A Convenient Method for the Determination of Hepatic Laurie Acid ω-Oxidation Based on Solvent Partition

Abstract

Assessment of hepatic ω-oxidation of fatty acids by cytochrome P450IV enzymes in toxicology studies can be a means of evaluating test compound effects on peroxisomal proliferation. Routine assay of ω-oxidation, however, requires a simpler method of enzymatic analysis than currently described GC, TLC, or HPLC methods. A method depending upon selective solvent separation of radiolabeled lauric acid from radiolabeled 11- or 12-hydroxydodecanoic acid has been developed. Following enzymatic incubation and addition of 15% methanol to the acidified postincubation mixtures, partitioning with an aJkane solvent such as iso-octane, cyclohexajie, or n-hexane separated the lauric acid and the hydroxylated products into two immiscible phases. Approximately 97% of the substrate partitioned into the organic phase, and approximately 84% of the hydroxylated products partitioned into the aqueous phase. Subsequent quantitation of the enzymatic activity required only liquid scintillation counting of the aqueous phase. Hepatic ho-mogenates from male rats treated with 0.01, 0.05, 0.125, and 0.25% clofibric acid in the diet for 7 days had enzyme levels 1.3, 6.1, 11.1, or 15.9 times control values, respectively, when assayed by a conventional TLC method, and 1.3, 5.3, 12.3, or 15.3 times control values when assayed by the solvent partition method. The data indicate that the partition method demonstrates comparable sensitivity and better precision and linearity than a more conventional TLC method when induction of hepatic microsomal enzymes in rats is studied.