VALIDATION OF THE (OMEGA-1)-HYDROXYLATION OF LAURIC ACID AS AN IN-VITRO SUBSTRATE PROBE FOR HUMAN LIVER CYP2E1

The (omega-1)-hydroxylation of lauric acid (11-OH-LA), a model substra te of fatty acids, was previously shown to be due to CYP2E1 in rat liv er microsomes. The present study examined changes in hepatic CYP2E1 co ntent and 11-OH-LA in a panel of 29 human liver microsomes. The 11-OH- LA activity was strongly correlated with the CYP2E1 content, quantitat ed by immunoblot (r = 0.75) and with four monooxygenase activities kno wn to be mediated by CYP2E1: chlorzoxazone-6-hydroxylation (r = 0.73), 4-nitrophenol hydroxylation (r = 0.84), N-nitrosodimethylamine demeth ylation (r = 0.79) and n-butanol oxidation (r = 0.73). The (omega-1)-h ydroxylation of lauric acid was inhibited by ethanol (K-i = 3.5 mM), a cetone (IC50 = 10 mM), dimethylsulfoxide, chlorzoxazone (competitive i nhibitors of CYP2E1), diethyldithiocarbamate, and diallylsulfide (both selective mechanism-based inactivators of CYP2E1). The weak value of ethanol K-i on the (omega-1)-hydroxylation of lauric acid suggested th at low levels of alcohol could modify fatty acid metabolism in the liv er. Furafylline and gestodene, suicide substrates of CYP1A and CYP3A4, respectively, did not modify the 11-hydroxylation of lauric acid. Pol yclonal antibody directed against rat CYP2E1 inhibited the formation o f 11-OH-LA without affecting 12-OH-LA activity. Taken together, these results suggest that CYP2E1 is involved in the (omega-1)hydroxylation of lauric acid in human liver microsomes, and omega-hydroxylation is m ediated by another enzyme. Finally, the use of yeasts and mammalian ce lls genetically engineered for expression of 9 human P450s demonstrate d that CYP2E1 was the one enzyme involved in the (omega-1)-hydroxylati on of lauric acid.