years ; and this requirement, if it were carried out mittee appointed for that purpose; at least two men strictly, especially the occasional inspection of-our mem¬ should be required for the inspection of any school, and bers, would at once strengthen and broaden the influence the report should be in writing for future reference. of this association and commend it to the state board of 3. The interests of the association demand that the examiners, do much to quiet the present state of unrest work be divided up so as to utilize as many men and and dissatisfaction existing in many medical schools and their influence as possible, if we would broaden our field elevate medical teaching in the eyes of the public out of of usefulness in both higher educational and moral or its present questionable status. ethical standing sufficiently to make membership an Standards acceptable to examining boards could be honor so distinctive that all good schools will desire the established and the variable demand of the different connection. states be gradually molded into a universal and much- 4. At present the four-year high school, or the four¬ needed standard agreeable to all. The result would be teen units for entrance and a four-year strictly graded better-prepared and more uniformly educated physicians, course of study of 4,000 hours is as high as the condi¬ and more ethical relations between the profession and tions of education will permit. the people. The time is ripe for some organized body to 5. If an additional year is wise, then a fifth year assume command and restore confidence by combining following the present four-year plan, which shall be the good accomplished by the several organizations labor¬ strictly practical and bedside in hospital wards, is pre¬ ing in the field. We should stand sponsor for as high ferable; and this association should insist that a hospital standard of medical education as present conditions will controlled by the college is essential to thorough training. permit, at the same time quieting the present feeling of If these suggestions are carried out then this associa¬ uncertainty which is agitating the modern school-world tion will be a great factor and the day will not be distant with the assurance that the schools would be given time when the medical men of America will be recognized all to establish and enforce the demands of to-day, and over the world. Then and not until then will this asso¬ adjust themselves to the changes, at least before some ciation have fulfilled its true mission. other radical change is forced on them. 150 North Eighth Avenue. While I would not detract from the great advances made in the last few years—and every force deserves credit for honest and faithful service—the conditions THE POISONS OF THE TROPICAL TOAD, above are the natural of lack of con¬ mentioned result BUFO AGUA certed effort and unity of action, and but illustrate the necessity of uniform standards applicable to the whole A PRELIMINARY COMMUNICATION country. JOHN J. ABEL, M.D., and DAVID I. MACHT, M.D. Now, I ask, what body is best equipped and qualified BALTIMORE for this work, or into what hands can be placed the Poisons of animal origin, this term in its widest and of to using elevation perfecting medical teaching properly sense, have always been of interest to the student of demands of the not our own meet the present, if Associa¬ medical science, and of late years they are beginning to tion of Medical Colleges? Indeed, we shall be derelict if be of actual value to the medical practitioner. Among we do not assume this responsibility. If every school poisons of animal origin1 may be named the various which is a member would live up to association stand¬ snake venoms, the poisons of amphibia, of fishes (e. g., ards and assume and perform its part willingly and the of of mussels would member¬ fugu poison Japan), (mytilotoxin), enthusiastically, every worthy school seek of scorpions, spiders, bees, ants, beetles (e. g., can- ship, and the vexed problem of standardizing medical tharidin), and other insects. Among poisons derived teaching would be solved. from the organs of mammals may be named those State laws can be made more uniform if the Amer¬ obtained from the thyroid and suprarenal glands as hav- ican Association of Medical Colleges and the Council on ing a very high therapeutic value. Medical Education of the American Medical Association While continuing our studies, in the autumn of 1910, join forces with state boards, and the unfortunate neces¬ on the convulsant action of certain organic dyestuffs, sity for the present differences in grading schools will acid fuchsin, betanaphthol, phenolsulphonaphthalein, have passed ; for there will be no excuse, even commer¬ and tropeolin, we had the opportunity of trying the cially, for the existence of low-grade schools, and the effect of these substances on a tropical toad, Bufo agua. m eh-needed reciprocity between the United States and While engaged in this study our interest was aroused by Canada will be assured, and with European countries the milky secretion which exudes from its "parotid" as well. glands when the animal is greatly irritated. Scraping In this short paper I have tried to point out some of off some of the secretion with a knife, we were struck the strong and important reasons for all three of the by the bluish-green discoloration which appeared on the educational organizations meeting here at this time. I blade soon after it had been used. This observation led us some diluted with predict that their coming together in a bond of unity of to test of the secretion ferric This was found purpose will, of affect every side of this chlorid. reagent to develop the char¬ necessity, vitally color of the great question. The combination of our interests and acteristic green pyrocatechin reaction. As energies for the future promises great things for the this reaction is given by the active principle of the medical profession, and, through them, for the people of suprarenal glands, further tests were at once undertaken this country. with the object of identifying the substance in the toad's secretion which reacts with ferric chlorid as described. In I would make the following suggestions : closing It was not a difficult task to demonstrate that we were 1. This association should a uniform standard adopt a substance which is identical or after with all and enforce here dealing with with, consultation forces interested, allied the Further work this standard by frequent inspections of members. closely to, suprarenal principle. 2. both and members should From the Pharmacological Laboratory of the Johns Hopkins Inspections of applicants University. be placed in the hands of a judicial council or a com- 1. Faust, Edwin Stanton: Die thierischen Gifte, 1905.

