Highly sensitive quantification of key regulatory oxysterols in biological samples by LC-ESI-MS/MS

Abstract

We describe a highly sensitive and specific method for the quantification of key regulatory oxysterols in biological samples. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After alkaline hydrolysis of human serum (5 ml) or rat liver microsomes (1 mg protein), oxysterols were extracted, derivatized into picolinyl esters, and analyzed by LC-MS/MS using the electrospray ionization mode. The detection limits of the picolinyl esters of 4b-hydroxycholesterol, 7a-hydroxycholesterol, 22R-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and 24S,25-epoxycholesterol were 2-10 fg (5-25 amol) on-column (signal-to-noise ratio 5 3). Reproducibilities and recoveries of these oxysterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.8% to 12.7% and 2.9% to 11.9%, respectively. The recovery experiments were performed using rat liver microsomes spiked with 0.05 ng to 12 ng of oxysterols, and recoveries of the oxysterols ranged from 86.7% to 107.3%, with a mean recovery of 100.6%. This method provides reproducible and reliable results for the quantification of oxysterols in small amounts of biological samples. Supplementary key words liquid chromatography-tandem mass spec-Biological samples contain a large number of oxysterols (1), and most of them are formed from cholesterol by enzymatic oxidation (2 -6) ( Fig. 1) or autoxidation (7). By contrast, the oxysterol 24S,25-epoxycholesterol is not derived from cholesterol but is produced de novo from acetyl-CoA via a shunt in the mevalonate pathway (8).These oxysterols are important molecules for preserving lipid homeostasis in the body. 7a-Hydroxycholesterol is a product of CYP7A1, which is the rate-limiting enzyme in the classic bile acid biosynthetic pathway. 27-Hydroxycholesterol, 24S-hydroxycholesterol, 4b-hydroxycholesterol, 22R-hydroxycholesterol, and 24S,25-epoxycholesterol are effective endogenous ligands of the nuclear receptors liver X receptor a (LXRa) and LXRb (9-11). In addition, 27-hydroxycholesterol (12), 25-hydroxycholesterol (13), and 24S,25-epoxycholesterol (14) are known to downregulate the cholesterol biosynthetic pathway, presumably by blocking the processing of the sterol-regulatory element binding protein.GC-MS has historically been used for the analyses of oxysterols in serum and tissues (1, 15) because the sensitivity and specificity of conventional GC with flame ionization detector is not sufficient to quantify oxysterols in biological samples. However, GC-MS is still not an ideal method, especially for the analysis of 24S,25-epoxycholesterol, because this epoxycholesterol does not survive the temperature required for GC analysis (16). Another approach to quantifying oxysterols in biological samples was HPLC with ultraviolet (UV) detection after derivatization to the D 4 -3-ketones (16-19). Thi...

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