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Metabolomics is a continuously dynamic field of research that is driven by demanding research questions and technological advances alike. In this review we highlight selected recent and ongoing developments in the area of mass spectrometry-based metabolomics. The field of view that can be seen through the metabolomics lens can be broadened by adoption of separation techniques such as hydrophilic interaction chromatography and ion mobility mass spectrometry (going broader). For a given biospecimen, deeper metabolomic analysis can be achieved by resolving smaller entities such as rare cell populations or even single cells using nano-LC and spatially resolved metabolomics or by extracting more useful information through improved metabolite identification in untargeted metabolomic experiments (going deeper). Integration of metabolomics with other (omics) data allows researchers to further advance in the understanding of the complex metabolic and regulatory networks in cells and model organisms (going further). Taken together, diverse fields of research from mechanistic studies to clinics to biotechnology applications profit from these technological developments.

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Alterations in metabolic activity contribute to the proliferation and survival of cancer cells. We investigated the effect of siRNA-mediated gene silencing of 222 metabolic enzymes, transporters, and regulators on the survival of 3 metastatic prostate cancer cell lines and a nonmalignant prostate epithelial cell line. This approach revealed significant complexity in the metabolic requirements of prostate cancer cells and identified several genes selectively required for their survival. Among these genes was 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), an isoform of phosphofructokinase 2 (PFK2). We show that PFKFB4 is required to balance glycolytic activity and antioxidant production to maintain cellular redox balance in prostate cancer cells. Depletion of PFKFB4 inhibited tumor growth in a xenograft model, indicating that it is required under physiologic nutrient levels. PFKFB4 mRNA expression was also found to be greater in metastatic prostate cancer compared with primary tumors. Taken together, these results indicate that PFKFB4 is a potential target for the development of antineoplastic agents.Significance: Cancer cells undergo several changes in their metabolism that promote growth and survival. Using an unbiased functional screen, we found that the glycolytic enzyme PFKFB4 is essential for prostate cancer cell survival by maintaining the balance between the use of glucose for energy generation and the synthesis of antioxidants. Targeting PFKFB4 may therefore present new therapeutic opportunities. Cancer Discov; 2(4); 328–43. ©2012 AACR.Read the Commentary on this article by Dang, p. 304This article is highlighted in the In This Issue feature, 288

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Metabolomics is a continuously dynamic field of research that is driven by demanding research questions and technological advances alike. In this review we highlight selected recent and ongoing developments in the area of mass spectrometry-based metabolomics. The field of view that can be seen through the metabolomics lens can be broadened by adoption of separation techniques such as hydrophilic interaction chromatography and ion mobility mass spectrometry (going broader). For a given biospecimen, deeper metabolomic analysis can be achieved by resolving smaller entities such as rare cell populations or even single cells using nano-LC and spatially resolved metabolomics or by extracting more useful information through improved metabolite identification in untargeted metabolomic experiments (going deeper). Integration of metabolomics with other (omics) data allows researchers to further advance in the understanding of the complex metabolic and regulatory networks in cells and model organisms (going further). Taken together, diverse fields of research from mechanistic studies to clinics to biotechnology applications profit from these technological developments.

Through evolution, eukaryote organisms have developed the ability to use different molecules as independent precursors to generate nicotinamide adenine dinucleotide (NAD

