A novel technique for assay of side-chain cleavage of exogenous and endogenous cholesterol in adrenal mitochondrial and submitochondrial preparations

Abstract

A mass fragmentographic technique for assay of side-chain cleavage of endogenous and exogenous cholesterol in adrenal mitochondria and in reconstituted systems containing cytochrome P-450 has been developed. Part of a chloroform extract of an incubation of [4- 14C]cholesterol with the adrenal preparation is subjected to thin-layer radiochromatography in order to determine the conversion of the exogenous cholesterol. Another part of the extract is converted into trimethylsilyl ether and subjected to combined gas chromatography-mass spectrometry. The ions at m e 388, 390, 368, and 370 are followed throughout the gas chromatography with a multiple ion detector. The ions at m e 388 and 390 correspond to the molecular peak in the mass spectrum of trimethylsilyl ether of unlabeled and 4- 14C-labeled pregnenolone, respectively. The ions at m e 368 and 370 correspond to the M-90 peak in the mass spectrum of trimethylsilyl ether of unlabeled and 4- 14C-labeled cholesterol, respectively. The ratio between labeled and unlabeled pregnenolone could be calculated from the tracings at m e 390 and 388. The ratio between labeled and unlabeled cholesterol could be calculated from the tracings at m e 370 and 368. From these ratios and from the percentage of conversion of the fixed amount of [4- 14C]cholesterol into [4- 14C]pregnenolone, the amount of exogenous and endogenous cholesterol as well as the amount of pregnenolone derived from these two pools of cholesterol could be calculated. It was shown that under the experimental conditions employed, the amount of endogenous pregnenolone present in the mitochondrial or submitochondrial preparation prior to incubation could be neglected. The effect of time and of enzyme and substrate concentration on side-chain cleavage of cholesterol by adrenal mitochondria and by partially purified cytochrome P-450 together with NADPH-cytochrome P-450 reductase were studied. Under most of the conditions used, the ratio between the amount of pregnenolone formed from exogenous and endogenous cholesterol was about constant.