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The aim of this original research is to evaluate the effect of SG on alcohol intake symptoms, blood alcohol content (BAC), and alcohol metabolite levels. At 0–6–12 months after SG, BAC of patients was measured at 0, 15, 30, and 60 min, and then every 30 min, and urinary metabolite (ethanol and acetaldehyde) levels were measured 2 h after consuming a standard red wine drink. Symptoms perceived by patients were evaluated using symptom alcoholization post-obesity surgery scores. Thirty obese patients (12 men/18 women; mean body mass index, 44 ± 4 kg/m2) who underwent SG were enrolled in this study. At 12 months after SG, no alcohol use disorder was observed and BAC tended to peak after 15 min, with alcohol intoxication symptoms (nausea/vomiting, flushing, and diaphoresis), and return to zero after 90 min of wine intake. Ethanol and acetaldehyde levels were significantly different at 12 months compared with the levels at time 0 (p

Vitamin E is a potent antioxidant that has been considered involved in fertility, but studies have mostly focused on α-tocopherol. Our study aimed at measuring, by an isotope dilution gas chromatography-mass spectrometry method, α- and γ-tocopherol concentration in human semen in a large and well-characterised population (134 men with different semen parameters and in varicocele patients), as well as their potential role in male fertility. We carried out freeze/thaw experiments in 15 samples with the two isomers in the cryoprotective medium. Moreover, our study included 10 subjects supplemented in vivo with α-tocopherol for 90 days. In seminal plasma, γ-tocopherol concentration was significantly lower in the varicocele group than in the normozoospermic group. We observed that γ-tocopherol, supplemented to cryopreservation medium, induced a higher post-thaw human sperm viability and motility than α-tocopherol. The results of in vivo α-tocopherol supplementation showed a decrease in γ-tocopherol concentration with increasing α-tocopherol level in blood. This is the first report related to γ-tocopherol distribution in human semen analysed by gas chromatography-mass spectrometry. γ-tocopherol would not seem to be related to semen parameters but to cellular oxidative condition. This tocopherol may contribute to human health in a yet unexplored way.

Oxysterols are bioactive lipids that act as regulators of lipid metabolism, inflammation, cell viability and are involved in several diseases, including atherosclerosis. Mounting evidence linked the atherosclerosis to endothelium dysfunction; in fact, the endothelium regulates the vascular system with roles in processes such as hemostasis, cell cholesterol, hormone trafficking, signal transduction and inflammation. Several papers shed light the ability of oxysterols to induce apoptosis in different cell lines including endothelial cells. Apoptotic endothelial cell and endothelial denudation may constitute a critical step in the transition to plaque erosion and vessel thrombosis, so preventing the endothelial damaged has garnered considerable attention as a novel means of treating atherosclerosis. Endoplasmic reticulum (ER) is the site where the proteins are synthetized and folded and is necessary for most cellular activity; perturbations of ER homeostasis leads to a condition known as endoplasmic reticulum stress. This condition evokes the unfolded protein response (UPR) an adaptive pathway that aims to restore ER homeostasis. Mounting evidence suggests that chronic activation of UPR leads to cell dysfunction and death and recently has been implicated in pathogenesis of endothelial dysfunction. Autophagy is an essential catabolic mechanism that delivers misfolded proteins and damaged organelles to the lysosome for degradation, maintaining basal levels of autophagic activity it is critical for cell survival. Several evidence suggests that persistent ER stress often results in stimulation of autophagic activities, likely as a compensatory mechanism to relieve ER stress and consequently cell death. In this review, we summarize evidence for the effect of oxysterols on endothelial cells, especially focusing on oxysterols-mediated induction of endoplasmic reticulum stress., Graphical abstract fx1, Highlights • Endothelial cells dysfunction is critical in the process of atherothrombosis. • Endoplasmic reticulum stress is a key component in endothelial cell dysfunction. • Oxysterols are oxidation products of cholesterol found in atherosclerosis lesions. • Oxysterols are potential modulators of endoplasmic reticulum stress.

Cholesterol is a main lipid component of sperm cell that is essential for sperm membrane fluidity, capacitation, and acrosomal reaction. Recent data obtained in bovine sperm showed that sperm capacitation is associated to the formation of oxysterols, oxidized products of cholesterol. The aim of this study was to profile oxysterol content in human semen, and to investigate their potential role in sperm pathophysiology. Among the 12 oxysterols analyzed, 25-hydroxycholesterol (25-HC) resulted the most represented in normozoospermic samples, and its concentration positively correlated with spermatozoa number. We detected Cholesterol 25-hydroxylase, the enzyme responsible for 25-HC production, in human spermatozoa at the level of the neck and the post acrosomal area. Upon incubation with spermatozoa, 25-HC induced calcium and cholesterol transients in connection with the acrosomal reaction. Our results support a role for 25-HC in sperm function. Keywords: Oxysterols, Cholesterol, Sperm function, Male infertility

