In humans, the brain accounts for about 20% of the body's free cholesterol, most of which is synthesized de novo in brain. To maintain cholesterol balance throughout life, cholesterol becomes metabolized to 24S-hydroxycholesterol, principally in neurons. In mouse, rat, and probably human, metabolism to 24S-hydroxycholesterol accounts for about 50% of cholesterol turnover; however, the route by which the remainder is turned over has yet to be elucidated. Here, we describe a novel liquid chromatography (LC) multistage fragmentation mass spectrometry (MS n ) methodology for the identification, with high sensitivity (low pg), of cholesterol metabolites in rat brain. The methodology includes derivatization to enhance ionization, exact mass analysis at high resolution to identify potential metabolites, and LC-MS n (n53) to allow their characterization. 24S-hydroxycholesterol was confirmed as a major oxysterol in rat brain, and other oxysterols identified for the first time in brain included 24,25-, 24,27-, 25,27-, 6,24,-7a,25-, and 7a,27-dihydroxycholesterols. In addition, 3b-hydroxy-5-oxo-5,6-secocholestan-6-al and its aldol, two molecules linked to amyloidogenesis of proteins, were characterized in rat

There is increasing evidence that modified phospholipid products of low density lipoprotein (LDL) oxidation mediate inflammatory processes within vulnerable atherosclerotic lesions. Lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) is present in vulnerable plaque regions where it acts on phospholipid oxidation products to generate the pro-inflammatory lysophsopholipids and oxidized non-esterified fatty acids. This association together with identification of circulating Lp-PLA 2 levels as an independent predictor of cardiovascular disease provides a rationale for development of Lp-PLA 2 inhibitors as therapy for atherosclerosis. Here we report a systematic analysis of the effects of in vitro oxidation in the absence and presence of an Lp-PLA 2 inhibitor on the phosphatidylcholine (PC) composition of human LDL. Mass spectrometry identifies three classes of PC whose concentration is significantly enhanced during LDL oxidation. Of these, a series of molecules, represented by peaks in the m/z range 594 -666 and identified as truncated PC oxidation products by accurate mass measurements using an LTQ Orbitrap mass spectrometer, are the predominant substrates for Lp-PLA 2 . A second series of oxidation products, represented by peaks in the m/z range 746 -830 and identified by LTQ Orbitrap analysis as non-truncated oxidized PCs, are quantitatively more abundant but are less efficient Lp-PLA 2 substrates. The major PC products of Lp-PLA 2 , saturated and mono-unsaturated lyso-PC, constitute the third class. Mass spectrometric analysis confirms the presence of many of these PCs within human atherosclerotic lesions, suggesting that they could potentially be used as in vivo markers of atherosclerotic disease progression and response to Lp-PLA 2 inhibitor therapy.

While the proteome defines the expressed gene products, the metabolome results from reactions controlled by such gene products. Plasma represents an accessible "window" to the metabolome both in regard of availability and content. The wide range of the plasma metabolome, in terms of molecular diversity and abundance, makes its comprehensive analysis challenging. Here we demonstrate an analytical method designed to target one region of the metabolome i.e. oxysterols. Since the discovery of their biological activity as ligands to nuclear receptors there has been a reawakening of interest in oxysterols and their analysis. In addition, the oxysterols, 24S-and 27-hydroxycholesterol, are currently under investigation as potential biomarkers associated with neurodegenerative disorders such as Alzheimer's disease and multiple sclerosis; widespread analysis of these lipids in clinical studies will require the development of robust, sensitive and rapid analytical techniques. In this communication we present results of an investigation of the oxysterols content of human plasma using a newly developed high-performance liquid chromatography -mass spectrometry (HPLC-MS) method incorporating charge-tagging and highresolution MS. The method has allowed the identification in plasma of monohydroxylated cholesterol molecules, 7α-, 24S-and 27-hydroxycholesterol; the cholestenetriol 7α,27-dihydroxycholesterol; and 3β-hydroxycholest-5-en-27-oic acid and its metabolite and 3β,7α-dihydroxycholest-5-en-27-oic acid. The methodology described is also applicable for the analysis of other sterols in plasma i.e. cholesterol, 7-dehydrocholesterol, and desmosterol, as well as cholesterol 5,6-seco-sterols and steroid hormones. Although involving derivatisation, sample preparation is straight forward and chromatographic analysis rapid (17 min), while the MS method offers high sensitivity (ng/mL of sterol in plasma, or pg on-column) and specificity. The methodology is suitable for targeted metabolomic analysis of sterols, oxysterols and steroid hormones opening a "window" to view this region of the metabolome.