Effect of 7-Ketolithocholic Acid on Bile Acid Metabolism in Humans

Transcription

1 GASTRONTROLOGY 1982;83:341-7 ffect of 7-Ketolithocholic Acid on Bile Acid Metabolism in Humans GRALD SALN, DAN VRGA, A. K. BATTA, G. S. TNT, and S. SHFR Department of Medicine, College of Medicine and Dentistry of New Jersey-New Jersey Medical School. Newark, New Jersey; Veterans Administration Medical Center, ast Orange, New Jersey; Cabrini Medical Center, New York, New York The effect of 7-ketolithocholic acid on biliary bile acid composition, cholesterol saturation, and as an intermediate in the conversion of chenodeoxycholic acid to ursodeoxycholic acid was investigated in 5 subjects with gallstones. After 7-ketolithocholic acid (400 mg/day) was administered orally for 14 days, biliary bile acid composition changed: The proportion of cholic acid decreased (from 45% to 19%), deoxycholic acid decreased (from 15% to 10%), chenodeoxycholic acid increased markedly (from 36% to 59%), ursodeoxycholic acid increased (from 2% to 7%), and lithocholic acid increased (fi'om 2% to 5%), while only trace amounts of 7-ketolithocholic acid were detected. During this treatment, the biliary lithogenic index fell from 2,6 to 0.9 and was accompanied by a pronounced drop in biliary cholesterol concentration. After biliary bile acid levels became constant [24-14 Clchenodeoxycholic acid was given intravenously as a pulse-label, and the resultant biliary ursodeoxycholic acid and lithocholic acid specific activity curves showed a precursorproduct relationship with chenodeoxycholic Similarly, when uniformly labeled 7-[24-14 Clketolithocholic acid was fed (400 mg/day, 1000 ± 100 dpm/mg) the specific activities of biliary chenodeoxycholic acid and ursodeoxycholic acid became constant and approximated each other, but these were only 75% as high as the fed 7-ketolithocholic These results indicate that 7-ketolithocholic acid is absorbed, and suppresses endogenous bile acid production and biliary cholesterol secretion. Both isotopic experiments infer that ursodeoxycholic acid and lithocholic acid are formed from chenodeoxycholic acid and not from 7-ketolithocholic The reduction in biliary lithogenic index and in cholesterol concentration suggest that low doses of 7-ketolithocholic acid may be effective in dissolving gallstones. 7-Ketolithocholic acid (Figure 1) is a secondary bile acid commonly found in the feces of humans and other animals. t is formed from chenodeoxycholic acid through the action of intestinal bacteria. Recently, Haslewood et a1. (1) and MacDonald et a1. (2) have isolated several strains of bacteria that 7 -dehydrogenate chenodeoxycholic acid to form 7-ketolithocholie acid rather than 7-dehydroxylate to produce lithocholic The ph optimum for the bacterial dehydrogenation is strongly alkaline, and this may explain why lithocholic acid rather than 7-ketolithocholic acid predominates in stool, as fecal ph is about 6. Recently, considerable interest has focused on the use of chenodeoxycholic acid and ursodeoxycholic acid as gallstone-dissolving agents in humans (3-14). Of particular note was the demonstration that large amounts of ursodeoxycholic acid were formed in some subjects during chenodeoxycholic acid treatment and the suggestion that 7 -ketolithocholic acid may serve as an intermediate in this reaction (15). This paper examines the effect offeeding 400 mg/day of 7 -ketolithocholic acid on endogenous bile acid synthesis, biliary cholesterol saturation, and the possible role of 7-ketolithocholic acid as a precursor of chenodeoxycholic acid and ursodeoxycholic Received November 3, Accepted April 2, Address requests for reprints to: G. Salen, M.D., Gastrointestinal Section, Veterans Administration Medical Center, ast Orange, New Jersey This work was supported by Public Health Service Grants AM 18707, AM 26756, and HL by the American Gastroenterological Association /82/ $02.50 Materials and Methods Clinical Studies were conducted in 5 male subjects with gallstones (mean age, 55 yr) who were hospitalized at the ast Orange Veterans Administration Hospital. nformed