Alzheimer's disease (AD) is a gradually debilitating disease that leads to dementia. The molecular mechanisms underlying AD are still not clear, and at present no reliable biomarkers are available for the early diagnosis. In the last several years, together with oxidative stress and neuroinflammation, altered cholesterol metabolism in the brain has become increasingly implicated in AD progression. A significant body of evidence indicates that oxidized cholesterol, in the form of oxysterols, is one of the main triggers of AD. The oxysterols potentially most closely involved in the pathogenesis of AD are 24-hydroxycholesterol and 27-hydroxycholesterol, respectively deriving from cholesterol oxidation by the enzymes CYP46A1 and CYP27A1. However, the possible involvement of oxysterols resulting from cholesterol autooxidation, including 7-ketocholesterol and 7β-hydroxycholesterol, is now emerging. In a systematic analysis of oxysterols in post-mortem human AD brains, classified by the Braak staging system of neurofibrillary pathology, alongside the two oxysterols of enzymatic origin, a variety of oxysterols deriving from cholesterol autoxidation were identified; these included 7-ketocholesterol, 7α-hydroxycholesterol, 4β-hydroxycholesterol, 5α,6α-epoxycholesterol, and 5β,6β-epoxycholesterol. Their levels were quantified and compared across the disease stages. Some inflammatory mediators, and the proteolytic enzyme matrix metalloprotease-9, were also found to be enhanced in the brains, depending on disease progression. This highlights the pathogenic association between the trends of inflammatory molecules and oxysterol levels during the evolution of AD. Conversely, sirtuin 1, an enzyme that regulates several pathways involved in the anti-inflammatory response, was reduced markedly with the progression of AD, supporting the hypothesis that the loss of sirtuin 1 might play a key role in AD. Taken together, these results strongly support the association between changes in oxysterol levels and AD progression., Highlights • Changes in brain oxysterol levels may influence AD progression. • Oxysterol accumulation in the brain may amplify neuroinflammation. • SIRT-1 loss during AD progression may favor neuroinflammation. • Oxysterols and SIRT-1 might be useful markers for early AD diagnosis.

International audience; Serum concentrations of lathosterol, the plant sterols campesterol and sitosterol and the cholesterol metabolite 5alpha-cholestanol are widely used as surrogate markers of cholesterol synthesis and absorption, respectively. Increasing numbers of laboratories utilize a broad spectrum of well-established and recently developed methods for the determination of cholesterol and non-cholesterol sterols (NCS). In order to evaluate the quality of these measurements and to identify possible sources of analytical errors our group initiated the first international survey for cholesterol and NCS. The cholesterol and NCS survey was structured as a two-part survey which took place in the years 2013 and 2014. The first survey part was designed as descriptive, providing information about the variation of reported results from different laboratories. A set of two lyophilized pooled sera (A and B) was sent to twenty laboratories specialized in chromatographic lipid analysis. The different sterols were quantified either by gas chromatography-flame ionization detection, gas chromatography- or liquid chromatography-mass selective detection. The participants were requested to determine cholesterol and NCS concentrations in the provided samples as part of their normal laboratory routine. The second part was designed as interventional survey. Twenty-two laboratories agreed to participate and received again two different lyophilized pooled sera (C and D). In contrast to the first international survey, each participant received standard stock solutions with defined concentrations of cholesterol and NCS. The participants were requested to use diluted calibration solutions from the provided standard stock solutions for quantification of cholesterol and NCS. In both surveys, each laboratory used its own internal standard (5alpha-cholestane, epicoprostanol or deuterium labelled sterols). Main outcome of the survey was, that unacceptably high interlaboratory variations for cholesterol and NCS concentrations are reported, even when the individual laboratories used the same calibration material. We discuss different sources of errors and recommend all laboratories analysing cholesterol and NCS to participate in regular quality control programs.

Oxysterols, oxidized derivatives of cholesterol found in LDL and atherosclerotic plaques, trigger several biological responses involved in the initiation and progression of atherosclerosis. Endothelial dysfunction, which occurs when vascular homeostasis is altered, plays a key role in the pathogenesis of several metabolic diseases. The contribution of endoplasmic reticulum (ER) stress to endothelial disfunction is a relatively recent area of investigation. There is a well-established link between LDL oxidation and ER stress but the role played by specific products of lipid oxidation into this interaction is still to be defined. The present study shows that secosterol-B (SEC-B), 3β-hydroxy-5β-hydroxy-B-norcholestane-6βcarboxaldehyde, a cholesterol autoxidation product recently identified in the atherosclerotic plaque, is able to induce ER stress in HUVEC cells, as revealed by significant expansion and change of structure. At low doses, i.e. 1 and 5 μM, cells try to cope with this stress by activating autophagy and the ubiquitin proteasome system in the attempt to restore ER function. However, at higher doses, i.e. 20 μM, cell apoptosis occurs in a pathway that involves early phosphorylation of eIF2α and NF-kB activation, suggesting that the adaptive program fails and the cell activates the apoptotic program. These findings provide additional insight about the role of oxysterols in endothelial dysfunction and its potential involvement in atherosclerotic pathophysiology.

Advanced prostate cancer (PCa) is a clinical challenge as no curative therapeutic is available. In this context, a better understanding of metastasis and resistance mechanisms in PCa is an important issue. As phosphatase and tensin homolog (PTEN) loss is the most common genetic lesion in such cancer, we investigate human data sets for mechanisms that can constrain cancer evolution in this setting. Here we report a liver X receptor (LXR) signature, which tightly correlates with PTEN loss, in PCa. Accordingly, the LXR pathway is deregulated in prostate carcinomas in Pten-null mice. Genetic ablation of LXRs in Pten-null mice, exacerbates PCa invasiveness and metastatic dissemination, which involves mesenchymal transition and accumulation of matrix metalloproteinases. Mechanistically, PTEN deletion governed LXR transcriptional activity through deregulation of cholesterol de novo synthesis, resulting in accumulation of endogenous LXR ligands. Our study therefore reveals a functional circuit linking PTEN and LXR, and highlights LXRs as metabolic gatekeepers that are able to constrain PCa progression. Treatment of prostate cancer, especially in its advanced stage, is still challenging; therefore, strategies to prevent metastatic dissemination are of great interest. Here the authors reveal a crucial role for liver X receptors in suppressing prostate carcinogenesis and metastatic progression in PTEN-null tumors.

