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 e nzymes involved in the metabolism of lauric acid and to establish cond itions in which it can be used as a model substrate for both cytochrom e P450 4A and cytochrome P450 2E1 in human liver microsomes. Studies o f enzyme kinetics of lauric acid omega-hydroxylation in human liver mi crosomes indicated the involvement of more than one enzyme in this pat hway, a relatively low K-m enzyme with a K-m of 22 mu M +/- 12 (n = 8) and a high K-m enzyme with a K-m an order of magnitude higher (550 mu M +/- 310, n = 7). The apparent V-max for this component correlated w ith the rate of cyclosporin metabolism and was highly sensitive to ket oconazole inhibition. These results indicated that this enzyme was a m ember of the 3A subfamily. The activity associated with the low K-m en zyme (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, sulfap henazole, or diethyldithiocarbamate (DDC). Lauric acid omega-1 hydroxy lation demonstrated simple Michaelis-Menten kinetics in each of the hu man liver microsomal samples examined, with a K-m of 130 mu M +/- 42 ( n = 8). This activity was highly correlated with chlorzoxazone 6-hydro xylation in human liver microsomes (r = 0.98, n = 14, p < 0.001) and w as inhibited by both DDC and chlorzoxazone. Additionally, rats treated with the P450 2E1 inducer isoniazid demonstrated a 3-fold increase in lauric acid alpha-1 hydroxylation relative to the control group. Thus , the lauric acid hydroxylation assay, at a substrate concentration of 20 mu M, appears to be an effective and specific P450 model substrate capable of determining simultaneously P450 4A and P450 2E1 related ac tivities in hepatic microsomal samples.