INCREASED RATIO OF GLYCINE- TO TAURINE-CONJUGATED BILE SALTS IN PATIENTS WITH ILEAL DISORDERS

Transcription

1 GASTROENTEROLOGY Copyright 1969 by The Williams & Wilkins Co. Vol. 56, No. 4 Printed in U.S.A. INCREASED RATIO OF GLYCINE- TO TAURINE-CONJUGATED BILE SALTS IN PATIENTS WITH ILEAL DISORDERS J. T. GARBUTT, M.D., K. W. HEATON, M.D., L. LACK, PH.D., AND M. P. TYOR, M.D. Department of Medicine and Department of Physiology and Pharmacology, Duke University Medical Center, Durham, North Carolina The relative conjugation of cholic acid with glycine and taurine (G : T ratio) was studied in duodenal fluid obtained from 13 patients with ileal disorders, 4 patients with disorders which involved the duodenojejunum primarily (2 with nontropical sprue and 2 with Whipple's disease), and 5 student volunteers. The enterohepatic recirculation of sodium taurocholate C was studied in all subjects, and the relative conjugation (G:T ratio) of deoxycholate- l4 C, derived from bacterial metabolism of the injected taurocholate- 14 C, was measured in appropriate duodenal samples. In selected experiments, unconjugated bile salts, cholic acid- l4 C and deoxycholic acid- l4 C, were each administered intravenously, and their relative conjugation (G: T ratio) was measured in duodenal samples obtained throughout an initial 3-hr period and serially thereafter. For both bile salts, the lowest G: T ratio exhibited by patients with a disorder of the ileum was higher than the highest G : T ratio observed in either student volunteers or patients with duodenojejunal disorders. These changes were related to the markedly decreased enterohepatic recirculation of injected taurocholate- 14 C and injected deoxycholate- l4 C in patients with ileal disorders. The G: T ratio of cholic acid- l4 C and deoxycholic acid- l4 C was elevated to comparable levels in duodenal fluid sampled throughout the 3-hr period following intravenous injection of these bile salts into patients with ileal disorders. Furthermore, serial determinations of G: T ratio for deoxycholate- 14 C were similar to initial values after injection of either deoxycholate- l4 C or taurocholate- l4 C. Thus, the hepatic origin of these altered G : T ratios has been demonstrated. Although the precise mechanism involved in the production of these changes was not determined, oral administration of taurine to patients with ileal disorders resulted in a marked decrease in G: T ratio, demonstrating a maintained capacity for conjugation with taurine. Patients with Whipple's disease exhibited G: T ratios for both bile Presented in part at a meeting of the Southern Society for Clinical Investigation, New Orleans, Louisiana, January 26, Address requests for reprints to: Dr. Malcolm P. Tyor, Duke University Medical Center, Durham, North Carolina This investigation was primarily supported by Research Grants AM and AM from the National Institutes of Health, United States Public 711 Health Service, Bethesda, Maryland. The services of the Clinical Research Unit were supported by Grant MO-1FR30 from the National Institutes of Health. The work of Dr. Garbutt was supported by Training Grant 2-A 5093 from the National Institute of Arthritis and Metabolic Diseases. Dr. Heaton's present address is: Department of Medicine, Royal Infirmary, Bristol 2, England.

