BIOLOGY LIBRARY G THE METABOLISM OF BILE ACIDS A THESIS ACCEPTED IN PARTIAL SATISFACTION OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY AT THE UNIVERSITY OF CALIFORNIA BY MARJORIE GREENE FOSTER. 1919 THE METABOLISM OF BILE ACIDS I. A QUANTITATIVE METHOD FOR ANALYSIS OF BILE ACIDS IN DOG'S BILE BY M. G. FOSTER AND C. W. HOOPER (FROM THE GEORGE WILLIAMS HOOPER FOUNDATION FOR MEDICAL RESEARCH, UNIVERSITY OF CALIFORNIA MEDICAL SCHOOL, SAN FRANCISCO) REPRINTED FROM THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. XXXVIII, No. 2, JUNE, 1919 B/OLOGY UBRfi RY G Reprinted from THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. XXSVil 1 ., No. 2, l c l&. THE METABOLISM OF BILE ACIDS. I. A QUANTITATIVE METHOD FOR ANALYSIS OF BILE ACIDS IN DOG'S BILE. BY M. G. FOSTER* AND C. W. HOOPER. (From the George Williams Hooper Foundation for Medical Research, Uni- versity of California Medical School, San Francisco.} (Received for, publication, April 28, 1919.) An unusual opportunity for a comprehensive study of the bile acids presented itself to us in this laboratory because of the pres- ence of a number of bile fistula dogs under careful routine observation. These bile fistula dogs were under observation for the study of bile pigments. They present normal factors of weight, activity, and appetite when under the laboratory routine care which has been described in detail by Hooper and Whipple (8). In order to study satisfactorily the metabolism of the bile acids it is necessary to have a method for the analysis of bile acids which is relatively simple and accurate and which does not require large quantities of material for analysis. The method must be specific for bile acids and react negatively with the other substances in bile and it must not be obscured by any substances which may be present in bile fistula bile. We know of no pub- lished method which meets all these requirements. As a preliminary step we undertook to test a number of the clinical methods for the determination of bile acids, some of which are known to be inaccurate and others of which are claimed to be merely qualitative. Without exception we have found these *This series of papers on Bile Acid Metabolism was completed just prior to the death of Miss Foster from influenza pneumonia. The work should stand as a memorial to her enthusiasm, patience, and spirit of truth- ful research. This work was submitted as a thesis for her degree of Doctor of Philosophy, University of California. 355 356" * Metabolism of Bile Acids. I V.: /:>:'";: : A '': feMrvdy 'siiiipfe elmical' methods to be grossly inaccurate and useless for analysis of bile salts. This statement applies to a re- cent method advocated by Hoover and Blankenhorn (9). No controls are given by these workers to show that many other substances present in abnormal sera may not have been respon- sible for the positive bile acid reaction noted in their experiments. For a discussion of these possibilities refer to the paragraph below on Pettenkofer's test. Some of the more elaborate chemical methods for the ex- traction of bile salts from bile are' relatively accurate, but the large quantity of material used and the amount of time required make frequent analyses at short intervals impracticable. These extraction methods are very expensive as, well as time-consuming. The older methods may be divided into three groups : (1) methods in which the bile acids are separated and weighed; (2) methods in which the bile acids are calculated from the sulfur content in whole bile; and (3) methods which depend on the color re- actions of cholic acid.* There are possibilities of error in all these methods, as will be pointed out later. Methods in Which the Bile Acids are Separated and Weighed. Huppert's Method 1864. Huppert (11) modified Neukomm's (17) meth- od for bile salts in urine and applied it to the bile and blood in the following way. The albumin was first coagulated with alcohol and the precipitate carefully extracted with additional amounts of alcohol. The whole extract was evaporated, dissolved in water, freed from fat by ether extraction, neutralized, and Ba(N0 3 )2 added to separate the fatty acids, soaps, and any remaining protein. The washed precipitate was supposedly free from bile acids. The filtrate and wash water were precipitated with lead acetate and the precipitate washed with water to free from excess acetate. The precipitate was then washed out with alcohol, heated, and NagCOa added. After it was evaporated to dryness, the residue was ex- tracted with absolute alcohol; the alcohol was evaporated to dryness; and the residue was dissolved in water. This water solution was filtered into a weighed dish and dried to constant weight. Socoloff's Method 1875. Socoloff (19) published a method in which the bile acids present were calculated by the amount of bile soluble in alcohol. This work was done in Hoppe-Seyler's laboratory using a method very much like the one described in the following paragraph. Hoppe-Seyler's Method 1881. Hoppe-Seyler's method (10) was used by Pfaff and Balch, Stadelmann, and others. The bile was dried to con- stant weight at 110C. and the residue completely extracted with boiling M. G. Foster and C. W. Hooper 357 absolute alcohol. The extract was allowed to stand 24 hours, filtered, and evaporated to dryness at 110C. The residue was dissolved in absolute alcohol, evaporated to a small volume, cooled, and precipitated with a large excess of absolute ether. The bile salts which crystallized out were dried at 110C. and weighed. Stadelmann (21) evaporated the bile to a thick syrup and allowed it to stand in contact with alcohol on the water bath some time in order to coagulate the protein and give a clear filtrate. He then repeated the extraction three times with boiling 96 per cent alcohol. He dissolved the final precipitate in water and dried to constant weight. Croftan's Method 1902 Croftan (3, 4) estimated the bile acids by. coagulating the albumin, evaporating the filtrate and washings, and pre- cipitating with absolute alcohol to free from salts. The filtrate was diluted with water, precipitated with basic lead acetate and ammonia, and the precipitate extracted with absolute alcohol and filtered hot. This solution of bile salts was dried to constant weight. (According to Croftan the mucin holds with it a large amount of bile acid and they are so closely bound that it is impossible to separate them even by repeated washings.) Goodman's Method 1906. Goodman (5) hydrolyzed 50 gm. of bile with 125 gm.of 60 per cent KOH in a reflux condenser for 24 hours. The solu- tion was extracted five times with 75 cc. of freshly distilled petroleum ether in order to extract the cholesterol. The last of the petroleum ether was evaporated off on a water bath and the solution precipitated with 5 per cent BaCl 2 to separate the higher fatty acids. The precipitate was ex- tracted with boiling water and the filtrate and washings evaporated to 200 to 300 cc. The solution was cooled in ice and salt, and acidified with HC1. After standing 2 hours the cholic acid which crystallized out was filtered off, washed, dried, and extracted in a Soxhlet apparatus with ace- tone. In about 5 hours all the cholic acid was dissolved and the solution was evaporated and dried to constant weight. Methods in Which the Bile Acids Are Calculated from the Sulfur Content in Whole Bile. Spiro's Method 1880. The sulfur method as used by Spiro (20) con- sisted in fusing a definite amount of bile (usually 50 cc.) in a silver dish with KOH and KNO 3 . The mixture was dissolved in water, supersatu- rated with HC1, and precipitated hot with BaCl 2 . This precipitate was filtered on a paper of known ash, washed well, and dried. The main part of the precipitate was transferred to a porcelain crucible. The paper with the remainder of the precipitate was ignited and weighed. The main part of the precipitate was weighed, a few drops of concentrated H2SO4 added, washed out well with water into another filter paper, and this was weighed. The difference in weight showed the amount of the impurity. From this difference was calculated the BaSO4 in the other portion of the precipitate which was weighed with the ash of the larger filter paper, and the total amount of sulfur was calculated from this corrected value. 358 Metabolism of Bile Acids. I Von Bergman's Method 1904- Von Bergman (2) precipitated a 24 hour collection of bile with many volumes of 96 per cent alcohol. The mu- cin precipitate was filtered off and washed six or eight times with alcohol. The combined washings and filtrate were evaporated to a definite volume and an aliquot part used for the determination of the combined sulfur. Methods Based on the Color Reactions of Cholic Acid. Pettenkofer's Method. This test as outlined in Hammarsten (6) is per- formed by dissolving a small amount of bile in concentrated sulfuric acid and warming to 60 or 70C. A 10 per cent solution of cane sugar is added drop by drop. A beautiful red color develops which turns bluish violet on standing. The red liquid shows a spectrum with two absorption bands, one at F and one between D and E . The test fails if the solution is heated too hot, or if too much sugar is added (on account of the sugar carboniz- ing). Also, if impurities are present in the acid, such as H 2 SO 3 or the lower oxides of nitrogen, the reaction fails. Proteins, amyl alcohol, oleic acid, morphine, etc., give a similar color so that it is necessary to carry out the spectroscopic examination also. Ville and Deriien (23) state that vanillin and anisaldehyde give the same color, and cholesterol gives a sim- ilar color. This method has been improved upon by Mylius and von Udran- szky, who advise the use of a 1 per cent solution of furfurol in place of the sugar. Von Udranszky (22) emphasizes the fact that pure bile acids are necessary and suggests decolorizing the bile with charcoal and using an alcoholic solution of the residue. To each cc. of alcoholic solution, add one drop of furfurol and 1 cc. of concentrated sulfuric acid, and warm gently, This will detect 0*0 to ^V mg. of cholic acid. According to Hammarsten, the protein and fat should also be removed by neutralizing the bile and adding alcohol to at least 85 volumes per cent pure alcohol. The solution is fil- tered and the protein extracted- with fresh alcohol, and the alcoholic ex- tract evaporated to dryness. This residue is extracted with absolute alcohol, filtered, and the extract evaporated to dryness. The residue is extracted with ether, dissolved in water, and the solution precipitated with basic lead acetate and NH 4 OH. The precipitate is washed and dissolved in boiling alcohol, filtered, and made alkaline with a few drops of ^SaOH. This solution is evaporated to dryness, the residue extracted with absolute alcohol, filtered, and precipitated with ether. This solution may be used for Pettenkofer's test, but even this may contain phosphatides and they give the same color reaction as do the bile acids. Inouye and Ito (12) reported that when vanillin and concentrated sul- furic acid are added to solutions of bile acids a red line is formed at the line of contact. If the fluids are then mixed, the solution changes to a red-brown and then violet. When this solution is diluted with glacial ace- tic acid, an adsorption band is shown at B. This reaction is sensitive with taurocholic acid in a dilution of 1 : 11,000 and with cholic acid 1 : 22,000. Jolles (13) published a method in which the bile acid (2 to 3 cc. of 1 per cent solution) is mixed with rhamnose (1 to 2 drops of 5 per cent solution) M. G. Foster and C. W. Hooper 359 and boiled with*2 to 3 cc. of concentrated HC1. A red color is produced fol- lowed by a green fluorescence which is due to the formation of methyl fur- furol aldehyde. The reaction can be carried out by 0.005 to 0.0001 gm. of pure acid. It is not affected by urea, albumin, carbohydrates, hydro- carbons, or acids of the aliphatic or aromatic series, glycocoll, taurine, or cholesterol. Authors' Method. Dog's bile contains only taurocholic and taurocholeic acids, and on hydrolysis these split into taurine and cholic and choleic acids. Taurine is amino ethyl sulfonic acid, CH 2 NH 2 CH 2 HSO 3 . It acts like an a-amino-acid, and gives off its NH 2 quantitatively in 3 minutes in the Van Slyke amino nitrogen apparatus. This method consists in hydrolyzing a definite aniount of bile with NaOH, thus splitting the bile acid, and then determining the amount of NH 2 in the taurine. Neither taurocholic nor taurocholeic acid gives off NH 2 before hydrolysis. In other animals both taurocholic and glycocholic acids are present, and it is therefore not certain that the method in its present form can be applied to the bile of animals other than the dog. For the determination 5 cc. of bile cleared in the centrifuge and measured in a calibrated pipette are precipitated with 40 cc. of 95 per cent alcohol, and heated to the boiling point to insure a complete solution of the bile acid which might be held with the mucin precipitate. After cooling, the mixture is made up to 50 cc. in a cylinder with 95 per cent alcohol, and passed through a dry filter paper. Two specimens of 20 cc. each are evaporated to dryness. One is washed out with water and made up to 10 cc. in a calibrated flask. 2 cc. samples of this are used to determine the amount of NH 2 present before hydrolysis. The other is washed out quantitatively with 6 cc. of 8 per cent NaOH into a test-tube. The test-tube is loosely stoppered and placed in a boiling water bath for 5 hours. The contents are washed out into a 10 cc. calibrated flask and made up to volume with distilled water. 2 cc. samples of this are used to determine the amount of amino nitrogen due to the hydrolysis of the bile acids. The nitrogen is then figured on the basis of 1 cc. of bile for both speci- mens by multiplying the mg. of amino nitrogen found by 2.5 and subtracting the unhydrolyzed amino nitrogen from that due to hydrolysis. This figure is multiplied by the total volume of 360 Metabolism of Bile Acids. I bile for the 6 hour collection, which gives the total output of amino nitrogen for 6 hours. The bile acids are figured as tauro- cholic acid by multiplying by 36.72, the factor obtained by divid- ing the molecular weight of taurocholic acid by the atomic weight of nitrogen. Quantitative Estimation of Taurine. Table I shows that taurine gives up its NH 2 quantitatively with 3 minutes shaking in the Van Slyke amino nitrogen appara- tus. The reaction is complete in that time even at as low a tem- perature as 13C. TABLE I. Quantitative Estimation of Taurine. Taurine. Nitrogen gas. Temper- ature. Pressure. Correction. NHz-N found. NH 2 -N theoretical. mg. cc. C. mm. cc. mg. mg. 46.00 9.11 18 760 0.10 5.157 5.152 9.10 9.785 2.06 22 . 756 0.10 1.095 1.096 2.07 10.58 2.17 13 759 0.14 1.189 1.185 2.17 Analysis of Sodium Taurocholate. To test out the method a specimen of sodium taurocholate was analyzed for N, S, and ash, and the NH 2 yielded after hydroly- sis. Table II shows that the content of both N and S of the sodium taurocholate used was about 85 per cent of their theoreti- cal values. The 13.3 per cent ash partly accounts for the low values of N and S. Sodium taurocholate should have had only 4.3 per cent sodium in the ash if one hydrogen atom was replaced by Na. The solutions used in Table III were hydrolyzed 5 hours in a boiling water bath and made up to 10 cc. in a calibrated flask. NH 2 determinations gave the following results. M. G. Foster and C. W. Hooper 361 Number. Amount used. Nitrogen gas. Temper- ature. Pressure. Correc- tion. Amino nitrogen in 10 cc. solution. Total NH?-N in 10 cc. Hydro- lysis NH 2 "11" cc. cc. C. mm. cc. mg. mg. per cent 1 I 1.83 20 758 0.10 9.74 10.29 94.7 1.82 2 1 1.28 20 758 0.10 6.62 6.86 96.5 1.26 3 2 1.31 20 758 0.10 3.42 3.43 99.8 1.31 4 2 0.33 20 '758 0.10 0.65 0.68 95.6 0.33 TABLE II. Analysis of Sodium Taurocholate. Sodium taurocholate. Total N. Theoretical N. Per cent of theo- retical value. Ash. mg. mg. per cent per- cent per cent per cent 779.6 17.15 2.20 2.60 84.62 13.3 Sodium taurocholate. BaSO 4 Total S. . Theoretical S. Per cent of theoretical value. mg. 250* mg. 92.6 per cent 5.09 per cent 5.96 per cent 85.42 * The sulfur determinations were very kindly carried out by Dr. C. L. A, Schmidt. TABLE III. Hydrolysis of Sodium Taurocholate. Number. Sodium taurocholate solution.