Free Oxysterols Research Articles

Association of circulating free and total oxysterols in breast cancer patients.

Oxysterols, a family of oxidized cholesterol derivates, are of increasing interest due to their role in cancer development and progression. Some oxysterols areestrogen receptor modulators and thus of particular interest in breast cancer research. In human studies, two forms of circulating oxysterols are commonly evaluated: "free" (unesterified) and "total" (esterified and unesterified). However, associations between free and total oxysterols arenot well established. We addressed this knowledge gapin a pilot study by evaluating correlations between the free and the total form of each of the circulating oxysterols (free vs. total), and pairwise associations within the panel oftotal oxysterols (total vs. total) and the panel of free oxysterols (free vs. free). Concentrations of oxysterols and other non-cholesterol sterols were quantified in blood samples of 27 breast cancer patients from the MARIE breast cancer patient cohort using liquid chromatography mass spectrometry. We used Spearman rank correlations to assess associations. Overall, 12 oxysterols (including 27-hydroxycholesterol (HC), 25-HC, 24S-HC, 7a-HC, 5a6a-epoxycholesterol) and five sterols (including lanosterol and desmosterol) were analyzed. Strong correlations (r≥0.82) were observed for seven circulating free and total oxysterols/sterols. The free and total form of 27-HC (r=0.63), 25-HC (r=0.54), and two more oxysterols were weaker correlated. Correlation patterns in the panel of total oxysterols/sterols and the panel of free oxysterols/sterols were similar. These findings demonstrate that concentrations of most free and total oxysterols/sterols are strongly correlated. We provide further insight into the interrelationships between oxysterols in breast cancer patients.

Effective sample preparation procedure for the analysis of free neutral steroids, free steroid acids and sterol sulfates in different tissues by GC–MS

Steroids play an important role in cell regulation and homeostasis. Many diseases like Alzheimer’s disease or Smith-Lemli-Opitz syndrome are known to be associated with deviations in the steroid profile. Most published methods only allow the analysis of small subgroups of steroids and cannot give an overview of the total steroid profile. We developed and validated a method that allows the analysis of free neutral steroids, including intermediates of cholesterol biosynthesis, free oxysterols, C19 and C21 steroids, free steroid acids, including bile acids, and sterol sulfates using gas chromatography-mass spectrometry. Samples were analyzed in scan mode for screening purposes and in dynamic multiple reaction monitoring mode for highly sensitive quantitative analysis. The method was validated for mouse brain and liver tissue and consists of sample homogenization, lipid extraction, steroid group separation, deconjugation, derivatization and gas chromatography-mass spectrometry analysis. We applied the method on brain and liver samples of mice (10 months and 3 weeks old) and cultured N2a cells and report the endogenous concentrations of 29 physiological steroids.

Elevated oxysterol levels in human and mouse livers reflect nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH), a primary cause of liver disease, leads to complications such as fibrosis, cirrhosis, and carcinoma, but the pathophysiology of NASH is incompletely understood. Epstein-Barr virus-induced G protein-coupled receptor 2 (EBI2) and its oxysterol ligand 7α,25-dihydroxycholesterol (7α,25-diHC) are recently discovered immune regulators. Several lines of evidence suggest a role of oxysterols in NASH pathogenesis, but rigorous testing has not been performed. We measured oxysterol levels in the livers of NASH patients by LC-MS and tested the role of the EBI2-7α,25-diHC system in a murine feeding model of NASH. Free oxysterol profiling in livers from NASH patients revealed a pronounced increase in 24- and 7-hydroxylated oxysterols in NASH compared with controls. Levels of 24- and 7-hydroxylated oxysterols correlated with histological NASH activity. Histological analysis of murine liver samples demonstrated ballooning and liver inflammation. No significant genotype-related differences were observed in Ebi2−/− mice and mice with defects in the 7α,25-diHC synthesizing enzymes CH25H and CYP7B1 compared with wild-type littermate controls, arguing against an essential role of these genes in NASH pathogenesis. Elevated 24- and 7-hydroxylated oxysterol levels were confirmed in murine NASH liver samples. Our results suggest increased bile acid synthesis in NASH samples, as judged by the enhanced level of 7α-hydroxycholest-4-en-3-one and impaired 24S-hydroxycholesterol metabolism as characteristic biochemical changes in livers affected by NASH.