2 712 GARBUTT ET AL. Vol. 56, No. 4 salts which were intermediate between those of student volunteers and patients with nontropical sprue, on the one hand, and patients with ileal disorders on the other, decreasing to a normal range after treatment. Studies l - 4 of several species of animals have demonstrated that primary and secondary bile salts are conjugated in the liver, primarily with glycine and taurine, prior to their secretion into bile. In normal man, the relative conjugation of bile salts with glycine and taurine attains a ratio which approximates 3: 1. 5 An increase in this ratio has been observed in patients with hepatobiliary disorders during 4 to 20 days of bile fistula T-tube drainage. 6 In these studies, bile salts and other constituents of bile were lost from the enterohepatic circulation. Observations in our laboratory7.8 and in others 9-12 have demonstrated that ileal disorders also result in a considerable loss of enterohepatic recirculation of bile salts. However, under these conditions it may be assumed that materials normally absorbed from more proximal regions may continue to circulate enterohepatically. We have reported previously that "available" cholate conjugates (glycine and taurine) accumulated in the biliary tree of 4 patients with ileal disorders fasted overnight were composed of a marked predominance of glycocholate. 8 The present studies provide confirmatory observations to this predominance, which has also been reported by McLeod and Wiggins. 13 In addition, the present report relates the markedly increased ratio of glycine to taurine conjugates of trihydroxy and dihydroxy bile salts in patients with ileal disorders to the reduction in enterohepatic circulation of these materials. These composite results were not only altered as compared with those obtained from student volunteers but also in comparison with those from patients with disorders which involved the duodenojejunum primarily. Although the precise mechanism involved in the production of these changes in the ratio of glycine to taurine conjugates of bile salts was not determined, it is apparent from the present observations that they were hepatic in origin. Materials and Methods Subjects. Thirteen patients had disorders of the distal small intestine (table 1). Measurements of the rate of disappearance of glycine and taurine conjugates of cholate-24- l4 C from the enterohepatic circulation have been reported previously in 6. 8 In 3, the jejunum, 30 inches from the ligament of Treitz, was anastomosed end-to-side to the transverse colon (approximately 73 inches of distal small intestine by-passed); the distal one-half and the distal one-third of the small intestine, respectively, had been resected for intestinal caused by volvulus in 2 other patients. The 6th patient exhibited a marked mucosal alteration of the distal one-half of the jejunoileum which was believed to be a result of extensive irradiation. The remaining 7 patients all had regional enteritis; 6 had a resection of the distal 50 to 100 cm of the small intestine with end-to-end anastomosis to the ascending colon. The 7th patient exhibited clinical and radiological disease compatible with involvement of the distal one-fourth of the small intestine; none had sigmoidoscopic or radiological evidence of colonic disease. Four patients had disorders which involved the duodenojejunum primarily (table 1). One (W. L.) of 2 patients with nontropical sprue had responded clinically to gluten withdrawal but continued to show gross mucosal changes in specimens obtained from the ligament of Treitz; another (J. B.) was studied prior to and 2 months subsequent to the institution of a gluten-free diet, at which time she was clinically well with moderate improvement in mucosal morphology. Two patients had Whipple's disease; both were studied prior to antibiotic therapy. One of these patients (V. E.) was severely malnourished (weight, 38 kg; weight loss, 30 kg). He was restudied 2 weeks and 5 months after institution of antibiotics and was essentially asymptomatic on the latter occasion (weight gain, 18 kg). The other patient with Whipple's disease (E. C.) had lost 16 kg but his general nutritional state appeared good. He was restudied 3 months after starting antibiotics and was essentially asymptomatic at that time, having gained 17 kg. There was no clinical or laboratory evidence of liver disease in any of

