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Background Exposure to perfluoroalkyl substances may affect offspring immune development and thereby increase risk of childhood asthma, but the underlying mechanisms and asthma phenotype affected by such exposure is unknown. Methods In the Danish COPSAC2010 cohort of 738 unselected pregnant women and their children plasma PFOS and PFOA concentrations were semi-quantified by untargeted metabolomics analyses and calibrated using a targeted pipeline in mothers (gestation week 24 and 1 week postpartum) and children (age ½, 1½ and 6 years). We examined associations between pregnancy and childhood PFOS and PFOA exposure and childhood infections, asthma, allergic sensitization, atopic dermatitis, and lung function measures, and studied potential mechanisms by integrating data on systemic low-grade inflammation (hs-CRP), functional immune responses, and epigenetics. Findings Higher maternal PFOS and PFOA exposure during pregnancy showed association with a non-atopic asthma phenotype by age 6, a protection against sensitization, and no association with atopic asthma or lung function, or atopic dermatitis. The effect was primarily driven by prenatal exposure. There was no association with infection proneness, low-grade inflammation, altered immune responses or epigenetic changes. Interpretations Prenatal exposure to PFOS and PFOA, but not childhood exposure, specifically increased the risk of low prevalent non-atopic asthma, whereas there was no effect on atopic asthma, lung function, or atopic dermatitis. Funding All funding received by COPSAC are listed on www.copsac.com. The Lundbeck Foundation (Grant no R16-A1694); The Novo Nordic Foundation (Grant nos NNF20OC0061029, NNF170C0025014, NNF180C0031764); The Ministry of Health (Grant no 903516); Danish Council for Strategic Research (Grant no 0603-00280B); and The Capital Region Research Foundation have provided core support to the COPSAC research center. COPSAC acknowle, Background: Exposure to perfluoroalkyl substances may affect offspring immune development and thereby increase risk of childhood asthma, but the underlying mechanisms and asthma phenotype affected by such exposure is unknown. Methods: In the Danish COPSAC2010 cohort of 738 unselected pregnant women and their children plasma PFOS and PFOA concentrations were semi-quantified by untargeted metabolomics analyses and calibrated using a targeted pipeline in mothers (gestation week 24 and 1 week postpartum) and children (age ½, 1½ and 6 years). We examined associations between pregnancy and childhood PFOS and PFOA exposure and childhood infections, asthma, allergic sensitization, atopic dermatitis, and lung function measures, and studied potential mechanisms by integrating data on systemic low-grade inflammation (hs-CRP), functional immune responses, and epigenetics. Findings: Higher maternal PFOS and PFOA exposure during pregnancy showed association with a non-atopic asthma phenotype by age 6, a protection against sensitization, and no association with atopic asthma or lung function, or atopic dermatitis. The effect was primarily driven by prenatal exposure. There was no association with infection proneness, low-grade inflammation, altered immune responses or epigenetic changes. Interpretations: Prenatal exposure to PFOS and PFOA, but not childhood exposure, specifically increased the risk of low prevalent non-atopic asthma, whereas there was no effect on atopic asthma, lung function, or atopic dermatitis. Funding: All funding received by COPSAC are listed on www.copsac.com. The Lundbeck Foundation (Grant no R16-A1694); The Novo Nordic Foundation (Grant nos NNF20OC0061029, NNF170C0025014, NNF180C0031764); The Ministry of Health (Grant no 903516); Danish Council for Strategic Research (Grant no 0603-00280B); and The Capital Region Research Foundation have provided core support to the COPSAC research center. COPSAC acknowledges the National Facility for Exposomics (SciLi

