Development of methods for analyzing phthalate-type plasticizers and bile acids in biological matrices : applications in different pathophysiological contexts

Ce travail de thèse s'articule autour de deux axes :1/ Les plastifiants, y compris les phtalates, ont été identifiés comme cancérigènes, mutagènes, reprotoxiques (CMR) de catégorie 1b et comme perturbateurs endocriniens. Le di-2-éthylhexyle phtalate (DEHP) est l'un des plastifiants les plus courants et est généralement associé au polychlorure de vinyle (PVC) dans les dispositifs médicaux. Le DEHP n'étant pas lié de manière covalente au PVC, il peut facilement migrer dans des matrices lipophiles et atteindre ensuite la circulation sanguine. Il est métabolisé par le foie en mono-2-éthylhexyle phtalate (MEHP), tout aussi toxique. Ces dernières années, des plastifiants alternatifs au DEHP ont été développés, notamment le di-2-éthylhexyl téréphtalate (DEHT), qui est métabolisé in vivo en mono-2-éthylhexyl téréphtalate (MEHT).La première partie de ce travail de thèse a consisté à développer des méthodes de dosage des plastifiants et de leurs métabolites dans diverses matrices biologiques, comme par exemple le plasma. Deux méthodes LC-MS/MS ont été développées pour la détermination du DEHP et du MEHP ainsi que des métabolites du DEHT. L'ionisation par spectrométrie de masse du DEHT étant très faible, une méthode LC-UV a été développée pour quantifier ce téréphtalate. Ces méthodes ont permis d'estimer le relargage du DEHP et du DEHT à partir de poches de sang et le dosage de leurs métabolites primaires dans les produits sanguins.2/ Les acides biliaires (AB) constituent une large famille de stéroïdes composée de nombreuses espèces. Ils sont synthétisés dans le foie et l'intestin et représentent la voie principale de catabolisme du cholestérol. Le 7a-hydroxy-4-cholesten-3-one (C4) est le précurseur des AB. Les AB jouent un rôle essentiel dans l'absorption des lipides mais également dans la signalisation cellulaire, étant ligands du récepteur nucléaire « Farnesoid X receptor » (FXR) et/ou du récepteur membranaire couplé aux protéines G, TGR5. Ces récepteurs, et donc leurs ligands, sont impliqués dans la régulation de l'homéostasie du glucose, des lipides et de la dépense énergétique. Toute modulation du profil des AB peut donc conduire à une modification de l'homéostasie métabolique.La deuxième partie de ce travail de thèse a consisté à développer deux méthodes de dosage par LC-MS/MS de 31 espèces d'AB et du C4 dans différentes matrices biologiques, dont le plasma. Une méthode spécifique permettant le dosage d'AB dérivés du LCA, récemment décrits, dans du contenu caecal est en cours d'optimisation. Ces méthodes ont permis d'analyser les variations du profil des AB dans divers contextes de la maladie cardiométabolique (obésité, insulino-résistance, diabète de type 2, NAFLD).Pour conclure, les méthodes d'analyse développées pour la quantification des plastifiants et des AB ont été validées et appliquées dans des études précliniques et cliniques. De manière intéressante, des données de la littérature ainsi que des essais préliminaires de transfection transitoire ont montré que des phtalates et leurs métabolites modulent l'activité du récepteur alpha activé par les proliférateurs de peroxysomes (PPARa) régulateur clé de l'homéostasie métabolique et de l'expression de l'enzyme CYP7A1 (enzyme majeure de la synthèse hépatique des AB). Les outils analytiques développés dans cette thèse permettent d'ouvrir des perspectives originales d'étude des effets des phtalates sur l'homéostasie métabolique via la régulation du métabolisme des AB.L'ensemble de ces travaux a permis de lier développements analytiques et applications dans le domaine de la biologie et de la santé.This thesis has two main focuses:1/ Plasticizers, including phthalates, have been identified as category 1b carcinogenic, mutagenic and reprotoxic (CMR) and as endocrine disruptors. Di-2-ethylhexyl phthalate (DEHP) is one of the most common plasticizers and is generally associated with polyvinyl chloride (PVC) in medical devices. As DEHP is not covalently bound to PVC, it can easily migrate into lipophilic matrices and then reach the bloodstream. It is metabolized by the liver into mono-2-ethylhexyl phthalate (MEHP), which is just as toxic. In recent years, alternative plasticizers to DEHP have been developed, notably di-2-ethylhexyl terephthalate (DEHT), which is metabolized in vivo to mono-2-ethylhexyl terephthalate (MEHT).The first part of this thesis involved developing methods for measuring plasticizers and their metabolites in various biological matrices, such as plasma. Two LC-MS/MS methods were developed for the determination of DEHP and MEHP as well as DEHT metabolites. As the ionization in mass spectrometry of DEHT is very low, a LC-UV method was developed to quantify this terephthalate. These methods have made it possible to estimate the release of DEHP and DEHT from blood bags and to measure their primary metabolites in blood products.2/ Bile acids (BA) are a large family of steroids made up of numerous species. They are synthesized in the liver and intestine and represent the main route of cholesterol catabolism. 7a-hydroxy-4-cholesten-3-one (C4) is the precursor of BA. BA play an essential role in lipid absorption but also in cell signaling, as they are ligands for the nuclear receptor 'Farnesoid X receptor' (FXR) and/or the G protein-coupled membrane receptor, TGR5. These receptors, and hence their ligands, are involved in glucose homeostasis, lipid homeostasis and energy expenditure. Any modulation of the BA profile can therefore lead to changes in metabolic homeostasis. The second part of this thesis involved developing two LC-MS/MS assay methods for 31 BA species and C4 in different biological matrices, including plasma. A specific method for the determination of recently described BA derived from LCA in caecal contents is currently being optimized. These methods have made it possible to analyze variations in the BA profile in various cardiometabolic disease contexts (obesity, insulin resistance, type 2 diabetes, NAFLD).In conclusion, the analytical methods developed for quantifying plasticizers and BA have been validated and applied in preclinical and clinical studies. Interestingly, data from the literature and preliminary transient transfection assays have shown that phthalates and their metabolites modulate the activity of the peroxisome proliferator-activated receptor alpha (PPARa), a key regulator of metabolic homeostasis and expression of CYP7A1 (a major enzyme in hepatic BA synthesis). The analytical tools developed in this thesis open up original perspectives for studying the effects of phthalates on metabolic homeostasis via the regulation of BA metabolism. All of this work has made it possible to link analytical developments and applications in the field of biology and health