3 April 1969 INCREASED GLYCINE-CONJUGATED BILE SALTS 713 TABLE 1. Summary of clinical data Patient Age. sex Diagnosis Indication for surgery Ileal disorders C. B.a E. D.a J. H.a E. R.a E. B.a N. S.a J. B. H. E. D. H. P. L. N.M. R. T. E. C. Duodenojejunal disorders J. B. W.L. V.E. E. C. 47, F 25, M 43, F 48, F 67, F 79, F 36, M 48, M 19, M 17, M 24, M 46, M 49, F 19, F 42, M 35, M 50, M Jejuno-transverse colostomy, distal two-thirds of small intestine bypassed Jejuno-transverse colostomy, distal two-thirds of small intestine bypassed Jejuno-transverse colostomy, distal two-thirds of small intestine bypassed Jejuno-ascending colostomy, distal one-half of small intestine resected Ileal-ascending colostomy, distal one-third of small intestine resected Amyloid of distal one-half of jejuno-ileum (postirradiation) Ileal-ascending colostomy, distal one-third of small intestine resected Jejuno-ascending colostomy, distal one-half of small intestine resected Ileal-ascending colostomy, distal one-third of small intestine resected Ileal-ascending colostomy, distal one-third of small intestine resected Ileal-ascending colostomy, distal one-third of small intestine resected Jejuno-ascending colostomy distal one-half of small intestine resected Regional enteritis, active distal one-fourth of small intestine involved Nontropical sprue Nontropical sprue Whipple's disease Whipple's disease Obesity Obesity Obesity Intestinal Intestinal Regional enteritis, intestinal Regional en teri tis, in tes tinal Regional enteritis, intestinal Regional enteritis, intestinal Regional enteritis, intestinal Regional enteritis, intestinal a The rate of disappearance of glycine and taurine conjugates of cholate-24-14c from the enterohepatic circulation has been reported previously.s the patients studied. Five male medical student volunteers served as controls. Study procedure. All patients were hospitalized in the Clinical Research Unit at Duke University Medical Center. They were placed on a 70- to 8O-g fat diet, with a caloric intake varying between 1700 and 2500 calories consumed during three meals per day. Their feeding schedule was restricted only as indicated. Student volunteers were ambulatory and

4 714 GARBUTT ET AL. Vol. 56, No. 4 their feeding schedules and daily habits were restricted only as indicated. In all instances, 5.0 to 7.0 Jl.c of sodium taurocholate C was administered intravenously at approximately 8: 30 AM, after an overnight fast. All subjects remained fasting for a 3-hr period following the administration of the labeled bile salt. The method of preparation and administration of the labeled bile salt as well as the collection of duodenal contents, which included intubation and aspiration following the intravenous injection of cholecystokinin (Cecekin, Vitrum Chemical Company, Stockholm, Sweden), have been described previously.? Duodenal samples obtained within 2 to 3 hr after injection of the labeled bile salt were combined and kept on ice. Upon completion of the sampling, 2 to 6 ml of the pooled duodenal contents were saved for analysis. This never amounted to more than 5% of the total aspirate. The remainder of the sample was delivered into the duodenum via the indwelling tube which was flushed with water and then removed. The entire procedure of intubation, cholecystokinin infusion, and duodenal aspiration was repeated in student volunteers and patients with duodenal-jejunal disorders after an overnight fast at 24, 48, 72, and in some instances 96 and 120 hr after the injection of the labeled bile salt. In patients with ileal disorders, duodenal sampling was repeated at 24 and 48 hr. Taurine, 3.0 to 5.0 g, was given orally three times each day to 2 patients with ileal disorders, 1 patient with nontropical sprue in clinical remission, and 1 control subject. Mter 2 to 3 days, sodium taurocholate- 14 C was again given intravenously and duodenal fluid was sampled at 3 hr and subsequently in the same manner and time intervals described previously. In selected subjects (3 with ileal disorders and 1 student volunteer), 5.0 to 6.0 Jl.C of sodium cholate-24-14c or sodium deoxycholate C were administered intravenously at 8 : 30 AM after an overnight fast. The dihydroxy bile salt was dissolved in 20 ml of 7.5% sodium bicarbonate. The method of collection of duodenal contents was as described following administration of cholate-conjugated bile salts, with the exception that samples were obtained continuously throughout the 3-hr period which followed the administration of labeled bile salts. Preparation and chromatographic analysis of duodenal samples. Duodenal samples were prepared for chromatographic analysis by protein precipitation and extraction with ethanol. Conjugated bile salts were separated by thin layer chromatography using two solvent systems. Isoamyl acetate-propionic acid-npropanol-water (30:30:20: 15 v/v) (A. F. Hofmann, personal communication) was used for the separation of glycine and taurine conjugates of cholic acid, and isoamyl acetatepropionic acid-n-propanol-water (40: 30: 20: 10 V/V)14 was used for separation of glycine and taurine conjugates of deoxycholic acid- 14C. The method of identification of cholate conjugates and subsequent extraction from silica gel have been described previously.? No attempt was made to separate glycine and taurine conjugates of deoxycholate from corresponding conjugates of chenodeoxycholate; their Rr was similar as judged by appropriate standards. However, glycodeoxycholate was clearly separable from unconjugated cholic acid in this solvent system and there was no colorimetric evidence of trihydroxy bile salts in the glycodeoxycholate fraction. The