Downloaded From: http://jama.jamanetwork.com/ by a Simon Fraser University User on 06/03/2015 consistence on the secretion of the demonstrated the small pots in which it gradually acquires the of glands presence curari. A further of the later he obtained of a second in to its supply poisons may body which, respect pharmacologie smeared on action, is to be classed with substances belonging to the from the toads thus treated. The poison is to the of tips of arrows which are shot into game from blowing tubes. group poisons. A small is killed a arrow in from two to has been from the stag by poisoned The toad regarded earliest times four minutes, a jaguar in from four to eight minutes. as a venomous animal. In the Talmud2 under the name And in to the toad which is engag¬ of tzab (from the root meaning to swell or puff up) it regard particular is differentiated from the frog and is classed with ani¬ ing our attention, Fillio" makes the following state¬ mals whose touch contaminates. Various have ments : peoples There in the of the Amazon made medicinal use of the toad. The Chinese have exists, more particularly regions long a which is a veritable toads and which ' used as a a derived from toadskins species giant among remedy preparation has described under the name of Bufo agua, whose venom which call senso. to a notice Spix they According preliminary it would be worth while to stud}'. It is very probable that this senso is an similar in by Hyashi,3 impure product its is the species from which the aborigines of the Amazon derive to to one more action digitalis, but fifty hundred times the poison with which they smear the points of their arrows, use toad powerful. Western nations also made of the in place of a sort of curara which certain other tribes use. for medicinal several centuries and purposes during Further details in to the use of the of various medical treatises and regard poison European pharmacopeias have not come to our but our of an to the dried toad a Bufo agua yet knowledge, earlier day give prominent own with it abundant that it Thus, in the "Thesau¬ experiments give proof place among therapeutic agents. be indeed when used as an arrow of would very deadly rus Pharmakologicus" Johannes Schröder, published poison. in Leyden in 1672, and in the "Pharmacologia" of humer- in London in Bufo agua (horridus, maculiventris, marinus, Samuel Dale, published 1692, powdered alis, ornatus. ictericus, Lazarus, Rana marina, Bombi- toad is highly recommended for dropsv, bleeding of the nator horridus, Neotes, Pseudobufo and nose and other ailments. In the "London Docidophryne Dispensatory" is the of the or tailless of edited William of agua) largest Anitra, amphibia, 1702, by Salmon, professor physick, a of or more centimeters and a are the neck in attaining length twenty we read, "They (the toads) hung up by breadth of twelve centimeters. It is found in all of the the air are and then for use. till they through dry, kept countries and most of the islands of South and Central Wierus saith that the of a dry'd toad taken 5ss poudre America, and in the warmer parts of Mexico.7 at a time or cures almost incurable more, dropsies, carry¬ The examined us were obtained from the water urine. I the ashes of specimens by ing away by suppose the of Jamaica, where they them burned is better." In an text4 of some neighborhood Montego Bay, abridged known as The color of of Michael Etmüller are popularly bullfrogs.8 general the medical writings (1644-S3). of the animal is to dark brown with dark at it is stated that light sooty professor of medicine Leipsic, "living and its back is covered with toads aroused to the of are but patches warty protuberances. point fury venomous, A feature is two oval, so-called found dead are devoid of Trans¬ striking large "parotid they entirely poison. behind each ear. It is from these "glands" that fixed in the month of July, dried, (the glands" (alive) powdered our is obtained. to these