Comparative characterization of the infant gut microbiome and their maternal lineage by a multi-omics approach.

The human gut microbiome establishes and matures during infancy, and dysregulation at this stage may lead to pathologies later in life. We conducted a multi-omics study comprising three generations of family members to investigate the early development of the gut microbiota. Fecal samples from 200 individuals, including infants (0-12 months old; 55% females, 45% males) and their respective mothers and grandmothers, were analyzed using two independent metabolomics platforms and metagenomics. For metabolomics, gas chromatography and capillary electrophoresis coupled to mass spectrometry were applied. For metagenomics, both 16S rRNA gene and shotgun sequencing were performed. Here we show that infants greatly vary from their elders in fecal microbiota populations, function, and metabolome. Infants have a less diverse microbiota than adults and present differences in several metabolite classes, such as short- and branched-chain fatty acids, which are associated with shifts in bacterial populations. These findings provide innovative biochemical insights into the shaping of the gut microbiome within the same generational line that could be beneficial in improving childhood health outcomes.

Urinary Metabolite Profiling to Non-Invasively Monitor the Omega-3 Index: An Exploratory Secondary Analysis of a Randomized Clinical Trial in Young Adults.

The Omega-3 Index (O3I) reflects eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content in erythrocytes. While the O3I is associated with numerous health outcomes, its widespread use is limited. We investigated whether urinary metabolites could be used to non-invasively monitor the O3I in an exploratory analysis of a previous placebo-controlled, parallel arm randomized clinical trial in males and females (n = 88) who consumed either ~3 g/d olive oil (OO; control), EPA, or DHA for 12 weeks. Fasted blood and first-void urine samples were collected at baseline and following supplementation, and they were analyzed via gas chromatography and multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS), respectively. We tentatively identified S-carboxypropylcysteamine (CPCA) as a novel urinary biomarker reflecting O3I status, which increased following both EPA and DHA (p < 0.001), but not OO supplementation, and was positively correlated to the O3I (R = 0.30, p < 0.001). Additionally, an unknown dianion increased following DHA supplementation, but not EPA or OO. In ROC curve analyses, CPCA outperformed all other urinary metabolites in distinguishing both between OO and EPA or DHA supplementation groups (AUC > 80.0%), whereas the unknown dianion performed best in discriminating OO from DHA alone (AUC = 93.6%). Candidate urinary biomarkers of the O3I were identified that lay the foundation for a non-invasive assessment of omega-3 status.

A novel multi-ancestry proteome-wide Mendelian randomization study implicates extracellular proteins, tubular cells, and fibroblasts in estimated glomerular filtration rate regulation.

Estimated glomerular filtration rate (eGFR) impacts the concentration of plasma biomarkers confounding biomarker association studies of eGFR with reverse causation. To identify biomarkers causally associated with eGFR, we performed a proteome-wide Mendelian randomization study. Genetic variants nearby biomarker coding genes were tested for association with plasma concentration of 1,161 biomarkers in a multi-ancestry sample of 12,066 participants from the Prospective Urban and Rural Epidemiological (PURE) study. Using two-sample Mendelian randomization, individual variants' effects on biomarker concentration were correlated with their effects on eGFR and kidney traits from published genome-wide association studies (GWAS). Genetically altered concentrations of 22 biomarkers were associated with eGFR above a Bonferroni-corrected significance threshold. Five biomarkers were previously identified by GWAS (UMOD, FGF5, LGALS7, NINJ1, COL18A1). Nine biomarkers were within 1 Mb of the lead GWAS variant but the gene for the biomarker was unidentified as the candidate for the GWAS signal (INHBC, TNFRSF11A, TCN2, PXN1, PRTN3, PSMD9, TFPI, ITGB6, CA3). Single-cell transcriptomic data indicated the 22 biomarkers are expressed in kidney tubules, collecting duct, fibroblasts, and immune cells. Pathway analysis showed significant enrichment of identified biomarkers in the extracellular kidney parenchyma. Thus, using genetic regulators of biomarker concentration via proteome-wide Mendelian randomization, we identified 22 biomarkers that appear to causally impact eGFR in either a beneficial or adverse manner. The current study provides rationale for novel therapeutic targets for eGFR and emphasized a role for extracellular proteins produced by tubular cells and fibroblasts for impacting eGFR.

