Hepatocyte heterogeneity in bile formation and hepatobiliary transport of drugs.

In the past two decades many studies have been devoted to the involvement of the periportal (zone-1) and perivenous (zone-3) hepatocytes in bile formation and hepatobiliary transport of endogenous and exogenous compounds. It became clear that such a heterogeneity in transport function can, in principle, be due to the different localization of the cells in the acinus with respect to the incoming blood, to intrinsic differences between the cells or to both. In this review we first discuss the techniques used to study hepatocyte heterogeneity in hepatobiliary transport function. Combinations of such techniques can be used to discriminate between cellular heterogeneity due to acinar localization as opposed to intrinsic differences. These techniques include: normal and retrograde perfusions of isolated perfused livers; autoradiographic, fluorimetric and histochemical localization of injected substrates; separation of isolated hepatocytes into fractions enriched in periportal and perivenous cells; measurements of fluorescent surface signals with microlight guides; selective zonal toxicity, and pharmacokinetic modelling and analysis. Subsequently, for each of the rate-limiting steps in the hepatobiliary transport of organic compounds, the basic mechanisms are summarized and the available knowledge on the involvement of the cells from the various zones in these transport steps is discussed. The available literature data indicate that heterogeneity in transport function is often due to the localization of the cells in the acinus: the periportal cells are the first to come into contact with the portal blood and are thus exposed to the highest substrate concentration. Consequently they obtain the most prominent task in further disposition of the particular compound. It follows that the extent of involvement of the perivenous cells in drug disposition is implicitly determined by the activity of the periportal cells. Because of the potential saturation of elimination processes in the periportal cells, the involvement of perivenous cells may vary with the input concentration. In addition, real intrinsic differences have been established in the hepatobiliary transport of some substrates. These are probably based on differences in the cellular content of carrier- and receptor-binding and/or metabolizing proteins. In some cases these intrinsic differences may be secondary to existing sinusoidal gradients of endogenous compounds, such as O2, amino acids, bile acids or monosaccharides. Yet, data on the heterogeneity of hepatocytes in the various transport steps are far from complete or are even totally lacking, especially for human liver. A multi-experimental approach and advanced technology will be needed in the future to gain more insight into the acinar organization of bile formation and hepatobiliary transport of drugs in the human.     

Chemical properties of bile acids. IV. Acidity constants of glycine-conjugated bile acids

The dissociation constants for the carboxyl group of a series of glycine (N-acyl)-conjugated and unconjugated bile acids were determined by potentiometric titration using dimethylsulfoxide-water and methanol-water mixtures of varying proportions. The pKa values in water were calculated by extrapolating the experimental values determined in different mole fractions of the organic solvent mixtures. The following values were obtained: 3.9 +/- 0.1 for glycine-conjugated bile acids and 5.0 +/- 0.1 for unconjugated bile acids, as general pKa values for the two classes of bile acids, respectively. The amidation of bile acids with glycine lowers the pKa value because of the proximity of the amide bond to the terminal carboxyl group. Bile acid dissociation constants are independent of the substituents in the steroid nucleus, since inductive effects of the hydroxyl groups on the steroid nucleus are too distant from the acidic group at the end of the side chain to influence its ionization.     

Serum concentrations of glucuronidated and sulfated bile acids in children with cholestasis

Serum concentrations of nonglucuronidated-nonsulfated, glucuronidated, and sulfated bile acids in 9 control children and 16 children with cholestasis were quantitated by mass fragmentography. Total bile acid levels in control children were 19.55 +/- 2.78 mumol/liter (mean +/- SEM), and glucuronidated and sulfated bile acids comprised 2.6 +/- 0.5 and 17 +/- 3.1%, respectively. In 9 patients with congenital biliary atrasia, total bile acid levels were 167.34 +/- 11.18 mumole/liter of which 2.1 +/- 0.3% were glucuronidated and 15 +/- 1.4% were sulfated. Lithocholic and 3 beta-hydroxy-5-cholenoic acids, which have hepatotoxic effects, were presented in only small amounts in cholestatic children, and they were almost all glucuronidated or sulfated. The percentages of glucuronidated bile acids in control and cholestatic children were lower than in healthy and cholestatic adults, which may be explained by the lower activity of UDP-glucuronyltransferase in neonatal liver.     

Aerobic catabolism of bile acids.

Seventy-eight stable cultures obtained by enrichment on media containing ox bile or a single bile acid were able to utilize one or more bile acids, as well as components of ox bile, as primary carbon sources for growth. All isolates were obligate aerobes, and most (70) were typical (48) or atypical (22) Pseudomonas strains, the remainder (8) being gram-positive actinomycetes. Of six Pseudomonas isolates selected for further study, five produced predominantly acidic catabolites after growth on glycocholic acid, but the sixth, Pseudomonas sp. ATCC 31752, accumulated as the principal product a neutral steroid catabolite. Optimum growth of Pseudomonas sp. ATCC 31752 on ox bile occurred at pH 7 to 8 and from 25 to 30 degrees C. No additional nutrients were required to sustain good growth, but growth was stimulated by the addition of ammonium sulfate and yeast extract. Good growth was obtained with a bile solids content of 40 g/liter in shaken flasks. A near-theoretical yield of neutral steroid catabolites, comprising a major (greater than 50%) and three minor products, was obtained from fermentor growth of ATCC 31752 in 6.7 g of ox bile solids per liter. The possible commercial exploitation of these findings to produce steroid drug intermediates for the pharmaceutical industry is discussed.     

