6α-hydroxylation of taurochenodeoxycholic acid and lithocholic acid by CYP3A4 in human liver microsomes

The aim of the present study was to identify the enzymes in human liver catalyzing hydroxylations of bile acids. Fourteen recombinant expressed cytochrome P450 (CYP) enzymes, human liver microsomes from different donors, and selective cytochrome P450 inhibitors were used to study the hydroxylation of taurochenodeoxycholic acid and lithocholic acid. Recombinant expressed CYP3A4 was the only enzyme that was active towards these bile acids and the enzyme catalyzed an efficient 6 alpha-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid. The V-max for 6 alpha-hydroxylation of taurochenodeoxycholic acid by CYP3A4 was 18.2 nmol/nmol P450/min and the apparent K-m was 90 mu M. Cytochrome b(5) was required for maximal activity. Human liver microsomes from 10 different donors, in which different P450 marker activities had been determined, were separately incubated with taurochenodeoxycholic acid and lithocholic acid. A strong correlation was found between 6 alpha-hydroxylation of taurochenodeoxycholic acid, CYP3A levels (r(2) =0.97) and testosterone 6 beta-hydroxylation (r(2)=0.9). There was also a strong correlation between 6 alpha-hydroxylation of lithocholic acid, CYP3A levels and testosterone 6 beta-hydroxylation (r(2) = 0.7). Troleandomycin, a selective inhibitor of CYP3A enzymes, inhibited 6 alpha-hydroxylation of taurochenodeoxycholic acid almost completely at a 10 mu M concentration. Other inhibitors, such as alpha-naphthoflavone, sulfaphenazole and tranylcypromine had very little or no effect on the activity. The apparent K-m for 6 alpha-hydroxylation of taurochenodeoxycholic by human liver microsomes was high (716 mu M). This might give an explanation for the limited formation of 6 alpha-hydroxylated bile acids in healthy humans. From the present results, it can be concluded that CYP3A4 is active in the 6 alpha-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid in human liver. (C) 1999 Elsevier Science B.V. All rights reserved.