2 342 SALN T AL. GASTRONTROLOGY Vol. 83, No. 2 cip"" HO"'" H 'hh Chenodeoxycholic acid (3o.7a-dthydroll.y-5,8-choloOic acid) 7-ketolithocholic acid (3a-hyc\roltY, 7oxo-S{j-cholonic ocid) Lithocholic acid (3a-monohydroJ,y-S,B-choionic odd) Figure 1. Structures of bile acids. Jtx: HO H r o Ursodeollycholic acid (3a. 7 (3-dihydroxy-S{3-cholonic acid) consent was obtained from each subject before initiation of the studies and the protocol was approved by the Human Studies Committee of the College of Medicine and Dentis.try of New Jersey-New Jersey Medical School. The presence of radiolucent gallstones was demonstrated by oral cholecystography. All subjects were fed regular hospital diets during the investigation and calorie intakes were adjusted to maintain constant body weight. Materials Preparation of unlabeled and 7-[24-14 CJketolithocholic 7-Ketolithocholic acid was prepared from chenodeoxycholic acid by the following method: chenodeoxycholic acid (1 g) in 75 ml of dioxane and 5 ml of water was reacted with N-bromosuccinimide (1.1 g) for 2 h; the reaction mixture was poured over ice, and the resultant white precipitate was washed with water and crystallized to yield 7-ketolithocholic 7-[24c 14 C]Ketolithocholic acid was synthesized from [24Y C]chenodeoxycholic acid as described above. The final specific activity of 7-[24-14 C]ketolithocholic acid was adjusted with unlabeled 7-ketolithocholic acid to 1000 ± 100 dpm/mg. The bile acids used in this study had a chemical and radiochemical purity of >98% as determined by thin-layer chromatography and gas-liquid chromatography-mass spectrometry. Radioactive compound. The [24-14 C]chenodeoxycholic acid was purchased from New ngland Nuclear Corp., Boston, Mass. (sp act, 50 LCi/lLmol). xperimental Design Two separate experiments were performed: (a) 400 mg of unlabeled 7-ketolithocholic acid was fed to each patient daily for 14 days. On the seventh day, a tracer dose of [24-14 C]chenodeoxycholic acid (3.0 x 10 6 dpm) was injected intravenously as a pulse-label. Bile specimens were collected before isotopic labeling and each day for the next 7 days. The specific activities of chenodeoxycholic acid, ursodeoxycholic acid, and lithocholic acid were measured and precursor-product relationships sought. (b) n.the second experiment, 400 mg/day of uniformly labeled 7-ketolithocholic acid (sp act, 1000 ± 100 dpm/mg) was fed to each patient daily for 2 wk and the specific activities of chenodeoxycholic acid and ursodeoxycholic acid in bile were measured every other day. This experiment examined the direct conversion of 7-ketolithocholic acid into chenodeoxycholic acid and ursodeoxycholic During both experiments, the effects of 7-ketolithocholic acid on biliary bile acid composition, bile acid conjugation, and cholesterol saturation were determined. Lipid and Steroid Analysis Methods. ntestinal bile specimens (5 ml) were collected over ice by duodenal intubation through a Rehfuss tube (Davol, nc., Providence, R..) that was positioned under fluoroscopic guidance. Sincalide (Kinevac;. R. Squibb & Sons, nc., New Brunswick, N.J.) was injected intravenously to stimulate gallbladder concentration and to facilitate bile flow. Bile was deproteinized by the addition of a tenfold volume of absolute methanol and then filtered. After thorough mixing, the aliquots were taken for individual lipid determinations and solvolysis. Cholesterol was determined as described by Shefer et al. (16), and total biliary bile acid composition was measured according to Salen et a1. (15). The phosphorus content of a Folch extract of bile was determined by the method of Chen et al. (17). Phospholipid concentration was calculated by multiplying the phosphorus content by 25. The masses