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JOURNAL OF C m m c EDUCATION ~~
FEBRUARY, 1926
THE PREPARATION AND CHEMISTRY OF INSULIN A. SBONLE, L ~ L R&SEARCH Y LAEORATORIBS, ELI L ~ L &YCO.,INDIANAPOLIS HORACE Introduction1 As early as 1856 it was known that the ligation of the pancreatic ducts caused the pancreas to degenerate without causing any symptoms of diabetes.' Some thirty years later Minkowski and v. Mehring3demonstrated that the pancreas was the seat of diabetes, when they were able to produce a diabetic condition in dogs similar to human diabetes by the complete extirpation of the pancreas. Somewhat later, it was demonstrated indirectly that an internal secretion of the pancreas was responsible for the normal metabolism of fats and carbohyd~ates.~Other investigators noted that when pancreatic degeneration occurred after the ligation of the ducts, all of the pancreatic tissue disappeared with the exception of the Islets of Langerhans? I t had been suggested previously that it must be these cells which produced the internal anti-diabetic secretion.' During the third of a century which had elapsed since the discovery of Minkowski and v. Mehring, many investigators vainly endeavored to isolate a substance from the pancreas which would alleviate or remove the symptoms of diabetes. Practically no advance had been made until Drs. Banting and Best reported in 1922 that they had succeeded in isolating the antidiabetic hormone from degenerated pancreas.' During this thirty years, numerous investigators, endeavoring to secure a direct proof of the existence of a pancreatic hormone, tested the pancreas and many pancreatic extracts of one sort or another. These preparations were usually administered to dogs rendered artificially diabetic by the removal of the pancreas, although in some instances they were given to diabetic people. Now and then an investigator reported that the use of pancreatic tissue or of an extract caused a diminution in the excretion of sugar in the urine. None of the preparations, however, could be used clinically since toxic symptoms always developed on repeated injections, and the results secured were either negative or else not favorable enough to justify their further use. The chief mode of administration of the pancreas itself or of extracts of it appears to have been by mouth. No results could have been secured by this method even if any of the extracts had been potent, since we now know that the enzymes of the alimentary tract destroy the hormone. Subcutaneous or intravenous injections of the minced pancreas or of extracts gave conflicting results which were finally interpreted as negative since the investigators one after another abandoned their experiments. It is worthwhile to mention a few of the investigators of this periodCaparellis was one of the first to report (1892) the preparation of a pancreatic extract which lessened the sugar excretion of a depancreatized dog.
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Rennie and Frazer9 gave subcutaneous injections of the separate islet tissue of the pancreas of certain bony fishes, but failed to secure favorable results. Zuelzer (1908),1° among other methods, minced pancreas with weak bicarbonate solutions and after allowing them to autolyze for several days extracted them. He precipitated the proteins in the extract with alcohol and concentrated the protein-free filtrate. In some instances the use of this preparation was able to alleviate part of the symptoms of diabetes but as it was too toxic for continued administrations, the experiment was abandoned. While Zuelzer carried out his extraction under conditions which favored the hydrolytic action of the trypsin, other investigators endeavored to inactivate the trypsin during the extraction without, however, succeeding in the preparation of active extracts. E. I,. Scott," in 1912, attempted to eliminate the enzymes of the external secretion of the pancreas by ligating the duct just as Banting and Best later did successfully. As he did not succeed in completely ligating the ducts, he did not secure a complete degeneration of the cells which secrete the enzymes. He then made alcoholic extractions of whole pancreas and also aqueous extraction after a preliminary alcoholic extraction, but he was unable to secure a preparation capable of being administered repeatedly. There was but slight evidence that the extracts were capable of lessening the sugar output and the work was given up. Knowlton and Starlingx2made aqueous acid extractions of the pancreas which enabled the heart of a diabetic dog to