Background: We recently conducted a double-blinded randomised controlled trial showing that fish-oil supplementation during pregnancy reduced the risk of persistent wheeze or asthma in the child by 30%. Here, we explore the mechanisms of the intervention. Methods: 736 pregnant women were given either placebo or n-3 long-chain polyunsaturated fatty acids (LCPUFAs) in the third trimester in a randomised controlled trial. Deep clinical follow-up of the 695 children in the trial was done at 12 visits until age 6 years, including assessment of genotype at the fatty acid desaturase (FADS) locus, plasma fatty acids, airway DNA methylation, gene expression, microbiome and metabolomics. Results: Supplementation with n-3 LCPUFA reduced the overall risk of non-atopic asthma by 73% at age 6 (relative risk (RR) 0.27 (95% CI 0.06 to 0.85), p=0.042). In contrast, there was no overall effect on asthma with atopic traits (RR 1.42 (95% CI 0.63 to 3.38), p=0.40), but this was significantly modified by maternal FADS genotype and LCPUFA blood levels (interaction p<0.05), and supplementation did reduce the risk of atopic asthma in the subgroup of mothers with FADS risk variants and/or low blood levels of n-3 LCPUFA before the intervention (RR 0.31 (95% CI 0.11 to 0.75), p=0.016). Furthermore, n-3 LCPUFA significantly reduced the number of infections (croup, gastroenteritis, tonsillitis, otitis media and pneumonia) by 16% (incidence rate ratio 0.84 (95% CI 0.74 to 0.96), p=0.009). Conclusions: n-3 LCPUFA supplementation in pregnancy showed protective effects on non-atopic asthma and infections. Protective effects on atopic asthma depended on maternal FADS genotype and n-3 LCPUFA levels. This indicates that the fatty acid pathway is involved in multiple mechanisms affecting the risk of asthma subtypes and infections. Trial registration number: NCT00798226.

Birth by cesarean section (CS) is linked to an increased risk of developing asthma, but the underlying mechanisms are unclear. To elucidate the link between birth by CS and asthma using newborn metabolomic profiles and integrating early life gut microbiome data and cord blood immunology. We investigated the influence of CS on liquid chromatography mass spectrometry (LC-MS) metabolomic profiles of dried blood spots from newborns of the two independent Copenhagen Prospective Studies on Asthma in Childhood cohorts, i.e. COPSAC2010 (n=677) and COPSAC2000 (n=387). We assessed the associations between the CS metabolic profile, age one-week gut microbiome data and frequency of cord blood Tregs. In COPSAC2010, a partial least square-discriminant analysis (PLS-DA) model showed that children born by CS versus natural delivery had different metabolic profiles (AUC=0.77, p=2.2e-16), which was replicated in COPSAC2000 (AUC=0.66, p=1.2e-5). The metabolic profile of CS was significantly associated with an increased risk of asthma at school-age in both COPSAC2010 (p=0.03) and COPSAC2000 (p=0.005). CS was associated with lower abundance of tryptophan, bile acid and phenylalanine metabolites, indicative of a perturbed gut microbiota. Further, gut bacteria dominating after natural delivery, i.e. Bifidobacterium and Bacteroides were correlated with CS-discriminative microbial metabolites, suggesting maternal microbial transmission during birth regulating the newborn's metabolism. Finally, the CS metabolic profile was associated with frequency of cord blood Tregs. These findings propose that CS is programming the risk of childhood asthma through perturbed immune responses and gut microbial colonization patterns reflected in the blood metabolome at birth.

Background: Intake of fish-oil and fatty fish during pregnancy has been shown to reduce the risk of childhood asthma but biomarkers of such intake are lacking. Objective: To establish biomarkers of prenatal fish-oil exposure from newborn dry blood spot metabolomics profiles and assess their relevance for childhood asthma risk stratification. Methods: The Danish COPSAC2010 mother–child cohort was utilized to investigate the effect of a double-blinded randomized controlled trial of fish-oil supplementation during pregnancy on dry blood spot liquid-chromatography mass spectrometry-based metabolomics profiles of 677 newborns. We thereafter investigated the association between fish-oil associated biomarkers in the newborn and development of asthma-related outcomes. Replication was sought in the independent observational COPSAC2000 cohort with 387 newborn metabolomics profiles. Results: The newborn metabolomics profiles differed between children in the fish-oil vs. placebo group in COPSAC2010 (area under the receiver operator curve = 0.94 ± 0.03, p <.001). The fish-oil metabolomics profile and the top biomarker, 3-carboxy-4-methyl-5-propyl-2-furan propanoic acid (CMPF) were both associated with a decreased risk of asthma by age 6 years (HR = 0.89, p =.002 and HR = 0.67, p =.005, respectively). In COPSAC2000, newborn CMPF level was also inversely associated with asthma risk by age 6 years (HR = 0.69, p =.01). Troublesome lung symptoms and common infections in the first 3 years were also inversely associated with newborn CMPF levels in both cohorts.Conclusions: Newborn children's blood levels of the furan fatty acid metabolite CMPF reflect fish-oil and fatty fish intake during pregnancy and are associated with a lower risk of asthma across two cohorts, which could aid newborn screening for childhood asthma.

