Metabolomic data presents challenges for epidemiological meta-analysis: a case study of childhood body mass index from the ECHO consortium.

INTRODUCTION: Meta-analyses across diverse independent studies provide improved confidence in results. However, within the context of metabolomic epidemiology, meta-analysis investigations are complicated by differences in study design, data acquisition, and other factors that may impact reproducibility. OBJECTIVE: The objective of this study was to identify maternal blood metabolites during pregnancy (> 24 gestational weeks) related to offspring body mass index (BMI) at age two years through a meta-analysis framework. METHODS: We used adjusted linear regression summary statistics from three cohorts (total N = 1012 mother-child pairs) participating in the NIH Environmental influences on Child Health Outcomes (ECHO) Program. We applied a random-effects meta-analysis framework to regression results and adjusted by false discovery rate (FDR) using the Benjamini-Hochberg procedure. RESULTS: Only 20 metabolites were detected in all three cohorts, with an additional 127 metabolites detected in two of three cohorts. Of these 147, 6 maternal metabolites were nominally associated (P < 0.05) with offspring BMI z-scores at age 2 years in a meta-analytic framework including at least two studies: arabinose (Coefmeta = 0.40 [95% CI 0.10,0.70], Pmeta = 9.7 × 10-3), guanidinoacetate (Coefmeta = - 0.28 [- 0.54, - 0.02], Pmeta = 0.033), 3-ureidopropionate (Coefmeta = 0.22 [0.017,0.41], Pmeta = 0.033), 1-methylhistidine (Coefmeta = - 0.18 [- 0.33, - 0.04], Pmeta = 0.011), serine (Coefmeta = - 0.18 [- 0.36, - 0.01], Pmeta = 0.034), and lysine (Coefmeta = - 0.16 [- 0.32, - 0.01], Pmeta = 0.044). No associations were robust to multiple testing correction. CONCLUSIONS: Despite including three cohorts with large sample sizes (N > 100), we failed to identify significant metabolite associations after FDR correction. Our investigation demonstrates difficulties in applying epidemiological meta-analysis to clinical metabolomics, emphasizes challenges to reproducibility, and highlights the need for standardized best practices in metabolomic epidemiology.

Associations of maternal plasma and umbilical cord plasma metabolomics profiles with birth anthropometric measures.

BACKGROUND: The metabolomics profiles of maternal plasma during pregnancy and cord plasma at birth might influence fetal growth and birth anthropometry. The objective was to examine how maternal plasma and umbilical cord plasma metabolites are associated with newborn anthropometric measures, a known predictor of future health outcomes. METHODS: Pregnant women between 24 and 28 weeks of gestation were recruited as part of a prospective cohort study. Blood samples from 413 women at enrollment and 787 infant cord blood samples were analyzed using the Biocrates AbsoluteIDQ® p180 kit. Multivariable linear regression models were used to examine associations of cord and maternal metabolites with infant anthropometry at birth. RESULTS: In cord blood samples from this rural cohort from New Hampshire of largely white residents, 13 metabolites showed negative associations, and 10 metabolites showed positive associations with birth weight Z-score. Acylcarnitine C5 showed negative association, and 4 lysophosphatidylcholines showed positive associations with birth length Z-score. Maternal blood metabolites did not significantly correlate with birth weight and length Z-scores. CONCLUSIONS: Consistent findings were observed for several acylcarnitines that play a role in utilization of energy sources, and a lysophosphatidylcholine that is part of oxidative stress and inflammatory response pathways in cord plasma samples. IMPACT: The metabolomics profiles of maternal plasma during pregnancy and cord plasma at birth may influence fetal growth and birth anthropometry. This study examines the independent effects of maternal gestational and infant cord blood metabolomes across different classes of metabolites on birth anthropometry. Acylcarnitine species were negatively associated and glycerophospholipids species were positively associated with weight and length Z-scores at birth in the cord plasma samples, but not in the maternal plasma samples. This study identifies lipid metabolites in infants that possibly may affect early growth.

Longitudinal associations of pre-pregnancy BMI and gestational weight gain with maternal urinary metabolites: an NYU CHES study.