* Water added. Sodium taurocholate. 16 per rent of NaOH added. NaOH in solution. cc. mg. cc. per cent cc. per cent 1 3 467.7 15 3 8 2 2 311.8 1 10 3 8 . 3 1 155.9 2 6 3 8 4 3t 31.2 1 3 8 * 3. 8980 gm. were made up to volume in a 25 cc. flask and definite amounts were used. t Solution prepared by diluting 1 cc. of the original solution with 14 cc. of distilled water. THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XXXVIII, NO. 362 Metabolism of Bile Acids. I Table III shows that the hydrolysis of this specimen of sodium taurocholate in from 1 to 15 per cent solutions was approxi- mately 100 per cent in 5 hours when the alkali present was 8 per cent during the hydrolysis. This complete analysis was carried out on another specimen of sodium taurocholate with the same results. Normal Constituents of Whole Bile. According to Hoppe-Seyler (10) the normal constituents of whole bile are bile salts, bile pigments, cholesterol, mucin, ethereal sulfates, conjugated glucuronic acids, fats, soaps, a trace of urea, jercorin and other phosphatides, hydrochloric and phosphoric acids, and sulfuric acid as Na, P, Ca, Mg, Fe, and Cu salts. Mar- shall and Davis (16) found the same amount of urea in the bile as in the blood 32 mg. of urea per 100 cc. 1 cc. of bile would then contain 0.149 mg. of N, but Van Slyke states that only 3 per cent of N in the urea is given off in the first 3 minutes, i.e. 0.00447 mg. of N, and that amount is not sufficient to cause an appreciable error in this method. Traces of amino-acids have been found in disease, but these would give off their NH 2 nitrogen in the unhydrolyzed specimen, and thus would be corrected for. Stadel- mann thinks that glycocholic acid may be present in dog's bile. The generally accepted opinion is that dog's bile contains no glycocholic acid or at the most a small trace. Even the pres- ence of glycocholic acid sufficient to make up 10 per cent of the hypothetical bile acid mixture, would introduce no appreciable error, figuring all the bile acids as taurocholic acid. This is because of the large size of the cholic acid molecule and the simi- larity of weight of the molecules of glycocol and taurine. None of the other constituents set free NH 2 groups on hydrolysis except mucin. To obviate this error we precipitated the mucin with 10 volumes of 95 per cent alcohol. In one set it was filtered immediately; in a second, the alcohol was allowed to remain in contact with the mucin for 24 hours; and in a third, the alcoholic mixture was heated to its boiling point, cooled, and filtered im- mediately. Since Croftan showed that the mucin precipitate could not be freed of bile acid by extraction, we made the mixture .to a definite volume with alcohol and then took an aliquot part of the filtrate. This would insure a uniform mixture of bile salts. M. G. Foster and C. W. Hooper 363 Table IV shows that muciri is best removed by alcohol heated to its boiling point. The NH 2 given off by the unhydrolyzed bile may be due to traces of urea or to some splitting of the bile acid. It is always proportional to the bile acid content, but not in exact ratio. The unhydrolyzed mucin-free bile gives a higher NH 2 in some cases than whole bile. This may be due to a slight splitting of the bile acid. The hydrolyzed whole bile gives off decidedly more NH 2 nitrogen than the mucin-free bile. This shows that the mucin is decomposed by hydrolysis into NH 2 - TABLE IV. Whole Bile and Mucin-Free Bile. Dog. Unhydrolyzed bile. Hydrolyzed bile. Whole. Mucin-free. Whole. Mucin-free. NH 2 -N*per cc. of bile. NH 2 -Nper cc. of bile. NH 2 -Nper cc. of bile. NH 2 -Nper cc. of bile filtered im- mediately. NH 2 -N per cc. of bile af- ter 24 hours. NH 2 ^Nper cc. of bile heated to boiling. mg. mg. mg. mg. mg. mg 17-151 0.194 0.245 1.210 0.924 1.012 0.915 16- 15 0.051 0.072 0.277 0.188 0.144 0.203 15- 22 0.205 0.245 1.386 1.185 1.214 1.276 18- 23 0.080 0.072 0.452 0.216 0.289 0.290 * The readings on the NH 2 apparatus have been omitted for simplicity. containing substances (amino-acids) . The mucin precipitated by heating the mixture of bile and alcohol to boiling seems to give the quickest and most satisfactory results. Sodium Taurocholate Added to Bile. To ascertain if the amino nitrogen determined in hydrolyzed bile is really specific for the bile acid, three specimens of the same bile were hydrolyzed after adding different known amounts of sodium taurocholate to each. A control was run on the bile and on the sodium taurocholate solution. Table V shows that known amounts of sodium taurocholate added to bile can be recovered quantitatively with this method. The theoretical increase is figured from No. 1. The error is about 6 to 8 per cent loss. Since our conclusions are based on 364 Metabolism of Bile Acids. I decided increases in bile acid excretion in our experiments, a loss due to the method would have little significance in the analy- sis of results. TABLE V. Known Amounts of Sodium Taurocholate Added to Bile. Number. Bile. Sodium taurocholate. (15 per cent solution). NH 2 -N increase. NH 2 -N in- crease due to sodium taurocholate. Theoretical increase. Error (loss). cc. cc. mg.* mg. mg. p*r cent 1 1 1.150 2 5 3 4.061 3.229 3.45 6.4 3 5 2 2.953 2.121 2.30 7.8 4 5 1 1.914 1.082 1.15 6.1 5 5 0.832 * The readings for the NH 2 have been omitted for simplicity. A Six Hour Collection of Bile. Table VI shows the same total for three periods of 2 hours each as for one period of 6 hours. This speaks for a thorough mixture of the thick viscous bile and its contained bile acids and shows that a single estimation is accurate for the total excretion. It also gives confirmatory evidence of the accuracy of this method. TABLE VI. Mixed Collection of Bile. Time. NH 2 -N per 1 Volume. Total NHz. Output. Per 6 hours. hrs. mg. cc. mg. mg. 1-2 1.174 15 17.61 3^ 0.553 14 7.74 -6 0.558 16 8.93 34. 28 Total. 1-6 0.753 45 33.88 33. 88 Total. Variations in the Concentration of Alkali. The amount and concentration of alkali used are of minor im- portance as can be seen in Table VII. The same specimen of bile was hydrolyzed with varying amounts and concentration of M. G. Foster and C. W. Hooper 365 NaOH and the results show 'that the bile acid was completely hydrolyzed in all the tubes. Table VII also shows that slight variations in the amount and concentration of alkali used for the hydrolysis do not affect the accuracy of the method. TABLE VII. Strength of NaOH Used. Tube. NaOH NH 2 -N cc. per cent mg. 1 5 8 0.364 2 6 8 0.364 3 7 8 0.350 4 6 6 0.350 5 6 12 0.364 DISCUSSION. Hammarsten (6 and 7) found jecorin, lecithin, and other phos- phatides in bile, and all contain nitrogen and sulfur. Since the nitrogen is in the choline radical, it does not interfere with the present method, as choline on hydrolysis with alkalies yields trimethylamine and glycol, and trimethylamine does not react with nitrous acid. But sulfur does interfere with the bile acid methods as determined by the sulfur content. The phospha tides cause a decided error in the methods in which the bile acids are weighed. Long and Gephart (15) have found it . impossible to separate bile acids from lecithin even with ace- tone. Bile salts can hold in stable solution 80 per cent of their weight of egg lecithin. Part of this can be separated by pre- cipitation, but the amount remaining with the bile acids and not separated by acetone is much in excess of that contained in any bile. Ethereal sulfates are present in some bile (human and shark) according to Hammarsten, and thus interfere with the sulfur determination. But von Bergman could not detect any in dog's bile. It is evident that there are many sources of error in all the older methods. The method outlined in this paper is not open to the criticisms of the methods previously used for the quantitative estimation of the bile salts. It is a simple procedure requiring careful tech- nique only in washing out the various residues and making up to volume. 366 Metabolism of Bile Acids. I The results, although 6 to 8 per cent below the theoretical values, are very constant, as we have demonstrated over and over again when duplicates were run through by several different people. The determinations can be carried out so that the results are available within 8 hours. SUMMARY. A method is given for the quantitative estimation of the bile acid present in dog's bile. It is based on the fact that taurine gives up its NH 2 nitrogen quantitatively in the Van Slyke ammo nitrogen apparatus. The taurocholic acid is hydrolyzed by NaOH into taurine and cholic acid. The amino nitrogen of the taurine is then determined by the gasometric method. We are indebted to both Dr. Alice Rohde and Dr. Donald D. Van Slyke for their advice and help in working out this method; also to Dr. C. L. A. Schmidt for sulfur determinations. BIBLIOGRAPHY. 1. Beddard, A. P., and Pemhrey, M. S., Brit Med. J., 1902, i, 702. 2. von Bergman, G., Beit. chem. Phys. u. Path., 1904, iv, 192. 3. Croftan, A. C., Am. J. Med. Sc., 1902, cxxiii, 150. 4. Croftan, A. C., Phil. Med. J., 1902, ix, 75, 142. 5. Goodman, E. H., Beitr.-chem. Phys. u. Path., 1907, ix, 91. 6. Hammarsten, O., text book of physiological chemistry, New York, 7th edition, 1914. 7. Hammarsten, O., Ergebn. Physiol., 1905, iv, 7. 8. Hooper, C. W., and Whipple, G. H., Am. J. Physiol., 1916, xl, 332. 9. Hoover, C. F., and Blankenhorn, M. A., Arch. Int. Med., 1916, xviii, 289. 10. Hoppe-Seyler, F., Handbuch der physiol. u. path-chem. Analyse., Berlin, 8th edition, 1909, 705; Z. physiol. Chem., 1881, v, 1. 11. Huppert, H., Arch. Heilk., 1864, v, 236. 12. Inouye, K., and Ito, H., Z. physiol. Chem., 1908, Ivii, 313. 13. Jolles, A., Ber. chem. Ges., 1908, xli, 2766. 14. Kunkel, A., Arch. ges. Physiol., 1877, xiv, 344. 15. Long, J. H., and Gephart, F., /. Am. Chem. Soc., 1908, xxx, 1312. 16. Marshall, E. K., Jr., and Davis, D. M., J. Biol. Chem., 1914, xviii, 60. 17. Neukomm, J., Ann. Chem., 1860, cxvi, 38. 18. Pfaff, F., and Balch, A. W., /. Exp. Med., 1897, ii, 49. 19. Socoloff, N., Arch. ges. Physiol., 1875, xi, 166. 20. Spiro, P., Arch. Physiol., 1880, Suppl. 50. 21. Stadelmann, E., Z. Biol., 1897, xxxiv, 1. 22. von Udranszky, L., Z. physiol. Chem., 1888, xii, 355. 23. Ville, J., and Derrien, E., Compt. rend. Soc. biol., 1909, Ixvi, 175. 24. Van Slyke, D. D., J. Biol. Chem., 1913-14, xvi, 127. THE METABOLISM OF BILE ACIDS II. NORMAL FLUCTUATIONS IN HEALTHY BILE FISTULA DOGS BY M. G. FOSTER, C. W. HOOPER, AND G. H. WHIPPLE (FKOM THE GEORGE WILLIAMS HOOPER FOUNDATION FOR MEDICAL RESEARCH, UNIVERSITY OF CALIFORNIA MEDICAL SCHOOL, SAN FRANCISCO) REPRINTED FROM THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. XXXVIII, No. 2, JUNE, 1919 Reprinted from THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. XXXVIII, No. 2, 1919 ^ ' , ( THE METABOLISM OF BILE ACIDS. II. NORMAL FLUCTUATIONS IN HEALTHY BILE FISTULA DOGS; BY M. G. FOSTER, C. W. HOOPER, AND G. H. WHIPPLE. (From the George Williams Hooper Foundation for Medical Research, Uni- versity of California Medical School, San Francisco.) (Received for publication, April 28, 1919.) As a preliminary to any series of bile acid experiments it is necessary to establish the normal curve of excretion. This curve of excretion may vary with the condition of the animal, with the diet administered, and with other factors which may not be sub- ject to control. Unless otherwise noted the dogs used in these experiments were in fine physical condition as shown by normal activity, vigorous appetite, a uniform weight curve, and normal blood picture. The operative procedure and general routine care of these animals have been described in detail in an earlier publication, Hooper and Whipple (1). It is very essential that these dogs be exercised, fed, set up, and drained regularly. Diet regulation is very important arid at times difficult. Diar- rhea is a troublesome feature which must be controlled, and this is often best done by a proper admixture of kaolin to the food. We have found it necessary to set up the dogs for collection of bile at least 30 minutes before the actual collection is started. This assures a complete drainage of the thick viscid night bile which escapes only slowly from the fistula. The presence of this concentrated bile in the first collection will cause high read- ings and introduce an error unless this precaution is taken. This explains some of the high readings in the first 2 hour periods of Tables VIII and IX. Unless otherwise stated, dogs were fed exactly 2 hours after the daily collection was started and again after the period of exercise following the collection. Weights were taken in the morning before the collection. 367 368 Metabolism of Bile Acids. II EXPERIMENTAL. The method of chemical analysis has been controlled and shown to be reasonably accurate. The method of bile collection has been described and may be assumed to be accurately con- trolled. The dogs were kept in a room in which they were under constant supervision, which assured an accurate and complete bile collection. Occasionally a dog may be restless and displace the rubber tube in the fistula allowing the escape of bile, but the binders are large and by experience carefully fitted, so that such accidents are rare. When there was loss of bile the material was discarded unless the loss was very small or could be measured. Notes are made of any deviations from the uniform routine com- plete collection. As explained in another place we feel that 6 or 8 hour collections are more satisfactory than the longer 12 to 24 hour collections used by Stadelmann and other workers. The longer collections are more trying to the animal and usually cause loss of weight, appetite, and strength. This immediately introduces the factor of disease with its many unknown vari- ables. We feel that these 6 hour collections continued over weeks and months in dogs which are in every respect healthy and active will give more truthful information about the normal bile acid metabolism. It is to be noted in the first two tables (Tables VIII and IX) that the dogs were not drained for 30 minutes before the collec- tions were begun. The first 2 hour periods show a bile which is more concentrated and contains more bile acids than does the second unit period. A part of this high excretion during the first 2 hours is undoubtedly to be explained by the presence of some of the concentrated night bile in the bile passages, not com- pletely drained off before collections were begun. One must keep in mind the normal fluctuations in bile acid excretion on a mixed diet so that proper care may be exercised in the interpre- tation of the fluctuations noted under experimental conditions. Fluctuations in bile volume are at tunes startling and inexplicable. The constitution of the mixed diet may account for some of the fluctuations in bile volume. It has been pointed out elsewhere by Whipple and Hooper (4) that a meat diet produces a thin, pale, voluminous bile excretion, poor in bile pigments; and further Foster, Hooper, and Whipple 369 TABLE VIII. Bile Acid Excretion 2 Hour Periods Mixed Diet. Dog 18-23. Simple Bile Fistula. Amino nitro- Tauro- Date. Hour. Bile. gen. cholic acid Weight. Remarks. Per cc. Out- output. of bile. put. 1918 cc. ing. mg. mg. Ibs. Mar. 5 1-2 20 0.374 7.48 Mixed diet. 3-4 26 0.231 6.00 5-6 29 0.158 4.58 1-6 75 18.06 664 32.0 Mar. 6 1-2 17 0.344 5.85 3-4 26 0.228 5.93 5-6 21 0.198 4.16 1-6 64 15.94 585 32.0 Mar. 7 1-2 25 0.253 6.32 Hb. 110 per cent. 3-4 28 0.170 4.76 R. B. C. 6,290,000. 5-6 32 0.114 3.65 1-6 85 14.73 541 33.3 Mar. 8 1-2 22 0.264 5.80 3-4 34 0.190 6.46 5-6 25 0.191 4.77 1-6 81 17.03 626 34.0 Mar. 11 1-2 34 0.247 , 8.40 3-4 32 0.160 5.11 5-6 25 0.189 4.72 1-6 91 18.23 670 32.8 Mar. 12 1-2 23 0.253 5.82 3^ 27 0.148 3.99 5-6 19 0.153 2.90 1-6 69 12.71 467 34.0 Mar. 13 1-2 28 0.368 10.30 3-4 29 0.368 10.67 5-6 25 0.225 5.63 1-6 82 26.60 978 35.0 Mar. 14 1-2 21 0.315 6.61 3-4 21 0.286 6.60 5-6 18 0.300 5.40 1-6 60 18.61 662 35.5 Mar. 15 1-2 34 0.390 13.26 3^t 22 0.277 6.09 t 5-6 26 0.223 5.80 1-6 82 25.15 923 34.8 370 Metabolism of Bile Acids. II TABLE IX. Bile Acid Excretion 2 Hour Periods- Mixed Diet. Dog 17-151. Simple Bile Fistula. Amino nitro- Tauro- Date Hour. Bile. cholic Weight. Remarks. Per cc. Out- output . of bile. put. 1918 cc. mg. mg. mg. Ibs. Mar. 5 1-2 7 0.828 5.79 3-4 26 0.675 17.55 5-6 28 0.329 9.21 1-6 61 32.55 1,196 42.5 Mar. 6 1-2 11 1.26 13.86 3-4 23 0.549 12.62 5-6 14 0.379 5.30 1-6 48 31.78 1,167 41.8 Mar. 7 1-2 24 0.409 9.81 Hb. 118 per cent. 3-4 25 0.267 6.67 R. B.C. 6,350,000. 