Biomedical subjects
Publications and source records attributed to A Radominska.
How does cholestasis develop when there is no obvious obstruction, destruction, or malformation of bile ducts? Recent findings on bile formation and flow are reviewed, and two models of intrahepatic cholestasis are presented: one of rapid onset, which is bile acid induced, and the other of slow onset, which is estrogen induced. Potential clinical correlations are discussed.
Milligram amounts of [3 beta-3H]lithocholic (3 alpha-hydroxy-5 beta-cholanoic) acid were administered by intravenous infusion to rats prepared with a biliary fistula. Analysis of sequential bile samples by thin-layer chromatography (TLC) demonstrated that lithocholic acid glucuronide was present in bile throughout the course of the experiments and that its secretion rate paralleled that of total isotope secretion. Initial confirmation of the identity of this metabolite was obtained by the recovery of labeled lithocholic acid after beta-glucuronidase hydrolysis of bile samples. For detailed analysis of biliary metabolites of [3H]lithocholic acid, pooled bile samples from infused rats were subjected to reversed-phase chromatography and four major labeled peaks were isolated. After complete deconjugation, the two major compounds in the combined first two peaks were identified as murideoxycholic (3 alpha, 6 beta-dihydroxy-5 beta-cholanoic) and beta-muricholic (3 alpha, 6 beta, 7 beta-trihydroxy-5 beta-cholanoic) acids and the third peak was identified as taurolithocholic acid. The major component of the fourth peak, after isolation, derivatization (to the methyl ester acetate), and purification by high pressure liquid chromatography (HPLC), was positively identified by proton nuclear magnetic resonance as lithocholic acid 3 alpha-O-(beta-D-glucuronide). These studies have shown, for the first time, that lithocholic acid glucuronide is a product of in vivo hepatic metabolism of lithocholic acid in the rat.
Biliary secretion of bile acid glucuronides was studied in control rats and in rats with a congenital defect in hepatobiliary transport of organic anions (GY rats). In control animals, hepatobiliary transport of [3H]lithocholic acid 3-O-glucuronide and [3H]cholic acid 3-O-glucuronide was efficient (greater than 95% in 1 h) and comparable to that of [14C]taurocholic acid. Secretion of both glucuronides was impaired in GY rats (24% and 71% at 1 h), whereas that of taurocholate was similar to control values. However, recovery of the glucuronides in bile was nearly complete within 24 h; virtually no radioactivity was found in urine. In control rats, biliary secretion of lithocholic acid 3-O-glucuronide, but not that of cholic acid 3-O-glucuronide or taurocholate, could be delayed by simultaneous infusion of dibromosulphthalein. In mutant rats, dibromosulphthalein infusion was also able to inhibit secretion of cholic acid 3-O-glucuronide. [3H]Hydroxyetianic acid, a C20 short-chain bile acid, was secreted by control rats as a mixture of 20% carboxyl-linked and 80% hydroxyl-linked (3-O-)glucuronide; secretion was very efficient (99% in 1 h). In GY rats, secretion was drastically impaired (16% at 1 h and 74% over a 24-h period). Initially, the mutant secreted more carboxyl- than hydroxyl-linked glucuronide, but the ratio reached that of control animals after 24 h. The rates of formation of both types of hydroxyetianic acid glucuronide by hepatic microsomes from mutant rats were similar or even slightly higher than those of control microsomes. These findings indicate that bile acid 3-O-glucuronides, but probably not carboxyl-linked glucuronides, are secreted into bile by a transport system shared with organic anions such as conjugated bilirubin and dibromosulphthalein, but different from that for amino acid-conjugated bile acids.
The hydroxylation of lithocholic acid (3 alpha-hydroxy-5 beta-cholanoic acid) by adult male Sprague-Dawley rat liver microsomes supplemented with NADPH was studied. Metabolites were separated by a combination of thin-layer chromatography and high pressure liquid chromatography, both with and without prior methylation and acetylation of the samples. The resulting products were characterized by thin-layer, gas-liquid, and high pressure liquid chromatography by comparison with authentic bile acid standards; final structure determination was by proton nuclear magnetic resonance spectroscopy and by mass spectrometry. The following reaction products were found: 3 alpha, 6 beta-dihydroxy-5 beta-cholanoic acid (80% of total metabolites) and 3 alpha, 6 alpha-dihydroxy-5 beta-cholanoic, 3 alpha, 7 alpha-dihydroxy-5 beta-cholanoic, 3 alpha, 6 beta,7 beta-trihydroxy-5 beta-cholanoic, and 3 alpha-hydroxy-6-oxo-5 beta-cholanoic acids (less than or equal to 5% each). In addition, one unidentified trihydroxylic bile acid and several minor compounds were present. It is concluded that four different hydroxylation reactions of lithocholic acid, namely the predominant 6 beta as well as the minor 6 alpha, 7 alpha, and 7 beta hydroxylations, are catalyzed by rat hepatic microsomes; 7 beta-hydroxylation may occur only with dihydroxylated bile acids but not with lithocholate itself. The presence of the 6-oxo bile acid can be explained either by direct oxidation of a hydroxyl group by cytochrome P-450, or by the action of microsomal dehydrogenase(s) which could also catalyze the epimerization of hydroxyl groups via their oxidation. The results form the basis of a proposed scheme of the oxidative metabolism of lithocholic acid in rat liver microsomes.
