Bile acid synthesis in cultured human hepatocytes:: Support for an alternative biosynthetic pathway to cholic acid

The biosynthesis of bile acids by primary cultures of normal human hepatocytes has been investigated. A general and sensitive method for the isolation and analysis of sterols and bile acids was used, based on anion exchange chromatography and gas chromatography-mass spectrometry (GC/MS). Following incubation for 5 days, 8 oxysterols and 8 C-27- or C-24-bile acids were identified in media and cells. Cholic and chenodeoxycholic acids conjugated with glycine or taurine were by far the major steroids found, accounting for 70% and 24% of the total, respectively, being consistent with bile acid synthesis in human liver. Small amounts of sulfated 3 beta-hydroxy-5-cholenoic acid and 3 beta,7 alpha-dihydroxy-5 beta -cholanoic acid were also detected. Nine steroids were potential bile acid precursors (2% of total), the major precursors being 7 alpha,12 alpha-dihydroxy-3-oxo-4-cholenoic acid and its 5 beta-reduced form. These 2 and 5 other intermediates formed a complete metabolic sequence from cholesterol to cholic acid (CA). This starts with 7 alpha-hydroxylation of cholesterol, followed by oxidation to 7 alpha-hydroxy-4-cholesten-3-one and 12 alpha-hydroxylation. Notably, 27-hydroxylation of the product 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one and further oxidation and cleavage of the side chain precede A-ring reduction. A-Ring reduction may also occur before side-chain cleavage, but after 27-hydroxylation, yielding 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid as an intermediate. The amounts of the intermediates increased in parallel to those of CA during 4 days of incubation. Suppressing 27-hydroxylation with cyclosporin A (CsA) resulted in a 10-fold accumulation of 7 alpha,12 alpha-dihydroxy-4-cholesten-3- one and a decrease of the production of CA and its acidic precursors. These results suggest that the observed intermediates reflect an alternative biosynthetic pathway to CA, which may be quantitatively significant in the cells.