Cortisol metabolism by human liver in vitro—I. Metabolite identification and inter-individual variability

Abstract

The measurement of urinary 6β-hydroxycortisol (6β-OHF) has been widely used as a non-invasive clinical test to detect cytochrome P450 induction. Although only a minor biotransformation, 6β-OHF formation represents a sensitive target for many P450-inducing drugs and environmental chemicals in man. There is good evidence that an isozyme of the P450IIIA subfamily is predominantly responsible for 6β-hydroxylase activity and therefore it has been suggested that urinary 6β-OHF is a marker of the induction of P450IIIA. The basis of the present study was that in order to realistically assign to 6β-OHF the status of a P450IIIA marker we should characterize all the metabolites of cortisol produced by human liver and assess inter-liver variability. Incubations at 37°C for 2 h contained [³H]cortisol (0.1 μCi, 1 or 50 μM), MgCl₂ (10 mM), microsomal or cytosolic protein (3 mg), an NADPH-regenerating system and 1/15 M phosphate buffer (pH 7.4) to give a final volume of 0.5 ml. Extraction with ethyl acetate (2 × 2 ml) was followed by radiometric HPLC analysis. Metabolites were identified by co-chromatography with authentic standards and mass spectrometry (electron impact and chemical ionization). All the microsomal incubations (n = 6 livers) produced 6α-hydroxycortisol (6α-OHF), 6β-OHF, 20β-dihydroxycortisol, 20β-dihydroxycortisone, cortisone, and 3α,5β-tetrahydrocortisone (3α,5β-THE), while five produced 6β-hydroxycortisone and four produced 3α,5β-tetrahydrocortisol (3α,5β-THF). The cytosolic incubations gave a much simpler metabolic profile, with 3α,5β-THF the major metabolite and 3α,5β-THE a minor metabolite. There was considerable inter-individual variability in metabolite profiles from microsomal incubations. 6β-OHF varied from 2.8 to 31.7%. Major metabolites were cortisone and 3α,5β-THE. Inter-liver variability was less for cytosolic incubations, the major metabolite always being 3α,5β-THF. In conclusion we have rigorously identified the hepatic metabolites of cortisol formed in vitro. The highly complex and variable hepatic metabolism of cortisol clearly limits the use of urinary 6β-OHF excretion as a marker of baselinem P450IIIA activity in man.