Cholesterol and its oxygenated metabolites, such as oxysterols, are intensively investigated as potential players in the pathophysiology of brain disorder. Altered oxysterol levels have been described in patients with numerous neuropsychiatric disorders, including Alzheimer's disease, Amyotrophic Lateral Sclerosis, Parkinson's disease, X-linked adrenoleukodystrophy, and Smith-Lemli-Opitz Syndrome. Recent studies have shown that Autism Spectrum Disorders are associated with disruption of cholesterol metabolism. The present study aimed at investigating the profile of oxysterols in plasma and their association with clinical parameters in patients with Autism Spectrum Disorders. Thirty-six children with Autism Spectrum Disorders and thirty-eight healthy children, from Sfax (a southern area of Tunisia) matched for age and sex, were included in the study. The severity of Autism Spectrum Disorders was evaluated using the childhood autism rating scale. Standard lipid profile (total cholesterol, triglycerides, and high-density lipoprotein-cholesterol), serum glucose, high-sensitive C-reactive protein and orosomucoid levels were measured utilizing standard techniques. Oxysterol levels were measured by isotope-dilution gas chromatography/mass spectrometry. Standard lipid profile, serum glucose, high-sensitive C-reactive protein and orosomucoid levels were similar between the two studied populations. Compared to the control group, children with Autism Spectrum Disorders showed a significant higher plasma level of 24-hydroxycholesterol, while borderline significance was observed for 7α-hydroxycholesterol, and 25-hydroxycholersterol. In patients, 24-hydroxycholesterol was inversely correlated with age. Multivariate analysis showed that high plasma levels of 24-hydroxycholesterol are independent risk factors for Autism Spectrum Disorders. On the other hand, an analysis of the receiver's operating characteristics proved that the measured parameters recorded satisfactory levels of specificity and sensitivity. The present study provides evidence that Autism Spectrum Disorders are associated with altered levels in circulating oxysterols. The finding that 24-hydroxycholesterol is an independent risk factor for the disease and suggests the use of this oxysterol as a diagnostic tool in Autism Spectrum Disorders.

Aims To evaluate the effect of ADH1B and ADH7 genotypes on blood acetaldehyde and ethanol levels after alcohol ingestion, and to measure the genotoxic effect of smoking and ethanol on the buccal cells, also controlling for ADH variants. Methods We recruited healthy Italian subjects with at least a moderate history of alcohol consumption. All subjects were given an alcoholic drink of 0.4 g ethanol /kg of body weight. Blood venous samples were collected at baseline, and 30, 60, 90 and 120 minutes after ingestion. Buccal cells were collected before ethanol ingestion. Results Sixty subjects were enrolled in the study. Individuals with the ADH1B GG genotype had median ethanol levels of 5.0 mM (IQR 3.4-7.2), and those with the ADH1B GT/TT genotype had 4.7 mM (IQR 4.2-4.8). Corresponding acetaldehyde levels were 1.5 μM (IQR 0.7-2.6) for ADH1B GG genotype and 1.6 μM (IQR 1.5-1.7) for ADH1B CG/GG genotype. Individuals with the ADH7 CC genotype had median ethanol levels of 5.0 mM (IQR 3.3-7.2), while 5.0 mM (IQR 4.7-5.6) was in those with the ADH7 CG/GG genotype. Corresponding acetaldehyde levels were 1.5 μM (IQR 0.7-2.6) for ADH7 CC genotype and 1.5 μM (IQR 1.4-1.6) for ADH7 CG/GG genotypes. A non significant increase in the frequency of karyolitic and pyknotic cells was found in the group of heavy drinkers and current smokers, when compared to the moderate drinkers and the non-smokers. Conclusions Our study does not support the hypothesis that ADH1B and ADH7 genotypes affect blood ethanol and acetaldehyde concentration.

International audience; Increasing numbers of laboratories develop new methods based on gas-liquid and high-performance liquid chromatography to determine serum concentrations of oxygenated cholesterol metabolites such as 7alpha-, 24(S)-, and 27-hydroxycholesterol. We initiated a first international descriptive oxycholesterol (OCS) survey in 2013 and a second interventional survey 2014 in order to compare levels of OCS reported by different laboratories and to define possible sources of analytical errors. In 2013 a set of two lyophilized serum pools (A and B) was sent to nine laboratories in different countries for OCS measurement utilizing their own standard stock solutions. In 2014 eleven laboratories were requested to determine OCS concentrations in lyophilized pooled sera (C and D) utilizing the same provided standard stock solutions of OCS. The participating laboratories submitted results obtained after capillary gas-liquid chromatography-mass selective detection with either epicoprostanol or deuterium labelled sterols as internal standards and high-performance liquid chromatography with mass selective detection and deuterated OCS as internal standard. Each participant received a clear overview of the results in form of Youden-Plots and basic statistical evaluation in its used unit. The coefficients of variation of the concentrations obtained by all laboratories using their individual methods were 58.5-73.3% (survey 1), 56.8-60.3% (survey 2); 36.2-35.8% (survey 1), 56.6-59.8, (survey 2); 61.1-197.7% (survey 1), 47.2-74.2% (survey 2) for 24(S)-, 27-, and 7alpha-hydroxycholesterol, respectively. We are surprised by the very great differences between the laboratories, even under conditions when the same standards were used. The values of OCS's must be evaluated in relation to the analytical technique used, the efficiency of the ample separation and the nature of the internal standard used. Quantification of the calibration solution and inappropriate internal standards could be identified as major causes for the high variance in the reported results from the different laboratories. A harmonisation of analytical standard methods is highly needed.

