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

Abstract

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.

Fig. 1.

NP-HPLC-APCI-MS-MS (Silica 4.6 mm × 25 cm column; 5μ; 1.0 ml/min; elution solvent: 10% 2-propanol in hexanes) chromatograms of the oxysterols from (A) control HFs and (B) SLOS HFs after being incubated in DMEM with lipid-deficient serum for 5 days. New peaks in B relative to control are marked with *.

Fig. 2.

NP-HPLC-APCI-MS-MS chromatograms of the oxysterols from (A) WT E20 mouse brain and (B) Dhcr7-KO E20 mouse brain. New peaks in B relative to control are marked with *.

Fig. 3.

Structure of a major oxysterol, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO) and NP-HPLC-MS-MS chromatogram of DHCEO in the presence of d₇-DHCEO standard from SLOS HF after being incubated in DMEM with lipid-deficient serum for 5 days.

Fig. 4.

Derivatization of d₀- or d₇-DHCEO with 1,4-dinitrophenyl hydrazine (DNPH) and the collision-induced dissociation (CID) mass spectrum of the d₀-adduct molecular ion in ESI-MS analysis.

Fig. 5.

RP-HPLC-ESI-MS-MS (150 × 2 mm C18 column; 3μ; 0.2 ml/min; elution solvent: acetonitrile/methanol = 70/30) chromatogram on the DNPH adducts of (A) d₀- and d₇-DHCEO standards, (B) SLOS HF in the presence of internal d₇-DHCEO standard, and (C) E20 Dhcr7-KO mouse brain in the presence of internal d₇-DHCEO standard. The SLOS samples were incubated in DMEM with lipid-deficient serum for 5 days.

Fig. 6.

Proposed formation mechanism of DHCEO.

Fig. 7.

NP-HPLC-APCI-MS-MS chromatograms of oxysterols from control HFs in DMEM media supplemented with 10% FBS (A) under control condition, and (B) after incubating with 5 μM of 5α,6α-epoxycholest-7-en-3β-ol (1) for 24 h. d₇-DHCEO was added when preparing the samples for HPLC-MS analysis to indicate the presence or absence of native d₀-DHCEO and to quantify it.

Fig. 8.

Effect of 10 μM antioxidant 3 on the level of DHCEO in SLOS HFs. **, p < 0.005 relative to SLOS-ctrl.