Elements and omega-3 fatty acids in fishes along a large, dammed river.

Damming of a river can trap and elevate levels of sediment-bound elements and alter food web dynamics in created reservoirs. It follows that dams may alter how elements and other nutrients, like the beneficial omega-3 fatty acids (n-3 FAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are accumulated in fish and thus the chemical composition of species above and below this barrier to migration. This study examined the spatial and species differences in contaminants and nutrients in fish from the Wolastoq | Saint John River (New Brunswick, Canada) in association with a large hydroelectric dam (Mactaquac Generating Station; MQGS), a river which supports both recreational fisheries and subsistence fishing by Indigenous communities. In 2020 and 2021, Smallmouth Bass, Yellow Perch, American Eel, and Striped Bass were collected from locations upstream (reservoir and river) and downstream of the MQGS and analyzed for mercury (Hg) and 30 other trace elements, n-3 FAs, δ15N, and δ13C. Fish from the reservoir were highest in the beneficial elements P, S, and K, while fish from upstream of the reservoir had lower levels of toxic elements, including Hg. The dam appeared to alter food web dynamics, as fish from the reservoir and immediately downstream of the dam had higher δ15N and reservoir fish were depleted in δ13C. DHA and Hg were positively corelated with δ15N, and EPA in Smallmouth Bass was higher in sites where fish had higher δ13C. Overall, this study suggests that the dam altered food web dynamics and the uptake of contaminants and nutrients by fish, and that location and species are important factors when examining the risks and benefits of consuming wild fish from a system impacted by a large dam.

Integrative multiomics analysis of infant gut microbiome and serum metabolome reveals key molecular biomarkers of early onset childhood obesity.

Evidence supports a complex interplay of gut microbiome and host metabolism as regulators of obesity. The metabolic phenotype and microbial metabolism of host diet may also contribute to greater obesity risk in children early in life. This study aimed to identify features that discriminated overweight/obese from normal weight infants by integrating gut microbiome and serum metabolome profiles. This prospective analysis included 50 South Asian children living in Canada, selected from the SouTh Asian biRth cohorT (START). Serum metabolites were measured by multisegment injection-capillary electrophoresis-mass spectrometry and the relative abundance of bacterial 16S rRNA gene amplicon sequence variant was evaluated at 1 year. Cumulative body mass index (BMIAUC) and skinfold thickness (SSFAUC) scores were calculated from birth to 3 years as the total area under the growth curve (AUC). BMIAUC and/or SSFAUC >85th percentile was used to define overweight/obesity. Data Integration Analysis for Biomarker discovery using Latent cOmponent (DIABLO) was used to identify discriminant features associated with childhood overweight/obesity. The associations between identified features and anthropometric measures were examined using logistic regression. Circulating metabolites including glutamic acid, acetylcarnitine, carnitine, and threonine were positively, whereas γ-aminobutyric acid (GABA), symmetric dimethylarginine (SDMA), and asymmetric dimethylarginine (ADMA) were negatively associated with childhood overweight/obesity. The abundance of the Pseudobutyrivibrio and Lactobacillus genera were positively, and Clostridium sensu stricto 1 and Akkermansia were negatively associated with childhood overweight/obesity. Integrative analysis revealed that Akkermansia was positively whereas Lactobacillus was inversely correlated with GABA and SDMA, and Pseudobutyrivibrio was inversely correlated with GABA. This study provides insights into metabolic and microbial signatures which may regulate satiety, energy metabolism, inflammatory processes, and/or gut barrier function, and therefore, obesity trajectories in childhood. Understanding the functional capacity of these molecular features and potentially modifiable risk factors such as dietary exposures early in life may offer a novel approach for preventing childhood obesity.