International audience; Breast cancer (BC) remains the primary cause of death from cancer among women worldwide. Cholesterol-5,6-epoxide (5,6-EC) metabolism is deregulated in BC but the molecular origin of this is unknown. Here, we have identified an oncometabolism downstream of 5,6-EC that promotes BC progression independently of estrogen receptor α expression. We show that cholesterol epoxide hydrolase (ChEH) metabolizes 5,6-EC into cholestane-3β,5α,6β-triol, which is transformed into the oncometabolite 6-oxo-cholestan-3β,5α-diol (OCDO) by 11β-hydroxysteroid-dehydrogenase-type-2 (11βHSD2). 11βHSD2 is known to regulate glucocorticoid metabolism by converting active cortisol into inactive cortisone. ChEH inhibition and 11βHSD2 silencing inhibited OCDO production and tumor growth. Patient BC samples showed significant increased OCDO levels and greater ChEH and 11βHSD2 protein expression compared with normal tissues. The analysis of several human BC mRNA databases indicated that 11βHSD2 and ChEH overexpression correlated with a higher risk of patient death, highlighting that the biosynthetic pathway producing OCDO is of major importance to BC pathology. OCDO stimulates BC cell growth by binding to the glucocorticoid receptor (GR), the nuclear receptor of endogenous cortisol. Interestingly, high GR expression or activation correlates with poor therapeutic response or prognosis in many solid tumors, including BC. Targeting the enzymes involved in cholesterol epoxide and glucocorticoid metabolism or GR may be novel strategies to prevent and treat BC.

International audience; 5,6α-epoxycholesterol (5,6α-EC) and 5,6β-epoxycholesterol (5,6β-EC) are oxysterols involved in the anticancer pharmacology of the widely used antitumor drug tamoxifen. They are both metabolized into cholestane-3β,5α,6β-triol (CT) by the cholesterol-5,6-epoxide hydrolase (ChEH) enzyme, and CT is metabolized by an as-yet uncharacterized enzyme into 6-oxo-cholestan-3β,5α-diol (OCDO). A recent feasibility study showed that the 5,6-ECs may represent surrogate markers of tamoxifen activity in breast cancer patients undergoing endocrine therapy, thus there is a growing interest in their accurate quantification. These oxysterols are usually quantified by gas-liquid chromatography coupled to mass spectrometry (GC/MS), using an isotope dilution methodology with the corresponding deuterated oxysterol. This method is considered to be relative quantitative since all of the standards used are deuterated oxysterols, however it is not known whether the preparation of each oxysterol is affected in the same way by the extraction, pre-purification by solid phase extraction (SPE) and trimethylsilylation steps, particularly when using biological samples that contain many other reactive compounds. Thus, in this study we investigated the yield of the 5,6-ECs, CT and OCDO recovery from patient serum samples at different stages of their work-up and trimethylsilylation prior to GC/MS analysis, using [14C]-labeled analogs to follow these oxysterols at each step. We measured a 40 to 60% loss of material for the 5,6-ECs and OCDO, however we also describe the conditions that improved their recovery. Our data also show that the use of deuterated 5,6α-EC, 5,6β-EC, CT and OCDO is an absolute requirement for their accurate quantification.

International audience; Accumulating evidence indicates that cholesterol oxygenation products, also known as oxysterols (OS), are involved in breast cancer (BC) promotion. The impact of Tam, as well as aromatase inhibitors (AI), an alternative BC endocrine therapy (ET), on OS metabolism in patients is currently unknown. We conducted a prospective clinical study in BC patients receiving Tam (n=15) or AI (n=14) in adjuvant or in metastatic settings. The primary end point was the feasibility of detecting and quantifying 11 different OS in the circulation of patients before and after 28days of treatment with Tam or AI. Key secondary end points were the measurements of variations in the concentrations of OS according to differences between patients and treatments. OS profiling in the serum of patients was determined by gas chromatography coupled to mass spectrometry. OS profiling was conducted in all patients both at baseline and during treatment regimens. An important inter-individual variability was observed for each OS. Interestingly 5,6β-epoxycholesterol relative concentrations significantly increased in the entire population (p=0.0109), while no increase in Cholestane-triol (CT) levels was measured. Interestingly, we found that, in contrast to AI, Tam therapy significantly decreased blood levels of 24-hydroxycholesterol (24-HC), 7α-HC and 25-HC (a tumor promoter) (p=0.0007, p=0.0231 and p=0.0231, respectively), whereas 4β-HC levels increased (p=0.0010). Interestingly, levels of 27-HC (a tumor promoter) significantly increased in response to AI (p=0.0342), but not Tam treatment. According to these results, specific OS are promising candidate markers of Tam and AI efficacy. Thus, further clinical investigations are needed to confirm the use of oxysterols as biomarkers of both prognosis and/or the efficacy of ET.

Cholesterol is a unique lipid molecule providing the building block for membranes, hormones, vitamin D and bile acid synthesis. Metabolism of cholesterol involves several enzymes acting on the sterol nucleus or the isooctyl tail. In the recent years, research interest has been focused on oxysterols, cholesterol derivatives generated by the addition of oxygen to the cholesterol backbone. Oxysterols can be produced enzymatically or by autoxidation. Autoxidation of cholesterol proceeds through type I or type II mechanisms. Type I autoxidation is initiated by free radical species, such as those arising from the superoxide/hydrogen peroxide/hydroxyl radical system. Type II autoxidation occurs stoichiometrically by non-radical highly reactive oxygen species such as singlet oxygen, HOCl, and ozone. The vulnerability of cholesterol towards high reactive species has raised considerable interest for mechanistic studies and for the potential biological activity of oxysterols, as well as for the use of oxysterols as biomarkers for the non-invasive study of oxidative stress in vivo.

