Early Metabolic Disruption and Predictive Biomarkers of Delayed-Cerebral Ischemia in Aneurysmal Subarachnoid Hemorrhage.

Delayed cerebral ischemia (DCI) following aneurysmal subarachnoid hemorrhage (aSAH) is a major cause of complications and death. Here, we set out to identify high-performance predictive biomarkers of DCI and its underlying metabolic disruptions using metabolomics and lipidomics approaches. This single-center prospective observational study enrolled 61 consecutive patients with severe aSAH; among them, 22 experienced a DCI. Nine patients without aSAH were included as validation controls. Blood and cerebrospinal fluid (CSF) were sampled within the first 24 h after admission. We identified a panel of 20 metabolites that, together, showed high predictive performance for DCI. This panel of metabolites included lactate, cotinine, salicylate, 6 phosphatidylcholines, and 4 sphingomyelins. The interplay of the metabolome and the lipidome found between CSF and plasma in our patients underscores that aSAH and its associated DCI complications can extend beyond cerebral implications, with a peripheral dimension as well. As an illustration, early biological disruptions that might explain the subsequent DCI found systemic hypoxia driven mainly by higher blood lactate, arginine, and proline metabolism likely associated with vascular NO and disrupted ceramide/sphingolipid metabolism. We conclude that targeting early peripheral hypoxia preceding DCI could provide an interesting strategy for the prevention of vascular dysfunction.

Prolonged dysbiosis and altered immunity under nutritional intervention in a physiological mouse model of severe acute malnutrition.

Severe acute malnutrition (SAM) is a multifactorial disease affecting millions of children worldwide. It is associated with changes in intestinal physiology, microbiota, and mucosal immunity, emphasizing the need for multidisciplinary studies to unravel its full pathogenesis. We established an experimental model in which weanling mice fed a high-deficiency diet mimic key anthropometric and physiological features of SAM in children. This diet alters the intestinal microbiota (less segmented filamentous bacteria, spatial proximity to epithelium), metabolism (decreased butyrate), and immune cell populations (depletion of LysoDC in Peyer's patches and intestinal Th17 cells). A nutritional intervention leads to a fast zoometric and intestinal physiology recovery but to an incomplete restoration of the intestinal microbiota, metabolism, and immune system. Altogether, we provide a preclinical model of SAM and have identified key markers to target with future interventions during the education of the immune system to improve SAM whole defects.

Deep phenotyping and biomarkers of various dairy fat intakes in an 8-week randomized clinical trial and 2-year swine study.

Health effects of dairy fats (DF) are difficult to evaluate, as DF intakes are hard to assess epidemiologically and DF have heterogeneous compositions that influence biological responses. We set out to find biomarkers of DF intake and assess biological response to a summer DF diet (R2), a winter DF diet (R3), and a R3 supplemented with calcium (R4) compared to a plant-fat-based diet (R1) in a randomized clinical trial (n=173) and a 2-year study in mildly metabolically disturbed downsized pigs (n=32). Conventional clinical measures were completed by LC/MS plasma metabolomics/lipidomics. The measured effects were modeled as biological functions to facilitate interpretation. DF intakes in pigs specifically induced a U-shaped metabolic trajectory, reprogramming metabolism to close to its initial status after a one-year turnaround. Twelve lipid species repeatably predicted DF intakes in both pigs and humans (6.6% errors). More broadly, in pigs, quality of DF modulated the time-related biological response (R2: 30 regulated functions, primarily at 6 months; R3: 26 regulated functions, mostly at 6-12 months; R4: 43 regulated functions, mostly at 18 months). Despite this heterogeneity, 9 functions overlapped under all 3 DF diets in both studies, related to a restricted area of amino acids metabolism, cofactors, nucleotides and xenobiotic pathways and the microbiota. In conclusion, over the long-term, DF reprograms metabolism to close to its initial biological status in metabolically-disrupted pigs. Quality of the DF modulates its metabolic influence, although some effects were common to all DF. A resilient signature of DF consumption found in pigs was validated in humans.