Early sex-dependent differences in metabolic profiles of overweight and adiposity in young children: a cross-sectional analysis.

BACKGROUND: Childhood obesity is a global health concern and can lead to lifetime cardiometabolic disease. New advances in metabolomics can provide biochemical insights into the early development of obesity, so we aimed to characterize serum metabolites associated with overweight and adiposity in early childhood and to stratify associations by sex. METHODS: Nontargeted metabolite profiling was conducted in the Canadian CHILD birth cohort (discovery cohort) at age 5 years (n = 900) by multisegment injection-capillary electrophoresis-mass spectrometry. Clinical outcome was defined using novel combined measures of overweight (WHO-standardized body mass index ≥ 85th percentile) and/or adiposity (waist circumference ≥ 90th percentile). Associations between circulating metabolites and child overweight/adiposity (binary and continuous outcomes) were determined by multivariable linear and logistic regression, adjusting for covariates and false discovery rate, and by subsequent sex-stratified analysis. Replication was assessed in an independent replication cohort called FAMILY at age 5 years (n = 456). RESULTS: In the discovery cohort, each standard deviation (SD) increment of branched-chain and aromatic amino acids, glutamic acid, threonine, and oxoproline was associated with 20-28% increased odds of overweight/adiposity, whereas each SD increment of the glutamine/glutamic acid ratio was associated with 20% decreased odds. All associations were significant in females but not in males in sex-stratified analyses, except for oxoproline that was not significant in either subgroup. Similar outcomes were confirmed in the replication cohort, where associations of aromatic amino acids, leucine, glutamic acid, and the glutamine/glutamic acid ratio with childhood overweight/adiposity were independently replicated. CONCLUSIONS: Our findings show the utility of combining measures of both overweight and adiposity in young children. Childhood overweight/adiposity at age 5 years has a specific serum metabolic phenotype, with the profile being more prominent in females compared to males.

The Relationship Between Diet, Gut Microbiota, and Serum Metabolome of South Asian Infants at 1 Year.

BACKGROUND: Diet is known to affect the gut microbiota and the serum metabolome in adults, but this has not been fully explored in infants. Infancy is an important developmental period that may influence a person's long-term health. Infant development can be affected by diet, which also interacts with the developing gut microbiota. OBJECTIVES: This study aimed to explore the associations between diet, the gut microbiota, and the serum metabolome of 1-y-old infants with the overarching goal of identifying serum biomarkers of diet and/or the gut microbiota. METHODS: We derived dietary patterns of 1-y-old infants (n = 182) participating in the Canadian South Asian Birth Cohort (START) study. We compared gut microbiota α-diversity and ß-diversity and taxa relative abundance from 16S rRNA gene profiles with dietary patterns (PERMANOVA, Envfit) and investigated diet-serum metabolite associations using a multivariate analysis (partial least squares-discriminant analysis) and univariate analysis (t test). We explored the effect of nondietary factors on diet-serum metabolite relationships by incorporating diet, the gut microbiota, and maternal, perinatal, and infant characteristics in a multivariable forward stepwise regression. We replicated this analysis in White European infants, from the CHILD Cohort Study (n = 81). RESULTS: A dietary pattern characterized by formula consumption and negatively associated with breastfeeding most strongly predicted variation in the gut microbiota (R2 = 0.109) and serum metabolome (R2 = 0.547). Breastfed participants showed higher abundance of microbes from the genera Bifidobacterium (3.29 log2-fold) and Lactobacillus (7.93 log2-fold) and higher median concentrations of the metabolites S-methylcysteine (1.38 µM) and tryptophan betaine (0.43 µM) than nonbreastfed participants. Formula consuming infants showed higher median concentrations of branched-chain/aromatic amino acids (average 48.3 µM) than non-formula-consuming infants. CONCLUSIONS: Formula consumption and breastfeeding most strongly predicted the serum metabolites of 1-y-old infants, even when the gut microbiota, solid food consumption, and other covariates were considered.

Dynamic Metabolic Signatures of Choline and Carnitine across Healthy Pregnancy and in Cord Blood: Association with Maternal Dietary Protein.