International audience; BackgroundConstitutional thinness (CT), a non‐malnourished underweight state with no eating disorders, is characterized by weight gain resistance to high fat diet. Data issued from muscle biopsies suggested blunted anabolic mechanisms in free‐living state. Weight and metabolic responses to protein caloric supplementation has not been yet explored in CT.MethodsA 2 week overfeeding (additional 600 kcal, 30 g protein, 72 g carbohydrate, and 21 g fat) was performed to compare two groups of CTs (12 women and 11 men) to normal‐weight controls (12 women and 10 men). Bodyweight, food intake, energy expenditure, body composition, nitrogen balance, appetite hormones profiles, and urine metabolome were monitored before and after overfeeding.ResultsBefore overfeeding, positive energy gap was found in both CT genders (309 ± 370 kcal in CT‐F and 332 ± 709 kcal in CT‐M) associated with higher relative protein intake per kilo (1.74 ± 0.32 g/kg/day in CT‐F vs. 1.16 ± 0.23 in C‐F, P < 0.0001; 1.56 ± 0.36 in CT‐M vs. 1.22 ± 0.32 in C‐M, P = 0.03), lower nitrogen (7.26 ± 2.36 g/day in CT‐F vs. 11.41 ± 3.64 in C‐F, P = 0.003; 9.70 ± 3.85 in CT‐M vs. 14.14 ± 4.19 in C‐M, P = 0.02), but higher essential amino acids urinary excretion (CT/C fold change of 1.13 for leucine and 1.14 for arginine) in free‐living conditions. After overfeeding, CTs presented an accentuated positive energy gap, still higher than in controls (675 ± 540 in CTs vs. 379 ± 427 in C, P = 0.04). Increase in lean mass was induced in both controls genders but not in CTs (a trend was noticed in CT women), despite a similar nitrogen balance after overfeeding (5.06 ± 4.33 g/day in CTs vs. 4.28 ± 3.15 in controls, P = 0.49). Higher anorectic gut hormones' tone, glucagon‐like peptide 1 and peptide tyrosine tyrosine, during test meal and higher snacking frequency were noticed before and after overfeeding in CTs.ConclusionsThe blunted muscle energy mechanism, previously described in CTs in free‐living state, is associated with basal saturated protein turn over suggested by the concordance of positive nitrogen balance and an increased urine excretion of several essential amino acids. This saturation cannot be overpassed by increasing this spontaneous high‐protein intake suggesting a resistance to lean mass gain in CT phenotype.

Nicotinamide adenine dinucleotide (NAD+) and its reduced form (NADH) are coenzymes employed in hundreds of metabolic reactions. NAD+ also serves as a substrate for enzymes such as sirtuins, poly(ADP‐ribose) polymerases (PARPs) and ADP‐ribosyl cyclases. Given the pivotal role of NAD(H) in health and disease, studying NAD+ metabolism has become essential to monitor ge-netic‐ and/or drug‐induced perturbations related to metabolic status and diseases (such as ageing, cancer or obesity), and its possible therapies. Here, we present a strategy based on liquid chroma-tography‐tandem mass spectrometry (LC‐MS/MS), for the analysis of the NAD+ metabolome in biological samples. In this method, hydrophilic interaction chromatography (HILIC) was used to sep-arate a total of 18 metabolites belonging to pathways leading to NAD+ biosynthesis, including pre-cursors, intermediates and catabolites. As redox cofactors are known for their instability, a sample preparation procedure was developed to handle a variety of biological matrices: cell models, rodent tissues and biofluids, as well as human biofluids (urine, plasma, serum, whole blood). For clinical applications, quantitative LC‐MS/MS for a subset of metabolites was demonstrated for the analysis of the human whole blood of nine volunteers. Using this developed workflow, our methodology allows studying NAD+ biology from mechanistic to clinical applications.

Fatty acids play a significant role in maintaining cellular and DNA protection and we previously found an inverse relationship between blood levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and DNA damage. The aim of this study was to explore differences in proteomic profiles, for 117 pro-inflammatory proteins, in two previously defined groups of individuals with different DNA damage and EPA and DHA levels. Healthy children and adolescents (n = 140) aged 9 to 13 years old in an urban area of Brazil were divided by k-means cluster test into two clusters of DNA damage (tail intensity) using the comet assay (cluster 1 = 5.9% ± 1.2 and cluster 2 = 13.8% ± 3.1) in our previous study. The cluster with higher DNA damage and lower levels of DHA (6.2 ± 1.6 mg/dL, 5.4 ± 1.3 mg/dL, p = 0.003) and EPA (0.6 ± 0.2 mg/dL, 0.5 ± 0.1 mg/dL, p &lt, 0.001) presented increased expression of the proteins CDK8–CCNC, PIK3CA–PIK3R1, KYNU, and PRKCB, which are involved in pro-inflammatory pathways. Our findings support the hypothesis that low levels of n-3 long-chain PUFA may have a less protective role against DNA damage through expression of pro-inflammatory proteins, such as CDK8–CCNC, PIK3CA–PIK3R1, KYNU, and PRKCB.