dihydroxy conjugate fractions were each scraped onto weighing paper (glassine) and then transferred to a 20-ml counting vial. The determination of specific activity, counts per minute per milligram, of the taurocholate present in aspirated duodenal contents has also been described previously.? In each instance, 0.1 ml of the extract containing taurocholic acid was transferred to a standard 20-ml counting vial. In addition, 0.5-ml aliquots were subjected to the modified Pettenkoffer reaction of Irvin et al. 15 which permitted the determination of taurocholic acid concentration. Measurements of glycine-taurine ratios of cholic acid. In all duodenal samples, 0.5-ml aliquots of the extracts containing the taurocholate and glycocholate fractions were measured for trihydroxy bile salt content using the modified Pettenkoffer reaction of Irvin et al. I5 Measurements of glycine-taurine ratios of deoxycholate- 14 C. The ratio of glycine to taurine conjugates of deoxycholate was obtained from the relative amount of 14C-radioactivity in each fraction. The scintillation mixture and counting methods used, including considerations of self-quenching, have been described in detail previously.? Chemicals. Sodium taurocholate C was obtained from Tracerlab Company, Waltham, Massachusetts. Between 1.0 and 1.5 mg were administered. Sodium cholate- 14 C (New England Nuclear Corporation, Boston, Massachusetts) and deoxycholic acid- 14 C (Mallinckrodt Nuclear, Orlando, Florida) were administered in amounts

5 April 1969 INCREASED GLYCINE-CONJUGATED BILE SALTS 715 varying between 0.04 and 0.77 mg. In each case, 1 ml was retained for analysis. Chromatographic analysis of these materials showed greater than 99% of the radioactivity recoverable in appropriate fractions. The standards used in these experiments were obtained from Maybridge Chemical Company, Cornwall, United Kingdom. Results Measurements of taurocholate- 14 e specific activity. The specific activity of taurocholate- 14 C in duodenal fluid obtained from student volunteers 24 hr after intravenous administration of the labeled bile salt ranged between 62 and 75% of the initial 2- to 3-hr specific activity. As reported previously,7, 8 such values are in sharp contrast to 24-hr values obtained from patients with ileal disorders. Here, the specific activity was virtually zero in 8 patients and between 1 and 5% of the initial specific activity in 4 patients. In 1 patient with regional enteritis who had not had a resection, the specific activity at 24 hr was 20% of the initial value. In 2 patients with nontropical sprue and 1 with Whipple's disease, the rate of disappearance of taurocholate- l4 C was similar to that of control subjects, i.e., 75, 64, and 73%, respectively, of the initial specific activity. The 2nd patient with Whipple's disease (E. C.) exhibited a specific activity of 40% 24 hr after taurocholate- 14 C injection. Values obtained at his second study period, during clinical remlsslon, were normal (specific activity of 80% 24 hr after taurocholate- l4 C injection), as were those obtained from all patients with duodenojejunal disorders studied following treatment. Measurements of glycine and taurine conjugates of cholic acid and deoxycholic acid- l4 e. Average values for the relative conjugation of cholic acid with glycine and taurine (G: T ratio) in patients with ileal disorders, those with disorders which involved the duodenojejunum primarily, and student volunteers are shown in table 2. The G: T ratio for the primary trihydroxy bile salt is clearly increased in patients with ileal disorders as compared with control subjects and patients with duodenojejunal disorders. It is also apparent that values for the G : T ratio of the secondary bile salt, deoxycholate- 14 C, derived from bacterial metabolism of the injected taurocholate C, although slightly higher, on the average bear an identical relationship between the three groups of subjects (table 2). For both bile salts, the lowest G: T ratio exhibited by a patient with a disorder of the ileum is higher than the highest G: T ratio observed either in student volunteers or in patients with duodenojejunal disorders. Patients with Whipple's disease may form an intermediate group in that the range of values for the G: T ratio appears some- TABLE 2. Relative conjugation of bile salts with glycine and taurine in duodenal fluid Cholate conjugates Deoxycholate-14C conjugatesa No. of subjectsb G:T G:T No. of Mean ± 1 SD Range subjects Mean ± 1 SD Range Ileal disorders : ' 16.8: Duodenojejunal disorders 4 3.4: b 5.1: (Nontropical sprue) (2) (1.6) ( ) (2) (2.8) ( ) (Whipple's disease) (2) (5.4) ( ) (2) (7.1) ( ) Student volunteers I 5 2.8: b 3.4: a Obtained following intravenous administration of sodium taurocholate-24-14c. See "Materials and Methods." b Each determination is the average of two to four values obtained during a single study period., Each determination is the average of two values in 1 patient and one value in 4 patients, obtained during a single study period.