Vitamin D3 Supplementation Alleviates Left Ventricular Dysfunction in a Mouse Model of Diet-Induced Type 2 Diabetes: Potential Involvement of Cardiac Lipotoxicity Modulation.

PURPOSE: To evaluate the effectiveness of vitamin D3 supplementation, in secondary prevention, on cardiac remodeling and function, as well as lipid profile, in a mouse model of diet-induced type 2 diabetes. METHODS: Mice were fed a high fat and sucrose diet for 10 weeks. Afterward, diet was maintained for 15 more weeks and two groups were formed, with and without cholecalciferol supplementation. A control group was fed with normal chow. Glucose homeostasis and cardiac function were assessed at baseline and at the 10th and 24th weeks. Animals were killed at the 10th and 25th weeks for plasma and cardiac sample analysis. Cardiac lipid profile was characterized by LC-MS/MS. RESULTS: After 10 weeks of diet, mice exhibited pre-diabetes, mild left ventricle hypertrophy, and impaired longitudinal strain, but preserved myocardial circumferential as well as global diastolic and systolic cardiac function. After 15 more weeks of diet, animals presented with well-established type 2 diabetes, pathological cardiac hypertrophy, and impaired regional myocardial function. Cholecalciferol supplementation had no effect on glucose homeostasis but improved cardiac remodeling and regional myocardial function. After 25 weeks, non-supplemented mice exhibited increased myocardial levels of ceramides and diacylglycerol, both of which were normalized by vitamin D3 supplementation. CONCLUSION: This work brought to light the beneficial effects of cholecalciferol supplementation, in secondary prevention, on cardiac remodeling and function in a mouse model of diet-induced type 2 diabetes. Those cardioprotective effects may be, at least in part, attributed to the modulation of myocardial levels of lipotoxic species by vitamin D.

The Complex ABCG5/ABCG8 Regulates Vitamin D Absorption Rate and Contributes to its Efflux from the Intestine.

SCOPE: Most people are vitamin D insufficient around the world. Vitamin D intestinal absorption should thus be optimized. The role of the ATP-binging cassette G5/G8 (ABCG5/G8) heterodimer in vitamin D intestinal efflux is investigated. METHODS AND RESULTS: Both cholecalciferol and 25-hydroxycholecalciferol apical effluxes are increased by ABCG5/G8 overexpression in human Griptite cells. Mice deficient in ABCG5/G8 at the intestinal level (I-Abcg5/g8-/- mice) display an accumulation of cholecalciferol in plasma in females and in liver in males compared to control animals. I-Abcg5/g8-/- mice display a delay in cholecalciferol postprandial response after gavage compared with controls. 25-Hydroxycholecalciferol transfer from plasma to lumen is observed in vivo in intestine-perfused mice, and the lack of intestinal ABCG5/G8 complex induces a decrease in this efflux, while vitamin D bile excretion remains unchanged. CONCLUSION: Overall, it is showed for the first time that the ABCG5/G8 heterodimer regulates the kinetics of absorption of dietary vitamin D by contributing to its efflux back to the lumen, and that it also participates in vitamin D transintestinal efflux.

Four days high fat diet modulates vitamin D metabolite levels and enzymes in mice.

Obesity is classically associated with low serum total and free 25(OH)D. Hypotheses have been advanced to explain this observation but mechanisms remain poorly understood, and notably priming events that could explain such association. We investigated the impact of short-term high fat (HF) diet to investigate early events occurring in vitamin D metabolism. Male C57BL/6J mice were fed with a control diet (control group) and HF diet for 4 days. HF fed mice displayed similar body weight to control mice but significantly increased adiposity, together with a decrease of free 25(OH)D concentrations, which could be explained at least in part by a decrease of Cyp2r1 and Cyp3a11 expression in the liver. An increase of 1,25(OH)2D concentration was also observed and could be explained by a decrease of Cyp24a1 expression observed in the kidney. In white adipose tissue (WAT), no modification of vitamin D metabolites quantity detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Nevertheless, an increase of Cyp2r1 and Cyp27a1 mRNA expression and a decrease of Cyp27b1 mRNA expression could suggest a possible storage of 25(OH)D in WAT at long-term. Our data are supportive of an active role of HF diet in mediating a priming effect leading the well-established perturbation of the vitamin D metabolism associated with obesity, including a decrease of free 25(OH)D and modulation of expression of genes involved in vitamin D metabolism.