BACKGROUND/OBJECTIVES: Excessive gestational weight gain (GWG) and pre-pregnancy obesity affect a significant portion of the US pregnant population and are linked with negative maternal and child health outcomes. The objective of this study was to explore associations of pre-pregnancy body mass index (pBMI) and GWG with longitudinally measured maternal urinary metabolites throughout pregnancy. SUBJECTS/METHODS: Among 652 participants in the New York University Children's Health and Environment Study, a longitudinal pregnancy cohort, targeted metabolomics were measured in serially collected urine samples throughout pregnancy. Metabolites were measured at median 10 (T1), 21 (T2), and 29 (T3) weeks gestation using the Biocrates AbsoluteIDQ® p180 Urine Extension kit. Acylcarnitine, amino acid, biogenic amine, phosphatidylcholine, lysophosphatidylcholine, sphingolipid, and sugar levels were quantified. Pregnant people 18 years or older, without type 1 or 2 diabetes and with singleton live births and valid pBMI and metabolomics data were included. GWG and pBMI were calculated using weight and height data obtained from electronic health records. Linear mixed effects models with interactions with time were fit to determine the gestational age-specific associations of categorical pBMI and continuous interval-specific GWG with urinary metabolites. All analyses were corrected for false discovery rate. RESULTS: Participants with obesity had lower long-chain acylcarnitine levels throughout pregnancy and lower phosphatidylcholine and glucogenic amino acids and higher phenylethylamine concentrations in T2 and T3 compared with participants with normal/underweight pBMI. GWG was associated with taurine in T2 and T3 and C5 acylcarnitine species, C5:1, C5-DC, and C5-M-DC, in T2. CONCLUSIONS: pBMI and GWG were associated with the metabolic environment of pregnant individuals, particularly in relation to mid-pregnancy. These results highlight the importance of both preconception and prenatal maternal health.

Chemical exposures assessed via silicone wristbands and endogenous plasma metabolomics during pregnancy.

BACKGROUND: Metabolomics is a promising method to investigate physiological effects of chemical exposures during pregnancy, with the potential to clarify toxicological mechanisms, suggest sensitive endpoints, and identify novel biomarkers of exposures. OBJECTIVE: Investigate the influence of chemical exposures on the maternal plasma metabolome during pregnancy. METHODS: Data were obtained from participants (n = 177) in the New Hampshire Birth Cohort Study, a prospective pregnancy cohort. Chemical exposures were assessed via silicone wristbands worn for one week at ~13 gestational weeks. Metabolomic features were assessed in plasma samples obtained at ~24-28 gestational weeks via the Biocrates AbsoluteIDQ® p180 kit and nuclear magnetic resonance (NMR) spectroscopy. Associations between chemical exposures and plasma metabolomics were investigated using multivariate modeling. RESULTS: Chemical exposures predicted 11 (of 226) and 23 (of 125) metabolomic features in Biocrates and NMR, respectively. The joint chemical exposures did not significantly predict pathway enrichment, though some individual chemicals were associated with certain amino acids and related metabolic pathways. For example, N,N-diethyl-m-toluamide was associated with the amino acids glycine, L-glutamic acid, L-asparagine, and L-aspartic acid and enrichment of the ammonia recycling pathway. SIGNIFICANCE: This study contributes evidence to the potential effects of chemical exposures during pregnancy upon the endogenous maternal plasma metabolome.

Identifier mapping performance for integrating transcriptomics and proteomics experimental results.

BACKGROUND: Studies integrating transcriptomic data with proteomic data can illuminate the proteome more clearly than either separately. Integromic studies can deepen understanding of the dynamic complex regulatory relationship between the transcriptome and the proteome. Integrating these data dictates a reliable mapping between the identifier nomenclature resultant from the two high-throughput platforms. However, this kind of analysis is well known to be hampered by lack of standardization of identifier nomenclature among proteins, genes, and microarray probe sets. Therefore data integration may also play a role in critiquing the fallible gene identifications that both platforms emit. RESULTS: We compared three freely available internet-based identifier mapping resources for mapping UniProt accessions (ACCs) to Affymetrix probesets identifications (IDs): DAVID, EnVision, and NetAffx. Liquid chromatography-tandem mass spectrometry analyses of 91 endometrial cancer and 7 noncancer samples generated 11,879 distinct ACCs. For each ACC, we compared the retrieval sets of probeset IDs from each mapping resource. We confirmed a high level of discrepancy among the mapping resources. On the same samples, mRNA expression was available. Therefore, to evaluate the quality of each ACC-to-probeset match, we calculated proteome-transcriptome correlations, and compared the resources presuming that better mapping of identifiers should generate a higher proportion of mapped pairs with strong inter-platform correlations. A mixture model for the correlations fitted well and supported regression analysis, providing a window into the performance of the mapping resources. The resources have added and dropped matches over two years, but their overall performance has not changed. CONCLUSIONS: The methods presented here serve to achieve concrete context-specific insight, to support well-informed decisions in choosing an ID mapping strategy for "omic" data merging.

