fell in 12 during vasopressin administration. In one subject, 13 determinations in 5 days by the N20 The maximum fall varied from 17 to 63 per cent of the method showed a mean deviation of + 11 per cent. baseline value. In 3 subj ects, there was no change in These results indicate that this method provides a re- HBF. Hepatic oxygen consumption did not change sig- liable measure of pulmonary blood flow in subjects with nificantly during vasopressin infusion. There was a good normal lungs. 1022 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING

The "Internal" pH of Mitochondria with Observations of different occupation and residence. Group A consisted on the Functiontal Significance of Mitochondrial Mem- of 69 San Francisco, Bay area women in key executive branes. EUGENE D. ROBIN, JOHN W. VESTER, ROBERT positionis characterized by immersion in competitive, J. WILSON and MARGARET H. ANDRUS, Pittsburgh, Pa. "deadline" and other socioeconomic stresses, and in- (introduced by Jack D. Myers). cluded 19 executive Catholic nuns. Group B consisted of 107 housewives and clerical secretaries and included 20 Measurements of the internal pH of isolated rat liver all Group B mitochondria have been attempted by using two buffer nuns teaching elementary school. Almost women and in central Costra County, an and NH3-NH4+. Rat liver mitochon- lived worked pairs, C02-HCOs- independent urban community of 35,000, notably devoid centrifugation at 40 C. dria were prepared by differential the well known characterizing the intense on mito- of attributes Measurements using the C02 system were done of and in densely populated urban tensions of gas. pace living working chondria equilibrated with known C02 and suburban communities. Total CO2 was determined manometrically and the The two groups of women were not found to differ Henderson-Hasselbalch equation applied (pK'a: 6.27; age, habitus, catamenia maturity history, Measurements using the NH3 system in average a: 0.093). were parental history of cardiovascular disease, exercise habits, done by equilibration with sucrose solutions of known or diet (calculated from individually kept diet diaries). pH and ammonia concentration. The serum cholesterol (SC) averaged 294 mg per 100 many implicit in these studies, Among the assumptions ml (SE + ..6) in Group A and 217 (± 3.5) in Group B. the most important is that external membranes of mito- The SC averaged 281 mg per 100 ml (SE ± 5.3) in chondria function in a manner similar to cellular mem- premenopausal, and 318 (± 8.8) in postmenopausal Group branes and that substances being measured are more or A women, compared to 211 (± 3.3) and 247 (+ 10.0) less in free solution in intramitochondrial water. This respectively, in Group B women. An average of 289 assumption was tested by determining the relationship (± 10) was observed in Group A nuns compared to 229 between supernatant pH and mitochondrial ammonia (± 9.5) in Group B nuns. These striking differences concentrations. An increase of supernatant pH produced were highly significant. a rise, and a decrease of supernatant pH produced a fall of mitochondrial ammonia. Thus intramitochondrial ammonia transfer appears to depend on non-ionic diffu- Hcemiodvnanmics of Systemiiic Arterial Hypertenision. G. sion suggesting a functional role for mitochondrial mem- G. ROWE, C. A. CASTILLO, G. M. MAXWELL and C. W. branes. The "internal" pH of mitochondria as deter- CRUMPTON,* Madison, Wis. mined by C02 measurements averaged 6.58 + 0.17 units In a series of 21 compensated subjects with systemic and intramitochondrial bicarbonate concentration averaged arterial hypertension, cardiac output was determined by 8.46 + 2.94 mmoles per L mitochondrial water. The the Fick principle and coronary blood flow by the nitrous mean pH as determined by the NH3 system averaged oxide saturation method. For purposes of comparison 6.25 + 0.24 units. The difference in pH as determined the series was divided into two groups. Group I con- by the two techniques is presumably related to technical tained 14 subjects with hypertension of Smithwick factors. grades 1 and 2. Group II contained 7 subjects in Smith- Of interest was the finding that liver mitochondria, wick grades 3 and 4. Statistical comparisons were done unequilibrated with NH3-containing solutions, contained by the t-test. In Group I, the cardiac index was normal quite large concentrations of NH3 (1.60+ 0.51 mmoles L but in Group II it was reduced (- 31 per cent, p < 0.01). mitochondrial water). If this represents uncombined The calculated total peripheral vascular resistance was NH3, this substance may be a quantitatively important elevated in Group I as compared to 30 normals (+ 47 intramitochondrial buffer. Mitochondrial pH values are per cent, p < 0.001) and in Group II as compared to at least 0.5 unit lower then the generally accepted internal Group I (+ 50 per cent, p < 0.001). Pulmonary vascular pH values of liver cells. It appears that one mechanism resistance was elevated in Group II as compared to for uneven H+ distribution within cells is the presence Group I (+ 32 per cent, p <0.05). The left ventricular of distinctly different pH values in subcellular structures. work index in Group I was increased as compared to normal and as compared to Group II (+ 39 per cent, Occupational and Environmental Influences on Serum p < 0.01), whereas right ventricular work index was Cholesterol: Comparative Study of Two Dissimilar normal in Group I and reduced in Group II (- 33 per Groups of Female Subjects. RAY H. ROSENMAN,* cent, p < 0.02). Coronary blood flow and cardiac oxy- San Francisco, Calif. gen usage per 100 g of LV per minute were not sig- nificantly different in the two groups nor was coronary Comparative studies of possible stressful occupational vascular resistance. However, as compared to normal, influences oni serum cholesterol levels often have failed coronary vascular resistance was increased in both to eliminate certain other stressful environmental factors groups. The data indicate that in Smithwick grades 1 or have compared groups differing so widely in their and 2 the primary abnormality appears to be elevated diet and in ethnic origin as to make conclusions impossi- peripheral vascular resistance. In grades 3 and 4 vascu- ble. Therefore two groups of 30 to 59 year old California lar resistance is still more elevated and cardiac index women were randomly selected for study on the basis reduced. AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 1023

Foci of Polyoma Virus Infection in House Mice. WAL- sectioned sprue and control biopsies. There was unani- LACE P. ROWE * and ROBERT J. HUEBNER, Bethesda, mous agreement that the characteristic sprue lesion de- Md. veloped in the vicinity of wheat instillation where the mucosa had been normal previously. Damage Infection with mouse polyoma virus is prevalent in produced certain laboratory mouse infection trans- distal to wheat instillation progressively diminished; the colonies, being lesion remained mitted primarily by urine and saliva of mice infected as proximal essentially unchanged. This sucklings. To further clarify the of this demonstration of intestinal mucosal damage by wheat epidemiology implicates as the to anatomic tumor virus, serological and virus isolation studies were gluten exposure precursor made on mice trapped in various parts of New York changes in idiopathic sprue. City. Infected house mice were found, and the infections were sharply localized to mice trapped in certain blocks Purification and Properties of a Pituitary Component in the Harlem district. There was a distinct tendency Which Produces Lipemia in the Rabbit. DANIEL for infections to be localized to certain apartment houses, RUDMAN, MARIO DI GIROLAMO, FLOYD SEIDMAN and and even to individual floors. In some foci, 40 per cent MARIA B. REID,- New York, N. Y. (introduced by of mice were serologically positive. Virus was recovered David Seegal). from organs of 25 per cent of antibody-positive mice, and Our previous studies showed that a single subcutaneous occasionally from excreta and floor sweepings. No virus injection of an aqueous extract of anterior pituitary was recovered from mites or roaches in infected apart- glands produces lipemia in the rabbit. Comparable lipe- ments. Humans, cats and rats from the infected areas mia is not produced by purified preparations of any of the did not have antibody to polyoma virus. It is hypothe- recognized anterior pituitary hormones. Fractionation of sized that the infection is maintained by urinary con- the crude pituitary extract has now yielded a fraction tamination of communal nesting areas. (labeled "Fraction, H") which produces lipemia in the rabbit. Bioassays have shown that Fraction H contains The Effect of Wheat Instillation into the Proximal Ileum 0.07 per cent thyroid-stimulating hormone, 0.002 per cent of Patients wcith Idiopathic Sprue. CYRUS E. RUBIN, oxytocin, and no detectable amounts of the 6 other pitu- LLOYD L. BRANDBORG, ARNOLD L. FLICK, CHERILL itary hormones. PARMENTIER, PATRICIA PHELPS and SALLY VAN NIEL, The injection of as little as 0.25 mg of Fraction H it) Seattle, Wash. (introduced by Clement A. Finch). the rabbit causes an increase in serum free fatty acid (FFA) concentration from 200 to 2,000 ,tEq per L with- The characteristic mucosal abnormality in idiopathic in 1 hour. This effect is not suppressed by the intra- sprue is obvious in the duodenum and proximal jejunum, venous infusion of glucose. The period of elevation of whereas normal areas are observed distally. Wheat or serum FFA concentration varies from 2 to 24 hours, rye gluten exacerbates this illness, but relationships be- depending on the dosage of Fraction H. The rapid tween gluten and the mucosal lesion are poorly under- elevation of serum FFA level is followed within 12 hours stood. The hypothesis that gluten is digested and ab- by a two- to fivefold increase in serum total lipid con- sorbed proximally might explain how the distal intestine centration. In a typical experiment, serum cholesterol is spared. This study was undertaken to determine the increased from 42 to 103 mg per 100 ml, serum phos- effect of exposing the normal distal intestine in sprue to pholipid from 65 to 189 mg per 100 ml, and serum wheat gluten. triglyceride from 35 to 1,270 mg per 100 ml. Two sprue patients in remission continued their gluten- The following properties of Fraction H have been free diets while wheat flour was instilled into their determined: nondialyzability, precipitation by 40 per cent proximal ileum 3 times daily for 9 days. Studies before (NH4)2 SO4 or 5 per cent CHCl8 COOH, solubility in and after wheat infusion included: clinical observations, the presence of high concentrations of ethanol or acetone, chemical fat balance, B12Co'° absorption and roentgeno- wreak affinity for anion and cation exchange resins, sta- graphically positioned peroral mucosal biopsies at 18 bility of the biological activity to partial hydrolysis by levels from terminal ileum to pylorus. Suitable control HCl or pepsin, and disappearance of the biological activity data were obtained. After the first day of ileal gluten, following partial hydrolysis by NaOH or trypsin. In- both sprue patients became irritable and anorexic; by the activation by trypsin proves the presence of peptide bonds third day they were obviously ill, and during succeeding in the hormone molecule. days experienced flatulence, distention, cramping and diarrhea. Fat absorption decreased from 92 to 65 per cent in the first patient and from 80 to 10 per cent in the Inferior Vena Cava Blockade: A Method for Studying second. B12Co'° absorption (Schilling) was unchanged in Circuilatory Dynamics. MARVIN A. SACKNER, TRUMAN the first, and in the second decreased from normal (30 G. SCHNABEL, JR.,* MARY B. ALLAN and DAVID H. per cent) to almost nothing (4 per cent). Wheat instilla- LEWIS, Philadelphia, Pa. tion in two normals did not affect fat or B22Co'° absorp- Reduction in venous return by vasodilators, venesec- tion; they felt well despite moderate flatulence and tion, tilting, or tourniquets has been utilized to investigate diarrhea. cardiovascular function in man. Obstruction of the vena Eight observers coded and blindly evaluated 116 serially cava (Farber and Eichna) offers certain advantages over 1024 PROCEEDINGS OF THE FIFTY-SECOND ANN\TUAL MEETING

these methods: it is mechanical, its application is not de- syndrome studied by us have had 45 and 47 chromosomes, tected by the subject, and it is associated with minimal respectively, as described by other workers. changes in pulse rate and oxygen consumption. In 7 2) Heteropycnosis and allocycly of the X-chromosome, normals and 13 patients with heart disease, a balloon not previously described, have been shown to exist in catheter was inflated in the inferior vena cava just below human subjects, most clearly in those with Turner's syn- the hepatic vein. Inflation was done gradually (10 to 20 drome. In some subjects with ovarian dysgenesis, espe- minutes) and usually until the balloon was not freely cially those with a chromosomal population of 46, differ- movable. Pressure measurements and Fick outputs were ences in size, staining intensity and pronounced allocycly done before, during, and after caval obstruction. In in the replication rate of the X-chromosome have become normals, maximum inflation produced an average rise of apparent. 14 mm Hg in caval pressure. Pulmonary artery pres- 3) In one of two subjects with precocious puberty sure (PAP) and cardiac index fell an average of 30 per (menses and breast development since birth) an abnormal cent. Right ventricular filling pressure (RVd) fell an number of chromosomes (47-48) has been demonstrated. average of 3 mm Hg, with a 50 per cent decline in right A normal diploid number of 46 with an XY constitution ventricular stroke work index (RVSWI). Both sys- has been demonstrated in a subject with intersex. temic (SVR) and pulmonary resistance (PVR) rose slightly. Fifteen minutes after complete deflation RVd Role of Immunmity in the Genesis of Experimental Chronic had risen to 2 mm Hg above control with a return of Pyelontephritis. JAY P. SANFORD, BETTY W. HUNTER, RVSWI to only 80 per cent of control. In patients with ROBERT E. WINDOM and JANET C. HOGAN, Dallas, Tex. heart disease, inflation and deflation caused similar altera- (introduced by Ralph Tompsett). tions in these parameters. In 5 of this group, LVSWI and LVd changed in the same manner as RVSWI and Factors influencing persistence of pyelonephritis have RVd. Elevated PVR secondary to mitral stenosis de- been extensively investigated. However, immune mech- creased with inflation. In 2 patients wNith atrial septal anisms which may determine healing have not been defects, RVd and PAP fell as in the others. However, defined. Immunity in pyelonephritis is of critical impor- pulmonary blood flow more than doubled and PVR fell tance, inasmuch as it may be a major determinant of the considerably. As a result, these patients showed an in- repetitive attacks which influence clinical progression. creased RVSWI in the face of a decreased RVd. These studies were undertaken to ascertain the role of Inferior vena cava blockade offers a safe and promis- immunity in establishment of experimental chronic pye- ing method for studying the determinants of cardiac per- lonephritis. Pyelonephritis was produced in rats by intra- formance and the pathophysiology of cardiac shunts and vascular injections of E. coli (0-111 : B-4) and renal pulmonary vascular reactivity. massage. While this procedure uniformly resulted in acute pyelonephritis demonstrable 2 weeks following a Chromosome Constitution in Human Gonadal Disorders. single injection (> 100,000 colonies per g of kidney), AVERY A. SANDBERG,* THEODORE S. HAUSCHKA, EDWIN