A comparative study of two procedures used in the determination of hepatic microsomal aniline hydroxylation

Abstract

The hepatic microsomal parahydroxylation of aniline is measured by the determination of its product p-aminophenol (pAP). The pAP formed in incubation mixtures is assayed either after ether extraction or after trichloroacetic acid (TCA) precipitation. The TCA-precipitation method is simpler and less time-consuming than the ether-extraction method, but it has been found that the apparent recovery of pAP was lower in TCA supernatants than with the ether-extraction method. In the present work, it was found that the recovery of pAP by the TCA-precipitation method depended on the nature of NADPH-generating system used in the incubation mixture. When “soluble fraction” was used as a source of glucose-6-phosphate dehydrogenase for the reduction of NADP to NADPH, the recovery of pAP using the TCA-precipitation method was less than that with the ether-extraction method. But when “soluble fraction” was replaced by yeast glucose-6-phosphate dehydrogenase or by chemically prepared NADPH, the recovery of pAP was approximately equal with both the methods. Lowered recovery of pAP when added to the soluble fraction or its dialyzate might be due to the presence of sulfhydryl reacting groups. The addition of mercuric chloride to the soluble fraction dialyzate resulted in almost “full” recovery of pAP by the TCA-precipitation method, i.e., the apparent loss of pAP was prevented.