Fast liquid chromatography-mass spectrometry reveals side chain oxysterol heterogeneity in breast cancer tumour samples

Oxysterols can contribute to proliferation of breast cancer through activation of the Estrogen Receptors, and to metastasis through activation of the Liver X Receptors. Endogenous levels of both esterified and free sidechain-hydroxylated oxysterols were examined in breast cancer tumours from Estrogen Receptor positive and negative breast tumours, using a novel fast liquid chromatography tandem mass spectrometry method. Multiple aliquots of five milligram samples of 22 tumours were analysed for oxysterol content to assess intra- and inter-tumour variation. Derivatization was performed with Girard T reagent (with and without alkaline hydrolysis) and sample clean-up was performed using a robust automatic on-line column switching system (“AFFL”). Oxysterols were separated isocratically on a 2.1 mm inner diameter column packed with ACE SuperPhenylHexyl core shell particles using a mobile phase consisting of 0.1% formic acid in H2O/methanol/acetonitrile (57/10/33, v/v/v) followed by a wash out step (0.1% formic acid in methanol/acetonitrile, 50/50, v/v). The total analysis time, including sample clean-up and column reconditioning, was 8 min (80% time reduction compared to other on-line systems). Analysis revealed large intra-tumour variations of sidechain oxysterols, resulting in no significant differences in endogenous oxysterols levels between Estrogen Receptor positive and Estrogen Receptor negative breast cancers. However, a correlation between esterified and free 27-hydroxycholesterol was observed. The same correlation was not observed for 24S-hydroxycholesterol or 25-hydroxycholesterol. The oxysterol heterogeneity of tumour tissue is a critical factor when assessing the role of these lipids in cancer.

Frontiers of Complex Disease Mechanisms: Membrane Surface Tension May Link Genotype to Phenotype in Glaucoma.

Although many monogenic diseases are understood based upon structural changes of gene products, less progress has been made concerning polygenic disease mechanisms. This article presents a new interdisciplinary approach to understand complex diseases, especially their genetic polymorphisms. I focus upon primary open angle glaucoma (POAG). Although elevated intraocular pressure (IOP) and oxidative stress are glaucoma hallmarks, the linkages between these factors and cell death are obscure. Reactive oxygen species (ROS) promote the formation of oxidatively truncated phosphoglycerides (OTP), free fatty acids, lysophosphoglycerides, oxysterols, and other chemical species that promote membrane disruption and decrease membrane surface tension. Several POAG-linked gene polymorphisms identify proteins that manage damaged lipids and/or influence membrane surface tension. POAG-related genes expected to participate in these processes include: ELOVL5, ABCA1, APOE4, GST, CYP46A1, MYOC, and CAV. POAG-related gene products are expected to influence membrane surface tension, strength, and repair. I propose that heightened IOP overcomes retinal ganglion cell (RGC) membrane compressive strength, weakened by damaged lipid accumulation, to form pores. The ensuing structural failure promotes apoptosis and blindness. The linkage between glaucoma genotype and phenotype is mediated by physical events. Force balancing between the IOP and compressive strength regulates pore nucleation; force balancing between pore line tension and membrane surface tension regulates pore growth. Similar events may contribute to traumatic brain injury, Alzheimer's disease, and macular degeneration.