kidneys of rats subjected to repetitive injections showed GORDY and GEORGE F. KOEPF, Buffalo, N. Y. no evidence of active infection 2 weeks following the fourth injection (< 1000 colonies per g). Such rats had The chromosomal patterns (number, morphology and antibodies against the 0 antigen (1: 256 to 1: 4096). frequency of chromosomal abnormalities) have been ex- These observations suggested that immunity resulting amined in tissue obtained from bone marrow aspirations from acute pyelonephritis protected against reinfection by of a large group of patients with gonadal disorders, espe- the original organism. To test this hypothesis, rats were cially in those in whom the somatic sex chromatin (as actively immunized by 1) producing acute pyelonephritis, determined from buccal mucosa and blood smears) did 2) i.v. injection of viable E. coli 0-111: B-4 without renal not coincide with the "apparent" external sexual charac- massage, 3) injection of 0-111 antigen in Freund's ad- teristics of these individuals. The important findings juvant and were passively immunized with rabbit E. coli would appear to be: 0-111 antiserum. Antibodies against the 0-111 antigen 1) Subjects with a negative sex chromatin pattern in conferred virtually complete resistance to induction of the buccal mucosa smear and with ovarian dysgenesis do homotypic E. coli pyelonephritis though resistance was not not necessarily have 45 chromosomes (XO) as reported conferred against infection with Klebsiella type C and by others. We have shown that in two subjects with the three heterotypic strains of E. coli. classical syndrome of ovarian dysgenesis the chromosomal In contrast, induction of acute pyelonephritis with a constitution is about half 46 and half 45, the former hav- strain of Klebsiella failed to evoke vigorous antibody re- ing two X-chromosomes. In one case of Turner's syn- sponses. The pyelonephritic process in these animals was drome the entire marrow cell population had the usual more persistent ( > 2000 colonies per g at 10 weeks) than diploid number of 46, with two X-chromosomes. In two subjects with Klinefelter's syndrome the preponderant that observed with strains of E. coli which evoked vigor- number of metaphases contained 46, with only one X- ous immune responses. chromosome being present (instead of the expected XXY In summary, the importance of immunity as a deter- condition). It should be pointed out, however, that 8 minant in experimental pyelonephritis has been shown subjects with ovarian dysgenesis and 6 with Klinefelter's both by prevention of acute hematogenous pyelonephritis AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 1025 in immune rats and by persistence of infection in rats not cholesterol levels as measured by the standard deviation eliciting vigorous immune responses. and coefficient of variation had decreased to one-third control values. The time required for the changes to The Effect of Changes in Atrial Systole on the Relation appear varied with the amount of DNA injected. The Between Mean Atrial Pressure and Stroke Work. animals receiving 2.5 mg changed inside of 2 months, STANLEY J. SARNOFF,* JERE H. MITCHELL and JOSEPH those receiving 500 ug in 5 months, and those receiving P. GILMORE, Bethesda, Md. 50,ug required 10 months. There was no return to control Experiments were designed to further clarify the rela- levels at any time after the serum cholesterol had declined tion between mean atrial pressure and stroke work. To significantly. The serum phospholipid and neutral fat this end, the relation between mean atrial pressure and showed similar changes, paralleling the cholesterol. ventricular end diastolic pressure was studied at constant A single injection, therefore, of minute quantities of heart rate. Continuous measurements were made of pure, undenatured heterologous DNA decreased the serum venous, right and left atrial, pulmonary artery, left ven- cholesterol and maintained a significantly lowered serum tricular and aortic pressures, cardiac output and heart cholesterol for at least 1.5 years in the rabbit. Con- rate, before and during stimulation of cardiac sympathetic sidering the known properties of biologically active DNA, and parasympathetic nerves. It was observed that: 1) the prolonged alteration may be a permanent effect. sympathetic stimulation augments and vagal stimulation diminishes the vigor of atrial systole; 2) at any given Vitamin D and the Active Transport of Calcium. DAVID stroke volume, the level of mean atrial pressure in rela- SCHACHTER, EUGENE B. DOWDLE, HARRIS SCHENKER tion to ventricular end diastolic pressure and ventricular and DANIEL V. KIMBERG, New York, N. Y. (introduced segment length is lowered by sympathetic stimulation and by John V. Taggart). elevated by vagal stimulation; 3) the higher the stroke The small intestine of several mammalian species can volume, the greater were the changes that could be in- transport calcium against concentration gradients, from duced by sympathetic and vagal stimulation. It was also fluid bathing the mucosal surface to fluid bathing the found that high heart rates, per se, elevated mean atrial serosa. The mechanism was studied with everted gut- pressure in relation to ventricular end diastolic pressure sacs and with slices of intestine incubated in vitro. The as well as to stroke work and stroke volume. These data active transfer is relatively specific for Ca++, is limited in show that the relation between mean atrial pressure and capacity, dependent on oxidative phosphorylation, and in stroke work is determined by the performance character- several species it is maximally active in the proximal istics of the atrium as well as the ventricle, whereas the duodenum. relation between ventricular end diastolic pressure and Adaptive changes in the active transport appear to ex- stroke work is determined only by the performance plain the facultative nature of calcium absorption in vivo. characteristics of the ventricle. Simultaneously observed The active transfer is maximal in younger, growing rats changes in venous pressure will be discussed. and decreases in older rats. Intestine from pregnant rats transfers calcium more readily than that from nonpreg- Quantitative Studies of a Prolonged Alteration of Serum nant controls. Maintenance on a low calcium diet en- Cholesterol Induced by Desoxyribonucleic Acid. J. hances the active transport. PHILIP SAVITSKY, New York, N. Y. (introduced by After 4 to 6 weeks on a vitamin D-free diet the active Robert Bloch). transport is severely impaired, but can be restored by It has been previously reported that a single injection feeding rats vitamin D or AT-10, or by ultraviolet irradi- of 1 mg of purified, undenatured desoxyribonucleic acids ation of the animal. Restoration of the mechanism (DNA) produced a significant decline in the serum lipids occurs in 1 to 4 hours following 50,000 IU of calciferol lasting at least 6 months. The lowered mean serum orally, or in 48 hours following 1 to 20 IU. The effect cholesterol reflected the decline (50 to 70 per cent) in of vitamin D is maximal in respiring, proximal duodenum. cholesterol levels of those animals with high normal The vitamin increases the maximal transport capacity for values. DNA preparations from homologous and heter- calcium. Thus, its principal action appears to be on the ologous mammalian tissues were equally effective. The active transport mechanism rather than on the permea- present study describes the effect in rabbits over 1.5 years bility to calcium of the mucosa. of a single injection of varying amounts of heterologous DNA. Groups of 10 normal rabbits received a single Lesions in Rats Caused by Serotonin and Histamine Be- injection intravenously of heterologous DNA in the fol- fore and After Amine Oxidase Inactivation and lowing amounts: 50 ,ug, 200 jug, 500 ,ug, 1 mg and 2.5 mg. Serotonin Inhibition. ARTHUR L. SCHERBEL, Roy L. Serum cholesterol determinations were made prior to McKirTRICK and WILLIAM A. HAWK, Cleveland, Ohio the injections and at monthly intervals thereafter. DNA- (introduced by A. Carlton Ernstene). injected and saline-injected control groups were indis- Serotonin and histamine possess multiple pharmaco- tinguishable in nutrition, weight gain and general health. logical actions. They are believed necessary for central At the end of 1.5 years, the mean serum cholesterol of nervous system function and may produce profound al- each DNA-injected group was significantly (30 to 40 terations in vascular tone and permeability. Their effect per cent) below control levels. The variation of the on connective tissue has not been completely defined. 1026 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING

Three hundred ten rats were studied from 1 to 4 weeks able and inconsistent. Total lipids, optical density and while serotonin or histamine, 1 to 30 mg daily, was ad- phospholipid phosphorus rose with lipemia. ministered subcutaneously. Connective tissue was studied The diluted thromboplastin generation test is a sensi- by the Ivalon sponge implantation technic. When sero- tive tool for study of the effects of lipemia on early phases tonin was administered, a wasting disease resembling the of coagulation. runting syndrome developed. Necropsies were performed weekly and the findings were compared with those in the The Iniduction of Heterologous Immutnity to Influenza by controls. Gross lesions included renal ischemia, gastric Aerosol Administration of Iniactivated Virus. JEROME ulcers, enterocolitis, focal areas of submucosal hemor- L. SCHULMAN and EDWIN D. KILBOURNE,* New York, rhage, cardiac dilatation, and splenic infarcts. Micro- N. Y. scopic lesions included renal tubular necrosis, myocarditis, Influenza viruses may be so treated by the application focal areas of hepatic necrosis, and occasionally vasculitis. of heat or ultraviolet or ionizing radiation that their capac- None of these lesions appeared in histamine-treated or in ity to multiply is inactivated yet their ability to inhibit control animals. Serotonin increased inflammation in the multiplication of infective virus is preserved. The sponges but did not alter fibroplasia from that seen in con- cellular resistance induced by inactivated viruses (viral trols; heavily granulated mast cells were present. His- interference) has been demonstrated in in vitro cell cul- tamine significantly increased fibroplasia, but inflammation tures and in the allantoic sac of the chick embryo. Such was only slightly increased; mast cells were degranulated. topical immunity is heterologous in that antigenically dis- Monoamine oxidase inhibitors stimulated fibroplasia in similar influenza viruses may interfere with the mul- the sponges. The effect of serotonin was greatly potenti- tiplication of one another. ated when animals were pretreated with an amine oxidase The virtual restriction of influenza virus infection to inhibitor and it was completely blocked when animals cells lining the mammalian respiratory tract presents a were pretreated with a serotonin antagonist (1-methyl- situation peculiarly amenable to the induction of topical methergine-tartrate, coded UML 491). immunity with nonreplicating virus. When CFW mice It is concluded that inflammation, alterations in fibro- received by aerosol an estimated 250 H A units of con- plasia and in mast-cell granulation, localized vasospasm centrated, partially purified suspensions of influenza B and vasodilatation; and increased vascular permeability (Lee) or influenza A (CAM) viruses inactivated with resulting in ulceration may occur from the administra- ultraviolet light, immunity to heterologous viral challenge tion of these biogenic amines. The lesions are increased 24 hours later (also by aerosol) was demonstrated. This when an amine oxidase inhibitor is administered simul- "immunity" results in 1 to 4 log reductions in pulmonary taneously, and conversely, they are prevented if a sero- virus (as determined by infectivity titrations of in- tonin antagonist is administered simultaneously. dividual mouse lungs) and in the prevention or reduction of pulmonary lesions. The concentrations of inactivated A SenGsitive Test for the Detection of Coagulation Ab- virus employed have induced no manifest harmful effects. normalities in Hyperlipemia. JOHANN SCHMIDr and The use of antigenically heterologous viruses in these GEORGE 0. CLIFFORD, Detroit, Mich. (introduced by experiments precludes the participation of specific anti- Richard J. Bing). body in the immunity induced. The relative nonspeci- ficity of the immunity of "interference" has special potential A study was done to investigate the mechanism of co- significance in influenza because strain-specific immunity agulation abnormalities possibly induced by lipemia. The may be circumvented by antigenic change in this highly thromboplastin generation test, modified by dilution of mutagenic virus. Furthermore, interference which modi- absorbed plasma 1: 10 and serum 1: 40, exhibits markedly fies but does not completely inhibit infection may result accelerated evolution of thromboplastin when lipemia is not only in immediate nonspecific resistance, but also in present. Dilution weakens the system and increases its lasting strain-specific immunity following the challenge sensitivity. infection so modified. Ten normal subj ects were studied fasting and at the peak of lipemia following 70 g of butter. Four hyper- Rclation of Hormone-Receptor Bonding and Antidiuretic lipemic patients (1 diabetic, 1 biliary cirrhotic, 2 essen- Actiont of Vasopressin. IRVING L. SCHWARTZ,* HOWARD tial) were studied fasting. At the height of lipemia in RASMUSSEN, MARY ANNE SCHOESSLER, CONRAD T. 0. normals, the diluted thromboplastin generation test con- FONG and LAWRENCE SILVER, Upton, N. Y. sistently revealed 50 to 100 per cent increase of thrombo- plastic activity during the first 5 minutes of incubation, An in vitro system has been used to study some aspects test was of the interaction of an antidiuretic hormone, arginine while the standard thromboplastin generation vasopressin, and a target organ, the toad bladder. only slightly accelerated. In the hyperlipemic patients, Bladders from Bufo marinus were tied as sacs onto glass activity of the diluted thromboplastin generation test was tubes and filled from within with Ringer's solution diluted proportional to elevation of serum lipids. Shortening of with water or concentrated above isotonicity with manni- the Stypven time was the most constant additional finding, tol. Then the assembly was placed in a bath of isotonic with platelet-poor lipemic plasma as active as fasting Ringer's solution and weighed periodically. Net water platelet-rich plasma. Other coagulation tests were vari- transfer assumed the direction of the osmotic gradient and AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 1027 increased 20- to 30-fold above control values when neuro- has certain advantages. Since no blood priming is re- hypophyseal hormones were added to the outside bath. quired, blood consumption is greatly reduced. Regional No difference was encountered in potency or in other heparinization reduces the danger of hemorrhage. Con- parameters of hormone action when the effect of arginine tinuous adjustment of electrolyte balance prevents the vasopressin (AVP) was compared with that of tritium- rapid and often dangerous shifts that can take place with labeled AVP (specific activity, 400 Ac per mg). intermittent dialysis. Continuous nitrogen removal pre- When the bladders were pretreated with reagents which vents recurrences of uremia even in highly catabolic bind SH groups (N-ethylmaleimide, 10' M; p-chloro- patients. mercuribenzoate, 10' M; CH8HgBr, 10' M), the action of AVP was completely inhibited, although a variety of The Metabolism of Variously C"-Labeled