5-6 31 0.213 6.70 1-6 80 23.18 852 42.5 Mar. 8 1-2 13 0.618 8.03 3-4 6 0.816 4.89 5-6 13 0.717 9.32 1-6 32 22.24 818 43.0 Mar. 11 1-2 13 0.437 5.68 3^ 15 0.278 4.17 5-6 10 0.101 1.01 1-6 38 10.86 400 41.5 Mar. 12 1-2 13 0.780 10.14 3-4 13 0.780 10.14 5-6 4 0.717 2.86 1-6 30 23.14 850 43.0 Mar. 13 1-2 10 1.00 10.00 3-4 3 0.691 2.07 5-6 15 0.663 9.94 1-6 28 22.01 809 42.5 Mar. 14 1-2 32 0.656 20.99 Ml 5-6J 8 0.457 3.65 2nd and 3rd collec- tions combined. 1-6 40 24.64 906 43.5 Mar. 15 1-2 13 0.717 9.32 3^ 5-6 5\ 4J 0.654 5.88 2nd and 3rd collec- tions combined. 1-6 22 15,20 559 44.0 Foster, Hooper, and Whipp]e 371 that a carbohydrate diet is associated with a thick, scanty bile excretion, rich in bile pigments. The mixed diet used in these experiments consists of a variable mixture of kitchen scraps con- taining meat, bones, bread, potato, rice, soup, etc. Table VIII shows the variations of bile salt excretion from day to day in 2 hour collections. No preliminary drainage. Table IX shows the variation in bile acid excretion from day to day in 2 hour collections. No preliminary drainage. Hourly Variations in Bile Acid Elimination. Dogs were set up for 8 and 9 hours to establish the hourly curve of daily excretion of bile acids. Compare Tables X and XI with Tables VIII and IX of this paper and subsequent tables in Paper III. These experiments are characteristic of many others .which need not be tabulated at this time, but some of these supplementary observations will be given in later papers to prove other points. TABLE x. Bile Acid Excretion- Hour Periods Mixed Diet. Dog 18-137. Simple Bile Fistula and Splenectomy. Hour. Volume . Amino nitnxren . Tauro- cholic acid per hour. Remarks. Per cc.of bile. Per hour. cc. mg. mg. mg. I . 9.0 0.317 2.85 104 October 1. 2 and 3 20.0 0.302 3.00 110* 4 9.0 0.274 2.47 90 5 12.0 0.230 2.76 101 End of 5th hour fed mixed diet. 6 10.5 0.230 2.41 88 7 and 8 22.0 0.216 2.37 87* Hb. 90 per cent. R. B. C. 3,830,000. 9 10.5 0.230 2.41 88 Weight 24.0 Ibs. * Average of 2 hours. Table X is quite typical of a group of experiments and shows a nearly uniform hourly elimination of bile acids. There is a general tendency for the bile acid excretion curve to fall slightly in the afternoon in spite of a midday feeding and careful pre- liminary drainage of the concentrated night bile before begin- ning the experiment. 372 Metabolism of Bile Acids. II TABLE XI. Bile Acid Excretion Hour Periods Meat Diet. Dog 17-84' Bile Fistula and Splenectomy. Hour. Volume. Amino nitrogen. Tauro- cholic acid per hour. Remarks. Per cc. of bile. Per hour. cc. mg. mg. mg. December 11. 1 2 3.8 9.1 0.686 0.438 2.61 3.98 96 147 Fed 500 gm. of meat. " 300 " " " 3 9.1 0.351 3.19 118 Weight 29. 8 Ibs. 4 6.4 0.614 3.93 143 5 4.1 0.614 2.52 92 6 5.7 0.614 3.50 129 7 5.1 0.658 3.36 124 8 3.9 0.731 2.85 106 9 6.0 0.686 4.10 150 TABLE Xl-a. Bile Acid Excretion 2 Hour Periods Fasting and Meat Diet. Dog 17-34. Bile Fistula and Splenectomy. Amino C5 C^I 00 nitrogen. '^ a - 3 H Hour. 1 8-a* a Remarks. 3 a > Per cc. of bile. In 2 hours. tL . 3 3 *' .C s"* cc. mg. mg. mg. mg. February 13. 1-2 22 0.440 9.68 355 15.3 Fasting. Mixed diet day before fasting. 3-4 20 0.332 6.64 242 '13.2 5-6 20 0.318 6.36 235 13.9 Weight 31. 8 Ibs. 7-8 18 0.303 5.45 201 13.1 9-10 19 0.318 6.04 220 7.0 February 19 to 26 Rice, potato, and milk diet. February 27. 1-2 21 0.189 3.96 146 11.0 Meat 250 gm. at beginning. 3-4 16 0.132 2.11 77 8.9 5-6 20.5 0.161 3.30 121 12.4 Weight 27.5 Ibs. 7-8 14 0.247 3.46 129 11.2 9-10 13 0.261 3.39 124 11.8 Foster, Hooper, and Whipple 373 Tables XI and Xl-a show slight fluctuations in bile acid out- put after feeding, but we do not attach any significance to this reaction. It will be noted (Table Xl-a) that the level of bile TABLE XII. Bile Acid Excretion Not Influenced by "Bile Exclusion."* Dog 18-23. Simple Bile Fistula. Amino CO U t 1 *-l nitrogen. 'o a .3 . Date. 1 1 In Ice. of bile. In 6 hours |a| 11 5 ss ,|a 1 8 Remarks. 1918 cc. mg. mg. mg. mg. Ibs. June 3 62 0.326 20.20 741 6.5 30.2 No bile exclusion. " 4 60 0.409 24.54 902 13.6 31.2 Mixed diet. " 5 61 0.422 25.74 932 20.9 31.2 " 6 82 0.371 30.42 1,115 11.2 " 7 75 0.191 14.33 526 24.8 32.2 " 8 58 0.284 16.57 608 16.2 32.0 Hb. 127 per cent. R. B. C. 6,665,000. " 10 75 0.224 16.80 618 22.2 30.2 * " 11 77 0.289 22.25 817 28.7 31.7 Average 21.4 782 No bile exclusion. June 12 80 0.199 15.92 584 23.7 32.0 Absolute bile exclusion. " 13 78 Lost. 16.3 32.0 Mixed diet. " 14 96 0.346 33.22 1,215 20.0 32.0 " 15 72 0.248 17.86 656 30.2 16 Dog set up and drained 2 hrs. " 17 66 0.180 11.88 436 24.1 30.5 " 18 83 0.238 19.75 725 23.3 32.0 - Average 19.72 723 Complete bile exclusion. * "Bile exclusion" means total inability of the dog to lick any bile from the fistula at any time. This is effected by means of a thick gauze pad and large binder. This acid excretion is influenced by the diet of the previous day. point will be taken up again. This method makes it possible to follow the hourly fluctuations in the bile acid output and to establish for the first time the actual hourly curve of bile acid elimination from hour to hour. 374 Metabolism of Bile Acids. II TABLE XIII. Bile Acid Excretion Not Influenced by "Bile Exclusion.' Dog 18-23. Simple Bile Fistula. Amino CO 3 pv_ j , ; nitrogen. la . i JJate. i 2 Inl cc. of bile. In 6 hours. jll IS p PI Jd Remarks. 1918 cc. mg. mg. mg. mg. Ibs. No bile exclusion. May 21 57 0.223 12.72 467 12.9 30.75 Diet 300 gm. of cracker meal, 65 gm. of meat. " 22 52 0.255 13.26 487 10.8 30.75 Diet 300 gm. of cracker meal, 65 gm. of meat. " 23 73 0.323 23.58 866 12.9 30.50 Diet 300 gm. of cracker meal, 65 gm. of meat. " 24 61 0.253 15.44 567 12.9 30.25 Diet 300 gm. of cracker meal, 65 gm. of meat. " 25-26 30.25 Mixed diet. " 27 66 30.25 300 gm. of cracker meal, 65 gm. of meat. " 28 72 0.221 15.90 574 7.5 30.50 300 gm. of cracker meal, 65 gm. of meat. " 29 82 0.334 27.40 1,005 11.2 30.25 300 gm. of cracker meal, 65 gm of meat. " 30 300 gm. of cracker meal, 65 gm. of meat. " 31 72 0.210 15.12 552 10.0 30.00 Average 17.6 645 No bile exclusion. Absolute bile exclusion. June 19 77 0.252 19.40 712 18.8 32.50 300 gm. of cracker meal, 65 gm. of meat. " 20 78 0.210 16.38 602 23.7 32.00 300 gm. of cracker meal, 65 gm. of meat. " 21 59 0.294 17.35 637 19.8 31.50 300 gm. of cracker meal, 65 gm. of meat. " 22 28 0.476 13.32 489 23.4 31.00 300 gm. of cracker meal, 65 gm. of meat. Average 16.6 610 Complete bile exclusion. The question of "bile exclusion" is a very important one for this entire series of experiments. The question resolves itself into the following: Does a bile fistula dog lick enough bile from Foster, Hooper, and Whipple 375 its fistula during the night to influence in any way the daily ex- cretion of bile or bile acids? According to Stadelmann (3) in his experiments this "bile exclusion" is necessary. The ex- clusion of any possible ingestion of the dog's own bile in his experi- ments would cause a decrease of about f the total bile acid ex- cretion. Because of our respect for Stadelmann's work we felt that it was necessary to control this point beyond the peradven- ture of a doubt. We submit a sufficient number of experiments in Paper V to prove that under the conditions of our experiments "bile exclusion" does not influence the output of bile acids. The reasons for this are discussed more in detail in that paper. These experiments (Tables XII and XIII) make the same point and are sufficient to inform the reader that this important factor has been properly controlled in experiments that follow. Absolute "bile exclusion" does not affect the output of bile acids on either a mixed diet or a known diet. The bile salt excretion can be partly controlled by diet, as will be shown in another paper. A diet rich in meat protein increases the output and a diet poor in meat protein reduces the excretion. Effect of Bile by Mouth on the Following Day's Excretion. In order to find out if bile by mouth in moderate amounts affected the following day's excretion several dogs were given bile at night and the bile acid excretion followed. Table XIV shows that bile feeding in the late afternoon does not influence the following day's excretion of bile acids. Many other experiments have been performed with identical results. It is apparent from experiments tabulated in Paper III that the greater part (about 80 per cent) of the bile acids ingested as bile will appear in the bile fistula bile within 4 hours. We may as- sume that only bile ingested during the early morning hours (5 to 8 a. m.) influences the bile collections in our experiments. There is no clinical evidence that the dogs lick any bile from their fistulas during this period and the experimental data confirm this point. 376 Metabolism of Bile Acids. II TABLE XIV. Bile Feeding at Night Mixed Diet. Simple Bile Fistula with Splenectomy . Amino O CO nitrogen. Dog Date. OJ |.s g - ^ Remarks. No. J3 Inl cc. of bile. In 6 hours. 3'5 *" if 17-181 Average previous- 10 days. 10.60 389 1918 CC. mg. mg. mg. Ibs. July 29 4 p.m. given 58 cc. of^bile by stomach tube. " 30 36 0.267 9.61 352 25.50 5 p.m. given 60 cc. of bile by stomach tube. " 31 25 0.418 10.45 384 26.25 5 p.m. given 60 cc. of bile by stomach tube. Aug. 1 38 0.285 10.82 397 25.00 No bile given. " 2 37 0.244 9.03 332 25.25 Hb. 100 per cent. R. B. C. 4,500,000. 18-137 Average previous 10 days. 14.9 547 July 29 22.75 4 p.m. given 58 cc. of bile by stomach tube. " 30 50 0.310 15.50 569 23.50 4 p.m. given 60 cc. of bile by stomach tube. " 31 42 0.401 16.80 617 24.0 5 p.m. given 60 cc. of bile by stomach tube. Aug. 1 43 0.395 16.55 608 23.5 No bile given. " 2 30 0.145 4.35 159 22.75 Hb. 100 per cent. R. B. C. 4,425,000. DISCUSSION. Loeb (2) stated that the sulfur and nitrogen excretion in the bile is higher during the first 4 hours than during the second 4 hours after eating. From the tables given in this paper it is clear that the bile acid excretion is greater during the early part of the day than later in the afternoon. There is always a ten- dency to fall off during the 5th and 6th hours of the collection in spite of the ingestion of food. But in an individual the amount Foster, Hooper, and Whipple 377 excreted throughout the day is fairly uniform provided a fore- period of 30 minutes or longer for drainage of the bile fistula has been a part of the routine preceding the usual 6 hour collection. It is evident that the amount of bile which these dogs normally lick from their fistulas during the hours they spend in their cages is insufficient to cause any variation in the following day's out- put (Tables XII and XIII). The only time our dogs are prone to lick their fistulas is just after the completion of the daily col- lection. But Table XIV shows that moderate amounts of bile at this time are excreted before the following day's collection is made, and do not affect the determination in any way. SUMMARY. Bile acid excretion in a healthy bile fistula dog given a mixed diet will show great variations from day to day. The amount of bile acid excreted is. usually somewhat higher in the morning than in the afternoon. This holds good even after complete drainage (J hour) of the concentrated night bile, and in spite of liberal feeding 2 hours after collections are begun. The amount of bile acid excreted hour by hour during any given day is fairly uniform. The amount of bile which a dog may lick from its fistula during the afternoon and night resting period is not sufficient in our experiments to cause any demonstrable variation in the following day's excretion. Moderate amounts of bile given by stomach in the late after- noon do not influence the following day's excretion of bile acids. BIBLIOGRAPHY. 1. Hooper, C. W., and Whipple, G. H., Am. J. PhysioL, 1916, xl, 332. 2. Loeb, A., Z. BioL, 1911, Iv, 167. 3. Stadelmann, E., Z. physiol. Chem., 1897, xxxiv, 1. 4. Whipple, G. H., and Hooper, C. W., Am. J. PhysioL, 1916, xl, 349. THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XXXVIII, NO. 2 THE WAVERLY PHE BALTIMORE. U. 0. A. THE METABOLISM OF BILE ACIDS III. ADMINISTRATION BY STOMACH OF BILE, BILE ACIDS, TAURINE, AND CHOLIC ACID TO SHOW THE INFLUENCE UPON BILE ACID ELIMINATION BY M. G. FOSTER, C. W. HOOPER, AND G. H. WHIPPLE (FROM THE GEORGE WILLIAMS HOOPER FOUNDATION FOR MEDICAL RESEARCH, UNIVERSITY OF CALIFORNIA MEDICAL SCHOOL, SAN FRANCISCO) REPRINTED FROM THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. XXXVIII, No. 2, JUNE, 1919 Reprinted from THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. XXXVIII, No. 2, 1919. THE METABOLISM OF BILE ACIDS. III. ADMINISTRATION BY STOMACH OF BILE, BILE ACIDS, TAUR- INE, AND CHOLIC ACID TO SHOW THE INFLUENCE UPON BILE ACID ELIMINATION. BY M. G. FOSTER, C. W. HOOPER, AND G. H. WHIPPLE. (From the George Williams Hooper Foundation for Medical Research, Uni- versity of California Medical School, San Francisco.) (Received for publication, April 28, 1919.) The cholagogue action of whole bile given by stomach is a well known fact. It has been established by many experiments that the curve of bile volume excretion does not necessarily parallel the curve of excretion of bile pigments (Whipple and Hooper, 9). In other words it is possible to- stimulate a free flow of bile which is poor in bile pigments, so much so that the total bile pigment out- put may fall to half normal during a period of active cholagogue excretion with twice normal output of bile. Hooper (3) has pointed out several factors which may contribute to this reaction. It is well to recall that taurocholic acid given by mouth has a marked cholagogue action experiments given below add more data to establish this statement. An ether extract of dried bile, which of course contains a mixture of substances, has no chola- gogue action, but inhibits the excretion of bile pigments. It is of considerable interest to know that certain substances can stimu- late or inhibit the total bile excretion while influencing the output of the various bile constituents in the same or opposite direction. In 1875 Socoloff (7) injected glycocholic acid into a dog and found an increased excretion but no increase in the per cent of bile acids. But his method was questionable and he used only one dog. Rutherford and Vignal (5) injected bile acids into the jejunum and found an increased output of bile acids, but they do not mention how the bile acids were estimated. To Schiff (6) is given the credit for having established the fact that there is a reabsorption of bile acid and this is often termed the "circulation 379 380 Metabolism of Bile Acids. Ill of the bile." This term "circulation of the bile" is used loosely by some writers and is at times misquoted to indicate a circulation of other of the substances found in the bile, for example, bile pig- ments. We have definite proof that bile pigments are not ab- sorbed from the intestine (Hooper and Whipple, 4). At present we have little if any accurate knowledge about the many other substances present in fresh bile they may be absorbed or not. In giving dog's bile by mouth, Stadelmann (8) found that with doses of 2.0 or 2.5 gm. of bile salt, the cholagogue action lasted up to 24 or 36 hours, but the bile salt was excreted within 24 hours, usually within 10 hours. With 1.5 gm. of pure sodium glycocholate the whole amount was excreted within 12 hours. The greatest cholagogue action was during the first 6 hours. From then on it began to abate and was over within the next 12 hours. The bile salts may have been excreted in less than 10 to 12 hours, for Stadelmann performed bile salt analyses only upon these large col- lections. With doses of 3 to 5 gm. morning and evening the salts and volume were tremendously increased but the excretion was not directly propor- tional to the amounts given. 