22-23-dihydroergosterol and 7-dehydrocholesterol

Cytochrome P450scc (P450scc) catalyzes the cleavage of the side chain of both cholesterol and the vitamin D(3) precursor, 7-dehydrocholesterol. The aim of this study was to test the ability of human P450scc to metabolize ergosterol, the vitamin D(2) precursor, and define the structure of the major products. P450scc incorporated into the bilayer of phospholipid vesicles converted ergosterol to two major and four minor products with a k(cat) of 53 mol · min(-1) · mol P450scc(-1) and a K(m) of 0.18 mol ergosterol/mol phospholipid, similar to the values observed for cholesterol metabolism. The reaction of ergosterol with P450scc was scaled up to make enough of the two major products for structural analysis. From mass spectrometry, NMR, and comparison of the NMR data to that for similar molecules, we determined the structures of the two major products as 20-hydroxy-22,23-epoxy-22,23-dihydroergosterol and 22-keto-23-hydroxy-22,23-dihydroergosterol. Molecular modeling and nuclear Overhauser effect (or enhancement) spectroscopy spectra analysis helped to establish the configurations at C20, C22, and C23 and determine the final structures of major products as 22R,23S-epoxyergosta-5,7-diene-3β,20α-diol and 3β,23S-dihydroxyergosta-5,7-dien-22-one. It is likely that the formation of the second product is through a 22,23-epoxy (oxirane) intermediate followed by C22 hydroxylation with the formation of strained 22-hydroxy-22,23-epoxide (oxiranol), which is immediately transformed to the more stable α-hydroxyketone. Molecular modeling of ergosterol into the P450scc crystal structure positioned the ergosterol side chain consistent with formation of the above products. Thus, we have shown that P450scc efficiently catalyzes epoxide formation with ergosterol giving rise to novel epoxy, hydroxy, and keto derivatives, without causing cleavage of the side chain.

Topics: Cholesterol Side-Chain Cleavage Enzyme; Dehydrocholesterols; Epoxy Compounds; Ergocalciferols; Ergosterol; Ethylene Oxide; Humans; Hydroxylation; Kinetics; Magnetic Resonance Spectroscopy; Mass Spectrometry; Phospholipids

We have determined the effect of the side chain on the uptake of sterols from micellar solutions by isolated rat jejunal villus cells, brush border membranes, and erythrocytes. From an equimolar mixture of 7-dehydrosterols, the uptake decreased with an increasing number of carbon atoms at C24 of the sterol side chain in a manner identical to that observed for the parent-delta 5-sterols. The brush border and erythrocyte membranes were found to absorb 4-5 times more 7-dehydrocholesterol than 7-dehydro-beta-sitosterol over a 60-min period of incubation. A somewhat lower specificity of sterol uptake by the villus cells was attributed to the exposure of large areas of the basolateral membrane, which apparently was less able to discriminate between sterols. The higher sterol selectivity was associated with higher membrane organization anticipated from the higher free cholesterol/phospholipid and protein/phospholipid ratios of the brush border and red blood cell membrane. On a mass basis the villus cells and brush borders absorbed 30-60 times more sterol than the erythrocytes. Assuming that the delta 5,7-sterols accurately represent the absorption of the delta 5 parent sterols, it is suggested that the 3- to 5-fold excess of absorbed cholesterol over beta-sitosterol that is typically found in the jejunal wall of the rat following feeding of radioactive sterols arises from an inability of beta-sitosterol to enter the brush border membrane as easily as cholesterol.

Topics: Animals; Cell Fractionation; Cholestadienols; Dehydrocholesterols; Ergosterol; Erythrocytes; In Vitro Techniques; Intestinal Absorption; Jejunum; Male; Microvilli; Rats; Rats, Inbred Strains; Sitosterols

To determine the role of the ring structure in the differential absorption of sterols, we have used rat jejunal brush border vesicles and erythrocytes to examine the uptake of cholesterol, campesterol, and sitosterol following successive chemical degradations of rings A and B. The cell and membrane preparations were incubated with the sterols and sterol analogues (about 30 micromolar each) dissolved in 7 mM sodium taurocholate and 0.6 mM egg phospholipid. The uptake of the analogues was analyzed by high performance liquid chromatography and capillary gas--liquid chromatography. In both membrane preparations, the uptake of the 7-dehydroanalogues of cholesterol, campesterol, and sitosterol was linear with time. 7-Dehydrocholesterol was absorbed 4-5 times faster than 7-dehydrositosterol by both preparations. The uptake of the campesterol analogue was intermediate between that of the analogues of cholesterol and sitosterol at all time points. Following conversion of the 7-dehydrosterols to their calciferol derivatives, the 27-carbon sterols were absorbed only 1.9 and 1.4 times faster than those of the 29-carbon sterols by the erythrocyte and brush border membranes, respectively. A similar degree of selectivity was expressed in the erythrocytes during the uptake of a steroid series possessing keto-4-ene ring system. Complete oxidation of the calciferol derivatives to the des-AB-8-ones resulted in a total loss of discrimination among the various side-chain homologues during absorption from micellar solutions. It is concluded that the selective absorption of animal and plant sterols depends upon the presence of a ring system having the bulk of the cholestane nucleus, although not necessarily a rigid or planar one containing a hydroxyl group.

Topics: Absorption; Animals; Cell Membrane; Cholesterol; Chromatography, High Pressure Liquid; Dehydrocholesterols; Ergocalciferols; Ergosterol; Erythrocytes; Gas Chromatography-Mass Spectrometry; Jejunum; Micelles; Microvilli; Phytosterols; Rats; Sitosterols; Structure-Activity Relationship