Detection of 13 emerging soil pollutant compounds using a dual extraction method (QuEChERS and solid phase extraction) and a liquid chromatography/mass spectrometry LC-MS/MS method

International audienceEmerging pollutants derived from human and animal sources, are present in soils and pose sig- nificant environmental and health impacts, even at low concentrations. Their detection in soil is analytically complex due to soil interference and the rapid degradation of compounds in the matrix. In this study, a protocol was optimized for quantifying hormonal steroids ( n = 7), human drugs ( n = 3), and antibiotics ( n = 3) by a dual-phase extraction using QuEChERS and Solid Phase Extraction (SPE), followed by analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The double extraction phase allows an accurate and effective purification of the tar- get compounds while eliminating the interference in the soil matrix. The method is optimized to detect environmental concentrations of these pollutants, to suit large-scale sampling campaigns and to maintain the efficiency of extraction while reducing analysis time. The limits of detection (LODs) of these compounds ranged between 0.0043 and 0.13 ng/g and recovery rates between 75.9 % and 105.39 %. • Enhanced Analyte Purification: Implements QuEChERS and SPE for robust removal of matrix interferences, optimizing target compound isolation. • Precision at Trace Levels: Secures LODs as minimal as 0.0043 ng/g, enabling accurate detec- tion of low-concentration contaminants. • Adapted for Broad-scale sampling: Modifies extraction and analysis durations to accommodate large-scale environmental assessments