The pregnancy period and first days of a newborn’s life is an important time window to ensure a healthy development of the baby. This is also the time when the mother and her baby are exposed to the same environmental conditions and intake of nutrients, which can be determined by assessing the blood metabolome. For this purpose, dried blood spots (DBS) of newborns are a valuable sampling technique to characterize what happens during this important mother-child time window. We used metabolomics profiles from DBS of newborns (age 2–3 days) and maternal plasma samples at gestation week 24 and postpartum week 1 from n = 664 mother-child pairs of the Copenhagen Prospective Studies on Asthma in Childhood 2010 (COPSAC2010) cohort, to study the vertical mother-child transfer of metabolites. Further, we investigated how persistent the metabolites are from the newborn and up to 6 months, 18 months, and 6 years of age. Two hundred seventy two metabolites from UPLC-MS (Ultra Performance Liquid Chromatography-Mass Spectrometry) analysis of DBS and maternal plasma were analyzed using correlation analysis. A total of 11 metabolites exhibited evidence of transfer (R > 0.3), including tryptophan betaine, ergothioneine, cotinine, theobromine, paraxanthine, and N6-methyllysine. Of these, 7 were also found to show persistence in their levels in the child from birth to age 6 years. In conclusion, this study documents vertical transfer of environmental and food-derived metabolites from mother to child and tracking of those metabolites through childhood, which may be of importance for the child’s later health and disease.

Prediction of retention times (RTs) is increasingly considered in untargeted metabolomics to complement MS/MS matching for annotation of unidentified peaks. We tested the performance of PredRet (http://predret.org/) to predict RTs for plant food bioactive metabolites in a data sharing initiative containing entry sets of 29–103 compounds (totalling 467 compounds, >30 families) across 24 chromatographic systems (CSs). Between 27 and 667 predictions were obtained with a median prediction error of 0.03–0.76 min and interval width of 0.33–8.78 min. An external validation test of eight CSs showed high prediction accuracy. RT prediction was dependent on shape and type of LC gradient, and number of commonly measured compounds. Our study highlights PredRet’s accuracy and ability to transpose RT data acquired from one CS to another CS. We recommend extensive RT data sharing in PredRet by the community interested in plant food bioactive metabolites to achieve a powerful community-driven open-access tool for metabolomics annotation.

Purpose: Autologous blood transfusion is performance enhancing and prohibited in sport but remains difficult to detect. This study explored the hypothesis that an untargeted urine metabolomics analysis can reveal one or more novel metabolites with high sensitivity and specificity for detection of autologous blood transfusion.Methods: In a randomized, double-blinded, placebo-controlled, cross-over design, exercise-trained males (n=12) donated 900 ml blood or were sham phlebotomized. After four weeks, RBCs or saline were reinfused. Urine samples were collected before phlebotomy and 2 h, 1, 2, 3, 5 and 10 days after reinfusion and analyzed by UPLC-QTOF-MS. Models of unique metabolites reflecting autologous blood transfusion were attained by partial least squares discriminant analysis.Results: The strongest model was obtained 2 h after reinfusion with a misclassification error of 6.3% and 98.8% specificity. However, combining only a few of the strongest metabolites selected by this model provided a sensitivity of 100% at days 1 and 2 and 66% at day 3 with 100% specificity. Metabolite identification revealed the presence of secondary di-2-ethylhexyl phtalate metabolites and putatively identified the presence of (iso)caproic acid glucuronide as the strongest candidate biomarker.Conclusion: Untargeted urine metabolomics revealed several plasticizers as the strongest metabolic pattern for detection of autologous blood transfusion for up to 3 days. Importantly, no other metabolites in urine appear of value for anti-doping purposes.

