Véronique Ferchaud-Roucher

Perinatal protein restriction affects milk free amino acid and fatty acid profile in lactating rats: potential role on pup growth and metabolic status

Perinatal undernutrition affects not only fetal and neonatal growth but also adult health outcome, as suggested by the metabolic imprinting concept. Although maternal milk is the only channel through which nutrients are transferred from mother to offspring during the postnatal period, the impact of maternal undernutrition on milk composition is poorly understood. The present study investigates, in a rat model of nutritional programming, the effects of feeding an isocaloric, low-protein diet throughout gestation and lactation on milk composition and its possible consequences on offsprings growth and metabolic status. We used an integrated methodological approach that combined targeted analyses of macronutrients, free amino acid and fatty acid content throughout lactation, with an untargeted mass-spectrometric-based metabolomic phenotyping. Whereas perinatal dietary protein restriction failed to alter milk protein content, it dramatically decreased the concentration of most free amino acids at the end of lactation. Interestingly, a decrease of several amino acids involved in insulin secretion or gluconeogenesis was observed, suggesting that maternal protein restriction during the perinatal period may impact the insulinotrophic effect of milk, which may, in turn, account for the slower growth of the suckled male offspring. Besides, the decrease in sulfur amino acids may alter redox status in the offspring. Maternal undernutrition was also associated with an increase in milk total fatty acid content, with modifications in their pattern. Altogether, our results show that milk composition is clearly influenced by maternal diet and suggest that alterations in milk composition may play a role in offspring growth and metabolic programming.

Maternal supplementation with n-3 long chain polyunsaturated fatty acids during perinatal period alleviates the metabolic syndrome disturbances in adult hamster pups fed a high-fat diet after weaning ☆

Perinatal nutrition is thought to affect the long-term risk of the adult to develop metabolic syndrome. We hypothesized that maternal supplementation with eicosapentaenoic acid and docosahexaenoic acid during pregnancy and lactation would protect offspring fed a high-fat diet from developing metabolic disturbances. Thus, two groups of female hamsters were fed a low-fat control diet, either alone (LC) or enriched with n-3 long chain polyunsaturated fatty acids (LC-PUFA) (LO), through the gestational and lactation periods. After weaning, male pups were randomized to separate groups that received either a control low-fat diet (LC) or a high-fat diet (HC) for 16 weeks. Four groups of pups were defined (LC-LC, LC-HC, LO-LC and LO-HC), based on the combinations of maternal and weaned diets. Maternal n-3 LC-PUFA supplementation was associated with reduced levels of basal plasma glucose, hepatic triglycerides secretion and postprandial lipemia in the LO-HC group compared to the LC-HC group. Respiratory parameters were not affected by maternal supplementation. In contrast, n-3 LC-PUFA supplementation significantly enhanced the activities of citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase compared to the offspring of unsupplemented mothers. Sterol regulatory element binding protein-1c, diacylglycerol O-acyltransferase 2, fatty acid synthase, stearoyl CoA desaturase 1 and tumor necrosis factor α expression levels were not affected by n-3 LC-PUFA supplementation. These results provide evidence for a beneficial effect of n-3 LC-PUFA maternal supplementation in hamsters on the subsequent risk of metabolic syndrome. Underlying mechanisms may include improved lipid metabolism and activation of the mitochondrial oxidative pathway.

Multiplexed peptide analysis for kinetic measurements of major human apolipoproteins by LC/MS/MS.

A multiplexed assay was developed by MS to analyze, in a single run, six major human Apos involved in lipoprotein metabolism: ApoA-I, ApoA-II, ApoB100, ApoC-II, ApoC-III, and ApoE. This method was validated in vivo in six subjects who received a 14 h constant infusion of [5,5,5-2H3]L-leucine at 10 μM/kg/h. Plasma lipoprotein fractions were isolated from collected blood samples and were digested with trypsin. Proteotypic peptides were subsequently analyzed by LC/MS/MS. Enrichment measurement data were compared with those obtained by the standard method using GC/MS. The required time to obtain the LC/MS/MS data was less than that needed for GC/MS. The enrichments from both methods were correlated for ApoA-I (r = 0.994; P < 0.0001) and ApoB100 (r = 0.999; P < 0.0001), and the Bland-Altman plot confirmed the similarity of the two methods. Intra- and inter-assay variability calculated for the six Apos of interest did not exceed 10.7 and 12.5%, respectively, and kinetic parameters were similar and/or in agreement with previously reported data. Therefore, LC/MS/MS can be considered as a useful tool for human Apo kinetic studies using stable isotopes.