BACKGROUND: In pregnancy, choline is deemed an essential nutrient and carnitine needs are increased, but amounts remain undefined. OBJECTIVES: We aimed to measure choline and total dietary protein and dairy protein intake from food and supplements across pregnancy and the response to intake by profiling choline and carnitine metabolites across pregnancy and in cord blood. METHODS: An exploratory analysis of choline and protein intake from 3-d diet records and measures of 36 serum choline and carnitine metabolites in early (12-17 wk) and late (36-38 wk) pregnancy was conducted in participants from the Be Healthy in Pregnancy study randomized to high dairy protein+walking exercise or usual care. Metabolites were measured in fasted maternal and cord serum using multisegment injection-capillary electrophoresis-mass spectrometry. Mixed ANOVA adjusted for body mass index was performed for comparison of metabolites across pregnancy and between intervention and control. RESULTS: In 104 participants, the median (Q1, Q3) total choline intake was 347 (263, 427) mg/d in early and 322 (270, 437) mg/d in late pregnancy. Only ∼20% of participants achieved the recommended adequate intake (450 mg/d) and ∼10% consumed supplemental choline (8-200 mg/d). Serum-free choline (µmol/L) was higher in late compared with early pregnancy [12.9 (11.4, 15.1) compared with 9.68 (8.25, 10.61), P < 0.001], but choline downstream metabolites were similar across pregnancy. Serum carnitine [10.3 (9.01, 12.2) compared with 15.9 (14.1, 17.9) µmol/L, P < 0.001] and acetylcarnitine [2.35 (1.92, 2.68) compared with 3.0 (2.56, 3.59), P < 0.001] were significantly lower in late pregnancy. High cord:maternal serum metabolite ratios were found in most measured metabolites. CONCLUSIONS: Despite inadequate choline intake, serum-free choline was elevated in late pregnancy and enriched in cord blood compared with maternal serum. Serum carnitine declined in late pregnancy despite a high protein diet. The higher cord:maternal concentrations in choline and carnitine metabolites suggest active uptake in late pregnancy, reflecting the importance of these circulating metabolites in fetal development. This trial was registered at clinicaltrials.gov as NCT01689961.

Parenteral BCG vaccine induces lung-resident memory macrophages and trained immunity via the gut-lung axis.

Aside from centrally induced trained immunity in the bone marrow (BM) and peripheral blood by parenteral vaccination or infection, evidence indicates that mucosal-resident innate immune memory can develop via a local inflammatory pathway following mucosal exposure. However, whether mucosal-resident innate memory results from integrating distally generated immunological signals following parenteral vaccination/infection is unclear. Here we show that subcutaneous Bacillus Calmette-Guérin (BCG) vaccination can induce memory alveolar macrophages (AMs) and trained immunity in the lung. Although parenteral BCG vaccination trains BM progenitors and circulating monocytes, induction of memory AMs is independent of circulating monocytes. Rather, parenteral BCG vaccination, via mycobacterial dissemination, causes a time-dependent alteration in the intestinal microbiome, barrier function and microbial metabolites, and subsequent changes in circulating and lung metabolites, leading to the induction of memory macrophages and trained immunity in the lung. These data identify an intestinal microbiota-mediated pathway for innate immune memory development at distal mucosal tissues and have implications for the development of next-generation vaccine strategies against respiratory pathogens.

Sources of Variation in Food-Related Metabolites during Pregnancy.

