α-Naphthylisothiocyanate (ANIT) Induced Cholestasis in Rats

In order to distinguish the disorder of bile acid and cholesterol metabolism in α-naphthylisothiocyanate (ANIT)-induced cholestasis, we examined changes in bile acid levels and compositions in bile, serum, feces and urine, as well as cholesterol levels in bile, serum, liver and feces in Wistar male rats (10-13 weeks) after a single oral administration of 100 mg/kg of ANIT. The bile flow and the biliary secretions of cholesterol, phospholipids and bile acids markedly decreased on days 1 and 2 but increased over the normal values on day 4 and then returned to the normal ranges. The fecal excretion of bile acids decreased after the treatment and remained low by day 4 but markedly increased thereafter. The urinary excretion of bile acids changed almost in parallel with serum bile acid level, increasing to 37 mg/day on day 2, 28 mg/day on days 3-4 but to a trace on days 5-6. The urinary bile acids on day 2 mainly consisted of cholic acid while those on days 3-4 and biliary bile acids on day 4 were mostly β-muricholic acid. The serum cholesterol level markedly increased maximally on day 2 and decreased thereafter. The fecal excretion of sterols, cholesterol and coprostanol, decreased on days 1-2 but rather increased thereafter. These data suggest that the cholestasis induced by ANIT is very similar to that in bile duct ligated rats for a short period but not to those ligated for long periods. In addition, the present data suggest that the bile acid independent bile flow is impaired and the daily synthesis of bile acids, especially β-muricholic acid, is increased in the ANIT induced cholestasis

Dehydrogenation of Conjugated Cholic Acid by Escherichia coli

7α-Dehydrogenation of taurocholic acid and glycocholic acid by Escherichia coli (E. coli) was examined in aerobic and anaerobic culture conditions. Bile acids in the culture medium of E. coli were extracted, separated into free, glycine-conjugate and taurine-conjugate fractions by piperidinohydroxypropyl dextran gel column chromatography, hydrolyzed in alkali and analyzed by gas-liquid chromatography. Both conjugated cholic acids were dehydrogenated to the corresponding 3α,12α-dihydroxy-7-oxo-5β-cholan oic acid without deconjugation and no deconjugation of both conjugated cholic acid was detected in aerobic cultures. But there was little transformation in anaerobic cultures. These data suggest that conjugated cholic acids are taken up by E. coli in an aerobic culture as conjugate forms, dehydrogenated without deconjugation and excreted from the cell as conjugate forms

Internal Standard Compounds for Quantitative Determination of Bile Acids by Gas Chromatography

Gas chromatography is well recognized as a useful tool with several advantages for the analysis of bile acids as well as various compounds. In gas chromatographic analysis, bile acids in an analytical sample are subjected to a number of complicated procedures involving many steps such as extraction, fractionation, solvolysis, hydrolysis, derivatization and injection to the gas chromatograph. These procedures result in the loss of bile acids in the analytical sample. The addition of suitable internal standard compound(s) into the analytical sample prior to the extraction of bile acids is indispensable for an accurate determination of bile acids. There are two methods for the quantitative determination of bile acids in a biological sample by gas chromatography: one is the determination of total bile acid amounts in the sample. The other is the determination of bile acid amounts in each fraction after group separation of bile acids in the biological sample using an ion exchange gel column. The addition of 7β,12α-dihydroxy-5β-cholanoic acid or 7β,12β-dihydroxy-5β-cholanoic acid as an internal standard compound is useful for the former method. On the other hand, the addition of 7β,12β-dihydroxy-5β-cholanoic acid, glyco-7α,12α-dihydroxy-5β-cholanoic acid, tauro-7α,12β-dihydroxy-5β-cholanoic acid and glyco-7β,12α-dihydroxy-5β-cholanoic acid 7-sulfate is a suitable combination as internal standard compounds for the latter method

Aerobic and Anaerobic Biotransformation of Bile Acids by Escherichia coli (III)