of biliary cholesterol and the individual bile acid were determined by gas-liquid chromatography on a Hewlett-Packard Model 7610A gas-chromatograph (Hewlett Packard Company, Palo Alto, Calif.) equipped with a flame ionization detector and an electronic integrator. Cholesterol. was injected as the trimethylsilyl ether derivative on a 6-ft glass column packed with 3% QF-l on mesh Gas Chrom Q (Applied Science Laboratories, nc., State College, Pa.). The column temperature was 230 C and the flash heater was set at 250 C. The trimethylsilyl ether derivatives of the methylated bile acids were injected on a column packed with 1 % HiFF 8BP on mesh Gas Chrom Q (Applied Science Laboratories, nc.). The operating conditions were: detector temperature, 260 C; column temperature, 240 C; and flash heater temperature, 280 C. Mass measurements were calculated from peak areas. Lithogenic index. Cholesterol saturation was determined by the method of Thomas and Hofmann (18) according to the limits of cholesterol solubility defined by Hegardt et al. (19) and Holzbach et al. (20). Bile acid conjugates. Keto bile acids may be unstable when undergoing alkaline hydrolysis. To ensure that no 7-ketolithocholic acid was lost during the extraction and subsequent alkaline hydrolysis of the conjugated biliary bile acid, duplicate assays were performed using cholylglycine hydrolase to hydrolyze the conjugated bile acids. The proportions of taurine and glycine conjugated biliary bile acids also were measured before and during treatment with 7-ketolithocholic Separate aliquots of bile were deproteinized by methanol, filtered, and applied

3 August KTOLTHOCHOLC ACD. BL ACDS, GALLSTONS x Q) ] 3-0 c: 0 " (/) e Q) "0.c 1-0 U 0 Control 7KLCA 400 mg /day Figure 2. ffect of 7-ketolithocholic acid on the biliary lithogenic index_ to glass plates coated with a 0.25-mm layer of silica gel (Analabs. nc.. North Haven. Conn.). Reference standards of the taurine and glycine conjugated bile acids. cholic chenodeoxycholic ursodeoxycholic lithocholic and 7-ketolithocholic acid were applied also. The plates were developed in chloroform/isopropanoll glacial acetic acid/water with a ratio of 30: 20: 4: 1 (vol/voll vol/vol). The conjugated bile acid bands were visualized with water. scraped. eluted with methanol. and evaporated to dryness. The free bile acids in each band were isolated after hydrolysis with cholylglycine hydrolase; they were then methylated. and mass measurements were made by gas-liquid chromatography. Bile acid-specific activities. A portion of the isolated bile acid methyl ester fraction was applied to 20 by 20-cm glass plates coated with a 0.25-mm layer of silica gel (Analabs. nc.). Reference standards of methyl chenodeoxycholic methyl ursodeoxycholic methyl lithocholic and methyl 7-ketolithocholic acid were also applied. The plates were developed in chloroform/methanollacetone with a ratio of 70: 20: 5 (vollvollvol). The bile acid bands were visualized with water. scraped. and eluted with methanol. The solvent was evaporated. and 5.0 ml of methanol containing 5a-cholestane as an internal standard were added. Aliquots were taken for mass determinations by gas-liquid chromatography and for radioactivity measurements in a Beckman Model LS-OO C liquid scintillation spectrometer. Appropriate corrections were made for quench and background. The efficiency for counting 14C was 91% and for 3H. 49%. Results ffect of 7-Ketolithocholic Acid on Biliary Bile Acid Composition, Conjugation. and Lithogenicity 7-Ketolithocholic acid (400 mg/day) was fed to 5 subjects for 2 wk, and biliary bile acid composition was determined and compared with pretreatment values. The results are given in Table 1. The proportion of chenodeoxycholic acid rose from 36% to 59%, and of ursodeoxycholic acid from 2% to 7% of the total bile acid composition. n contrast, the molar percentage of cholic acid fell from 45% to 19% and its