Micronutrients and their metabolites are cofactors in proteins involved in lipid metabolism. The present study was a subproject of the Harmonized Micronutrient Project (ClinTrials.gov # NCT01823744). Twenty participants were randomly selected from 136 children and adolescents that consumed a daily dose of 12 vitamins and 5 minerals supplementation for 6 weeks. The 20 individuals were divided into two pools of 10 individuals, according to their lipid profile at baseline (Pool 1 with lower triglycerides, LDL, and VLDL). The individuals were analyzed at baseline, after 6 weeks of daily supplementation, and after 6 weeks of a washout period in relation to anthropometric, body composition, food intake, lipid profile, micronutrient levels, and iTRAQ proteomic data. Genetic ancestry and its association with vitamin serum levels were also determined. After supplementation, LDL levels decreased while alpha‐tocopherol and pantothenic acid levels increased in pool 2; lipid profiles in pool 1 did not change but had higher plasma levels of pantothenic acid, pyridoxal, and pyridoxic acid. In pool 2, expression of some proteins increased, and expression of other ones decreased after intervention, while in pool 1, the same proteins responded inversely or did not change their levels. Plasma alpha‐tocopherol and Native American genetic ancestry explained a significant fraction of LDL plasma levels at baseline and in response to the intervention. After intervention, changes in expression of alpha‐1 antitrypsin, haptoglobin, Ig alpha‐1 chain C region, plasma protease C1 inhibitor, alpha‐1‐acid glycoprotein 1, fibrinogen alpha, beta, and gamma‐chain in individuals in pool 2 may be associated with levels of LDL and vitamin E. Vitamin E and Native American genetic ancestry may also be implicated in changes of vitamin E and LDL levels. The results of this pilot study must be validated in future studies with larger sample size or in in vitro studies., Changes in expression of alpha‐1 antitrypsin, haptoglobin, Ig alpha‐1 chain C region, plasma protease C1 inhibitor, alpha‐1‐acid glycoprotein 1, fibrinogen alpha, beta and gamma chain in response to a micronutrient intervention may be associated with levels of LDL and vitamin E in children and adolescents. Baseline vitamin E levels and Native American genetic ancestry may also be implicated in changes of vitamin E and LDL levels.

Objective A decay in intracellular NAD+ levels is one of the hallmarks of physiological decline in normal tissue functions. Accordingly, dietary supplementation with NAD+ precursors can prevent, alleviate, or even reverse multiple metabolic complications and age-related disorders in diverse model organisms. Within the constellation of NAD+ precursors, nicotinamide riboside (NR) has gained attention due to its potent NAD+ biosynthetic effects in vivo while lacking adverse clinical effects. Nevertheless, NR is not stable in circulation, and its utilization is rate-limited by the expression of nicotinamide riboside kinases (NRKs). Therefore, there is a strong interest in identifying new effective NAD+ precursors that can overcome these limitations. Methods Through a combination of metabolomics and pharmacological approaches, we describe how NRH, a reduced form of NR, serves as a potent NAD+ precursor in mammalian cells and mice. Results NRH acts as a more potent and faster NAD+ precursor than NR in mammalian cells and tissues. Despite the minor structural difference, we found that NRH uses different steps and enzymes to synthesize NAD+, thus revealing a new NRK1-independent pathway for NAD+ synthesis. Finally, we provide evidence that NRH is orally bioavailable in mice and prevents cisplatin-induced acute kidney injury. Conclusions Our data identify a new pathway for NAD+ synthesis and classify NRH as a promising new therapeutic strategy to enhance NAD+ levels., Highlights • A reduced form of nicotinamide riboside (NRH) is a potent NAD+ precursor in cultured cells and mouse tissues. • NRH leads to NAD+ synthesis through a new, independent path to that of NR. • NRH is orally bioavailable and not degraded in plasma. • NRH alleviates cisplatin-induced acute kidney injury.