Glucose and an nonthiol-binding metabolic inhibitors did not block hor- Estimation of the Extent of Glucose Metabolism by mone action. In other experiments in which bladders the Hexosemonophosphate Shunt Pathway in Man. were exposed to H3-AVP and freed of electrovalently- STANTON SEGAL, MONES BERMAN and ALBERTA BLAIR, bonded radioactivity, 60 to 90 per cent of the hormone Bethesda, Md. (introduced by Donald S. Fredrickson). appeared to be attached to the bladders through disulphide Previous studies of glucose metabolism in man have linkage as determined by the removal of radioactivity employed uniformly C"-labeled glucose. Because the in- under mild conditions specific for the reduction of di- dividual carbon atoms of glucose have different metabolic sulphide bonds (L-cysteine, 10' M, pH 8). However, fates in the known pathways of glucose catabolism, fur- pretreatment with SH binding reagents prior to exposure ther information may be obtained by studying the meta- to H3-AVP greatly decreased the amount of hormone bolic fate of C' derived from glucose labeled in a single attached to the bladder through -S-S- linkage. carbon atom. These findings, in conj unction with concordant data Glucose-1-C" and glucose-6-C" were administered i.v. reported previously for the rat kidney, support the hy- to 4 normal subjects. In 2 of these subjects, experi- pothesis that the mechanism of action of autidiuretic ments with glucose-2-C" and glucose-U-C" were also hormone on its receptor involves a thiol-disulphide inter- performed. Expired C"02 was collected for 5 hours. change reaction. Marked differences have been observed in the C"02 ex- cretion curves derived from metabolism of each of the Continuous Hemodialysis as a Method of Preventing sugars. A characteristic time lag was seen for C"02 Uremia in Acute Renal Failure. BELDING H. SCRIBNER,* excretion from glucose-6-C", and the C" yield was RACHIT BURi and JOHN E. Z. CANER, Seattle, Wash. smaller than from glucose-i-C'. From blood collected A number of investigators now believe that critically at intervals up to 3.5 hours glucose was isolated as ill patients who develop acute renal failure should not be gluconate from 2 subj ects who received glucose-1 and allowed to develop the uremic syndrome. Preventing the 6-C" and degraded so that C-1 and C-6 were isolated uremic syndrome often requires frequent, even daily, individually. The turnover rate of these carbon atoms hemodialyses, and this large task led us to attempt con- was identical. Moreover, up to 3 hours after injection tinuous hemodialysis. To date 14 patients have been little randomization of C" in blood glucose occurred. treated by continuous hemodialysis for a total of 48 Examination of C" incorporation into plasma lipids re- patient-days. One patient was treated 14 days. A vealed 40 per cent greater incorporation from glucose-6 Skeggs-Leonards dialyzer with 6 layers arranged in than from glucose--C"4. parallel was used because of its low resistance and ade- The C"'02 excretion and lipid C" data indicate the quate clearance. Its low volume obviated priming with operation of the hexosemonophosphate shunt pathway in blood. The external circuit was cooled to near O° C to man. A biological model system has been constructed minimize clotting, platelet consumption, and bacterial based on a three-compartment glucose and a two-com- growth. Preliminary experience demonstrated that a partment bicarbonate system as well as on the reactions blood flow of only 30 ml per minute permitted removal of of the glucose pathways. Kinetic solution of the model 15 to 30 g of nitrogen per 24 hours with the BUN stabi- has revealed that about 8 per cent of glucose is catabo- lized at or below 100 mg per 100 ml. All fluid and elec- lized via the shunt pathway. Glucose metabolism for trolyte imbalances were readily corrected. At this low various endocrine states is now under study employing blood flow, a constant infusion of heparin into the arterial the above methods of analysis. cannula at about 6 mg per hour usually prevented clot- ting in the extracorporeal circuit while producing little, Effects of Glucose, Sulfonylureas and Other Hypogly- if any, prolongation of the patient's clotting time. No cemic Agents on Insulin Activity in Pancreatic Venous blood pump was used. The 300 L dialysate tank was Blood. HOLBROOKE S. SELTZER and WALTER L. SMITH, changed no more often than once a day. These features Dallas, Tex. (introduced by Ben Friedman). resulted in a simplified technique that was monitored by This study compared respective insulin-secreting poten- the special nurse on the case. No serious complications cies of glucose and sulfonylureas, and investigated pos- attributable to the procedure have been encountered. sible insulinogenesis by other hypoglycemia-inducing Continuous hemodialysis appears to be a safe and agents. Phlorizinized dogs were anesthetized, laparot- practical alternative to repeated intermittent dialysis and omized and heparinized. Pancreatico-duodenal vein can- t028 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING

nulation enabled continuous collection of effluent from 178/103, 183/105, and 187/106 for the three tests, respec- the right pancreatic limb after acute injection of test tively, in contrast to 164/101, 172/105, and 178/108 mm agents by femoral vein. Insulin activity of four suc- Hg in the untreated group. Responses of 33/15, 40/29, cessive 10 minute aliquots (baseline, A, B, C) was de- and 26/7, on1 the three tests, respectively, in the treated termined by Vallance-Owen's method (rat diaphragm), patients, contrasted with responses of 27/13, 32/26, and and mean flow rates were expressed in microunits ef- 17/6 mm Hg in the untreated group. Apparent dif- fective insulin concentration per minute. ferences were statistically significant only between initial Control substances did not increase pancreatic venous systolic pressures in Test 1 (p=0.05). insulin activity. Saline: baseline =42; postinjection = 0. Two explanations for failure of drugs to depress re- Sulfadiazine (50 mg per kg): baseline = 27; A = 29; sponses are suggested: 1) dose was insufficient and the B = 0. Glucose (15 g) elicited immediate, maximal, apparently satisfactory management resulted primarily sustained outpouring of insulin activity: baseline = 59; from placebo effects; 2) during chronic adminstration, A= 3,530 (p < 0.001); B = > 5,000 (p < 0.001); C= the drugs, by sympatholytic effects, may enhance re- > 5,000 (p < 0.001). sponsiveness to humoral components of the pressor re- Sulfonylureas increased secretory rate promptly but sponse, as acute studies in animals and man suggest. To only transiently. Tolbutamide (50 mg per kg): base- further elucidate these observations, paired studies in line=44; A=785 (p<0.02); B=713 (p<0.05); C= the same patient before and during treatment are indi- 68. Chlorpropamide (50 mg per kg): baseline = 66; cated. A=988 (p<0.02); B=1,045 (p<0.025); C=654 (p< 0.05). Metahexamide (10 mg per kg): baseline = 84; Type 2 Poliovirus Infection and Disease in Panamiia City. A = 937 (p < 0.01); B = 1,350 (p < 0.02); C = 282 (p < ALEXIS I. SHELOKOV, JOHN CRAIGHEAD and JACOB 0.05). BRODY, Balboa Heights, Canal Zone (introduced by Pancreatic venous insulin activity remained unchanged Joseph E. Smadel). after injection of sodium salicylate (50 mg per kg); indole-3-acetic acid (50 mg per kg); L-leucine (50 mg Outbreaks of paralytic poliomyelitis caused by Type 2 per kg); and "Tris" buffer or THAM (0.3 M). strains are uncommon, but such occurred in Panama City The data indicate that 1) both glucose and sulfonyl- in late 1959. Data from an enterovirus survey on chil- ureas cause prompt insulin release; 2) the greater po- dren under 3 years of age in Panama City during the tency of hyperglycemia suggests that it may actually half year preceding the outbreak and following it were stimulate new insulin formation, while sulfonylureas may of particular interest. Thirty to 35 rectal swabs obtained only release preformed insulin; 3) salicylates, indole-3- from pediatric clinic patients every 2 weeks were tested acetic acid, L-leucine and "Tris" buffer produce hypo- for presence of poliovirus. glycemia via extrapancreatic mechanisms. Two of the clinic children had intestinal infection with Type 2 poliovirus, one in June and the other in Septem- Effect of Hypotensive Drugs on the Pressor Response to ber. Thus, only two polioviruses were found in the 292 Noxious Stimuli. ALVIN P. SHAPIRO, Pittsburgh, Pa. children examined during the 4 months. The number of (introduced by I. Arthur Mirsky). polio isolates, all Type 2, increased progressively, i.e., October, 2; November, 7; December, 8; then dropped to In spite of numerous clinical assays of hypotensive 4 in January. During the peak of the spread of virus in drugs in therapy, little attention has been given to the the community, about 11 per cent (8/71) of the children effect of their chronic administration in man on responsiv- examined were shedding Type 2. ity to pressor stimuli. Therefore, during studies of the Paralytic polio appeared in Panama City in October variables influencing the magnitude and quality of pressor with 1 case reported, in November there were 2 cases, in responses to different psychophysiological stimuli, changes December there were 7, and in January there was a drop in a group of 30 hypertensive patients receiving rau- to 4. Eleven of the 14 paralytic patients studied yielded wolfia, chlorothiazide, and mecamylamine in the usual Type 2 virus; no poliovirus was isolated from 3 cases. doses, singly or in combinations, were compared with The rough correlation found between the appearance of those in 40 untreated patients. Type 2 virus in the community, its rapid spread and de- An automatic indirect recorder which permits unat- cline, and the increase and decrease in paralytic disease tended determinations of blood pressure at 2-minute inter- in the inhabitants is in accord with other epidemiologic vals was used. Results of three tests, designed to be aspects of this disease, particularly the fact that paralysis simple, readily reproducible, and to contain minimal occurs in only a small proportion of infections. "doctor-patient interaction," were evaluated: 1) maxi- mum rise in blood pressure after the investigator en- A New Syndrome of Post-Transfusion Immunologic tered room of the resting patient and injected 10 ml of normal Purpura. N. RAPHAEL SHULMAN, RICHARD H. ASTER, saline intravenously; 2) rise during a modified ALFRED LEITNER and MERILYN C. HILLER, Bethesda, cold pressor test; 3) rise during performance of a diffi- Md. A. cult "color reading" task. Most subj ects received all (introduced by James Shannon). three tests. Two patients who developed fulminant thrombocyto- Pretest blood pressures in the treatment group were: penic purpura with abundant megakaryocytes 1 week after AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 1029

blood transfusion had a circulating antibody which re- jects. An additional asymptomatic carrier of the orotic acted with platelets of normal individuals to cause com- aciduric trait has been discovered in control studies. plement fixation, agglutination and inhibition of clot re- Preliminary studies indicate that the enzymatic defects traction. One patient was treated successfully with can also be demonstrated in saliva. Orotic aciduria can exchange transfusion, the other recovered spontaneously. be produced experimentally in animals with azauracil, Platelets obtained from both patients after recovery did which inhibits 05P-decarboxlase. Orotic aciduria repre- not react with the antibody of either patient, yet no sents a genetic block in 05P'ase and 05P-decarboxylase, blocking agent was detectable on their platelets. Rather which seems to be transmitted in the pattern of a Men- it was found by using platelets obtained from 40 relatives delian recessive trait. Which, if either, enzymatic defect and 90 other individuals that there are 2 antigenically has primacy has not been established. The finding of different platelet types unrelated to erythrocyte groups. an additional carrier in a small control study suggests The most common phenotype (genotypes P1APlA in that the trait may not be uncommon, but that the homo- approximately 74 per cent of the population and P1APlB zygous state is generally lethal. in 24 per cent) reacts with the antibody, and the rarer patients' phenotype (genotype PlBPlB in 2 per cent) Qualitative Differences in the Immune Response of In- does not. pI' is an autosomal recessive. Immunization fants and Adults Receiving Salmonella Vaccines. studies in animals suggest that P1BPlB platelets simply RICHARD T. SMITH,* DONALD V. EITZMAN and BILLI- lack the P1I antigen. EVELYN MILLER, Gainesville, Fla. The described syndrome is unrelated to classical idio- pathic thrombocytopenic purpura (ITP), for 5 consecu- Previous observations showed that neonatal infants tive cases of ITP tested had PI' platelets. Although without passively acquired agglutinins produce "H" approximately 2 per cent of all transfusions involve a agglutinins in high titer after immunization with Salmo- PlBPlB individual receiving PI' platelets, immunization nella vaccines. This study describes the characterization does not occur with the expected frequency. Moreover, of this agglutinin and comparison of its properties with one patient received 15 transfusions subsequent to the agglutinins which appear in the serum of older children initial immunization without stimulating further antibody and adults after a comparable immunological stimulus. formation, and in both patients recovery took place in Salmonella vaccines and diagnostic antigens were pre- the presence of a significant antibody titer. This sug- pared from strains having a single monophasic flagellar gests that the donor's blood which provokes the syndrome antigen. The antigens for immunization were selected as must be unique. It may contain an unusual amount or those for which no detectable flagellar or somatic ag- form of PI' antigen which both immunizes a PlBPl' glutinins were present in a preimmunization specimen. recipient and coats PlBPl' platelets to render them sus- Serial bleedings were examined by tube agglutination, ceptible to the antibody. and for specificity, heat-stability, electrophoretic mobil- ity, sedimentation characteristics, and the effect of thiol The Enzymatic Defect reagents on the agglutinin. of Orotic Aciduria. LLOYD H. The infants examined were found to produce for SMITH, JR. and CHARLES M. HUGULEY, JR., Boston, the Mass. first 2 to 4 months of life only "H" agglutinins, which and Atlanta, Ga. (introduced by Anne P. were in each instance heat-stable, highly specific, 'yi, 19 Forbes). to 20 S globulins. The agglutinin activity was intact The propositus and thus far the only reported patient after treatment with 2-mercaptethanol, but in smaller with orotic aciduria was a child with refractory megalo- active fragments (approximately 7 S). In older infants blastic anemia associated with large amounts of orotic this neonatal type of antibody was found during the first acid in the urine. A hematologic remission and reduc- days of the response, but soon thereafter a 7 S, -y2, heat- tion in orotic acid excretion followed the administration stable globulin antibody-resisting thiol reagent also ap- of mixed pyrimidine nucleotides. It was suggested that peared. the patient had an enzymatic block in the further metabo- Young adults were found to produce early in the im- lism of orotic acid. The patient died several years ago mune response a yrL, heat-stable, macroglobulin, which of varicella. variably lost agglutinin activity when treated with thiol Assay procedures have been developed for orotidylic reagent. After 7 days agglutinins of lower sedimentation pyrophosphorylase (05P'ase) and orotidylic decarbox- coefficient, probably 7 S appeared. "Naturally" occur- ylase (05P-decarboxylase), sequential enzymes which ring Salmonella agglutinins in adults were found to be convert orotic acid to uridine-5'-phosphate. These en- of the thiol labile macroglobulin type, as has been pre- zymes can be measured in circulating erythrocytes and viously reported. leukocytes, but are absent from serum. A heterozygous These data suggest that the neonatal infant differs defect in both 05P'ase and 05P-decarboxylase was qualitatively in his immune response to this stimulus from demonstrated in erythrocytes from both parents and two the mature individual. siblings of the propositus. A third sibling demonstrated no enzymatic abnormality. Levels of erythrocyte aspar- Reticuloendothelial Clearance of Blood Thromboplastin tate carbamyltransferase and dihydroorotase, enzymes in Rats. THEODORE H. SPAET,* HERBERT I. HOROWITZ prior to orotic acid formation, were normal in all sub- and DORarHEA ZUCKER-FRANKLIN, New York, N. Y. 1030 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING

The hypothesis has been proposed that blood clotting Esterase activity was significantly increased, however, in intermediates are cleared during in vivo circulation, and postepinephrine and post-ECS specimens when strep- that this clearance is a major factor in the preservation tokinase was added to the reaction mixture. Since strep- of blood fluidity. The present study concerns the re- tokinase alone lacks this activity, these results indicate moval of blood thromboplastin. that euglobulin following epinephrine and ECS contains Experimental animals were 250 g Sprague-Dawley rats, an increase in material activated by streptokinase, either and thromboplastic reagents used were of rat origin. proactivator or plasminogen. Further studies demon- Reagents were: rat blood thromboplastin (RBT), a strated that the material increased was not plasminogen. thromboplastin generation mixture prepared in the usual This material was assayed by conversion to plasmin and manner; and sedimented rat blood thromboplastin the activator used for this conversion removed either by (SRBT), similar to product II of Bergsagel and Hougie acid precipitation or heat. Esterase activity of the acti- except that activity was sedimented on crude "cephalin" vator-free plasmin was unchanged following epinephrine instead of platelets. RBT given into the jugular vein or or ECS. It was consistent regardless of the activator aorta caused a marked increase in the prothrombin time used or the method of its removal, and was regularly and drop in fibrinogen, but injection into the mesenteric less than that due to streptokinase when activator was vein had a greatly reduced effect. The protective effect not removed. These findings indicate that streptokinase- of mesenteric injection was partially reversed by previous induced esterase is due to the conversion of both pro- reticuloendothelial blockade with Pelikan Ink. Addition activator and plasminogen and that the former alone is of IP1`-labeled albumin to RBT showed that the injected increased by epinephrine and ECS. The findings also material was not grossly trapped in the liver following indicate the separate identities of these substances. The mesenteric injection. Similar results were obtained when value of an artificial substrate, without clot lysis, in the the RBT was prepared with crude "cephalin," aqueous measurement of these components of the fibrinolytic sys- platelet extract, or intact platelets. SRBT was labeled tem is demonstrated. with P"31, injected into the jugular vein, and radioactivity was determined in various organs. About 75 per cent Cholesterolemiia, Hypertension and Coronary Disease in was found in the liver. With crude "cephalin" lacking Negroes Compared with Whites. JEREMIAH STAMLER,* thromboplastic activity only about 50 per cent was found HOWARD A. LINDBERG, DAVID M. BERKSON, YOLANDA in the liver. Injection of SRBT during a carbon clear- HALL and WILDA A. MILLER, Chicago, Ill. ance produced significant depression of carbon removal; the effect of crude "cephalin" without thromboplastic ac- It has been clearly demonstrated that hypertension is tivity was considerably less. associated with a several-fold increase in the risk of The data indicate that blood thromboplastin as prepared coronary disease in middle-aged men. It has also been above is cleared from the circulation as a "foreign" unequivocally established that prevalence rates of hyper- particle, by the reticuloendothelial system. Although tension are grossly higher in Negroes than in whites in these findings are in conformity with the original hy- the United States. Nevertheless, available data indicate pothesis, it remains to be determined what relationship that the incidence of coronary heart disease is no greater, the laboratory thromboplastin has to coagulant material and may even be lower, in middle-aged Negro men than that develops in vivo under physiological conditions. in white men. This apparent paradox stimulated an epidemiologic investigation on patterns of cholesterolemia inl Induiced -another major variable associated with susceptibility Demonstration of Increased Proactivator to heart disease-in Negroes and whites aged BURTON D. coronary Fibrinolytic States. NORTON SPRITZ, 40 to 59 in Chicago. Population samples for study were COHEN and ALBERT L. RUBIN, New York, N. Y. (in- obtained from the labor force of a Chicago industrial troduced by Robert F. Watson). corporation (1,245 white males, 84 Negro males) and The present concept of the fibrinolytic system is that from a community survey in a residence area of lower plasminogen is converted to the fibrinolytic agent, plas- income Negroes. Data were obtained on such variables min, by a group of proteolytic substances (activators). as age, sex, race, height, weight, occupation, industry, Both plasniin and plasminogen activators possess lysine employment status, place of birth and time of migration ester-splitting activity. Streptokinase lacks this activity, North, if born in the South. Mean serum cholesterol and its capacity to activate plasminogen results from its level in the white males was 239 mg per 100 ml; in the interaction with an inactive precursor (proactivator) Negro men employed in the Chicago company, 227 mg producing an activator with esterase activity. It is con- per 100 ml; in the Negro men in the community, 207 troversial whether or not proactivator and plasminogen mg per 100 ml. The white men in different socioeco- are separate substances. nomic subgroups had generally similar mean serum Epinephrine and electroconvulsive shock (ECS) have cholesterol values; for the Negro males, serum cholesterol been shown to produce increased euglobulin fibrinolytic levels were lower in the unemployed than in the em- activity, resulting from an increase of plasminogen acti- ployed, lower in the "blue collar" than in the "white vation. Lysinemethyl esterase activity of the euglobulin collar" workers. These lower levels of serum cholesterol fraction was not increased by these procedures, although in middle-aged Negro men compared with those in xwhite such activity is characteristic of plasminogen activators. men may be significantly related to the comparative pat- AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 1031

terns of coronary disease incidence rates in the two 1958, 193, 34). One of each pair was incubated with races. 02 (370 C); the other was refrigerated at 20 C with N2. The medium was changed after 1 hour and incuba- The Effects of Acetylcholine on Pulmonary Circulation tion or refrigeration continued 3 hours longer in fresh and Alveolar Gas Exchange. C. ALPHEUS STANFIELD, medium. C", stable cholesterol contents of media and MILTON N. LURIA, JAMES K. FINLAYSON, FRANK W. fixed tissue were determined. In vitro, while mucosa LOVEJOY, JR. and PAUL N. Yu, Rochester, N. Y. (in- actively absorbed cholesterol from medium, there was troduced by Nolan L. Kaltreider). transfer of recently absorbed (in vivo) cholesterol to Pulmonary blood flow, pulmonary vascular pressures, medium from mucosa. Transfer from mucosa to medium arterial blood gases, ventilation and pulmonary diffusing averaged 3 per cent per hour for 370 C specimens; re- capacity for carbon monoxide were measured in 17 pa- frigerated anoxic specimens had less than half this rate. tients with rheumatic or congenital heart disease before Transfer rate during the 3 hour second period was nearly and during the continuous infusion of acetylcholine into as rapid as that during the first hour. In general, our the pulmonary artery at rates of 2 to 5 mg per minute. data concerning absorption confirm those of Treadwell The changes in oxygen consumption, pulmonary blood et al. Since segments could not transfer cholesterol flow, respiratory frequency and minute ventilation were through serosa, accumulation of sterol might favor secre- inconsistent during acetylcholine infusion. Pulmonary tion through mucosa. As an explanation desorption artery mean pressure declined significantly in 6 of 9 could not be excluded although it is unlikely in view of patients with elevated control values, although pulmonary simultaneous active absorption. Cholesterol secretion "capillary" mean pressure decreased in only 2 patients. by intestinal mucosa may be an important means by which No change in pulmonary vascular pressures was observed body cholesterol content is regulated. in patients with normal pulmonary hemodynamics. There was no significant change in pulmonary diffusing ca- Alterations of Cardiopulmonary Physiology Following pacity for carbon monoxide. Acute Experimental Pulmonary Embolism. MYRON A decline in arterial oxygen saturation which oc- STEIN, CLAUDE E. FORKNER, JR., E. MARTIN SPENCER, curred in 8 patients was not necessarily accompanied by EUGENE D. ROBIN and GEORGE S. KURLAND, Boston, a significant fall in pulmonary artery pressure, although Mass. and Pittsburgh, Pa. (introduced by A. Stone pulmonary vascular resistance usually decreased slightly. Freedberg). Inhalation of 100 per cent oxygen while acetylcholine in- Many discrepancies exist between the physiologic and fusion was continued resulted in an increment in arterial pathologic findings in acute experimental pulmonary em- oxygen content of more than 2 vol per cent in each of bolism. Using the technique of serum-induced the 8 patients. throm- bosis for the production of emboli, multiple parameters Our observations confirm the reports by other workers of pulmonary and cardiac function were measured be- that acetylcholine decreases pulmonary artery pressure fore and after pulmonary embolization in 32 anesthetized without appreciable change in the rate of blood flow in dogs. Massive embolization resulted in a some patients with prompt de- pulmonary hypertension. The exact crease in cardiac output and a fall in arterial 02 satura- mechanism for this reduction is not clear but most likely tion; arterial 02 content did not rise to control levels the drug causes direct vasodilatation of the pulmonary arterioles. after ventilation with 100 per cent 02 for 20 minutes. One to three hours after embolization, arterial con- The occurrence of arterial desaturation 02 during acetyl- tent following 100 per cent 02 for 20 minutes returned to choline infusion in a certain number of patients deserves control values. These physiologic alterations occurred special comment. Available evidence indicates that there only in animals in which is neither impairment of alveolar more than 50 per cent of the ventilation nor increase main lobar arteries were obstructed by emboli as in direct pulmonary arteriovenous shunts. deter- The most mined at necropsy. In 16 dogs, dissemination of similar likely explanation for the arterial desaturation is an in- total quantities of emboli to a single lobar artery or creased perfusion of blood through pre-existing hypo- ventilated areas. diffuse seeding to smaller peripheral pulmonary arteries of several lobes was not associated with a fall in arterial Secretion In of oxygen saturation or a decrease in cardiac output. In Vitro Recently Absorbed Cholesterol and both Absorption of Cholesterol In Vivo and In Vitro by the groups, acute pulmonary embolization resulted in Rat Small Intestine. MALCOLM M. STANLEY,* an increase in rate, depth and minute volume of ventila- C. ED- tion, a WIN BALL and KENNETH R. JAEGERs, Louisville, disproportionate increase in dead space ventilation, Ky. and an arterial-alveolar C02 tension gradient. These Fasting rats given free C"-cholesterol by stomach tube data, obtained with emboli of the animal's own blood sug- were killed after 4 hours. Each small intestine was di- gest that the physiologic abnormalities induced depend vided into 24 approximately equal segments. After not only on the quantity of emboli but also on the spe- eversion and washing, every third segment was fixed as cific pattern of the embolic distribution. The decreased a control and the others studied by Wilson's method (J. arterial 02 content breathing 100 per cent 02 iS consistent appl. Physiol. 1958, 12, 145) using medium containing with the development of a right to left shunt. Its rapid stable cholesterol (Treadwell, et al., Amer. J. Physiol. reversal reflects the anesthetized animal's capacity to 1032 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING compensate for an acutely-induced abnormality in pul- terol accumulated to a fairly constant level, accounting monary perfusion. for 20 to 25 per cent of the circulating sterols in the serum and 17 per cent of the red blood cell sterols. Four Hepatic and Systemic Blood Flow in Man during Intra- hours after the inj ection of 2-C14-mevalonic acid the venous Infusion of Ethanol. SAMUEL W. STEIN, specific radioactivity of the circulating 24-dehydrocholes- WALTER H. ABELMANN,* CHARLES S. LIEBER, CARROLL terol was greater than the specific radioactivity of the M. LEEVY and GILBERT R. CHERRICK, Boston, Mass. circulating cholesterol. This, and similar data from ani- mal studies, are compatible vith a precursor-product There is conflicting evidence concerning the effect of relationship. This conclusion is supported by direct ethanol on the hepatic circulation. In the present study demonstration of the rapid conversion of C14-labeled 24- hepatic blood flow and cardiac output were measured dehydrocholesterol to cholesterol after intravenous in- simultaneously in patients without liver disease. Cardiac jection into rats and after incubation with whole rat liver output was measured by indicator dilution, using Evans homogenates. The conversion is blocked by addition of blue. Hepatic flow was determined by the Fick principle MER-29 to the homogenate. It is concluded that the with constant infusion of indocyanine green. After one major site of action of MER-29 is in the final step in hour of saline, intravenous infusion was maintained un- cholesterol biosynthesis, namely, in the reduction of 24- changed for a second hour in some patients and was dehydrocholesterol to cholesterol. changed to ethanol in saline at the rate of 0.6 to 0.7 g The color yield of 24-dehydrocholesterol at 635 m, in per minute in a second group. the Liebermann-Burchard reaction is approximately 60 In 6 patients mean hepatic flow during the first hour per cent of the color yield of cholesterol. Consequently of saline infusion was 798 ml per minute per m2, with most standard methods give a falsely low value for the a range of 450 to 1,120 ml per minute per m2. Hepatic total serum sterol level. The importance of this in prop- flow averaged 24.1 per cent of cardiac output, with a erly evaluating clinical results with MER-29 will be range of 21.5 to 26.4 per cent. In 2 of the 3 patients in discussed in relation to a group of treated