Microsomal preparations from livers of Sprague-Dawley rats catalyze the glucuronidation of 3 alpha-hydroxy-5 beta-H (3 alpha, 5 beta) short-chain bile acids (C20-C23), predominantly at the hydroxyl group, while the glucuronidation of 3 beta, 5 beta short-chain bile acids occurs exclusively at the carboxyl group. A similar pattern of conjugation was also observed in Wistar rats having normal levels of 3-hydroxysteroid UDP-glucuronosyltransferase. Significant reductions of formation rates for hydroxyl-linked, but not carboxyl-linked, short-chain bile acid glucuronides were observed in hepatic microsomes from Wistar rats with low 3-hydroxysteroid UDP-glucuronosyltransferase activity. 3-Hydroxysteroid UDP-glucuronosyltransferase, purified to homogeneity from Sprague-Dawley liver microsomes, catalyzed the 3-O-glucuronidation of 3 alpha, 5 beta C20-23 bile acids, as well as of lithocholic and isolithocholic acids (C24). The apparent Michaelis constants (KM) for short-chain bile acids were similar to the value obtained for androsterone. 3 alpha, 5 beta-C20 and 3 beta, 5 beta-C20 competitively inhibited glucuronidation of androsterone by the purified 3-hydroxysteroid UDP-glucuronosyltransferase. Purified 17 beta-hydroxysteroid and p-nitrophenol UDP-glucuronosyltransferases did not catalyze the glucuronidation of bile acids. In addition, none of the purified transferases catalyzed the formation of carboxyl-linked bile acid glucuronides. The results show that 3-hydroxysteroid UDP-glucuronosyltransferase, an enzyme specific for 3-hydroxyl groups of androgenic steroids and some conventional bile acids, also catalyzes the glucuronidation of 3 alpha-hydroxyl (but not carboxyl) groups of 3 alpha, 5 beta short-chain bile acids.
The glucuronidation of 6-hydroxylated bile acids by rat liver microsomes was studied in vitro; for comparison, several major bile acids lacking a hydroxyl group in position 6 were also investigated. The highest reaction rates were found for lithocholic and deoxycholic acid (10.2 +/- 0.2 and 7.3 +/- 1.4 nmol/mg.min, respectively); our results for these substrates agree well with published values. Glucuronidation rates for the 6 beta-hydroxylated bile acids 3 alpha, 6 beta-dihydroxy-5 beta-cholanoate (murideoxycholate) and 3 alpha, 6 beta, 7 beta-trihydroxy-5 beta-cholanoate (beta-muricholate) were only slightly lower (3.7 +/- 0.3 and 3.6 +/- 0.3 nmol/mg.min). 6 alpha-Hydroxylated bile acids were glucuronidated at rates that were lower than those for their 6 beta-hydroxy counterparts. Rigorous product identification by high-field proton NMR of methyl/acetyl derivatives revealed that while bile acids lacking a 6-hydroxyl group gave rise exclusively to the typical 3-O-glucuronide, the presence of a hydroxyl group in position 6 led to the formation, in ratios depending on the substrate, of three types of conjugate: the 3-O-, the 6-O-, and the carboxyl-linked (acyl-) glucurnide. The latter is the first example of an acyl glucuronide of a bile acid of conventional (C24) size.
The reaction of the 6-hydroxylated bile acid, hyodeoxycholic acid, and its 6-O-glucuronide conjugate with 3 alpha-hydroxysteroid dehydrogenase was examined. A standard end-point assay and determination of the initial rates of reaction showed only minimal activity of the enzyme toward hyodeoxycholate-6-glucuronide in spite of the presence of a free 3 alpha-hydroxyl group. It was established that 6-hydroxylation itself did not significantly affect the enzyme reaction. It is concluded that the 6-glucuronide either blocks or hinders enzyme access to the 3-hydroxyl group.
The glucuronidation of monohydroxylated bile acids and their analogs with a shortened side chain (short-chain bile acids) by human liver microsomes and by two UDP-glucuronosyltransferases purified therefrom has been studied in vitro. In microsomes, all 18 substrates tested underwent glucuronidation; the rate of reaction and the site of attachment of glucuronic acid (hydroxyl group in position 3, side chain carboxyl group, or various ratios of both products) were strongly dependent on the length of the side chain, the configuration of the 3-hydroxyl group, and the configuration of the A/B ring junction (5 alpha-H/5 beta-H). Two UDP-glucuronosyltransferases (UDPGTs) purified from human microsomes, designated "pl 7.4" and "pl 6.2" according to their behavior in chromatofocusing, accounted for the formation of hydroxyl-linked glucuronides of a different pair of bile acids each. The pl 7.4 human liver UDPGT catalyzed the glucuronidation of C20 and C22 with the 3 alpha-OH, 5 beta-H configuration, while the pl 6.2 human liver UDPGT catalyzed the glucuronidation of either 3-OH epimer of C21 and C24 acids with the 5 alpha-H configuration. The enzymes displayed a relatively high selectivity in that they did not accept any of the remaining 14 bile acids as substrates; none of the enzymes led to the formation of a carboxyl-linked glucuronide. In addition, purified human liver UDPGT did not catalyze the glucuronidation of cholate, deoxycholate, chenodeoxycholate, ursodeoxycholate, lithocholate, hyocholate, hyodeoxycholate, bilirubin, morphine, or 4-hydroxybiphenyl. The above results suggest that several bile-acid UDP-glucuronosyltransferases of high specificity exist in human liver.