burn sugar. Since this result could not be duplicated later, the experiment was abandoned. Murlin and KramerlJ prepared aqueous acid extracts of the pancreas which were made alkaline and injected. The sugar excretion of diabetic dogs was lessened but they concluded that this was due to the action of the alkalinity of the material injected. Kleiner14 reported a marked decrease in blood sugar after the intravenous injection into animals of an unfiltered aqueous pancreatic extract. No attempt was made to purify the extract so that i t would be devoid of toxic effects. Exact and rapid micro methods of determining the amount of sugar in the blood had, in the mean time, been developed. By observing the changes occurring in the blood sugar of diabetic dogs after the administration of pancreatic extracts, i t was now possible to determine whether or not the injected extract contained the hormone, since the presence of the hormone is shown by a lessening of the amount of sugar in the blood. Use was made of this method in the discovery of insulin. I n 1920, Dr. F. G.Banting conceived the idea that the previous failures to isolate the antidiabetic hormone from the pancreas were due to its destruction by the proteolytic enzymes of the pancreas during the process
of extraction. As stated above, by completely ligating the pancreatic ducts for a long period of time the total degeneration and disappearance of the enzyme secreting cells occurs, leaving only the cells of the Islets of Langer-
Dr. I>.G. Uanting, lsolator ol Insulin
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hans which are believed to secrete the internal principle. He reasoned that it ought to be possible to extract the active principle of the internal secretion from the degenerated enzyme-free pancreas. With the cooperation of Mr. C . H. Best the idea was tested out in the department of physiology of the University of Toronto under the direction of Professor J. J. R. Macleod. A simple extraction of the minced degenerated pancreas with cold saline solution gave an extract, which was capable, on injection into dogs, made diabetic by the total removal of the pancreas, of reducing the blood sugar and lessening the sugar excretion in the 24-hour urine of the animal.' Similar extracts of the pancreas of foetal calves removed a t a stage of development before the enzymes had formed, were similarly effective in reducing the blood sugar on injection.15 Extracts from normal adult, pancreas made with acidulated alcohol were found to contain the effective substance. By the use of acidulated alcohol, the enzymes were destroyed and the amount of solids in the extract decreased. With the cooperation of Dr. J. B. Collip a process for the purification of the acid alcoholic extract from the adult pancreas was perfected.l6 This process, which forms the basis for the methods now used in this country, consists of extracting the fresh minced pancreas with an approximately equal volume of acidulated alcohol, and the separation from this extract of that fraction which is soluble in 80 per cent, but not in 93 per cent alcohol. This fraction which contains the active substance also still contains a great amount of inert extracted material and must be p d e d untilthe solids are reduced to one to two per cent of the amount originally present.
The Preparation of Insulin" Numerous methods have been described practically all of which are based on the extraction of the minced pancreas with an acidulated water-miscible organic solvent such as acetone, or methyl and ethyl alcohol. The use of acid or alkaline aqueous extractions has been reported favorably but other laboratories have not been able to duplicate the results, especially on a larger scale. While the fresh pancreas of any animal will yield msulin, the pancreas of hogs and cattle are used for its commercial production. The glands, freed from fat and connective tissue, are placed in a refrigerator a t the slaughter houses and transported in iced containers to prevent autolysis. On arriving a t the factory they are inspected, weighed, and ground up by can be 'handled large power choppers. Hundreds of pounds of daily. The finely ground glands may either.drop into an equal volume of alcohol (ethyl alcohol containing 10 per cent, of pure methyl alcohol) acidified with about one per cent sulfuric acid or they may be mixed with '
a quarter of their weight of acidified water and the alcohol then added. Since it is difficult to remove traces of metals from the pancreatic extract,
SCIIN*1.-Bciorc
alcoholic rrtractiun the pancreas glands are ruu ihrough grindrrs.