The oxidation/reduction reactions of bile acids by Escherichia coli (E. coli) K-12 were examined in both Davis and brain-heart infusion (BHI) media under aerobic and anaerobic conditions. The pH in the Davis medium changed by almost the same amount, around pH 6.5?7.0 in both aerobic and anaerobic cultures, but the pH in the BHI medium was different in both cultures, that is, about pH 9.0 in the aerobic culture but only about 6.5 in the anaerobic culture. The growth curve of E. coli in the Davis medium showed a similar pattern in both conditions. Cholic acid (CA) was oxidized to 3α12α-dihydroxy-7-oxo-5β-cholanoic acid (3α12α7=O) in both cultures, but the reaction in the anaerobic culture was somewhat slower than that in the aerobic culture. On the other hand, reduction of 3α12α7=O to CA did not occur in the aerobic culture, but about 10% reduction was observed in the anaerobic culture after 4 days. These data suggest that the oxidation/reduction reaction of E. coli was oxidative in aerobic culture but reductive in anaerobic culture and these characteristics were not due to the changes in the pH of the medium. The reactions of CA and glycocholic acid to crude 7α-HSDH prepared from E. coli were examined and it was found that both free and conjugated CA as a substrate for the 7α-HSDH showed similar Km values

Effect of Partial Ileal Bypass on Cholesterol and Bile Acid Metabolism in Rats

In order to clarify the effect of ileal bypass on cholesterol and bile acid metabolism, partial (20 cm) ileal bypass rats were fed a 2% cholesterol supplemented diet for a week after 4 weeks of the operation. The serum and liver cholesterol and phospholipid levels, biliary cholesterol, phospholipid and bile acid secretions, and fecal cholesterol, coprostanol and bile acid excretions were examined. The serum cholesterol level in ileal bypass rats was lower than in normal rats and no hypercholesterolemia was brought about in ileal bypass rats by feeding them the cholesterol diet. The liver cholesterol level increased by feeding the cholesterol diet even in ileal bypass rats but the increase was far less than that in normal rats (22% versus 57%). Biliary bile acid secretion decreased and fecal bile acid excretion increased markedly in ileal bypass rats. Deoxycholic acid increased remarkably in both bile and feces and resulted in an increase in the ratio of the sum of bile acids derived from cholic acid over the sum of bile acids derived from chenodeoxycholic acid (CA/CDCA ratio) in the feces. These results suggest that the absorption of bile acids is impaired, the pool size of bile acids decreases and the hepatic synthesis of bile acids, especially that of cholic acid, increases in ileal bypass rats. As a result, cholesterol feeding to ileal bypass rats produces neither hypercholesterolemia nor a further increase in bile acid synthesis

Biotransformation of Bile Acids by Bacteroides sp. Strain T-40 Isolated from Human Microflora

The effects of Bacteroides sp. strain T-40 isolated from human feces on the biotransformation of bile acids were examined in an anaerobic culture system. Bacteroides sp. T-40 oxidized cholic acid (CA) and chenodeoxycholic acid (CDCA) to 3α,12α-dihydroxy-7-oxo-5β-cholanoic acid and 3α-hydroxy-7-oxo-5β-cholanoic acid, and reduced these oxo-bile acids to CA and CDCA, respectively. However, the reduction activities were lower than the oxidation activities. Hyocholic acid was dehydrogenated, but to a lesser extent than CA or CDCA. On the other hand, α-muricholic acid, which has a hydroxyl group at the position of 7α, was not dehydrogenated. Glycocholic acid was converted to free 3α,12α-dihydroxy-7-oxo-5β-cholanoic acid but any glycine conjugated 7-oxo product was not detected. These data indicate that Bacteroides sp. T-40 possesses bile acid hydrolase and 7α-hydroxysteroid dehydrogenase, by which conjugated bile acids are initially deconjugated, and then undergo oxidization of the 7α-hydroxy group

Utility of 19-Hydroxycholesterol as an Internal Standard Compound for the Quantitative Determination of Sterols Using Capillary Gas Chromatograph

Gas chromatography is able to detect many kinds of sterols simultaneously. In order to identify and estimate accurately many kinds of sterols in biological samples by gas chromatography, it is indispensable to add an internal standard compound into the analytical samples prior to the analysis. However, there is no report on a suitable internal standard compound for the determination of sterols. In this study, 19-hydroxycholesterol was proved to be a suitable internal standard compound for simultaneous and quantitative determination of several sterols in various analytical samples by capillary gas chromatography, including the quantitative determination of β-cholestanol in the human plasma. Moreover, the excellent condition of gas chromatographic analysis for quantitative determination of several sterols in various analytical samples was found