bacterial metabolite. deoxycholic fell from 15% to 10% of total bile acids. There was a modest rise in the amount of free lithocholic acid from 2% to 5% of total bile acids without any increase in sulfate esters. Only trace amounts of 7- ketolithocholic acid were detected in the bile. These results indicate that 7 -keto lithocholic acid was absorbed from the intestine and converted predominantly to chenodeoxycholic ndogenous bile acid production was probably suppressed as evidenced by the decrease of cholic acid and deoxycholic acid concentrations. Table 2 illustrates the effect of 7-ketolithocholic acid on the formation of conjugated bile acids. Before treatment, 55% of the total bile acids in 5 subjects were conjugated with glycine and 45% with taurine. During 7-ketolithocholic acid administration, however, there was a marked increase in the proportion of glycine conjugates. which rose to 73% of the total conjugated bile acids. Thus, the administration of 400 mg/day of unconjugated 7 -ketolithocholic acid depleted the hepatic taurine pool and resulted in increased formation of glycine conjugates. Also. during treatment with 7-ketolithocholic biliary cholesterol saturation and lithogenic index were measured in 5 subjects; the results are given in Figure 2 and Table 1. Before treatment. bile was markedly lithogenic (mean lithogenic index, 2.6 ± 0.9). However. when 7-ketolithocholic acid was fed, the cholesterol saturation index fell to 0.8 ± 0.3, and the difference was statistically significant (p < 0.01). The reduction in lithogenic index resulted from a substantial decrease in biliary cholesterol concentration (Table 1). Conversion of [24-14 C]Chenodeoxycholic Acid into Ursodeoxycholic Acid When unlabeled 7-ketolithocholic acid (400 mg/day) was fed to 5 subjects for 2 wk. biliary bile acid composition became constant, as checked by gas-liquid chromatography after 7 days. On the seventh day, [24-14 C]chenodeoxycholic acid was injected intravenously as a pulse-label, and the specific activities of biliary chenodeoxycholic acid, ursodeoxycholic acid, and lithocholic acid were determined five times over the next week (Figure 3). The specific activities of chenodeoxycholic acid decayed linearly over this period. which indicated that a single pool of chenodeoxycholic acid existed despite the two sources for the bile acid: Chenodeoxycholic acid was produced endogenously both from cholesterol and from the reduction of 7-ketolithocholic Radiolabeled ursodeoxycholic acid was also detected in the bile. and the specific activities

4 344; SALN T AL. GASTRONTROLOGY Vol. 83, No.2 Table 1. ffect of 7-Ketolithocholic Acid on Cholesterol and Biliary Bile Acid Composition Bile acid concentration a (% of total bile acids) Cholesterol concentration Patient Treatment a CA DCA CDCA LCA UDCA (%) H.J. Pretreatment Trace b Trace 6 7-KLCN W.H. Pretreatment KLCA W.R. Pretreatment KLCA O.R. Pretreatment Trace KLCA A.S. Pretreatment KLCA Mean:!: S Pretreatment 45 ± ± 9 36 ± 15 2 ± 1 2 ± 1 12 ± 6 7-KLCA 19 ± 5 10 ± 6 59 ± 11 5 ± 3 7 ± 5 5 ± 4 P < 0.05 NS p < 0.05 P < 0.05 NS p < 0.01 a Abbreviations: 7-KLCA, 7-ketolithocholic acid; CA, cholic acid; DCA, deoxycholic acid; CDCA, chenodeoxycholic acid; LCA, lithocholic acid; UDCA, ursodeoxycholic acid; NS, not significant. b "Trace" amounts to < 1%. c 400 mg of 7-ketolithocholic acid were administered daily for 14 days. rose and intersected with the decay curve of chenodeoxycholic acid at a maximum that suggested a precursor-product relationship between the two bile acids. As expected, lithocholic acid was labeled, and its specific activity curve declined in parallel with that of chenodeoxycholic No 7-[14C]ketolithocholic acid was detected in the bile. These results suggest that chenodeoxycholic acid was transformed into ursa deoxycholic acid and lithocholic Further, because the specific activities of ursodeoxycholic acid and lithocholic