Oxysterols from Free Radical Chain Oxidation of 7-Dehydrocholesterol: Product and Mechanistic Studies

Abstract

Free radical chain oxidation of highly oxidizable 7-dehydrocholesterol (7-DHC) initiated by 2,2′-azobis(4-methoxy-2,4-dimethylvaleronitrile) was carried out at 37°C in benzene for 24 hours. Fifteen oxysterols derived from 7-DHC were isolated and characterized with 1D- and 2D-NMR spectroscopy and mass spectrometry. A mechanism that involves abstraction of hydrogen atoms at C-9 and/or C-14 is proposed to account for the formation of all of the oxysterols and the reaction progress profile. In either the H-9 or H-14 mechanism, a pentadienyl radical intermediate is formed after abstraction of H-9 or H-14 by a peroxyl radical. This step is followed by the well-precedented transformations observed in peroxidation reactions of polyunsaturated fatty acids such as oxygen addition, peroxyl radical 5-exo cyclization, and SHi carbon radical attack on the peroxide bond. The mechanism for peroxidation of 7-DHC also accounts for the formation of numerous oxysterol natural products isolated from fungal species, marine sponges, and cactaceous species. In a cell viability test, the oxysterol mixture from 7-DHC peroxidation was found to be cytotoxic to Neuro2a neuroblastoma cells in the micromolar concentration range. We propose that the high reactivity of 7-DHC and the oxysterols generated from its peroxidation may play important roles in the pathogenesis of Smith-Lemli-Opitz syndrome (SLOS), X-linked dominant chondrodysplasia punctata (CDPX2), and cerebrotendinous xanthomatosis (CTX), all of these being metabolic disorders having an elevated level of 7-DHC.

Conversion of 7-Dehydrocholesterol to 7-Ketocholesterol Is Catalyzed by Human Cytochrome P450 7A1 and Occurs by Direct Oxidation without an Epoxide Intermediate

“…A 7-hydroxycholesterol dehydrogenase was purified from hamster liver microsomes, although the enzyme is apparently not present in rats or (most) humans (36). 7-Ketocholesterol was not among the many identified oxysterols in the free radical oxidation of 7-dehydrocholesterol (12). However, in a recent study, 7-ketocholesterol was one of the major oxysterols observed in a rat model for SLOS, in which 7-dehydrocholesterol is the dominant sterol (37).…”

“…Some humans are deficient in 7-dehydrocholesterol reductase (DHCR7) and exhibit Smith-Lemli-Opitz syndrome (SLOS), 2 with elevated levels of 7-dehydrocholesterol and attenuated levels of cholesterol (3,(7)(8)(9)(10). 7-Dehydrocholesterol is exceptionally susceptible to free radical oxidation (11), and more than one dozen biologically active oxysterol products have been characterized from 7-dehydrocholesterol oxidation in solution (12)(13)(14). In particular, we have been interested in the enzymatic conversion of 7-dehydrocholesterol to oxysterols.…”

A koleszterin-bioszintézis veleszületett zavara: a Smith–Lemli–Opitz-szindróma

“…A lipidperoxidáció során számos oxiszterol keletkezik belőle, amelyek feltételez-hetően kóroki szerepet játszanak a szindrómában megfigyelhető központi idegrendszeri tünetek kialakulásában [16,17,18].…”

“…A legújabb kutatások során a 7-DHC bizonyult a legreaktívabb lipidmolekulának szabad gyökökkel szemben, lipidperoxidációs folyamatok során számos oxiszterol keletkezik belőle in vitro és in vivo [16,17], amely terápiás szempontból az antioxidánsok potenciálisan előnyös hatását feltételezi [18]. SLO-szindrómás betegek fi broblastjaiban és DHCR-7-KO egerekben a vizsgált antioxidánsok közül az E-vitamin önmagában hatékonynak bizonyult, gátolta a 7-DHC-ből az oxiszterolok képző-dését és ezen keresztül helyreállította a lipid-bioszintézist érintő génexpressziós változásokat.…”

“…A készítményt az Európai Unió számos országában forgalmazzák. A Debreceni Egyetem SLOmunkacsoportja 2014. május 1-jén kezdeményezett klinikai tanulmányt a fenti vizsgálat és az említett közlések [16,17,18,44] alapján. A bevont betegek koleszterinpótlás mellett kizárólag E-vitamin-cseppeket kapnak.…”