2',7'-dichlorofluorescin-based analysis of Fenton chemistry reveals auto-amplification of probe fluorescence and albumin as catalyst for the detection of hydrogen peroxide.

Fluorophore 2',7'-dichlorofluorescin (DCF) is the most frequently used probe for measuring oxidative stress in cells, but many aspects of DCF remain to be revealed. Here, DCF was used to study the Fenton reaction in detail, which confirmed that in a cell-free system, the hydroxyl radical was easily measured by DCF, accompanied by the consumption of H2O2 and the conversion of ferrous iron into ferric iron. DCF fluorescence was more specific for hydroxyl radicals than the measurement of thiobarbituric acid (TBA)-reactive 2-deoxy-D-ribose degradation products, which also detected H2O2. As expected, hydroxyl radical-induced DCF fluorescence was inhibited by iron chelation, anti-oxidants, and hydroxyl radical scavengers and enhanced by low concentrations of ascorbate. Remarkably, due to DCF fluorescence auto-amplification, Fenton reaction-induced DCF fluorescence steadily increased in time even when all ferrous iron was oxidized. Surprisingly, the addition of bovine serum albumin rendered DCF sensitive to H2O2 as well. Within cells, DCF appeared not to react directly with H2O2 but indirect via the formation of hydroxyl radicals, since H2O2-induced cellular DCF fluorescence was fully abolished by iron chelation and hydroxyl radical scavenging. Iron chelation in H2O2-stimulated cells in which DCF fluorescence was already increasing did not abrogate further increases in fluorescence, suggesting DCF fluorescence auto-amplification in cells. Collectively, these data demonstrate that DCF is a very useful probe to detect hydroxyl radicals and hydrogen peroxide and to study Fenton chemistry, both in test tubes as well as in intact cells, and that fluorescence auto-amplification is an intrinsic property of DCF.

A Combination of Single Nucleotide Polymorphisms is Associated with the Interindividual Variability of Cholesterol Bioavailability in Healthy Adult Males.

SCOPE: Cholesterol bioavailability displays a high interindividual variability, partly due to genetic factors. Existing studies have focused on single nucleotide polymorphisms (SNPs) analyzed individually, which only explained a minor fraction of the variability of this complex phenotype. The aim is to identify a combination of SNPs associated with a significant part of the variability in cholesterol bioavailability. METHODS AND RESULTS: Thirty-nine healthy adult males are given a standard test snack containing 80 mg heptadeuterated (D7) cholesterol. The plasma D7-cholesterol concentration is measured at equilibrium 40 h after test snack intake. The D7-cholesterol response (D7-cholesterol/total cholesterol concentration) exhibits a relatively high interindividual variability (CV = 32%). The association of exonic SNPs in candidate genes (188 genes involved in or related to cholesterol metabolism) with the plasma D7-cholesterol response is assessed by univariate statistics followed by partial least squares regression. A significant model (p-value after cross-validation ANOVA = 1.64 × 10-7 ) that includes 8 SNPs (SOAT2-rs9658625, DNAH11-rs11768670, LIPC-rs690, MVK-rs2287218, GPAM-rs10787428, APOE-rs7412, CBS-rs234706, and WRN-rs1801196) explains 59.7% of the variance in cholesterol bioavailability (adjusted R²). CONCLUSION: Here a combination of SNPs is significantly associated with the variability in dietary cholesterol bioavailability in healthy adult males.