acid were greater than that of chenodeoxycholic acid, unlabeled 7 -ketolithocholic acid was not a major precursor of either bile f unlabeled 7-ketolithocholic acid was converted directly into ursodeoxycholic acid or lithocholic acid, their respective specific activities would fall below that of chenodeoxycholic Transformation of 7-[24-14 CKetolithocholic Acid into Chenodeoxycholic Acid n a separate experiment, 5 subjects were given 400 mg/day of uniformly labeled 7-[24-14 C]ke- Table 2. Treatment Pretreatment 7-KLCA b ffect of 7-Ketolithocholic Acid on Biliary Bile Acid Conjugation Glycine 55 ± ± 10 Bile acid conjugates (%) Taurine 45 ± 6 27 ± 5 a The numbers in the table denote the mean value from 5 patients ± standard deviation. b 400 mg of 7-ketolithocholic acid (7- KLCA) were administered daily for 14 days. to lithocholic acid for 2 wk. The specific activities of the ursodeoxycholic acid and chenodeoxycholic acid were measured from specimens of bile obtained over the 2-wk period. The results are given in Figure 4. Biliary bile acid composition and the specific activities of chenodeoxycholic acid an.d ursodeoxycholic acid became constant by day 7 as seen by gasliquid chromatography. This finding suggested that 0' " Ē a. '0 1,000 1/ /1 / / /,/ /X Day 7 Tracer Dose of C24- '4 CJ COCA, 3'0 x 10 6 dpm COCA x--x UDCA &--& LCA OL- L L -L Days after pulse labeling i Pulse administered Figure 3. Conversion of [24-14 C)chenodeoxycholic acid into ursodeoxycholic A pulse-label of [24-14 C]chenodeoxycholic acid WaS administered intravenously after bile acid composition became constant. Specific activity decay curves of ursodeoxycholic acid and lithocholic acid intersect with that of chenodeoxycholic acid, which suggest precursor-product relationships.

5 August KTOLTHOCHOLC ACD, BL ACDS, GALLSTONS 345 '" " 600 Q.." 400 Specific octivity of [24_ 14 C] 7KLCA OOOdpml mg Days Figure 4. Transformation of 7-[24-" 4 C)ketolithocholic acid into chenodeoxycholic Uniformly labeled 7-[24-14C)ketolithocholic acid was fed (400 mg/day) and the specific activities of chenodeoxycholic acid and ursodeoxycholic acid measured. Bile acid specific activities became constant by day 7; the specific activity of chenodeoxycholic acid was 20% lower than that of the 7-ketolithocholic acid administered, but it did not differ significantly from the specific activity of ursodeoxycholic isotopic equilibrium and a steady state had been attained. During the last 7 days, the mean specific activity of chenodeoxycholic acid was 760 ± 30, and the mean specific activity of ursodeoxycholic acid was 720 ± 20. The difference between these values was not statistically significant. The results suggest that ursodeoxycholic acid was formed from chenodeoxycholic acid and not directly from 7-ketolithocholic Discussion The results of this investigation show that 7- ketolithocholic acid was absorbed and converted principally to chenodeoxycholic During the administration of 7 -ketolithocholic acid, endogenous bile acid synthesis was suppressed as evidenced by the fall in the proportions of biliary cholic acid and deoxycholic Thus, 7-ketolithocholic acid may act similarly with other bile acids (chenodeoxycholic acid, ursodeoxycholic acid, and cholic acid) in exerting feedback inhibition of hepatic bile acid synthesis. Because 7-ketolithocholic acid was not detected in the bile, however, it is possible that the suppression of endogenous bile acid synthesis was not a direct effect of 7-ketolithocholic acid, but rather was mediated by chenodeoxycholic acid, which increased substantially in the bile (Table 1). Similarly, biliary cholesterol concentrations and the lithogenic index fell when 7-ketolithocholic acid was administered. This beneficial effect of bile cholesterol excretion may also be a direct action of 7- keto lithocholic acid, or it could alternatively be due to the increased concentrations of chenodeoxycholic