The emerging field of metabolomics, in which a large number of small-molecule metabolites from body fluids or tissues are detected quantitatively in a single step, promises immense potential for ...early diagnosis, therapy monitoring and for understanding the pathogenesis of many diseases. Metabolomics methods are mostly focused on the information-rich analytical techniques of NMR spectroscopy and mass spectrometry (MS). Analysis of the data from these high-resolution methods using advanced chemometric approaches provides a powerful platform for translational and clinical research and diagnostic applications. In this review, the current trends and recent advances in NMR- and MS-based metabolomics are described with a focus on the development of advanced NMR and MS methods, improved multivariate statistical data analysis and recent applications in the area of cancer, diabetes, inborn errors of metabolism and cardiovascular diseases.
Mitochondrial trifunctional protein deficiency, due to mutations in hydratase subunit A (HADHA), results in sudden infant death syndrome with no cure. To reveal the disease etiology, we generated ...stem cell-derived cardiomyocytes from HADHA-deficient hiPSCs and accelerated their maturation via an engineered microRNA maturation cocktail that upregulated the epigenetic regulator, HOPX. Here we report, matured HADHA mutant cardiomyocytes treated with an endogenous mixture of fatty acids manifest the disease phenotype: defective calcium dynamics and repolarization kinetics which results in a pro-arrhythmic state. Single cell RNA-seq reveals a cardiomyocyte developmental intermediate, based on metabolic gene expression. This intermediate gives rise to mature-like cardiomyocytes in control cells but, mutant cells transition to a pathological state with reduced fatty acid beta-oxidation, reduced mitochondrial proton gradient, disrupted cristae structure and defective cardiolipin remodeling. This study reveals that HADHA (tri-functional protein alpha), a monolysocardiolipin acyltransferase-like enzyme, is required for fatty acid beta-oxidation and cardiolipin remodeling, essential for functional mitochondria in human cardiomyocytes.
Regeneration requires cells to regulate proliferation and patterning according to their spatial position. Positional memory is a property that enables regenerating cells to recall spatial information ...from the uninjured tissue. Positional memory is hypothesized to rely on gradients of molecules, few of which have been identified. Here, we quantified the global abundance of transcripts, proteins, and metabolites along the proximodistal axis of caudal fins of uninjured and regenerating adult zebrafish. Using this approach, we uncovered complex overlapping expression patterns for hundreds of molecules involved in diverse cellular functions, including development, bioelectric signaling, and amino acid and lipid metabolism. Moreover, 32 genes differentially expressed at the RNA level had concomitant differential expression of the encoded proteins. Thus, the identification of proximodistal differences in levels of RNAs, proteins, and metabolites will facilitate future functional studies of positional memory during appendage regeneration.
Broad-based, targeted metabolite profiling using mass spectrometry (MS) has become a major platform used in the field of metabolomics for a variety of applications. However, quantitative MS analysis ...is challenging owing to numerous factors including (1) the need for, ideally, isotope-labeled internal standards for each metabolite, (2) the fact that such standards may be unavailable or prohibitively costly, (3) the need to maintain the standards’ concentrations close to those of the target metabolites, and (4) the alternative use of time-consuming calibration curves for each target metabolite. Here, we introduce a new method in which metabolites from a single serum specimen are quantified on the basis of a recently developed NMR method Nagana Gowda et al. Anal. Chem. 2015, 87, 706 and then used as references for absolute metabolite quantitation using MS. The MS concentrations of 30 metabolites thus derived for test serum samples exhibited excellent correlations with the NMR ones (R 2 > 0.99) with a median CV of 3.2%. This NMR-guided-MS quantitation approach is simple and easy to implement and offers new avenues for the routine quantification of blood metabolites using MS. The demonstration that NMR and MS data can be compared and correlated when using identical sample preparations allows improved opportunities to exploit their combined strengths for biomarker discovery and unknown-metabolite identification. Intriguingly, however, metabolites including glutamine, pyroglutamic acid, glucose, and sarcosine correlated poorly with NMR data because of stability issues in their MS analyses or weak or overlapping signals. Such information is potentially important for improving biomarker discovery and biological interpretations. Further, the new quantitation method demonstrated here for human blood serum can in principle be extended to a variety of biological mixtures.
•51 phospholipids are significantly different between SCZ patients and controls.•These significant phospholipids include PCs, LPCs, PEs, LPEs and SMs.•Extensive disturbances of phospholipids may be ...involved in the development of SCZ.•A panel of 6 metabolites could discriminate SCZ patients from healthy controls.