enameled or earthenware apparatus must be used in every step after the glands leave the choppers. After several hours' thorough mixing, the alcoholic extract is removed
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by centrifuging in a large basket centrifuge having a rubber-lined basket. The residual gland material is removed from the centrifuge and reextracted with 60 per cent alcohol and this extract is likewise separated by the centrifuge. The two extracts which are now mixed have an alcoholic content of about 60 per cent and contain fine particles of suspended glandular material. Clarification may be effected either by filtering through large funnels containing fluted filter papers or by passing i t through high speed Sharples centrifuges. The clarified alcoholic extract, the acidity of which has been partially neutralized, is concentrated in hundred-gallon, steamheated, enameled vacuum stills to about one-twelfth to one-fifteenth of its original volume. During the distillation, extreme care must be exercised to prevent overheating which will destroy the insulin. Batteries of huge rotary vacuum pumps and the use of cold brine in the condensing columns enables the thousands of gallons of alcohol to be distilled off a t a temperature not exceeding 25% With the complete removal of the alcohol a fat fraction separates out on warming and is removed by skimming and filtration. From this step either of the two following procedures may be used without making any marked difference in the yields. In the method used a t the Connanght Laboratories of the University of Toronto, ammonium sulfate is added to the fat-free, concentrated extract until a point of half saturation is reached.ls This concentration of ammonium sulfate causes the insulin together with all proteins and their primary split products to flocculate out of solution and come to the surface, leaving the (lower) proteoses, peptides, etc., in solution. The flocculent material is skimmed off, yielding about 200 grams of moist precipitate from 300 pounds of glands. This precipitate is dissolved in warm, acidified alcohol and the solution is then neutralized. This causes the insulin fraction to precipitate. After standing several days a t 5 T . , the dark colored supernatant alcohol, containing a considerable amount of impurities and no insulin, is discarded 'and the percipitated insulin fraction is dried, in vacua, and then dissolved in acidified water. This solution is then made alkaline to a pH of 7.3 to 7.5, a t which degree of alkalinity a dark colored precipitate of inert protein material settles out and is removed. The clear solution is acidified to a flH of 5.0 which causes the precipitation of the insulin. After standing a t 5°C. for some days to insure the complete precipitation of the insulin, the snpernatant liquor, containing inert materials, is discarded and the precipitated insulin is again dissolved in acidified water and again enough alkali is added to bring the pH to 5.0, thereby precipitating the insulin. After standing a t 5°C. for several days, the precipitated insulin is removed and
is now of a sufficient degree of purity to be used. This process of purification is the so-called isoelectric precipitation method. The insulin is then dissolved in acidified water a t a pH of 2.6, to which 0.1% tricresole is added as a preservative and enough sodium chloride added to make the solution isotonic with the blood. The solution is now tested for potency on rabbits, sterilized by passing through a Berkfelt filter and ampuled. The other methods used follow more closely the procedure of C ~ l l i p . ' ~
Scrmr: 2.-?';inks
oi cxtractcrl alcol~olicliqnid a w a i t h g clnrilicntion '!,I
centiiIugc.
After the removal of fat from the concentrated extract, alcohol is added until the concentration reaches 80 per cent. The heavy precipitate, consisting of inert protein material, is removed by filtration and is discarded. The concentration of alcohol in the filtrate is increased to 93 per cent which causes a second precipitate to form. This precipitate, which contains the insulin together with much inert protein material, is removed by filtration and dissolved in acidified water.
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On the careful addition of dilute alkali to the acid solution until a pH of about 5.0 is reached, the insulin precipitates, leaving much of the inert substances in solution. By repeating this isoeledric method of precipitation the insulin is secured of an adequate purity for clinical use. One unit of the insulin represents but 0.05 to 0.07 mg. of organic solids. The final precipitate is dissolved in acidified water to which are added 0.1% tricresole and sufficient sodium chloride to make the solution isotonic. The solution is tested, sterilized by passing through the Berkefeld filter, retested, and ampuled. The more important of the other reported methods follow. Best and Scott," who extracted pancreas with both hot and cold acidified water, found that while the method was successful in the laboratory, it could not be successfully applied to large scale production. Other investigators report similar results. After preparing crude insulin according to Collip's procedure, Dudley18 purified it by preparing the insoluble picrate, removing the water-soluble impurities and then converting the insulin picrate into insulin hydrochloride. Dudley and StarlingZodescribe a modificationof the preceding process which involves the mixing of the minced pancreas with sodium bicarbonate, followed by extraction with alcohol. By this method less inert material is extracted. The crnde insulin is then purified by the picric acid method. The British Drug Houses, Ltd., are reported to make use of the alkaline alcoholic extraction method followed by the picric acid method of purif i c a t i ~ n . Dodds ~~ and DickensZ2extract the pancreas with a 1 per cent formic acid solution containing 5 per cent paraldehyde. The crude insulin is then purified by the picric acid method. They also have developed a picric acid-acetone method of extraction, finding that insulin picrate is more soluble in 70 per cent acetone than are the picrates of the inert proteins which accompany it. The method of Doisy, Somogyi, and ShaEer which has already been referred tol8 is based upon an acid alcoholic extraction of the pancreas followed, after the removal of the alcohol and fat, by a precipitation with ammonium sulfate. Maloney and Findlayt3 describe a method in which the crnde insulin is purified by adsorption on charcoal. After removing the charcoal and washing, the insulin is released by alcoholic benzoic acid. MurlinZ4has found that insulin can be secured in a purified state by perfusing the pancreas with an acidified Ringers' solution.
