The Oxidation of Cholesterol in Rat Liver Sub‐Cellular Particles

Abstract

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.

Studies on Rat‐Liver Microsomal Cholesterol 7α‐Hydroxylase

“…As has been shown in an earlier paper, cholesterol incubated in the presence of liver microsomes, NADPH and oxygen tends to give rise to a wide variety of oxidation products, which do not occur normally in W;vo [3]. To circumvent the difficulties raised by these "autoxidation" products, all incubations were conducted in the presence of IOmM cysteamine which inhibits their formation and allows the 7%-hydroxylation of cholesterol to proceed without the complications of the coexistence of other oxidation products.…”

“…Incubations were buffered with 2 ml 0.1 M phosphate buffer pH 7.4 (NaH,PO,/Na,HPO,). 10 mM cysteamine was included in the incubation mixture to prevent the formation of autoxidation products of cholesterol [3].…”

“…This reaction was first shown to occur in the 1800Oxg supernatant fraction of rat liver [Z], and has since been shown to be associated with the microsomal fraction [3]. The reaction requires the presence of NADPH and oxygen and therefore falls into the "mixed function oxidation" class of reactions [4].…”

The effect of portacaval transposition on hepatic cholesterol‐7α‐hydroxylase activity in the rat

“…The animals were anaesthetised with ether and a blood sample taken from the heart. The liver was rapidly removed, weighed and homogenized in 0.154 molar KC1 (20% w/v) and microsomes prepared in the usual way [5].…”

5α,6α‐Epoxy‐cholestan‐3β‐ol (cholesterol α‐oxide): A specific substrate for rat liver glutathione transferase B

“…Almost all studies of GSH conjugation by these enzymes have been concerned with exogeneous compounds, but the recent discovery of the structure of leukotrienes [4,5] shows that GSH conjugation of reactive endogenous metabolites also occurs. Another possible endogenous reactant is 5cu,&-epoxy-cholestan-3&01 (cholesterol a-oxide), a possible carcinogen [6,7] which is formed during microsomal lipid peroxidation [8]. This is converted to the GSH conjugate by the soluble supernatant of rat liver homogenate [9].…”