struc¬ head and entrails and administered in poison According Seeck,9 being removed) tures consist of doses of twelve on alternate days they furnish an simple, elongated glands closely packed grains the whole a resemblance to a honey¬ excellent cure for Others administer this remedy together, bearing dropsv. AVhen the are hardened in alcohol their in fever at its Powdered toad is also comb. glands burning height. lumen is filled with a brownish mass. an effective incontinence of urine, and compact remedy against in a short note in Science™ is said to be efficacious because of its anodyne character, Bristol and Bartelmez, Avhile its volatile, penetrating salt acts as a diuretic. say : found on the surface of the From it an anodyne oil is also prepared with the aid of The poison glands are only upper sea-salt and sweet almonds." body, while mucus glands are found all over the skin, and are in behind each ear. as are further mentioned in the crowded together large parotoid "glands" Toads remedial agents the and extend Universalis" of R. James. London, They are much larger than mucus glands deep "Pharmacopeia M.D., down into the corium layer. They are surrounded by in the of John Quincy, compact 1747, "English Dispensatory" a thin layer of loose connective tissue which contains nerve and as late as but here in a M.D., London, 1749, 1833, fibers and a dense net-work of capillaries. There is an almost skeptical way only, by J. A. Paris in his "Pharm¬ continuous layer of smooth muscle fibers about the gland. The acologia." cells of the glandular epithelium develop to an enormous size, Further testimony that very powerful principles are and when they mature they disintegrate, their entire plasm found in toads is seen in the fact that primitive peoples becoming the secretion, so that when a poison gland has full it is a reservoir of have made use of their skin secretion as arrow poisons. reached its development simply poison. Thus, writes :5 When the poison is discharged the remains of the gland are Pagenstecher resorbed, and at the same time one of the five or six undevel¬ to made the botanist André, wliicli liave According reports by around the mouth of the functioning been substantiated by the Choco Indians of the oped glands, grouped Saffray, prime¬ down the remains of the discharged val forests of the Sierra Templada of New Granada at eleva¬ gland, grows alongside pushing it aside to occupy its former place. tions up to 2,000 meters employ for this purpose the secretion gland, of a species of pelobates ("spade-footed" toad). The animal When the animal is irritated as by the bite of a dog is placed in a tube of bamboo, the hands of the operator being or by some other sufficiently powerful stimulus, mechan- protected with leaves, and when some of the poison is desired the tube the toad is suspended high over a fire. 6. Filho, Lacerdo: Algumas Experiencias com o veneno do Bufo containing ictericus do Mus, Nacion. do Rio Janeiro, 1878, with a which is (Spix), Archivos The toad soon becomes covered yellow juice iii. 33. allowed to drop into bowls from which it is transferred to 7. Brehm, A. C. : Thierleben, Allgemeine Kunde des Thierreichs, 1878, vii. 602; Waite, F. C.: Bufo Agua in the Bermudas, Science, Naturalist's in Jamaica. Das 342. 1901, xiii, 342. Gosse, P. H. : A Sojurn 2. Hoffmann, D. : Buch Leviticus, Berlin, 1905, i, 431. 15 a; v. 1. London, 1851. p. Babylonian Talmud : Kethuboth Mishna, Teharoth, Prof. E. A. of this for 3. Deutsch, med. No. 13, p. 624. 8. We are indebted to Andrews university Wchnschr., 1911, a sufficient of these animals. 4. Michaelis Etmulleri Opera in Compendium redacta, Londini, much assistance in obtaining supply 147. 9. Seeck, Oscar : Ueber die Hautdr\l=u"\seneiniger Amphibien, Diss. 1701, p. 1891. 5. Pagenstecher : Allgemeine Zoologie, as cited by Kobert, Dorpat, Lehrbuch der Intoxikationen, Edition 2, 1906, ii. 470. 10. Bristol and Bartelmez: Science, 1908, xxvii, 455.