Simple Fast Quantification of Cholecalciferol, 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D in Adipose Tissue Using LC-HRMS/MS.

Vitamin D metabolism is actively modulated in adipose tissue during obesity. To better investigate this process, we develop a specific LC-HRMS/MS method that can simultaneously quantify three vitamin D metabolites, i.e., cholecalciferol, 25-hydroxyvitamin D3 (25(OH)D3), and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in a complex matrix, such as mouse adipose tissue and plasma. The method uses pretreatment with liquid-liquid or solid-phase extraction followed by derivatization using Amplifex® reagents to improve metabolite stability and ionization efficiency. Here, the method is optimized by co-eluting stable isotope-labelled internal standards to calibrate each analogue and to spike biological samples. Intra-day and inter-day relative standard deviations were 0.8-6.0% and 2.0-14.4%, respectively for the three derivatized metabolites. The limits of quantification (LoQ) achieved with Amplifex® derivatization were 0.02 ng/mL, 0.19 ng/mL, and 0.78 ng/mL for 1,25(OH)2D3, 25(OH)D3 and cholecalciferol, respectively. Now, for the first time, 1,25(OH)2D3 can be co-quantified with cholecalciferol and 25(OH)D3 in mouse adipose tissue. This validated method is successfully applied to study the impact of obesity on vitamin D status in mice.

Effects of dietary fat on insulin secretion in subjects with the metabolic syndrome.

Objective Impaired insulin secretion and action contribute to the development of type 2 diabetes. Dietary fat modification may improve insulin sensitivity, whereas the effect on insulin secretion is unclear. We investigated the effect of dietary fat modification on insulin secretion in subjects with the metabolic syndrome. Design In a 12-week pan-European parallel, randomized controlled dietary intervention trial (LIPGENE), 486 subjects were assigned to four isoenergetic diets: high-fat diets rich in saturated fat (HSFA) or monounsaturated fat (HMUFA) or low-fat, high-complex carbohydrate diets with (LFHCC n-3) or without (LFHCC control) 1.2 g/day of n-3 PUFA supplementation. Insulin secretion was estimated as acute insulin response to glucose (AIRg) and disposition index (DI), modeled from an intravenous glucose tolerance test. Results There were no overall effect of the dietary intervention on AIRg and DI in the total cohort, in neither the high-fat nor LFHCC groups. We observed significant diet*fasting glucose category interactions for AIRg (P = 0.021) and DI (P = 0.001) in the high-fat groups. In subjects with normal fasting glucose and preserved first phase insulin secretion, the HMUFA diet increased, whereas the HSFA diet reduced AIRg (P = 0.015) and DI (P = 0.010). Conclusions The effects of dietary fat modification on insulin secretion were minor, and only evident in normoglycemic subjects. In this case, the HMUFA diet improved AIRg and DI, as compared to the HSFA diet.

ABCB1 (P-glycoprotein) regulates vitamin D absorption and contributes to its transintestinal efflux.

Efficient intestinal absorption of dietary vitamin D is required in most people to ensure an adequate status. Thus, we investigated the involvement of ATP binding cassette subfamily B member 1 (ABCB1) in vitamin D intestinal efflux. Both cholecalciferol (D3) and 25-hydroxycholecalciferol [25(OH)D3] apical effluxes were decreased by chemical inhibition of ABCB1 in Caco-2 cells and increased by ABCB1 overexpression in Griptites or Madin-Darby canine kidney type II cells. Mice deficient for the 2 murine ABCB1s encoded by Abcb1a and Abcb1b genes ( Abcb1-/-) displayed an accumulation of 25(OH)D3 in plasma, intestine, brain, liver, and kidneys, together with an increased D3 postprandial response after gavage compared with controls. 25(OH)D3 efflux through Abcb1-/- intestinal explants was markedly decreased compared with controls. This reduction of 25(OH)D3 transfer from plasma to lumen was further confirmed in vivo in intestine-perfused mice. Docking experiments established that both D3 and 25(OH)D3 could bind with high affinity to Caenorhabditis elegans P-glycoprotein, used as an ABCB1 model. Finally, in a group of 39 healthy male adults, a single-nucleotide polymorphism (SNP) in ABCB1 (rs17064) was significantly associated with the fasting plasma 25(OH)D3 concentration. Thus, we showed here for the first time that ABCB1 is involved in neo-absorbed vitamin D efflux by the enterocytes and that it also contributes to vitamin D transintestinal excretion and likely impacts vitamin D status.-Margier, M., Collet, X., le May, C., Desmarchelier, C., André, F., Lebrun, C., Defoort, C., Bluteau, A., Borel, P., Lespine, A., Reboul, E. ABCB1 (P-glycoprotein) regulates vitamin D absorption and contributes to its transintestinal efflux.