Plasma PCSK9 measurement by liquid chromatography-Tandem mass spectrometry and comparison with conventional ELISA

The combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and trypsin proteolysis is an effective tool for accurate quantitation of multiple proteins in a single run. However, expensive samples pre-treatment as immunoenrichment are often required to analyze low abundant proteins. Plasma proprotein convertase subtilisin/kexin type 9 (PCSK9), a circulating regulator of low-density lipoprotein metabolism, was studied as an example of a low abundant plasma protein. We investigated post-proteolysis solid-phase extraction (SPE) as an alternative strategy to improve its detection. After optimization of pretreatment, including denaturation, reduction, alkylation, tryptic digestion and selective SPE concentration, 91±7% of PCSK9 was recovered from human plasma samples and coefficients of variation were less than 13.2% with a lower limit of quantification of 37.5ng/ml. This LC-MS/MS method was compared with standard enzyme-linked immunosorbent assay in 30 human plasma samples with a broad range of PCSK9 concentrations. Both methods were significantly correlated (r=0.936, p<0.001) with less than 7% of the values out of the 95% confidence interval and similar concentrations were measured using either LC-MS/MS or ELISA methods (514.2±217.2 vs. 504.2±231.0ng/ml, respectively- p=NS). This method involving SPE is an effective measurement tool for low abundant plasma protein analysis that could be easily included in multiplexed assays.

P040 Existe-t-il des biomarqueurs lipidiques prédictifs du diabète de type 2 ? Application sur deux cohortes d’origine différente

Introduction Des modifications du lipidome chez les diabetiques de type 2 (DT2) ont ete rapportees deja presentes chez les pre-diabetiques (MEIKLE, 2013 PloS One, 8 : 74341). Ces biomarqueurs lipidiques predictifs permettraient d’identifier les patients a risque d’evoluer vers le DT2 et proposer des interventions therapeutiques ciblees. Le but de cette etude etait de confirmer ces biomarqueurs predictifs de DT2 chez des patients issus de deux cohortes independantes ? Patients et methodes Pour la cohorte iranienne ICS, 20 sujets non diabetiques en 2001 et en 2007 ont ete selectionnes et compares a 20 sujets non diabetiques en 2001devenus diabetiques en 2007. Pour l’etude francaise IT-DIAB, 72 sujets prediabetiques ont ete selectionnes et analyses en deux groupes comparables dont 36 sont restes prediabetiques et 36 sont devenus diabetiques en 2 annees. L’analyse lipidomique globale plasmatique a ete realisee par UHPLC-ESI-HRMS. Resultats Les analyses multivariees n’ont pas mis en evidence la separation des differents groupes pour les etudes ICS et IT-DIAB. Concernant ICS, aucun biomarqueur lipidique specifique et predictif du diabete de type 2 n’a ete mis en evidence. Pour IT-DIAB, 20 lipides differents entre les deux groupes ont ete identifies mais sans lien direct avec les donnees anterieures. Conclusion Ces donnees ne permettent pas de confirmer les resultats prealablement publies sur le lipidome pour la prediction du diabete de type 2. Differentes approches methodologiques, statistiques ou specifiques aux groupes de patients etudies pourraient cependant expliquer ce resultat. Cette approche de detection de biomarqueurs predictifs est prometteuse mais complexe et meriterait d’etre standardisee. Declaration d’interet Les auteurs declarent ne pas avoir d’interet direct ou indirect (financier ou en nature) avec un organisme prive, industriel ou commercial en relation avec le sujet presente.