The Oxidation of Cholesterol in Rat Liver Sub‐Cellular Particles

• Published in: European journal of biochemistry Vol. 20; no. 4; pp. 569 - 579
• Main Authors: Mitton, John R., Scholan, Norma A., Boyd, George S.
• Format: Journal Article
• Language: English; Japanese
• Published: Oxford, UK Blackwell Publishing Ltd 01.06.1971
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1971.tb01429.x

Various sub‐cellular fractions of rat liver exert catalytic effects on the oxidation of [4‐14C]‐cholesterol to 7α‐hydroxy‐[4‐14C]cholesterol and to other [4‐14C]cholesterol oxidation products. Some of these oxidation products created in these studies in vitro appear to be aberrant oxidation products in that there is no evidence that they are intermediates in the physiological oxidation of cholesterol. These aberrant oxidation products have been classified as autoxidation products despite the fact that the autoxidation reactions in these cell fractions appear to require the participation of NADPH, native proteins and perhaps certain nucleotides for the production of these compounds. The enzyme system catalysing the oxidation of [4‐14C]cholesterol to 7α‐hydroxy‐[4‐14C]cholesterol could be assayed in a 18000 ×g supernatant fraction of rat liver. This enzymic reaction was adversely affected by extensive homogenisation of liver tissue and by prolonged incubation periods, both of which tended to increase the autoxidation reactions. The cholesterol‐7α‐hydroxylase enzyme showed optimal activity in the presence of NADPH and oxygen. In the washed liver microsomal fraction, [4‐14C]cholesterol was extensively oxidised in the presence of NADPH, ADP and ferrous ions to 7β‐hydroxycholesterol, cholestan‐3β, 5α, 6β‐triol and 7‐ketocholesterol. Further investigation showed that another compound was also formed, having the same mobility as 7‐ketocholesterol in the thin layer solvent systems used. Reduction and further metabolism of this compound compared with reference standards suggested that it may be a cholesterol oxide, formed under conditions where peroxidation of microsomal lipids could occur. A comparison of the effects of Tris and phosphate buffers, and various nucleotides suggests that cholesterol autoxidation in the microsomal fraction may be related to the general lipid peroxidation systems. The boiled 100000 ×g liver supernatant preparation or 2‐mercaptoethylamine both effectively quenched cholesterol peroxidation, at the same time stimulating the cholesterol‐7α‐hydroxylase system. A scheme has been suggested to explain the liver microsomal cholesterol autoxidation or peroxidation and to contrast this process with the mixed function oxidase involved in the enzymic cholesterol‐7α‐hydroxylase reaction.