Phospholipids in lipoproteins: compositional differences across VLDL, LDL, and HDL in pregnant women.

OBJECTIVE: The aim of this study was to analyse the differences in the phospholipid composition of very low density (VLDL), low density (LDL) and high density lipoprotein (HDL) monolayers in pregnant lean and obese women. METHODS: LDL, HDL, and VLDL were isolated from plasma samples of 10 lean and 10 obese pregnant women, and their species composition of phosphatidylcholines (PC) and sphingomyelins (SM) was analysed by liquid-chromatography tandem mass-spectrometry. Wilcoxon-Mann-Whitney U test and principal component analysis (PCA) were used to investigate if metabolite profiles differed between the lean/obese group and between lipoprotein species. RESULTS: No significant differences have been found in the metabolite levels between obese and non-obese pregnant women. The PCA components 1 and 2 separated between LDL, HDL, and VLDL but not between normal weight and obese women. Twelve SM and one PCae were more abundant in LDL than in VLDL. In contrast, four acyl-alkyl-PC and two diacyl-PC were significantly higher in HDL compared to LDL. VLDL and HDL differed in three SM, seven acyl-alkyl-PC and one diacyl-PC (higher values in HDL) and 13 SM (higher in VLDL). We also found associations of some phospholipid species with HDL and LDL cholesterol. CONCLUSION: In pregnant women phospholipid composition differs significantly in HDL, LDL and VLDL, similar to previous findings in men and non-pregnant women. Obese and lean pregnant women showed no significant differences in their lipoprotein associated metabolite profile.

Early Programming of Obesity Throughout the Life Course: A Metabolomics Perspective.

BACKGROUND: Over the last decades, research on early life risk factors for obesity and its comorbidities in early life has gained attention within the field of developmental origins of health and diseases. Metabolomics studies that are trying to find early life biomarker and intervention targets for the early development of obesity and associated cardiovascular diseases could help break the inter-generational cycle of obesity. SUMMARY: Metabolomics studies in the field of early programming are scarce and causality is lacking at this stage, as most of the studies are cross-sectional. The main metabolites in the focus of obesity are branched-chain and aromatic amino acids, long-chain polyunsaturated fatty acids, lysophosphatidylcholines, and sphingomyelins. Sex and puberty have not been considered in most of the biomarker studies, but show differences in the metabolite associations to obesity. Key Messages: There is still a lot unknown about the associations between early programming exposures, metabolite concentrations, and the development of obesity. The few studies focusing on this topic find similar metabolite classes in the same age groups being associated with rapid early growth or obesity; but due to differences in the methodological and statistical approaches, the single species often differ. Therefore, more research, preferably with standardized approaches, is needed.

Long-Term Health Impact of Early Nutrition: The Power of Programming.

The Power of Programming conference 2016 at Ludwig-Maximilians-Universität Munich brought together about 600 researchers and other stakeholders from around the world who reviewed the recent evidence on the lasting health impact of environment and nutrition during early life, from pre-pregnancy to early childhood. The conference was hosted by the Early Nutrition Project, a multidisciplinary research collaboration funded by the European Commission with collaborating researchers from 35 institutions in 15 countries in Europe, the United States and Australia. The project explores the early origins of obesity, adiposity and associated non-communicable diseases, underlying mechanisms and opportunities for prevention. The project also proactively supports translational application of research findings. In fact, some existing evidence has already been rapidly adopted into policy, regulatory standards and practice. Further, broad dissemination of findings is achieved through the established digital eLearning platform of the Early Nutrition eAcademy, video clip-based learning and graphically supported messaging to consumers. The project demonstrated powerful effects of early metabolic programming on later health. Compared to other common prevention strategies, modifying risk trajectories in early life can achieve a much larger risk reduction and be more cost-effective. While some effective prevention strategies have been promptly implemented in policy and guidelines, legislation and practice, in other areas, the uptake is limited by a paucity of quality human intervention trials and insufficient evaluation of the feasibility of implementation and econometric impact. This needs to be strengthened by future collaborative research work.