patients in whom ethanol was not employed, both hepatic and sys- whom levels of both cholesterol and 24-deyhdrocholes- temic flow in the second hour changed less than 10 per terol have been determined. cent from initial values, leaving the ratio of hepatic to In the third, hepatic flow in- systemic flow unaffected. KENNETH creased by 47 per cent and total systemic flow by 28 per Free and Bound Thyroxine of Serum. STERLING* and MILTON TABACHNICK, New York, cent. This was the only patient who demonstrated a change in the hepatic fraction of systemic flow, from N. Y. 26.4 to 30.2 per cent. Thyroxine in serum has been found to migrate with During infusion of ethanol in 3 patients mean hepatic albumin, a-globulin and prealbumin on electrophoresis, as flow increased by 43 per cent, to 1,127 ml per minute per reported by others. The existence of a small moiety of M2. Simultaneously determined cardiac output, however, "free" or unbound thyroxine has been previously postu- increased by 48 per cent. Thus, the fraction of total lated. In the present work, free thyroxine has been systemic flow going to the liver remained essentially un- demonstrated by dialysis through cellophane. Tracer changed at 23.8 per cent. amounts of I"1-labeled thyroxine added to serum yielded Under the conditions of study the hepatic fraction of radioactivity in the dialysate which was identified as cardiac output remained remarkably constant. Since in- thyroxine chromatographically. Equilibrium dialysis fusion of ethanol did not affect arterial blood pressure, studies suggested an association constant between serum the data indicate that it decreased both splanchnic albumin and thyroxine of the order of 105. Experiments and extrasplanchnic vascular resistance proportionately. with serum on both sides of the cellophane membrane also There was no evidence for a specific effect on the hepatic indicated transfer of free thyroxine, as reported by circulation. Christensen. The sera of thyrotoxic patients exchanged labeled thyroxine faster, while sera of myxedematous The Mechanism of Action of MER-29. DANIEL STEIN- patients exchanged the tracer more slowly than normal BERG, JOEL AvIGAN and EUGENE B. FEIGELSON, Be- sera. thesda, Md. (introduced by Jack Orloff). The uptake of added tracer thyroxine from serum by ion exchange resins varied with the state of thyroid func- MER-29 (1- [p-,8-diethylaminoethoxy) -phenyl] -1- (p- tion. With triiodothyronine as a tracer, the uptake by tolyl) -2- (p-chlorophenyl) ethanol) has been shown by IRA-400, formate cycle, was adapted as a diagnostic test Blohm and MacKenzie to be a potent inhibitor of choles- which could be used with sera despite iodine contamina- terol biosynthesis in animals and to lower serum and tion. tissue levels of cholesterol. Studies in this laboratory, both in animals and in man, show that administration of the drug is accompanied by accumulation in the serum Polycythemic Response to Erythropoietine Compared with and in the red blood cells of large amounts of 24-de- a Short-Termi Assay. FREDERICK STOHLMAN, JR.,* hydrocholesterol (desmosterol). In patients on a dosage GEORGE BRECHER and ARCHIE A. MACKINNEY, of 100 mg per day, it was found that 24-dehydrocholes- Bethesda, Md. AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 1033

The relationship between the dose of erythropoietine goiters, and 2 patients with adolescent goiter. The sec- producing a positive response in the fasted rat and that tions treated with the sera of the 5 normal persons were necessary to induce polycythemia in the intact animal was negative. Sections treated with the sera of 2 patients studied. Sources of erythropoietine were rats exposed with Hashimoto's disease obtained 7 months after thyroid- for 18 hours to a barometric pressure of 310 mm of Hg, ectomy were also negative, although a serum obtained rats with phenylhydrazine-induced anemia, and extracts from one of these preoperatively had been positive. Pre- of urine from a child with congenital erythrocytic hypo- cipitins (agar diffusion) to thyroid extract were found in plasia (J. B. urine). In a short-term assay (starved varying amounts in 9 out of 10 sera of patients with rat), the iron incorporation in the fasted recipients of thyroid disease and in none of 5 controls. The significance normal plasma was 5 to 8 per cent; that for recipients of of these results will be discussed. high altitude plasma, 21 per cent; phenylhydrazine plasma, 26 per cent; J. B. urine, 33 per cent. A one to three The Hyponatremia Associated zwith Marked Overhydra- dilution of high altitude plasma resulted in an iron in- tion and Water Intoxication. JAMES M. STORMONT and corporation of 10 per cent; a similar dilution of phenyl- CHRISTINE WATERHOUSE,* Rochester, N. Y. hydrazine plasma, 13 per cent; and of J. B. urine, 24 per cent. When these substances were administered to normal Although hyponatremia in patients without salt deple- rats over a 3 to 4 week period, the values for red cell tion is nearly always attributable to increased total body mass were: normal plasma, 4.9 cc; altitude plasma, 5.2 water and dilution, alterations of serum sodium concen- cc; phenylhydrazine plasma, 5.7 cc; J. B. urine, 6.9 cc. tration unaccounted for by water or cation balance have Injection of a one to three dilution of J. B. urine resulted been postulated in certain diseases. We have obtained in average red cell mass of 4.9 cc. Thus an amount of balance data on 2 patients (pneumonia and cerebral erythropoietine capable of increasing the output of red vascular lesion) during both the development of and cells in a starved animal failed to produce a sustained recovery from severe hyponatremia (103,108 mEq per L) increase in red cell mass in the normal animal. However, in the absence of salt depletion. Over 50 per cent of the if the amount of erythropoietine was substantially in- change in serum sodium concentration below 116 mEq per creased, polycythemia ensued. The failure of erythro- L could not be accounted for by fluid or electrolyte poietine in lower doses to produce polycythemia indicates balance, whereas above 116 mEq per L the rise in serum that some other regulant re-established homeostasis. This sodium was almost entirely accounted for by balance. in turn supports our concept of a dual regulation of To evaluate the role of overhydration in this process, erythropoiesis. fluid retention with hyponatremia and clinical water in- toxication was induced in 5 control patients receiving a Fluorescence Microscopy in Diseases of the Thyroid constant diet (16 to 86 mEq per day sodium) on a meta- Gland. IRWIN L. STOLOFF, Philadelphia, Pa. (intro- bolic ward. Vasopressin in oil was administered for 6 duced by W. Paul Havens, Jr.). to 12 days. Severe symptoms of water intoxication were correlated with serum sodium concentrations of 114 to Hemagglutinins, precipitins, and complement-fixing 100 mEq per L, negative sodium balance (11 to 109 mEq antibodies for antigens prepared from aqueous extractions per day), and negative potassium balance (20 to 31 mEq of normal human thyroid glands have been found in the per day after correction for nitrogen). In these patients sera of patients with a variety of conditions due to or over 50 per cent of the fall in serum sodium concentra- associated with injury of the thyroid. The possibility that tion below 114 mEq per L could not be accounted for the demonstration of affinity by fluorescence microscopy by cation or fluid balance. With serum sodium above 114 between normal human thyroid and substances found in mEq per L, potassium balance was less negative and the the sera of patients with disease of the thyroid might be a change in serum sodium concentration was largely ac- better indication of the role of autoimmunity in diseases of counted for by balance. Reciprocal changes were con- the thyroid prompted the use of this technique in the fol- sistently noted during recovery from severe hyponatremia. lowing studies. Both symptomatology and metabolic abnormalities were Sections (cryostat) of normal human thyroid were prevented by fluid restriction (1,200 to 1,500 ml per day), covered with the sera to be tested for 30 minutes; washed or reversed by intravenous 10 per cent mannitol or dis- for 10 minutes with phosphate buffer (pH 7.3); covered continuance of vasopressin. In 5 other patients with an with rabbit antihuman y-globulin labeled with fluorescein equivalent degree of vasopressin-induced overhydration, isothiocyanate for 30 minutes; and washed in running tap urine volume increased and fluid equilibrium was estab- water overnight. The sera of 5 normal persons and 10 lished with serum sodium concentrations above 114 mEq patients with a variety of diseases of the thyroid were per L. Symptomatology and metabolic abnormalities tested. Brilliant fluorescence was seen in the acinar cells were less marked or absent. and interstitial cells of the sections treated with the sera of 3 patients with Hashimoto's disease and 1 patient with These data are interpreted to indicate altered cellular Riedel's struma. Strikingly little fluorescence was seen metabolism due to overhydration which is etiologically on the colloid. Traces of fluorescence were seen in the related to the unusually low serum sodium concentration sections treated with the sera of 2 patients with idiopathic (below 114 mEq per L) and severe symptomatology in hypothyroidism without goiter, 2 patients with euthyroid certain patients. 1034 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING Antimetabolite-Metabolite Cancer Chemotherapy: Effects mercury but showed considerable variation between dif- of Intra-Arterial Methotrexate-Intramuscular Citro- ferent wedged segments. A serotonin-induced increase in vorunm Factor Therapy. ROBERT D. SULLIVAN, EDWARD pressure gradient measured at constant blood flow demon- MILLER, A. MICHAEL WOOD, P. P. CLIFFORD and JOHN strated the vascular components of the segment to be re- DUFF, Nairobi, Kenya and New York, N. Y. (intro- active. Provided that the effective perfusion pressure was duced by Joseph H. Burchenal). less than 50 mm of mercury, no evidence of damage in A method of therapy has been developed to enhance the the perfused area was noted. antitumor activity of chemotherapeutic compounds in the In several humans with pulmonary hypertension, no common forms of "solid" neoplastic disease. This consists reduction in the pressure gradient was associated with of the continuous 24 hour delivery of massive doses of an addition of acetylcholine to the infusion medium perfused antimetabolite into the arterial supply of tumors, to- at constant flow rate. gether with the intermittent, intramuscular administration This method offers a simple assessment of pressure-flow of the appropriate metabolite. Clinical studies of the relations in the pulmonary vascular bed and permits the effects of methotrexate-citrovorum factor (CF) therapy study of drug effects and physiological responses. are reported. Twenty-two patients with incurable epidermoid carci- Excretion of the Radioactive Catabolic End Products of noma or sarcoma of the head and neck or carcinoma of I"1-Albumin in Human Sweat. Y. TAKEDA, I. C. the bladder received intra-arterial methotrexate in mas- PLOUGH and E. B. REEVE, Denver, Colo. (introduced sive doses for as long as 15 days. Sufficient CF was by Gordon Meiklejohn). given by the intermittent, intramuscular route to prevent When satisfactory I'31-albumin is injected intravenously, severe systemic toxicity. The usual dosage schedule em- the rate of catabolism of plasma albumin can be deter- ployed was: methotrexate, 50 mg per 24 hours (if bi- mined either from the change in specific activity of the lateral catheterizations, 25 mg per 24 hours per side); plasma albumin over several weeks (Method 1) or from CF, 3 to 9 mg every 4 to 6 hours intramuscularly. The this and the concurrent excretion in the urine of the 1" usual total volume of the infusion was 2,000 cc per 24 split from the labeled albumin during catabolism (Method hours. Antitumor effects were noted as early as the 2). For accurate results with Method 2 the radioactivity third day of therapy. There was progressive decrease in released during catabolism either must be completely size of visible tumor masses, and as ulcerated oral excreted, or a known constant fraction must be excreted, growths regressed, their surfaces became cleaner and less in the urine. Ordinarily in subjects leading a sedentary necrotic. Therapy was continued until complete tumor life the two methods give results that show good agree- regression had occurred or until early signs of systemic ment and Method 2 then has a number of advantages. toxicity were noted. Partial or complete tumor regres- However, in subjects doing heavy work in hot environ- sion was noted in 11 patients. ments, the second method gave significantly lower values The enhancement of the antitumor effect of metho- than the first. A possible explanation was the excretion trexate appears to be related to its continuous arterial of catabolic radioactivity through other channels than the administration, possibly increasing the concentration of urine. When IP1`-iodide or IP1-albumin was given by "antimetabolic effect" in the tumor, and the use of CF, mouth to subjects made to sweat, I"-1 was excreted in the permitting the administration of massive doses of the anti- sweat, and when sweating was near maximal the rate of metabolite. excretion in the sweat approached one-fourth of the rate Results suggest that antimetabolite-metabolite chemo- of urinary excretion. Thus, in conditions when sweating therapy, using methotrexate and CF, may prove to be of is increased, measurements by the second method may practical value in the management of certain localized give underestimates of the catabolic rate of albumin. incurable cancer. Arylsulfatase Activity of Human Leukocytes: Distinctive Pressure-Flow Relations in a Portion of the Pulmonary Pattern in Eosinophils. KouICHI R. TANAKA, WILLIAM Vascular Bed, the Wedge Segment. H. J. C. SWAN N. VALENTINE* and ROBERT E. FREDRICKS, Los and ARTHUR H. KITCHIN, Rochester, Minn. (intro- Angeles, Calif. duced by Ward S. Fowler). Arylsulfatase activity of human leukocytes as deter- Cardiac catheters (no. 6L to 8L) were wedged in mined by using 2-hydroxy-5-nitrophenyl sulfate as sub- peripheral arteries of the lung. With a constant-rate strate has not previously been reported to our knowledge. syringe, autogenous blood or saline was infused at known Because of a report of markedly increased activity in the rates and the resulting pressure gardient across the urine of chronic granulocytic leukemia (CGL) patients "wedged segment" measured. Variations in perfusion and because sulfates are metabolically important, aryl- pressure at constant flow were noted, the gradient being sulfatase activity has been assayed (spectrophotometri- reduced during inspiration. In 27 experiments in 10 dogs cally, 0.006 M substrate concentration, pH 5.7) on sepa- the relation between pressure and flow was apparently rated leukocytes from over 400 patients with a variety of linear in the transmural pressure range of 5 to 60 mm diseases. Values in 16 normal subjects ranged from 10 to of mercury. The average of the mean flow rates in dogs 39 with a mean of 26 (expressed as milligrams of 4- perfused with blood was 0.43 ml per minute per mm of nitrocatechol liberated per 1010 WBC per hour at 370 C). AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 1035