Free oxysterols and bile acids including conjugates - Simultaneous quantification in human plasma and cerebrospinal fluid by liquid chromatography-tandem mass spectrometry

A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI(+)-MS/MS) assay was developed and qualified for analyzing 35 analytes of the cholesterol metabolism, including free cholesterol, 17 free, non-esterified oxysterols and 17 free and conjugated bile acids in plasma and cerebrospinal fluid. As internal standards, 25 commercially available stable deuterium-labeled analogs of the analytes were used. Pre-analytical investigations included stability tests of analyte concentrations affected by different anticoagulation additives: lithium heparin-, citrate-, EDTA-K3-stabilized plasma and serum, and the stability in EDTA whole blood at RT. This LC-ESI(+)-MS/MS method was successfully applied for the analysis of paired serum/cerebrospinal fluid samples of patients with and without blood-brain barrier disturbance, as well as of 100 plasma samples of a LIFE-Adult study sub-cohort.A fast and simple sample preparation including protein precipitation and on-line solid-phase extraction was developed. As little as 55 μL of human plasma/serum or cerebrospinal fluid were needed for the analysis. It was possible to separate isomeric oxysterols and bile acids within 23 min using a C18 core-shell column. The assay is capable of quantifying in a linear range of 0.8–250 ng mL−1 for free hydroxycholesterols, 0.2–10 ng mL−1 for dihydroxycholesterols, 0.2–500 ng mL−1 for bile acids and 16–2000 μg mL−1 for cholesterol with acceptable accuracy and precision.In cerebrospinal fluid one free oxysterols, five free and five conjugated bile acids could be quantified. No significant differences between patients with and without blood-brain barrier disturbance were obtained.In the LIFE-Adult sub-cohort two free oxysterols, four free and seven conjugated bile acids could be quantified in EDTA plasma. Men showed significantly higher concentrations of 26-OHC than women (p = 0.035). Furthermore, in women lower levels of cholic acid, glycocholic acid, glycodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, glycoursodeoxycholic acid, glycolithocholic acid and higher levels of taurocholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid/hyodeoxycholic acid were quantified.

Simvastatin reduces circulating oxysterol levels in men with hypercholesterolaemia

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.

New method to determine free sterols/oxysterols in food matrices using gas chromatography and ion trap mass spectrometry (GC–IT-MS)

Sterols/oxysterols in food may be free or bound i.e. esterified with fatty acids. Methods commonly applied to determine those compounds in such matrices (based on various analytical techniques) usually start with hydrolysis of the food lipid fraction, which means that the results are no good indication of concentration of free sterols/oxysterols only. But only free oxysterols are proatherogenic factors, bound ones are not. There are some published methods selectively sensitive to free oxysterols only, but they are capable to determine only a few compounds and feature very low recovery rates.The aim of this work was to develop a method to determine various free (non-esterified) sterols/oxysterols in various food matrices. The developed method is based on the GC–IT-MS technique used in the chemical ionization mode. It was applied to determine 16 different free sterols/oxysterols in egg powder, cheese, butter, milk and salami. Fat extracted from the given matrix is purified on a specially prepared silica-gel bed to separate the sterol fraction from the oxysterol one. Sterols are silylated using N,O-bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane BSTFA:TMCS, then GC–IT-MS analysed. The method features high recovery rates (75–95%), high reproducibility (RSD<20%), and sensitivity within the 0.01–0.3mg 100g−1 range, depending on the analysed compound.The method is ideally suited for determination of free sterols/oxysterols. Besides, should total concentration of both free and bound forms be of interest, food lipids may be transesterificated before the silica-gel bed purification step.

PP66 - 7-keto-cholesterol: a free radical-derived oxysterol of human adipose tissue with anti-adipogenic activity