Investigating the early metabolic fingerprint of celiac disease - a prospective approach.

OBJECTIVES AND STUDY: In the development of Celiac Disease (CD) both genetic and environmental factors play a crucial role. The Human Leukocyte Antigen (HLA)-DQ2 and HLA-DQ8 loci are strongly related to the disease and are necessary but not sufficient for the development of CD. Therefore, increasing interest lies in examining the mechanisms of CD onset from the early beginning. Differences in serum and urine metabolic profiles between healthy individuals and CD patients have been reported previously. We aimed to investigate if the metabolic pathways were already altered in young, 4 month old infants, preceding the CD diagnosis. METHODS: Serum samples were available for 230 four month old infants of the PreventCD project, a multicenter, randomized, double-blind, dietary intervention study. All children were positive for HLA-DQ2 and/or HLA-DQ8 and had at least one first-degree relative diagnosed with CD. Amino acids were quantified after derivatization with liquid chromatography - tandem mass spectrometry (MS/MS) and polar lipid concentrations (acylcarnitines, lysophosphatidylcholines, phosphatidylcholines, and sphingomyelins) were determined with direct infusion MS/MS. We investigated the association of the metabolic profile with (1) the development of CD up to the age of 8 years (yes/no), (2) with HLA-risk groups, (3) with the age at CD diagnosis, using linear mixed models and cox proportional hazards models. Gender, intervention group, and age at blood withdrawal were included as potential confounder. RESULTS: By the end of 2014, thirty-three out of the 230 children (14%) were diagnosed with CD according to the ESPGHAN criteria. Median age at diagnosis was 3.4 years (IQR, 2.4-5.2). Testing each metabolite for a difference in the mean between healthy and CD children, we (1) could not identify a discriminant analyte or a pattern pointing towards an altered metabolism (Bonferroni corrected P > 0.05 for all). Metabolite concentrations (2) did not differ across the HLA-risk groups. When investigating the age at diagnosis using (3) survival models, we found no evidence for an association between the metabolic profile and the risk of a later CD diagnosis. CONCLUSION: The metabolic profile at 4 months of age was not predictive for the development of CD up to the age of 8 years. Our results suggest that metabolic pathways reflected in serum are affected only later in life and that the HLA-genotype does not influence the serum metabolic profile in young infants before introduction of solid food.

Rapid growth and childhood obesity are strongly associated with lysoPC(14:0).

BACKGROUND: Despite the growing interest in the early-origins-of-later-disease hypothesis, little is known about the metabolic underpinnings linking infant weight gain and childhood obesity. OBJECTIVE: To discover biomarkers reflective of weight change in the first 6 months and overweight/obesity at age 6 years via a targeted metabolomics approach. DESIGN: This analysis comprised 726 infants from a European multicenter randomized trial (Childhood Obesity Programme, CHOP) for whom plasma blood samples at age 6 months and anthropometric data up to the age of 6 years were available. 'Rapid growth' was defined as a positive difference in weight within the first 6 months of life standardized to WHO growth standards. Weight change was regressed on each of 168 metabolites (acylcarnitines, lysophosphatidylcholines, sphingomyelins, and amino acids). Metabolites significant after Bonferroni's correction were tested as predictors of later overweight/obesity. RESULTS: Among the overall 19 significant metabolites, 4 were associated with rapid growth and 15 were associated with a less-than-ideal weight change. After adjusting for feeding group, only the lysophosphatidylcholine LPCaC14:0 remained significantly associated with rapid weight gain (ß = 0.18). Only LPCaC14:0 at age 6 months was predictive of overweight/obesity at age 6 years (OR 1.33; 95% CI 1.04-1.69). CONCLUSION: LPCa14:0 is strongly related to rapid growth in infancy and childhood overweight/obesity. This suggests that LPCaC14:0 levels may represent a metabolically programmed effect of infant weight gain on the later obesity risk. However, these results require confirmation by independent cohorts.

