An oxysterol biomarker for 7-dehydrocholesterol oxidation in cell/mouse models for Smith-Lemli-Opitz syndrome

The level of 7-dehydrocholesterol (7-DHC) is elevated in tissues and fluids of Smith-Lemli-Opitz syndrome (SLOS) patients due to defective 7-DHC reductase. Although over a dozen oxysterols have been identified from 7-DHC free radical oxidation in solution, oxysterol profiles in SLOS cells and tissues have never been studied. We report here the identification and complete characterization of a novel oxysterol, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), as a biomarker for 7-DHC oxidation in fibroblasts from SLOS patients and brain tissue from a SLOS mouse model. Deuterated (d₇)-standards of 7-DHC and DHCEO were synthesized from d₇-cholesterol. The presence of DHCEO in SLOS samples was supported by chemical derivatization in the presence of d ₇-DHCEO standard followed by HPLC-MS or GC-MS analysis. Quantification of cholesterol, 7-DHC, and DHCEO was carried out by isotope dilution MS with the d₇-standards. The level of DHCEO was high and correlated well with the level of 7-DHC in all samples examined (R = 0.9851). Based on our in vitro studies in two different cell lines, the mechanism of formation of DHCEO that involves 5α,6α-epoxycholest-7-en-3β-ol, a primary free radical oxidation product of 7-DHC, and 7-cholesten-3β, 5α,6β-triol is proposed. In a preliminary test, a pyrimidinol antioxidant was found to effectively suppress the formation of DHCEO in SLOS fibroblasts.