Simvastatin reduces circulating oxysterol levels in men with hypercholesterolaemia

Abstract

Oxysterols (OHC) are biologically active cholesterol metabolites circulating in plasma that may be formed enzymatically (e.g. 24S-OHC, 25-OHC and 27-OHC) or by autoxidative mechanisms (e.g. 7-ketocholesterol, 7β-OHC and 25-OHC). Oxysterols are more soluble than cholesterol and are reported to exert inflammatory, cytoprotective and apoptotic effects according to concentration and species. Esterified oxysterols have been analysed in people with dementia and cardiovascular diseases although there is no consistent relationship between oxysterol esters and disease. However, oxysterol esters are held in lipoprotein core and may not relate to the concentration and activity of plasma free oxysterols. Methodological limitations have challenged the analysis of free oxysterols to date. We have developed a fast, sensitive and specific quantitative LC-MS/MS, multiple reaction monitoring (MRM) method to target five oxysterols in human plasma with analyte recoveries between 72% and 82% and sensitivities between 5 and 135 pg/ml. A novel method was used to investigate the hypothesis that simvastatin may reduce the concentrations of specific plasma free oxysterols in hypercholesterolaemia. Twenty healthy male volunteers were recruited (aged 41-63 years); ten were asymptomatic with high plasma cholesterol > 6.5 mM and ten were healthy with normal plasma cholesterol (< 6.5 mM). Simvastatin (40 mg/day) was prescribed to those with hypercholesterolaemia. Plasma samples were taken from both groups at baseline and after three months. Simvastatin reduced plasma cholesterol by ~35% (p < 0.05) at the end of three months. Oxysterols generated by autoxidation (but not enzymatically) were elevated up to 45 fold in hypercholesterolaemic midlife men. Plasma oxysterols were restored to those of healthy controls after simvastatin intervention suggesting that autoxidation is either prevented by simvastatin directly or that autoxidation is less prevalent when plasma cholesterol concentrations are within the normal range.

Keywords: Dementia; Hydroxycholesterol; Mass spectrometry; Statin; Vascular disease.

Fig. 1

Chromatographic separation of oxysterols and cholesterol mixture (5 ng) in a 48 min run time. 24-hydroxycholesterol (385/161: RT = 11.43 min); 25-hydroxycholesterol (385/147: RT = 11.93 min); 27-hydroxycholesterol (385/161: RT = 12.88 min); 7β-hydroxycholesterol (385/81: RT = 13.80 min); 7keto-cholesterol (401/196: RT = 14.79 min); cholesterol (369/81: RT = 19.87 min).

Fig. 2

Free oxysterol concentrations adjusted to free cholesterol in plasma from hypercholesterolaemic patients before and after Simvastatin intervention (n = 10 in each group). Box plots show the plasma oxysterols; (A) 24S-OHC (B) 25-OHC (C) 27-OHC (D) 7β-OHC (E) 7-KC. Data was analysed by Wilcoxon matched pair t-test.