Modeling blood metabolite homeostatic levels reduces sample heterogeneity across cohorts.

Blood metabolite levels are affected by numerous factors, including preanalytical factors such as collection methods and geographical sites. These perturbations have caused deleterious consequences for many metabolomics studies and represent a major challenge in the metabolomics field. It is important to understand these factors and develop models to reduce their perturbations. However, to date, the lack of suitable mathematical models for blood metabolite levels under homeostasis has hindered progress. In this study, we develop quantitative models of blood metabolite levels in healthy adults based on multisite sample cohorts that mimic the current challenge. Five cohorts of samples obtained across four geographically distinct sites were investigated, focusing on approximately 50 metabolites that were quantified using 1H NMR spectroscopy. More than one-third of the variation in these metabolite profiles is due to cross-cohort variation. A dramatic reduction in the variation of metabolite levels (90%), especially their site-to-site variation (95%), was achieved by modeling each metabolite using demographic and clinical factors and especially other metabolites, as observed in the top principal components. The results also reveal that several metabolites contribute disproportionately to such variation, which could be explained by their association with biological pathways including biosynthesis and degradation. The study demonstrates an intriguing network effect of metabolites that can be utilized to better define homeostatic metabolite levels, which may have implications for improved health monitoring. As an example of the potential utility of the approach, we show that modeling gender-related metabolic differences retains the interesting variance while reducing unwanted (site-related) variance.

Intracellular pyruvate-lactate-alanine cycling detected using real-time nuclear magnetic resonance spectroscopy of live cells and isolated mitochondria.

Pyruvate, an end product of glycolysis, is a master fuel for cellular energy. A portion of cytosolic pyruvate is transported into mitochondria, while the remaining portion is converted reversibly into lactate and alanine. It is suggested that cytosolic lactate and alanine are transported and metabolized inside mitochondria. However, such a mechanism continues to be a topic of intense debate and investigation. As a part of gaining insight into the metabolic fate of the cytosolic lactate and alanine; in this study, the metabolism of mouse skeletal myoblast cells (C2C12) and their isolated mitochondria was probed utilizing stable isotope-labeled forms of the three glycolysis products, viz. [3-13 C1 ]pyruvate, [3-13 C1 ]lactate, and [3-13 C1 ]alanine, as substrates. The uptake and metabolism of each substrate was monitored, separately, in real-time using 1 H-13 C 2D nuclear magnetic resonance (NMR) spectroscopy. The dynamic variation of the levels of the substrates and their metabolic products were quantitated as a function of time. The results demonstrate that all three substrates were transported into mitochondria, and each substrate was metabolized to form the other two metabolites, reversibly. These results provide direct evidence for intracellular pyruvate-lactate-alanine cycling, in which lactate and alanine produced by the cytosolic pyruvate are transported into mitochondria and converted back to pyruvate. Such a mechanism suggests a role for lactate and alanine to replenish mitochondrial pyruvate, the primary source for adenosine triphosphate (ATP) synthesis through oxidative phosphorylation and the electron transport chain. The results highlight the potential of real-time NMR spectroscopy for gaining new insights into cellular and subcellular functions.

Labile Metabolite Profiling in Human Blood Using Phosphorus NMR Spectroscopy.

Phosphorus metabolites occupy a unique place in cellular function as critical intermediates and products of cellular metabolism. Human blood is the most widely used biospecimen in the clinic and in the metabolomics field, and hence an ability to profile phosphorus metabolites in blood, quantitatively, would benefit a wide variety of investigations of cellular functions in health and diseases. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy are the two premier analytical platforms used in the metabolomics field. However, detection and quantitation of phosphorus metabolites by MS can be challenging due to their lability, high polarity, structural isomerism, and interaction with chromatographic columns. The conventionally used 1H NMR, on the other hand, suffers from poor resolution of these compounds. As a remedy, 31P NMR promises an important alternative to both MS and 1H NMR. However, numerous challenges including the instability of phosphorus metabolites, their chemical shift sensitivity to solvent composition, pH, salt, and temperature, and the lack of identified metabolites have so far restricted the scope of 31P NMR. In the current study, we describe a method to analyze nearly 25 phosphorus metabolites in blood using a simple one-dimensional (1D) NMR spectrum. Establishment of the identity of unknown metabolites involved a combination of (a) comprehensively analyzing an array of 1D and two-dimensional (2D) 1H/31P homonuclear and heteronuclear NMR spectra of blood; (b) mapping the central carbon metabolic pathway; (c) developing and using 1H and 31P spectral and chemical shift databases; and finally (d) confirming the putative metabolite peaks with spiking using authentic compounds. The resulting simple 1D 31P NMR-based method offers an ability to visualize and quantify the levels of intermediates and products of multiple metabolic pathways, including central carbon metabolism, in one step. Overall, the findings represent a new dimension for blood metabolite analysis and are anticipated to greatly impact the blood metabolomics field.