Diet induced obesity modifies vitamin D metabolism and adipose tissue storage in mice.

Low circulating levels of total and free 25-hydroxyvitamin D (25(OH)D) indicative of vitamin D status have been associated with obesity in humans. Moreover, obesity is thought to play a causal role in the reduction of 25(OH)D levels, and several theories have been put forward to explain this relationship. Here we tested the hypothesis that obesity disrupts vitamin D homeostasis in key organs of vitamin D metabolism. Male C57BL6 mice were fed for 7 or 11 weeks on either a control diet (control, 10% energy from fat) or a high-fat diet (HF, 60% energy from fat) formulated to provide equivalent vitamin D3 intake in both groups. After 7 weeks, there was a transient increase of total 25(OH)D together with a significant decrease of plasma vitamin D3 that could be related to the induction of hepatic genes involved in 25-hydroxylation. After 11 weeks, there was no change in total 25(OH)D but a significant decrease of free 25(OH)D and plasma vitamin D3 levels. We also quantified an increase of 25(OH)D in adipose tissue that was inversely correlated to the free 25(OH)D. Interestingly, this accumulation of 25(OH)D in adipose tissue was highly correlated to the induction of Cyp2r1, which could actively participate in vitamin D3 trapping and subsequent conversion to 25(OH)D in adipose tissue. Taken together, our data strongly suggest that the enzymes involved in vitamin D metabolism, notably in adipose tissue, are transcriptionally modified under high-fat diet, thus contributing to the obesity-related reduction of free 25(OH)D.

Quantification of trans-resveratrol and its metabolites in human plasma using ultra-high performance liquid chromatography tandem quadrupole-orbitrap mass spectrometry.

Trans-resveratrol is a stilbene polyphenol with a large spectrum of biological activities. This is why it is widely studied in terms of activities, bioavailability and quantitation in different foods, beverages and biological matrices. Different analytical methods are employed for its quantitation. In this study a quadrupole-orbitrap tandem mass spectrometer coupled to a reverse phase ultra-high performance liquid chromatography is applied to a quantitation of trans-resveratrol and its metabolites trans-resveratrol-3-O-ß-d-glucuronide, trans-resveratrol-4'-O-ß-d-glucuronide, trans-resveratrol-3-O-sulfate, a,b-dihydroresveratrol, a,b-dihydroresveratrol-glucuronide, a,b-dihydroresveratrol-glucuronide-sulfate, a,b-dihydroresveratrol-sulfate, trans-resveratrol-3,5-O-ß-d-diglucuronide, trans-resveratrol-3,4'-O-d-ß-diglucuronide, trans-resveratrol-3-O-ß-d-glucuronide-sulfate and trans-resveratrol-4'-O-ß-d-glucuronide-sulfate in human plasma. MS/MS experiments coupled to a high resolving power and accurate mass measurements as well as the use of labeled internal standards enabled the achievement of linear calibration curves across the four orders of magnitude concentration ranges. The method was validated in terms of specificity and selectivity, accuracy and precision, sensitivity and matrix effect and can be now applied to pharmacokinetic studies or routine analysis. In addition, the application of quadrupole-orbitrap mass spectrometer to the quantitation of trans-resveratrol and its metabolites provides acquisition of full collision induced dissociation spectra of analyzed compounds giving place to the structural characterization and sensitivity and linear concentration ranges respecting the accuracy and precision, specificity and selectivity requirements.

