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The Fate of the 6-Hydrogen of 5-Cholest-7-en-3ß-ol in the Conversion to 7-Dehydrocholesterol by Rat Liver Microsomes

From the ¹ From the Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts, 01545

2-¹⁴C-Mevalonic acid has been incubated with a rat liver homogenate in the presence of the inhibitor trans-1,4-bis(2-chlorobenzylaminoethyl) - cyclohexanedihydrochloride yielding 1,7,15,22,26-¹⁴C₅-cholesta-5,7-dien-3ß-ol which was reduced with lithium and ammonia to ¹⁴C₅-5-cholest-7-en-3ß-ol (⁷-cholestanol). Separation from endogenous cholesterol was achieved by oxidation with chromic acid, and subsequent borohydride reduction of the isolated ⁷-3-ketone.

6ß-³H-5-Cholest-7-en-3ß-ol has been converted in 3% yield to 6-³H-cholesta-5,7-dien-3ß-ol by a rat liver microsomal preparation containing (trans-1,4-bis(2-chlorobenzylaminoethyl)-cyclohexane dihydrochloride.

6-³H-¹⁴C₅-5-Cholest-7-en-3ß-ol has been incubated with a rat liver microsomal preparation, being converted in 5.5% yield into ¹⁴C₅-cholesta-5,7-dien-3ß-ol with loss of tritium. Less than 1% of the liberated tritium was found in isolated NADH, the remainder being isolated as ³H₂O.

In a control experiment, synthetic NADH-4-³H₂ was incubated with a microsomal preparation as before. The resulting NADH, isolated in 30% yield, contained 70% of the ³H (corrected to 100% isolation); the remainder of the radioactivity was isolated as ³H₂O.

Clearly then in the conversion of 5-cholest-7-en-3ß-ol into 7-dehydrocholesterol, the 6-hydrogen is removed as a proton, and is not directly transferred to NAD⁺. Had NADH-³H been formed, not more than a 30% loss of ³H would have been expected during the isolation of the NADH.

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