6 716 GARBUTT ET AL. Vol. 56, No.4 what higher than any value shown by patients with nontropical sprue or student volunteers (table 2). Additional evidence on this point may be obtained from a comparison of G: T ratios of both bile salts in patients with Whipple's disease before and after treatment (table 3). During clinical remission, values decreased TABLE 3. Relative conjugation of bile salts with glycine and taurine in duodenal fluid, during the course of illness Patient Intervala Cho- J~ b -- Ileal disord 3rsd E. R year 13.5 E. B months 16.3 J. B months months 21.0 N.M months 10.2 E. C months months 12.9 Duodenojejunal disorders Nontropical sprue' J. B months 2.0 Whipple's disease! V. E weeks months 2.4 E. C months 1.6 Deoxycholate-"C G:T" a Interval between initial and subsequent study. b Ratio of glycine to taurine conjugates of cholate. c Ratio of glycine to taurine conjugates of deoxycholate-l4 C obtained following intravenous administration of taurocholate-24-l4 C. d Clinical course un< hanged during interval between studies. E. C. was mildly active clinically. e Gluten-free diet during interval between studies.! Taking antibiotics, penicillin and streptomycin (2 weeks) and tetracycline thereafter, during interval between studies. to a level comparable to those seen in normal controls. In contrast, repeat values for the G: T ratio of both bile salts remained in the same range during the course of illness of patients with ileal disorders and in the patient with nontropical sprue who was studied during a remission after withdrawal of dietary gluten (table 3). No patient or subject studied exhibited significant radioactivity in glycine or taurine conjugates of deoxycholate-h C in samples of duodenal fluid obtained 3 hr after intravenous administration of taurocholate C. Table 4 shows the G : T ratio of deoxycholate- 14 C obtained at 24 and 48 hr in the 2 patients with ileal disorders who exhibited sufficient radioactivity 48 hr after intravenous injection of taurocholate C to accomplish such measurements. It is apparent that the G : T ratios obtained at 24 and 48 hr were similar and did not show a consistent increase with time. Similarly, values for the G: T ratio of deoxycholate- 14 C obtained from patients with duodenojejunal disorders and from student volunteers were similar during the 72- to 120-hr period of study (table 4). Measurements of conjugation of cholic acid-h e and deoxycholic acid_ 14 e with glycine and taurine. When cholate- 14 C and or deoxycholate-hc were administered intravenously to patients with ileal disorders, values for the G: T ratio of both labeled bile salts obtained at 3 hr were at the same level (12.1 to 26.0, table 5) as those shown in table 2. In 1 patient studied, (J. B., table 5), the G:T ratio for deoxycholate- 14 C at 24 and 48 hr was essentially identical with the 3-hr value. There was no detectable HC-radioactivity present in duodenal fluid obtained 24 hr after administration of deoxycholate- 14 C to R. T. and 72 hr after its administration to J. B. A single student volunteer was studied following intravenous administration of deoxycholate- 14 C with similar values for the G: T ratio at 3, 24, 48, and 96 hr (table 5). Measurements of glycine and taurine conjugates of cholic acid and deoxycholic acid-he, following an oral taurine load.