Gene Expression Pattern in Response to Cholecalciferol Supplementation Highlights Cubilin as a Major Protein of 25(OH)D Uptake in Adipocytes and Male Mice White Adipose Tissue.

It is well established that the active form of vitamin D (i.e., 1,25-dihydroxyvitamin D [1,25(OH)2D]) regulates the expression of genes involved in its own metabolism and transport in the kidney and possibly in the liver. However, little is known about the transcriptional impact of cholecalciferol supplementation on white adipose tissue (WAT) and adipocytes, which are a major site of vitamin D and 25-hydroxyvitamin D [25(OH)D] storage in the organism. To fill this gap, we investigated the impact of cholecalciferol supplementation in WAT via a panel of genes coding for enzymes and proteins involved in vitamin D metabolism and uptake. Mice supplemented with cholecalciferol (15,000 IU/kg of body weight per day) for 4 days showed decreased messenger RNA (mRNA) levels of proteins involved in cholecalciferol metabolism (Cyp24a1, Cyp27a1) and decreased cubilin mRNA levels in WAT. These data were partly confirmed in 3T3-L1 adipocytes incubated with 1,25(OH)2D. The downregulation of cubilin mRNA observed in WAT and in 3T3-L1 was confirmed at the protein level in WAT and at the mRNA level in human primary adipocytes. Vitamin D receptor (VDR) agonist (EB1089) and RNA interference approaches demonstrated that VDR was involved in this regulation. Furthermore, chemical inhibitor and RNA inference analysis demonstrated that cubilin was involved in 25(OH)D uptake by adipocytes. This study established an overall snapshot of the genes regulated by cholecalciferol in mouse WAT and cell-autonomously in adipocytes. We highlighted that the regulation of cubilin expression was mediated by a VDR-dependent mechanism, and we demonstrated that cubilin was involved in 25(OH)D uptake by adipocytes.

APOE genotype influences insulin resistance, apolipoprotein CII and CIII according to plasma fatty acid profile in the Metabolic Syndrome.

Metabolic markers associated with the Metabolic Syndrome (MetS) may be affected by interactions between the APOE genotype and plasma fatty acids (FA). In this study, we explored FA-gene interactions between the missense APOE polymorphisms and FA status on metabolic markers in MetS. Plasma FA, blood pressure, insulin sensitivity and lipid concentrations were determined at baseline and following a 12-week randomized, controlled, parallel, dietary FA intervention in 442 adults with MetS (LIPGENE study). FA-APOE gene interactions at baseline and following change in plasma FA were assessed using adjusted general linear models. At baseline E4 carriers had higher plasma concentrations of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and apolipoprotein B (apo B) compared with E2 carriers; and higher TC, LDL-C and apo B compared with E3/E3. Whilst elevated plasma n-3 polyunsaturated FA (PUFA) was associated with a beneficially lower concentration of apo CIII in E2 carriers, a high proportion of plasma C16:0 was associated with insulin resistance in E4 carriers. Following FA intervention, a reduction in plasma long-chain n-3 PUFA was associated with a reduction in apo CII concentration in E2 carriers. Our novel data suggest that individuals with MetS may benefit from personalized dietary interventions based on APOE genotype.

Insulin resistance determines a differential response to changes in dietary fat modification on metabolic syndrome risk factors: the LIPGENE study.