Certain B-vitamins and vitamin A may be involved in inflammatory pathways associated with homocysteine and omega-3 fatty acids. The aims of this study were (i) to determine whether different metabolic profiles of B-vitamins and vitamin A in Brazilian children and adolescents were positively or negatively related to homocysteine and omega-3 fatty acids using k-means clustering analysis, (ii) compare nutrient intakes and metabolites between the different metabolic profiles, (iii) evaluate if the statistically significant metabolites found between the metabolic groups, can predict the variation of leukotriene A4 hydrolase (LTA4H) levels, a biomarker of low-grade inflammation, in the total studied population. This cross-sectional study included 124 children and adolescents, aged 9-13 y old. Dietary intake was assessed by the food frequency questionnaire and 24-hour recall. Biomarkers for vitamins B2, B6, B12, folate and vitamin A were measured in plasma. Omega-3 fatty acids and homocysteine were measured in red blood cells (RBC). Two different metabolic profiles were found. Thirty of these individuals had overall average higher riboflavin, pyridoxal, and vitamin B12 plasma levels (metabolic group 1) compared to 94 individuals (group 2). Group 2 had lower dietary intake of vitamin B2, vitamin A, and vitamin B12 and higher RBC levels of homocysteine. EPA and DHA erythrocyte levels were not different between metabolic groups. Multiple linear regression analyses showed that blood cobalamin, riboflavin, pyridoxal and homocysteine combined, explained 9.0% of LTA4H levels variation in the total studied population. The metabolic group that had low plasma levels of riboflavin, pyridoxal, and cobalamin also had a lower dietary intake of B-vitamin and higher RBC homocysteine. The combined levels of the riboflavin, pyridoxal, cobalamin and homocysteine biomarkers can predict the variation of LTA4H in the total population studied, but it is not clear how this regulation occurs.

Polyphenols, natural products present in plant-based foods, play a protective role against several complex diseases through their antioxidant activity and by diverse molecular mechanisms. Here we develop a network medicine framework to uncover mechanisms for the effects of polyphenols on health by considering the molecular interactions between polyphenol protein targets and proteins associated with diseases. We find that the protein targets of polyphenols cluster in specific neighbourhoods of the human interactome, whose network proximity to disease proteins is predictive of the molecule’s known therapeutic effects. The methodology recovers known associations, such as the effect of epigallocatechin-3-O-gallate on type 2 diabetes, and predicts that rosmarinic acid has a direct impact on platelet function, representing a novel mechanism through which it could affect cardiovascular health. We experimentally confirm that rosmarinic acid inhibits platelet aggregation and α-granule secretion through inhibition of protein tyrosine phosphorylation, offering direct support for the predicted molecular mechanism. Our framework represents a starting point for mechanistic interpretation of the health effects underlying food-related compounds, allowing us to integrate into a predictive framework knowledge on food metabolism, bioavailability and drug interaction. Molecular interactions between polyphenol targets and proteins associated with disease are explored through a network medicine framework. The network proximity of polyphenol protein targets to disease proteins can predict therapeutic effects, highlighting more broadly the potential of network medicine as a tool for nutritional sciences.