Plasma asymmetric dimethylarginine concentrations are not related to differences in maximal oxygen uptake in endurance trained and untrained men

International audienceWhat is the central question of this study? Is there an association of plasma concentration of asymmetric dimethylarginine, which is related to exercise capacity in patients with cardiovascular diseases, with oxygen delivery and subsequently exercise capacity in healthy subjects in the absence of the potentially confounding influence of inflammation and oxidative stress? What is the main finding and its importance? Plasma asymmetric dimethylarginine concentrations are not related to exercise capacity in healthy subjects, while O2Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthesis that could limit oxygen (O2-1-1-1-12-1-1-1-1-1-1-1-1-1-1

A pharmaco-metabolomics approach in a clinical trial of ALS: Identification of predictive markers of progression.

There is an urgent and unmet need for accurate biomarkers in Amyotrophic Lateral Sclerosis. A pharmaco-metabolomics study was conducted using plasma samples from the TRO19622 (olesoxime) trial to assess the link between early metabolomic profiles and clinical outcomes. Patients included in this trial were randomized into either Group O receiving olesoxime (n = 38) or Group P receiving placebo (n = 36). The metabolomic profile was assessed at time-point one (V1) and 12 months (V12) after the initiation of the treatment. High performance liquid chromatography coupled with tandem mass spectrometry was used to quantify 188 metabolites (Biocrates® commercial kit). Multivariate analysis based on machine learning approaches (i.e. Biosigner algorithm) was performed. Metabolomic profiles at V1 and V12 and changes in metabolomic profiles between V1 and V12 accurately discriminated between Groups O and P (p<5×10-6), and identified glycine, kynurenine and citrulline/arginine as the best predictors of group membership. Changes in metabolomic profiles were closely linked to clinical progression, and correlated with glutamine levels in Group P and amino acids, lipids and spermidine levels in Group O. Multivariate models accurately predicted disease progression and highlighted the discriminant role of sphingomyelins (SM C22:3, SM C24:1, SM OH C22:2, SM C16:1). To predict SVC from SM C24:1 in group O and SVC from SM OH C22:2 and SM C16:1 in group P+O, we noted a median sensitivity between 67% and 100%, a specificity between 66.7 and 71.4%, a positive predictive value between 66 and 75% and a negative predictive value between 70% and 100% in the test sets. This proof-of-concept study demonstrates that the metabolomics has a role in evaluating the biological effect of an investigational drug and may be a candidate biomarker as a secondary outcome measure in clinical trials

Control of lipid metabolism by the dynamic and nutrient‐dependent post‐translational modification O‐GlcNAcylation

Abstract O‐GlcNAcylation is a post‐translational modification belonging to the large group of glycosylations. It consists of the modification of cytoplasmic, nuclear, and mitochondrial proteins with a single N‐acetylglucosamine residue by O‐GlcNAc transferase (OGT). Despite its structural simplicity, O‐GlcNAcylation orchestrates many functions inside the cell. This modification regulates fatty acids synthesis, fat storage, and utilization. The generation of white and brown adipocyte‐OGT knock‐out mice has highlighted the marked interference of O‐GlcNAcylation in adiposity and, as a consequence, in metabolic pathologies. OGT is more especially involved in the regulation of lipolysis, and thermogenesis in brown adipose tissue. In addition, O‐GlcNAcylation directly regulates fatty acid synthase, the main enzyme responsible for fatty acids synthesis, and other lipogenic enzymes and transcription factors. Nevertheless, only a few studies reported connections between O‐GlcNAcylation and homeostasis of cholesterol or its derivatives. This knowledge gap is surprising due to the crucial importance of cholesterol in structuring animal biological membranes and as a precursor of a wide variety of biological compounds. Here, we review the current literature about this topic and discuss future prospects in the field. Key points As a PTM, O‐GlcNAcylation exponentially expands protein functions. O‐GlcNAcylation orchestrates many biological functions in living beings including metabolic fluxes. O‐GlcNAcylation is crucial for fat storage and mobilization, and for fatty acid synthesis but its function in the metabolism of other lipid compounds is less documented