There was no detectable activity in the erythrocytes by phate 18 per cent, and produced greater rise of pH and our method. CO2. The mean of 57 assays on 26 patients with CGL was The foregoing suggests, but does not prove, that Tris- 52.4. Thirteen polycythemia vera (PV) patients had a hypoglycemia is mediated by an islet and/or peripheral nean of 36.9, while the mean of 10 myeloid metaplasia insulin mechanism. The slight lowering of glucose in (MM) patients was 41.1. In contrast, 27 assays on 18 diabetics can be ascribed to alkalinity since NaHCO3 patients with chronic lymphocytic leukemia revealed a causes similar lowering. Furthermore, the hypoglycemia mean of 5.4. Results in acute and subacute leukemia were in normals is not pH-dependent since NaHCOa does not variable. The highest arylsulfatase values occurred in produce it in normals. That the hypoglycemia is partly those patients with eosinophilia of various etiologies. conditioned by pH is shown by its greater magnitude and Twenty-one patients with a mean eosinophil percentage of duration after alkaline in comparison to acidified Tris. 31.2 had a mean value of 129.5. There was fairly good correlation between the percentage of eosinophils and A Physiological and Biochemical Comparison of Crystal- degree of activity. line Dihydrotachysterol (AT-10) with Hytakerol. A The data indicate that the eosinophil is extremely active RAYMOND TEREPKA and PHILIP S. CHEN, JR., in arylsulfatase activity, whereas the basophil is much Rochester, N. Y. (introduced by Ralph F. Jacox). less active, but more so than the neutrophil. Lymphocytes and lymphoblasts show essentially no activity. The in- The "calcemic" action of calciferol and that of the creased mean values in CGL, PV, and MM appear to be structurally closely related dihydrotachysterol (AT-10) due mainly to the increased percentage of basophils and/ have been extensively utilized in the treatment of hy- or eosinophils. poparathyroidism. Although originally suggested as the The function of the eosinophil is virtually unknown. ideal substitute for parathyroid hormone, the commer- However, the finding of high arylsulfatase activity in cially available preparation of the latter compound (Hy- eosinophils suggests that they may play a role in sulfate takerol) has fallen into disfavor in recent years due to metabolism and may provide a clue as to their func- its greater cost and apparently variable potency. tion (s). In a comparative study of the relative actions of vitamin D, crystalline AT-10, and Hytakerol in rats, it On the Nature of the Hypoglycemic Effect of Tris- was noted that Hytakerol had significantly less calcium (hydroxymethyl) aminomethane (Tris Buffer). R. and phosphorus mobilizing activity when compared with TARAIL and T. E. BENNETT, Buffalo, N. Y. (introduced pure crystalline AT-10. Since the results suggested that by David K. Miller). Hytakerol did not contain the labeled quantity of AT-10, reverse-phase paper chromatographic analyses of the con- Explanation of our previous finding that alkaline Tris stituents of 3 different lots of Hytakerol were made. A (CH20H)3 - C-NH2 ("THAM") produces significant hy- compound exhibiting the typical AT-10 ultraviolet ab- poglycemia in man, massive quantities reducing blood sorption spectrum could be isolated. Surprisingly, glucose in dogs virtually to zero, may help solve theo- spectrophotometric assay of this compound, based on the retical and clinical problems of carbohydrate metabolism. molar absorbance of crystalline AT-10, revealed that Eight to 12 mmoles per kg of 0.3 M Tris or 0.3 M Hytakerol contained 2 to 3 times more "AT-10" than NaHCOs was given intravenously to unanesthetized, fast- the label indicated. In addition, 2 substances more polar ing normal and diabetic dogs in about 17 minutes. Mean than AT-10 and giving positive antimony trichloride tests maximum fall of peripheral plasma glucose concentration were found. One exhibited a strong ultraviolet absorp- wvas 39 per cent with persistent depression for 1 hour tion at 250 m, with lesser peaks at 283 and 291 my. The after alkaline Tris (R-NH2; pH 10) in 4 studies of other showed weak absorption at 236 and 286 m,i. normals. Arterialized blood pH rose by 0.27 with marked By running mixed paper chromatograms, the AT-10- ultraviolet absorbing in elevation of plasma CO2 content. Plasma inorganic phos- like material Hytakerol was definitively shown to be chromatographically different phate declined 66 per cent. In contrast, Tris acidified to from crystalline AT-10. In each of 4 different reverse- pH 6 (R-NH3+) produced a lesser, transient fall in plasma phase systems, crystalline AT-10 migrated faster than the glucose of 23 per cent, in 4 studies, with slight reduction Hytakerol sterol. in pH and CO. content and a 43 per cent fall in phosphate. It is concluded that the commercially available Hy- NaHCO, (4 experiments) caused no change in plasma takerol does not contain AT-10 but rather an isomer of glucose despite increase in pH and CO2 similar to that this sterol which is of significantly lower potency. Avail- after alkaline Tris; phosphate fell by 14 per cent. able evidence suggests that the compound is dihydro- Alkaline Tris transiently reduced plasma glucose by vitamin D2 II. only 17 per cent in 5 studies of 2 diabetic (pancreatecto- mized) dogs deprived of crystalline insulin for 18 to 24 Prevention of Stokes-Adams Attacks with Chlorothiazide hours. pH rose by 0.25, CO2 increased strikingly, and and Salt. Louis TOBIAN,* Minneapolis, Minn. phosphate fell 52 per cent. NaHCO3 given to diabetics It seemed feasible to prevent Stokes-Adams attacks by (3 studies) lowered plasma glucose 17 per cent, phos- mildly depleting body potassium with ehlorothiazide and 1036 PROCEEDINGS OF THE FIFTY-SECOND ANNUAI MEETING sodium salts. Six patients were all having frequent was prefed, mean value 2.8 + 2.8 per cent. Similar re- syncopal episodes which occurred whenever their atrial sults were obtained when 4.0 g of Viokase (11 subjects) impulses were temporarily blocked at the auriculo-ven- was mixed with the labeled lipid prior to intraduodenal tricular nodal area. None had permanent complete heart administration, mean value 11.0 ± 7.5 per cent. Of block. When 5 of these patients begani taking 500 to particular interest was the significant reduction in fecal 1,000 mg of chlorothiazide and 4 to 9 g of NaCl daily, radioactivity which followed the intravenous administra- the Stokes-Adams attacks ceased to occur in every one tion of secretin and pancreozymin-cholecystokinen. When of them. When the medication wras temporarily discon- these materials were given over a 20 minute period prior tinued in 2 patients, the attacks began to reappear. Re- to the I31-triolein infusion (7 subjects), fecal radio- sumption of medication again completely prevented the activity averaged 3.41 + 1.83 per cent. This value bore attacks. In the one other patient, the combination of similar statistical significance to lipid prefeeding when 750 mg of chlorothiazide and 10 g of NaHCOs daily compared with other groups. Blood radioactivity, meas- completely prevented the syncopal seizures without pro- ured from 1 to 6 hours, was significantly greater when ducing any significant changes in plasma pH, bicarbonate, lipid was prefed than with any other maneuver. or potassium. So far the regimen has provided excellent These observations provide further evidence in support improvement in every patient with Stokes-Adams seizures of the digestive nature of this induced defect and point up who does not have complete heart block (6 out of 6). the importance of intestinal hormones in the normal diges- Moreover, in all 6 patients there was electrocardiographic tion of IP1`-triolein. The data also suggest that the secre- evidence of improved conduction. The regimen has not tagogue effect of carbohydrates and peptone is sig- caused any annoying side effects. The medications pro- nificantly less than that of fat. duced no significant alterations in pH or potassium or sodium concentration in plasma. Only 2 patients had a The Hormonial Role of Enidogenous Glucagon in Blood: slight elevation of plasma bicarbonate, never higher than Glucose Homeostasis as Demonstrated by a Specific 33 mEq per L. Balance studies indicated that the Inmmunoassay. R. H. UNGER, A. M. EISENTRAUT, regimen caused mild depletion of body potassium. A MARY S. MCCALL and L. L. MADISON, Dallas, Tex. lowering of potassium in the cardiac conduction tissue (introduced by R. W. Berliner). may be partly responsible for the improved cardiac con- duction. To summarize, patients whose conduction sys- Despite extensive knowledge of the actions of exoge- tem is not irrevocably damaged can often obtain striking nous glucagon, the lack of a specific method for measuring relief from Stokes-Adams syncopal seizures by taking circulating endogenous glucagon has left its hormonal generous amounts of chlorothiazide and NaCl or status and physiological role in doubt. The recent de- NaHCO8. The efficacy of the regimen may or may not velopment in this laboratory of a specific glucagon radio- be related to a mild depletion of potassium in the con- chromatographic immunoassay, sensitive to 50 ,u,g, made duction fibers. possible the following studies of glucagon secretion dur- ing acute and chronic hypoglycemia. Absorption of ]Y'-Labeled Triolein Following Intraduo- Acute hypoglycemia was induced in 9 dogs by i.v. in- denal Administration: The Relative Effect of Intestinal jection of glucagon-free insulin, and chronic hypogly- Hormones, Secretagogues and Pancreatic Enzymes. cemia in 4 dogs by phloridzinization. Glucagon concen- MALCOLM P. TYOR, RICHARD R. HORSWELL and ED- tration was measured radioimmunologically in pancreatic WARD E. OWEN, Durham, N. C. (introduced by Julian and femoral venous blood. In normal dogs fasting gluca- M. Ruffin). gon levels in pancreatic plasma averaged 653 u,ug per ml (50 to 1,300), always exceeding the corresponding fem- The intraduodenal administration of I"1'-labeled triolein oral levels which averaged 443 (0 to 900). Insulin (3 ml per minute) to normals results in increased fecal hypoglycemia below 45 mg per 100 ml was invariably and decreased blood radioactivity. This induced defect, followed by a rise in pancreatic venous glucagon to a peak which is primarily digestive, may be corrected by the averaging 160 per cent (36 to 290 per cent) above pre- oral administration of a fat meal 30 minutes before the injection values. This hyperglucagonemia coincided with intraduodenal infusion. The present report deals with the blood glucose rebound; it was abruptly reversed by rapid effect of intestinal hormones, carbohydrate, peptone and glucose injection. Chronically hypoglycemic phlorid- pancreatic enzymes on intraduodenal administration of zinized dogs had high fasting glucagon levels averaging labeled triglyceride. 1,993 ,LAg per ml (680 to 3,100) which rose further after When carbohydrate (12 subjects) and peptone (11 sub- insulin inj ection. In 8 normal and 11 diabetic humans jects) were given orally 30 minutes before the intraduo- peripheral venous fasting glucagon averaged 273 ,u,g per denal infusion of P`3-triolein, 72 hour fecal radioactivity ml (0 to 700). Only a small rise followed insulin hypo- + 9.5 and 10.8 + 9.0 per cent, respectively. averaged 12.7 in a Both values were significantly reduced (p = <0.05) when glycemia in normals but a tenfold rise occurred hy- compared with those obtained from subj ects who were perlabile insulin-sensitive diabetic. not prefed, mean value 22.8 + 12.5 per cent. However, The hormonal status of glucagon and its role in glucose fecal radioactivity in each group was significantly greater homeostasis have been demonstrated by direct specific (p = <0.01) than the mean value observed when lipid measurements which indicate its pancreatic origin, its AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 1037 secretory responsiveness to hypoglycemia, and its counter- fell in 3 hours below 600, then rose sharply. Ketonemia regulatory action thereupon. increased at one-half hour, then fell toward normal. Glucose intolerance was not observed. Human growth The Pathogenesis of Viruria: Animal Kidney Infection hormone, therefore, appeared to speed mobilization of by Viruses Isolated from Human Urine. JOHN P. UTZ, fatty acids, increase fasting ketosis, and revert to normal Bethesda, Md. (introduced by Vernon Knight). the influence of glucose administration on ketonemia. Using a technique of ultracentrifugation of urine, we Studies on the have been able to demonstrate viruria in over 30 patients Physiological antd Pharmacological Revers- with infection due to 7 viruses: mumps, Coxsackie B2, ibility of Endotoxin Shock in Dogs. JAMES A. VIcK, B3, B4, ECHO 9, ECHO 14 and another untyped en- Minneapolis, Minn. (introduced by Wesley W. Spink). terovirus. Virus has been found as early as the first day Peripheral vascular collapse (endotoxin shock) in hu- of illness and as late as the thirteenth day in patients with mans is most frequently due to coliform bacteria. mumps. Five urinary isolates representing ECHO 9 (2 Management is difficult to evaluate because combinations isolates), Coxsackie B2, Coxsackie B3, and Coxsackie B4 of procedures and drugs are employed simultaneously in viruses were inoculated subcutaneously into suckling mice critically ill patients. More precise information has been to determine whether animal kidneys could be infected. obtained in adult mongrel male dogs anesthetized with Ten2` to 10" TCID5o per mg (infective dose for 50 per sodium pentobarbital, given lethal doses of endotoxin (E. cent of tissue cultures per milligram of wet tissue) coli), and then treated with a single agent or combina- amounts of virus were found in kidneys of animals killed tions after the onset of progressive hypotension. Over a 6 to.8 days after inoculation. This amount was less than period of 12 hours systemic blood pressures were recorded that found in the hind leg, approximately the same con- continuously; urinary outputs were evaluated qualita- centration as that found in brain and heart, and 10 to 300 tively and quantitatively; and hematocrit concentrations times that found in blood of the same animals. A tech- and femoral arterial pH were determined at intervals. nique of perfusion of the living animal with saline to 1) The average survival time for 12 control dogs was remove blood from viscera before determining the degree 11.5 hours. Irreversible shock usually occurred when the of viral infection provided additional evidence that the hematocrit exceeded 60 vol per cent, and the blood pH presence of virus was not accounted for on the basis of fell below 7.0. Renal failure was a prominent feature. the viral content of blood in the kidney. The finding of 2) The survival time of 10 dogs given 250 to 750 cc of viruria in man and the evidence that viral propagation canine plasma was 14.7 hours. 3) When a pressor agent, may occur in renal tissue of animals points to the pos- metaraminol, was administered to 10 dogs so that sibility that renal viral infection may be a concomitant of systolic pressures were maintained around 90 to 100 mm several acute viral illnesses of man. Hg, the survival time was 13.1 hours. 4) The survival time of 11 dogs given large intravenous doses of cortisol Restoration by Growth Hormone of Normal Responses to was 15.1 hours. 5) The most significant results were Fasting in a Pituitary Dwarf. W. P. VANDERLAAN* obtained with a combination of cortisol and metaraminol. and D. P. SIMPsoN, La Jolla, Calif. When a large infusion of cortisol was administered, only one-tenth the usual amounts metaraminol were neces- The effects of 48 to 60 hours of fasting on free fatty of sary to sustain pressure. renal function was also acid (FFA), blood ketone, and sugar levels were meas- Good sustained in the surviving dogs. Seven out ured in 22 volunteers. There were rises in FFA and throughout of 10 not resumed their normal ketone levels to average maxima of 1,850 ,uEq and 2,426 animals only survived, but activities. ,amoles per L. Blood sugars fell as low as 45 mg per The results in dogs with a combination of large doses 100 ml. The fast was broken with an oral glucose toler- ance test, FFA, ketone and glucose levels being observed of hydrocortisone and metaraminol simulate observations in as long as 7 hours. After glucose administration, FFA made human patients. fell sharply (2 hours after glucose average = 723 /AEq per L). The extent- of fall varied directly with dosage. De- The Role of Nutrition in the Alcoholic Neurological spite 100 g of glucose after 5 to 7 hours, FFA levels Diseases. MAURICE ViCrOR, Boston, Mass. (introduced again rose sometimes to exceed the fasting peak. Ketone by Raymond D. Adams). levels frequently rose one-half hour after glucose, then This communication proposes to define the etiological fell and again rose later. Occasionally glucose intoler- role of nutritional factors in 1) delirium tremens and re- ance occurred. lated disorders and 2) the Wernicke-Korsakoff syndrome. A pituitary dwarf, receiving thyroid U.S.P. and 10 mg Two types of data were secured: 1) dietary history and cortisone, fasted 48 hours and had a maximal rise in FFA nutritional status on examination; 2) observations on the levels to 480 ,uEq per L. Ketonemia was minimal until mode of recovery under strict dietary control. 50 g of glucose was given; then a fourfold rise occurred Of 213 patients with delirium tremens and related dis- over 3 hours. In a subsequent fast he received human orders, symptoms developed on a background of inade- growth hormone, 4 mg on alternate days; a rise in FFA quate diet in 148, but adequate in 65. Signs of nutritional levels to 1,620 ,uEq per L and a ketonemia tenfold greater deficiency were present in 37 of the former, in none of than before occurred. After 50 g of glucose, FFA levels the latter. Thirty patients (3 groups of 10) with de- 1038 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING

lirium tremens recovered completely in 24 to 96 hours, The Removal of Norepinephrine (NE) from the Blood while receiving a diet lacking in all vitamins, protein and Stream by the Extremities of Man. JOHN M. WALLACE, fat (water ad. lib., 5 per cent glucose/saline, 45 ml Durham, N. C. (introduced by Eugene A. Stead, Jr.). whiskey in water every 4 hours. Ten patients with acute Plasma levels and identity of small amounts of material auditory hallucinosis recovered in 1 to 8 days while in- measured as NE in normal human blood are uncertain. gesting only water ad. lib. (3 patients), 20 per cent Infused NE disappears rapidly from blood. Observations dextrose/water (6 patients), and 45 ml whiskey in water on arterio-venous differences of material present in every 3 hours (1 patient). These rates of recovery did normal plasma and comparison of the behavior of this not differ significantly from those of 101 patients with material with NE have not been available. delirium tremens and 65 patients with acute auditory An ethylenediamine method sufficiently sensitive to deal. hallucinosis who received a full diet and all vitamins. quantitatively with known epinephrine and NE in con- In 186 patients with Wernicke's disease prolonged centrations believed present in normal plasma was de- dietary inadequacy had been present in all; 171 showed veloped. For NE determinations, standard solutions con- signs of malnutrition on examination. Nine patients were tain 0.004 to 0.008 ,zg NE per 5 cc plasma (0.8 to 1.6 ug given a diet composed only of glucose, minerals and per L); 0.004 Ag gives 23 galvanometer deflections above water, and 27 an unfortified rice diet, for periods up to plasma. NE infusions of 4 ,tg per minute were given to 14 days. Only when thiamine was added did the apathy, 5 subjects. Slight increases in blood pressure anid decreases ophthalmoplegia, nystagmus and ataxia improve. With in pulse rate occurred. Two sets of samples were drawn respect to memory defect and confabulation (Korsakoff's from a brachial artery (BA), antecubital vein (ACV) psychosis), observations were made on 12 patients given and femoral vein (FV) before and during infusion. the purified diet for 8 weeks. This defect remained un- Resting plasma contents of apparent NE did not differ, changed in 4, improved in 6, and disappeared completely mean values in jug per 5 cc plasma being 0.0038 (BA), in 2, an outcome similar to that in 29 patients given a 0.0033 (ACV) and 0.0040 (FV). During infusion mean full diet and all vitamins. levels of added NE (control levels subtracted) in Ag per 5 cc plasma were 0.0088 (BA), 0.0063 (ACV) and Reciprocal Relationship Between Magntesiumtl anid Calcium 0.0015 (FV). In every subject ACV and FV plasmas Metabolism in Tetany. WARREN E. C. WACKER and contained significantly less added NE than BA plasma DAVID D. ULMER, Boston, Mass. (introduced by Bert and in 4 subjects FV plasma contained significantly less L. Vallee). than ACV plasma. In 2 subjects FV plasma content Magnesium or calcium deficiency tetany can only be during infusion showed no increase over resting levels. distinguished by chemical means. The establishment of When arterial levels of NE are increased to produce simple and direct relationships in the biological response minimal changes in pulse and arterial pressure most of and interactions of these elements has encountered great the NE is destroyed in the leg; less is destroyed in the difficulty due to the multiple and unknown equilibria be- arm. Behavior of infused NE in both arm and leg is tween their various phases and compartmental allocations. different from that of normal plasma material commonly Specific and reciprocal relationships between the metab- called NE. NE might be both removed and added by olism of magnesium and calcium under marginal nutri- tissues. Efficient destruction of NE by the leg makes it tional conditions have now been identified. questionable whether NE made there escapes destruction In intestinal malabsorption concurrent magnesium and and appears in effluent blood. calcium deficiency are observed as evidenced by the low concentrations of both ions in serum. The administration Changes in Phage Types of Staphylococcus aureus in a of calcium will restore the concentration of this element General Hospital, 1952 to 1959. GoSTA WALLMARK and in serum to normal values, but simultaneously the con- MILDRED W. BARNES, Boston, Mass. (introduced by centration of magnesium is depressed further and tetany Maxwell Finland). eventually ensues. The correlation of these events is Four series of coagulase-positive strains of Staphylococ- reflected in a concomitant and marked loss of magnesium Clts autreuts collected from various sources in patients at in urine. Reciprocally, parenteral administration of mag- the Boston City Hospital in the years 1952, 1955, 1958, nesium, while abolishing tetany, similarly results in en- 1959 and 1960 were subjected to phage-typing. The same hanced urinary losses of calcium. set of phages was used and all the tests were done within The therapeutic response to the administration of a the last few months. The results indicate that about one- metabolite is a potential method to diagnose a deficiency half of the 400 strains isolated in 1952 were lysed by state clinically. In the present instance, however, calcium phages of Group III and that a few phage patterns pre- operates as a conditioning factor, aggravating the pre- dominated. This is in contrast to the later series in which existent subclinical, magnesium deficiency. The observed those of phage Group III accounted for only about one- potentiation is an example of biological antagonism which fifth of the strains. Staphylococci lysed by phages of presents a therapeutic hazard. Therapy cannot be relied Group I, on the other hand, increased in frequency from upon to serve a diagnostic function here-diagnosis, there- about 15 per cent among the 1952 strains to 50 to 60 fore, depends on precise, quantitative measurements of per cent in the later series. There were only 2 strains of both ions. type 80/81 among those tested in 1952, whereas in 1955 AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 1039 at least one-third of the strains were of this type, and this sulfide, form (PSSP). Fully active, reduced papain incidence has since remained about the same. The strains (PSH) has no effect on cartilage in vivo. Studies with were also tested for susceptibility to the antibiotics that isolated rabbit ear cartilage in vitro have shown that are in common use, and the results correlated with the normal rabbit serum impairs the entry into cartilage of phage types will be presented. Of special interest was the PSH, but not of PSSP. For these reasons, the rate of almost universal resistance of type 80/81 strains to peni- disappearance from serum of PSH and PSSP in vivo cillin, streptomycin and tetracycline in contrast to the has been studied using papain labeled with I'. Approxi- rarity with which strains of this type were found to be mately 50 per cent of the injected enzyme is lost from resistant to erythromycin. ' serum within 30 minutes after an intravenous injection of 2 to 5 mg of PSH or PSSP in 1 kg rabbits. However, Hypoxia antd Ammonia Toxicity. KENNETH S. WARREN only a minute fraction of the injected material enters and STEVEN SCHENKER, Bethesda, Md. (introduced by cartilage, the concentration being 1 to 2 ,ug per g of wet Robert S. Gordon). cartilage in the case of PSSP, and less than 0.5 ,ug fol- Ammonia toxicity was strikingly potentiated by short lowing PSH. On the basis of immunoelectrophoresis and periods of hypoxia. Although the toxicity (LD50) of zone electrophoresis in barbital buffer, 0.1 M, pH 8.0 and ammonium chloride administered intravenously to mice 8.6, papain was found to be in the a-globulin region of after 2 minutes in 15 per cent oxygen was the same as serum. The localization was identical for PSH and that in 21 per cent oxygen, its toxicity began to increase PSSP, both after intravenous administration, and follow- when they were exposed to 13 per cent oxygen. The ing incubation with serum in vitro. Under these con- lethal effect from that point on was markedly enhanced ditions, the electrophoretic mobility of papain alone was by each 2 per cent decrement in oxygen concentration. toward the cathode, and it was not modified by albumin In comparison with 21 per cent oxygen, 2 minutes in 7 or y-globulin obtained by Cohn fractionation of serum. per cent oxygen increased the toxicity of ammonium However, the mobility of papain was modified by isolated chloride 4.4 times. a2-globulin exactly as in whole serum. In vitro studies The lethal effect of a dose causing 100 per cent mortal- with a variety of human sera have shown a similar ity in 7 per cent oxygen could be completely reversed in a localization of papain in the a-globulin region. Since in- matter of seconds by transferring the mice to 21 per cent hibition of trypsin, hyaluronidase and deoxyribonuclease oxygen. Oxygen concentrations as high as 99 per cent is associated with such a-globulins, it is suggested that did not alter the LD50, but significantly prolonged life. binding of papain by serum proteins may represent a The effect of hypoxia on the toxicity of another compound similar mechanism preventing damage to tissue by a po- with analeptic properties, Metrazol, was relatively slight. tentially harmful enzyme entering the circulation. Blood pH was not altered by the short periods of hypoxia utilized in these experiments. In addition, measurements Bile Salt Transport in the Dog. HENRY 0. WHEELER, of brain ammonia concentrations following a standard PIER L. MANCUSI-UNGARO and ROBERT T. WHITLOCK, dose of ammonium chloride at 21, 11, and 7 per cent New York, N. Y. (introduced by Stanley E. Bradley). oxygen revealed that hypoxia had no effect on ammonia Bile salt transport and its effect on bile flow and elec- uptake or detoxication by the brain. It appeared there- trolyte composition and on transport of anionic dyes was fore, that hypoxia directly potentiated the toxic effect of studied in 4 unanesthetized, cholecystectomized dogs (20 ammonia on the brain. The problem of hepatic coma will be to 25 kg) equipped with duodenal Thomas cannulae. discussed in view of these findings. Intravenous infusions of 1.5 per cent sodium taurocholate The presence of elevated blood ammonia concentrations at rates of 170 to 230 ,umoles per minute resulted in in with patients severe liver disease has often been related arterial plasma concentrations of 0.6 to 1.2 mmoles to the genesis of hepatic coma. per Hypoxemia and reduced L at 30 minutes which continued to rise, reaching 1.0 to cerebral blood flow are also frequent concomitants of 2.3 mmoles per L at 90 minutes. A constant and repro- marked liver pathology. In addition, there are definite ducible maximal rate of biliary taurocholate excretion was neurological similarities between hepatic coma and chronic achieved within 30 minutes (mean 132 ,Amoles per pulmonary insufficiency. Therefore, measures to improve minute, range 111 to 141 ,umoles per cerebral minute). The re- oxygenation might be a useful adj unct to the sulting choleresis (0.70 to 1.35 ml per minute) was ac- therapy of hepatic coma. companied by chloride (17.9 to 90.0 ,umoles per minute) and bicarbonate (9.3 to 41.5 ,umoles per minute), outputs Binding of Papain Protease by Alpha-globulin of Rabbit much greater than those and reported previously at lower Human Serum. GERALD WEISSMANN, JACOBUS L. taurocholate excretory rates. POTTER, FRANK McELLIGOTT, MARTIN MELTZER and The excretory transport maximum of sulfobromophtha- ROBERT T. MCCLUSKEY, New York, N. Y. (introduced lein sodium (BSP) -mean 4.0 Atmoles per minute-was by Lewis Thomas). not reduced when BSP was infused during periods of It has been shown previously that crystalline papain maximal taurocholate output. However, the uptake of protease produces- widespread depletion of cartilage BSP from plasma (or the hepatic BSP storage) was matrix when injected intravenously into young rabbits, altered by taurocholate since 1) taurocholate administra- provided that the enzyme is injected in an inactive, di- tion during constant BSP infusion produced abrupt in- 1040 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL ME:ETING creases in plasma BSP concentration, and 2) unexpectedly Patients with essential hypertension have normal high plasma concentrations and delayed achievement of peripheral venous tone despite the increase in arteriolar maximal BSP excretion occurred when BSP was given tone; normal subjects with acute hypertension induced during taurocholate infusions. The hepatic uptake of by infusions of angiotensin II have increased venous tone. indocyanine green was similarly affected. Effects of these The effect of infusions of synthetic angiotensin II (Ciba) dyes on taurocholate uptake and excretion were equivocal. upon responses of the peripheral veins of patients with Thus biliary taurocholate secretion is active, rate- essential hypertension were studied. limited and not obviously related to BSP secretion, al- Eight normotensive subj ects received infusions of though taurocholate does interfere with hepatic uptake of angiotensin II of 2 to 4 ,ug per minute for 30 minutes. BSP and indocyanine green. In response to secretion of Arterial blood pressure averaged 123/75, a 8/6 before osmotically active bile salt, water and diffusible electro- infusion and 165/108, a 20/9 during infusion. Venous lytes probably enter bile passively. Diminished reabsorp- volume of the leg measured plethysmographically in tion of these constituents could also contribute to their milliliters per 100 ml of leg tissue per 30 mm Hg rise enhanced excretion during taurocholate choleresis. in local venous pressure averaged 4.3, a 1.0 before in- fusion and 3.4, a 0.9 during infusion. Twelve patients Blood ACTH in CGushing's Disease. W. C. WILLIAMS, with essential