acid in the bile. Nevertheless, because relatively small doses of 7-ketolithocholic acid were given and the effect on biliary cholesterol saturation and secre- tion was great, further studies on the effect of 7- ketolithocholic acid as a gallstone-dissolving agent seem warranted. Another interesting observation concerned the marked decrease in taurine conjugates in the bile when 7-ketolithocholic acid was given. The depletion of the taurine pool has been noted previously during administration of unconjugated chenodeoxycholic acid and ursodeoxycholic acid, and this results in a major increase in the proportion of glycine conjugated bile The physiologic significance of this change is still unknown, but gimi and Carey (21) have suggested that glycine conjugate of ursodeoxycholic acid may be a less effective detergent and thus may reduce the capacity of the bile to solubilize cholesterol. Thus, the reduction in biliary taurine conjugate of ursodeoxycholic acid may adversely affect the capacity of the bile to dissolve cholesterol. A major part of this investigation concerned the fate of the ingested 7-ketolithocholic ncreased amounts of chenodeoxycholic acid, ursodeoxycholic acid, and lithocholic acid were detected in the bile of our subjects. Similar findings have been observed earlier by Mahowald et al. (22) and Samuelsson et al. (23) in the rat, and Sal en et al. (15) and Fromm et al. in humans (24). These investigators administered labeled 7-ketolithocholic acid and found radioactivity in isolated chenodeoxycholic acid and ursodeoxycholic Thus, it was postulated that the 7-keto group of 7-ketolithocholic acid could be reduced directly to both chenodeoxycholic acid and ursodeoxycholic However, our two studies suggest a different pathway, namely that 7-ketolithocholic acid was reduced primarily to chenodeoxycholic acid and that little ursodeoxycholic acid or lithocholic acid was formed directly from 7-ketolithocholic The evidence in favor of this interpretation can be summarized as follows: When [24-14 C)chenodeoxycholic acid was given as a pulse-label during the administration of labeled 7-ketolithocholic acid, the decay curves of ursodeoxycholic acid and lithocholic acid intersected with the chenodeoxycholic acid curve, revealing a precursor-product relationship. The specific activities of ursodeoxycholic acid and lithocholic acid were consistently higher than the corresponding value for chenodeoxycholic f 7 -keto lithocholic acid was directly converted to ursodeoxycholic acid or lithocholic acid, the specific activities of these two bile acids would be lower than that of chenodeoxycholic acid, reflecting a dilution with unlabeled bile Rather, the specific activity curves suggest that both bile acids originated from chenodeoxycholic Similarly, when uniformly labeled 7-[24-14 C)ketolithocholic acid was fed daily, the specific activities of chenodeoxycholic

6 346 SALN T AL. GASTRONTROLOGY Vol. 83, No.2 Cholesterol,,,,, CDCA /Lr //, 7 KLCA UDCA Figure 5. Conversion of chenodeoxycholic acid into 7-ketolithocholic acid, lithocholic acid, and ursodeoxycholic The solid lines represent conclusions regarding the pathway that were derived from data in the present study and the dashed lines represent conclusions derived from literature. acid and ursodeoxycholic acid reached a plateau by day 7. The mean specific activity of chenodeoxycholic acid for the next 7 days was 760 dpm and 720 dpm for ursodeoxycholic acid as compared with 1000 dpm for fed 7-ketolithocholic This experiment suggests that about 76% of the chenodeoxycholic acid was derived from the reduction of 7 -ketolithocholic acid and 24% was produced endogenously. However, the consistently lower specific activities suggested that very little ursodeoxycholic acid was derived from 7 -ketolithocholic f that had occurred, then the specific activity of the ursodeoxycholic acid would be higher than that of chenodeoxycholic