Aims Oxidative stress of adipose tissue (AT) emerges as an instigator of metabolic dysfunctions in obese insulin-resistant patients. Here we test the hypothesis whether free radical-derived cholesterol oxidation products (i.e., oxysterols) are: 1) generated in the dysfunctional “diabetic” AT, 2) secreted in AT interstitial fluid, 3) and active as modulators of AT stem cell (ASC) adipogenic differentiation. 7-ketocholesterol (7k-C) and 7β-hydroxycholesterol (7β OH-C) were assessed in AT microdialysis samples of healthy subjects as well as in subcutaneous abdominal AT of type 2 diabetic obese patients. Adipogenic differentiation and “canonical” Wnt and MAPK signaling were investigated in primary cultures of human ASCs treated with 7k-C or with the autoxidation-type II oxysterol 5,6-secosterol (5,6-S) for comparison. Results 7k-C and 7β OH-C were detected in human AT interstitial fluid (0.27±0.03 and 0.91±0.13 μM, respectively). When compared with non-diabetic individuals, diabetic obese patients showed increased levels of 7k-C and 7β OH-C, as well as of 4-HNE-protein adducts, in subcutaneous AT. Challenging ASCs with 7k-C at levels resembling those found in vivo (1 μM), resulted in a lowered adipogenic differentiation by the transient increase of cellular ROS and sequential activation of Wnt/β-catenin, ERK1/2, p38-MAPK and JNK signaling. Signaling data differentiated the anti-adipogenic response of 7k-C and 5,6-S. Innovation and Conclusion Free radical-derived oxysterols were identified in the human AT secretome and may act as novel adipokines with regulatory effects on ASC differentiation. Pathogenic effects of these molecules and particularly of 7k-C in the impaired de novo adipogenesis of insulin-resistant patients is worth of further investigation.

Mitotane Inhibits Sterol-O-Acyl Transferase 1 Triggering Lipid-Mediated Endoplasmic Reticulum Stress and Apoptosis in Adrenocortical Carcinoma Cells.

Adrenocortical carcinoma (ACC) is a rare malignancy that harbors a dismal prognosis in advanced stages. Mitotane is approved as an orphan drug for treatment of ACC and counteracts tumor growth and steroid hormone production. Despite serious adverse effects, mitotane has been clinically used for decades. Elucidation of its unknown molecular mechanism of action seems essential to develop better ACC therapies. Here, we set out to identify the molecular target of mitotane and altered downstream mechanisms by combining expression genomics and mass spectrometry technology in the NCI-H295 ACC model cell line. Pathway analyses of expression genomics data demonstrated activation of endoplasmic reticulum (ER) stress and profound alteration of lipid-related genes caused by mitotane treatment. ER stress marker CHOP was strongly induced and the two upstream ER stress signalling events XBP1-mRNA splicing and eukaryotic initiation factor 2 A (eIF2α) phosphorylation were activated by mitotane in NCI-H295 cells but to a much lesser extent in four nonsteroidogenic cell lines. Lipid mass spectrometry revealed mitotane-induced increase of free cholesterol, oxysterols, and fatty acids specifically in NCI-H295 cells as cause of ER stress. We demonstrate that mitotane is an inhibitor of sterol-O-acyl-transferase 1 (SOAT1) leading to accumulation of these toxic lipids. In ACC tissue samples we show variable SOAT1 expression correlating with the response to mitotane treatment. In conclusion, mitotane confers adrenal-specific cytotoxicity and down-regulates steroidogenesis by inhibition of SOAT1 leading to lipid-induced ER stress. Targeting of cancer-specific lipid metabolism opens new avenues for treatment of ACC and potentially other types of cancer.