Biomarkers for Duchenne muscular dystrophy progression: impact of age in the mdx tongue spared muscle.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a severe form of muscular dystrophy without an effective treatment, caused by mutations in the DMD gene, leading to the absence of dystrophin. DMD results in muscle weakness, loss of ambulation, and death at an early age. Metabolomics studies in mdx mice, the most used model for DMD, reveal changes in metabolites associated with muscle degeneration and aging. In DMD, the tongue muscles exhibit unique behavior, initially showing partial protection against inflammation but later experiencing fibrosis and loss of muscle fibers. Certain metabolites and proteins, like TNF-α and TGF-ß, are potential biomarkers for dystrophic muscle characterization. METHODS: To investigate disease progression and aging, we utilized young (1 month old) and old (21-25 months old) mdx and wild-type tongue muscles. Metabolite changes were analyzed using 1H nuclear magnetic resonance, while TNF-α and TGF-ß were assessed using Western blotting to examine inflammation and fibrosis. Morphometric analysis was conducted to assess the extent of myofiber damage between groups. RESULTS: The histological analysis of the mid-belly tongue showed no differences between groups. No differences were found between the concentrations of metabolites from wild-type or mdx whole tongues of the same age. The metabolites alanine, methionine, and 3-methylhistidine were higher, and taurine and glycerol were lower in young tongues in both wild type and mdx (p < 0.001). The metabolites glycine (p < 0.001) and glutamic acid (p = 0.0018) were different only in the mdx groups, being higher in young mdx mice. Acetic acid, phosphocreatine, isoleucine, succinic acid, creatine, and the proteins TNF-α and TGF-ß had no difference in the analysis between groups (p > 0.05). CONCLUSIONS: Surprisingly, histological, metabolite, and protein analysis reveal that the tongue of old mdx remains partially spared from the severe myonecrosis observed in other muscles. The metabolites alanine, methionine, 3-methylhistidine, taurine, and glycerol may be effective for specific assessments, although their use for disease progression monitoring should be cautious due to age-related changes in the tongue muscle. Acetic acid, phosphocreatine, isoleucine, succinate, creatine, TNF-α, and TGF-ß do not vary with aging and remain constant in spared muscles, suggesting their potential as specific biomarkers for DMD progression independent of aging.

Anomalous Dynamics of Labile Metabolites in Cold Human Blood Detected Using 1H NMR Spectroscopy.

Recent efforts in our laboratory have enabled access to an unprecedented number (∼90) of quantifiable metabolites in human blood by a simple nuclear magnetic resonance (NMR) spectroscopy method, which includes energy coenzymes, redox coenzymes, and antioxidants that are fundamental to cellular functions [ J. Magn. Reson. Open 2022, 12-13, 100082]. The coenzymes and antioxidants, however, are notoriously labile and are extremely sensitive to specimen harvesting, extraction, and measurement conditions. This problem is largely underappreciated and carries the risk of grossly inaccurate measurements and incorrect study outcomes. As a part of addressing this challenge, in this study, human blood specimens were comprehensively and quantitatively investigated using 1H NMR spectroscopy. Freshly drawn human blood specimens were treated or not treated with methanol, ethanol, or a mixture of methanol and chloroform, and stored on ice or on bench, at room temperature for different time periods from 0 to 24 h, prior to storing at -80 °C. Interestingly, the labile metabolite levels were stable in blood treated with an organic solvent. However, their levels in blood in untreated samples increased or decreased by factors of up to 5 or more within 3 h. Further, surprisingly, and contrary to the current knowledge about metabolite stability, the variation of coenzyme levels was more dramatic in blood stored on ice than on bench, at room temperature. In addition, unlike the generally observed phenomenon of oxidation of redox coenzymes, reduction was observed in untreated blood. Such preanalytical dynamics of the labile metabolites potentially arises from the active cellular metabolism. From the metabolomics perspective, the massive variation of the labile metabolite levels even in blood stored on ice is alarming and stresses the critical need to immediately quench the cellular metabolism for reliable analyses. Overall, the results provide compelling evidence that warrants a paradigm shift in the sample collection protocol for blood metabolomics involving labile metabolites.

Demographic, Health and Lifestyle Factors Associated with the Metabolome in Older Women.

Demographic and clinical factors influence the metabolome. The discovery and validation of disease biomarkers are often challenged by potential confounding effects from such factors. To address this challenge, we investigated the magnitude of the correlation between serum and urine metabolites and demographic and clinical parameters in a well-characterized observational cohort of 444 post-menopausal women participating in the Women's Health Initiative (WHI). Using LC-MS and lipidomics, we measured 157 aqueous metabolites and 756 lipid species across 13 lipid classes in serum, along with 195 metabolites detected by GC-MS and NMR in urine and evaluated their correlations with 29 potential disease risk factors, including demographic, dietary and lifestyle factors, and medication use. After controlling for multiple testing (FDR < 0.01), we found that log-transformed metabolites were mainly associated with age, BMI, alcohol intake, race, sample storage time (urine only), and dietary supplement use. Statistically significant correlations were in the absolute range of 0.2-0.6, with the majority falling below 0.4. Incorporation of important potential confounding factors in metabolite and disease association analyses may lead to improved statistical power as well as reduced false discovery rates in a variety of data analysis settings.

