Smith-Lemli-Opitz syndrome (SLOS) is a metabolic and developmental disorder caused by mutations in the gene encoding the enzyme 7-dehydrocholesterol reductase (Dhcr7). This reductase catalyzes the last step in cholesterol biosynthesis, and levels of 7-dehydrocholesterol (7-DHC), the substrate for this enzyme, are elevated in SLOS patients as a result of this defect. Our group has previously shown that 7-DHC is extremely prone to free radical autoxidation, and we identified about a dozen different oxysterols formed from oxidation of 7-DHC. We report here that 7-DHC-derived oxysterols reduce cell viability in a dose- and time-dependent manner, some of the compounds showing activity at sub-micromolar concentrations. The reduction of cell survival is caused by a combination of reduced proliferation and induced differentiation of the Neuro2a cells. The complex 7-DHC oxysterol mixture added to control Neuro2a cells also triggers the gene expression changes that were previously identified in Dhcr7-deficient Neuro2a cells. Based on the identification of overlapping gene expression changes in Dhcr7-deficient and 7-DHC oxysterol-treated Neuro2a cells, we hypothesize that some of the pathophysiological findings in the mouse SLOS model and SLOS patients might be due to accumulated 7-DHC oxysterols.

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 d₇-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.

HNE (4-hydroxynonenal), a byproduct of lipid peroxidation, reacts with nucleophilic centers on proteins. A terminal alkynyl analog of HNE (alkynyl HNE, aHNE) serves as a surrogate for HNE itself, both compounds reacting with protein amine and thiol functional groups by similar chemistry. Proteins modified with aHNE undergo reaction with a click reagent that bears azido and biotin groups separated by a photocleavable linker. Peptides and proteins modified in this way are affinity purified on streptavidin beads. Photolysis of the beads with a low intensity UV light releases bound biotinylated proteins or peptides, i.e. proteins or peptides modified by aHNE. Two strategies, (a) protein catch and photorelease and (b) peptide catch and photorelease, are employed to enrich adducted proteins or peptide mixtures highly enriched in adducts. Proteomics analysis of the streptavidin-purified peptides by LC-MS/MS permits identification of the adduction site. Identification of 30 separate peptides from human serum albumin by peptide catch and photorelease reveals 18 different aHNE adduction sites on the protein. Protein catch and photorelease shows that both HSA and ApoA1 in human plasma undergo significant modification by aHNE.