OP2-6 - Oxysterols induce mitochondrial impairment and hepatocellular toxicity in non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is a chronic hepatic disorder affecting up to 25% of the general population. Several intracellular events leading to NAFLD and progression to non-alcoholic steatohepatitis (NASH) have been identified, including lipid accumulation, mitochondrial dysfunction and oxidative stress. Emerging evidence links both hepatic free fatty acids (FFAs) and cholesterol (FC) accumulation in NAFLD development; in particular oxysterols, the oxidative products of cholesterol, may contribute to liver injury. We performed a targeted lipidomic analysis of oxysterols in the liver of male Wistar rats fed a high-fat (HF), high-cholesterol (HC) or high-fat/high-cholesterol (HF/HC) diet. Both HF and HC diets caused liver steatosis, but the HF/HC diet resulted in steatohepatitis with associated mitochondrial dysfunction. Above all, the oxysterol cholestane-3beta,5alpha,6beta-triol (triol) was particularly increased in the liver of rats fed diets rich in cholesterol. To verify the molecular mechanism involved in mitochondrial dysfunction and hepatocellular toxicity, Huh7 and primary rat hepatocytes were exposed to palmitic acid (PA) and/or oleic acid (OA), with or without triol. This compound induced apoptosis in cells co-exposed to both PA and OA, and this was associated with impaired mitochondrial respiration as well as down-regulation of PGC1-alpha, mTFA and NRF1.In conclusion, our data show that hepatic free fatty acid or oxysterols accumulation per se induce low hepatocellular toxicity. On the contrary, hepatic accumulation of both fatty acids and toxic oxysterols such as triol are determinant in the impairment of mitochondrial function and biogenesis, contributing to liver pathology in NAFLD.

Preanalytical standardization for reactive oxygen species derived oxysterol analysis in human plasma by liquid chromatography–tandem mass spectrometry

The analysis of the oxysterols 7-keto-, 7-α/β-hydroxy-, 5α,6α-epoxy-, 5β,6β-epoxycholesterol and cholestane-3β,5α,6β-triol derived from reactive oxygen species (ROS) is of interest as biomarkers in the field of atherosclerosis. Preanalytical validation is a crucial point to minimize the susceptibility of oxysterols to in vitro autoxidation. The aim of this study was to standardize a preanalytical protocol for ROS-derived oxysterol analysis by liquid chromatography–tandem mass spectrometry in human plasma.Sample matrices were compared and stability of free oxysterols in whole blood and EDTA-plasma was investigated with regard to short-term storage until sample preparation, freeze–thaw cycles, addition of butylated hydroxytoluene and long-term storage up to 1year at different temperatures (−20°C, −80°C and −130°C) as well as different storage containers (safe-lock tubes, cryo tubes and straws). Sample preparation prior LC–MS/MS analysis was reduced to a simple concentration and protein precipitation step.Storing EDTA-whole blood for 30min at room temperature resulted in <25% concentration changes, within acceptable change limits (ACL). In freshly prepared plasma samples, free oxysterols were stable for 90min stored at 4°C with concentration changes <23.5% (within ACL). Up to nine freeze–thaw cycles did not affect analyte concentrations (concentration change −8.5% to +5.0%). 7-Ketocholesterol was stable for 2years stored <−80°C; concentration changes below 20.5% (within ACL). The remaining oxysterols were stored for a maximum of 2–4weeks without exceeding ACL. The addition of BHT did not reveal improvement in analyte stability for storage at −80 or −130°C.We developed a standardized preanalytical protocol for oxysterol analysis based on LC–MS/MS, compared cryobanking conditions for each oxysterol and present data for long-term storage up to 2years.

Sulphatation Does Not Appear to Be a Protective Mechanism to Prevent Oxysterol Accumulation in Humans and Mice

24S- and 27-hydroxycholesterol (24OHC and 27OHC) are potent regulators of different biochemical systems in vitro and are the major circulating oxysterols. A small fraction of these oxysterols has been reported to be sulphated but there are no detailed studies. We considered the possibility that sulphatation is a protective mechanism preventing accumulation of free oxysterols. Using an accurate assay we found the sulphated fraction of 24OHC and 27OHC in circulation of adults to be less than 15% of total. In two patients with a mutation in CYP7B1 and markedly increased levels of 27OHC the sulphated fraction was 8% and 10% respectively. Infants with severe neonatal cholestasis had however markedly increased sulphate fraction of the above oxysterols. In untreated mice the degree of sulphatation of 24OHC and 27OHC in serum varied between 0 and 16%. Similar degree of sulphatation was found in two mouse models with markedly increased levels of 27OHC and 24OHC respectively. Bile duct ligated mice had higher levels of oxysterols than sham-operated controls but the sulphate fraction was not increased. We conclude that a primary increase in the levels of the oxysterols due to increased synthesis or reduced metabolism in adults and mice does not induce increased sulphatation.

