Lauric acid as a model substrate for the simultaneous determination of cytochrome P450 2E1 and 4A in hepatic microsomes.

In vitro techniques have been utilized to investigate the microsomal enzymes involved in the metabolism of lauric acid and to establish conditions in which it can be used as a model substrate for both cytochrome P450 4A and cytochrome P450 2E1 in human liver microsomes. Studies of enzyme kinetics of lauric acid omega-hydroxylation in human liver microsomes indicated the involvement of more than one enzyme in this pathway, a relatively low Km enzyme with a Km of 22 microM +/- 12 (n = 8) and a high Km enzyme with a Km an order of magnitude higher (550 microM +/- 310, n = 7). The apparent Vmax for this component correlated with the rate of cyclosporin metabolism and was highly sensitive to ketoconazole inhibition. These results indicated that this enzyme was a member of the 3A subfamily. The activity associated with the low Km enzyme (P450 4A) did not correlate with P450 1A2, 2A6, 2C9/8, 2C19, 2D6, 2E1, or 3A activities in a bank of human liver microsomes and was not appreciably inhibited by ketoconazole, furafylline, quinidine, sulfaphenazole, or diethyldithiocarbamate (DDC). Lauric acid omega-1 hydroxylation demonstrated simple Michaelis-Menten kinetics in each of the human liver microsomal samples examined, with a Km of 130 microM +/- 42 (n = 8). This activity was highly correlated with chlorzoxazone 6-hydroxylation in human liver microsomes (r = 0.98, n = 14, p < 0.001) and was inhibited by both DDC and chlorzoxazone. Additionally, rats treated with the P450 2E1 inducer isoniazid demonstrated a 3-fold increase in lauric acid omega-1 hydroxylation relative to the control group. Thus, the lauric acid hydroxylation assay, at a substrate concentration of 20 microM, appears to be an effective and specific P450 model substrate capable of determining simultaneously P450 4A and P450 2E1 related activities in hepatic microsomal samples.