Novel oxysterols observed in tissues and fluids of AY9944-treated rats: a model for Smith-Lemli-Opitz syndrome

Abstract

Treatment of Sprague-Dawley rats with AY9944, an inhibitor of 3β-hydroxysterol-Δ(7)-reductase (Dhcr7), leads to elevated levels of 7-dehydrocholesterol (7-DHC) and reduced levels of cholesterol in all biological tissues, mimicking the key biochemical hallmark of Smith-Lemli-Opitz syndrome (SLOS). Fourteen 7-DHC-derived oxysterols previously have been identified as products of free radical oxidation in vitro; one of these oxysterols, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), was recently identified in Dhcr7-deficient cells and in brain tissues of Dhcr7-null mouse. We report here the isolation and characterization of three novel 7-DHC-derived oxysterols (4α- and 4β-hydroxy-7-DHC and 24-hydroxy-7-DHC) in addition to DHCEO and 7-ketocholesterol (7-kChol) from the brain tissues of AY9944-treated rats. The identities of these five oxysterols were elucidated by HPLC-ultraviolet (UV), HPLC-MS, and 1D- and 2D-NMR. Quantification of 4α- and 4β-hydroxy-7-DHC, DHCEO, and 7-kChol in rat brain, liver, and serum were carried out by HPLC-MS using d(7)-DHCEO as an internal standard. With the exception of 7-kChol, these oxysterols were present only in tissues of AY9944-treated, but not control rats, and 7-kChol levels were markedly (>10-fold) higher in treated versus control rats. These findings are discussed in the context of the potential involvement of 7-DHC-derived oxysterols in the pathogenesis of SLOS.

Fig.1.

A: NP-HPLC-UV (silica 4.6 mm × 25 cm column; 5 µ; 1.0 ml/min; elution solvent: 10% 2-propanol in hexanes) and (B) RP-HPLC-UV (150 × 2 mm C18 column; 3 µ; 0.2 ml/min; elution solvent: acetonitrile-methanol, 70:30, v/v) analysis of the sterol fraction extracted from AY9944-treated rat brain. Detection wavelengths: solid line, 210 nm; dashed line, 246 nm; dotted line (gray), 281 nm.

Fig.2.

Oxysterols identified in brain tissues from AY9944-treated rats.

Fig.3.

HMBC, COSY, and NOESY correlations of selected oxysterols isolated from brain tissues of AY9944-treated rats.

Fig.4.

Primary products of photooxidation (¹O₂) of 7-DHC (49, 50).

Fig.5.

NP-HPLC-APCI-MS-MS (silica 4.6 mm × 25 cm column; 5 μ; 1.0 ml/min; elution solvent: 10% 2-propanol in hexane) analysis of the oxysterols from brains of 2 month-old (A) control and (B) AY9944-treated rats. New peaks observed in AY9944-treated rats relative to control rats are indicated by asterisks (*).

Fig.6.

NP-HPLC-APCI-MS-MS chromatograms of (A) oxysterols from liver and (B) KOH-hydrolyzed serum from 2 month-old AY9944-treated rats.