Fast LC–MS/MS analysis of free oxysterols derived from reactive oxygen species in human plasma and carotid plaque

BackgroundA rapid liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed and validated for the quantification of reactive oxygen species (ROS) derived free oxysterols and cholesterol in human plasma and atherosclerotic plaque. MethodIn vitro autoxidation of cholesterol during sample pretreatment was avoided by applying only one protein precipitation and re-concentration step using 80μl plasma. For preparation of 10mg atherosclerotic plaques an additional liquid–liquid extraction was included. Free 7-keto-, 7-α/ß-hydroxy-, 5,6-α-epoxy-, 5,6-β-epoxycholesterol, cholestane-3ß,5α,6ß-triol and cholesterol were separated within 7min on a monolithic column. An API 4000 tandem mass spectrometer was applied in positive ionization mode using atmospheric pressure chemical ionization. ResultsThe detection limit was 0.1ng/ml and the linearity ranged from 0.5 to 0.75 to 2000ng/ml for the oxysterols and from 50 to 1000μg/ml for cholesterol. Recovery was between 80.9 and 107.9%. Between-run imprecision ranged from 7.9 to 11.7%. Analysis of plasma samples from additional 50 middle-aged volunteers revealed a large inter-individual variability (e.g. 7-ketocholesterol 2.63-30.47ng/ml). Oxysterol concentrations normalized to cholesterol were about 43 times higher in carotid plaque compared to plasma (n=5). ConclusionThis rapid LC–MS/MS method enables reliable quantification focused on especially ROS-derived oxysterols in human plasma and atherosclerotic plaque samples under high-throughput conditions.

Impaired MEK Signaling and SERCA Expression Promote ER Stress and Apoptosis in Insulin-Resistant Macrophages and Are Reversed by Exenatide Treatment

Accumulation of toxic lipids evokes the unfolded protein response (UPR) and apoptotic death of macrophages and vascular cells in atherosclerotic plaques. Primary macrophages from insulin-resistant ob/ob and insulin receptor (Insr)−/− mice display increased apoptosis in response to loading with free cholesterol or oxysterol, but underlying mechanisms have not been elucidated. We show increased activation of all three major branches of the UPR in response to free cholesterol or oxysterol loading in insulin-resistant macrophages. Inhibition and rescue experiments revealed that defective MEK/extracellular signal\\x{2013}related kinase (ERK)/cAMP-responsive element–binding protein (CREBP) signaling in insulin-resistant macrophages leads to decreased expression of sarcoplasmic endoplasmic reticulum (ER) Ca2+-ATPase, depletion of ER calcium stores, PKR-like ER kinase activation, and ER stress–associated apoptosis. Activation of macrophage glucagon-like peptide 1 (GLP-1) receptor via the antidiabetic drug exenatide led to improvements in both ERK and AKT signaling and reversed the increase in UPR and apoptosis of insulin-resistant macrophages in atherosclerotic lesions of ob/ob.Ldlr−/− and Insr−/−.Ldlr−/− mice. Increased signaling via GLP-1 receptor or the CREBP activator protein kinase A thus offers a way to rescue insulin-resistant macrophages from excessive ER stress responses and apoptosis in insulin resistance and type 2 diabetes.

A sensitive and specific LC-MS/MS method for rapid diagnosis of Niemann-Pick C1 disease from human plasma