7 April 1969 INCREASED GLYCINE-CONJUGATED BILE SALTS 717 TABLE 4. Relative conjugation of deoxycholate- 14 C with glycine and taurine in duodenal fluid following intravenous administration of taurocholate C, serial determinations during a single study Subject Time DeoxycholateJ'C G:T" Ileal disorders E. C.b hr J. B Duodenojejunal disordersc Nontropical sprue J. B W.L Whipple's disease V.E E. C Student volunteerd S. L G Ratio of glycine to taurine conjugates. b Two studies separated by 6 months. c Initial study. d Representative study. The oral administration of taurine to a student volunteer resulted in a marked reduction in the G: T ratio of both bile salts (table 6), as reported previously.16 A similar reduction was obtained in 1 patient with nontropical sprue who was in clinical remission. It is also apparent from table 6 that oral taurine administration resulted in a marked reduction in the G: T TABLE 5. Relative conjugation of cholate C and deoxycholate-24-14c with glycine and taurine in duodenal fluid G Subject Time Cholate-14C G:'fb --- hr Ileal disorders E. D ?t:1~tC8~j-b J. B R. T.c Student volunteer W.E G In these studies, the 14C-Iabeled unconjugated bile salt was injected intravenously. b Ratio of glycine to taurine conjugates. c Cholate-14C and deoxycholate-14c studies separated by 72 hr. TABLE 6. Relative conjugation of bile salts with glycine and taurine in duodenal fluid before and after oral administration of taurinea Subject Cholate G:Tb Deoxycholate-HC G:Tc Before After Before After Ileal disorders E. B N. S.d Nontropical sprue W.L Student volunteer S. L G Patients with ileal disorders received taurine, 5 g three times daily, for 2 days prior to study; others were given 3 g three times daily for 3 days. b Ratio of glycine to taurine conjugates of cholate. c Ratio of glycine to taurine conjugates of deoxycholate- 14 C obtained 24 hr after intravenous administration of taurocholate-24-14c. d Morning sample. No significant radioactivity present 24 hr after taurocholate-14c administration.

8 718 GARBUTT ET AL. Vol. 56, No. 4 ratio of both bile salts in patients with ileal disorders. Discussion These studies demonstrate an increased concentration of glycine relative to taurine conjugates of trihydroxy and dihydroxy bile salts in cholecystokinin-stimulated duodenal fluid obtained from patients with ileal disorders. The results confirm and enlarge our previous preliminary report, 8 in which the "available" cholate conjugates (glycine and taurine) accumulated in the biliary tree of 4 patients with ileal disorders after an overnight fast were each estimated by isotope dilution, and also the observations of McLeod and Wiggins.13 In addition, the present study clearly relates the increased G: T ratio to the markedly decreased enterohepatic circulation of injected sodium taurocholate-h C in these patients with ileal disorders. We have demonstrated previously a similar loss of glycocholate-hc from the enterohepatic circulation of such patients. 8 Deoxycholate, derived from bacterial hydrolysis and reduction of cholate conjugates, is presumably reabsorbed in normal man principally by passive diffusion processes, returning to the liver via the portal circulation and reconjugated with glycine or taurine prior to secretion into the bile. 17 The presence of significant concentrations of conjugates of deoxycholic acid in duodenal fluid obtained from normal subjects l8-20 implies enterohepatic recirculation of this secondary bile salt. The absence of deoxycholate conjugates in duodenal fluid obtained from 4 of 6 patients with ileal disorders, reported by others, suggests impaired absorption. The present observations provide direct evidence of this point. Deoxycholate-HC when administered intravenously to a student volunteer provided sufficient radioactivity in duodenal samples obtained at 96 hr (a later sample was not attempted) to permit measurements of the G : T ratio of deoxycholate-hc; whereas HC-radioactivity was virtually zero in duodenal fluid obtained at 24 hr in 1 patient (R. T.) and at 72 hr in another (J. B.) with ileal disorders (table 5). Previous observations by Ekdahl and Sjova1l 6 of patients with hepatobiliary disorders and T-tube drainage have demonstrated an increase in the ratio of glycine to taurine conjugates of bile salts during 4 to 20 days of bile fistula drainage. In these studies, bile salts and other constituents of bile were lost to enterohepatic circulation, and the origin of such altered conjugation of bile salts is clearly hepatic. Under the present conditions, it may be assumed that such materials normally absorbed from ' more proximal regions may continue to circulate enterohepatically. If proximal intestinal absorption of glycineconjugated bile salts were significant, as has been suggested,21 then the elevated G : T ratios exhibited by patients with ileal disorders could by attributed to such preferential reabsorption of glycineconjugated bile salts.13 However, the markedly increased G : T ratios observed in duodenal fluid obtained throughout the 3-hr period which followed intravenous administration