The extent to which variation in food-related metabolites are attributable to non-dietary factors remains unclear, which may explain inconsistent food-metabolite associations observed in population studies. This study examined the association between non-dietary factors and the serum concentrations of food-related biomarkers and quantified the amount of variability in metabolite concentrations explained by non-dietary factors. Pregnant women (n = 600) from two Canadian birth cohorts completed a validated semi-quantitative food frequency questionnaire, and serum metabolites were measured by multisegment injection-capillary electrophoresis-mass spectrometry. Hierarchical linear modelling and principal component partial R-square (PC-PR2) were used for data analysis. For proline betaine and DHA (mainly exogenous), citrus foods and fish/fish oil intake, respectively, explained the highest proportion of variability relative to non-dietary factors. The unique contribution of dietary factors was similar (15:0, 17:0, hippuric acid, TMAO) or lower (14:0, tryptophan betaine, 3-methylhistidine, carnitine) compared to non-dietary factors (i.e., ethnicity, maternal age, gestational age, pre-pregnancy BMI, physical activity, and smoking) for metabolites that can either be produced endogenously, biotransformed by gut microbiota, and/or derived from multiple food sources. The results emphasize the importance of adjusting for non-dietary factors in future analyses to improve the accuracy and precision of the measures of food intake and their associations with health and disease.

Serum metabolomic signatures of gestational diabetes in South Asian and white European women.

INTRODUCTION: This study aimed to identify serum metabolomic signatures associated with gestational diabetes mellitus (GDM), and to examine if ethnic-specific differences exist between South Asian and white European women. RESEARCH DESIGN AND METHODS: Prospective cohort study with a nested case-control analysis of 600 pregnant women from two Canadian birth cohorts; using an untargeted approach, 63 fasting serum metabolites were measured and analyzed using multisegment injection-capillary electrophoresis-mass spectrometry. Multivariate logistic regression modeling was conducted overall and by cohort. RESULTS: The proportion of women with GDM was higher in South Asians (27.1%) compared with white Europeans (17.9%). Several amino acid, carbohydrate, and lipid pathways related to GDM were common to South Asian and white European women. Elevated circulating concentrations of glutamic acid, propionylcarnitine, tryptophan, arginine, 2-hydroxybutyric acid, 3-hydroxybutyric acid, and 3-methyl-2-oxovaleric acid were associated with higher odds of GDM, while higher glutamine, ornithine, oxoproline, cystine, glycine with lower odds of GDM. Per SD increase in glucose concentration, the odds of GDM increased (OR=2.07, 95% CI 1.58 to 2.71), similarly for metabolite ratios: glucose to glutamine (OR=2.15, 95% CI 1.65 to 2.80), glucose to creatinine (OR=1.79, 95% CI 1.39 to 2.32), and glutamic acid to glutamine (OR=1.46, 95% CI 1.16 to 1.83). South Asians had higher circulating ratios of glucose to glutamine, glucose to creatinine, arginine to ornithine, and citrulline to ornithine, compared with white Europeans. CONCLUSIONS: We identified a panel of serum metabolites implicated in GDM pathophysiology, consistent in South Asian and white European women. The metabolic alterations leading to larger ratios of glucose to glutamine, glucose to creatinine, arginine to ornithine, and citrulline to ornithine in South Asians likely reflect the greater burden of GDM among South Asians compared with white Europeans.

Metabolite profiles and the risk of metabolic syndrome in early childhood: a case-control study.

BACKGROUND: Defining the metabolic syndrome (MetS) in children remains challenging. Furthermore, a dichotomous MetS diagnosis can limit the power to study associations. We sought to characterize the serum metabolite signature of the MetS in early childhood using high-throughput metabolomic technologies that allow comprehensive profiling of metabolic status from a biospecimen. METHODS: In the Family Atherosclerosis Monitoring In earLY life (FAMILY) prospective birth cohort study, we selected 228 cases of MetS and 228 matched controls among children age 5 years. In addition, a continuous MetS risk score was calculated for all 456 participants. Comprehensive metabolite profiling was performed on fasting serum samples using multisegment injection-capillary electrophoresis-mass spectrometry. Multivariable regression models were applied to test metabolite associations with MetS adjusting for covariates of screen time, diet quality, physical activity, night sleep, socioeconomic status, age, and sex. RESULTS: Compared to controls, thirteen serum metabolites were identified in MetS cases when using multivariable regression models, and using the quantitative MetS score, an additional eight metabolites were identified. These included metabolites associated with gluconeogenesis (glucose (odds ratio (OR) 1.55 [95% CI 1.25-1.93]) and glutamine/glutamate ratio (OR 0.82 [95% CI 0.67-1.00])) and the alanine-glucose cycle (alanine (OR 1.41 [95% CI 1.16-1.73])), amino acids metabolism (tyrosine (OR 1.33 [95% CI 1.10-1.63]), threonine (OR 1.24 [95% CI 1.02-1.51]), monomethylarginine (OR 1.33 [95% CI 1.09-1.64]) and lysine (OR 1.23 [95% CI 1.01-1.50])), tryptophan metabolism (tryptophan (OR 0.78 [95% CI 0.64-0.95])), and fatty acids metabolism (carnitine (OR 1.24 [95% CI 1.02-1.51])). The quantitative MetS risk score was more powerful than the dichotomous outcome in consistently detecting this metabolite signature. CONCLUSIONS: A distinct metabolite signature of pediatric MetS is detectable in children as young as 5 years old and may improve risk assessment at early stages of development.