Downloaded From: http://jama.jamanetwork.com/ by a Simon Fraser University User on 06/03/2015 ical, chemical or electric, the parotid glands exude a large that this principle is highly toxic when injected by itself, amount of a cretsny secretion having a pungent aromatic either suhcutaneously or intravenously, so small a quan¬ odor. The glands are certainly under the control of the tity as 1.5 mg., nearly causing the death of a very large central nervous system, as their secretion is discharged cat. in consequence of a peripheral irritation of sufficient Injected intravenously into a large cat, a quantity of strength. Budget!11 states that "the enormous parotid the emulsion containing less than 0.020 gm. of the crude glands are discharged like squirts when the creature is dried venom induced a tremendous rise of blood-pressure roughly handled." We have not ourselves observed any¬ followed immediately by a fall due to a sudden and thing of this kind, though we have repeatedly "milked" complete stand-still of the heart. as many as sixty of the animals at a time. Under chem¬ When a large quantity of the crude venom is intro¬ ical and mechanical stimulation we have only noticed duced into the stomach more marked symptoms are pro¬ free exudation from the openings of the gland. We duced. Thus, 0.1 gm. of the poison enclosed in a cap¬ obtained the needful amounts of this secretion by the sule was given on an empty stomach to a dog weighing simple device of squeezing (or "milking") the parotid 5.8 kg. In ten minutes a clear bile-stained fluid con¬ glands with a curved hysterectomy forceps, catching the taining most of the poison was ejected. This was fol¬ secretion as it spurted out from the numerous orifices lowed by retching and repeated vomiting and profuse into a large glass bowl held inverted over the toad. The salivation, all of which continued at frequent intervals semifluid substance thus obtained quickly dries in the for twenty minutes. For an hour following, during air to form hard, brittle scales of a yellow color, present¬ which time the animal was kept under observation, it ing much similarity in appearance to dried . was much depressed. On the following morning it bad When the air-dried scales are brought into contact with quite recovered. In regard to this experiment we would water they swell up to gelatinous masses, and in the say that the amount of epincphrin present in the venom presence of much water an opalescent, neutral foamy given to this dog would alone suffice to induce vomiting emulsion is obtained of a most nauseating bitter taste if given on an empty stomach. and pungent odor. In its physical properties, the secre¬ It may further be mentioned that an emulsion of tbe tion presents great similarity to some other animal crude venom is a powerful and rapidly acting aggluti¬ poisons, such as the Habu snake venom described by nating agent for the red corpuscles of the rabbit, the Ishizaka.12 only animal's blood tested in this connection. EXPERIMENTS MADE WITH THE CRUDE POISON THE PRESENCE OF EPINEPHRIN IN THE SECRETION OF A little of the emulsified secretion, well diluted with THE POISON-GLANDS 0.8 per cent, solution of sodium chlorid and instilled Introductory Note by the Senior Author (A.) into the conjunctival sac of the dog or cat, will quickly Fortune has thrown in our way an animal secretion which cause an extreme constriction of its blood-vessels so that most unexpectedly is found to contain a principle which has the conjunctiva becomes blanched. This action is due hitherto appeared only in the suprarenal glands and in homolo¬ mainly to the epinenhrin contained in the secretion, but gous chromaphil structures. In collaboration with A. ('. small also to the When this Crawford, I isolated the active principle of the suprarenal in part digitalis-like body. in the form of active latter substance, however, is to the in the glands of sheep and beeves physiologically applied eye salts which 1 named salts of epinephrin, for example, epin- form a of the it causes at first of solution pure crystals ephrin bisulphate, picrate, etc. The elementary .composition a of the smaller vessels of the momentary constriction of several compounds was established by analysis and for a dila¬ conjunctiva, but this is soon followed by marked epinephrin bisulphate, for example, was found to he repre¬ tation, so that great injection and irritation result. No sented by the formula13, C17H„NO,. H.SO,. dilatation of the vessels of the eve is observed when only In all of my published papers14 of that period (1897-1899) very dilute solutions of the crude poison are used, as in it was constantly emphasized by me that epinephrin is an this case the action of the epinephrin preponderates. unstable, basic substance, precipitatile from solutions of its A little of the emulsion injected into the abdominal active salts by ammonia and capable of being separated from the other constituents of the the proper of a frog will soon cause slowing of suprarenal glands by lymph-sac pithed use of chlorid.15 And this at a time, the with of the diastolic henzoyl may perhaps heart, apparent prolongation be permitted to say, when von Forth," another investigator rause and increase in the ventricular contractions. The in this field, was upholding the supposition that epinephrin is final is of the heart in effect complete stand-still systole. either tetrahydrodioxypyridin,

Downloaded From: http://jama.jamanetwork.com/ by a Simon Fraser University User on 06/03/2015 tion to those which belong to the entirely unaltered native wise there would be danger of injuring the epinephrin, compound and which were likewise given by my active salts. since the filtrate becomes alkaline. this work came the the unavoidably slightly Following next step, preparation of Then too, the with basic lead acetate must an amorphous indigo-colored iron compound of the active precipitation be undertaken with dilute solutions of the venom principle by von Fiirth,18 for which, however, at this time no highly as too an data were from which a molecular formula could only, otherwise large amount of epinephrin analytic given will in be calculated. Very soon after came the work of Takamine19 be retained the precipitate of inert substances. and Aldrich20 who succeeded in precipitating the base from When the filtrate, which has been freed from lead sul- concentrated extracts of the gland by the use of ammonia phid, has been sufficiently concentrated by distillation without the assistance of such agents as benzoyl chlorid or under diminished pressure it is only necessary to add chlorid of iron. The formula C,HuNOs, first proposed by ammonia to obtain the epinephrin in crystalline form. Aldrich, has to be the one which the proved truly represents By dissolving in acetic acid, adding a small amount of composition of our substance, but, as that chemist has sodium and with remarked, "it is to note in this connection that if sulphite again precipitating ammonia, interesting a ashless and almost colorless subtract a residue from Abel's formula for very pure, product is we benzoyl epi- obtained. obtain a formula—C10HloNO3—which is nephrin—CnH15N04—we of combustion not far removed from that of adrenalin." Such a result is The results analyses with products not due to chance and could have been obtained only from the obtained in this way are as follows : of individuals. study and analysis chemical I II III Next came the brilliant researches of the chemists, Dakin, Product Analyzed Product Analyzed Theoretical Require- Friedmann, Stolz and Flächer which have After Second Pre- After Third Pre- ments for Formula, Jowett, Pauly, cipitation ; No Sul- cipitation ; Sul- C0H13NO3 finally culminated in the synthetic production, first of the phite Used. phite Used. racemic and now of the levogyrate form as produced by C = 58.02 C = 58.68 C = 59.02 H = 6.87 H = 7.56 H = 7.10 Thus the combined labors of = Nature herself. by utilizing physi¬ 7.88; 7.88 = 7.05 ologists, pharmacologists, biologic chemists and organic chem¬ ists has science again unravelled one of Nature's secrets. It will be seen that the analytical results here given, The Council on Pharmacy and Chemistry of the American especially those obtained with the more highly purified Medical Association has lately adopted21 the name epinephrin preparation, stand in good agreement with the theo¬ for the active principle of the suprarenal glands in preference retical requirements for the accepted formula and prove to using a commercial or protected name and it is for this that the elementary composition of the epinephrin from reason that I am using the word in this paper instead of the toad is identical with that found in the suprarenal epinephrin hydrate, a name which I have had to abandon and of the animals. which I with the idea of the free base as glands higher proposed bringing Like the obtained from the isolated by Takamine and Aldrich into line with my mono- principle suprarenal benzoyl epinephrin. glands, Bufo epinephrin turns the plane of polarized to the left. As indicated the fine color light Epinephrin (0.078 gm.) obtained already green produced by from our toads and three times when dis¬ the addition of ferric chlorid to a solution of the crude crystallized, solved in 8 c.c. of water acidulated with acetic acid con¬ of our toad with the fact that such a poison together a trace of sodium a solution turns when to the air and taining sulphite, gave polarimetrie finally pink exposed of —1.00 in a two decimeter tube with that it exerts a action sufficed reading degree powerful vasoconstricting sodium light, whence : to show that we had discovered a substance closely allied 20° to, if not identical with the epinephrin of the suprarenal 1 =— 51.30°, the specific rotation. The of tests with D glands. results additional made solu¬ This rotation is in that tions of the crude confirmed this conclusion. perfect agreement with venom obtained Flächer22 with the For the addition of ammonia or other alkali by (—51.40°) highly puri¬ example, fied natural and differs from that obtained out a fine color which product, by brought pink appeared instantly Abderhalden and for the and was intensified the addition of a trace of iodin or Guggenheim23 synthetic levo- by the error of the instrument other and on with product by only. oxidizing agent, boiling Fehling's In to its solution or with ammoniacal silver nitrate solution regard physiologic activity—we need only reduction of both solutions occurred promptly. state that the rapid injection of so small a quantity as Two methods of isolating the newly found substance 0.000004 gm. (1 c.c. of a 0.0004 per cent, solution) 'into were one of which will here be outlined. the femoral vein of a dog weighing 6 kg. caused a rise employed, only of From 6 to 15 gm. of the air-dried venom were rubbed blood-pressure amounting to 20 mm. of mercury as measured the up with water until a thin, foamy, opalescent solution, in femoral artery and that the injection of 0.000008 c.c. of a or rather emulsion, was obtained. This was repeatedly gin. (2 (5.0004 per cent, solution) shaken with fresh quantities of a mixture of ether and caused a rise of 26 mm. The physiologic activity of the chloroform (1 : 4) by which means the digitalis-like substance is seen, therefore, to equal that24 of the purest body was removed. The solution was then diluted with specimens of epinephrin that have been hitherto isolated. much water, whereon the mucoid and other contami¬ It may also be stated that this epinephrin acts nating substances were quickly removed by precipitation promptly as a vasoconstrictor when applied to the blood¬ with basic lead acetate and the pinkish filtrate was vessels of the toad from which it is derived. In other immediately treated with hydrogen sulphid, freed from words, the animal has not acquired an immunity against the precipitated sulphid of lead and concentrated to a this poison ; small hemorrhages in this toad are as easily small bulk under diminished pressure. The above opera¬ controlled by a local application as in other animals, and tions from the moment of adding the solution of basic when a pithed toad is perfused with Locke's solution lead acetate to that of introducing the sulphuretted containing a small amount of the new epinephrin, its on with as other- hydrogen must be carried great celerity, arterioles are found to be just as responsive to the drug as are of or 18. Von F\l=u"\rth: Ztschr. f. physiol. Chem. (Hoppe-Seyler's), 1899- those the frog (R. clamata pipiens). So, 1900, xxix, 105. 19. Takamine: Therap. Gaz., 1901, xxv, 221; Am. Jour. Pharm., 22. Ztschr. f. physiol. Chem. (Hoppe-Seyler's), 1908-1909, lviii, 1901, lxxiii, 523. 189. 20. Aldrich: Am. Jour. Physiol., 1901. v, 457. 23. Ztschr. f. physiol. Chem. (Hoppe-Seyler's), 1908, lvii, 329. 21. Proprietary versus Unprotected Names, The Journal A. M. A., 24. Schultz, W. H.: Bulls. 55 and 61, U. S. Pub. Health and March 25, 1911,p. 910. Marine-Hosp Service, Washington, D. C.