5 gm. twice a day for 3 successive days kept both the volume and salts above normal for more than 8 days, even though they were continually decreasing. These experiments of Stadelmann's are in harmony with those tabulated below, and supplement our experi- mental data. One is not surprised to observe in the experiments given below that the curves of whole bile excretion and bile acid excretion may run parallel after the oral administration of fresh dog's bile moreover, on the contrary, that these curves may be widely dis- sociated. A moderate dose of whole dog's bile given by stomach will cause a distinct cholagogue action and a parallel increase in bile acids. With the fall in bile volume after 3 to 5 hours there is a fall in the bile acid output. It is possible to give sugar with con- centrated bile by stomach tube and completely inhibit the chola- gogue action while a great rise in output of bile acids is taking place. We believe this procedure gives a maximum concentration of bile acids in dog's bile, as high even as 7 to 9 per cent by weight. Perhaps this represents the maximum power of the liver cells to concentrate bile acids in whole bile, at least under the conditions of the experiment. Foster, Hooper, and Whipple 381 EXPERIMENTAL. The first four tables (Tables XV to XVIII) are to be taken as a unit because they illustrate the uniformity of reaction which fol- lows the ingestion of moderate amounts of fresh dog's bile. The taurocholic acid content varies from 0.63 to 1.83 gm. in any given single dose. The hourly curve of bile acid excretion is remarkably TABLE xv. Bile Feeding. Dog 17-181. Bile Fistula and Splenectomy. Hour. Volume. Amino nitrogen. Tauro- cholic acid in 1 hour. Remarks. Per cc. of bile. In 1 hour. cc. mg. mg. mg. August 27. 1 8.0 0.319 2.55 93 End of 1st hour given 50 cc. of whole dog's bile con- taining 752 mg. of tauro- cholic acid. 2 18.5 0.667 12.30 452 3 11.0 0.662 7.28 267 4 7.0 0.596 4.17 153 5 6.5 0.333 2.16 79 End of 5th hour fed mixed diet. 6 7.0 0.222 1.55 56 7' 10.5 0.222 2.33 85 8 10.0 0.210 2.10 77 9 9.0 0.207 1.86 68 Hb. 100 per cent. R. B. C. 4,810,000. 10 10.0 0.221 2.21 81 Weight 25.3 Ibs. constant and usually shows that the largest elimination takes place during the first 3 hours after ingestion. Some experiments show the maximum elimination of bile acids during the first hour, again during the second or third hour. The bile acid concentra- tion per cc. of the bile eliminated usually closely parallels the curve of total excretion. The cholagogue action is marked with the larger doses but is practically absent following the smallest dose. Table XV shows that about 90 per cent of bile acid in whole bile given by mouth is excreted within 3 hours. 382 Metabolism of Bile Acids. Ill TABLE XVI. Bile Feeding. Dog 18-137. Bile Fistula and Splenectomy. Hour. Volume. Amino nitrogen. Tauro- cholic acid inl hour. Remarks. Per cc. of bile. In 1 hour. cc. mg. mg. mg. September 5. 1 10.0 0.442 4.42 162 2 7.5 0.313 2.35 86 End of 2nd hour 75 cc. of bile containing 628 mg. of taurocholic acid. 3 11.0 0.470 5.17 189 4 9.0 0.666 6.00 220 5 8.5 0.696 5.92 217 End of 5th hour fed mixed diet. 6 7.0 0.464 3.25 119 7 10.0 0.275 2.75 101 8 8.0 0.261 2.25 82 Hb. 120 per cent. R. B. C. 5,060,000. 9 8.0 0.248 1.98 72 Weight 22.25 Ibs. 10 7.0 0.221 1.55 56 TABLE XVII. Bile Feeding. Dog 18-23. Simple Bile Fistula. Hour. Volume. Amino nitrogen. Tauro- cholic acid. Remarks. Per cc. of bile. In 1 hour. cc. mg. mg. mg. August 6. 1 7.5 0.364 2.73 100 End 1st hour given 100 cc. of bile containing 1.825 gm. of taurocholic acid. 2 22.0 0.757 16.65 612 3 17.0 0.980 16.65 612 4 20.5 0.925 18.95 696 5 13.5 0.582 7.86 288 End of 5th hour fed mixed diet. 6 11.0 0.373 4.10 150 7 10.5 0.276 2.90 106 Hb. 120 per cent. R. B. C. 6,375,000. 8 9.0 0.345 3.10 114 Weight 31 .5 Ibs. TABLE XVIII. Bile Feeding. Dog 18-137. Bile Fistula and Splenectomy. Hour. Volume. Amino nitrogen. Tauro- cholic acid in 1 hour. Remarks. Per cc. of bile. In 1 hour. cc. mg. mg . mg. September 24. 1 8.5 0.530 4.51 165 2 11.0 0.430 4.73 173 End of 2nd hour 100 cc. of bile containing 951 mg. of taurocholic acid. 3 18.0 0.788 14.18 520 4 16.0 0.845 13.51 499 5 10.0 0.516 5.16 189 End of 5th hour fed mixed diet. 6 10.5 0.508 5.33 195 7 11.0 0.326 3.58 131 8 11.0 0.312 3.43 126 Hb. 90 per cent. R. B. C. 3,830,000. 9 10.0 0.260 2.60 95 Weight 23.3 Ibs. TABLE XIX. Concentrated Bile Feeding. Dog 17-34. Bile Fistula and Splenectomy. TT^,,, Amino nitrogen. Tauro- xlour. Volume. Per cc. of bile. In 1 hour. cholic acid in 1 hour. Remarks. cc. mg. mg. mg. October 15. i 7.5 0.546 4.09 150 2 6.4 0.445 2.85 104 End 2nd hour 170 cc. of con- centrated bile solution* containing 11.50 gm. of taurocholic acid given by stomach tube. 3 21.0 0.968 20.31 745 4 26.0 1.430 37.20 1,365 Vomited about 200 cc. of fluid. 5 29.0 1.360 39.44 1,445 End 5th hour fed mixed diet. 6 34.3 1.220 41.88 1,535 7 54.0 1.000 54.00 1,980 8 18.0 0.657 11.82 433 Hb. 108 per cent. R. B. C. 5,700,000. 9 12.0 0.299 3.59 132 Weight 31.5 Ibs. Oct. 16 60. 0.313 18.80 690 Usual 6 hour collection. Oct. 17 64. 0.328 21.00 752 Usual 6 hour collection. Oct. 18 56. 0.300 16.80 616 Usual 6 hour collection. * Solution prepared by evaporating bile to dryness, extracting with water, and centrifuging. The qlear supernatant fluid is this bile solution. 383 384 Metabolism of Bile Acids. Ill Table XVI shows that about 60 per cent of bile acid adminis- tered by mouth was excreted within 4 hours. Note little if any cholagogue action. Table XVII shows that about 90 per cent of bile acid adminis- tered by mouth was excreted in 4 hours. Table XVIII shows that about 85 per cent of bile acid given in bile by mouth is excreted within 4 hours. TABLE xx. Concentrated Bile Feeding. Dog 18-137. Simple Bile Fistula and Splenectomy. Hour. Volume. Aminq nitrogen. Tauro- cholic acid in 1 hour. Remarks. Perec, of bile. In 1 hour. cc. mg. mg. mg. September 18. 1 7.5 0.187 1.40 51 2 12.0 0.330 3.96 145 End 2nd hour 370 cc. of bile solution f containing 11.60 gm. of taurocholic acid given by stomach tube. 3 16.5 0.729 12.00 440 Vomited 60 cc. 4 20.0 1.111 22.22 816 5 16.0 1.200 19.20 704 End 5th hour fed mixed diet. . 6 14.0 1.010 14.15 519 Hb. 120 per cent. R. B. C. 5,060,000. 7, 8, and 45.0 0.750 11.24* 413* Weight 23.5 Ibs. 9 * Average of 3 hours. t Solution prepared by evaporating bile 'to dryness, extracting with water, and centrifuging. The clear supernatant fluid is this bile solution. Tables XIX and XX show remarkably well the intense reaction which may follow large doses of taurocholic acid by mouth. Whole bile was not given, but a crude watery extract of dried dog's bile which is rich in taurocholic acid. Vomiting occurred and the amount regurgitated is not known. The cholagogue action is noted immediately and is sustained many hours and even days (Table XIX). The concentration of bile acids per cc. of bile is much above normal and the actual out- put of almost 2 gm. per hour is reached in one experiment. Large Foster, Hooper, and Whipple 385 amounts of bile acids seem to cause no ill effects, immediate or delayed, except some nausea and vomiting. What effect if any is produced in the general body metabolism is unknown. Table XIX shows that bile acid fed by mouth in high concentra- tion causes a great increase in bile acid output for 6 hours, and is a TABLE XXI. Concentrated Bile Feeding Plus Sugar. Dog 15-22. Simple Bile Fistula. Amino nitrogen. Tauro- Hour. Volume. Per cc. of bile. In 1 hour. cholic acid in 1 hour. Remarks. cc. mg. mg. mg. September 26. 1 21.5 0.675 14.50 532 2 22.0 0.380 8.36 301 End of 2nd hour 125 cc. of bile solution* containing 8.770 gm. of taurocholic acid 100 gm. of sugar- water. Little vomiting. 3 15.0 1.54 23.10 848 4 9.5 1.93 18.35 673 5 14.5 2.31 33.50 1,230 End of 5th hour fed mixed diet. 6 15.5 2.35 36.45 1,340 7 15.0 2.26 33.90 1,245 8 11.0 1.80 19.80 727 Hb. 110 per cent. R. B. C. 5,280,000. 9 8.5 1.77 15.05 552 Weight 32.8 Ibs. Sept. 27. 37.0 1.19 44.00 1,615 Usual 6 hour collection. Sept. 30. 12.0 1.18 14.10 518 Usual 6 hour collection. * Bile evaporated to dryness, the residue extracted with water, and centrifuged. The clear supernatant fluid is this bile solution. cholagogue for a much longer period. About 65 per cent was excreted in 6 hours. Table XX shows that concentrated bile acid by mouth may cause a long delayed bile acid excretion. Table XXI shows that bile acid plus sugar by mouth causes a delayed bile acid excretion with fairly low volume of bile. The extreme cholagogue action of bile acids may be completely 386 Metabolism of Bile Acids. ' III inhibited by simultaneous administration of a sugar solution by stomach. Sugar solutions alone will cause the excretion of a very concentrated bile but will not modify the bile acid curve. This TABLE XXII. The Effect of Taurine and Cholic Acid Separately and Combined. Dog 17-151.* Simple Bile Fistula. Date. I Volume. Amino nitrogen. Taurocholic acid in 6 hours . <> if MJ3 fc I Urinary N. Remarks. Inl cc. of bile. In 6 hours. 1918 cc. mg. mg. mg. mg. Ibs. gm. Average 11 days. 15 8.0* 294 3.3 Hb. 138 per cent. R. B.C. 7,670 ,000. Mar. 30 34 0.277 9.42 346 26.6 36.75 2.52 Before collection: 4 gm. of cholic acid in capsule. " 31 29 0.133 3.86 142 19.4 36.60 3.25 Apr. 1 18 0.312 5.61 206 23.5 36.50 2.41 Before collection: 0.75 gm. of tau- rine in 30 cc. of salt solution in- travenously. 2 28 0.353 9.89 366 17.5 36.25 2.63 3 45 0.797 35.85 1,316 17.3 35.80 2.63 Before collection: 3 gm. of mix- ture f cholic acid, | taurine in capsule. " 4 19 0.467 8.88 326 18.2 35.75 2.30 5 47 1.141 53.60 1,967 20.9 35.50 2.58 Before collection: , 4 gm. of tau- rocholic acid in capsule. " 6 15 0.402 6.03 220 35.00 2.52 Hb. 125 per cent. R.B.C. 6,980,000. * This dog was kept on a diet of 75 gm. of cane sugar and 100 gm. of glucose during this entire experiment, as well as during the fore-period of 11 days. experiment gives the highest concentration of bile acids per cc. of fistula bile (86 mg. per cc.). The sugar seems responsible for a definite delay in output of bile acids; but the output is enormous in spite of the low volume and slight delay in elimination. Foster, Hooper, and Whipple 387 Tables XXII and XXIII are companion experiments and show great uniformity of reaction. From these two and many other experiments it is established that taurine by mouth or intraven- TABLE XXIII. The Effect of Taurine and Cholic Acid Separately and Combined. Dog 18-23* Simple Bile Fistula. Date. Volume. Amino nitrogen. Taurocholic acid in 6 hours. Pigments in 6 hours. 1 I Urinary N. Remarks . Inl cc. of bile. In 6 hours. 1918 Average 12 days. Mar. 30 cc. 24 45 34 40 30 55 29 56 37 mg. 0.551 0.183 0.142 0.240 0.698 0.354 0.867 0.171 mg. 8.7* 24.75 6.22 5.68 7.20 38.4 10.25 48.53 6.33 mg. 319 909 228 208 264 1,300 376 1,780 232 mg. 18.0 24.6 18.8 14.6 10.5 11.1 22.4 14.8 Ibs. 27.7 27.4 27.1 27.0 26.6 26.5 26.4 25.8 gm. 3.10 3.27 3.25 2.57 2.74 2.55 2.69 2.58 3.30 Hb. 120 per cent. R. B.C. 7,895,000. Before collection: 3 gm. of cholic acid in capsule. Before collection: 0.75 gm. of tau- rine in 25 cc. of salt solution in- travenously. Before collection: 2.6 gm. of mix- ture I cholic acid, 1 taurine in capsule. Before collection: 3 gm. of tau- rocholic acid in capsule. Hb. 120 per cent. R. B.C. 7,300,000. " 31 Apr. 1 2 " 3 4 " 5 " 6 * This dog was kept on a diet of 75 gm. of cane sugar and 75 gm. of glucose during this entire experiment, as well as during the fore-period of 12 days. ously does not in any way influence the output of bile acids. This is true during fasting periods as well as for full diets. Taurocholic acid by mouth is known to be a cholagogue and this 388 Metabolism of Bile Acids. Ill action is well shown in our experiments. About 40 to 50 per cent of the taurocholic acid is excreted in the bile during the 6 hour collection period. There may have been slight delay in excretion owing to the use of capsules rather than solutions. Taurine plus cholic acid fed by mouth exerts the same influence on the bile fistula dog as does the pure taurocholic acid. In other words this synthesis can take place in the body with great ease and rapidity. The cholagogue action and increased bile acid output are identical, whether the mixture of taurine plus cholic acid is given or the pure taurocholic acid. Cholic acid alone has less than usual influence upon the bile acid output if given after a long fasting period. There may be a little increase in bile acid excretion or there may be a decided increase. The reaction seems to depend upon the amount of taurine present in the body which is available to combine with the cholic acid radical. Note additional data in Table XXIV. Table XXIV furnishes more interesting data concerning the feeding of cholic acid and its influence upon the bile acid excretion. A small dose (2.0 gm.) of cholic acid acts as a cholagogue but does not increase the output of bile acid after an 11 day fasting period. A larger dose (4.0 gm.) of cholic acid after 9 days fasting produces a cholagogue action and a definite increase in the taurocholic acid of the bile an increase of about 100 per cent. The next day the same dose repeated gives the same cholagogue action, but little if any increase in the bile acid output. We may assume that the first dose of cholic acid combined with all the available taurine in the body and formed taurocholic acid to be eliminated in the bile. The second large dose given the next day found no taurine to com- plement the cholic acid and no taurocholic acid resulted. We see therefore that either taurine or cholic acid can act as limiting factors in the over-production of taurocholic acid. This is par- ticularly true of cholic acid, which is probably the normal deter- mining factor. Cholic acid given by mouth during periods of full diet is usually associated with a strong cholagogue action and a great increase in output of bile acids (Table XXIV Dog 17-34). This indicates an abundant exogenous source of taurine in the common mixed diet. Table XXV shows the curve which results from the feeding of sodium taurocholate. Its reaction seems to be identical with that. Foster, Hooper, and Whipple 389 TABLE XXIV. Cholic Acid Feeding. Amino O CO nitrogen. Dog . J3-2 "R \r No! JJate. a Inl cc.of bile. In 6 hours jil f 1918 cc. mg. mg. mg. Ibs. 15-22 Average. 16 10.0 367 Average 3 days after 11 days fasting. May 4 35 0.231 8.09 297 28.0 2.0 gm. of cholic acid in capsule at beginning. June 3 16 0.510 8.16 299 29.5 After 8 days fasting. " 4 38 0.535 20.33 746 29.0 4.0 gm. of cholic acid in emulsion at beginning. " 5 35 0.250 8.75 321 28.4 4.0 gm. of cholic ' acid in emulsion at beginning. 17-34 Oct. 9 24 0.537 12.90 473 31.5 Mixed diet. " 10 114 0.579 66.00 2,420 31.5 4.0 gm. of cholic acid in 15 cc. of alcohol at be- ginning. " 11 128 0.478 61.2 2,250 31.5 4.0 gm. of cholic acid in 15 cc. of alcohol at begin- ning. TABLE XXV. Bile Salt Feeding. Dog 15-22. Simple Bile Fistula. Amino O CO a TT-,,_ nitrogen. Hour. 1 In 1 cc. In ill 3 CP O o> ^ Remarks. > of bile. 2 hours. d ^ cc. mg. mg. mg. mg. September 4, 1917. 1-2 18 0.800 14.14 529 6.8 End 2nd hour 10 gm. of crude sodium taurocholate given by stomach. 