Background: Knowledge regarding the plasma fatty acid (FA) pattern in patients with liver cirrhosis is fragmentary. Objective: We evaluated plasma FA lipidome and its association with the prognosis of cirrhosis and severity of liver graft damage after transplantation. Design: In this observational study, plasma FA lipidome was investigated in 51 cirrhotic patients before liver transplantation and in 90 age- and sex-matched healthy control subjects. In addition, we studied ischemia-reperfusion damage in the liver of 38 patients for whom a graft biopsy was available at transplantation. With the use of logistic regression, we modeled the presence of cirrhosis, the dichotomized model for end-stage liver disease score below and above the median, and the presence of severe liver graft ischemiareperfusion damage. Results: The FA pattern was markedly altered in cirrhotic patients, who showed, compared with healthy controls, higher monounsaturated FAs, lower n2 6a nd n23 polyunsaturated FAs, and undetectable cerotic acid. Plasma di-homo-g-linolenic acid was independently associated with the presence of cirrhosis (OR: 0.026; 95% CI: 0.004, 0.196; P , 0.0001), severity of its prognosis (OR: 0.041; 95% CI:0.005, 0.376; P = 0.006), postreperfusion graft hepatocellular necrosis (OR: 0.921; 95% CI: 0.851, 0.997; P = 0.043), and sinusoidal congestion (OR: 0.954; 95% CI: 0.912, 0.998; P = 0.039). Associations of di-homo-g-linolenic acid with the presence of cirrhosis and severity of its prognosis were confirmed also after false discovery rate correction. Conclusions: Cerotic and di-homo-g-linolenic acids may serve as markers of disease and prognosis in liver cirrhosis. Dietary supplementation with di-homo-g-linolenic acid could be a reasonable interventional strategy to delay disease progression in liver cirrhosis. This trial was registered at clinicaltrials.gov as NCT01389115. Am J Clin Nutr doi: 10.3945/ajcn.113.074427.

Polyunsaturated fatty acids (PUFA) of the n-3 series have been linked to brain physiology and cognitive decline, but little is known about the other components of the complex fatty acids category. Here, we compared 30 molecular species pertaining to saturated, monounsaturated, polyunsaturated, and trans fatty acids, measured in plasma by gas chromatography, in 14 patients with a diagnosis of amnestic single domain mild cognitive impairment (aMCI), 30 patients with mild Alzheimer's disease (AD), and 30 healthy controls (HC). As no participants showed neuroimaging evidence of cerebrovascular disease, patients could be considered as purely neurodegenerative. We found differences in specific components of almost all fatty acid classes except n-3-polyunsaturated fatty acids. Compared with HC, aMCI and AD patients had higher levels of arachidic (C20:0), erucic (C22:1, n-9), and vaccenic acid (C18:1, n-9) and lower levels of cerotic (C26:0) and linoleic acid (C18:2, n-6). In particular, level of linoleic acid decreased and level of mead acid increased progressively from HC to aMCI to AD patients, and they were also inversely correlated in AD and aMCI patients. In conclusion, we found a previously unrecognized linoleic acid deficiency in the early phase of neurodegeneration that was strongly supported by an increased, compensatory mead acid level. These findings suggest the importance of creating new dietary manipulation strategies to counteract disease progression. © 2013 - IOS Press and the authors. All rights reserved.

Lipid accumulation is the hallmark of Non-alcoholic Fatty Liver Disease (NAFLD) and has been suggested to play a role in promoting fatty liver inflammation. Previous findings indicate that during oxidative stress conditions excess cholesterol autoxidizes to oxysterols. To date, the role of oxysterols and their potential interaction with fatty acids accumulation in NASH pathogenesis remains little investigated. We used the nutritional model of high fatty acids (HFA), high cholesterol (HCh) or high fat and high cholesterol (HFA+FCh) diets and explored by a lipidomic approach, the blood and liver distribution of fatty acids and oxysterols in response to dietary manipulation. We observed that HFA or HCh diets induced fatty liver without inflammation, which was otherwise observed only after supplementation of HFA+HCh. Very interestingly, the combination model was associated with a specific oxysterol fingerprint. The present work provides a complete analysis of the change in lipids and oxysterols profile induced by different lipid dietary model and their association with histological alteration of the liver. This study allows the generation of interesting hypotheses on the role of interaction of lipid and cholesterol metabolites in the liver injury during NAFLD development and progression. Moreover, the changes in the concentration and quality of oxysterols induced by a combination diet suggest a novel potential pathogenic mechanism in the progression from simple steatosis to steatohepatitis., Graphical abstract fx1, Highlights • Dietary fatty acids or cholesterol induce specific liver histological alterations. • A lipidomic approach allows the identification of steatotic oxysterol fingerprint. • FA and Ch interaction is a potential pathogenic mechanism for steatohepatitis.