acid, and it would approach that of 7- ketolithocholic Similar conclusions regarding the pathway of ursodeoxycholic acid have been reached by Fedorowski et al. (25). These investigators incubated [7lPHlchenodeoxycholic acid with intestinal bacteria and found that 3H-abel was retained in the ursodeoxycholic acid that was produced. They proposed that 7-ketolithocholic acid was not involved in the formation of ursodeoxycholic acid because the oxidation of the hydroxyl to a ketone would eliminate the tritium atom. nstead, they suggested an alternative pathway from chenodeoxycholic acid to ursodeoxycholic acid that includes the putative intermediate 3a-hydroxy-6-cholenoic However, it should be emphasized that 3a-hydroxy-6-cholenoic acid has not been identified in the feces or the bile of these subjects. However, Matkovics and Samuelsson (26) have proposed a similar pathway in the conversion of cholic acid to ursocholic The hypothetical intermediate in this pathway was 3a,12a-dihydroxy-6-cholenoic Unfortunately, the present study does not deal with the site where 7-ketolithocholic acid was trans- formed. Both hepatic and intestinal bacterial enzymes may be responsible. Fedorowski et al. (25) and MacDonald et al. (27) have reported the bacterial conversion of chenodeoxycholic acid into ursodeoxycholic Also, Fedorowski et al. have shown that in humans ursodeoxycholic acid can be transformed into chenodeoxycholic acid, and they have suggested that 7-ketolithocholic acid may be an intermediate (28). Fromm et al. have presented convincing evidence that 7-ketolithocholic acid is wellabsorbed from the upper intestine (24). Therefore, it is probable that the transformation of 7-ketolithocholic acid occurred within the liver. n the light of these findings and our present results, it is conceivable that hepatic enzymes reduce 7-ketolithocholic acid preferentially to chenodeoxycholic acid and produce only small amounts of ursodeoxycholic The intestinal bacteria may reduce 7-ketolithocholic acid to both chenodeoxycholic acid and ursodeoxycholic acid (Figure 5). We cannot, however, rule out the possibility that feeding 7 -ketolithocholic acid changes the bacterial flora and inhibits the growth of bacteria capable of reducing 7-ketolithocholic acid to ursodeoxycholic acid, which may be the reason why we could not observe a precursorproduct relationship for 7-ketolithocholic acid and ursodeoxycholic References 1. Haslewood GAD, Murphy GM, Richardson JM. A direct enzymatic assay for 7 a-hydroxy bile acids and their conjugates. Clin Sci 1973;44: MacDonald la, Williams CN, Mahony D. Behavior of 3aand 7 a-hydroxysteroid dehydrogenases on chenodeoxycholate substituted sepharose. Steroids 1976;28: Danzinger RG, Hofmann AF, Schoenfield LS, et al. Dissolution of cholesterol gallstones by chenodeoxycholic N ngl J Med 1972;286: Bell GD, Whitney B, Dowling RH. Gallstone dissolution in man using chenodeoxycholic Lancet 1972;2: Thistle JL, Hofmann AF. fficacy and specificity of chenodeoxycholic acid therapy for dissolving gallstones. N ngl J Med 1973;289: lser JH, Dowling RH, Mok HY, et al. Chenodeoxycholic acid treatment of cholesterol gallstones: a follow-up report and analysis of factors influencing response to therapy. N ngl J Med 1975;293: Barbara L, Roda, Roda A, et al. The medical treatment of cholesterol gallstones: experience with chenodeoxycholic Digestion 1976;14: Nakagawa S, Makino l, lshizaki T. Dissolution of cholesterol gallstones by ursodeoxycholic Lancet 1977;2: Kutz K, Schulte A. ffectiveness of ursodeoxycholic acid in gallstone therapy. Gastroenterology 1977;73: Marton PN, Murphy GM, Dowling RH. Ursodeoxycholic acid treatment of gallstones. Dose response study and possible mechanism of action. Lancet 1977;2: Makino l, Nakagawa S. Changes in biliary lipid and biliary bile acid composition in patients after administration of ursodeoxycholic J Lipid Res 1979;19:723-8.