A Cross-Platform Metabolomics Comparison Identifies Serum Metabolite Signatures of Liver Fibrosis Progression in Chronic Hepatitis C Patients.

Metabolomics offers new insights into disease mechanisms that is enhanced when adopting orthogonal instrumental platforms to expand metabolome coverage, while also reducing false discoveries by independent replication. Herein, we report the first inter-method comparison when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS) and nuclear magnetic resonance (NMR) spectroscopy for characterizing the serum metabolome of patients with liver fibrosis in chronic hepatitis C virus (HCV) infection (n = 20) and non-HCV controls (n = 14). In this study, 60 and 30 serum metabolites were detected frequently (>75%) with good technical precision (median CV < 10%) from serum filtrate samples (n = 34) when using standardized protocols for MSI-CE-MS and NMR, respectively. Also, 20 serum metabolite concentrations were consistently measured by both methods over a 500-fold concentration range with an overall mean bias of 9.5% (n = 660). Multivariate and univariate statistical analyses independently confirmed that serum choline and histidine were consistently elevated (p < 0.05) in HCV patients with late-stage (F2-F4) as compared to early-stage (F0-F1) liver fibrosis. Overall, the ratio of serum choline to uric acid provided optimal differentiation of liver disease severity (AUC = 0.848, p = 0.00766) using a receiver operating characteristic curve, which was positively correlated with liver stiffness measurements by ultrasound imaging (r = 0.606, p = 0.0047). Moreover, serum 5-oxo-proline concentrations were higher in HCV patients as compared to non-HCV controls (F = 4.29, p = 0.0240) after adjustment for covariates (age, sex, BMI), indicative of elevated oxidative stress from glutathione depletion with the onset and progression of liver fibrosis. Both instrumental techniques enable rapid yet reliable quantification of serum metabolites in large-scale metabolomic studies with good overlap for biomarker replication. Advantages of MSI-CE-MS include greater metabolome coverage, lower operating costs, and smaller sample volume requirements, whereas NMR offers a robust platform supported by automated spectral and data processing software.

Urinary Metabolites Enable Differential Diagnosis and Therapeutic Monitoring of Pediatric Inflammatory Bowel Disease.

Rates of pediatric Crohn's disease (CD) and ulcerative colitis (UC) are increasing globally. Differentiation of these inflammatory bowel disease (IBD) subtypes however can be challenging when relying on invasive endoscopic approaches. We sought to identify urinary metabolic signatures of pediatric IBD at diagnosis, and during induction treatment. Nontargeted metabolite profiling of urine samples from CD (n = 18) and UC (n = 8) in a pediatric retrospective cohort study was performed using multisegment injection-capillary electrophoresis-mass spectrometry. Over 122 urinary metabolites were reliably measured from pediatric IBD patients, and unknown metabolites were identified by tandem mass spectrometry. Dynamic changes in sum-normalized urinary metabolites were also monitored following exclusive enteral nutrition (EEN) or corticosteroid therapy (CS) in repeat urine samples collected over 8 weeks. Higher urinary excretion of indoxyl sulfate, hydroxyindoxyl sulfate, phenylacetylglutamine, and sialic acid were measured in CD as compared to UC patients, but lower threonine, serine, kynurenine, and hypoxanthine (p < 0.05). Excellent discrimination of CD from UC was achieved based on the urinary serine:indoxylsulfate ratio (AUC = 0.972; p = 3.21 × 10-5). Urinary octanoyl glucuronide, pantothenic acid, and pyridoxic acid were also identified as specific dietary biomarkers of EEN in pediatric IBD patients who achieved clinical remission. This work may complement or replace existing strategies in the diagnosis and early management of children with IBD.