Summary Previous reports showed altered fatty acid content in subjects with altered sperm parameters compared to normozoospermic individuals. However, these studies focused on a limited number of fatty acids, included a short number of subjects and results varied widely. We conducted a case–control study involving 155 patients allocated into four groups, including normozoospermia (n = 33), oligoasthenoteratozoospermia (n = 32), asthenozoospermia (n = 25), and varicocoele (n = 44). Fatty acid profiling, including 30 species, was analyzed by a validated gas chromatography (GC) method on the whole seminal fluid sample. Multinomial logistic regression modeling was used to identify the associations between fatty acids and the four groups. Specimens from 15 normozoospermic subjects were also analyzed for fatty acids content in the seminal plasma and spermatozoa to study the distribution in the two compartments. Fatty acids lipidome varied markedly between the four groups. Multinomial logistic regression modeling revealed that high levels of palmitic acid, behenic acid, oleic acid, and docosahexaenoic acid (DHA) confer a low risk to stay out of the normozoospermic group. In the whole population, seminal fluid stearic acid was negatively correlated (r = −0.53), and DHA was positively correlated (r = 0.65) with sperm motility. Some fatty acids were preferentially accumulated in spermatozoa and the highest difference was observed for DHA, which was 6.2 times higher in spermatozoa than in seminal plasma. The results of this study highlight complete fatty acids profile in patients with different semen parameters. Given the easy-to-follow and rapid method of analysis, fatty acid profiling by GC method can be used for therapeutic purposes and to measure compliance in infertility trials using fatty acids supplements.

Context:Increased oxidative stress in adipose tissue emerges as an inducer of obesity-linked insulin resistance. Here we tested whether free-radical derived oxysterols are formed by, and accumulate in, human adipocytes. Moreover, we asked whether increased accumulation of oxysterols characterizes the adipose cells of obese patients with type 2 diabetes (T2D) (OBT2D) compared with lean, nondiabetic controls (CTRLs). Finally, we studied the effects of the free radical–derived oxysterols on adipogenic differentiation of adipose-derived stem cells (ASCs).Main Outcome Measures:Adipocytes and ASCs were isolated from sc abdominal adipose tissue biopsy in four OBT2D and four CTRL subjects. Oxysterols in adipocytes were detected by gas chromatography/mass spectrometry. The cellular and molecular effects of oxysterols were then evaluated on primary cultures of ASCs focusing on cell viability, adipogenic differentiation, and “canonical” WNT and MAPK signaling pathways.Results:7-ketocholesterol (7κ-C) and 7β-hydroxycholesterol were unambiguously detected in adipocytes, which showed higher oxysterol accumulation (P < .01) in OBT2D, as compared with CTRL individuals. Notably, the accumulation of oxysterols in adipocytes was predicted by the adipose cell size of the donor (R2 = 0.582; P < .01). Challenging ASCs with free radical–derived type I (7κ-C) and type II (5,6-Secosterol) oxysterols led to a time- and concentration-dependent decrease of cell viability. Meaningfully, at a non-toxic concentration (1μM), these bioactive lipids hampered adipogenic differentiation of ASCs by sequential activation of WNT/β-catenin, p38-MAPK, ERK1/2, and JNK signaling pathways.Conclusion:Free radical–derived oxysterols accumulate in the “diabetic” fat and may act as novel adipokines modulating the adipogenic potential of undifferentiated adipose precursor cells.

International audience; Background: Obesity and the related entity metabolic syndrome are characterized by altered lipid metabolism and associated with increased morbidity risk for cardiovascular disease and cancer. Oxysterols belong to a large family of cholesterol-derived molecules known to play crucial role in many signaling pathways underlying several diseases. Little is known on the potential effect of obesity and metabolic syndrome on oxysterols in human. Objectives: In this work, we questioned whether circulating oxysterols might be significantly altered in obese patients and in patients with metabolic syndrome. We also tested the potential correlation between circulating oxysterols and fatty acids. Methods: 60 obese patients and 75 patients with metabolic syndrome were enrolled in the study along with 210 age- and sex-matched healthy subjects, used as control group. Plasma oxysterols were analyzed by isotope dilution GC/MS, and plasma fatty acids profiling was assessed by gas chromatography coupled with flame ionization detection. Results: We found considerable differences in oxysterols profiling in the two disease groups that were gender-related. Compared to controls, males showed significant differences only in 4 alpha- and 4 beta-hydroxycholesterol levels in obese and metabolic syndrome patients. In contrast, females showed consistent differences in 7-oxocholesterol, 4 alpha-hydroxycholesterol, 25-hydroxycholesterol and triol. Concerning fatty acids, we found minor differences in the levels of these variables in males of the three groups. Significant changes were observed in plasma fatty acid profile of female patients with obesity or metabolic syndrome. We found significant correlations between various oxysterols and fatty acids. In particular, 4 beta-hydroxycho1esterol, which is reduced in obesity and metabolic syndrome, correlated with a number of saturated and mono-unsaturated fatty acids that are end-products of de novo lipogenesis. Conclusions: Our data provide the first evidence that obesity and metabolic syndrome are associated with major, gender-specific, changes in circulating oxysterols and fatty acids. These findings suggest a metabolic link between oxysterols and fatty acids, and that oxysterols may contribute to the epidemic diseases associated with obesity and metabolic syndrome in female.