3-4 58 1.711 99.26 3,640 18.4 5-6 45 1.597 71.85 2,635 11.7 7-8 22 0.642 14.12 518 8.4 Weight 35.0 Ibs. 390 Metabolism of Bile Acids. Ill observed after taurocholic acid feeding. The cholagogue effect is pronounced and about 50 per cent of the taurocholate is re- covered in the first 4 hours of collection. The crude taurocholate was not carefully prepared or analyzed, so that the actual per- centage output cannot be estimated. The reaction subsides rapidly and falls almost to normal within 6 hours. DISCUSSION. Von Bergman (1) fed sodium cholate to dogs and found a decided increase in the sulfur content when only 1.0 gm. was given. But his dog weighed only 4.5 kilograms. A dose of 2.0 gm. caused an even greater increase, but did not double it. He also fed a dog of 8.5 kilograms sodium cholate (2.0 gm.) every day for 3 days. The first day there was a sufficient amount of sulfur excreted to account for all the cholate being changed into taurocholic acid. But each successive day the sulfur excreted was less. The third day it was not back to normal although it was much lower. He explains this by saying that the body could not furnish sufficient taurine to unite with the abnormally high amount of sodium cholate given. Our experiments show that a minimal amount of taurine is avail- able after a long fasting period (Table XXIV). Our mixed diet was probably richer in sulfur than von Bergman's diet of 200 gm. of meat, 150 gm. of rice, and 30 gm. of casein. Note the high out- put with cholic acid feeding and a liberal mixed diet (Table XXIV). Goodman (2) gave 0.6 gm. of cholic acid to a dog on one day and found a decided increase in the amount of bile and the cholic acid excreted. This experiment was done once on a dog of 4.5 kilo- grams weight on a diet of dog biscuit. The cholic acid appears to be the important determining factor in the output of taurocholic acid, and it is of some importance to learn the source of cholic acid in the body, its true metabolic history, and its usefulness and ultimate fate in the body. The solution of these and other questions relating to cholic acid is not easy but will repay further investigation. Feeding cholic acid causes a minimal reaction after long periods of fasting. Repeated doses of cholic acid during fasting result in complete failure of subsequent doses to call out an increase of bile acid excretion. This may be interpreted to mean that under Foster, Hooper, and Whipple 391 these conditions of fasting and repeated ingestion of cholic acid the available taurine is reduced close to zero. The cholic acid is unable to combine with taurine in the usual way and is eliminated in some other form, perhaps in the bile or elsewhere. Feeding cholic acid without taurine in a liberal mixed diet will give a maximal cholagogue action and output of bile acids. Pre- sumably under these conditions there is ample taurine to combine with the cholic acid. The result is a large increase in taurocholic acid in the bile. Probably under normal conditions of diet and health there is always available an excess of taurine so that the normal determining factor is the cholic acid radical. Depending upon the available supply of cholic acid there is a high or low out- put of bile acids in fistula bile. It is evident that certain foods favor a high bile acid output, and presumably furnish considerable amounts of cholic acid in their metabolic history. Much more data on this point will be furnished in other publications. SUMMARY. When moderate amounts of bile are given by mouth (less than 1.8 gm. of taurocholic acid) about 90 per cent of the contained taurocholic acid is excreted in the first 4 hours. Larger amounts of concentrated bile (8.0 to 11.0 gm. of tauro- cholic acid) may prolong the cholagogue action for many hours or even days. A large amount of concentrated bile given with sugar causes a very high concentration of bile acids in the bile excreted (7 to 9 per cent). There may even be an absence of cholagogue action. This may represent the maximum effort of the liver cell to con- centrate bile acids in bile. Taurocholic acid and sodium taurocholate given by mouth have the familiar cholagogue action and a large amount will appear in the bile fistula bile (40 to 80 per cent) within 4 to 6 hours, depend- ing upon the dose given. Taurine intravenously has no effect on the excretion of bile acids. Taurine plus cholic acid by mouth causes a marked increase in bile secretion and bile acid output as much as does taurocholic acid itself. 392 Metabolism of Bile Acids. Ill Cholic acid by mouth usually causes a distinct cholagogue effect. Cholic acid fed during long periods of fasting gives a minimal output of bile acids, but fed during full diet periods gives a maximal output of bile acids. This reaction probably depends upon the available supply of taurine, which is much reduced after fasting, but is abundantly available during full diet periods. We are indebted to Dr. C. L. A. Schmidt for a large amount of taurine which was prepared in his laboratory. BIBLIOGEAPHY. 1. von Bergman, G., Beitr. chem. Physiol. u. Path., 1904, iv, 192. 2. Goodman, E. H., Beitr. chem. Physiol. u. Path., 1907, ix, 91. 3. Hooper, C. W., Am. J. Physiol., 1917, xlii, 280. 4. Hooper, C. W., and Whipple, G. H., Am. J. Physiol., 1917, xlii, 264. 5. Rutherford, W., and Vignal, M., J. Anal, and Physiol., 1876, x, 253; ibid., 1877, xi, 61, 623. 6. Schiff, M., Arch. ges. Physiol., 1870, iii, 598. 7. Socoloff, N., Arch. ges. Physiol., 1875, xi, 166. 8. Stadelmann, E., Z. physiol. Chem., 1897, xxxiv, 1. 9. Whipple, G. H., and Hooper, C. W., Am. J. Physiol., 1916, xl, 349. THE WAVERLY P 0ALTIMORH, U. . A. THE METABOLISM OF BILE ACIDS IV. ENDOGENOUS AND EXOGENOUS FACTORS BY M. G. FOSTER, C. W. HOOPER, AND G. H. WHIFFLE (FROM THE GEORGE WILLIAMS HOOPER FOUNDATION FOR MEDICAL RESEARCH, UNIVERSITY OF CALIFORNIA MEDICAL SCHOOL, SAN FRANCISCO) REPRINTED FROM THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. XXXVIII, No. 2, JUNE, 1919 Reprinted from THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. XXXVIII, No. 2, 1919. THE METABOLISM OF BILE ACIDS. IV. ENDOGENOUS AND EXOGENOUS FACTORS. BY M. G. FOSTER, C. W. HOOPER, AND G. H. WHIPPLE. (From the George Williams Hooper Foundation for Medical Research, University of California Medical School, San Francisco.) (Received for publication, April 28, 1919.) This paper gives the results of experiments to show the excre- tion of bile acids during periods of fasting, of sugar feeding, and of standard diets. The total daily urinary nitrogen excretion is given in some experiments to show that the output of bile acids runs an interesting parallel to the body's' endogenous nitrogen metabolism. Limited diets may profoundly influence the curve of bile acid excretion, and the nitrogenous portion of the diet is most important. It is obvious that certain meat proteins added to the diet profoundly modify the excretion of bile acids. So there seems to be an interesting relationship between the metab- olism of meat proteins from the food and of bile acids excreted in the bile. It is at least possible that a similar relationship may hold for the tissue proteins of the body and the bile acid excretion. Much more experimental data must be submitted but the experiments outlined below make certain fundamental points quite clear. Bidder and Schmidt (1) followed the solid constituents of the bile and concluded that there was an increase on a meat diet. Spiro (3) followed the sulfur in the food and in the bile and found that increasing amounts of meat increased the sulfur excreted, but not proportionately. He also stated that there was a continuous sulfur excretion during fasting, but at a much lower level than when fed. Also, feeding carbohydrates de- creases the amount of sulfur excreted. These experiments are in accord with our results. Kunkel (2) followed the sulfur partition in a single day and found that the sulfur increased in the bile. on starvation at one time, and not at another. Bread and milk decreased the sulfur while various amounts of meat did not have any effect at all. Twice that amount of blood by stomach decreased the bile sulfur. The dog lived only about a month after the experiment, so that very little importance can be attached to these experiments. orvo 393 THE JOURNAL OP BIOLOGICAL, CHEMISTRY, VOL. XXXVIII, NO. 2 394 Metabolism of Bile Acids. IV EXPERIMENTAL. These dogs were kept on the usual routine already described for the previous articles of this series. The experiments given in Tables XXXI to XXXIV show the urinary nitrogen excretion TABLE XXXI. Sugar Feeding. Dog 17-151. Simple Bile Fistula. Amino CO CJ I" a nitrogen. O.S 1 * &B in x 3 Date. I In 1 O 8^ 5 aS $2 4* .C KemarKs . 3 2 cc.of bile. In 6 hours. I'iJi ,2 o is i 1918 cc. mg. mg. mg. mg. gm. Ibs. Mar. 18 13 0.815 10.59 389 38.4 43.0 75 gm. of cane sugar, 50 gm. of glucose given daily by stomach tube. " 19 16 0.618 9.88 363 69.5 4.76 40.7 Hb. 125 per cent. R. B. C. 6,840,000. " 20 11 0.972 10.69 392 42.0 3.05 40.8 " 21 10 0.802 8.02 294 20.9 3.16 39.7 No bile exclusion in this experiment. " 22 3.89 38.7 " 23 13 0.780 10.14 372 36.6 2.71 39.0 75 gm. of cane sugar, 100 gm. of glucose given *- daily by stomach tube. " 24 12 0.482 5.78 212 37.6 3.33 38.4 " 25 11 0.556 6.11 224 21.0 3.92 38.5 " 26 15 0.379 5.68 209 26.1 3.42 38.0 " 27 30 0.236 7.08 260 14.6 3.41 37.9 " 28 20 0.279 5.58 205 29.2 3.17 37.25 Hb. 138 per cent. R. B. C. 7,670,000. " 29 19 0.472 8.98 329 30.8 3.11 37.0 Average 8.05 295 3.45 86 mg. of bile acid per 1.0 gm. of urinary nitrogen. per 24 hour periods. In these experiments the dogs were kept at all times in 'standard metabolism cages arranged for complete collection of the urine with elimination of the feces. Diarrhea was never present and no fecal nitrogen is included in these figures. The night bile of course was included in urine collections, but the nitrogen concerned is constant and rarely exceeds 0.2 gm. Foster, Hooper, and Whipple 395 per 24 hours. The dogs were catheterized at the same hour each day, weighed, and given the sugar solutions or water by stomach tube. The cage was then washed out and the washings added to the cage urine, bladder urine, and bladder washings, which were made up to a unit volume. Duplicate specimens were analyzed by the Kjeldahl method and the total nitrogen calcu- TABLE XXXII. Fasting. Dog 17-151. Simple Bile Fistula. Amino on c nitrogen. .2 eo gj . %* II 3 ' Date. 1 Inl cc. of bile. In 6 hours. o 2 H 3 ' I Remarks. 1918 cc. mg. mg. mg. mg. gm. 76s. April 23 38 0.513 19.50 716 13.2 40.7 No bile exclusion in this experiment. " 24 18 0.510 9.18 337 41.3 2.24 38.6 Hb. 132 per cent. R. B. C. 6,800,000. " 25 15 0.704 10.56 388 33.9 4.14 37.4 " 26 15 0.508 7.62 279 24.9 3.97 36.6 " 27 11 0.615 6.76 248 15.2 4.20 36.0 Set up 3 hours. " 28 27 16.0 4.62 34.5 " 3 " 29 25 0.543 13.68 502 13.1 4.20 35.0 " 30 15 0.641 9.61 352 12.0 3.83 34.5 May 1 18 0.711 12.78 468 12.5 3.55 34.13 " 2 16 0.899 14.39 528 9.5 3.78 33.56 " 3 15 0.676 10.15 373 11.8 3.97 33.13 Hb. 130 per cent. R. B. C. 7,235,000. Average 11.42 419 3.85 109 mg. of bile acid per 1.0 gm- of urinary nitrogen. lated. One of the dogs (15-22, Table XXXIV) has an obstruction in his urethra which makes catherization impossible. The regular 24 hour collections were made as usual, and the average of several days will correct for the daily variations which are in part due to variable amounts of bladder urine retained on dif- ferent days. Tables XXXI and XXXII are to be compared, as these experi- ments were performed upon the same healthy, vigorous dog under 396 Metabolism of Bile Acids. IV identical experimental conditions. The amount of sugar given in the first experiment was not large for a dog of this size, and was increased from 125 gm. per day to 175 gm. during the last hah of the experiment. It may be merely a coincidence, but during the second period of higher sugar intake the bile acid output fell somewhat. This dog averaged 3.45 gm. of urinary nitrogen per day and 295 mg. of taurocholic acid per 6 hours. The 6 hour amount of taurocholic acid per gm. of daily nitrogen is therefore 86 mg. When this dog is put on fasting after a suitable resting period with liberal mixed diet we note a higher urinary nitrogen output, 3.85 gm. per day- and 419 mg. of taurocholic acid per 6 hours. The amount per gm. of 24 hour nitrogen is therefore 109 mg. taurocholic acid per 6 hour period. As the endogenous urinary nitrogen excretion rises with fasting, as compared with sugar feeding, we note a parallel or slightly greater rise in the output of taurocholic acid. We wish to point out an interesting fact in Tables XXXII and XXXIV. The first day in each table gives a remarkably high taurocholic acid figure, and this may not be clear until one remembers that a full mixed diet preceded this fasting period. The first day's bile acid output results from the mixed diet of the preceding day, but it is significant that after the first 24 hours the base line is reached and maintained. This is somewhat dif- ferent from the basal nitrogen excretion, which does not reach its lowest level on fasting until the third or fourth day. Tables XXXIII and XXXIV are like the preceding two ex- periments and correspond in almost every detail. These two experiments were done on two different dogs of approximately the same weight. Both were in excellent condition. The fast- ing dog of course shows a higher output of urinary nitrogen and also of taurocholic acid per 6 hour period. The fasting dog shows a urinary nitrogen of 3.76 gm. per 24 hours and taurocholic acid 407 mg. per 6 hours. The sugar fed dog presents a urinary ni- trogen of 3.23 gm. per 24 hours and taurocholic acid 318 mg. per 6 hours. The amount of 6 hour taurocholic acid per gm. of 24 hour nitrogen is almost identical, 108 mg. in fasting and 98 mg. with sugar. It seems sufficiently clear that there is a close relationship Foster, Hooper, and Whipple 397 between the endogenous nitrogen metabolism and the excre- tion of taurocholic acid in bile fistula bile. During fasting periods we may assume that more body protein is broken down and more taurocholic acid results from this process. We may assume that TABLE XXXIII. Sugar Feeding. Dog 18-23. Simple Bile Fistula. Amino I* a nitrogen. J.s . 1 1 * a Date. II bo ^ Remarks. Tn 1 Oo ^ 1 in i cc.of bile. In 6 hours JH - s s 1* I 1918 cc. mg. mg. mg. mg. gm. Ibs. Mar. 18 52 0.204 10.60 389 26.2 33.3 75 gm. of cane sugar, 50 gm. of glucose given * daily by stomach tube. " 19 27 0.329 8.88 326 17.4 4.51 31.75 Hb. 118 per cent. R. B. C. 6,130,000. " 20 31 0.324 10.04 368 15.6 3.72 31.06 No bile exclusion in this experiment. " 21 17 0.507 8.62 316 10.1 3.08 30.75 " 22 20 0.414 8.28 304 13.9 3.88 29.19 " 23 22 0.288 6.33 232 10.7 3.22 29.75 75 gm. of cane sugar, 75 gm. of glucose given daily by stomach tube. " 24 17 0.512 8.70 319 14.2 2.04 29.56 " 25 23 0.394 9.06 333 22.0 3.64 29.44 " 26 26 0.325 8.45 310 11.3 3.13 28.0 " 27 33 0.194 6.40 235 12.6 3.19 28.56 " 28 20 0.363 7.26 266 10.4 2.74 27.38 Hb. 120 per cent. R. B. C. 7,895,000. " 29 34 0.332 11.30 415 14.2 2.38 27.06 Average 8.66 318 3.23 98 mg. of bile acid per 1.0 gm. of urinary nitrogen. sugar feeding enables the body to conserve its protein at the source, or enables the body to conserve its protein end-products and re- construct these into body cells. When less bile acids are excreted during sugar periods we may wish to assume some such con- servation of bile acid or its parent substance for other uses in 398 Metabolism of Bile Acids. IV body metabolism. Refer also to Table XL, where there is even- more conservation of bile acids or substances from which they are derived. Fasting does not decrease the bile acid output to a remarkably low level. Sugar feeding causes a drop of bile acid excretion below the fasting level. These two points emphasize the fact TABLE XXXIV. Fasting. Dog 15-22. Simple Bile Fistula. Amino 9 g nitrogen. ^3 11 M o g H 5 Date. a JM bo -^ Remarks. "o Inl cc. of bile. In 6 hours . Jll fto ra-2 5 M j 1918 cc. mg. mg. mg. mg. ffw. Ibs. April 23 50 0.668 33.40* 1,225* 21.8 34.7 Dog not catheterized during experiment. " 24 20 0.602 12.04 442 20.9 5.71 33.0 Hb. 150 per cent. R. B. C. 6,390,000. " 25 27 0.470 12.68 465 32.9 3.75 32.38 No bile exclusion in this experiment. " 26 18 0.486 8.77 322 21.9 3.16 31.5 " 27 28 0.536 15.00 550 4.4 2.91 31.4 Set up 3 hours. " 28 27 0.552 14.90 547 10.3 4.59 30.56 (( Q (( " 29 22 0.493 10.85 398 4.2 3.58 30.20 " 30 20 0.404 8.09 297 22.6 2.94 29.63 May 1 17 0.505 8.58 315 26.6 3.19 29.20 " 2 12 0.744 8.93 328 16.3 4.03 28.70 Average 11.09 407 3.76 108 mg. of bile acid per 1.0 gm. of urinary nitrogen. * Not included in average. that there is an important endogenous factor in the bile acid metabolism. Tables XXXV and XXXVI are identical in practically every re- spect and show a remarkable parallelism between the food in- take nitrogen and the output of taurocholic acid. It will be shown later that this reaction depends in part upon the type of food protein. The first dog (Table XXXV) was in perfect con- dition during the entire experiment, maintained a constant weight, Foster, Hooper, and Whipple 399 TABLE XXXV. Nitrogen in Food and Bile Acid Excretion. Dog 17-151. Simple Bile Fistula. Amino 5 . nitrogen. la - Remarks. s 3 3 In 1 cc. of bile. In 6 hours. |l! 5.50 -S.2 W 1918 cc. mg. mg. mg. mg. Ibs. Jan. 7 42 0.272 11.02 405 40.9 33.0 Diet 325 gm. of cracker meal, 80 gm. of beef heart contain- ing 0.5 gm. of nitrogen and 100 calories per kilo. ". 