The maternal serum metabolome by multisegment injection-capillary electrophoresis-mass spectrometry: a high-throughput platform and standardized data workflow for large-scale epidemiological studies.

A standardized data workflow is described for large-scale serum metabolomic studies using multisegment injection-capillary electrophoresis-mass spectrometry. Multiplexed separations increase throughput (<4 min/sample) for quantitative determination of 66 polar/ionic metabolites in serum filtrates consistently detected (coefficient of variance (CV) <30%) with high frequency (>75%) from a multi-ethnic cohort of pregnant women (n = 1,004). We outline a validated protocol implemented in four batches over a 7-month period that includes details on preventive maintenance, sample workup, data preprocessing and metabolite authentication. We achieve stringent quality control (QC) and robust batch correction of long-term signal drift with good mutual agreement for a wide range of metabolites, including serum glucose as compared to a clinical chemistry analyzer (mean bias = 11%, n = 668). Control charts for a recovery standard (mean CV = 12%, n = 2,412) and serum metabolites in QC samples (median CV = 13%, n = 202) demonstrate acceptable intermediate precision with a median intraclass coefficient of 0.87. We also report reference intervals for 53 serum metabolites from a diverse population of women in their second trimester of pregnancy.

Lyophilized fecal short-chain fatty acid and electrolyte determination by capillary electrophoresis with indirect UV detection for assessment of pediatric inflammatory bowel disease.

Short-chain fatty acids (SCFAs) and electrolytes are major constituents of human feces involved in maintaining gastrointestinal homeostasis that underlie complex diet, host and microbiome interactions. Reliable quantification of SCFAs and electrolytes is challenging given the heterogeneity of stool specimens from pediatric patients with diarrhea-predominate inflammatory bowel disease (IBD). Herein, we introduce two validated methods for determination of 3 SCFAs and 5 electrolytes consistently quantified from fecal extracts when using capillary electrophoresis with indirect UV detection (CE-iUV), where concentrations are normalized to total dried weight (mmol/kg d.w.). Lyophilization facilitates sample handling and extraction of heterogeneous stool specimens (∼ 15 mg) from a cohort of children with Crohn's disease (CD, n = 12) and ulcerative colitis (UC, n = 10) treated with exclusive enteral nutrition (EEN) or corticosteroid (CS) therapy to induce remission, respectively. Good technical precision (mean CV = 13 %, n = 14) and accuracy (recovery from 84 to 116%) is demonstrated for SCFAs and electrolytes from freeze dried stool extracts using a modified Bligh-Dyer protocol with low micromolar detection limits (∼ 2-15 µM). Fecal butyrate is 2.6-fold higher in CD as compared to UC patients (effect size = 1.51; p = 0.00291), and there is a strong co-linearity between fecal butyrate and acetate (r = 0.835) unlike propionate, which is correlated with fecal calprotectin (r = 0.517), a protein biomarker of intestinal inflammation. Also, a longitudinal study of matching stool samples collected from a sub-set of IBD patients revealed about a 7-fold enrichment in magnesium and calcium following 4 weeks of EEN as compared to baseline (F > 4.1 ; p < 0.05) unlike the CS treatment arm with no changes in other fecal SCFAs and electrolytes, including sodium, potassium, and ammonium. CE-iUV enables rapid fecal SCFA and electrolyte determination as required for new insights into the role of gut dysbiosis in IBD, as well as treatment monitoring of nutritional interventions that stabilize the disease course in affected children.

