The dehydrogenation of cholest‐7‐en‐3β‐ol to cholesta‐5,7‐dien‐3β‐ol by rat liver microsomal acetone powder has been known to involve the abstraction of 5α and 6α hydrogens, which were found in the water, of the medium. It was proposed by other investigators that an enzyme‐oxygen‐metal complex may be involved in this dehydrogenation. This could then result in the reduction of the molecular oxygen to hydrogen peroxide. We have evaluated the possibility of the formation of hydrogen peroxide in the course of the transformation of 5α‐[3α‐3H]cholest‐7‐en‐3β‐ol to [3α‐3H]cholesta‐5,7‐dien‐3β‐ol by a rat liver microsomal acetone powder in the presence of NAD+ and O2. Hydrogen peroxide was not detected in the reaction mixture even though the sensitivity of the H2O2 detection method exceeded four times the calculated amount of hydrogen peroxide which could be produced in this reaction. We have determined that the reaction is mitigated by a metal‐dependent enzyme complex and the process requires the presence of EDTA. The involvement of a metal‐enzyme complex is supported by the. observation that the desaturation is inhibited by 2,2′‐dipyridyl, 1,10‐phenanthroline and diethyldithiocarbamate. The reaction was also inhibited by horse radish peroxidase which suggested the participation of an enzyme‐metal‐oxygen complex. It was found that NADH promoted the dehydrogenation better than NAD+. Attempts to dehydrogenate enzymatically 5α‐[3α‐3H]cholestan‐3 β‐ol failed. This tends to indicate the specificity of the enzyme system for 5α‐cholest‐7‐en‐3β‐ol. The free radical inhibitors diphenylamine, N‐methylaniline and p‐cresol exhibited a stimulatory effect on the reaction. Based on these results, a tentative mechanism for the dehydrogenation of cholest‐7‐en‐3β‐ol is proposed.
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Reddy et al. (1976) studied this question.
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