8 62 0.158 9.79 359 27.0 33.3 " 9 72 0.073 5.22 191 25.0 33.0 Hb. 115 per cent. R. B. C. 7,024,000. " 10 67 0.206 13.80 506 29.4 33.0 " 11 69 0.158 10.93 401 17.6 32.8 No bile exclusion in this ex- periment. " 14 67 0.143 9.58 453 20.8 33.0 " 15 76 0.088 6.68 245 26.4 33.0 " 16 56 0.211 11.81 433 14.6 33.5 " 17 74 0.057 4.21 154 18.1 33.1 " 18 70 0.184 12.85 472 12.2 33.3 " 21 75 0.202 15.13 555 18.3 33.5 " 22 68 0.226 15.40 566 19.9 33.5 Average .... 10.53 395 22.5 Diet 0.5 gm. of nitrogen per kilo. Jan. 23 79 0.3431 27.1 994 15.2 33.8 602 gm. of beef heart, 100 calories and 1.0 gm. of nitro- gen per kilo. " 24 77 0.246 18.93 685 18.8 33.6 " 25 82 0.217 17.77 552 16.7 33.0 " 28 43 0.392 16.85 618 11.0 32.0 350 gm. not eaten. Diarrhea. " 29 58 0.416 24.12 885 9.1 31.8 250 " " " " " 30 55 0.489 26.89 986 22.0 30.5 110 " " " " 31 70 0.424 29.68 1,089 20.7 30.5 on n Feb. 1 68 0.380 25.84 948 18.2 30.3 " 4 80 0.312 24.96 916 18.1 29.5 Diarrhea. " 5 60 0.395 23.70 870 14.6 29.0 " Noon meal omitted. " 6 48 0.317 15 21 558 8.4 29.5 " 7 74 0.213 15 76 578 14.1 29.0 " 8 47 0.307 14.42 529 11.5 28.5 Average 21.63 794 15.2 Diet 1.0 gm. of nitrogen per kilo. Foster, Hooper, and Whipple 401 and ate all food. The bile pigment figures are included and show remarkable and inexplicable fluctuation, particularly with the beef heart diet. The taurocholic acid output is quite uniform each day with occasional fluctuations. The average daily output of bile acid per 6 hour period shows a very remarkable increase when the dog was suddenly changed from the diet rich in carbohydrate (0.5 gin. of nitrogen per kilo) to the beef heart diet (1.0 gm. of nitrogen per kilo). The increase in bile acid corresponds to the increase in food nitrogen that is, about 100 per cent increase. The sharp rise in taurocholic acid following the change to a rich pro- tein diet (beef heart) is well shown in both these experiments. The reaction even goes above the average figures of taurocholic acid output on the very first day of rich protein diet. The second dog (Table XXXVI) shows a general reaction which is in every respect similar to the preceding experiment but for some diarrhea in the beef heart period. Associated with this was some loss of weight and appetite. These abnormal factors, however, did not influence the uniform reaction to the change in food pro- tein. Additional data which confirm these experiments will be found in Paper V of this series. Tables XXXVII and XXXVIII add some interesting data to that of the preceding experiments. In both these experiments the food nitrogen was decreased to 0.25 gm. per kilo by decreas- ing the cracker meal and replacing the beef heart with fat. The caloric value was held unchanged at 100 calories per kilo. One dog (Table XXXVII) showed practically no reaction to this change in diet and excreted almost the same amount of tauro- cholic acid as formerly upon a diet of 0.5 gm. of nitrogen per kilo. The other dog (Table XXXVIII) showed a decided drop in excre- tion of taurocholic acid but not to one-half the output on the diet containing 0.5 gm. of nitrogen per kilo. The level of bile acid output on this cracker meal-fat diet approaches the fasting excretion level of bile acid. This shows at once that there is no hard and fast parallel between the nitrogen intake and bile acid excretion, but it is clear that there is an important exogen- ous factor. The mixed diet periods show a level of bile acid excretion which is below that of the beef heart diet. The elements in the 402 Metabolism of Bile Acids. IV TABLE XXXVII. Nitrogen in Food and Bile Acid Excretion. Dog 17-151. Simple Bile Fistula. Amino CO .2 . nitrogen. a c g Date. fP a 11 Remarks. S In 1 T! 3 "CLCD 43 In 6 -s a 1. cc. of bile. hours. I 8 - '" I 1918 cc. mg. mg. mg. mg. Ibs. Feb. 11 32 0.525 16.80 617 38.8 40.0 262 gm. of cracker meal, 10 gm. of butter, 30 gm. of lard, 105 gm. of cane sugar, 0.25 gm. of nitro- gen, and 100 calories per kilo. " 12 30 0.536 16.08 590 28.2 39.5 t " 13 11 0.602 6.62 243 10.4 39.8 Hb. 125 per cent. R. B. C. 6,240,000. " 14 32 0.425 13.60 499 20.6 40.0 " 15 24 0.527 12.66 465 20.9 40.0 " 18 32 0.385 12.32 452 10.0 40.0 No bile exclusion in this experiment. " 19 20 0.493 9.86 362 13.2 40.0 " 20 45 0.345 15.52 570 14.9 " 21 23 0.695 15.98 586 5.2 Average 13.27 487 18.0 Diet 0.25 gm. of nitrogen per kilo. Mar. 5 61 0.534 32.55 1,196 16.6 42.5 Mixed diet. " 6 48 0.662 .31.79 1,167 21.5 41.8 7 80 0.290 23.19 852 36.9 42.5 Hb. 118 per cent. R. B. C. 6,350,000. 8 32 0.695 22.25 818 10.1 43.0 " 11 38 0.286 10.86 399 21.9 41.5 " 12 30 0.771 23.14 850 3.9 43.0 : " 13 28 0.786 22.00 809 11.3 42.5 " 14 40 0.616 24.64 906 5.8 43.5 " 15 22 0.691 15.20 559 12.6 44.0 Average 22.85 839 15.6 Mixed diet. Foster, Hooper, and Whipple 403 TABLE' XXXVIII. Nitrogen in Food and Bile Acid Excretion. Dog 18-23. Simple Bile Fistula. Amino CO -2 . nitrogen. Q S 3 Date. o 6 Inl In 6 '0.0 I Remarks. 1 cc. of bile. hours. | 8J -.2 fe 1918 cc. mg. mg. 'mg. mg. Ibs. Feb. 11 48 0.231 11.08 407 18.4 30.3 30 gm. of lard, 59 gm. of cane sugar, 198 gm. of cracker meal, 10 gm. of butter, 0.25 gm. of nitro- gen, and 100 calories per kilo. " 12 43 0.174 7.47 274 16.1 29.5 " 13 68 0.100 6.80 253 16.6 29.5 Hb. 115 per cent. R. B. C. 7,928,000. " 14 58 0.027 1.60 58 18.2 29.3 " 15 63 0.129 8.10 297 18.4 29.3 No bile exclusion in this ex- periment. " 18 35 0.114 3.97 145 13.4 29.0 " 19 64 0.197 12.64 464 11.1 29.2 " 20 55 0.172 9.48 348 19.1 " 21 51 0.085 4.34 159 Average 7.27 267 16.4 Diet 0.25 gm. of nitrogen per kilo. Mar. 5 75 0.241 18.06 664 31.8 32.0 Mixed diet. 6 64 0.249 15.93 585 36.3 7 85 0.173 14.73 541 37.0 33.3 Hb. 110 per cent. R. B. C. 6,290,000. " 8 81 0.210 17.04 626 37.5 34.0 " 11 91 0.200 18.22 670 27.9 32.8 " 12 69 0.184 12.72 467 29.0 34.0 " 13 82 0.324 26.60 978 25.3 35.0 " 14 60 0.300 18.01 662 27.6 35.5 " 15 82 0.307 25.14 923 29.5 34.8 Average 18.50 679 31.3 Mixed diet. 404 Metabolism of Bile Acids. IV mixed diet are of course variable. This diet is a mixture of kitchen scraps bones, meat, bread, potato, rice, etc. Table XXXIX shows a third dog which was placed upon these same diets containing 0.5, 1.0, and 0.25 gm. of nitrogen per kilo. This dog remained in perfect condition during the entire experi- ment and adds confirmatory data to the other experiments. This bile fistula dog has been under observation for 3 years, and it is known that there is a small communication between the com- mon duct and duodenum. This allows a small amount of bile to enter the duodenum when the fistula is not draining freely, .for example at night. This dog shows only a 50 per cent rise in bile acid output when the diet is changed from 0.5 to 1.0 gm. of nitrogen per kilo. Further change in diet from 1.0 to 0.25 gm. of nitrogen per kilo causes a fall from 1,262 mg. of taurocholic acid to 684 mg. per 6 hour period. These fluctuations are not proportional to the nitrogen content of the diets, but it is obvious that a rise in the food nitrogen intake does cause a rise in the taurocholic acid excretion and vice versa. Table XL gives the data on two experiments which show the influence of a preceding period of fasting upon subsequent excretion of bile acids with a standard diet of 0.5 gm. of nitrogen per kilo. It is to be recalled that these same two dogs on a previous occasion showed an output of bile acids on this same diet which was much higher (550 and 875 mg. of taurocholic acid per 6 hour period Tables XXXV and XXXIX) . Compare with this high output the low excretion in Table XL on the same diet (376 and 419 mg. of taurocholic acid per 6 hour period). The only factor which can explain this difference is the preceding fasting period of 10 and 11 days. This low level of excretion is actually that of the fasting period or even lower. It is evident that the fasting period has caused a clianged reaction in the body so that much less bile acid is permitted to escape in the bile. It may be a correct assumption that some of the material which under usual diet conditions goes to form the bile acid fraction is deviated for other uses in the body. This reaction must be kept in mind whenever any diet experi- ments are planned for these bile fistula dogs. For certain experi- ments it might be assumed that a preliminary period of fasting might give an ideal simple base line from which to estimate the change brought about by a given diet. But the reaction may be Foster, Hooper, and Whipple 405 TABLE XXXIX. Nitrogen in ^ood and Bile Acid Excretion. Dog 15-22. Simple Bile Fistula. Amino CO 1 nitrogen. 1.3 Bo Date. 1 Inl In 6 ill 22 i Remarks. 1 cc.of bile. hours. i 8 * :.S 1 1918 cc. mg. mg. mg. mg. Ibs. Jan. 7 13 0.835 10.85 398 10.0 32.0 325 gm. of cracker meal, 75 gm. of beef heart. 100 calories and 0.5 gm. of nitrogen per kilo. 8 20 0.992 19.84 728 3.5 32.3 9 32 0.662 21.18 778 3.3 32.3 " 10 26 0.591 15.36 551 3.6 32.5 No bile exclusion in this experiment. " 11 38 0.373 14.17 520 7.3 32.3 " 14 35 1.071 37.48 1,375 8.5 32.3 Large amount not eaten. " 15 34 0.751 25.53 936 4.1 32.0 100 gm. not eaten. -'' 16 14 0.920 12.88 473 3.4 32.0 100 " " " " 17 46 0.630 28.98 1,065 6.4 32.5 150 " " " " 18 43 0.822 35.35 1,297 7.5 32.0 " 21 60 0.759 45.54 1,670 4.2 32.3 25 " " " " 22 42 0.453 19.00 698 9.5 32.0 Average 23.84 875 5.9 Diet 0.5 gm. of nitrogen per kilo. Jan. 23 38 0.915 34.77 1,277 5.3 32.0 571 gm. of beef heart con- taining 100 calories and 1.0 gm. of nitrogen per kilo. ;i 24 45 0.750 33.75 1,238 8.6 31.5 " 25 62 0.700 43.40 1,593 5.4 31.0 " 28 35 0.566 19.81 727 24.2 30.5 Diarrhea. " 29 22 1.400 30.80 1,130 10.3 30.8 " 30 30 .730 51.90 1,905 7.8 31.0 " 31 46 .100 50.60 1,855 11.2 30.8 Feb. 1 28 .394 29.03 1,065 6.8 30.8 4 29 .131 32.80 1,205 13.6 31.0 5 39 .015 39.58 1,453 6.0 30.8 Noon meal omitted. 6 12 1.896 22.75 835 6.3 31.0 7 42 0.698 29.31 1,075 6.6 30.5 8 29 0.980 28.42 1,042 11.6 30.3 Average . 34.37 1,262 9.5 Diet 1.0 gm. of nitrogen per kilo. 406 Metabolism of Bile Acids. IV TABLE XXXIX- Concluded. Amino CO j nitrogen. 3 4 - g|' Tt 1 Date. 1 Inl cc. of In 6 hours. ill .2.2 * jf a; Remarks . > bile. 3 ^ 1918 cc. mg. mg. mg. mg. At. Feb. 11 29 1.105 32.05 1,175 11.6 32.0 209 gm. of cracker meal, 10 gm. of butter, 30 gm. of lard, 65 gm. of cane sugar, 0.25 gm. of nitrogen and 100 calories per kilo. " 12 23 0.654 15.04 550 16.0 31.3 " 13 47 0.403 18.95 696 8.4 32.0 " 14 23 0.633 14.56 534 36.0 31.5 Hb. 124 per cent. R. B. C. 5,632,000. " 15 48 0.658 31.58 1,160 10.8 31.8 " 18 45 0.296 13.32 488 24.3 30.5 " 19 28 0.331 9.26 340 14.6 30.5 " 20 12 1.410 16.92 620 13.0 31.0 " 21 17 0.922 14.97 599 17.2 31.3 Average 18.50 684 16.8 Diet 0.25 gm. of nitrogen per kilo. very different toward the same diet factor depending upon whether a fasting period or a carbohydrate diet period had preceded. To get a complete understanding of a single diet factor it will be neces- sary to observe any change in the bile acid excretion curve which may be associated with the administration of any such substance after short fasting periods as well as after carbohydrate or high protein diet periods. Table XLI gives data to indicate that the formation of bile acids depends in part upon the functional capacity of the liver. The Eck fistula liver is produced by an anastomosis between the portal vein and vena cava and a ligature on the portal vein above this anastomosis which limits the blood supply of the liver to the he- patic artery and deviates all of the portal blood directly into the vena cava. The Eck fistula liver is known to be smaller than normal, to exhibit a moderate degree of fatty degeneration, to show a marked decrease in production of bile pigments (Whipple and Hooper, 4) , and to present a distinct impairment of its normal capacity to Foster, Hooper, and Whipple 407 TABLE XL. Nitrogen in Food and Bile Acid Excretion after Fasting Period. Amino el -r Dog No. Date. ' nitrogen. |.s II Remarks. In 1 In 6 2^2 g C.O ,a M 3 cc. of bile. hours. |8- |' 2 1 17-151 Average 10 days fasting 11.8 433 1918 cc. mg. mg. mg. mg. Ibs. May 4 37 0.454 16.80 616 11.0 32.6 Diet 330 gm. of cracker meal, 75 gm. of beef heart, containing 0.5 gm. of nitrogen and 100 calories per kilo. " 6 19 0.349 6.64 224 1.6 36.25 " 7 20 2.4 35.5 " 8 27 0.367 9.91 364 3.8 35.8 Hb. 130 per cent. R. B. C. 7,235,000. " Q 25 0.350 8.75 321 6.1 34.7 " 10 24 0.407 9.77 359 6.7 35.0 Average 10.37 376 Diet 0.5 gm. of nitrogen per kilo. 15-22 Average 11 days fasting 11.09 407 May 6 21 0.587 12.32 452 2.9 29.2 Diet 285 gm. of cracker meal, 60 gm. of beef heart, containing 0.5 gm. of nitrogen and 100 calories per kilo. , " 7 32 5.6 29.5 " 8 36 0.353 12.70 466 9.4 29.2 Hb. 125 per cent. R.B.C.7,335,.000. " 9 29 0.365 10.58 388 6.1 29.0 " 10 24 0.421 10.10 371 4.2 29.2 Average 11.42 419 Diet 0.5 gm. of nitrogen per kilo. 408 Metabolism of Bile Acids. IV excrete phenoltetrachlorphthalein (Whipple, Peightal, and Clark, 5). We may assume that the Eck fistula liver is functionally deficient. The observations in Table XLI harmonize with those TABLE XLI. Nitrogen in Food and Bile Acid Excretion. Eck Fistula. Dog 16-15. Simple Bile and Eck Fistula. Amino CO fl 0Q nitrogen. S 3 Date. "o a "I S II . Remarks. S In 1 OT3 3 2 3 In 6 S'S o - ~ 1 cc. of bile. hours. 1" 1 1918 cc. mg. mg. mg. mg. Ibs. Jan. 7 16 0.314 5.03 183 4.5 20.5 Diet 200 gm. of cracker meal, 55 gm. of beef heart, containing 0.5 gm. of nitrogen and 100 calories per kilo. " 8 25 0.258 6.45 236 5.7 20.0 Hb. 99 per cent. R. B. C. 6,240,000. " 10 12 0.219 2.62 96 1.9 19.8 Not eating well. " 11 16 0.144 2.30 84 5.3 19.8 u " 14 12 0.155 1.86 68 3.3 20.0 " 15 6 0.116 0.69 25 20.2 " 16 10 0.141 1.41 52 1.6 20.7 " 17 14 0.330 4.62 169 3.2 19.8 " 18 21 0.273 5.73 210 3.7 20.3 50 gm. not eaten. " 21 20 0.310 6.20 227 2.1 20.0 " 22 20 0.212 4.24 155 2.1 20.0 Average 3.74 137 3.34 Diet 0.5 gm. of nitrogen per kilo. Jan. 24 30 0.447 13.41 492 3.7 19.8 Diet 355 gm. of beef heart, containing 1.0 gm. of ni- trogen and 100 calories per kilo. 1 gm. of yeast twice a day. " 25 17 0.534 9.07 333 20.8 outlined above and show that an Eck fistula dog upon a standard diet will excrete not over one-half the normal amount of bile acids. Compare this dog (Table XLI), weight 20 Ibs. and output on standard diet of 137 mg. of taurocholic acid per 6 hour period, with the dog of Table XXXV, weight 40 Ibs. and output on the same Foster, Hooper, and Whipple 409 standard diet of 550 mg. of taurocholic acid per 6 hour period. Also compare with dog of Table XXXIX, weight 33 Ibs. and out- put on the same standard diet of 875 mg. of taurocholic acid per 6 hour period. When the Eck fistula dog was changed to a rich protein diet we observe a considerable jump in the output of bile acids, as in the other dogs. It is not possible to keep an Eck fistula dog on a pure beef heart diet for any length of time without precipitating the characteristic Eck fistula intoxication which usually results in death. DISCUSSION. There is sufficient experimental data given above to make the point that both endogenous and exogenous factors are concerned in the metabolism of bile acids. There is a reasonably constant output of taurocholic acid during fasting periods, and this output may be somewhat diminished by administration of pure carbo- hydrate. The relative diminution of urinary nitrogen and tauro- cholic acid excretion may show a certain similarity under these experimental conditions. This may indicate a certain relationship between the metabolism of the body protein and the production of taurocholic acid. There is obviously a very important endog- enous factor in the metabolism of bile acids. It is equally clear that there is an important relationship be- tween the output of taurocholic acid and the intake of certain food proteins. On certain diets a uniform level of bile acid excretion may be observed for days and a sudden shift to a similar diet con- taining double the amount of food nitrogen may cause a sudden doubling of bile acid excretion. This fact comes out clearly in several experiments in this paper but we wish to refer to additional evidence submitted in Paper V of this series. It is certain that some food proteins act very differently from others as to their value in modifying the bile acid excretion. It is to be noted that the