Proteomic analysis of stage I endometrial cancer tissue: identification of proteins associated with oxidative processes and inflammation.

OBJECTIVE: The present study aimed to identify differentially expressed proteins employing a high resolution mass spectrometry (MS)-based proteomic analysis of endometrial cancer cells harvested using laser microdissection. METHODS: A differential MS-based proteomic analysis was conducted from discrete epithelial cell populations gathered by laser microdissection from 91 pathologically reviewed stage I endometrial cancer tissue samples (79 endometrioid and 12 serous) and 10 samples of normal endometrium from postmenopausal women. Hierarchical cluster analysis of protein abundance levels derived from a spectral count analysis revealed a number of proteins whose expression levels were common as well as unique to both histologic types. An independent set of endometrial cancer specimens from 394 patients were used to externally validate the differential expression of select proteins. RESULTS: 209 differentially expressed proteins were identified in a comparison of stage I endometrial cancers and normal post-menopausal endometrium controls (Q<0.005). A number of differentially abundant proteins in stage I endometrial cancer were identified and independently validated by western blot and tissue microarray analyses. Multiple proteins identified with elevated abundance in stage I endometrial cancer are functionally associated with inflammation (annexins) and oxidative processes (peroxiredoxins). PRDX1 and ANXA2 were both confirmed as being overexpressed in stage I cancer compared to normal endometrium by independent TMA (Q=0.008 and Q=0.00002 respectively). CONCLUSIONS: These data provide the basis for further investigation of previously unrecognized novel pathways involved in early stage endometrial carcinogenesis and provide possible targets for prevention strategies that are inclusive of both endometrioid and serous histologic subtypes.

Synovial fluid proteins differentiate between the subtypes of juvenile idiopathic arthritis.

OBJECTIVE: Juvenile idiopathic arthritis (JIA) is a heterogeneous group of inflammatory diseases, and no clinically useful prognostic markers to predict disease outcome in children with JIA are currently available. Synovial fluid likely reflects the proteins present in the inflamed synovium. The purpose of this study was to delineate the synovial fluid proteome and determine whether protein expression differs in the different subtypes of JIA. METHODS: Synovial fluid samples obtained from children with oligoarticular JIA, polyarticular JIA, or systemic JIA were compared. Two-dimensional gel electrophoresis for protein separation and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and quadripole time-of-flight mass spectrometry for protein identification were used for this study. Synovial fluid cells were analyzed by polymerase chain reaction (PCR) for the presence of haptoglobin messenger RNA (mRNA). RESULTS: The synovial fluid proteome of the samples was delineated. The majority of proteins showed overexpression in JIA synovial fluid as compared with noninflammatory control samples. There were 24 statistically significantly differentially expressed spots (>2-fold change; P < 0.05) between the subtypes of JIA. PCR analysis revealed haptoglobin mRNA, suggesting that haptoglobin is locally produced in an inflamed joint in JIA. CONCLUSION: Despite the similar histologic appearance of inflamed joints in patients with different subtypes of JIA, there are differences in protein expression according to the subtype of JIA. Haptoglobin is differentially expressed between the subtypes of JIA and is locally produced in an inflamed joint in JIA. Haptoglobin and other differentially expressed proteins may be potential biomarkers in JIA.

Two methods for assessment of choline status in a randomized crossover study with varying dietary choline intake in people: isotope dilution MS of plasma and in vivo single-voxel magnetic resonance spectroscopy of liver.