Assessing vitamin E status in humans is critical for nutritional evaluation and verification of clinical and biological compliance of supplemented subjects. An accurate analytical method for measuring the two main vitamin E isoforms, i.e. α- and γ-tocopherol (α- and γ-TOH) in small volumes of plasma can facilitate the application of this analysis to clinical trials and in situations where a limited amount of sample is available.We have developed a micro method, which uses only 5 μL plasma, based on isotope dilution, trimethylsilation and GC-MS. The method was validated according to the guidelines of the International Conference on Harmonization of analytical procedures. The method was also applied to 5 μL of whole blood for the potential use in conditions were the availability of specimens is limited.Accurate quantitation of α-TOH and γ-TOH was achieved at levels ≥ 0.417 μM and ≥ 0.007 μM, respectively. Within-day coefficient of variation was 1.31% and 4.70% for α-TOH and γ-TOH, respectively. Between-day coefficient of variation was 1.32% and 2.88% for α-TOH and γ-TOH, respectively. Recovery, assessed at three concentration levels, ranged 98-103% and 100-102% for α-TOH and γ-TOH, respectively. The method allowed the detection of α-TOH and γ-TOH in 5 μL whole blood and in membranes of red blood cells washed from 5 μL of blood as well. The analytical performance was assessed in plasma from a cohort of Italian healthy subjects (n = 205). The mean plasma concentrations were 28.01 ± 6.31 and 0.68 ± 0.48 μM (mean ± SD) for α-TOH and γ-TOH, respectively. Alpha-TOH correlated with total cholesterol (r = 0.617, p0.0001) and triglycerides (r = 0.420, p0.0001) while γ-TOH correlated modestly with total cholesterol (r = 0.213, p0.0001) but not with triglycerides. γ-TOH, but not α-TOH, was significantly lower in smokers than in non-smokers (0.72 ± 0.50 vs. 0.56 ± 0.37, μM, mean ± SD, p = 0.017). Given the high sensitivity, the method allowed to be applied to 5 μM whole blood without specific modification.This micro-method represents an analytical advancement in α- and γ-TOH assay that is available to accurately verify the nutritional status and compliance after supplementation in large-scale settings, and to measure the two vitamers in conditions where sample availability is limited.

7-Ketocholesterol and 5,6-secosterol are cholesterol autoxidation products generated under oxidative stress by two distinct mechanisms. They are present in atherosclerotic plaques and are candidate players in the disease initiation and progression. While 7-ketocholesterol affects at cellular level, in particular apoptosis, are well known and reported on diverse cell lines, 5,6-secosterol is a recently discovered oxysterol with relatively few reports on the potential to affect endothelial cell functions. Endothelial cells have a central role in cardiovascular disease as they provide the barrier between blood and the vessel wall where atherosclerosis starts and progresses. Insults to endothelial cells provoke their dysfunction favoring pro-atherogenic and pro-thrombotic effects. In the present work, we tested 7-ketocholesterol and 5,6-secosterol on endothelial cells - focusing on apoptosis and the associated mitochondrial/lysosome alterations - and on endothelial function using the in vitro model of arterial relaxation of aortic rings. Our data provide evidence that 7-ketocholesterol and 5,6-secosterol are efficient instigators of apoptosis, which for 5,6-secosterol is associated to PKC and p53 up-regulation. In addition 5,6-secosterol is a potent inhibitor of endothelial-dependent arterial relaxation through PKC-dependent mechanisms. This may contribute to pro-atherogenic and pro-thrombotic mechanisms of 5,6-secosterol and highlights the role of cholesterol autoxidation in cardiovascular disease.

The oxysterol 27-hydroxycholesterol (27-OH) is increasingly considered to be involved in a variety of pathophysiological processes, having been shown to modulate cell proliferation and metabolism, and also to exert proinflammatory and proapoptotic effects. This study aimed to elucidate the molecular pathways whereby 27-OH may generate survival signals in cells of the macrophage lineage, and to clarify whether its known prooxidant effect is involved in that process. A net up-regulation of survival signaling, involving the extracellular signal-regulated kinase (ERIC) and phosphoinositide 3-kinase (PI3K)/Akt phosphorylation pathways, was observed in U937 promonocytic cells cultivated over time in the presence of a low micromolar concentration of the oxysterol. Interestingly, the up-regulation of both kinases was shown to be closely dependent on an early 27-OH-induced intracellular increase of reactive oxygen species (ROS). In turn, stimulation of ERR and PI3K/Akt both significantly quenched ROS steady state and markedly phosphorylated Bad, thereby determining a marked delay of the oxysterol's proapoptotic action. The 27-OH-induced survival pathways thus appear to be redox modulated and, if they occur within or nearby inflammatory cells during progression of chronic diseases such as cancer and atherosclerosis, they could significantly impact the growth and evolution of such diseases. (C) 2014 Elsevier Inc. All rights reserved.

An abnormal accumulation of cholesterol oxidation products in the brain of patients with Alzheimer's disease (AD) would further link an impaired cholesterol metabolism in the pathogenesis of the disease. The first evidence stemming from the content of oxysterols in autopsy samples from AD and normal brains points to an increase in both 27-hydroxycholesterol (27-OH) and 24-hydroxycholesterol (24-OH) in the frontal cortex of AD brains, with a trend that appears related to the disease severity. The challenge of differentiated SK-N-BE human neuroblastoma cells with patho-physiologically relevant amounts of 27-OH and 24-OH showed that both oxysterols induce a net synthesis of Aβ1-42 by up-regulating expression levels of amyloid precursor protein and β-secretase, as well as the β-secretase activity. Interestingly, cell pretreatment with N-acetyl-cysteine (NAC) fully prevented the enhancement of β-amyloidogenesis induced by the two oxysterols. The reported findings link an impaired cholesterol oxidative metabolism to an excessive β-amyloidogenesis and point to NAC as an efficient inhibitor of oxysterols-induced Aβ toxic peptide accumulation in the brain.

