Determination of cytochrome P450 enzymes involved in the metabolism of (−)-terpinen-4-ol by human liver microsomes

Abstract

1. The in vitro metabolism of (−)-terpinen-4-ol was examined in human liver microsomes and recombinant enzymes.

2. The biotransformation of (−)-terpinen-4-ol was investigated by gas chromatography–mass spectrometry. (−)-Terpinen-4-ol was found to be oxidized to (−)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol, major metabolic product by human liver microsomal P450 enzymes. The formation of metabolites of (−)-terpinen-4-ol was determined by relative abundance of mass fragments and retention times on GC.

3. CYP2A6 in human liver microsomes was a major enzyme involved in the oxidation of (−)-terpinen-4-ol by human liver microsomes, based on the following lines of evidence. First, of 11 recombinant human P450 enzymes tested, CYP2A6 had the highest activity for oxidation of (−)-terpinen-4-ol. Second, oxidation of (−)-terpinen-4-ol was inhibited by (+)-menthofuran. Finally, there was a good correlation between CYP2A6 maker activity and (−)-terpinen-4-ol oxidation activities in liver microsomes of 10 human samples.

4. Kinetic analysis showed that the V_max/K_m values for (−)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol catalysed by liver microsomes of human sample HH-18 was 2.49 μL/min/nmol.

5. Human recombinant CYP2A6 catalysed (−)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol with V_max values of 13.9 nmol/min/nmol P450 and apparent K_m values of 91 μM.

Keywords::

• (−)-Terpinen-4-ol
• terpene
• P450
• CYP2A6
• microsomes
• human