The in utero environment during pregnancy has important implications for the developing health of the child. We aim to examine the potential impact of maternal metabolome at two different timepoints in pregnancy on offspring respiratory health in early life. In 685 mother-child pairs from the Vitamin D Antenatal Asthma Reduction Trial, we assessed the prospective associations between maternal metabolites at both baseline (10–18 weeks gestation) and third trimester (32–38 weeks gestation) and the risk of child asthma or recurrent wheeze by age three using logistic regression models accounting for confounding factors. Subgroup analyses were performed by child sex. Among 632 metabolites, 19 (3.0%) and 62 (9.8%) from baseline and third trimester, respec-tively, were associated with the outcome (p-value < 0.05). Coffee-related metabolites in the maternal metabolome appeared to be of particular importance. Caffeine, theophylline, trigonelline, quinate, and 3-hydroxypyridine sulfate were inversely associated with asthma risk at a minimum of one timepoint. Additional observations also highlight the roles of steroid and sphingolipid metabolites. Overall, there was a stronger relationship between the metabolome in later pregnancy and offspring asthma risk. Our results suggest that alterations in prenatal metabolites may act as drivers of the development of offspring asthma.

Rationale: A link among sphingolipids, 17q21 genetic variants, and childhood asthma has been suggested, but the underlying mechanisms and characteristics of such an asthma endotype remain to be elucidated.Objectives: To study the sphingolipid-associated childhood asthma endotype using multiomic data.Methods: We used untargeted liquid chromatography-mass spectrometry plasma metabolomic profiles at the ages of 6 months and 6 years from more than 500 children in the COPSAC2010 (Copenhagen Prospective Studies on Asthma in Childhood) birth cohort focusing on sphingolipids, and we integrated the 17q21 genotype and nasal gene expression of SPT (serine palmitoyl-CoA transferase) (i.e., the rate-limiting enzyme in de novo sphingolipid synthesis) in relation to asthma development and lung function traits from infancy until the age 6 years. Replication was sought in the independent VDAART (Vitamin D Antenatal Asthma Reduction Trial) cohort.Measurements and Main Results: Lower concentrations of ceramides and sphingomyelins at the age of 6 months were associated with an increased risk of developing asthma before age 3, which was also observed in VDAART. At the age of 6 years, lower concentrations of key phosphosphingolipids (e.g., sphinganine-1-phosphate) were associated with increased airway resistance. This relationship was dependent on the 17q21 genotype and nasal SPT gene expression, with significant interactions occurring between the genotype and the phosphosphingolipid concentrations and between the genotype and SPT expression, in which lower phosphosphingolipid concentrations and reduced SPT expression were associated with increasing numbers of at-risk alleles. However, the findings did not pass the false discovery rate threshold of <0.05.Conclusions: This exploratory study suggests the existence of a childhood asthma endotype with early onset and increased airway resistance that is characterized by reduced sphingolipid concentrations, which are associated with 17q2

Asthma, allergic rhinitis, food allergy, and atopic dermatitis are common childhood diseases with several different underlying mechanisms, i.e., endotypes of disease. Metabolomics has the potential to identify disease endotypes, which could beneficially promote personalized prevention and treatment. Here, we summarize the findings from metabolomics studies of children with atopic diseases focusing on tyrosine and tryptophan metabolism, lipids (particularly, sphingolipids), polyunsaturated fatty acids, microbially derived metabolites (particularly, short-chain fatty acids), and bile acids. We included 25 studies: 23 examined asthma or wheezing, five examined allergy endpoints, and two focused on atopic dermatitis. Of the 25 studies, 20 reported findings in the pathways of interest with findings for asthma in all pathways and for allergy and atopic dermatitis in most pathways except tyrosine metabolism and short-chain fatty acids, respectively. Particularly, tyrosine, 3-hydroxyphenylacetic acid, N-acetyltyrosine, tryptophan, indolelactic acid, 5-hydroxyindoleacetic acid, p-Cresol sulfate, taurocholic acid, taurochenodeoxycholic acid, glycohyocholic acid, glycocholic acid, and docosapentaenoate n-6 were identified in at least two studies. This pathway-specific review provides a comprehensive overview of the existing evidence from metabolomics studies of childhood atopic diseases. The altered metabolic pathways uncover some of the underlying biochemical mechanisms leading to these common childhood disorders, which may become of potential value in clinical practice.