BACKGROUND Global sterol analysis is challenging owing to the extreme diversity of sterol natural products, the tendency of cholesterol to dominate in abundance over all other sterols, and the structural lack of a strong chromophore or readily ionized functional group. We developed a method to overcome these challenges by using different isotope-labeled versions of the Girard P reagent (GP) as quantitative charge-tags for the LC-MS analysis of sterols including oxysterols. METHODS Sterols/oxysterols in plasma were extracted in ethanol containing deuterated internal standards, separated by C18 solid-phase extraction, and derivatized with GP, with or without prior oxidation of 3β-hydroxy to 3-oxo groups. RESULTS By use of different isotope-labeled GPs, it was possible to analyze in a single LC-MS analysis both sterols/oxysterols that naturally possess a 3-oxo group and those with a 3β-hydroxy group. Intra- and interassay CVs were CONCLUSIONS This method allows the profiling of the widest range of sterols/oxysterols in a single analytical run and can be used to identify inborn errors of cholesterol synthesis and metabolism.

Obesity has been proposed as an energy balance disorder in which the expansion of adipose tissue (AT) leads to unfavorable health outcomes. Even though adiposity represents the most powerful driving force for the development of insulin resistance (IR) and type 2 diabetes, mounting evidence points to "adipose dysregulation", rather than fat mass accrual per se, as a key pathophysiological trigger of the obesity-linked metabolic complications. The dysfunctional fat, besides hypertrophic adipose cells and inflammatory cues, displays a reduced ability to form new adipocytes from the undifferentiated precursor cells (ie, the preadipocytes). The failure of adipogenesis poses a "diabetogenic" milieu either by promoting the ectopic overflow/deposition of lipids in non-adipose targets (lipotoxicity) or by inducing a dysregulated secretion of different adipose-derived hormones (ie, adipokines and lipokines). This novel and provocative paradigm ("expandability hypothesis") further extends current "adipocentric view" implicating a reduced adipogenic capacity as a missing link between "unhealthy" fat expansion and impairment of metabolic homeostasis. Hitherto, reactive oxygen species have been implicated in multiple forms of IR. However, the effects of stress on adipogenesis remain controversial. Compelling circumstantial data indicate that lipid peroxidation by-products (ie, oxysterols and 4-hydrononenal) may detrimentally affect adipose homeostasis partly by impairing (pre)adipocyte differentiation. In this scenario, it is tempting to speculate that a fine tuning of the adipose redox status may provide new mechanistic insights at the interface between fat dysregulation and development of metabolic dysfunctions. Yet, in humans, the molecular "signatures" of oxidative stress in the dysregulated fat as well as the pathophysiological effects of adipose (per)oxidation on glucose homeostasis remain poorly investigated. In this review we will summarize the potential mechanisms by which increased oxidative stress in fat may impair (pre)adipocyte differentiation and promote the adipose dysfunction. We will also attempt to highlight the conundrum with the adipose redox changes and the regulation of glucose homeostasis. Finally, we will briefly discuss the scientific rationale for proposing the adipose redox state as a potential target for novel therapeutic strategies to curb/prevent adiposity-linked insulin resistance.

Dipyridamole was introduced on the market as coronary vasodilator drug more than half a century ago and is still used as antithrombotic and vasodilator. Among cellular targets, it inhibits phosphodiesterases and raises extracellular levels of adenosine through inhibition of adenosine reuptake by red blood cells. As a consequence, endocellular levels of cyclic nucleotides are upregulated. The rise of cGMP in vascular smooth mu scle cells and of cAMP in platelets provide the mechanism of vasodilation and antithrombosis, which are further potentiated by the release of PGI 2 consequent on the increase in endothelial cell cAMP. These effects support the use of dipyridamole in cardiovascular diseases in which the drug is approved for the secondary prevention of cerebrovascular events. On the other hand, dipyridamole has been shown to possess a potent, little perceived, antioxidant activity of potential use in the several fields where pathophysiological pathways are dependent on oxidative stress, including those occurring in atherosclerosis, thrombosis, CNS-related diseases, and cancer.

Throughout life, stress stimuli act upon the brain leading to morphological and functional changes in advanced age, when it is likely to develop neurodegenerative disorders. There is an increasing need to unveil the molecular mechanisms underlying aging, in a world where populations are getting older. Egr-1 (early growth response 1), a transcriptional factor involved in cell survival, proliferation and differentiation - with a role also in memory, cognition and synaptic plasticity, can be implicated in the molecular mechanism of the aging process. Moreover, Heme Oxygenase-1a (HO), a 32 kDa heat-shock protein that converts heme to iron, carbon monoxide and biliverdin, is a key enzyme with neuroprotective properties. Several in vitro and in vivo studies reported that HO-1 could regulate the metabolism of oxysterols, oxidation products of cholesterol that include markers of oxidative stress. Recently, a link between Egr-1 and HO-1 has been demonstrated in mouse lung cells exposed to cigarette smoke. In view of these data, we wanted to investigate whether Egr-1 can be implicated also in the oxysterol metabolism during brain aging. Our results show that Egr-1 expression is differently expressed in the cortex and hippocampus of old mice, as well as the oxysterol profile between these two brain areas. In particular, we show that the cortex experiences in an age-dependent fashion increasing levels of the Egr-1 protein, and that these correlate with the level of HO-1 expression and oxysterol abundance. Such a situation was not observed in the hippocampus. These results are further strenghtened by our observations made with Egr-1 KO mice, confirming our hypothesis concerning the influence of Egr-1 on oxysterol production and accumulation via regulation of the expression of HO-1 in the cortex, but not the hippocampus, of old mice. It is important to notice that most of the oxysterols involved in this process are those usually stimulated by oxidative stress, which would then represent the triggering factor for this mechanism. Keywords: Egr-1, aging brain, oxysterols, HO-1, oxidative stress, cortex, hippocampus