Multiple Selection Criteria for Probiotic Strains with High Potential for Obesity Management

International audienceSince alterations of the gut microbiota have been shown to play a major role in obesity, probiotics have attracted attention. Our aim was to identify probiotic candidates for the management of obesity using a combination of in vitro and in vivo approaches. We evaluated in vitro the ability of 23 strains to limit lipid accumulation in adipocytes and to enhance the secretion of satiety-promoting gut peptide in enteroendocrine cells. Following the in vitro screening, selected strains were further investigated in vivo, single, or as mixtures, using a murine model of diet-induced obesity. Strain Bifidobacterium longum PI10 administrated alone and the mixture of B. animalis subsp. lactis LA804 and Lactobacillus gasseri LA806 limited body weight gain and reduced obesity-associated metabolic dysfunction and inflammation. These protective effects were associated with changes in the hypothalamic gene expression of leptin and leptin receptor as well as with changes in the composition of gut microbiota and the profile of bile acids. This study provides crucial clues to identify new potential probiotics as effective therapeutic approaches in the management of obesity, while also providing some insights into their mechanisms of action

One-week cocoa flavanol intake increases prefrontal cortex oxygenation at rest and during moderate-intensity exercise in normoxia and hypoxia.

During exercise in hypoxia, O2 delivery to brain and muscle is compromised, and oxidative stress is elicited. Cocoa flavanols (CF) have antioxidant capacities and can increase blood flow by stimulating endothelial function. We aimed to examine the effects of 7-day CF intake on oxidative stress, nitric oxide production, and tissue oxygenation in response to exercise in normobaric hypoxia (14.3% O2). In a randomized, double-blind, cross-over study, 14 well-trained male cyclists completed four trials: exercise in normoxia or hypoxia, after 7-day CF or placebo intake. Flow-mediated dilation (FMD) was measured before intake of the last dose CF or placebo. One hundred minutes later, 20-min steady-state (SS; 45% V̇o2max) and 20-min time trial (TT) (cycling) were performed. Blood samples were taken. Prefrontal and muscular oxygenation was assessed by near-infrared spectroscopy. At baseline, FMD was increased by CF. Hypoxia increased exercise-induced elevations in lipid peroxidation and antioxidant capacity. CF suppressed exercise-induced lipid peroxidation but did not influence antioxidant capacity. At rest and during SS, prefrontal and muscular oxygenation was decreased by hypoxia. CF elevated prefrontal oxygenation but did not impact muscular oxygenation. During TT, hypoxia accelerated the exercise-induced decrease in prefrontal oxygenation, but not in muscular oxygenation. During TT, CF did not alter prefrontal and muscular oxygenation. CF did not change plasma nitrite, nitrate, and arginine:citrulline. During high-intensity exercise, CF improved neither tissue oxygenation nor performance in well-trained athletes. At rest and during moderate-intensity exercise, CF reduced exercise-induced lipid peroxidation and partially restored the hypoxia-induced decline in prefrontal oxygenation. NEW & NOTEWORTHY For the first time, we showed that CF had beneficial effects on endothelial function at rest, as well as on prefrontal oxygenation at rest and during moderate-intensity exercise, both in normoxia and hypoxia. Moreover, we showed that CF intake inhibited oxidative stress during exhaustive exercise in hypoxia.info:eu-repo/semantics/publishe

Circulating biomarkers of nitric oxide bioactivity and impaired muscle vasoreactivity to exercise in adults with uncomplicated type 1 diabetes