Niemann-Pick type C1 (NPC1) disease is a rare, progressively fatal neurodegenerative disease for which there are no FDA-approved therapies. A major barrier to developing new therapies for this disorder has been the lack of a sensitive and noninvasive diagnostic test. Recently, we demonstrated that two cholesterol oxidation products, specifically cholestane-3β,5α,6β-triol (3β,5α,6β-triol) and 7-ketocholesterol (7-KC), were markedly increased in the plasma of human NPC1 subjects, suggesting a role for these oxysterols in diagnosis of NPC1 disease and evaluation of therapeutics in clinical trials. In the present study, we describe the development of a sensitive and specific LC-MS/MS method for quantifying 3β,5α,6β-triol and 7-KC human plasma after derivatization with N,N-dimethylglycine. We show that dimethylglycine derivatization successfully enhanced the ionization and fragmentation of 3β,5α,6β-triol and 7-KC for mass spectrometric detection of the oxysterol species in human plasma. The oxysterol dimethylglycinates were resolved with high sensitivity and selectivity, and enabled accurate quantification of 3β,5α,6β-triol and 7-KC concentrations in human plasma. The LC-MS/MS assay was able to discriminate with high sensitivity and specificity between control and NPC1 subjects, and offers for the first time a noninvasive, rapid, and highly sensitive method for diagnosis of NPC1 disease.

Vitamin E and Enzymatic/Oxidative Stress-Driven Oxysterols in Amnestic Mild Cognitive Impairment Subtypes and Alzheimer's Disease

Oxidative stress,which contributes to neuronal damage, is thought to be a pathophysiologicalmechanism of Alzheimer's disease (AD). Markers of oxidative stress may appear early in the preclinical, mild cognitive impairment (MCI) phase of AD.We investigated the interaction among enzymatic-derived oxysterols (24S-hydroxycholesterol and 27-hydroxycholesterol), markers of oxidative stress, including free radical-related oxysterols (7 hydroxycholesterol and 7-ketocholesterol), and vitamin E in AD patients and two amnestic MCI subtypes, amnestic single-domain MCI (a-MCI) subjects, and multidomain MCI (md-MCI) subjects, compared to healthy control subjects (HC). The study included 37 patients with AD, 24 with a-MCI, 29 with md-MCI, and 24 HC. Plasma assessments were made using isotope dilution-mass spectrometry. Although we found no significant change in free radical- or enzymatic-derived oxysterol concentrations in AD or MCI patients, vitamin E levels corrected for cholesterol were reduced in AD patients compared to HC. Results suggest that AD patients have upregulated cerebral oxidative stress or a nutritional deficit of vitamin E. The oxysterols investigated here are not useful markers for diagnosing AD or MCI.

Cholesterol oxides inhibit cholesterol esterification by lecithin: cholesterol acyl transferase

Cholesterol oxides are atherogenic and can affect the activity of diverse important enzymes for the lipidic metabolism. The effect of 7β-hydroxycholesterol, 7-ketocholesterol, 25-hydroxycholesterol, cholestan-3β,5α,6β-triol,5,6β-epoxycholesterol, 5,6α-epoxycholesterol and 7α-hydroxycholesterol on esterification of cholesterol by lecithin:cholesterol acyl transferase (LCAT, EC 2.3.1.43) and the transfer of esters of cholesterol oxides from high density lipoprotein (HDL) to low density lipoproteins (LDL) and very low density lipoproteins (VLDL) by cholesteryl ester transfer protein (CETP) was investigated. HDL enriched with increasing concentrations of cholesterol oxides was incubated with fresh plasma as source of LCAT. Cholesterol and cholesterol oxides esterification was followed by measuring the consumption of respective free sterol and oxysterols. Measurements of cholesterol and cholesterol oxides were done by gas-chromatography. 14C-cholesterol oxides were incorporated into HDL2 and HDL3 subfractions and then incubated with fresh plasma containing LCAT and CETP. The transfer of cholesterol oxide esters was followed by measuring the 14C-cholesterol oxide-derived esters transferred to LDL and VLDL. All the cholesterol oxides studied were esterified by LCAT after incorporation into HDL particles, competing with cholesterol by LCAT. Cholesterol esterification by LCAT was inversely related to the cholesterol oxide concentration. The esterification of 14C-cholesterol oxides was higher in HDL3 and the transfer of the derived esters was greater from HDL2 to LDL and VLDL. The results suggest that cholesterol esterification by LCAT is inhibited in cholesterol oxide-enriched HDL particles. Moreover, the cholesterol oxides-derived esters are efficiently transferred to LDL and VLDL. Therefore, we suggest that cholesterol oxides may exert part of their atherogenic effect by inhibiting cholesterol esterification on the HDL surface and thereby disturbing reverse cholesterol transport.