Standardizationz6 Insulin is required for the normal burning of carbohydrates in the body, but if an excess is present in one's system, the m&abolism or burning of the carbohydrate goes on so rapidly that the normal sugar content of the blood is greatly lowered. When this lowering (hypoglycemia) reaches a
certain minimum, the animal or person is seized with convulsions, which can be antidoted instantly by the administration of sugar hypodermically or orally. In rabbits, the usual test animals, the convulsions occur when the blood sugar content has dropped from the normal value of about 0.1 per cent to 0.045 per cent. The unit of insulin is defined as one-thud of the amount required to lower
the blood sugar of a 2.0 kgm. fasted rabbit to 0.045 per cent over a period of 5 hours in 60 to 70 per cent of the animals employed for the test. Insulin is standardized by three methods, first the amount of insulin solution required, on subcutaneous injection, to produce convulsions per kilogram of rabbit is found. Then the amount per kilogram required to lower the blood sugar to 0.045 per cent during a period of 5 hours after injection is determined. Finally the insulin is given to diabetic patients to determine actually its ability to burn carbohydrate in order to be
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absolutely certain that the lot of insulin will enable the diabetic to metabolize properly the carbohydrate of his diet. As many as a thousand rabbits are used for the complete standardization of a large commercial lot of insulin. The Chemistry of Insulin Insulin is associated with proteins and none of the methods of purification reported have succeeded in separating an active uon-protein substance possessing the properties of insulin. I t is true that several investigators reported that they secured negative tests for protein in physiologically active solutions, but this was because the sensitivity of the physiological test was greater than the sensitivity of the protein test. Since the publication of the paper with Waldo,2ewe prepared in 1924 a number of samples of insulin having a purity of 50 to 60 units per mg. and one which was active at 100 units per mg. The purification was carried out by using various modifications of the isoelectric precipitation method. It has not been found possible to secure a higher degree of purity by these methods, nor do all lots of insulin permit a purification to the degree of 100 units per mg. In the purest products tested, nothing other than protein substances could be detected. Whether insulin is itself of a protein nature or whether it is merely associated with a protein fraction of the pancreas still remains to be determined. It was early noted by numerous investigators2' that the physiological activity of insulin was destroyed by proteolytic enzymes, such as pepsin, papain, trypsin, or erepsin. The complexity of the protein molecule in the insulin, therefore, must be greater than that of the active principle of the pituitary gland, which is not attacked by pepsin and which KammZ8 has shown has a molecular weight of about 1000. De6nite proof is available20.3" to show that the destruction by enzymes is due to the splitting apart (hydrolysis) of the protein molecule, rather than to an inactivation due to the formation of a physical complex of the insulin and enzymes, although the latter undoubtedly is the first step in the process of destruction by enzymes. The insulin of commerce cannot be considered as an unaltered natural protein, since the process of extraction is such that it would undoubtedly have converted the natural proteins of the pancreas into such derivatives as acid proteins and even the higher proteoses. While pnrified insulin has some of the characteristics both of proteins and primary proteoses, it has seemed more fitting to us to characterize the fraction as a primary proteose. Insulin will not coagulate with heat, it will slowly dialyze through a parchment membrane, and the precipitate which occurs with the addition of nitric or trichloroacetic acid disappears on warming to reappear again on cooling. All of these tests are characteristic of the