Polyphenols, natural products present in plant-based foods, play a protective role against several complex diseases through their antioxidant activity and by diverse molecular mechanisms. Here we developed a network medicine framework to uncover the mechanistic roles of polyphenols on health by considering the molecular interactions between polyphenol protein targets and proteins associated with diseases. We find that the protein targets of polyphenols cluster in specific neighborhoods of the human interactome, whose network proximity to disease proteins is predictive of the molecule’s known therapeutic effects. The methodology recovers known associations, such as the effect of epigallocatechin 3-O-gallate on type 2 diabetes, and predicts that rosmarinic acid (RA) has a direct impact on platelet function, representing a novel mechanism through which it could affect cardiovascular health. We experimentally confirm that RA inhibits platelet aggregation and alpha granule secretion through inhibition of protein tyrosine phosphorylation, offering direct support for the predicted molecular mechanism. Our framework represents a starting point for mechanistic interpretation of the health effects underlying food-related compounds, allowing us to integrate into a predictive framework knowledge on food metabolism, bioavailability, and drug interaction.

This study evaluated the validity of nutrient and food group intakes estimated by an FFQ against biomarkers. A 71-item semiquantitative FFQ was administered to 210 Brazilian children and adolescents aged 9–13 years. Intakes were correlated with biomarkers in plasma and red blood cells. Correlations between nutrients and their biomarkers were presented for animal protein, myristic acid (C14:0), EPA, DHA, β-carotene, folate, and vitamins B3, B5 and B6. Food groups and biomarkers were correlated as follows: fish products with EPA and DHA; milk and dairy with C14:0, pyridoxal 5'-phosphate and vitamin B12; total vegetables and dark green and orange vegetables with β-carotene; 5-methyltetrahydrofolate with green vegetables; and flour products with para-aminobenzoylglutamic acid. This FFQ is a valid tool for ranking Brazilian children and adolescents according to their intake of several nutrients and food groups.

Polyphenol-rich foods are part of many nutritional interventions aimed at improving health and preventing cardiometabolic diseases (CMDs). Polyphenols have oxidative, inflammatory, and/or metabolic effects. Research into the chemistry and biology of polyphenol bioactives is prolific but knowledge of their molecular interactions with proteins is limited. We mined public data to (i) identify proteins that interact with or metabolize polyphenols, (ii) mapped these proteins to pathways and networks, and (iii) annotated functions enriched within the resulting polyphenol-protein interactome. A total of 1,395 polyphenols and their metabolites were retrieved (using Phenol-Explorer and Dictionary of Natural Products) of which 369 polyphenols interacted with 5,699 unique proteins in 11,987 interactions as annotated in STITCH, Pathway Commons, and BindingDB. Pathway enrichment analysis using the KEGG repository identified a broad coverage of significant pathways of low specificity to particular polyphenol (sub)classes. When compared to drugs or micronutrients, polyphenols have pleiotropic effects across many biological processes related to metabolism and CMDs. These systems-wide effects were also found in the protein interactome of the polyphenol-rich citrus fruits, used as a case study. In sum, these findings provide a knowledgebase for identifying polyphenol classes (and polyphenol-rich foods) that individually or in combination influence metabolism.

Introduction: Phenolic acids are important phenolic compounds widespread in foods, contributing to nutritional and organoleptic properties. Factors affceting individual variability: The bioavailability of these compounds depends on their free or conjugated presence in food matrices, which is also affected by food processing. Phenolic acids undergo metabolism by the host and residing intestinal microbiota, which causes conjugations and structural modifications of the compounds. Human responses, metabolite profiles and health responses of phenolics, show considerable individual variation, which is affected by absorption, metabolism and genetic variations of subjects. Opinion: A better understanding of the gut-host interplay and microbiome biochemistry is becoming highly relevant in understanding the impact of diet and its constituents. It is common to study metabolism and health benefits separately, with some exceptions; however, it should be preferred that health responders and non-responders are studied in combination with explanatory metabolite profiles and gene variants. This approach could turn interindividual variation from a problem in human research to an asset for research on personalized nutrition.