increased excretion of bile acids ap- pears very promptly when the diet is changed to beef heart. It may be that the formation of bile acids by the liver cell is an automatic response to the proper stimulus, just as these cells respond to a protein digestion stimulus by urea formation. In the case of urea formation we know that even in the greatest food THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XXXVIII, NO. 2 410 Metabolism of Bile Acids. IV shortage emergency the liver will form urea from amino-acids, even though the body may need all the food amino-acids, for example, after long periods of fasting. In the case of taurocholic acid we have shown that long periods of fasting followed by high protein feeding will show little rise in the taurocholic acid output contrary to what we might expect if this substance was purely a waste product to be eliminated from the split products of protein digestion. The body evidently conserves the taurocholic acid or its parent substance under certain conditions, perhaps for use elsewhere in the body in the reconstruction of its depleted body protein. It is generally accepted that bile acids are dependent upon the ndrrnal liver cell function for their production. There has been little dispute in medical literature concerning this point. Yet there is very little available direct proof of this statement if one wished to argue that the bile acids were formed elsewhere in the body and eliminated in the bile. For example there is convincing evidence that bile pigments may be formed outside of the liver and excreted in the bile secondarily (Whipple and Hooper). We have evidence in the Eck fistula experiment given above that bile acid output will be much subnormal in the Eck fistula liver which is functionally subnormal. This indicates that in a general way the bile acid output may fall with impaired functional ca- pacity of the liver cell. Other experiments in which the liver is injured by chloroform and other poisons give similar results which will be published in the near future. All this evidence gives some direct and positive proof that the bile acids are actually produced by liver cell activity. SUMMARY. There is a uniform excretion of taurocholic acid in the bile fistula dog during fasting periods. There is a uniform and slightly lower excretion of taurocholic acid in the same dog during similar periods of carbohydrate (sugar) feeding. This fall in taurocholic acid excretion is much like the fall in urinary nitrogen excretion under the same conditions. Th6re is an important endogenous factor in the bile acid metabolism and this may concern the body protein metabolism. Foster, Hooper, and Whipple 411 The output of bile acid in bile fistula bile may be influenced at will by suitable control of the diet. Meat protein seems to be of the greatest importance and a pure meat diet will give the highest output of bile acid per 6 hour period. There is an important exogenous factor in bile acid metabolism which is concerned especially with the food protein. After a long fasting period the bile fistula dog will not react to a high protein diet with the usual high bile acid output. There is evidently a deviation of certain precursors of the bile acid to serve other purposes in the body perhaps to supply some im- portant substances relating to body protein which have been depleted by the fasting period. A functionally deficient liver (Eck fistula) produces less than one-half the normal amount of bile acid during a standard diet period. This is direct evidence (of which there has been little available) that the bile acids are formed essentially by liver cell activity. BIBLIOGKAPHY. 1. Bidder, F., and Schmidt, C., Die Verdauungssafte u. der Stoffevechsel, Mitau, 1852. 2. Kunkel, A., Arch. ges. PhysioL, 1877, xiv, 344. 3. Spiro, P., Arch. PhysioL, 1880, Suppl. 50. 4. Whipple, G. H., and Hooper, C. W., Am. J. PhysioL, 1917, xlii, 544. 5. Whipple, G. H., Peightal, T. C., and Clark, A. H., Johns Hopkins Hosp. Bull., 1913, xxiv, 343. THE WAVERI.Y P BALTIMORE, U. 6. A. THE METABOLISM OF BILE ACIDS V. CONTROL OF BILE INGESTION AND FOOD FACTORS BY M. G. FOSTER, C. W. HOOPER, AND G. H. WHIFFLE (FROM THE GEORGE WILLIAMS HOOPER FOUNDATION FOR MEDICAL RESEARCH, UNIVERSITY OP CALIFORNIA MEDICAL SCHOOL, SAN FRANCISCO) REPRINTED FROM THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. XXXVIII, No. 2, JUNE, 1919 Reprinted from THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. XXXVIII, No. 2, 1919 THE METABOLISM OF BILE ACIDS. V. CONTROL OF BILE INGESTION AND FOOD FACTORS. BY M. G. FOSTER, C. W. HOOPER, AND G. H. WHIPPLE. (From the George Williams Hooper Foundation for Medical Research, Uni- versity of California Medical School, San Francisco.) (Received for publication, April 28, 1919.) The experiments tabulated below serve two purposes. They furnish additional data to make absolutely certain that the amount of bile acid excreted can be increased with the increase of meat protein nitrogen in the diet. These experiments in addition show that absolute exclusion of every drop of bile by mouth does not modify the excretion of bile acids under the conditions of the experiment. It was necessary to submit these control experi- ments because Stadelmann, who has done exceptional experi- mental work in this field, has stated that bile exclusion will cause a fall of bile acid excretion. He assumes therefore that a muzzle at night is necessary for accurate work, and that the bile fistula dogs lick sufficient bile from their fistulas to modify the follow- ing day's excretion of bile and bile acids. Our dogs are set up each morning after a period of exercise followed by a 30 minute period of free drainage before collections are started. After the collection period of 6 hours the dogs are al- lowed to exercise in the yard before being fed in their cages. They are kept during the night in large cages of wire mesh beneath which are pans for the collection of excreta. The dogs, of course, during the night drain bile from their fistulas into the pans, but no bile collects where the dogs have access to it because the wire mesh retains no fluids. The dogs usually lick their fistulas when the collection is finished but it seems to be due in part to the fact that the skin itches where it is in contact with the abdominal binder. These dogs rarely lick bile from their fistulas in the earry morning and at this time the flow is at a minimum. We feel that the experiments given make it quite clear that under 413 414 Metabolism of Bile Acids. V this laboratory routine the amount of bile which may be obtained by a given dog licking its own fistula is not sufficient to modify the excretion curve of bile acids. We cannot attempt to explain Stadelmann's results, but two factors may be concerned. It is well known that dogs which have had bile fistulas for many months or years do not lick their fistulas as much as do dogs who are less accustomed to the bile fistula. One of our dogs has been under observation with a bile fistula for 3J years. Furthermore, in our cages, as stated above, bile could not accumulate during the night where the dog had access to it at any time, as the bile flowed through the mesh cage floor. If bile accumulated on the floor of a room or cage it is very probable that a dog would lick some of this bile in the morn- ing and obtain a sufficient amount to make a decided difference in the output of bile acids. It can be deduced from the experiments given in Paper III that moderate amounts of bile given late in the afternoon will not influence the output of bile acids on the following day. Bile given in the forenoon will cause a cholagogue action lasting several hours, but usually a rapid elimination of the excess of bile acids within 6 hours at least 80 to 90 per cent elimination within this time. EXPERIMENTAL. Bile exclusion in these experiments indicates that the dog in question was unable to gain access at any time to any bile either from its own fistula or elsewhere. During the routine 6 hour collection all the bile is collected in a small rubber bag. At the end of the collection a muzzle is put securely on the dog, which is then permitted to run in the yard for a few minutes under care- ful observation. The dog is then brought in and dressed with its night binder which is worn until the next morning. The muzzle was not worn during the night except in one instance, where the dog tried to chew off the straps of the abdominal binder. The night binder was made of light canvas and fitted to the individual to cover the thorax and abdomen completely. Anteriorly it was held from slipping backward by soft webbing which en- circles the neck and fore legs. The binder was held about the abdomen by soft webbing or straps. A large gauze pad was Foster, Hooper, and Whipple 415 placed over the bile fistula and served to absorb all the night bile. With this routine we are absolutely certain that no bile was ingested at any time during periods of "bile exclusion." The animals were comfortable and maintained their usual con- ditions of diet and activity. Tables XLII, XLIII, and XLIV are to be considered as a unit. These three experiments were done at the same time under identical conditions, and the results are remarkably uniform. They are to be compared with Tables XXXV, XXXVI, and XXXIX in Paper IV of this series. In each of the three experiments tabulated (XLII to XLIV) the dog was placed upon a diet of cracker meal and beef heart containing 0.5 gm. of nitrogen and 100 calories per kilo. This diet with complete bile exclusion obtained for 1 week, and the average daily output per 6 hours shows a fairly uniform figure of 400 to 500 mg. of taurocholic acid per 6 hour period. The dog which is slightly heavier shows a slightly greater output. The three dogs kept on this same diet for the second week were not prevented from licking their own fistulas in the cage during the night. It will be seen that the bile acid output remains about the same it is actually somewhat less during this second week, 340 to 440 mg. of taurocholic acid per 6 hours. During the third week each dog was given the first day with no bile exclusion, the second and third day with complete bile ex- clusion, and the fourth day with no bile exclusion. Individual: fluctuations appear but this week in general agrees with th& data of the first 2 weeks. The fourth week was continued with strict bile exclusion but the diet was changed to a mixture of beef heart and a little cracker meal, giving 1.0 gm. of nitrogen and 100 calories per kilo. The figures from this group of experiments resemble those referred to in Paper IV. The increase in beef heart stimulates the out- put of bile acids from a level of 400-500 mg. to 700-900 mg. per 6- hours. Two points are made by these and other experiments. Bile exclusion does not modify the excretion of bile acids under the conditions of our experiments. Certain food proteins in the diet have a marked influence on the excretion of bile acids in bile fistula bile. 416 Metabolism of Bile Acids. V TABLE XLII. Known Diet With and Without Bile Exclusion. Dog 18-93. Simple Bile Fistula. Amino nitrogen. It Date. 3 Inl In 6 111 "S Remarks. 1 cc.of bile. hours. | 8>S 1 1918 CC. mg. mg. mg. Ibs. Aug. 5 57 0.256 14.59 536 * 31.8 Absolute bile exclusion. Diet 330 gm. of cracker meal. 60 gm. o ? beef heart = 0.5 gm. of nitrogen and 100 calories per kilo. " 6 46 0.320 14.70 540 31.5 * " 7 38 0.254 9.66 355* 32.0 " 8 56 0.238 13.33 489 32.0 " 9 59 0.255 15.05 552 32.3 Average 13.50 494 Complete bile exclusion. Aug. 12 54 0.224 12.08 443 30.8 No bile exclusion. " 13 61 0.210 12.81 470 31.5 " 14 58 0.242 14.05^ 516 * 31.5 " 15 61 0.158 9.64 354 31.3 " 16 64 0.181 11.58 425 31.3 Average 12.00 442 No bile exclusion. Aug. 19 70 0.266 18.62 683 31.0 No bile exclusion. " 20 71 0.196 13.90 510 30.5 Bile exclusion 12 hours pre- viously. " 21 64 0.230 14.72 540 30.8 Bile exclusion. " 23 77 0.185 14.25 523 30.5 No bile exclusion. Aug. 28 72 0.326 23.48 862 30.0 Absolute bile exclusion. Diet 467 gm. of beef heart, 100 gm. of cracker meal = 1.0 gm. of nitrogen and 100 calories per kilo. 29 72 0.293 21.08 774 29.8 " 30 76 0.335 25.48 934 29.5 Hb. 126 per cent. R. B. C. 6,430,000. Average 23.30 857 Complete bile exclusion. * 2-3 cc. of bile lost. Foster, Hooper, and Whipple 417 TAB'LE XLIII. Known Diet With and Without Bile Exclusion. Dog 18-54- Bile Fistula and Splenectomy . Amino nitrogen. 1-3 Date. | 1 Inl cc. of bile. In 6 hours. JiJ .C i Remarks. 1918 cc. nig. mg. mg. Ibs. Aug. 5 65 0.170 11.05 405 27.5 Absolute bile exclusion. Diet 280 gm. of cracker meal, 60 gm. of beef heart = 0.5 gm. of nitrogen and 100 calories per kilo. " 6 62 0.18*1 11.23 412 27.8 " 7 41 0.100 4.10 150 28.0 Binder chewed off night of the 6th. " 8 47 0.182 8.56 314 27.5 " 9 79 0.212 16.75 614 27.0 Average 10.33 379 Complete bile exclusion. Aug. 12 77 = 0.224 17.25=*= 633 27.3 No bile exclusion. 5 hour collection. " 13 51 0.140 7.14 262 27.3 " 14 43 0.172 7.40 271 27.0 " 15 57 0.115 6.56 241 27.0 " 16 53 0.126 6.68 245 27.5 Average 9.00 330 No bile exclusion. Aug. 19 77 0.222 17.10 628 27.5 No bile exclusion. 20 76 0.167 12.68 465 27.3 Bile exclusion 12 hrs. pre- viously. " 21 71 0.187 13.26 486 27.5 Bile exclusion. " 23 80 0.128 10.24 376 27.3 No bile exclusion. Aug. 27 46 27.0 Absolute bile exclusion. Diet 417 gm. of beef heart and 100 gm. cracker meal = 1.0 gm. of nitrogen and 100 calories per kilo. " 28 81 0.288 23.31 856 26.3 " 29 75 0.311 23.32 856 26.5 " 30 70 0.380 26.60 976 26.3 Hb. 110 per cent. R. B. C. 5,515,000. Average . . . 24.40 896 Complete bile exclusion. 418 Metabolism of Bile Acids. V TABLE XLIV. Known Diet With and Without Bile Exclusion. Dog 18-137. Bile Fistula and Splenectomy. Date. Volume. Amino nitrogen. C 5O "o c jM t M '5 Remarks. Inl cc. of bile. In 6 hours. 1918 cc. mi. mg. mg. Ibs. Aug. 5 53 0.313 16.58 608 23.0 Absolute bile exclusion. Diet 245 gm. of cracker meal, 45 gm. of beef heart = 0.5 gm. of nitrogen and 100 cal- " 6 56 0.264 14.78 542 23.0 ories pgr kilo. " 7 38 0.365 13.86 509 23.3 " 8 37 0.250 9.25 340 23.0 " 9 34 0.198 6.73 12.25 247 23.0 Average - ... 450 Complete bile exclusion. Aug. 12 56 0.196 10.96 402 22.8 No bile exclusion. " 13 46 0.266 12.24 449 22.3 " 14 40 0.214 8.56 314 22.0 " 15 43 0.129 5.50 202 22.0 " 16 44 0.198 8.71 320 22.0 Average .... 9.20 338 No bile exclusion. Aug. 19 50 0.404 20.20 742 22.0 3 hour collection. " 20 57 0.292 16.65 611 22.3 Bile exclusion 12 hours pre- viously. " 21 68 0.188 12.78 22.3 Bile exclusion. " 23 40 0.099 3.96 145 22,3 No bile exclusion. Aug. 28 21.8 Absolute bile exclusion. Diet 327 gm. of beef heart and 100 gm.' of cracker meal = 1.0 gm. of nitrogen and 100 cal- ories per kilo. " 29 50 0.335 16.75 611 22.0 " 30 57 0.376 21.42 786 22.0 Hb. 120 per cent. R. B. C. 5,060,000. Average 19.07 698 Complete bile exclusion. Foster, Hooper, and Whipple 419 Tables XLV and XLVI supply more data on bile exclusion and fasting excretion of bile acids. These tables are to be com- pared with Tables XXXII and XXXIV in Paper IV and the average of these last two tables is added to Tables XLV and XLVI now under consideration. Table XLV shows practically the same output of bile acids per 6 hours whether bile is excluded or not. This is in harmony with all the other experiments given in this and other papers as well as many experiments which are unpublished. TABLE XLV. Bile Exclusion- Fasting. Dog 17-151.