Personalization of Aspirin Therapy Ex Vivo in Patients with Atherosclerosis Using Light Transmission Aggregometry.

Acetylsalicylic acid (ASA), also known as aspirin, appears to be ineffective in inhibiting platelet aggregation in 20-30% of patients. Light transmission aggregometry (LTA) is a gold standard platelet function assay. In this pilot study, we used LTA to personalize ASA therapy ex vivo in atherosclerotic patients. Patients were recruited who were on 81 mg ASA, presenting to ambulatory clinics at St. Michael's Hospital (n = 64), with evidence of atherosclerotic disease defined as clinical symptoms and diagnostic findings indicative of symptomatic peripheral arterial disease (PAD), with an ankle brachial index (ABI) of <0.9 (n = 52) or had diagnostic features of asymptomatic carotid arterial stenosis (CAS), with >50% stenosis of internal carotid artery on duplex ultrasound (n = 12). ASA compliance was assessed via multisegmented injection-capillary electrophoresis-mass spectrometry based on measuring the predominant urinary ASA metabolite, salicyluric acid. LTA with arachidonic acid was used to test for ASA sensitivity. Escalating ASA dosages of 162 mg and 325 mg were investigated ex vivo for ASA dose personalization. Of the 64 atherosclerotic patients recruited, 8 patients (13%) were non-compliant with ASA. Of ASA compliant patients (n = 56), 9 patients (14%) were non-sensitive to their 81 mg ASA dosage. Personalizing ASA therapy in 81 mg ASA non-sensitive patients with escalating dosages of ASA demonstrated that 6 patients became sensitive to a dosage equivalent to 162 mg ASA and 3 patients became sensitive to a dosage equivalent to 325 mg ASA. We were able to personalize ASA dosage ex vivo in all ASA non-sensitive patients with escalating dosages of ASA within 1 h of testing.

Maternal Diet and the Serum Metabolome in Pregnancy: Robust Dietary Biomarkers Generalizable to a Multiethnic Birth Cohort.

BACKGROUND: Advances in metabolomics are anticipated to decipher associations between dietary exposures and health. Replication biomarker studies in different populations are critical to demonstrate generalizability. OBJECTIVES: To identify and validate robust serum metabolites associated with diet quality and specific foods in a multiethnic cohort of pregnant women. DESIGN: In this cross-sectional analysis of 3 multiethnic Canadian birth cohorts, we collected semiquantitative FFQ and serum data from 900 women at the second trimester of pregnancy. We calculated a diet quality score (DQS), defined as daily servings of "healthy" minus "unhealthy" foods. Serum metabolomics was performed by multisegment injection-capillary electrophoresis-mass spectrometry, and specific serum metabolites associated with maternal DQSs were identified. We combined the results across all 3 cohorts using meta-analysis to classify robust dietary biomarkers (r > ± 0.1; P < 0.05). RESULTS: Diet quality was higher in the South Asian birth cohort (mean DQS = 7.1) than the 2 white Caucasian birth cohorts (mean DQS <3.2). Sixty-six metabolites were detected with high frequency (>75%) and adequate precision (CV <30%), and 47 were common to all cohorts. Hippuric acid was positively associated with healthy diet score in all cohorts, and with the overall DQS only in the primarily white Caucasian cohorts. We observed robust correlations between: 1) proline betaine-citrus foods; 2) 3-methylhistidine-red meat, chicken, and eggs; 3) hippuric acid-fruits and vegetables; 4) trimethylamine N-oxide (TMAO)-seafood, meat, and eggs; and 5) tryptophan betaine-nuts/legumes. CONCLUSIONS: Specific serum metabolites reflect intake of citrus fruit/juice, vegetables, animal foods, and nuts/legumes in pregnant women independent of ethnicity, fasting status, and delays to storage across multiple collection centers. Robust biomarkers of overall diet quality varied by cohort. Proline betaine, 3-methylhistidine, hippuric acid, TMAO, and tryptophan betaine were robust dietary biomarkers for investigations of maternal nutrition in diverse populations.