Schizophrenia (SCZ) is a multifactorial psychiatric disorder. However, the molecular pathogenesis of SCZ remains largely unknown, and no reliable diagnostic test is currently available. Phospholipid metabolism is known to be disturbed during disease processes of SCZ. In this study, we used an untargeted liquid chromatography-mass spectrometry (LC-MS)-based metabolic profiling approach to measure lipid metabolites in serum samples from 119 SCZ patients and 109 healthy controls, to identify potential lipid biomarkers for the discrimination between SCZ patients and healthy controls. 51 lipid metabolites were identified to be significant for discriminating SCZ patients from healthy controls, including phosphatidylcholines (PCs), lysophosphatidylcholines (LPCs), phosphatidylethanolamines (PEs), lysophosphatidylethanolamines (LPEs) and sphingomyelins (SMs). Compared to healthy controls, most PCs and LPCs, as well as all PEs in patients were decreased, while most LPEs and all SMs were increased. A panel of six lipid metabolites could effectively discriminate SCZ patients from healthy controls with an area under the receiver-operating characteristic curve of 0.991 in the training samples and 0.980 in the test samples. These findings suggest that extensive disturbances of phospholipids may be involved in the development of SCZ. This LC-MS-based metabolic profiling approach shows potential for the identification of putative serum biomarkers for the diagnosis of SCZ.
In most organisms, dietary restriction (DR) increases lifespan. However, several studies have found that genotypes within the same species vary widely in how they respond to DR. To explore the ...mechanisms underlying this variation, we exposed 178 inbred Drosophila melanogaster lines to a DR or ad libitum (AL) diet, and measured a panel of 105 metabolites under both diets. Twenty four out of 105 metabolites were associated with the magnitude of the lifespan response. These included proteinogenic amino acids and metabolites involved in α-ketoglutarate (α-KG)/glutamine metabolism. We confirm the role of α-KG/glutamine synthesis pathways in the DR response through genetic manipulations. We used covariance network analysis to investigate diet-dependent interactions between metabolites, identifying the essential amino acids threonine and arginine as "hub" metabolites in the DR response. Finally, we employ a novel metabolic and genetic bipartite network analysis to reveal multiple genes that influence DR lifespan response, some of which have not previously been implicated in DR regulation. One of these is CCHa2R, a gene that encodes a neuropeptide receptor that influences satiety response and insulin signaling. Across the lines, variation in an intronic single nucleotide variant of CCHa2R correlated with variation in levels of five metabolites, all of which in turn were correlated with DR lifespan response. Inhibition of adult CCHa2R expression extended DR lifespan of flies, confirming the role of CCHa2R in lifespan response. These results provide support for the power of combined genomic and metabolomic analysis to identify key pathways underlying variation in this complex quantitative trait.
Chronic kidney disease (CKD) is a public health problem with very high prevalence and mortality. Yet, there is a paucity of effective treatment options, partly due to insufficient knowledge of ...underlying pathophysiology. We combined metabolomics (GCMS) with kidney gene expression studies to identify metabolic pathways that are altered in adults with non-diabetic stage 3–4 CKD versus healthy adults. Urinary excretion rate of 27 metabolites and plasma concentration of 33 metabolites differed significantly in CKD patients versus controls (estimate range−68% to +113%). Pathway analysis revealed that the citric acid cycle was the most significantly affected, with urinary excretion of citrate, cis-aconitate, isocitrate, 2-oxoglutarate and succinate reduced by 40–68%. Reduction of the citric acid cycle metabolites in urine was replicated in an independent cohort. Expression of genes regulating aconitate, isocitrate, 2-oxoglutarate and succinate were significantly reduced in kidney biopsies. We observed increased urine citrate excretion (+74%, p=0.00009) and plasma 2-oxoglutarate concentrations (+12%, p=0.002) in CKD patients during treatment with a vitamin-D receptor agonist in a randomized trial. In conclusion, urinary excretion of citric acid cycle metabolites and renal expression of genes regulating these metabolites were reduced in non-diabetic CKD. This supports the emerging view of CKD as a state of mitochondrial dysfunction.
•Urinary excretion rate and plasma concentration of 60 metabolites differed significantly in CKD patients versus controls.•Pathway analysis revealed that the citric acid cycle was the most significantly affected.•Expression of genes regulating TCA cycle was significantly reduced in kidney biopsies.
