CYTOCHROME P450-DEPENDENT DESATURATION OF LAURIC ACID - ISOFORM SELECTIVITY AND MECHANISM OF FORMATION OF 11-DODECANOIC ACID

Cytochrome P450-catalyzed desaturation reactions have been reported in frequently in the literature. Previously, we documented the formation of the terminal olefinic metabolite of valproic acid by various member s of the CYP2B and CYP4B sub-families. However, despite the extensive use of fatty acid substrates in drug metabolism studies, other example s of terminal desaturation at non-activated carbon centers are lacking . The goals of the present studies were to determine whether the arche typal P450 substrate, lauric acid (dodecanoic acid; DDA), also undergo es desaturation reactions, identify specific rabbit P450 isoforms whic h catalyze this reaction and examine its mechanism. A highly sensitive , capillary GC/MS assay was developed to separate and quantitate the t rimethylsilyl derivatives of 11-ene-DDA, cis- and trans-10-ene-DDA and cis- and trans-9-ene-DDA. Among all of these potential olefinic metab olites, only 11-ene-DDA was formed at a significant rate by rabbit liv er microsomes. The formation of 11-ene-DDA was NADPH-dependent, and wa s induced markedly by acetone pre-treatment, but not by phenobarbital, rifampin or Arochlor 1254. Studies with seven purified, reconstituted rabbit P450 isoforms showed that the most rapid rates of desaturation were obtained with CYP2E1, CYP4A5/7 and CYP4B1. Non-competitive, inte rmolecular isotope effect experiments, conducted with [12,12,12-H-2(3) ]DDA and [11,11-H-2(2)]DDA, demonstrated further that CYP4B1-mediated terminal desaturation of DDA is initiated by removal of a hydrogen ato m from the omega-1 rather than the omega position. (C) 1998 Elsevier S cience Ireland Ltd. All rights reserved.