Associations of maternal and fetal SCD-1 markers with infant anthropometry and maternal diet: Findings from the ROLO study.

BACKGROUND: Elevated stearoyl-CoA desaturase 1 (SCD-1) activity showed associations with obesity in cross-sectional studies. In non-pregnant populations, nutrition regulates SCD-1 transcription and activity. OBJECTIVE: To investigate the longitudinal associations of maternal and fetal SCD-1 activity markers with infant anthropometry up to 2 years of age, and to explore how selected dietary intakes modulate SCD-1 activity in pregnancy. METHODS: As a secondary analysis from the ROLO intervention study, which was conducted in a population at risk for macrosomia, non-esterified fatty acids (NEFA) from maternal plasma at 13 and 28 weeks' gestation and in cord blood were measured via liquid-chromatography-mass-spectrometry. Fatty acid ratios 18:1/18:0 and 16:1/16:0 were used as markers for SCD-1 activity ('desaturation indices', DIs). Relationships of DIs with infant anthropometry up to 2 years of age and maternal dietary parameters during pregnancy were investigated using adjusted linear regression models and p-values correction for multiple testing. RESULTS: 18:1/18:0, but not 16:1/16:0, was associated with measures of infant anthropometry at birth (maternal and fetal markers) and up to 2 years of age (maternal markers only). Dietary intakes did not show strong associations with 18:1/18:0, but 16:1/16:0 was associated with absolute and relative dietary intakes. CONCLUSIONS: In a population at risk for macrosomia, maternal SCD-1 activity measured via 18:1/18:0 was involved in the fetal programming of infant obesity, but could not be substantially modulated by short-term diet in pregnancy. CLINICAL TRIAL REGISTRATION: ISRCTN Registration number: ISRCTN54392969 (http://www.isrctn.com/ISRCTN54392969).

Prolonged monitoring of postprandial lipid metabolism after a western meal rich in linoleic acid and carbohydrates.

Today, awareness has been raised regarding high consumption of n-6 polyunsaturated fatty acids (PUFA) in western diets. A comprehensive analysis of total and individual postprandial fatty acids profiles would provide insights into metabolic turnover and related health effects. After an overnight fast, 9 healthy adults consumed a mixed meal comprising 97 g carbohydrate and 45 g fat, of which 26.4 g was linoleic acid (LA). Nonesterified fatty acids (NEFA), phospholipid fatty acids (PL-FA) and triacylglycerol fatty acids (TG-FA) were monitored in plasma samples, at baseline and hourly over a 7-h postprandial period. Total TG-FA concentration peaked at 2 h after the meal and steadily decreased thereafter. LA from TG18:2n-6 and behenic acid from TG22:0 showed the highest response among TG-FA, with a biphasic response detected for the former. PL-FA exhibited no change. Total NEFA initially decreased to nadir at 1 h, then increased to peak at 7 h. The individual NEFA showed the same response curve except LA and some very-long-chain saturated fatty acids (VLCSFA, ≥20 carbon chain length) that markedly increased shortly after the meal intake. The similarities and dissimilarities in lipid profiles between study subjects at different time points were visualized using nonmetric multi-dimensional scaling. Overall, the results indicate that postprandial levels of LA and VLCSFA, either as NEFA or TG, were most affected by the test meal, which might provide an explanation for the health effects of this dietary lifestyle characterized by high intake of mixed meals rich in n-6 PUFA.

Variation and Interdependencies of Human Milk Macronutrients, Fatty Acids, Adiponectin, Insulin, and IGF-II in the European PreventCD Cohort.