7 August KTOLTHOCHOLC ACD, BL ACDS, GALLSTONS Tokyo Cooperative Gallstone Study Group. fficacy and indications of ursodeoxycholic acid treatment for dissolving gallstones. A multicenter double-blind trial. Gastroenterology 1980;78: Salen G, Colalillo A, Verga 0, et al. ffect of high and low doses of ursodeoxycholic acid on gallstone dissolution in humans. Gastroenterology 1980;78: Tint GS, Colalillo A, Sal en G, et al. Ursodeoxycholic acid is a safe and effective gallstone dissolving agent in humans (abstr). Gastroenterology 1981;80: Salen G, Tint GS, liav B, et al. ncreased formation of ursodeoxycholic acid in patients treated with chenodeoxycholic J Clin nvest 1974;53: Shefer S, Hauser S, Lapar V, et al. Regulatory effects of sterols and bile acids on hepatic 3-hydroxy-3-methylglutaryl CoA reductase and cholesterol 7a-hydroxylase in the rat. J Lipid Res 1973;14: Chen PS Jr, Toribara TY, Warner H. Microdetermination of phosphorus. Anal Chern 1956;28: Thomas PJ, Hofmann AF. A simple calculation of the lithogenic index of bile. xpressing biliary lipid composition on rectangular coordinates. Gastroenterology 1973;65: Hegardt FG, Dam H, Andersen G. Solubility of cholesterol in aqueous solutions of bile salts and lecithin. Z rnaehrungswiss 1971;10: Holzbach RT, Marsh M, Olszewski M, et al. Cholesterol solubility in bile. vidence that supersaturated bile is frequent in healthy man. J Clin nvest 1973;52: gimi H, Carey MC. ph Solubility relations of chenodeoxycholic acid and ursodeoxycholic acid: Physical-chemical basis for dissimilar solution and membrane phenomena. J Lipid Res 1980;21: M ahowald TA, Yim AM, Matschimer JT, et al. Bile acids. V. Metabolism of 7-ketolithocholic acid-24-c' 4 in the rat. J Bioi Chern 1958;230: Samuelsson B. The metabolism of 7-ketolithocholic acid-24- l C in the rat. Acta Chern Scand 1959;13: Fromm MH, Carlson GL, Hofmann AF, et al. Metabolism in man of 7-ketolithocholic acid: precursor of cheno- and ursodeoxycholic acids. Am J Physio1980;239:G Fedorowski T, Salen G, Tint GS, et al. Transformation of chenodeoxycholic acid and ursodeoxycholic acid by human intestinal bacteria. Gastroenterology 1979;77: Matkovics B, Samuelsson B. Synthesis and metabolism of 3a,12a-dihydroxY-d 6 -cholenic acid-24- ' C: bile acids and steroids 117. Acta Chern Scand 1962;16: MacDonald la, Hutchison OM, Forrest TP. Formation of ursoand ursodeoxycholic acids from primary bile acids by Clostridium obsonum. J Lipid Res 1981;22: Fedorowski T, Salen G, Colallilo A, et al. Metabolism of ursodeoxycholic acid in man. Gastroenterology 1977;73: