PATTERNS OF BILE ACIDS AND MICROFLORA IN THE HUMAN SMALL INTESTINE

Transcription

1 G ASTROENTEROLOG Y Copyright 1973 by The Williams & Wilkins Co. Vol. 64, No.1 Printed in U.S.A. PATTERNS OF BILE ACIDS AND MICROFLORA IN THE HUMAN SMALL INTESTINE I. Bile acids ANDREW MALLORY, M.D., FRED KERN, JR., M.D., JAMES SMITH, M.D., AND DWAYNE SAVAGE, PH.D. Division of Gastroenterology, Department of Medicine, University of Colorado School of Medicine, Denver, Colorado Bile acids were studied in the intestinal content of 12 control subjects, 8 patients with ileal resection, and 3 patients with intestinal stasis. Intestinal fluids were aspirated from the duodenum after cholecystokinin stimulation and from four additional sites in the small intestine, 50 cm apart, 4 to 7 hr after a meal. The total concentrations of bile acids and their conjugates were determined by enzymatic assay. The distribution of bile acids was measured by gas liquid chromatography. The results were examined in relation to the intestinal microfloracultured from the same samples, described in a separate paper. Bile acid concentrations were lower in the duodenum of patients with ileal resection. Throughout the small intestine there was a marked decrease in bile salts conjugated with taurine and a smaller decrease of glycine-conjugated bile salts. In addition, the percentage of chenodeoxycholic acid was clearly increased at all levels, while the percentage of deoxycholic acid was decreased. In the 2 patients with any deoxycholic or lithocholic acid, anaerobic organisms, such as bacteroides, were abundant in most areas in the small intestine. In the 3 patients with intestinal stasis the total duodenal bile acid concentrations were similar to those in the control subjects, but throughout their small intestine there was a modest decrease in taurine-conjugated bile acids and a definitely increased proportion of deoxycholic and lithocholic acids. The proportion of unconjugated bile acids was not clearly increased in any patient studied. Received May 30, Accepted August 10, Address requests for reprints to: Dr. Fred Kern, Jr., University of Colorado Medical Center, 4200 East Ninth Avenue, Denver, Colorado This investigation was supported by United States Public Health Service Research Grants AM (Dr. Kern), RR 69 (Dr. Savage), and the General Clinical Research Centers program of the National Institutes of Health, FR Dr. Mallory was supported by a Training Grant in 26 An intact enterohepatic circulation of bile acids is necessary for efficient absorption of dietary fat, cholesterol, and fat-soluble vitamins. The enterohepatic circulation is diminished in patients with ileal resection and is altered in patients with obstructed intestinal segments. In both of these disorders, especially the latter, Gastroenterology, AM 05122, National Institute of Arthritis and Metabolic Diseases, United States Public Health Service, and Dr. Smith was supported by Veterans Administration Training Grant in Gastroenterology, TR 110. Dwayne Savage, Ph.D., is presently at the Department of Microbiology, University of Texas, Austin, Texas. The authors gratefully acknowledge the excellent technical assistance of Mrs. Diann Miller, Miss Patricia Coan, and Mr; Philip Showalter.

2 January 1973 BILE ACIDS AND MICROFLORA IN SMALL INTESTINE. I 27 intestinal microorganisms able to alter bile acids influence intestinal bile acid concentration and composition. There exist intestinal bacteria that deconjugate bile acids, dehydroxylate them at the 7a position, and modify them in various other ways. 1-4 Unfortunately, the biological significance of most of the metabolites is not known. It has been proposed that some intestinal bacteria may transform deoxycholic acid into a substance carcinogenic for the colon and rectum. 5, 6 It is better documented at this time, however, that the concentrations of certain of the microbial metabolites sharply increase or decrease in some disease states. For example, bile acids dehydroxylated at the 7a position are detectable only at very low concentrations or not at all in some patients with small intestinal resection or colon resection and ileostomy. 7-9 By contrast, deconjugated bile acids are found at higher than normal concentrations in patients with obstructed or stagnant intestinal segments_ Such data are limited, however; the microflora and bile acid composition have bee:! studied in detail in only a few such patients. In the accompanying paper, we describe the microflora, and in this paper we report the bile acid composition of intestinal fluids collected from multiple areas throughout the small intestine in normal subjects, and in patients with ileal resection or small bowel stasis. We have examined in detail the relationships between the intestinal microbes and the bile acids. Materials and Methods Patients and subjects. Patients and control subjects were hospitalized in the clinical research center. The control subjects were six male and six female healthy students, laboratory workers, nurses, and hospital personnel, 18 to 45 years old, without gastrointestinal or hepatobiliary disorders. None had had a cholecystectomy. The clinical details of the 8 patients with intestinal resection and the 3 with intestinal stasis are shown in table 1. Procedures. A 300-cm double lumen, polyvinyl tube with a mercury-containing balloon (3 ml) on the end and with five 3-mm holes 1 cm apart at the distal end was passed through the nose into the stomach on the evening prior to the study. Early on the day of the study, with the patient fasting and the tube in the duodenum, as shown by fluoroscopy and an alkaline ph of aspirate, 75 U of cholecystokinin (Karolinska Institutet) were given by vein. When the duodenal aspirate became uniformly dark, the first sample was collected. Thereafter, a standard hospital diet was eaten and the tube was allowed to pass down the small intestine. When it had advanced approximately 50 cm, intestinal fluid was again collected. This was repeated every 50 cm until the terminal small intestine was reached. The period of study was from 20 to 72 hr. The individual sample size, for both microbiological and bile acid studies, was 2 to 10 ml. The position of the tube was determined from calibrations marked on the tube, and from fluoroscopy. As the end of the tube approached the colon, its position was checked very carefully. It entered the colon only once, and that study was repeated. The time of collection of intestinal fluids in relation to meals should be emphasized. The duodenal sample was obtained while the patient was fasting, but after the intravenous administrationof cholecystokinin. The samples from more distal locations were collected 4 or more hr after the previous meal. Although it was recognized that most of the bile acid pool would be stored in the gallbladder, except after cholecystokinin, and that intestinal bile add concentration would be low, the alternative procedure of obtaining specimens at shorter intervals after eating would have been more unsatisfactory for our purpose. There would have been a highly variable relationship between the enterohepatic circulation of bile acids and intestinal content, depending upon position of the tube, its rate of movement, and transit time of intestinal content, thereby making comparisons among groups unreliable. Bile acid analytical techniques. The intestinal fluid (2 to 10 ml) for bile acid assay was added to 10 volumes of 95% ethanol, heated for 20 to 30 min at 70 C, and centrifuged. The sediment was washed twice with 3 ml of 95% ethanol and the protein-free supernatants were pooled. The supernatant was then evaporated to dryness, redissolved in ethanol, and used for the enzymatic measurement of the total bile acid concentration, for the determination of bile acid conjugates, and for gas liquid chromatography (GLC). For total bile acid assay and for GLC, the ethanolic solution was added to 20 ml of 2 N NaOH and hydrolyzed at 120 C, 14 psi, for 90 min. After cooling it was acidified to ph 1.0

3 28 MALLORY ET AL. Vol. 64, No. 1 with concentrated HCl, extracted four times with 20 ml of peroxide-free diethyl ether, evaporated to dryness, and dissolved in absolute methanol. Prior to enzymatic assay, the bile acids were separated from free fatty acids by silica gel thin layer chromatography in a solvent system of hexane-diethyl ether-glacial acetic acid (80: 20 : 1).13 Standard solutions of purified free bile acids (lithocholic, cholic. chenodeoxycholic. and deoxycholic) and free fatty acids were applied to the same plate. The silica gel containing the bile acid was scraped into a test tube and the bile acids were extracted by thorough mixing with 5 ml of 95% ethanol five times. The ethanol rinses were separated by centrifugation, pooled, and evaporated. The residue was dissolved in methanol, and bile acid concentration was determined by the 3- steroid dehydrogenase assay of Talalay, as modified. 14 A series of standard bile acid solutions (standard curve) was always assayed in an identical manner. Recovery of conjugated bile salts added to the intestinal fluid averaged 96.0 ± 3%. Duplicate analyses agreed with ± 6.0%. For GLC, methyl ester trifluoroacetate derivatives were prepared. I5 The methanol containing the hydrolyzed bile acids was used without preliminary thin layer chromatography. The bile acids were esterified with freshly distilled ethereal diazomethane. After 30 min, the solvents were removed under nitrogen. The methyl esters were dissolved in benzene (pesticide grade, Fisher Scientific Co., Medford, Mass.) and three drops of dry. redistilled pyridine, and 1.2 to 2 ml of trifluoroaceticanhydride (Eastman, Rochester, N. Y.) were added and allowed to react for 30 min at 40 C or overnight at room temperature. The excess solvent was evaporated under nitrogen. The methyl ester trifluoroacetate derivatives were brought to an appropriate volume for injection TABLE 1. Patient data Small Chole- Length intestine Schil- Patient Age Sex Diagnosis cystec of ileal involved Carotene' Stool d-xylose d ling tomy resec by fat' tiona test' disease em em M/IOO ml g/day g/5 hr %/24 hr R. H. 26 M Regional enteritis No V. L. 25 M Regional enteritis No K.R. 35 F Regional enteritis No NDI ND A. C. 45 F Regional enteritis No ND V. P. 63 F Ileal obstruction,? Yes l(jo' cause B. C. 43 F Radiation enteritis Yes H.B. 67 F Ileal obstruction duo- Yes denal diverticulum D. A. 45 F Actinomycosis Intestinal stasis A. P. 68 M Multiple diverticula, No duodenum, jejunum, ileum M. P. 49 F Duodenal diverticulum; No Billroth I gastrectomy M. U. 49 F Scleroderma No a Denotes most distal part of Ileum unless otherwise noted. All patients except V. P. had a partial or total ascending colectomy at time of ileal resection. b Normal: > 70 Ilg per 100 m\. C Normal: < 5 g per day. d Normal: > 5 g per 5 hr. Normal with intrinsic factor: > 5.5% per 24 hr. I ND, not done. g Six centimeters of terminal ileum remaining.

4 January 1973 BILE ACIDS AND MICROFLORA IN SMALL INTESTINE. I 29 with carbon disulfide. The GLC was carried out using a Beckman Model GC5 with 6-foot spiral glass columns, 2 mm in internal diameter, filled with 1% QF-l on Gas Chrome Q, 100 to 120 mesh (Applied Science Laboratories, State College, Pa.). 16 A hydrogen flame ionization detector was used and the detector response was calibrated for each bile acid. Column temperature was maintained at 210 to 215 C. When solutions of a known amount of bile acid were carried through the entire procedure, recovery varied with the amount of bile acid present. With 1 to 2 mg of bile acid, the recovery was 75%; with 2.5 mg it was 88%; and with 3 mg, 97%. For determination of bile acid conjugates, a portion of the ethanolic extract of intestinal fluid was streaked on 1 mm thick silica gel plates, bile acid standards for identification of bands were spotted on the plate and taurine conjugates, glycine conjugates and free bile acids were separated using a solvent system described by Hofmann. 17 The bile acid bands were detected by iodine vapor, extracted from the silica gel, and the concentration assayed enzymatically as described above. Statistical methods. Differences between groups were compared by the Wilcoxon Rank Sum Test. 's Results Concentrations and conjugation of intestinal bile acids. The concentrations and distributions of bile acids in duodenal samples are shown for each normal subject and the patients in figure 1 and tables 2, 3, and 4. A correlation (r = 0.7) between magnitude of steatorrhea and duodenal bile acid concentration is apparent (tables 1 and 2). The mean total bile acid concentration was lower in the patients with ileal resection than in the control subjects (P < 0.05). In 2 of the 8 patients, however, the concentrations of 23.0 and 24.0.umoles per ml were within the normal range. These 2 patients, K. R. and D. A., had the smallest ileal resections, 30 and 28 cm, respectively. The duodenal bile acid concentration in the other 6 patients was 3 to 8.6.umoles per ml compared with a mean of 20 in the normal subjects (P < 0.01). In the Q "",tipnt<;, who hluj Imnpr!!One chole- }' molesl ml T Percent 100 U 10 : G 50 G G CONTROL RESECTION STA SIS CONTROl RtSECTION STASIS FIG. 1. The distribution of bile acids in the duodenum of control subjects, patients with resection of the ileum, and those with intestinal stasis. The left panel shows the mean concentrations of glycine conjugates (G), taurine conjugates (T), and unconjugated bile acids (U). The right panel shows the mean percentages of cholic (C), chenodeoxycholic (CDC), deoxycholic (D), and lithocholic (LC) acids. TABLE 2. Concentration and distribution of bile acid in the duodenum (normal subjects) Subject Total Bile acids Conjugated Distribution" Uncon- Gly- Taucinn rine 'ugated LC DC D C I'moleslml % J. S J. G H.A J. B T. J S. F. " J. V L. C C. C M. H J. S Mean LC, lithocholic acid; DC, deoxycholic acid; CDC, chenodeoxycholic acid; C, cholic acid. centrations were the same as in the patients with intact gallbladders. In the 3 patients with intestinal stasis duodenal bile acid concentrations were similar to those in the control subjects. In small bowel areas distal to the duodenum, bile acid concentrations were lower in all subjects and patients and c c

5 30 MALLORY ET AL. Vol. 64, No. 1 TABLE 3. Concentration and distribution of bile acids in the duodenum patients with ileal resection and intestinal stasis Patient Bile acids Conjugated Distribution" Total Uncon- Gly- Tau- 'ugated cine rine LC DC D C p.moleslml. % ileal resection R. H V. L.. " " K.R A. C V. P B. C H.B. 0, D. A. '" Mean Intestinal stasis A. P M. P M.U Mean "LC, lithocholic acid; DC, deoxycholic acid; CDC, chenodeoxycholic acid; C, cholic acid. (table 4). Unconjugated bile acids were present in very low concentrations in most samples. In the patients with ileal resections, the percentage of bile acids conjugated with taurine was markedly lower than normal (P < 0.05) in all areas of the small intestine except at 300 cm. Taurine conjugation was diminished even in the patients (K. R. and D. A.) with small ileal resections and normal duodenal bile acid concentrations. The 2 patients with stasis also had less bile acid conjugated with taurine than control subjects. Distribution of bile acids. The distribution of bile acids differed among the groups at all levels of the small bowel (tables 2, 3, and 4, fig. 1). Mean percentages of the four major bile acids varied only slightly from area to area in normal subjects (cholic 44 to 52%, chenodeoxycholic 27 to 34%, deoxycholic 17 to 20%, and lithocholic 1 to 3%). In patients with ileal resections, percentages of chenodeoxycholic were higher (P < 0.02) in all areas, and deoxycholic lower (P < 0.01) in all areas. Deoxycholic acid was present in only 2 of the patients. By contrast, a larger percentage of the major secondary bile acids was detected in the stasis patients than in the control subjects. No other bile acid metabolites were identified. In some chromatograms, there were tiny peaks which could not be differentiated from partial derivatives of major bile acids (as occasionally found in G LC of standard solutions); these never accounted for more than 2% of the bile acids present. TABLE 4. Concentration and distribution of bile acids in duodenum (jejunum and ileum in patients with ileal resection and intestinal stasis)" Bile acids Duodenum Conjugated Distribution' Uncon- Total Gly- Tau- 'ugated cine rine LC DC D C p.moleslml % 100 cm Normal Stasis cm Normal Stasis..,., cm Normal Stasis cm Normal ileal resection Stasis cm Normal Stasis " Mean values. b LC, lithocholic acid; DC, deoxycholic acid; CDC, chenodeoxycholic acid; C, cholic acid.

6 January 1973 BILE ACIDS AND MICROFLORA IN SMALL INTESTflVE. I 31 Discussion The total bile acid concentrations and their distribution patterns in normal subjects were similar to those reported, except for the regular finding of a small amount of unconjugated bile acid in most samples from all areas. This was usually less than 1 J.lmole per ml. McLeod and Wiggins 8 found unconjugated bile acids in jejunal aspirates in 1 of 3 normal subjects and Sjovall 19 found "trace" amounts in a "few" of his normal subjects. Patients with ileal resection. Patients with resection had significantly lower total bile acid concentrations in the duodenum after gallbladder emptying, as reported by others In the patient (A. C.), whose steatorrhea was greatest (51 g per day), the concentration was clearly below the critical micellar concentration. It is likely that this would have been so in several others had the duodenal bile been diluted by a meal. Although many factors, such as location of the sampling tube, timing of the collection, and gallbladder-concentrating ability undoubtedly influence the concentrations obtained, there was a correlation between total duodenal bile acid concentration and steatorrhea (P = 0.05). The only patients (K. R. and D. A.) with resections less than 50 cm had normal duodenal bile acid concentrations; 1 of them had very slight steatorrhea (6.6 g per day) and the other had none. VanDeest et al. 20 found that some ileal resection patients have normal duodenojejunal bile acid concentrations after the first meal of the day, but low concentrations after succeeding meals due to progressive depletion of the bile salt pool. 20 The bile acid concentrations in resection patients were similar to those in control subjects in the small bowel distal to the duodenum. No differences were detected, undoubtedly because samples from these areas were obtained more than 4 hr after the last gallbladder stimulus, at a time when most bile acid has been absorbed from the intestine, excreted by the liver, and stored in the gallbladder. The increase in the percentage of bile acids conjugated with glycine in patients with ileal resection, as reported by others is probably due to less jejunal and colonic absorption of taurine than of glycine conjugates and to depletion of the relatively scarce taurine. Glycine, in contrast, is abundantly available for conjugation of bile acids. Marked alterations in the distribution of bile acids were detected in all areas of the small bowel. The percentage of chenodeoxycholic acid was significantly increased in 7 of the 8 resection patients and deoxycholic acid was absent in 6 of 8. Similar changes in composition of the bile salt pool in such patients have been described by other investigators The proportion of chenodeoxycholic acid may be increased because chenodeoxycholic acid is absorbed from the jejunum by passive diffusion more efficiently than cholic acid. 23 In subjects without intestinal disease, Vlahcevic et al. 24 found that the average daily production of cholic acid was twice as great and the turnover rate approximately half that of chenodeoxycholic acid. Thus, in patients with ileal resection, the turnover of cholic acid is very rapid and the bile acid pool is probably maintained to a large extent by enterohepatic circulation of chenodeoxycholic acid. Secondary bile acids were absent in 6 of 8 patients, a finding that is generally consistent with that of others. No deoxycholic acid was detected by Hardison and Rosenberg 7 in the duodenal aspirate of 2 of 4 patients with ileal resection, or by McLeod and Wiggins 8 in 3 of 5 such patients (the 3 with the largest resection). Likewise, Hofmann and Poley 9 reported that patients with less than 100 cm of ileum resected had greatly reduced secondary bile acids in jejunal contents. By contrast, Thaysen et al. 2 5 found deoxycholic acid in small amounts in all 6 of his patients. Garbutt et al. 26 found that 24 hr after the administration of taurocholate C, duodenal fluid contained labeled deoxycholate, as the glycine conjugate, in 4 of 6 patients with ileal resection, without stasis. Similarly, in an earlier study, we found deoxycholic acid in 4 patients. 2 2

7 32 MALLORY ET AL. Vol. 64, No.1 One of those patients, V. L., was also studied in this investigation; deoxycholic acid was present both times, 3 years apart. Thus, of a total of 11 patients with ileal resection examined in this laboratory, 6 had no detectable deoxycholic or other secondary bile acids and 5 had normal amounts. The absence of secondary bile acids in some of these patients, but not others, has not been satisfactorily explained. Stasis patients. The 3 patients with stasis and without ileal resection exhibited markedly uniform findings. All 3 had pronounced bacterial overgrowth throughout the small bowel, normal total bile acid concentrations, elevated glycine to taurine ratios, and alterations of bile acid distribution consisting of increased percentages of lithocholate and deoxycholate with corresponding decreases in chenodeoxycholate and cholate. Most investigators I 0-12 have reported amounts of unconjugated bile salts in such patients, larger than we found in ours, probably because our distal samples were collected many hours after a meal, when bile acid concentrations were low and the freely diffusable un conjugated bile acids had been absorbed. Moreover, since the duodenal samples were collected almost immediately after cholecystokinin administration, there was very little time for deconjugation. It should be noted also that none of our 3 patients had the fully developed "stagnant loop syndrome" with vitamin B 12 malabsorption and steatorrhea. The changes in bile salt composition and their significance in patients with intestinal stasis have been discussed in detail by others REFERENCES 1. Midtvedt T, Norman A: Bile acid transformation by microbial strains belonging to genera found in intestinal contents. Acta Pathol Microbiol Scand 71: , Hill MJ, Drasar BS: Degradation of bile salts by human intestinal bacteria. Gut 9:22-27, Aries V, Crowther JS, Drasar BS, et al: Degradation of bile salts by human intestinal bacteria. Gut 10: , Aries V, Hill MJ: Degradation of steroids by intestinal bacteria. II. Enzymes catalysing the oxidoreduction of the 3a-, 7a and 12a-hydroxyl groups in cholic acid, and the dehydroxylation of the 7-hydroxyl group. Biochim Biophys Acta 202: , Aries V, Crowther JS, Drasar BS, et al: Bacteria and the aetiology of cancer of the large bowel. Gut 10: , Hill MJ, Crowther JS, Drasar BS, et al: Bacteria and aetiology of cancer of large bowel. Lancet 1:95-100, Hardison WGM, Rosenberg IH: Bile-salt deficiency in the steatorrhea following resection of the ileum and proximal colon. N Engl J Med 277: , McLeod GM, Wiggins HS: Bile-salts in small intestinal contents after ileal resection and in other malabsorption syndromes. Lancet 1: , Hofmann AF, Poley JR: Cholestyramine treatment of diarrhea associated with ileal resection (abstr). Gastroenterology 56:1168, Tabaqchali S, Hatzioannou J, Booth CC: Bilesalt deconjugation and steatorrhoea in patients with the stagnant-loop syndrome. Lancet 2:12-16, Rosenberg IH, Hardison WG, Bull DM: Abnormal bile-salt patterns and intestinal bacteria overgrowth associated with malabsorption. N Engl J Med 276: , Krone CL, Theodor E, Sleisenger MH, et al: Studies on the pathogenesis of malabsorption. Medicine 47:89-106, Gordon SG, Philippon F, Borgen KS, et al: Formation of fatty acid ethyl esters during lipid ex traction and storage: an important artifact. Biochim Biophys Acta 218: , Woodbury JF, Kern F: Fecal excretion of bile acids: a new technique for studying bile acid kinetics in patients with ileal resection. J Clin Invest 50: , Kuksis A: Gas-liquid chromatography of bile acids. J Am Oil Chern Soc 42: , Sjovall J: Gas chromatography of bile acids, Biomedical Applications of Gas Chromatography. Edited by HA Szymanski. New York, Plenum Press, 1964, p Hofmann AF: Thin-layer adsorption chromatography of free and conjugated bile acids on silicic acid. J Lipid Res 3: , Siegel S: Nonparametric statistics. New York, McGraw-Hill Book Co, 1956, p Sjovall J: On the concentration of bile acids in the human intestine during absorption. Acta Physiol Scand 46: , Van Deest BW, Fordtran JS, Morawski SG, et al: Bile salt and micellar fat concentration in proxi-

8 Janunry 1973 BILE ACIDS AND MICROFLORA IN SMALL INTESTINE. I 33 mal small bowel contents of ileectomy patients. J Clin Invest 47: , Garbutt JT, Heaton KW, Lack L, et al: Increased ratio of glycine- to taurine-conjugated bile salts in patients with ileal disorders. Gastroenterology 56: , Abaurre R, Gordon SG, Mann JG, et al : Fasting bile salt pool size and composition after ileal resection. Gastroenterology 57: , Hislop IG, Hofmann AF, Schoenfield JL: Determinants of the rate and site of bile acid absorption in man. J Clin Invest 46: , Vlahcevic ZR, Miller JR, Farrar JT, et al: Kinetics and pool size of primary bile acids in man. Gastroenterology 61 :85-90, Thaysen EH, Bruusgaard A, Eriksen B: Evaluation of the efficiency of bile salt recirculation in patients with terminal ileopathies by means of deoxycholate determination in duodenal aspirates. Scand J Gastroenterol 5:39-47, Garbutt JT, Wilkins RM, Lack L, et al: Bacterial modification of taurocholate during enterohepatic recirculation in normal man and patients with small intestinal disease. Gastroenterology 59: , Gorbach SL, Tabaqchali S: Bacteria, bile, and the small bowel. Gut 10: , Rosenberg IH: Influence of intestinal bacteria on bile acid metabolism and fat absorption. Am J Clin Nutr 22: , Donaldson RM: Small bowel bacterial overgrowth. Adv Intern Med 16: , 1970