primary protein. Insulin is insoluble in water although its hydrochloride and sodium salt are soluble just as is the case with many proteins and proteoses. It is insoluble in ether, benzene, and chloroform, but is soluble in glacial acetic acid and phenol. Protein precipitants such as tungstic, tannic, and picric acids precipitate it. The active fraction can be recovered from these precipitates by suitable methods. Half saturation with ammonium or zinc sulfate in acid solution causes the insulin to flocculate out of solution. Insulin is readily adsorbed by kaolin and charcoal; Lloyd's reagent and positive and nega-
SCENE4.-The
scpaiation oi the final product by filtration
tive colloids precipitate it. Since the inert substances accompanying the insulin are likewise adsorbed, i t has not been possible to carry the purification of insulin very far by differential adsorption. Insulin is slowly destroyed by boiling with 0.25 per cent-sulfuric acid and rapidly destroyed by boiling in a solution barely alkaline (pH 8.69.0). This destruction of activity parallels in a remarkable manner the splitting of the primary proteose molecule of the insulin into a secondary proteo~e.~~ The same parallel occurs when insulin is incubated with trypsin. The slightest disintegration of the proteose molecule seems to be sufficient to
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destroy the insulin. We have been unable to re-activate the insulin, if actual disintegration of the molecule has wcurred. When insulin has been destroyed by any of the above methods the resulting solution gives much the same general precipitation reactions as does the original solution. Only an exact quantitative analysis will detect the distinct but slight change which has occurred in the proteose molecule. Complete hydrolysis of the insulin to its constituent amino acids and the separation of these amino acids into groups by the method of Van Slyke has been carried out on some ten insulin samples of varying degrees of purity. It has not been possible to detect by this method any significant difference in the composition of insulin of a purity of 10 to 15 units a mg. or of a purity of 50 units per milligram, although widely Merent methods were used in the purification. The composition as shown by this method is similar to that of serum albumen and serum globulin and apparently only amino acids are present. Purified insulin contains neither carbohydrate nor phosphorous. It gives a positive test for protein, a positive test for tyrosine, a positive test for histidine, a positive test for cystine, a positive test for arginine, and a negative test for tryptophane, purines, and pyrimidines. A positive test for reduced sulfur is secured after boiling the insulin with alkali. So far, then, we have found nothing which is not of a protein nature. It is possible that our purest insulin still contains such a large preponderance of inert proteose that it is impossible to obtain any test other than those for amino acids. Physiological tests often are many times more sensitive than chemical tests. Drs. Abel and Geiling3' have purified insulin by the use of phenol and pyridine. They report that insulin readily splits off reduced sulfur on mild alkaline treatment in a manner which is not characteristic of proteins. Insulin is readily destroyed by oxidation with dilute solutions of hydrogen peroxide or potassium permanganate. Reducing agents such as sodium bisulfite, sulfur dioxide, etc., destroy it. Mnrlin3= recently reported on the recovery of the activity of reduced insulin by aeration. Insulin is inactivated by formaldehyde,provided that the reaction occurs in a neutral medium. Iodine slowly inactivates it after tirst causing a precipitation. The benzoyl derivative is inactive. Treating an etherial suspension with acetyl chloride does not destroy the activity. Coupling insulin with such substances as diazotized sulfanilic acid destroys the activity.33 Conclusion Dr. Banting with the coijperation of C. H. Best was able to avoid the pitfalls which checked other investigators during a thirty-year search for insulin. After successfully demonstrating its presence in the pancreas,