Influence of dietary bile acids on formation of bile acids in rat

Various taurine-conjugated bile acids were fed to rats at the 1%-level in the diet for 3 or 7 days and the effect on several hydroxylations involved in the biosynthesis and metabolism of bile acids was studied. The hydroxylations studied were all catalyzed by the microsomal fraction of liver homogenate fortified with NADPH. The 7α-hydroxylation of cholesterol was inhibited by feeding taurocholic acid, taurocheno-deoxycholic acid and taurodeoxycholic acid for 3 as well as 7 days. No marked inhibition was obtained with taurohyodeoxycholic acid or taurolithocholic acid. The 12α-hydroxylation of 7α-hydroxy-4-cholesten-3-one was inhibited after 3 as well as 7 days by all bile acids except taurohyodeoxycholic acid. With this acid a marked stimulation of 12α-hydroxylation was observed. The effects of the different bile acids on the 7α-hydroxylation of taurodeoxycholic acid were not very marked. The 6β-hydroxylation of lithocholie acid and taurochenodeoxycholic acid was stimulated by taurocholic acid and taurodeoxycholic acid. The reaction was inhibited by taurochenodeoxycholic acid, at least after 7 days. Taurohyodeoxycholic acid inhibited the 6β-hydroxylation slightly and taurolithocholic acid had no effect. The results were discussed in the light of present knowledge concerning mechanisms of regulation of formation and metabolism of bile acids and it was suggested that the mechanisms may be more complex than previously thought.     

A proposed nomenclature for bile acids.

A proposal is made for a system of nomenclature of the more common unconjugated and conjugated bile acids. Acceptable trivial names for bile acids are tabulated, and guidelines are proposed for using these existing trivial names as roots to create acceptable semi-systematic names for other bile acids, as well as for new natural bile acids that will be discovered in the future. The term alpha-hyocholic is recommended to replace hyocholic, and beta-hyocholic to replace omega-muricholic. The term murideoxycholic acid is recommended for 3 alpha,6 beta-dihydroxy-5 beta-cholan-24-oic acid. Proposals are also made for bile acids with epimeric hydroxy groups, for unsaturated bile acids, and for bile acids with oxo- and/or hydroxy-oxo- substituents on the nucleus and/or on the side chain. For conjugated bile acids, the term "aminoacyl amidates" is recommended to replace "amidates" for bile acids conjugated in N-acyl linkage with amino acids. Nomenclature for other types of conjugates (sulfates, glucuronides, glucosides) is included as well as abbreviations. It is recommended that the historic tradition of naming a newly discovered bile acid after the species from which it was isolated be abandoned, and that in the future such a bile acid should be named using the principles contained in this paper.     

Identification of unconjugated bile acids in human bile

Unconjugated bile acids in the bile of healthy and diseased humans were determined qualitatively and quantitatively by means of gas-liquid chromatography and gas-liquid chromatography-mass spectrometry, after their isolation by ion-exchange chromatography. In a healthy person and three patients with cholelithiasis, unconjugated bile acids comprised 0.1-0.4% of total biliary bile acids. The bile acid composition of the unconjugated fraction was quite different from that of the glycine- or taurine-conjugate fraction, in that it contained a relatively large proportion of unusual bile acids including C23 and C27 bile acids. In two patients with cerebrotendinous xanthomatosis, C22 and C23 bile acids were the major constituents of the biliary unconjugated bile acids, and comprised about 0.8% of total bile acids; no detectable amounts of C27 bile acids were found in their bile. The analysis of biliary unconjugated bile acids may be useful for the diagnosis of metabolic diseases concerning bile acids, particularly the accumulation or disappearance of unusual bile acids.     

Potential bile acid metabolites. 17. Synthesis of 2 beta-hydroxylated bile acids

The 2 beta-hydroxylated derivatives of lithocholic, chenodeoxycholic, deoxycholic, and cholic acids were synthesized from the respective parent bile acids by established procedures. The principal reactions involved were (1) bromination of 3-oxo formylated bile acids in N,N-dimethylformamide, (2) rearrangement and substitution of the resulting 4 beta-bromo-3-oxo derivatives to the 2 beta-acetoxy-3-oxo compounds with potassium acetate, and (3) reduction to the 2 beta-acetoxy-3 alpha-hydroxy compounds with tert-butylamine-borane complex. As for the prepared 2 beta-hydroxylated bile acids with a diequatorial trans-glycol structure, proton and carbon-13 nuclear magnetic resonance spectroscopic and gas-liquid chromatographic/mass spectrometric properties are discussed.     

Identification of bile acids in the serum and urine in cholestasis. Evidence for 6alpha-hydroxylation of bile acids in man.

In this qualitative study of the pattern of bile acid excretion in cholestasis, methods are described for the isolation of bile acids from large volumes of urine and plasma. The bile acids were subjected to a group separation and identified by combined gas chromatography-mass spectrometry. The techniques were developed to allow identification of the minor components of the bile acid mixture. Four bile acids that have not previously been described in human urine and plasma were detected, namely 3beta, 7alpha-dihydroxy-5beta-cholan-24-oic acid, 3alpha, 6alpha-dihydroxy-5beta-cholan-24-oic acid (hyodeoxycholic acid), 3alpha, 6alpha, 7alpha-trihydroxy-5beta-cholan-24-oic acid (hyocholic acid) and 3alpha, 7beta, 12alpha-trihydroxy-5beta-cholan-24-oic acid. In addition three C27 steroids were found; 26-hydroxycholesterol and a trihydroxy cholestane, probably 5 beta-cholestane-3alpha, 7alpha, 26-triol were found in the sulphate fraction of plasma and urine. In the plasma sample, a sulphate conjugate of 24-hydroxycholesterol was found. The presence of these compounds probably reflects the existence of further pathways for bile acid metabolism. It is not yet known whether this is a consequence of the cholestasis or whether they are also present in normal man, at much lower concentrations.