The relationship between developmental milestone achievement in infancy and later cognitive function and mental health is well established, but underlying biochemical mechanisms are poorly described. Our study aims to discover pathways connected to motor milestone achievement during infancy by using untargeted plasma metabolomic profiles from 571 six-month-old children in connection with age of motor milestones achievement (Denver Developmental Index) in the Copenhagen Prospective Studies on Asthma in Childhood 2010 (COPSAC2010) mother-child cohort. We used univariate regression models and multivariate modelling (Partial Least Squares Discriminant Analysis: PLS-DA) to examine the associations and the VDAART (Vitamin D Antenatal Asthma Reduction Trial) cohort for validation. The univariate analyses showed 62 metabolites associated with gross-motor milestone achievement (p<0.05) as well as the PLS-DA significantly differentiated between slow and fast milestone achievers (AUC = 0.87,p= 0.01). Higher levels of tyramine-O-sulfate in the tyrosine pathway were found in the late achievers in COPSAC (p= 0.0002) and in VDAART (p= 0.02). Furthermore, we observed that slow achievers were characterized by higher levels of fatty acids and products of fatty acids metabolism including acyl carnitines. Finally, we also observed changes in the lysine, histidine, glutamate, creatine and tryptophan pathways. Observing these metabolic changes in relation to gross-motor milestones in the first year of life, may be of importance for later cognitive function and mental health.

Heat treatment is a widely used method for food processing, and the compounds formed by heat processes may serve as biomarkers of heated food intake in nutrition studies. Therefore, we aimed to characterize the differential metabolic signatures resulting from intake of different potato products and identify potential intake biomarkers. In a randomized, controlled, crossover meal study, healthy volunteers consumed boiled rice, boiled potatoes, and two deep-fried potato products, potato chips and French fries. The urine metabolome was acquired by LC-MS-based untargeted metabolomics. Twenty-two selected metabolites were found for deep-fried potatoes, two for potato intake in general, and one for boiled rice. Fourteen of the 22 selected metabolites were tentatively identified as furan-, pyrrole- and pyrazine-derived compounds indicative of Maillard reactions. With further validation, these candidate biomarkers will be important tools to investigate the influence of heated foods on human health.

Scope: Self-reported dietary intake does not represent an objective unbiased assessment. We investigate the effect of the New Nordic Diet (NND) versus Average Danish Diet (ADD) on plasma metabolic profiles to identify biomarkers of compliance and metabolic effects.Methods and Results: In a 26-week controlled dietary intervention study, 146 subjects followed either NND, a predominantly organic diet high in fruit, vegetables, whole grains, and fish, or ADD, a diet higher in imported and processed foods. Fasting plasma samples were analyzed with untargeted UPLC-QTOF. We demonstrate that supervised machine learning with feature selection can separate NND and ADD samples with an average test set performance of up to 0.88 Area Under the Curve. The NND plasma metabolome was characterized by diet related metabolites such as pipecolic acid betaine (whole grain), trimethylamine oxide and prolyl hydroxyproline (both fish intake) while theobromine (chocolate) and proline betaine (citrus) were associated with ADD. Amino acid (i.e., indolelactic acid and hydroxy-3-methylbutyrate) and fat metabolism (butyryl carnitine) characterized ADD while NND was associated with higher concentrations of polyunsaturated phosphatidylcholines.Conclusions: The plasma metabolite profiles were predictive of dietary patterns and reflected good compliance while indicating effects of potential health benefit, including changes in fat metabolism and glucose utilization. This article is protected by copyright. All rights reserved.