Are side-chain oxidized oxysterols regulators also in vivo?

Oxsterols are oxygenated metabolites of cholesterol that are short-lived intermediates or end products in cholesterol excretion pathways. They are present in very low concentrations in mammalian systems, always accompanied by a high excess of cholesterol. According to current concepts, side-chain oxidized oxysterols may be mediators of many cholesterol-induced regulatory effects. When added to cultured cells in vitro, side-chain oxidized oxysterols limit intracellular cholesterol levels by at least three different mechanisms: 1) binding to Insig with subsequent block of the sterol regulatory element-binding proteins (SREBP)-mediated mechanism for regulation of sterol sensitive genes; 2) increasing degradation of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, eventually by a mechanism involving binding of Insig to the enzyme; 3) activation of LXR-mediated stimulation of cholesterol transporters and cholesterol metabolism. Addition of pure unesterified oxysterols to cell cultures is however highly unphysiological, and the in vivo relevance of such experiments is questionable. Transgenic mouse models with markedly reduced or increased concentration of some specific oxysterols do not present marked disturbances in cholesterol turnover and homeostasis. Oxysterol-binding proteins such as LXR have been conclusively shown to be of importance for cholesterol turnover in vivo, but their physiological ligands have not yet been defined with certainty. During the last few years, new experimental data has accumulated supporting the contention that side-chain oxysterols are involved in some LXR-mediated regulation in vivo, at least in some biological systems. The new findings will be critically reviewed here.

Relevance and mechanism of oxysterol stereospecifity in coronary artery disease

Cholesterol oxidation products (oxysterols) are markers for in vitro LDL oxidation. They are potent inducers of programmed cell death and are also found in high concentrations inside atherosclerotic lesions. Among physiologically occurring oxysterols, 7β-OH-cholesterol suggests an increase of lipid peroxidation in vivo. In the underlying study, we quantified free plasma oxysterols by means of gas chromatography in patients with stable coronary artery disease (CAD). Total free plasma oxysterols were elevated more than 2-fold in patients with stable CAD (233 ± 49 vs 108 ± 19 ng/ml, n = 22, P < 0.05) compared to a control group (n = 20) with similar atherogenic risk profile and angiographically normal coronary arteries. We found that 7-ketocholesterol, as well as the β-isomers of epoxide (25.7 ± 10.0 vs 7.3 ± 1.4 ng/ml, P = 0.07) and 7β-OH-cholesterol (65.1 ± 15.7 vs 19.4 ± 8.9 ng/ml, P < 0.01), was mainly responsible for this increase. To elucidate a potential relevance of oxysterol stereospecificity in regard to endothelial damage, we further conducted in vitro experiments using human arterial endothelial cells (HAECs). Surprisingly, β-isomers exerted an up to 10-fold higher amount of cell death in equivalent doses when compared to α-isomers. The greater cytotoxic potential of β-isomers was due to increased apoptosis, preceded by mitochondrial release of cytochrome c with subsequent caspase-3 activation. Stereospecific release of cytochrome c depended on the presence of an intact cytoplasmic membrane, hinting at the existence of a putative oxysterol receptor or a direct stereospecific effect on membrane biology. Finally, both isoforms of oxysterols directly released cytochrome c only in conjunction with protein containing cytosol and endoplasmatic reticulum. Free plasma oxysterol levels, particularly 7-ketocholesterol, β-epoxide and 7β-OH-cholesterol, are elevated in patients with stable CAD, independent of their LDL cholesterol levels. Due to the highly increased cytotoxicity of oxysterol β-isomers in vitro, they may represent important atherogenic risk factors.