Downloaded From: http://jama.jamanetwork.com/ by a Simon Fraser University User on 06/03/2015 also, dilatation of the pupil is as easily produced in the AMOUNT OF BUFAGIN" PRESENT IN THE VENOM excised of the toad as in that of the eye frog. have shown us that It is that the results Quantitative experiments 0.734 seen, therefore, obtained by of to 0.1 of chemical use gm. bufagin correspond gm. epinephrin analysis, by the of the polarimeter, by in and if and present the venom, that the epinephrin content quantitative qualitative physiologic experiments is 5 cent, we assume that the venom as have demonstrated that the substance per may secreted conclusively contains not far from 36 per cent, of isolated by us from the poison glands of Bufo agua is bufagin. identical with that produced by the suprarenal glands METHOD OF ISOLATING BUFAGIN of the animals. higher It is not difficult to isolate bufagin from the crude PERCENTAGE OF EPINEPHRIN CONTAINED IN THE VENOM venom. The poison is extracted from its solution in water with a mixture of ether and chloroform It is of interest to learn how an amount of by shaking large epi¬ as stated and the solvents are removed dis¬ nephrin may be isolated from a certain of the already by weight tillation until a small bulk of fluid remains. This is crude venom. from 5.42 gm. of venom, weighed only Thus, then into a volume of ether one hour alter it was out of the we poured large petroleum squeezed glands, which the as a white resin while obtained 0.243 of a of precipitates bufagin gm. crystalline epinephrin, yield the small amount of in 1.4 S cent. we holding fatty substances solution. per On the assumption that obtain by this The resinous method about two-thirds of the precipitate is taken up in chloroform and only epinephrin actually with much whereon in the crude venom the true content of again precipitated petroleum ether, present epi¬ it be from absolute alcohol. As would be nearly 7 per cent. (6.72 per cent.). may crystallized bufagin nephrin is soluble in this can from other in which the was fairly reagent crystallization only Judging experiments yield be effected from hot concentrated solutions. somewhat smaller, we conclude that the secretion prob¬ For of an ably varies in its percentage of epinephrin with the sea¬ purposes obtaining analytically pure product a more elaborate method was a son of the and other circumstances. But as the slightly employed, year of which will be later in the Journal method of isolation is a description given of not, strictly speaking, quantita¬ and The tive one, exact statements as to the of the Pharmacology Experimental Therapeutics. variability obtained this was content cannot be made. product by method reerystallized from epinephrin alcohol and from hot water. is but little' If we assume that the venom as secreted the animal finally Bufagin by soluble in water to the but dis¬ contains from 20 to 30 per cent, of water there would (0.0002 gm. c.c), by still be in accordance with the above analysis, solving the substance in a small volume of alcohol and present, this into much hot water an about 5 per cent, of epinephrin. In comparison with stirring drop by drop solution 1 or more this we would recall that one25 of us estimated that the aqueous (alcoholic) containing gram amount found in the of beeves is 0.3 in the liter may be obtained. From such solutions the suprarenal glands in clusters of whetstone- per cent. poison crystallizes glistening and which often attain a con¬ We would here state that we propose to examine the shaped crystals prisms poison glands of this toad by histologie methods for the siderable size. presence of the so-called chromaphil tissue, a tissue that PROPERTIES OF BUFAGIN has hitherto been in only found structures that elaborate As thus isolated, bufagin is found to contain no nitro¬ epinephrin, and we hope also to examine the skin secre¬ gen and to consist only of the elements, carbon, hydrogen tion of other species of toads for epinephrin, as we sus¬ and oxygen. Its melting point is 188°C. The substance pect that this principle is not confined to the poison is easily soluble in chloroform and acetone, fairly soluble glands of B. agua, but is probably to be found in the in absolute alcohol, but only very little soluble in petro¬ skin glands of other toads and perhaps also in those of leum ether and carbon tetrachlorid and also but little other amphibia. soluble in cold benzene. It is a "neutral" compound THE PRESENCE OF BUFAGIN IN THE SECRETION OF THE insoluble in alkalis or acids and in this respect, as in POISON-GLANDS most of its solubilities, it resembles the bufonin