methods for its commercial production in a highly purified state were developed through cooperative work with an industrial laboratory. Thorough investigations of purified insulin by different laboratories have been unable to separate an active fraction free from protein, nor has careful investigation of the active proteose fraction itself shown the presence of other than amino acids-the building stones of the proteins. Insulin itself may be a biologically specific proteose, or it may be that the presence of a protein or proteose structure is necessary for the successful functioning of some substance of a non-protein nature which as yet has defied detection. RBFERBNCSS 1. A more complete review can be found in the fallowing references:-Dale, H. H., Lancet, 1, 989 (1923); Macleod, J. J. R.,Br. Med. Jr., 2, 833 (1922); Best, C. H., and Scott, D. A,, J . Biol. Ckem., 57,709 (1923); and Murlin, J. R.,End., 7, 519 (1923). 2. Bernard, Claude, Lccons de physiol. Exp., 2, 274 (1856); and Schiff, Med. Centrdb., 790 (1872). 3. Minkowski, O., and v. Mehring, F. J., Centralb.j. klin. Med., 10, 393 (1889), and Arch. j. exper. Path. u. Phermakol., 26, 371 (1889); Ibid., 31, 85 (1893). 4. Lepine, Lynn Medical, 62, 630 (1889); Minkowski, O., Ber. klin. Wochenschr., 90 (1892); Hedon, Arch. de Physzoiogie, Oct., 1892. 5. Amozan and Vaillard, Arch. de Physiol. n o m et Path., 3, 287 (1884); and Schultz, Arck. j. Mikroskop. Annt., 56,491 (1900). 6. Laguesse, E. G., Compt. rend. Sac. de Biol., 44 (1893); and Schafer and Diamare, Internat. Monut. j. A m t . u. Phys., 16, 155, 177 (1899). 7. Banting, F. G., and Best, C. H., Jr. Lab. and C h . Med., 7, 251 (1921-22). 8. Caparelli. B i d . Zentralb., 12, 606 (1892). 9. Rennie, J., and Frazer, T., Biockem. J . , 2, 7 (1907). 10. Zuelzer, G., Zeitschr. f. exper. Path. u. Thwafi., 5,307 (190849); and Zuelzer, G., Dohm, M.,.and Manrer, A., Deutsch. Med. Wochenschr., 34, 1380 (1908). 11. Scott, E. L., Am. I. Physiol., 29, 306 (1911-12). 12. Knowlton, E. P., and Starling, E. H., J. Physiol., 45, 146 (1912-13). 13. Mnrlin, J. R.,and Kramer, B., 5 . B i d . Chem., 15,365 (1913). 14. Kleiner, I. S., Ibid., 40, 153 (1919). 15. Banting, F. G., and Best, C. H., J. Lab. and Clin. Med. 7, 464 (1921-22). 16. Banting, F. G., Best, C. H., and Collip, J. B., Canad. Med. Assn. J., 12, 71 (1923); and Banting, F. G., Best, C. H., Collip, J. B., and Macleod, J. J. R., Trans. Roy. Soc. Cannda, 16, 1 (1922). 17. The work on the large scale production of insulin a t the LiUy Research Laboratories was carried out by Geo. B. Walden under the direction of Dr. G. H. A. Clowes. The first portion is an abstract of the process as described by Best, C. H., and Scott, D. A., Biol. Ckem., 57, 709 (1923), and Ind. Eng. Chem., 17, 238 (1925). 18. The use of ammonium sulfate for the purification of insulin is also described by Doisey, E. A., Somogyi, M., and Shaffer, P. A,, J. B i d . Ckem., 60,3 (1924). 19. Dudley, H. W., B i o c k . J., 17, 376 (1923). 20. Dudley, H. W., and Starling, E. H., Ibid., 18, 147 (1924). 21. Renshaw, A:, J . Sac. Chem. I d , 44, 95T (1925). 22. Dodds, E. C., and Dickens, F., Br. J. Exp. Path., 5 , 115 (1924). 23. Maloney, P. J., and Findlay, D. M., Amer. J. Phys. Chem., 28,402 (1924). 24. Murlin, J. R., Clough, H. D., Gibbs, C. B. F.. and Stone, N. C., Amcr. J. Physiol., 64, 348 (1923).
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by Macleod, J, J. R.. and 25. A thorourrh discussion of standardization is ziven Orr, M. D., 3. ~ e end i CLin. Med., 9, 591 (1924). 26. Shonle. H. A,, and Waldo,~. 5. H., 3. Biol. Chen., 58, 731 (19%). 27. See references 16, 17, 18, and 19, and Witzemann, E. J., and Livshis, L., Ibid., 57, 425 (1923). 28. Kamm, O., in paper presented a t Washington meeting, A. C. S., 1924. 29. Scott, D. A., 3. Bid. Chem., 63, 641 (1925); and Epstein, A. A,, and Rosent h a t N., Amer. 3. Physiol., 70, 225 (1924). 30. Shonle, H. A., and Waldo, J. H., in papers presented a t the A. A. A. S. meeting, Cincinnati, 1923, and a t Washington meeting, A. C. S., 1924. 31. Abel, J. J., and Geiling, E. M. K., 3. Pherm. a d E r t . Therap., 25,423 (1925). 32. Allen, R. S., and Murlin, J. R., Proc. Soc. Exp. Bid. Med., 22, 492 (1925). 33. Unpublished data from this laboratory. ~