of l4c-iabeled unconjugated bile salts to patients with ileal disorders (table 5) provide convincing evidence of their hepatic origin. Furthermore, significant reabsorption of glycodeoxycholate from the proximal intestine might be expected to result in progressive increases in the G: T ratio of deoxycholate-14c obtained from bacterial modification of injected taurocholate- 14C. Observations obtained serially in student volunteers, in patients with ileal disorders, and also in patients with disorders involving the duodenojejunum primarily do not show such progressive increases in the G: T ratio (table 4). Similarly, serial determinations of the G : T ratio following intravenous administration of deoxycholate-l4c demonstrate values for the G: T ratio which are essentially identical with the 3-hr value (table 5). Therefore, these constant values for the G: T ratio, which were observed in all cases, are in accord with the concept that the biological turnovers for both glycine and taurine con-

9 April1969 INCREASED GLYCINE-CONJUGATED BILE SALTS 719 jugates of deoxycholic acid are essentially the same. The present experimental design permits only speculation on the mechanism of this enhanced hepatic conjugation of bile salts with glycine relative to taurine. In the steady state situation, bile salt synthesis is equal to daily fecal loss, approximating 0.8 g per day.1 7 Synthesis is enhanced in patients with ileal disorders who exhibit a markedly reduced enterohepatic recirculation of bile salts. It has been suggested that such synthesis may be highly variable in individual patients.12 Estimates in our laboratory indicate an enhanced synthesis which approximates 3- to 4-fold.8 This would result in a consequently increased demand for conjugation with glycine and taurine. An increased demand for such conjugation might also be operative in situations of increased bacterial modification of bile salts within the lumen of the small intestine. Here, unconjugated bile salts may be absorbed along the entire intestinal length with reconjugation prior to their secretion into bile. This sequence may have been operative in the patients recently reported by Tabaqchali et al. 22 who had the "stagnant loop syndrome" and exhibited a relative increase in glycine conjugates. Several factors other than enhanced requirements for conjugation may effect the relative partitioning between glycine and taurine. A preponderance of glycineconjugated bile salts has been reported in duodenal fluid obtained from hypothyroid patients. 25 Here, too, the mechanism involved in the production of these changes in relative conjugation of bile salts is not clear. However, the present observations, which demonstrate a marked decrease in the G: T ratio following oral administration of taurine to patients with ileal disorders, suggest that a decrease in the relative availability of taurine may be present. Although this thesis requires further documentation, these observations do show a maintained capacity for conjugation with taurine. The G: T ratios of cholate and deoxycholate-14c conjugates in the 2 patients with Whipple's disease were found to be slightly above normal, and they fell to within the normal range after treatment (table 3). Perhaps the first consideration toward an explanation of these observations should make reference to the recognized involvement of the ileum in some patients with Whipple's disease and also in an occasional patient with nontropical sprue Such involvement in the present patients with Whipple's disease could lead to a decrease in bile salt enterohepatic recirculation with consequently increased demand for hepatic conjugation. Although this thesis is consonant with the observations in patient E. C., who exhibited a 40% specific activity 24 hr after administration of taurocholate- l4 C, these results were normal in the other patient (V. E.), who was severely ill clinically at the time of his initial study. Additional studies in these and other patients have been directed toward serial measurements of total 14C-radioactivity in duodenal fluid following administration of taurocholate-14c and toward the accumulation of bacterial metabolites of the injected labeled bile salt. Such observations (to be published in detail) suggest an increased luminal metabolism of injected labeled bile salt in patients with Whipple's disease, rather than an abnormal loss of label from the enterohepatic circulation. Finally, the involvement of multiple organs in Whipple's disease, including the liver, 28 requires mention as a factor in the mechanism of the present alterations. REFERENCES 1. Haslewood, G. A. D The biological significance of chemical differences in bile salts. Bioi. Rev. 39: Bremer, J Species differences in the conjugation of free bile acids with taurine and glycine. Biochem.J. 63: Prange, I., F. Christensen, and H. Dam Alimentary production of gallstones in hamsters. II. Relation between diet and composition of the bladder bile. Z. Ernaehrungswiss 3: Bergstrom, S., H. Danielsson, and A. Goransson.

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