Human milk composition is variable. The identification of influencing factors and interdependencies of components may help to understand the physiology of lactation. In this study, we analyzed linear trends in human milk composition over time, the variation across different European countries and the influence of maternal celiac disease. Within a multicenter European study exploring potential prevention of celiac disease in a high-risk population (PreventCD), 569 human milk samples were donated by women from five European countries between 16 and 163 days postpartum. Some 202 mothers provided two samples at different time points. Protein, carbohydrates, fat and fatty acids, insulin, adiponectin, and insulin-like growth factor II (IGF-II) were analyzed. Milk protein and n-6 long chain polyunsaturated fatty acids decreased during the first three months of lactation. Fatty acid composition was significantly influenced by the country of residence. IGF-II and adiponectin concentrations correlated with protein content (r = 0.24 and r = 0.35), and IGF-II also correlated with fat content (r = 0.36), suggesting a possible regulatory role of IGF in milk macronutrient synthesis. Regarding the impact of celiac disease, only the level in palmitic acid was influenced by this disease, suggesting that breastfeeding by celiac disease mothers should not be discouraged.

An individual participant data meta-analysis on metabolomics profiles for obesity and insulin resistance in European children.

Childhood obesity prevalence is rising in countries worldwide. A variety of etiologic factors contribute to childhood obesity but little is known about underlying biochemical mechanisms. We performed an individual participant meta-analysis including 1,020 pre-pubertal children from three European studies and investigated the associations of 285 metabolites measured by LC/MS-MS with BMI z-score, height, weight, HOMA, and lipoprotein concentrations. Seventeen metabolites were significantly associated with BMI z-score. Sphingomyelin (SM) 32:2 showed the strongest association with BMI z-score (P = 4.68 × 10-23) and was also closely related to weight, and less strongly to height and LDL, but not to HOMA. Mass spectrometric analyses identified SM 32:2 as myristic acid containing SM d18:2/14:0. Thirty-five metabolites were significantly associated to HOMA index. Alanine showed the strongest positive association with HOMA (P = 9.77 × 10-16), while acylcarnitines and non-esterified fatty acids were negatively associated with HOMA. SM d18:2/14:0 is a powerful marker for molecular changes in childhood obesity. Tracing back the origin of SM 32:2 to dietary source in combination with genetic predisposition will path the way for early intervention programs. Metabolic profiling might facilitate risk prediction and personalized interventions in overweight children.

Metabolic Regulation of Pre- and Postnatal Growth.

Growth characteristics during periods of early developmental plasticity are linked with later health outcomes and with disease risks. Infant growth is modulated by genetic and exogenous factors including nutrition. We try to explore their underlying mechanisms using targeted metabolomic profiling of small molecules in biological samples using high-performance liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) to quantify hundreds of molecules in small biosamples, e.g., 50 µL plasma. In the large German LISA birth cohort study, cord blood lysophosphatidylcholines and fatty acids were closely associated with infant birth weight, with a nonsignificant trend towards an association with infant weight gain and later BMI. Studies in infants randomized to different protein intakes in the European CHOP Study show conventional high protein intakes to markedly increase plasma-indispensable amino acids (AA), particularly branched-chain AA (BCAA), while exceeding the infant's capacity of BCAA breakdown, and an increase in the dispensable AA tyrosine previously associated with insulin resistance. In a path model analysis of the relationship of infant plasma AA, growth factors, and infant growth, AA were generally found to induce a stronger response of insulin than IGF-I although effects of individual AA were very different. We conclude that targeted improvement in nutrient supply in pregnancy and infancy may offer large opportunities for promoting desirable child growth patterns and long-term health.

The impact of human breast milk components on the infant metabolism.