BACKGROUND: Choline deficiency has numerous negative health consequences; although the preponderance of the US population consumes less than the recommended Adequate Intake (AI), clinical assessment of choline status is difficult. Further, several pathways involved in primary metabolism of choline are estrogen-sensitive and the AI for premenopausal women is lower than that for men. OBJECTIVES: We sought to determine whether in vivo magnetic resonance spectroscopy (MRS) of liver and/or isotope-dilution MS of plasma could identify biomarkers reflective of choline intake (preregistered primary outcomes 1 and 2, secondary outcome 1). Determination of whether biomarker concentrations showed sex dependence was a post hoc outcome. This substudy is a component of a larger project to identify a clinically useful biomarker panel for assessment of choline status. METHODS: In a double-blind, randomized, crossover trial, people consumed 3 diets, representative of ∼100%, ∼50%, and ∼25% of the choline AI, for 2-wk periods. We measured the concentrations of choline and several metabolites using 1H single-voxel MRS of liver in vivo and using 2H-labeled isotope dilution MS of several choline metabolites in extracted plasma. RESULTS: Plasma concentrations of 2H9-choline, unlabeled betaine, and 2H9-betaine, and the isotopic enrichment ratio (IER) of betaine showed highly significant between-diet effects (q < 0.0001), with unlabeled betaine concentration decreasing 32% from highest to lowest choline intake. Phosphatidylcholine IER was marginally significant (q = 0.03). Unlabeled phosphatidylcholine plasma concentrations did not show between-diet effects (q = 0.34). 2H9 (trimethyl)-phosphatidylcholine plasma concentrations (q = 0.07) and MRS-measured total soluble choline species liver concentrations (q = 0.07) showed evidence of between-diet effects but this was not statistically significant. CONCLUSIONS: Although MRS is a more direct measure of choline status, variable spectral quality limited interpretation. MS analysis of plasma showed clear correlation of plasma betaine concentration, but not plasma phosphatidylcholine concentration, with dietary choline intake. Plasma betaine concentrations also correlate with sex status (premenopausal women, postmenopausal women, men).This trial was registered at clinicaltrials.gov as NCT03726671.

Identification of Novel Proteins Interacting with Proprotein Convertase Subtilisin/Kexin 9.

High levels of cholesterol, especially as low-density lipoprotein (LDL), are a well-known risk factor for atherosclerotic-related diseases. The key atherogenic property of LDL is its ability to form atherosclerotic plaque. Proprotein convertase subtilisin/kexin-9 (PCSK9) is an indirect regulator of plasma LDL levels by controlling the number of LDL receptor molecules expressed at the plasma membrane, especially in the liver. Herein, we performed a combination of affinity chromatography, mass spectrometry analysis and identification, and gene expression studies to identify proteins that interact with PCSK9. Through these studies, we identified three proteins, alpha-1-antitrypsin (A1AT), alpha-1-microglobulin/bikunin precursor (AMBP), and apolipoprotein H (APOH) expressed by C3A cells that interact with PCSK9. The expression levels of A1AT and APOH increased in cells treated with MITO+ medium, a condition previously shown to affect the function of PCSK9, as compared to treating with Regular (control) medium. However, AMBP expression did not change in response to the treatments. Additional studies are required to determine which of these proteins can modulate the expression/function of PCSK9. The identification of endogenous modulators of PCSK9's function could lead to the development of novel diagnostic tests or treatment options for patients suffering hypercholesterolemia in combination with other chronic metabolic diseases.

C16-ceramide is a natural regulatory ligand of p53 in cellular stress response.

Ceramides are important participants of signal transduction, regulating fundamental cellular processes. Here we report the mechanism for activation of p53 tumor suppressor by C16-ceramide. C16-ceramide tightly binds within the p53 DNA-binding domain (Kd ~ 60 nM), in close vicinity to the Box V motif. This interaction is highly selective toward the ceramide acyl chain length with its C10 atom being proximal to Ser240 and Ser241. Ceramide binding stabilizes p53 and disrupts its complex with E3 ligase MDM2 leading to the p53 accumulation, nuclear translocation and activation of the downstream targets. This mechanism of p53 activation is fundamentally different from the canonical p53 regulation through protein-protein interactions or posttranslational modifications. The discovered mechanism is triggered by serum or folate deprivation implicating it in the cellular response to nutrient/metabolic stress. Our study establishes C16-ceramide as a natural small molecule activating p53 through the direct binding.