hypertension received angiotensin II in- JR., R. A. A. OLDFIELD, JR., D. P. ISLAND and G. W. fusions of 2 to 4 ug per minute for 30 minutes. Arterial LIDDLE,* Nashville, Tenn. blood pressure averaged 163/90, a 30/17 before infusion and 208/122, a 25/14 during infusion. Venous volume The present study was undertaken in search of a defini- averaged 4.0, a 1.0 before infusion and 3.7, a 1.1 during tive answer to the long-debated question of whether the infusion. Venous volume was lowered significantly pituitary secretes abnormal quantities of ACTH in Cush- (p < 0.01) in the normal individuals by angiotensin II; ing's disease (hypercortisolism due to bilateral adrenal this material failed to alter venous volume significantly hyperfunction). It was reasoned that if the primary dis- in the hypertensive patients. Rise of venous pressure order in Cushing's disease were a derangement of with infusion of angiotensin II in the normotensive indi- pituitary function, then it would not be corrected by re- viduals averaged 9, range 4 to 15 cm of water and moval of the target organs, the adrenal glands. It was averaged 2, range 0 to 3 cm of water in the hypertensive postulated that the reason the adrenals were overactive in individuals. Angiotensin infusions invariably caused this disease was that ACTH secretion could not be sup- headache, dyspnea, chest discomfort, nausea or abdominal pressed by normal levels of cortisol. Two groups of cramps in normotensive subjects. Hypertensive subjects patients were studied: 6 patients who had been treated for suffered none of these symptoms with infusion. Venocon- Cushing's disease by bilateral adrenalectomy and 4 pa- strictor responses to norepinephrine infusions in 6 pa- tients who had Addison's disease without prior history of tients with essential hypertension were comparable to Cushing's disease. Both groups were maintained at those of normotensive individuals. normal blood levels of cortisol by administration of oral These experiments indicate that the veins of patients cortisol every 8 hours. None of the patients exhibited with essential hypertension are specifically insensitive to evidence of pituitary tumor. angiotensin, implying that these responses are so modified ACTH was extracted from plasma by a method utiliz- by previous experience with a substance at least similar ing adsorption onto and elution from an ion exchange to synthetic angiotensin II. resin, Amberlite XE-64. ACTH activity in this extract was determined in hypophysectomized rats by a bioassay Serum STANLEY and technique in which corticosterone was measured in adrenal Normal Hemolysins. YACHNIN vein blood following injection of graded doses of standard FRANK H. GARDNER,* Boston, Mass. ACTH or plasma extract into the femoral vein. With Normal human red cells (RBC) treated with various this method 0.06 mU of ACTH could regularly be de- agents which alter the stroma become susceptible to tected and a rectilinear log-dose response relationship acid hemolysis in human serum (HS). Such hemolysis was obtained with doses up to 0.25 mU per rat. is complement- and properdin-dependent. Sera vary Although cortisol treatment and plasma 17-hydroxy- widely in their capacity for hemolysis. Eighty HS sam- corticoid values were simnilar for both groups, blood ples have been studied for hemolysis and agglutination ACTH levels were 23 + 16 (mean and standard error) with treated RBC. These altered RBC are to varying mU per 100 ml for the patients with Cushing's disease degrees panagglutinable. The agglutinin titer of sera has and 0.3 + 0.2 mU per 100 ml for the patients with simple been related to their capacity to hemolyze altered RBC. Addison's disease. Conclusion: in Cushing's disease RBC treated with receptor-destroying enzyme (RDE) ACTH secretion is not suppressed by normal levelt of and periodate are most susceptible to hemolysis in sera cortisol. having the highest agglutinin titer. Bromelin and ficin- treated RBC are less consistently correlated, and trypsin- Absence of the Normal Peripheral Venoconstrictor Re- ized RBC show poor correlation between serum hemoly- sponse to Infusions of Synthetic Angiotensin II in tic and agglutinating capacity. Serum hemolysis is not Patients with Essential Hypertension. J. EDWIN WOOD, related to complement titer or lysis of paroxysmal noc- Augusta, Ga. (introduced by Thomas Findley). turnal hemoglobinuria (PNH) RBC. There is some AMERICAN SOCIETY FOR CLINICAL INVESTIGATION 1041 relationship between serum hemolytic capacity for RDE much less marked than those following glucose in the and trypsin-treated red cells. Bromelin and ficin-treated same patients. RBC are more closely related as regards hemolysis, but The high endogenous insulin secretion rate observed this relationship is not absolute. in maturity-onset diabetics on glucose loading suggests Serum hemolytic activity can be inhibited by absorbing that deficient glucose utilization in this condition is not serum with specific types of treated RBC at 20 C. due to inadequate synthesis or release of insulin but is Eluates of these RBC can be bound to the identical related to poor tissue responsiveness and/or inhibitors treated RBC in saline media. Suspension of these eluate- of the hormonal action of endogenous insulin. coated RBC in absorbed acid serum results in restoration of hemolysis. Marked specificity of serum factors for The MIechanismn of the Steroid Inhibition of Pyruvate each type of altered red cell may be demonstrated in HS. Oxidation. K. LEMONE YIELDING, GORDON M. TOM- These antibody-like substances are distinct from proper- KINS and JANET S. MUNDAY, Bethesda, Md. (intro- din and complement, but dependent on these agents for duced by Harry Eagle). acid hemolysis. These factors for treated red cells do not participate in PNH hemolysis. Such natural HS The DPN-dependent oxidation of pyruvic acid to hemolysins emphasize the potential opportunity for patho- acetyl coenzyme A and C02 iS the metabolic step which logic RBC destruction in diseased states. In addition the permits the entry of glucose carbon into the Krebs cycle, recognition of these HS factors may aid in understand- and its rate might be important in the regulation of carbo- ing the use and pitfalls of enzyme-treated RBC in sero- hydrate metabolism. Steroid hormones have been shown logic tests. to inhibit this reaction in vitro, and the elevated blood levels of pyruvic acid in patients with -Cushing's disease Immunoassay of Plasma Insulin Concentrations in Nor- and upon steroid administration suggest some such action mal and Diabetic Man: Insulin Secretory Response to of these hormones in vtvo. Glucose and Other Agents. ROSALYN S. YALOW and We have shown recently [Proc. Nat. Acad. Sci. SOLOMON A. BERSON,* Bronx, N. Y. (Wash.) 1959, 45, 1730] that the oxidation of DPNH to DPN by the DPNH oxidase reaction is strongly in- An immunoassay procedure requiring only 10 to 20 Al hibited by various steroid hormones. Since DPN is plasma and capable of detecting less than 10' U insulin essential for the oxidative decarboxylation of pyruvate, has been employed to measure plasma insulin concen- these findings suggested that the steroid inhibition of trations in more than 100 subjects during a standard glu- pyruvate oxidation might be due to curtailment of DPN cose tolerance test. Human insulin added to plasma in arising from DPNH oxidation. Pyruvate oxidation in vitro is recovered quantitatively and 2- to 100-fold dilu- homogenates of rat tissues was stimulated by the addition tion of plasma reveals no evidence for nonspecific plasma of cytochrome C and/or liver microsomes, which spe- substances reacting like insulin or inhibiting insulin in the cifically stimulate DPNH oxidation. This showed that immunologic reaction. Endogenous plasma insulin and the rate of pyridine nucleotide oxidation could control insulin-I' were similarly adsorbed by cellulose columns that of pyruvate. Alpha tocopherol, which prevented and destroyed by cysteine. steroid inhibition of the DPNH oxidase, also overcame Fasting insulin concentrations did not differ signifi- the steroid suppression of the pyruvate reaction. When cantly in untreated maturity-onset diabetic and nondia- DPNH oxidation was impeded by other inhibitors of betic subjects (range, 0 to 70 ,uU per ml; means 27, 21 electron transport, such as amytal or antimycin A, there ,AU per ml); 100 g glucose p.o. elicited peak insulin con- was a corresponding depression of the oxidation of centrations in nondiabetics at 0.5 hour (mean, 136 ,uU pyruvate. per ml) or 1 hour (mean, 128 AtU per ml) with decline These findings illustrate that steroids can regulate the by 2 hours (mean, 105 ,U per ml). Insulin concentra- oxidative decarboxylation of pyruvic acid, by virtue of tions in diabetics were lower at 0.5 hour (mean, 97 AU their ability to inhibit oxidation of DPNH. per ml) but continued to rise to a high peak at 2 hours (mean, 243 ,uU per ml) showing a significantly greater anid than normal insulin Glycolytic Citric Acid Cycle Metabolites Related to integrated output. Diabetics showed Intracellutlar Ion Composition in the Experimental still higher insulin concentrations on further glucose load- Nephrotic ing. In 5 of 7 patients with functioning islet cell tumors Syndrome. T. YOSHIDA, F. YAMASHITA, E. KAISER and J. METCOFF,* Chicago, Ill. (courtesy Drs. H. Epstein, J. Field, E. D. Furth, E. Gordon, A. Renold, J. Steinke) fasting insulin exceeded The experimental nephrotic syndrome, induced in rats 118 ,uU per ml but response to glucose was normal in 1 by 6-dimethylamino-9-(3-amino 3-deoxy 8-d-ribofurano- patient studied. syl)-purine (aminonucleoside), causes disturbance of pro- Plasma insulin increased abruptly following L-leucine tein, lipid and electrolyte metabolism and of some en- p.o. in 6 of 9 studies in 6 leucine-sensitive hypoglycemic zyme activities in kidney tissue. The present study was patients (courtesy Drs. A. DiGeorge, M. Goldner, M. designed to explore the influence of intracellular electro- Grumbach, I. Rosenthal and S. Weisenfeld). Increases lyte concentration on intermediary cellular metabolism. in plasma insulin were observed consistently following Fourteen nephrotic rats are compared with their pair- sodium tolbutamide (i.v. or p.o.) in all subjects but were fed controls (nephrotic versus control value). In muscle, 1042 PROCEEDINGS OF THE FIFTY-SECOND ANNUAL MEETING the intracellular water content (352 > 260 ml per 100 g and formation of urine iso- or hypotonic to plasma during DFFS) and composition in the edematous nephrotic rats osmotic diuresis, despite adequate vasopressin. With differed significantly from the controls (mmoles per L normocalcemia, GFR and U/Posm increased and hyper- ICW: Na 50 > 19, K 121 < 182, Mg 19 < 22, Porganie tonic urine was produced during osmotic diuresis. At 46 < 76, Pinorgante 44 < 54). Suggestive differences in high flow rates, however, TH2o0/GFR tended to decrease cellular contents of glycolytic and Krebs cycle inter- significantly. mediates also occurred (,umoles per g NCN phosphoenol- One patient with hypercalcemia and normal GFR pro- pyruvate 3 < 7, pyruvate 18 > 12, oxalacetatic 21 > 15). duced persistently hypotonic urine despite large amounts Lactate, citrate and a-keto-glutarate contents were simi- of vasopressin, resulting in a free water clearance of 5.0 lar. In vitro uptakes of pyruvic acid by isolated dia- ml per minute. This study suggests that the defect in phragm (9.2 > 3.7 ,umoles per g NCN per minute), and renal concentrating ability is not dependent upon changes by slices of kidney (10.9> 8.0) and liver (6.6> 5.1) of in glomerular filtration and may be related to relative nephrotic rats were increased. insensitivity of the distal tubule to antidiuretic hormone, Significant differences in activities (/Lmoles per g as well as to changes in intramedullary osmolality. NCN per minute) of glutamic dehydrogenase (8.4 > 5.6) and glutamic-pyruvic transaminase (8.7> 6.2) were The Poorly-Permeable Membrane as an Alternative to found. Pyruvic kinase, lactic dehydrogenase, isocitric the Carrier Hypothesis of Facilitated Diffusion. KEN- dehydrogenase and malic dehydrogenase activities were NETH L. ZIERLER,* Baltimore, Md. similar. Substrates were constructed containing ion con- centrations approximating those found in ICW of Diffusion facilitated by unidentified carrier molecules nephrotic and control rat muscle and activity of purified is a process hypothesized for movement of certain sub- pyruvic kinase tested. At appropriate dilutions, activity stances across cell membranes in order to account for in the substrate simulating the "nephrotic" muscle was three phenomena-saturation, competition and specificity significantly less than that in "control" substrate. -which occur even though net movement is in the direc- Apparently, distortion of essential intracellular electro- tion of the chemical or electrochemical potential gradi- lyte concentrations characterizing edema of the experi- ent. However, this hypothesis may be unnecessary be- mental nephrotic syndrome is associated with, and may cause saturation and competition are inherent properties condition, altered pathways of terminal glycolysis and of poorly-permeable membranes, and biological mem- subsequent metabolism of intermediates in the Krebs branes, in general, are poorly permeable. For example, it cycle. has been estimated that for mammalian skeletal muscle only 10' of the membrane surface is available for dif- fusion of water-soluble substances. When a relatively The Effects of Hypercalcemia on the Renal Concen- large number of molecules, whether of the same or dif- trating Mechanism in Man. J. LESTER ZEFFREN and ferent species, bombard such a membrane there will be HENRY 0. HEINEMANN, New York, N. Y. (intro- competition, not for a limited amount of carrier, but for duced by Alfred Gellhorn). a limited area of permeable sites. Saturation is exhibited The effects of hypercalcemia on the renal concentrating when the molecules are of a single species; competition mechanism has been studied in 7 patients with neoplastic when there are two species. Specificity occurs when the disease and skeletal metastases, and a patient with milk molecules and the' permieble sites are matched conforma- alkali syndrome. Qbservations were made during periods tionally. This hypothesis was tested in a simple mechani- of hypercalcemia and normocalcemia in 5 patients at inter- cal analog in which steel bearing balls were shaken in vals of 1 to 10 weeks. The results indicate that hyper- a chamber separated from a second chamber by a wall calcemia is associated with a reversible defect in the in which were drilled one or more holes. With increas- renal concentrating mechanism which is disproportionate ing concentration, flux became constant; there was satu- to the simultaneous reduction in glomerular filtration. ration. Addition of a second size of bearing balls reduced The renal concentrating ability was defined by the the flux of the first species; there was competition. Both maximal and minimal osmolal U/P ratio, U/Posm, and saturation and competition increased: 1) as mean velocity by the calculation of TH2OC during osmotic diuresis with of particles decreased, 2) as the ratio of area of a single 10 per cent mannitol in a hydropenic state. TH200 ex- permeable site to particle diameter decreased, and 3) as pressed as a percentage of glomerular filtration rate total permeable area decreased. It is concluded that the (TH2OC/GFR) permitted comparison between different observations for which the facilitated diffusion hypothesis observations. was invoked can be explained more simply by the proper- Hypercalcemia resulted in reduced GFR, lowering of ties of a system in which the membrane is poorly maximal U/Posm, preservation of the minimal U/Posm, permeable.