- Simple Bile Fistula. Date. Volume. Amino nitrogen. O 1 C f,~ 2"23 3'e o C3X h +2 II W>J3 'aCH (CH 2 ) 5 CH CHT~CH 2 OH EXPERIMENTAL. The experiments were conducted under conditions similar to those described above. The dogs were set up for J hour for free drainage of the bile fistula before the collections were started. Only a part of the experimental data is given, but it is uniform in character and the evidence all points the same way. Table L presents two similar experiments in which cystine was given intravenously with no positive influence on the bile acid output, but with a decided effect upon the bile unhydrolyzed amino nitrogen fraction. This may indicate an excretion in the bile of cystine or taurine under these conditions. On the follow- ing day a similar injection of cystine followed by cholic acid by stomach resulted in a great increase in taurocholic acid excretion and a return of the unhydrolyzed amino nitrogen to normal. This gives evidence that under these conditions the body can change cystine rapidly to taurine, which is then available to combine with the cholic acid. It may be objected that on a mixed diet the feeding of cholic acid will increase the output of taurocholic acid because plenty of taurine is available from the food. Refer to Table XXIV, Paper III, where it is seen that the rise in tauro- Foster, Hooper, and Whipple 425 cholic acid does actually reach a higher level after administration of 4.0 gm. of cholic acid with a full mixed diet. It would be desir- able to repeat these observations during a fasting period. Taken together with the experiments of von Bergman and Wohlgemuth we feel reasonably secure in the statement that cystine can produce taurine under physiological conditions, and TABLE L. Cystine Intravenously Reacts with Cholic Acid. Amino nitro- i: Dog KJ Date. a gen. i;i j Remarks. JNO. 3 "g In Ice. In 6 ill 1 > of bile. hours. * 1 1918 cc. mg. mg. mg. Ibs. 15-22 Nov. 25 53 0.227 12.04 442 33.3 Mixed diet. " 26 28 0.456* 12.75 468 33.0 0.7 gm. of cystine in- travenously. , " 27 51 0.746 38.05 1,395 32.5 0.7 gm. of cystine in- travenously + 2.1 gm. of cholic acid by stomach. 17-34 Nov. 25 35 0.198 6.93 254 30.3 Mixed diet. " 26 40 0.356* 14.24 522 30.3 0.7 gm. of cystine in- travenously. Vom- ited. " 27 76 0.453 34.45 1,264 30.3 0.7 gm. of cystine in- travenously + 2.1 gm. of cholic acid by stomach. Vomited a little of cholic acid solution. * The unhydrolyzed bile gave a high unhydrolyzed NH2 showing the presence of an excess of cystine or taurine uncombined with cholic acid. The following day when cholic acid was given this unhydrolyzed NH 2 had returned to normal which would indicate that all the cystine Had been synthesized into taurocholic acid. this taurine is available to combine with an excess of cholic acid. This holds good apparently whether the cystine is fed by mouth (von Bergman) or given intravenously (Table L). In comparing Table LI with Tables XXXI and XXXIII of Paper IV, one can see that these variations are within the normal fluctuations of these same dogs on a sugar diet. THE JOURNAL OP BIOLOGICAL CHEMISTRY, VOL XXXVIII, NO 2 426 Metabolism of Bile Acids. VI Tables LI and LII are to be considered together and they furnish strong evidence that cholesterol alone or fed with taurine exerts no influence upon the excretion of taurocholic acid. This holds for periods of fasting as well as for periods of mixed diet. The amounts of cholesterol administered (3 to 4 gm.) are sufficient to convince any investigator that this substance does not play a part in bile acid metabolism. There is no immediate reaction nor any TABLE LI. Cholesterol Feeding Sugar Diet Bile Acids Unchanged. Amino 3 CO A nitrogen. 1 C a Dog No Date. -o ;- S O 3 3 >> 09 Remarks. G 3 0_QJ 3 Ss C3 3 "S O 5" G hH ;H ^ P O) 1918 CC. mg. m0. m<7. mg. gm. s. 17-151 Average 11 days. 8.05 295 3.45 Diet of 75 gm. of cane sugar, 100 gm. of glucose. Apr. 6 15 0.402 6.03 220 2.52 35.0 7 17 0.556 9.45 347 18.6 2.13 35.1 4 gm. of cholesterol.* " 8 17 0.416 7.08 260 14.2 2.57 34.4 18-23 Average 11 days. 8.66 318 3.23 Diet of 75 gm. of cane sugar, 75 gm. of .glu- cose. Apr. 6 37 0.171 6.32 232 14.2 3.30 25.8 " 7 31 0.269 9.34 343 13.0 2.58 25.7 4 gm. of cholesterol.* " 8 37 0.125 4.62 169 18.7 2.18 25.7 * Given in gelatin capsule at beginning of 6 hour collection. delayed effect to be observed. In Table LII the taurine was given to insure an excess of this substance in the body to combine with any amount of cholic acid available. Table LII illustrates the inhibition of bile flow due to sugar by mouth (Dog 15-22). Vomiting on certain occasions may be associated with a decreased flow of bile in these fistula dogs. The amount of ether used to dissolve the cholesterol (8 to 10 cc.) will have no influence upon the bile excretion. Foster, Hooper, and Whipple 427 Tables LIII and LIV are similar experiments which show that red blood cells by mouth have no effect upon the excretion of bile acids. The control periods of 5 days and the red blood cell feed- ing periods give figures which are practically identical. The TABLE LII. Taurine Plus Cholesterol Feeding Bile Acids Unchanged. Amino o nitrogen. I.S , Dog Date. 0) if" 2 j Remarks. S j3 In 1 cc. of bile. In 6 hours 111 1 1918 CC, mg. mg. mg. Ibs. Mixed diet. 18-93 Sept. 11 76 0.210 15.95 586 31.5 " 12 58 0.320 18.55 681 32.5 1 gm. of taurine,* 3 gm. of cholesterol. Some diarrhea. r Intoxicated by ether. " 13 76 0.225 17.10 628 34.0 Hb. 126 per cent. R. B. C. 6,430,000. 15-22 6.40 235 Average 10 days fasting. June 6 3.6 0.642 2.31 84 28.2 100 gm. of cane sugar and 25 gm. of glucose after 10 days fasting with bile exclusion. Vomiting. " 7 10 0.616 6.16 226 27.4 2 gm. of taurine,* 3 gm. of cholesterol. Mixed diet. 18-54 Sept. 3 12 0.170 7.14 262 27.0 Hb. 110 per cent. R. B. C. 5,515,000. " 4 46 0.141 6.49 238 27.4 1 gm. of taurine* and 3 gm. of cholesterol. Intoxicated by ether. " 5 58 0.113 6.56 241 28.0 * Taurine dissolved in water. Cholesterol dissolved in ether and given by stomach tube at beginning of 6 hour collection. mixed diet gives considerable daily fluctuation, but the averages are nearly uniform. The addition of 10 gm. of dried red blood cells does not influence the curve of bile acid excretion. This amount of red cells contains an appreciable amount of cholesterol, but of course not comparable to the large amounts used in Tables 428* Metabolism of Bile Acids. VI LI and LII. We have other experiments with brain feeding which show the same negative influence on bile acid excretion. Table LV shows the negative reaction following the intravenous injection of laked red blood cells. This again illustrates how easily we may dissociate the excretion curves of bile pigments and TABLE LIII. Red Blood Cell Feeding' Bile Acids Unchanged. Dog 18-23. Simple Bile Fistula. Amino . nitrogen. I.S Date. 3 Inl In 6 22 5 bt Remarks. I cc. of bile. hours. i-S 1 1918 CC. mg. mg. mg. Ibs. July 15 56 0.272 15.23 559 31.8 Mixed diet. Absolute bile exclu- sion. " 16 63 0.254 16.00 587 33.3 Hb. 115 per cent.- R. B. C. 6,200,000. " 17 67 0.297 19.90 731 32.5 " 18 71 0.410 29.10 1,068 32.0 " 19 62 0.324 20.18 741 32.3 Average 20.00 734 July 22 53 0.254 13.47 495 31.0 Mixed diet + 10 gm. of R. B. C.* " 23 60 0.262 15.72 576 32.3 it tt + 10 " 24 57 0.302 17.20 628 32.5 tf i -if\ K u " 25 64 0.271 17.35 637 32.8 <( _J_ 1Q " 26 62 0.326 20.20 742 .. 1 1Q ., Averag B ... 16.80 617 * Prepared by washing red blood cells in normal saline 3 times. The residual cells were dried by warm air and ground to a powder, 10 gm. of red cells made into an indefinite solution emulsion mixture, flavored with a little sugar and salt, made up to 400 cc. with water, and given by stomach tube. bile acids. The intravenous injection of large amounts of laked red blood cells will cause a prompt and large increase in the output of bile pigments but 110 increase in bile acid excretion. There is no immediate and no delayed bile acid reaction which we have been able to observe. Some investigators have recorded a drop in bile acid excretion following intravenous injections of hemoglobin but our experiments are clear cut and negative in this respect. One Foster, Hooper, and W hippie 429 observes only the normal physiological fluctuations. A possible explanation of the observed depression of bile acid excretion is. the febrile reaction which sometimes is observed following hemoglobin injections. No such reaction was observed in these experiments. It is well to note that the whole laked cells were given that is, stroma and hemoglobin. After the blood had been laked by dis- TABLE LIV. Red Blood Cell Feeding- Bile Acids Unchanged. Dog 18-54. Bile Fistula and Splenectomy. Amino nitrogen. *O fl f e+ .^- g 3 Inl In 6 83s 43 hO ! cc.of bile. hours. ** 3 ' 1918 CC. mg. mg. mg. Ibs. July 22 37 0.268 9.91 363 28.0 Mixed diet. Bile exclusion. " 23 41 0.213 8.71 319 28.3 " 24 18 0.315 5.67 208 27.8 " 25 33 0.299 9.87 362 27.5 " 26 45 0.282 12.68 466 Average 9 40 345 July 30 65 0.125 8.13 299 28.0 Hb. 100 per cent. R. B. C. 5,075,000. Mixed diet + 10 gm. of R. B. C.* " 31 48 0.237 11.36 417 28.3 " " + 10 " " " Aug. 1 50 0.284 14.20 522 27.8 (( i -ir\ (( . " 2 37 0.145 5.36 196 27.5 + 10 Tube out. About 10 cc. lost. Average 9.7 356 * Red blood cells prepared as in Table LIII. tilled water it was shaken thoroughly to insure a complete injec- tion of stroma and cell fragments. It has been suggested repeat- edly that the red cell stroma and perhaps the contained cholesterol were waste products which normally came to the liver to be trans- formed into bile acids and eliminated in the bile. This is an attractive hypothesis which is delightfully simple, but like many others it has no basis of experimental fact and should be put aside even if with regret. 430 Metabolism of Bile Acids. VI Since Schrotter, Weitzenbock, and Witt were able to make rhizocholic acid from cholic acid, cholesterol, turpentine, or camphor, we thought it might be possible to produce cholic acid in the animal body by feeding either turpentine or camphor. Taurine was given on the last day in each experiment (Table LVI) so that any cholic acid which might be formed in the organism would unite with the taurine and be excreted in the bile as tauro- TABLE LV. Laked Red Cells Intravenously' Bile Acids Unchanged. Ami no nitrogen. .2 o Dog No. Date. a la I Remarks. In 1 3 cc. of bile. In 6 hours. JSJ ^ 1918 cc. mg. mg. mg. Ibs. 18-23 July 29 66 0.262 17.28 637 30.5 Mixed diet. " 30 72 0.223 16.05 590 32.3 Laked blood.* " 31 58 0.349 20.25 744 32.3 Hb. 120 per cent. R.B.C, 6,375,000. Aug. 1 63 0.287 18.18 678 31.8 Laked blood.* " 2 51 0.413 21.03 772 32.3 18-93 July 29 68 0.207 14.07 517 30.3 Mixed diet. " 30 66 0.241 15.90 584 31.8 Laked blood.* " 31 53 0.280 14.85 545 32.3 Hb. 135 per cent. R. B. C. 6,380,000. Aug. 1 70 0.229 16.02 588 32.3 Laked blood.* " 2 64 0.315 20.15 740 33.3 * 100 cc. of sterile defibrinated normal blood, centrifuged, red cells washed and laked with water, made up to 100 cc., and kept on ice over night; rendered isotonic, warmed, shaken, and given intravenously by hypodermic needle 2 hours after start of collection. cholic acid. This was probably an unnecessary precaution when we recall the great output of taurocholic acid which follows a cholic acid feeding during periods of mixed diet. This indicates an abundant source of taurine in the body during periods of liberal feeding, more than enough to combine with any expected excess of cholic acid. Careful scrutiny of Table LVI reveals no fluctuations other than those observed in control periods, whether terpene hydrate alone or combined with taurine was given by stomach. There is no Foster, Hooper, and Whipple 431 TABLE LVI. Terpene Hydrate With and Without Taurine by Mouth. Amino nitrogen. ^5 Dog No. Date. flj 1 l-sf 'So Remarks. Inl In 6 "o cc. oi bile. hours I s - 2 '* 1918 cc. mg. mg. mg. Z6s. Mixed diet. 15-22 Sept.. 3 22 1.150 25.30 929 33.3 Hb. 150 per cent. R. B. C. 7,455,000. " 4 47 0.214 10.05 369 34.5 1 gm. of taurine, 1 gm. of ter- pene hydrate.* K 45 0.381 17.15 630 33.5 1 gm. of taurine, 1 gm. of ter- * pene hydrate.* " 6 38 0.388 14.73 541 32.8 Mixed diet. ' 18-23 Aug. 27 57 0.261 14.86 545 30.8 " 28 57 0.343 19.55 718 31.8 0.25 gm. of terpene hydrate.* " 29 70 0.309 21.63 794 33.3 0.5 gm. of terpene hydrate.* 0.5 gm. of taurine. " 30 53 0.434 23.00 844 33.3 Mixed diet. 18-54 Sept. 10 61 0.112 6.84 751 28.8 " 11 82 0.238 19.50 716 29.0 " 12 81 0.186 15.05 552 28.5 1 gm. of terpene hydrate,* 1 gm. of taurine by stomach tube. Diarrhea. " 13 47 0.155 7.29 267 27.8 Mixed diet. 17-181 Sept. 10 49 0.292 14.30 525 25.0 " 11 42 0.280 11.75 431 25.3 " 12 67 0.267 17.85 655 26.0 1 gm. of taurine, 1 gm. of ter- pene hydrate.* Diarrhea. " 13 42 0.324 13.60 499 25.0 . Mixed diet. 18-93 Sept. 3 64 0.285 18.25 670 30.5 Hb. 126 per cent. R. B. C. 6,430,000. " 4 44 0.073 3.94 144 33.5 1 gm. of taurine and 1 gm. of terpene hydrate.* Diuresis. " 5 54 0.183 9.89 363 32.5 1 gm. of taurine, 1 gm. of ter- pene hydrate.* " 6 64 0.291 8.61 684 * Terpene hydrate dissolved in a little dilute alcohol ; taurine dissolved in water, given by stomach tube at beginning of 6 hour collection. 432 Metabolism of Bile Acids. VI cholagogue action and no change in the bile concentration and total amounts of bile acids. Table LVII shows the results of administration of camphor by mouth. Repeated doses of spirits of camphor have no effect upon the bile acid excretion. Similar observations have been made on TABLE LVII. Camphor Feeding With and Without Taurine by Mouth. Dog 15-22. Simple Bile Fistula. Date. Volume. Amino nitrogen. Taurocholic acid in 6 hours. js M i Remarks. Inl cc.of bile. In 6 hours. 1918 cc. mg. mg. mg. Ibs. July 29 48 0.290 13.92 511 33.5 Mixed diet. " 30 29 0.155 4.49 165 34.3 0.2 gm. of camphor* by stomach tube. Hb. 135 per cent. R. B. C. 6,430,000. " 31 35 0.292 10.22 375 34.3 0.4 gm. of camphor by stomach tube. Aug. 1 38 0.430 16.34 600 33.5 0.6 gm. of camphor by stomach tube. Considerable salivation. " 2 14 1.030 14.41 529 33.5 " 5 47 33.8 0.4 gm. of camphor by stomach tube. " 6 35.0 0.4 gm. of camphor by stomach tube. " 7 53 0.269 14.25 523 34.3 0.4 gm. of camphor by stomach tube. " 8 63 0.222 14.00 514 33.3 0.4 gm. of camphorand.l gm.taurine by stomach tube. " 9 40 0.439 17.56 644 34.0 0.4 gm. of camphor by stomach tube. * Camphor given as a 10 per cent solution spirits of camphor. Diu- resis was uniformly noted. other dogs. Obviously there is no immediate and no delayed reaction which can be attributed to the action of this drug. It is evident that neither camphor nor terpene hydrate have any effect on the excretion of taurocholic acid. Cholesterol, turpen- tine, cholic acid, and camphor may all belong to the same group chemically, but physiologically they certainly are not closely related, at least as regards bile acid metabolism. Foster, Hooper, and Whipple 433 SUMMARY. Taurine as found in the body is derived in all probability from the cystine of the food or body protein. Taurine appears to be present in excess of the amount needed to combine with the cholic acid of normal metabolism. Cholic acid is the limiting factor which determines the level of bile acid excretion in the bile. The origin and fate of cholic acid have not been satisfactorily determined. Cholesterol fed alone or combined with taurine causes no change in the excretion of bile acids. This gives no evidence of any physi- ological relationship between cholesterol and cholic acid. Red blood cells fed by mouth or hemolyzed and injected intra- venously have no influence upon the level of bile acid excretion. Terpene hydrate and camphor fed alone or combined with taurine do not influence the curve of bile acid excretion. BIBLIOGRAPHY. 1. d'Amato, L., Biochem. Z., 1915, Ixix, 217. 2. von Bergman, G., Beitr. chem. Physiol. u. Path., 1904, iv, 192. 3. Flury, F., Arch. Exp. Path. u. Pharm., 1911, Ixvi, 221. 4. Friedman, E., Beitr. chem. Physiol. u. Path., 1903, iii, 25. 5. Gibson, R. B., J. Biol. Chem., 1909, vi, p. xvi. 6. Goodman, E. H., Beitr. chem. Physiol. u. Path., 1907, ix, 91. 7. Hammarsten, O., Z. physiol. Chem., 1898, xxiv, 322. 8. Lifschiitz, J., Ber. chem. Ges., 1914, xlvii, 1459. 9. Moreschi, Redn. soc. chim. ital., 1913, v, 242. 10. Pregl, F., Z. physiol. Chem., 1910, Ixv, 157. 11. Schrotter, H., Weitzenbock, R., and Witt, R., Monatshr. Chem., 1908, xxix, 245. 12. Schrotter, H., and Weitzenbock, R., Monatschr. Chem., 1908, xxix, 395. 13. Windaus, A., Uber Cholesterin; Habilitationsschrift, Freiburg i. Br. Speyer u. Karner, 1903-Sep: v. Vf. Abstracted in Chem. Zentr., 1903, i, 814. 14. Wohlgemuth, J., Z. physiol. Chem., 1903-04, xl, 81. THE WAVERLY PRESS BALTIMORE. U. 8. A. THIS BOOK IS DUE ON THE LAST DATE STAMPED BELOW AN INITIAL FINE OF 25 CENTS WILL BE ASSESSED FOR FAILURE TO RETURN THIS BOOK ON THE DATE DUE. THE PENALTY WILL INCREASE TO SO CENTS ON THE FOURTH DAY AND TO $1.OO ON THE SEVENTH DAY OVERDUE. MAR 8 1933 WAR 201935 OCT 1 t937 OCT 14 1937 APR 10 1939 JAN 25 1943 APR 26 131950 AUG 6 1952 DEC 6 J964 WRR 22 1973 KIAR14 197319 LD 21-50m-l,'3S U.C. BERKELEY LIBRARIES C02bl73Dfi3 UNIVERSITY OF CAUFORNIA UBRARY