BACKGROUND & AIMS: Breastfeeding is beneficial for mothers and infants. Underlying mechanisms and biochemical mediators thus need to be investigated to develop and support improved infant nutrition practices promoting the child health. We analysed the relation between maternal breast milk composition and infant metabolism. METHODS: 196 pairs of mothers and infants from a European research project (PreventCD) were studied. Maternal milk samples collected at month 1 and month 4 after birth were analysed for macronutrient classes, hormone, and fatty acid (FA) content. Phospholipids, acylcarnitines, and amino acids were measured in serum samples of 4-month old infants. Associations between milk components and infant metabolites were analysed with spearman correlation and linear mixed effect models (LME). P-values were corrected for multiple testing (PLME). RESULTS: Month 1 milk protein content was strongly associated with infant serum lyso-phosphatidylcholine (LPC) 14:0 (PLME = 0.009). Month 1 milk insulin was associated to infant acetylcarnitine (PLME = 0.01). There were no associations between milk protein content and serum amino acids and milk total fat content and serum polar lipids. Middle- and odd-chain FA% in breast milk at both ages were significantly related to serum LPC and sphingomyelins (SM) species in infant serum (all PLME<0.05), while FA% 20:5n-3 and 22:6n-3 percentages were significantly associated to serum LPC 22:6 (PLME = 1.91×10-4/7.93×10-5) in milk only at month 4. Other polyunsaturated fatty acids and hormones in milk showed only weak associations with infant serum metabolites. CONCLUSIONS: Infant serum LPC are influenced by breast milk FA composition and, intriguingly, milk protein content in early but not late lactation. LPC 14:0, previously found positively associated with obesity risk, was the serum metabolite which was the most strongly associated to milk protein content. Thus, LPC 14:0 might be a key metabolite not only reflecting milk protein intake in infants, but also relating high protein content in milk or infant formula to childhood obesity risk.

Effects of Early Nutrition on the Infant Metabolome.

Breastfeeding induces a different metabolic and endocrine response than feeding conventional infant formula, and it has also been associated with slower weight gain and reduced disease risk in later life. The underlying programming mechanisms remain to be explored. Breastfeeding has been reported to induce lower levels of insulin, insulin-like growth factor-1 and some amino acids (AAs) than formula feeding. In the Childhood Obesity Project (CHOP), infants fed a conventional protein-rich formula had a higher BMI at 2 and 6 years than those fed a protein-reduced formula. At 6 months, higher protein intakes induced increased plasma concentrations of branched-chain AAs (BCAAs) and their oxidation products, short-chain acylcarnitines. With increasing BCAA levels, these short-chain acylcarnitines increased proportionally only until a break point was reached, after which BCAAs seemed to escape their degradation. The resulting marked elevation in BCAA levels with high-protein (HP) intakes appears to contribute to increased insulin levels and to affect ß-oxidation of fatty acids. The ratios of long-chain acylcarnitines to free carnitine decreased in infants who received a HP formula, which indicates a reduced initiation of ß-oxidation. We conclude that HP intakes inducing high BCAA plasma levels may inhibit fat oxidation and thereby enhance body fat deposition and adiposity.

Dietary protein intake affects amino acid and acylcarnitine metabolism in infants aged 6 months.

CONTEXT: The protective effect of breast-feeding against later obesity may be explained by the lower protein content compared with formula milk. However, the metabolic mechanisms remain unknown. OBJECTIVE: We studied the metabolic response to a higher or lower protein supply in infancy. DESIGN AND SETTING: The Childhood Obesity Project study is a double-blind, randomized, multicenter intervention trial. Infants were randomized to receive a higher (HP) or lower protein (LP) content infant formula or were breast-fed. PATIENTS AND INTERVENTIONS: Plasma samples of 691 infants who received formula milk with different protein content (HP, 2.05 g per 100 mL; LP, 1.25 g per 100 mL) or were breast-fed were collected. MAIN OUTCOME MEASURES: Changes in plasma amino acid and acylcarnitine concentrations of 6-month-old infants according to different dietary protein supply were determined by liquid chromatography coupled to tandem mass spectrometry. RESULTS: Twenty-nine metabolites differed significantly between the formula groups. Branched-chain amino acids (BCAAs) were the most discriminant metabolites. Their degradation products, the short-chain acylcarnitines C3, C4, and C5, were also significantly elevated in the HP group. A breakpoint analysis confirmed that with increasing BCAAs, the ratio between acylcarnitines and BCAAs decreases. Long-chain acylcarnitines were decreased in HP infants. CONCLUSIONS: BCAAs seem to play a pivotal role in the effect of a high-protein diet on ß-oxidation and fat storage. We provide new evidence for a possible saturation of the BCAA degradation pathway that may represent the mechanism by which high-protein intake affects the metabolic regulation. Moreover, it appears to inhibit the initial step of the ß-oxidation, thus leading to high early weight gain and body fat deposition.