BACKGROUND: Previous data support the benefits of reducing dietary saturated fatty acids (SFAs) on insulin resistance (IR) and other metabolic risk factors. However, whether the IR status of those suffering from metabolic syndrome (MetS) affects this response is not established. OBJECTIVE: Our objective was to determine whether the degree of IR influences the effect of substituting high-saturated fatty acid (HSFA) diets by isoenergetic alterations in the quality and quantity of dietary fat on MetS risk factors. DESIGN: In this single-blind, parallel, controlled, dietary intervention study, MetS subjects (n = 472) from 8 European countries classified by different IR levels according to homeostasis model assessment of insulin resistance (HOMA-IR) were randomly assigned to 4 diets: an HSFA diet; a high-monounsaturated fatty acid (HMUFA) diet; a low-fat, high-complex carbohydrate (LFHCC) diet supplemented with long-chain n-3 polyunsaturated fatty acids (1.2 g/d); or an LFHCC diet supplemented with placebo for 12 wk (control). Anthropometric, lipid, inflammatory, and IR markers were determined. RESULTS: Insulin-resistant MetS subjects with the highest HOMA-IR improved IR, with reduced insulin and HOMA-IR concentrations after consumption of the HMUFA and LFHCC n-3 diets (P < 0.05). In contrast, subjects with lower HOMA-IR showed reduced body mass index and waist circumference after consumption of the LFHCC control and LFHCC n-3 diets and increased HDL cholesterol concentrations after consumption of the HMUFA and HSFA diets (P < 0.05). MetS subjects with a low to medium HOMA-IR exhibited reduced blood pressure, triglyceride, and LDL cholesterol levels after the LFHCC n-3 diet and increased apolipoprotein A-I concentrations after consumption of the HMUFA and HSFA diets (all P < 0.05). CONCLUSIONS: Insulin-resistant MetS subjects with more metabolic complications responded differently to dietary fat modification, being more susceptible to a health effect from the substitution of SFAs in the HMUFA and LFHCC n-3 diets. Conversely, MetS subjects without IR may be more sensitive to the detrimental effects of HSFA intake. The metabolic phenotype of subjects clearly determines response to the quantity and quality of dietary fat on MetS risk factors, which suggests that targeted and personalized dietary therapies may be of value for its different metabolic features. This study was registered at clinicaltrials.gov as NCT00429195.

Can we trust untargeted metabolomics? Results of the metabo-ring initiative, a large-scale, multi-instrument inter-laboratory study.

The metabo-ring initiative brought together five nuclear magnetic resonance instruments (NMR) and 11 different mass spectrometers with the objective of assessing the reliability of untargeted metabolomics approaches in obtaining comparable metabolomics profiles. This was estimated by measuring the proportion of common spectral information extracted from the different LCMS and NMR platforms. Biological samples obtained from 2 different conditions were analysed by the partners using their own in-house protocols. Test #1 examined urine samples from adult volunteers either spiked or not spiked with 32 metabolite standards. Test #2 involved a low biological contrast situation comparing the plasma of rats fed a diet either supplemented or not with vitamin D. The spectral information from each instrument was assembled into separate statistical blocks. Correlations between blocks (e.g., instruments) were examined (RV coefficients) along with the structure of the common spectral information (common components and specific weights analysis). In addition, in Test #1, an outlier individual was blindly introduced, and its identification by the various platforms was evaluated. Despite large differences in the number of spectral features produced after post-processing and the heterogeneity of the analytical conditions and the data treatment, the spectral information both within (NMR and LCMS) and across methods (NMR vs. LCMS) was highly convergent (from 64 to 91 % on average). No effect of the LCMS instrumentation (TOF, QTOF, LTQ-Orbitrap) was noted. The outlier individual was best detected and characterised by LCMS instruments. In conclusion, untargeted metabolomics analyses report consistent information within and across instruments of various technologies, even without prior standardisation.

Effect on blood lipids of two daily servings of Camembert cheese. An intervention trial in mildly hypercholesterolemic subjects.