Chronic kidney disease (CKD) is very common and carries a high risk of complications and death. Patients with advanced disease have severe fatigue, organ dysfunction beyond the kidneys, and disturbances in sugar, protein and fat metabolism. We found many metabolites that differed significantly in kidney patients versus healthy controls. Analysis revealed that the citric acid cycle was the most significantly affected metabolic pathway. The citric acid cycle is performed in the cells` mitochondria and is the central metabolic hub where fuel molecules are converted into energy. This supports the view of CKD as a state of mitochondrial dysfunction.
Rapamycin, an inhibitor of mTOR signaling, has been shown to reverse diastolic dysfunction in old mice in 10 weeks, highlighting its therapeutic potential for a poorly treatable condition. However, ...the mechanisms and temporal regulation of its cardiac benefits remain unclear. We show that improved diastolic function in old mice begins at 2-4 weeks, progressing over the course of 10-week treatment. While TORC1-mediated S6 phosphorylation and TORC2 mediated AKT and PKCα phosphorylation are inhibited throughout the course of treatment, rapamycin inhibits ULK phosphorylation and induces autophagy during just the first week of treatment, returning to baseline at two weeks and after. Concordantly, markers of mitochondrial biogenesis increase over the first two weeks of treatment and return to control levels thereafter. This transient induction of autophagy and mitochondrial biogenesis suggests that damaged mitochondria are replaced by newly synthesized ones to rejuvenate mitochondrial homeostasis. This remodeling is shown to rapidly reverse the age-related reduction in fatty acid oxidation to restore a more youthful substrate utilization and energetic profile in old isolated perfused hearts, and modulates the myocardial metabolomein vivo. This study demonstrates the differential and dynamic mechanisms following rapamycin treatment and highlights the importance of understanding the temporal regulation of rapamycin effects.
In mass spectrometry (MS)-based metabolomics, missing values (NAs) may be due to different causes, including sample heterogeneity, ion suppression, spectral overlap, inappropriate data processing, ...and instrumental errors. Although a number of methodologies have been applied to handle NAs, NA imputation remains a challenging problem. Here, we propose a non-negative matrix factorization (NMF)-based method for NA imputation in MS-based metabolomics data, which makes use of both global and local information of the data. The proposed method was compared with three commonly used methods: k-nearest neighbors (kNN), random forest (RF), and outlier-robust (ORI) missing values imputation. These methods were evaluated from the perspectives of accuracy of imputation, retrieval of data structures, and rank of imputation superiority. The experimental results showed that the NMF-based method is well-adapted to various cases of data missingness and the presence of outliers in MS-based metabolic profiles. It outperformed kNN and ORI and showed results comparable with the RF method. Furthermore, the NMF method is more robust and less susceptible to outliers as compared with the RF method. The proposed NMF-based scheme may serve as an alternative NA imputation method which may facilitate biological interpretations of metabolomics data.
Colorectal cancer (CRC) tends to occur at older age; however, CRC incidence rates have been rising sharply among young age groups. The increasing prevalence of obesity is recognized as a major risk, ...yet the mechanistic underpinnings remain poorly understood. Using a diet-induced obesity mouse model, we identified obesity-associated molecular changes in the colonic epithelium of young and aged mice, and we further investigated whether the changes were reversed after weight loss. Transcriptome analysis indicated that obesity-related colonic cellular metabolic switch favoring long-chain fatty acid oxidation happened in young mice, while obesity-associated downregulation of negative feedback regulators of pro-proliferative signaling pathways occurred in older mice. Strikingly, colonic DNA methylome was pre-programmed by obesity at young age, priming for a tumor-prone gene signature after aging. Furthermore, obesity-related changes were substantially preserved after short-term weight loss, but they were largely reversed after long-term weight loss. We provided mechanistic insights into increased CRC risk in obesity.
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•Obesity reprograms mouse colonic DNA methylome, leading to gene expression changes•Obesity triggers a metabolic switch favoring long-chain fatty acids at young age•Obesity induces a tumor-prone gene signature in the colonic epithelium after aging•Obesity-related changes are reversed after long-term, but not short-term, weight loss
Li et al. find that obesity-induced DNA methylation changes reprogram the colonic transcriptome, leading to a metabolic switch favoring long-chain fatty acid oxidation in young mice and a more tumor-prone gene signature after aging. Obesity-related changes are substantially preserved after short-term weight loss, but they are largely reversed after long-term weight loss.
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