Coeliac disease and gluten avoidance in New Zealand children.

OBJECTIVES: Although gluten avoidance is thought to be common among New Zealanders, the prevalence of gluten avoidance and of actual coeliac disease (CD) in children is uncertain. Our aims were: (1) to determine the prevalence of doctor-diagnosed CD and of gluten avoidance in New Zealand children; and (2) among children without CD, to identify independent predictors of gluten avoidance. DESIGN: The New Zealand Asthma and Allergy Cohort Study has detailed information on participants' demographic, pregnancy-related and neonatal factors. The authors surveyed parents regarding their child's history of lactose intolerance and gluten-related issues (eg, gluten avoidance, history of wheat or gluten allergy in first degree relatives, testing and doctor diagnosis of CD). After excluding children with doctor-diagnosed CD, the authors identified independent predictors of gluten avoidance. RESULTS: Among 916 children, most (78%) were of European ethnicity. The authors identified nine (1.0%, 95% CI 0.5% to 1.9%) who had doctor-diagnosed CD, while 48 (5.2%, 95% CI 4.0% to 6.9%) avoided gluten. Among children without diagnosed CD, significant independent predictors for gluten avoidance were Christchurch site (OR 2.2, 95% CI 1.02 to 4.7), prior testing for CD (OR 9.0, 95% CI 4.1 to 19.5) and doctor-diagnosed lactose intolerance (OR 5.2, 95% CI 2.0 to 13.9). CONCLUSIONS: CD affected 1% of these New Zealand children, but 5% reported gluten avoidance. The predictors of gluten avoidance in children without doctor-diagnosed CD suggest important regional differences in community belief or medical practice regarding implementation of gluten avoidance and the contributory role of non-specific subjective abdominal complaints.

Randomized trial of vitamin D supplementation and risk of acute respiratory infection in Mongolia.

OBJECTIVE: Observational studies suggest that serum levels of 25-hydroxyvitamin D (25[OH]D) are inversely associated with acute respiratory infections (ARIs). We hypothesized that vitamin D supplementation of children with vitamin D deficiency would lower the risk of ARIs. METHODS: By using cluster randomization, classrooms of 744 Mongolian schoolchildren were randomly assigned to different treatments in winter (January-March). This analysis focused on a subset of 247 children who were assigned to daily ingestion of unfortified regular milk (control; n = 104) or milk fortified with 300 IU of vitamin D(3) (n = 143). This comparison was double-blinded. The primary outcome was the number of parent-reported ARIs over the past 3 months. RESULTS: At baseline, the median serum 25(OH)D level was 7 ng/mL (interquartile range: 5-10 ng/mL). At the end of the trial, follow-up was 99% (n = 244), and the median 25(OH)D levels of children in the control versus vitamin D groups was significantly different (7 vs 19 ng/mL; P < .001). Compared with controls, children receiving vitamin D reported significantly fewer ARIs during the study period (mean: 0.80 vs 0.45; P = .047), with a rate ratio of 0.52 (95% confidence interval: 0.31-0.89). Adjusting for age, gender, and history of wheezing, vitamin D continued to halve the risk of ARI (rate ratio: 0.50 [95% confidence interval: 0.28-0.88]). Similar results were found among children either below or above the median 25(OH)D level at baseline (rate ratio: 0.41 vs 0.57; P(interaction) = .27). CONCLUSIONS: Vitamin D supplementation significantly reduced the risk of ARIs in winter among Mongolian children with vitamin D deficiency.