Mitochondrial acyl-coenzyme A species are emerging as important sources of protein modification and damage. Succinyl-CoA ligase (SCL) deficiency causes a mitochondrial encephalomyopathy of unknown pathomechanism. Here, we show that succinyl-CoA accumulates in cells derived from patients with recessive mutations in the tricarboxylic acid cycle (TCA) gene succinyl-CoA ligase subunit-β (SUCLA2), causing global protein hyper-succinylation. Using mass spectrometry, we quantify nearly 1,000 protein succinylation sites on 366 proteins from patient-derived fibroblasts and myotubes. Interestingly, hyper-succinylated proteins are distributed across cellular compartments, and many are known targets of the (NAD+)-dependent desuccinylase SIRT5. To test the contribution of hyper-succinylation to disease progression, we develop a zebrafish model of the SCL deficiency and find that SIRT5 gain-of-function reduces global protein succinylation and improves survival. Thus, increased succinyl-CoA levels contribute to the pathology of SCL deficiency through post-translational modifications., The pathomechanism of succinyl-CoA ligase (SCL) deficiency, a hereditary mitochondrial disease, is not fully understood. Here, the authors show that increased succinyl-CoA levels contribute to SCL pathology by causing global protein hyper-succinylation.

Supplementation with the NAD+ precursor nicotinamide riboside (NR) ameliorates and prevents a broad array of metabolic and aging disorders in mice. However, little is known about the physiological role of endogenous NR metabolism. We have previously shown that NR kinase 1 (NRK1) is rate-limiting and essential for NR-induced NAD+ synthesis in hepatic cells. To understand the relevance of hepatic NR metabolism, we generated whole body and liver-specific NRK1 knockout mice. Here, we show that NRK1 deficiency leads to decreased gluconeogenic potential and impaired mitochondrial function. Upon high-fat feeding, NRK1 deficient mice develop glucose intolerance, insulin resistance and hepatosteatosis. Furthermore, they are more susceptible to diet-induced liver DNA damage, due to compromised PARP1 activity. Our results demonstrate that endogenous NR metabolism is critical to sustain hepatic NAD+ levels and hinder diet-induced metabolic damage, highlighting the relevance of NRK1 as a therapeutic target for metabolic disorders., Nicotinamide adenine dinucleotide (NAD+) sustains cellular energy metabolism, functions as a substrate of Sirt and PARP enzymes, and its supplementation is explored therapeutically in aging and other contexts. Here the authors provide insight into the role of endogenous NAD+ metabolism by studying nicotinamide riboside kinase 1 (NRK1) deficient mice.

In Figure 4, the panels showing the variability by cycle phases for Glycine, Serine, Methionine, Asparagine, Proline, Glutamine, Tyrosine, Gamma-glutamyl-alanine, Citrulline, O-Acetyl-serine, Alpha-aminobutyric acid and Gamma-glutamylglutamine were omitted. The correct Figure 4 appears below as Figure 1.

Polymorphisms in genes related to the metabolism of vitamin B12 haven’t been examined in a Brazilian population. To (a) determine the correlation between the local genetic ancestry components and vitamin B12 levels using ninety B12-related genes; (b) determine associations between these genes and their SNPs with vitamin B12 levels; (c) determine a polygenic risk score (PRS) using significant variants. This cross-sectional study included 168 children and adolescents, aged 9–13 years old. Total cobalamin was measured in plasma. Genotyping arrays and whole exome data were combined to yield ~ 7000 SNPs in 90 genes related to vitamin B12. The Efficient Local Ancestry Inference was used to estimate local ancestry for African (AFR), Native American, and European (EUR). The association between the genotypes and vitamin B12 levels were determined with generalized estimating equation. Vitamin B12 levels were driven by positive (EUR) and negative (AFR, AMR) correlations with genetic ancestry. A set of 36 variants were used to create a PRS that explained 42% of vitamin level variation. Vitamin B12 levels are influenced by genetic ancestry and a PRS explained almost 50% of the variation in plasma cobalamin in Brazilian children and adolescents.