International audienceAims/hypothesisEarly compromised endothelial function challenges the ability of individuals with type 1 diabetes to perform normal physical exercise. The exact mechanisms underlying this vascular limitation remain unknown, but may involve either formation or metabolism of nitric oxide (NO), a major vasodilator, whose activity is known to be compromised by oxidative stress.MethodsMuscle microvascular reactivity (near-infrared spectroscopy) to an incremental exhaustive bout of exercise was assessed in 22 adults with uncomplicated type 1 diabetes (HbA1c 64.5 ± 15.7 mmol/mol; 8.0 ± 1.4%) and in 21 healthy individuals (18–40 years of age). NO-related substrates/metabolites were also measured in the blood along with other vasoactive compounds and oxidative stress markers; measurements were taken at rest, at peak exercise and after 15 min of recovery. Demographic characteristics, body composition, smoking status and diet were comparable in both groups.ResultsMaximal oxygen uptake was impaired in individuals with type 1 diabetes compared with in healthy participants (35.6 ± 7.7 vs 39.6 ± 6.8 ml min−1 kg−1, p < 0.01) despite comparable levels of habitual physical activity (moderate to vigorous physical activity by accelerometery, 234.9 ± 160.0 vs 280.1 ± 114.9 min/week). Compared with non-diabetic participants, individuals with type 1 diabetes also displayed a blunted exercise-induced vasoreactivity (muscle blood volume at peak exercise as reflected by ∆ total haemoglobin, 2.03 ± 5.82 vs 5.33 ± 5.54 μmol/l; interaction ‘exercise’ × ‘group’, p < 0.05); this was accompanied by lower K+ concentration (p < 0.05), reduced plasma L-arginine (p < 0.05)—in particular when HbA1c was high (mean estimation: −4.0, p < 0.05)—and lower plasma urate levels (p < 0.01). Nonetheless, exhaustive exercise did not worsen lipid peroxidation or other oxidative stress biomarkers, and erythrocytic enzymatic antioxidant resources were mobilised to a comparable extent in both groups. Nitrite and total nitrosation products, which are potential alternative NO sources, were similarly unaltered.Conclusions/interpretationParticipants with uncomplicated type 1 diabetes displayed reduced availability of L-arginine, the essential substrate for enzymatic nitric oxide synthesis, as well as lower levels of the major plasma antioxidant, urate. Lower urate levels may reflect a defect in the activity of xanthine oxidase, an enzyme capable of producing NO from nitrite under hypoxic conditions. Thus, both canonical and non-canonical NO production may be reduced. However, neither of these changes exacerbated exercise-induced oxidative stress

Omics to Explore Amyotrophic Lateral Sclerosis Evolution: the Central Role of Arginine and Proline Metabolism

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Bile acids associate with glucose metabolism, but do not predict conversion from impaired fasting glucose to diabetes

International audienceObjective: Bile acids (BAs) are signaling molecules controlling lipid and glucose metabolism. Since BA alterations are associated with obesity and insulin resistance, plasma BAs have been considered candidates to predict type 2 diabetes (T2D) risk. We aimed to determine (1) the association of BAs with glucose homeostasis parameters and (2) their predictive association with the risk of conversion from prediabetes to new-onset diabetes (NOD) in a prospective cohort study.Design: 205 patients with impaired fasting glucose (IFG) were followed each year during 5 years in the IT-DIAB cohort study. Twenty-one BA species and 7α-hydroxy-4-cholesten-3-one (C4), a marker of BA synthesis, were quantified by LC/MS-MS in plasma from fasted patients at baseline. Correlations between plasma BA species and metabolic parameters at baseline were assessed by Spearman's coefficients and the association between BAs and NOD was determined using Cox proportional-hazards models.Results: Among the analyzed BA species, total hyocholic acid (HCA) and the total HCA/total chenodeoxycholic acid (CDCA) ratio, reflecting hepatic BA 6α-hydroxylation activity, negatively correlated with BMI and HOMA-IR. The total HCA/total CDCA ratio also correlated negatively with HbA1C. Conversion from IFG to NOD occurred in 33.7% of the participants during the follow-up. Plasma BA species were not independently associated with the conversion to NOD after adjustment with classical T2D risk factors.Conclusions: Fasting plasma BAs are not useful clinical biomarkers for predicting NOD in patients with IFG. However, an unexpected association between 6α-hydroxylated BAs and glucose parameters was found, suggesting a role for this specific BA pathway in metabolic homeostasis. IT-DIAB study registry number: NCT01218061