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Bile acid synthesis in cultured human hepatocytes: support for an alternative biosynthetic pathway to cholic acid
Author(s) -
Axelson Magnus,
Ellis Ewa,
Mörk Birgitta,
Garmark Kristina,
Abrahamsson Anna,
Björkhem Ingemar,
Ericzon BoGöran,
Einarsson Curt
Publication year - 2000
Publication title -
hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.488
H-Index - 361
eISSN - 1527-3350
pISSN - 0270-9139
DOI - 10.1053/jhep.2000.7877
Subject(s) - cholic acid , hydroxylation , cyp8b1 , taurine , bile acid , chemistry , chenodeoxycholic acid , biochemistry , glycine , amino acid , enzyme
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 3β‐hydroxy‐5‐cholenoic acid and 3β,7α‐dihydroxy‐5β‐cholanoic acid were also detected. Nine steroids were potential bile acid precursors (2% of total), the major precursors being 7α,12α‐dihydroxy‐3‐oxo‐4‐cholenoic acid and its 5β‐reduced form. These 2 and 5 other intermediates formed a complete metabolic sequence from cholesterol to cholic acid (CA). This starts with 7α‐hydroxylation of cholesterol, followed by oxidation to 7α‐hydroxy‐4‐cholesten‐3‐one and 12α‐hydroxylation. Notably, 27‐hydroxylation of the product 7α,12α‐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 3α,7α,12α‐trihydroxy‐5β‐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 7α,12α‐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.