which Faust has isolated from the skin of the . It has been intimated that the venom of B. already If a mere trace of concentrated acid be agua owes its as an arrow poison in a sulphuric efficacy large added to acetic a trace of degree to the presence of a digitalis-like substance, anhydrid containing bufagin, a fine pure blue color is at once If the although the large amount of epinephrin present is an developed. in this action. And it is to the reaction is reversed so that a fragment of a crystal is dis important auxiliary solved in concentrated acid and a mere trace of former constituent or to substances closely allied to it, sulphuric acetic is then added a fine is obtained such as have been described by Faust,20 that toad skins anhydrid pink which soon into a color which for a owe their efficacy as a cure for dropsy, a method of treat¬ passes plum persists time. With iodin and concentrated acid ment which would perhaps still be in vogue had not long sulphuric William Withering, in 1775, introduced for this very the crystals give a play of colors ranging from red as is seen is purpose the foxglove, the active constituent of an old through green to blue, when cholesterin Avives' remedy. Now that science has begun to study the similarly treated. ancient remedy, toadskin, we may confidently expect Tf a dilute solution in chloroform is stratified over further discoveries in this field, possibly with results of concentrated sulphuric acid, a pink to deep red zone value for practical medicine. develops at the border line between the fluids, and when We have named the digitalis-like substance "bufagin" they are well mixed and then allowed to separate, the in order to have a short term sufficiently indicative of its chloroform solution gives up all of its color to the origin. sulphuric acid which now shows a greenish-yellow 25. Abel: Am. Jour. Pharm., 1903, lxxv, 301. See also Hunt, fluorescence. Reid: The Comparative Physiologic Activity of Some Commercial A observation shows that is dex¬ Suprarenal Preparations, The Journal A. M. A., Sept. 8, 1906, preliminary bufagin p. 790. trogyrate. Crystallized from water and dissolved in 8 26. Arch. f. exper. Pathol. u. Pharmakol., 1902, xlvii, 278; 1903, xlix, 1. c.c. of chloroform, 0.160 gm. gave a polarimetrie reading

Downloaded From: http://jama.jamanetwork.com/ by a Simon Fraser University User on 06/03/2015 of -4-0.44 degrees in a two decimeter tube with sodium The substances bufonin, C34H540,, and , C.u light from which it is seen that the specific rotatory H4COJ0, which Faust has isolated from the skin of the power is common toad, are no doubt also cholesterin-like bodies, as that has but their [.]"'=+ U· investigator suggested, relationship to cholesterin is not so immediately apparent as in the Combustion analyses27 show that the elementary com¬ case of bufagin, nor can it be expressed in terms of a position of bufagin can only be expressed in the formula, quantitative replacement of one element by another. or in terms of a this such C„ H12 02, multiple of formula, PHAJiMACOLOGIC ACTION OF PURE BUFAGIN as C27 3ß 0„. The following tables give some of our analytical results. As this communication has already exceeded the limits of an we shall not here ordinary "preliminary" paper, Obtained directly from venom, Product obtained by crystal- offer a detailed account of our numerous experiments on but crystallized from dilute sofu- lization from alcohol after a the action of this newest member of the tion in petrol ether -f- chloro- preliminary process of purifiea- pharmacologie form without any other process tion. "digitalis series", but will content ourselves with a bare of purification. C = 70.98 = of our (' 70.74 11= 8.55 statement work in this field. We have already said H = 8.67 that the substance exerts a vasoconstrictor action when an - III IV aqueous solution is applied to the conjunctival sac. This Product same as in II, only Theoretical requirements for action is also well demonstrated in was further purified by crystal- the formula. (\,II12Oä or for perfusion experiments lization from water. C^ll ™ ß. = (C,lll1„0o)a with the pithed frog or with the pithed toad. agua, C = 71.07 I 70.92 (' Bufo ="71.01 in case a dose of is H= 8.28 I 8.25 II = 7.05 only that this large bufagin required since the toad shows a high degree of immunity towards It will be seen that our analytical results, especially its own poison. Injected intravenously into a decere- those obtained with the pure product III, are in brate cat whose spinal cord is destroyed, a pronounced perfect rise of the is agreement with the elementary formula, ,, ., ., or pressure obtained. with some multiple of it as C27H3B06. The marked action of bufagin on the heart, its vaso¬ action and its The results of a single molecular weight determination constrictor powerfully stimulating action on the centre action which is com¬ by the boiling point method kindly made for us by Dr. E. cardio-inhibitory (an 'Emmet Eeid in the laboratory of Physical Chemistry in pletely annulled by section of the vagi and by atropin) us class with the mem¬ this University, are in favor of the formula, compel to this drug most effective simple bers of the series. In two out C9H1202. The molecular weight as calculated from the digitalis of six experiments rise of the boiling point of chloroform (0.144eC. with on dogs, in which large quantities of the drug were and in which standstill was corrections) with bufagin in solution was 168, whereas injected intravenously heart that calculated for C9H1202 is 152. The determination induced, this organ was found on opening the chest, to be was not to be because of the firmly contracted in systole. Stoppage of the heart in thought entirely satisfactory in series is rather limited amount of pure substance (0.578 gm. systole mammals after bodies of the digitalis an in our observation in bufagin in 87.2 gm. CHCL). Should molecular weight uncommon experience except determinations in other solvents substantiate the value experiments in which the organ is transfused by the and we make mention of the fact here given, it would appear that we are dealing not with Langendorff method,29 here as an evidence of the marked action of on an oxycholesterin as we at first surmised, but rather with bufagin the heart A more detailed with an oxyproduct of a cholesterin nucleus, for certainly the muscle. comparison chemical and physical properties of the substance point other members of tbe digitalis group will be given later, to an undoubted and intimate with choles¬ as we propose to continue our chemical and pharma¬ relationship studies with this terin from which there is no escape. cologie drug. Cholesterin, C27H4t;0, is a monatomic alcohol found in all structures. It is a con¬ protoplasmic fairly large EQUIPMENT AND INSTRUCTION OF THE stituent of the nervous system, of sebum and other oily secretions of the skin, of bile and other fluids and tissues LABORATORY YEARS of the body. Of late years this substance has begun to E. P. LYON, Ph.D. play a large rôle in the studies on immunology and Professor of Physiology and Dean. St. Louis University School of and the of its Medicine serology problem unraveling complex ST. LOUIS chemical constitution is now the attention of absorbing Before to discuss the set before us, a number of chemists. attempting subject organic it be well if we as to the function of a medical Our studies above outlined would lead one to surmise may agree and as to the aim of all of us who devote our that is a When school, bufagin depolymerized oxycholesterin. lives to medical education. In an address last the two molecular formulas, year, Dr. C,7H3eOe= (C9H1202).,, Welch1 said that our was "to make doctors." and C27H400 are it is seen that object good (cholesterin), compared Professor Minot,2 in his com- in the former five atoms of oxygen have taken the Washington University place mencement a more exact formulation in of an ten atoms of in the latter. So address, gives equivalent hydrogen these words: "The chief function of a medical school also a is indicated between our substance relationship is to Whichever definition we and the saturated a- and produce practitioners." hydrocarbons ß-cholestan, C27 we doubtless on the main and I ask obtained from cholesterin Diels and Linn.28 We prefer, agree idea; H4S, by therefore, to bear in mind that our purpose is to are not aware that the molecular of these choles- you, weight make or and that is a tans has been determined, but if we assume for them the produce something, something lowest possible molecular formula, C9Hltl, then here good physician or practitioner. Arch. f. Pathol. Pharmakol., we come the formula if 29. Gottlieb and Magnus: exper. p. again to for bufagin, C9H]20,, 1904, li, 30. we substitute two atoms of oxygen for four .hvdrogen Read before the seventh annual conference of the Council on Medical Education of the American Medical Association, Chicago, March 1, 1911. 1. Proceedings of the Association of American Medical Colleges, 27. Complete data for these analyses will be given in a later paper. 1910, p. 62. 28. Ber. d. deutsch. chem. Gesellsch., xli, 544. 2. The Journal A. M. A., 1909, liii, 502.

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