20a-Hydroxycholesterol

S. Lieberman (1974). Biosynthetic conversion of cholesterol into pregnenolone. Side chain cleavage of some 20-p-tolyl analogs of cholesterol and 20a-hydroxycholesterol. J Biol. Chem. 249,1274-1285. 

The conversion of cholesterol to corticosteroids was further confirmed by in vitro experiments. The first reaction in this biochemical transformation is NADP and ATP dependent. It results in the splitting of the cholesterol side chain to yield pregnenolone and isoca-proic acid (see Fig. 8-11). Pregnenolone is a 21-carbon compound with a jS-carboxyl group in position 3 and an unsaturated bond between carbons 5 and 6. The exact mechanism of the splitting reaction is not clear, although it would appear that 20a-hydroxycholesterol and 22a-hydroxycholesterol can serve as intermediates. Their role has been questioned [192]. 

The most commonly postulated mechanism for the desmolase reaction has been the suggestion of the enzymatic consecutive reaction sequence cholesterol 20a-hydroxycholesterol — (22R)-20a,22-dihydroxycholesterol pregnenolone (Shimizu et al., 1961, 1962 Constantopoulos and Tchen, 1961). This hypothetical scheme was actually advanced on the basis of three suppositions (1) cholesterol is cleaved between C-20 and C-22, as was discussed in a previous section, (2) 20a-hydroxycholesterol was formed from cholesterol-4- C by a cow adrenocortical homogenate in the presence of 20a-hy-droxycholesterol as a trapping agent (Solomon et al., 1956), and (3) both 20a-hydroxycholesterol and (22R)-20a,22-dihydroxycholesterol are much more efficiently transformed to pregnenolone than is cho-... 

The further transformation of 20a-hydroxycholesterol, (22R)-22-hydroxycholesterol, and (22R)-20a,22-dihydroxycholesterol led to pregnenolone, and the two monohydroxycholesterols were transformed to (22R)-20a,22-dihydroxycholesterol. 

The efificiency of various hydroxylated cholesterol derivatives to form pregnenolone is summarized in Table III. At the substrate concentrations studied, first-order reaction kinetics were realized the fractions formed, therefore, represent fractional rates. As may be seen, the (22R)-22-hydroxycholesterol served as a much better precursor of pregnenolone than the 20a-hydroxycholesterol. (It is noteworthy that in Digitalis labeled 22 -hydroxycholesterol or 22-oxocho-lesterol do not appear to be converted to tigogenin or gitogenin... 

Conversion of Cholesterol to 20a-HYDROxYCHOLESTEROL, (22R)-22-Hydroxycholesterol, (22R)-20a, 22-Dihydroxycholesterol and Pregnenolone by Adrenal Acetone-Dried Powders -"... 

It is noteworthy that cholesterol derivatives with a hydroxyl at C-20 appear not to have been isolated from natural sources, with the exception of the studies of K. D. Roberts et al. (1969) who, by double isotope dilution techniques, have isolated approximately a total of 100 /ag/kg of the free and esterified 20a-hydroxycholesterol from bovine adrenal glands. These minute amounts are in line with the extremely small conversions observed by us as arising from cholesterol... 

In the isolation of steroids from natural sources one must, of course, consider their stability prior to enzyme inactivation, which usually occurs at the commencement of the isolation procedure, when the tissue is extracted with organic solvents. Kahnt and Neher (1965) could observe in bovine adrenal homogenates only trace amounts of 20a-hydroxycholesterol and (22R)-20,22-dihydroxy-cholesterol, as well as pregnenolone or progesterone (< figlgm). As stated earlier the absence of certain derivatives may be due to either their lack of formation or their rapid disappearance from the system. Until recently, primarily because of the lack of appropriate substrates of high specific activity, quantitative studies on the elucidation of the mechanism of the desmolase reaction could not be performed. The study of Ichii et al. (1967a) appears to entail the assumption of a steady state, for which no proof seems to have been provided. Also,... 

Typical results of the individual rates obtained with a bovine soluble preparation under conditions of first-order kinetics are summarized in Fig. 1. The rate of formation of 20a-hydroxycholesterol, as pointed out earlier, is probably to be considered maximal. As may be seen, the rate of formation of (22R)-22-hydroxycholesterol from cholesterol... 

Dehydration of 20a-hydroxycholesterol with a catalytic amount of hydrochloric acid in methanol gave 20(22)-dehydrocholesterol. This product is a mixture of the 20(22)-double bond isomers in which the cis isomer preponderates. The identical product was obtained from the reaction of pregnenolone acetate with isohexylidenetriphenyl-phosphorane in dimethyl sulfoxide (Gut, 1970). 

Dehydrocholesterol was tritiated to give cholesterol-1,2- H. 20a-Hydroxycholesterol-16- H was produced from pregnenolone-16- H. 3/3-Hydroxycholest-5-en-22-one-16,17- H, (22R)-22-hydroxycholes-terol-16,17- H, and (22S)-22-hydroxycholesta-5,16,dien-22-one, (22R)-and (22S)-22-hydroxycholesterol-l,2- H were prepared from cholesta-l,4-diene-3,22-dione (Burstein et al., 1970). 

In the course of incubations with 20a-hydroxycholesterol-7-H , -22-C, Shimizu et al. (1961) noted an accumulation in trace quantities of a compound X containing H and C in the same ratio as the starting material. It was isolated and partially identified as a metabolite... 

The enzymic activity for the conversion of 20a-hydroxycholesterol to pregnenolone is present in rat testis, bovine ovaries, and human term placenta, but not in liver (Shimizu et al., 1961). Liver in contrast has the capacity for the oxidative removal of the three terminal carbon atoms of the cholesterol side chain, an ability not shared by the adrenal. 

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