As a concentrated source of saturated fat, cheese consumption is considered to be associated with increased cholesterolemia and generally forbidden in dietary guidelines for adults with hypercholesterolemia. The aim of this study was to evaluate the impact of saturated fatty acids on lipid parameters and blood pressure with regards to different types of dairy products: Camembert and full-fat yoghurt. One-hundred and fifty-nine moderate hypercholesterolemic subjects without treatment were instructed to consume two full-fat yoghurts (2 × 125 g) per day for 3 weeks (run-in period) and then for a further period of 5 weeks, either two full-fat yoghurts or two 30 g servings of Camembert cheese per day. We observed that over the 5-week daily consumption of two servings of Camembert cheese, blood pressure and serum lipids did not change in moderate hypercholesterolemic subjects. These results suggest that fermented cheese such as Camembert could be consumed daily without affecting serum lipids or blood pressure.

Impact of geographical region on urinary metabolomic and plasma fatty acid profiles in subjects with the metabolic syndrome across Europe: the LIPGENE study.

The application of metabolomics in multi-centre studies is increasing. The aim of the present study was to assess the effects of geographical location on the metabolic profiles of individuals with the metabolic syndrome. Blood and urine samples were collected from 219 adults from seven European centres participating in the LIPGENE project (Diet, genomics and the metabolic syndrome: an integrated nutrition, agro-food, social and economic analysis). Nutrient intakes, BMI, waist:hip ratio, blood pressure, and plasma glucose, insulin and blood lipid levels were assessed. Plasma fatty acid levels and urine were assessed using a metabolomic technique. The separation of three European geographical groups (NW, northwest; NE, northeast; SW, southwest) was identified using partial least-squares discriminant analysis models for urine (R² X: 0·33, Q²: 0·39) and plasma fatty acid (R² X: 0·32, Q²: 0·60) data. The NW group was characterised by higher levels of urinary hippurate and N-methylnicotinate. The NE group was characterised by higher levels of urinary creatine and citrate and plasma EPA (20 : 5 n-3). The SW group was characterised by higher levels of urinary trimethylamine oxide and lower levels of plasma EPA. The indicators of metabolic health appeared to be consistent across the groups. The SW group had higher intakes of total fat and MUFA compared with both the NW and NE groups (P≤ 0·001). The NE group had higher intakes of fibre and n-3 and n-6 fatty acids compared with both the NW and SW groups (all P< 0·001). It is likely that differences in dietary intakes contributed to the separation of the three groups. Evaluation of geographical factors including diet should be considered in the interpretation of metabolomic data from multi-centre studies.

Top single nucleotide polymorphisms affecting carbohydrate metabolism in metabolic syndrome: from the LIPGENE study.

RATIONALE: Metabolic syndrome (MetS) is a high-prevalence condition characterized by altered energy metabolism, insulin resistance, and elevated cardiovascular risk. OBJECTIVES: Although many individual single nucleotide polymorphisms (SNPs) have been linked to certain MetS features, there are few studies analyzing the influence of SNPs on carbohydrate metabolism in MetS. METHODS: A total of 904 SNPs (tag SNPs and functional SNPs) were tested for influence on 8 fasting and dynamic markers of carbohydrate metabolism, by performance of an intravenous glucose tolerance test in 450 participants in the LIPGENE study. FINDINGS: From 382 initial gene-phenotype associations between SNPs and any phenotypic variables, 61 (16% of the preselected variables) remained significant after bootstrapping. Top SNPs affecting glucose metabolism variables were as follows: fasting glucose, rs26125 (PPARGC1B); fasting insulin, rs4759277 (LRP1); C-peptide, rs4759277 (LRP1); homeostasis assessment of insulin resistance, rs4759277 (LRP1); quantitative insulin sensitivity check index, rs184003 (AGER); sensitivity index, rs7301876 (ABCC9), acute insulin response to glucose, rs290481 (TCF7L2); and disposition index, rs12691 (CEBPA). CONCLUSIONS: We describe here the top SNPs linked to phenotypic features in carbohydrate metabolism among approximately 1000 candidate gene variations in fasting and postprandial samples of 450 patients with MetS from the LIPGENE study.