Evaluation of health related effects of beer intake is hampered by the lack of accurate tools for assessing intakes (biomarkers). Therefore, we identified plasma and urine metabolites associated with recent beer intake by untargeted metabolomics and established a characteristic metabolite pattern representing raw materials and beer production as a qualitative biomarker of beer intake. In a randomized, crossover, single-blinded meal study (MSt1) 18 participants were given one at a time four different test beverages: strong, regular and non-alcoholic beers and a soft drink. Four participants were assigned to have two additional beers (MSt2). In addition to plasma and urine samples, test beverages, wort and hops extract were analyzed by UPLC-QTOF. A unique metabolite pattern reflecting beer metabolome, including metabolites derived from beer raw material (i.e. N-methyl tyramine sulfate and the sum of iso-α-acids and tricyclohumols) and production process (i.e. pyro-glutamyl proline and 2-ethyl malate) were selected to establish a compliance biomarker model for detection of beer intake based on MSt1. The model predicted the MSt2 samples collected before and up to 12 h after beer intake correctly (AUC = 1). A biomarker model including four metabolites representing both beer raw materials and production steps provided a specific and accurate tool for measurement of beer consumption.

A previous study has shown effects of the New Nordic Diet (NND) to stimulate weight loss and lower systolic and diastolic blood pressure in obese Danish women and men in a randomized, controlled dietary intervention study. This work demonstrates long-term metabolic effects of the NND as compared to an Average Danish Diet (ADD) in blood plasma and reveals associations between metabolic changes and health beneficial effects of the NND including weight loss. A total of 145 individuals completed the intervention and blood samples were taken along with clinical examinations before the intervention started (week 0), and after 12 and 26 weeks. The plasma metabolome was measured using GC-MS and the final metabolite table contained 144 variables. Significant and novel metabolic effects of the diet, resulting weight loss, gender, and intervention study season were revealed using PLS-DA and ASCA. Several metabolites reflecting specific differences in the diets, especially intake of plant foods and seafood, and in energy metabolism related to ketone bodies and gluconeogenesis, formed the predominant metabolite pattern discriminating the intervention groups. Among NND subjects higher levels of vaccenic acid and 3-hydroxybutanoic acid were related to a higher weight loss, while higher concentrations of salicylic, lactic and N-aspartic acids, and 1,5-anhydro-D-sorbitol were related to a lower weight loss. Specific gender- and seasonal differences were also observed. The study strongly indicates that healthy diets high in fish, vegetables, fruit, and wholegrain facilitated weight loss and improved insulin sensitivity by increasing ketosis and gluconeogenesis in the fasting state.

Berries may improve health; however, accurate assessment of berry intake is still problematic. The discovery of objective biomarkers for intake of berries is therefore important in assessing both intake and compliance. We aimed to identify urinary exposure markers of two very different berries, strawberry and sea buckthorn, in humans. A randomized controlled single-blinded three-way cross-over meal study was conducted in 16 overweight men. The intervention meals consisted of sea buckthorn puree, strawberry puree or an iso-caloric control drink. Urine samples were collected on each test day at t = -15 min, t = 0–1 h, t = 1–2 h, and t = 2–24 h and were analyzed by untargeted metabolomics. Multivariate analysis was applied to discover markers, followed by molecular fragmentation to ease their chemical identification. Only a few common markers appeared for the two berries, and all except one were in very low concentrations and therefore not identified. Nine and 11 markers were (tentatively) identified for strawberry and sea buckthorn, respectively, the most specific being conjugates of aroma compounds and coloring flavonoids. Metabolites reflecting the step-wise aromatization of quinic to hippuric acid were observed after sea buckthorn intake. Three of the identified compounds validate previously proposed exposure biomarkers for the New Nordic Diet, which is high in berries. Potentially common berry exposure markers, as well as an aromatization pathway have been distinguished by human urine profiling after consumption of sea buckthorn or strawberries. Combinations of these markers are proposed as potentially specific for intake of each of these berries, but need further validation in larger and less-controlled study settings.