Monitoring live mitochondrial metabolism in real-time using NMR spectroscopy.

Investigation of mitochondrial metabolism is gaining increased interest owing to the growing recognition of the role of mitochondria in health and numerous diseases. Studies of isolated mitochondria promise novel insights into the metabolism devoid of confounding effects from other cellular organelles such as cytoplasm. This study describes the isolation of mitochondria from mouse skeletal myoblast cells (C2C12) and the investigation of live mitochondrial metabolism in real-time using isotope tracer-based NMR spectroscopy. [3-13 C1 ]pyruvate was used as the substrate to monitor the dynamic changes of the downstream metabolites in mitochondria. The results demonstrate an intriguing phenomenon, in which lactate is produced from pyruvate inside the mitochondria and the results were confirmed by treating mitochondria with an inhibitor of mitochondrial pyruvate carrier (UK5099). Lactate is associated with health and numerous diseases including cancer and, to date, it is known to occur only in the cytoplasm. The insight that lactate is also produced inside mitochondria opens avenues for exploring new pathways of lactate metabolism. Further, experiments performed using inhibitors of the mitochondrial respiratory chain, FCCP and rotenone, show that [2-13 C1 ]acetyl coenzyme A, which is produced from [3-13 C1 ]pyruvate and acts as a primary substrate for the tricarboxylic acid cycle in mitochondria, exhibits a remarkable sensitivity to the inhibitors. These results offer a direct approach to visualize mitochondrial respiration through altered levels of the associated metabolites.

Whole Body Distribution of Labile Coenzymes and Antioxidants in a Mouse Model as Visualized Using 1H NMR Spectroscopy.

Coenzyme A, acetyl coenzyme A, coenzymes of cellular energy, coenzymes of redox reactions, and antioxidants mediate biochemical reactions fundamental to the functioning of all living cells. There is an immense interest in measuring them routinely in biological specimens to gain insights into their roles in cellular functions and to help characterize the biological status. However, it is challenging to measure them ex vivo as they are sensitive to specimen harvesting, extraction, and measurement conditions. This challenge is largely underappreciated and carries the risk of grossly inaccurate measurements that lead to incorrect inferences. To date, several efforts have been focused on alleviating this challenge using NMR spectroscopy. However, a comprehensive solution for the measurement of the compounds in a wide variety of biological specimens is still lacking. As a part of addressing this challenge, we demonstrate here that the total pool of each group of unstable metabolites offers a starting place for the representation of labile metabolites in biological specimens. Based on this approach, in this proof-of-concept study, we determine the distribution of the labile compounds in different organs including heart, kidney, liver, brain, and skeletal muscle of a mouse model. The results were independently validated using different specimens and a different metabolite extraction protocol. Further, we show that both stable and unstable metabolites were distributed differentially in different organs, which signifies their differential functional roles, the knowledge of which is currently lacking for many metabolites. Intriguingly, the concentration of taurine, an amino sulfonic acid, in skeletal muscle is >30 mM, which is the highest for any metabolite in a mammalian tissue known to date. To the best of our knowledge, this is the first study to profile the whole body distribution of the labile and other high-concentration metabolites using NMR spectroscopy. The results may pave ways for gaining new insights into cellular functions in health and diseases.

Quantitative NMR Methods in Metabolomics.

Nuclear Magnetic Resonance (NMR) spectroscopy is one of the two major analytical platforms in the field of metabolomics, the other being mass spectrometry (MS). NMR is less sensitive than MS and hence it detects a relatively small number of metabolites. However, NMR exhibits numerous unique characteristics including its high reproducibility and non-destructive nature, its ability to identify unknown metabolites definitively, and its capabilities to obtain absolute concentrations of all detected metabolites, sometimes even without an internal standard. These characteristics outweigh the relatively low sensitivity and resolution of NMR in metabolomics applications. Since biological mixtures are highly complex, increased demand for new methods to improve detection, better identify unknown metabolites, and provide more accurate quantitation continues unabated. Technological and methodological advances to date have helped to improve the resolution and sensitivity and detection of a larger number of metabolite signals. Efforts focused on measuring unknown metabolite signals have resulted in the identification and quantitation of an expanded pool of metabolites including labile metabolites such as cellular redox coenzymes, energy coenzymes, and antioxidants. This chapter describes quantitative NMR methods in metabolomics with an emphasis on recent methodological developments, while highlighting the benefits and challenges of NMR-based metabolomics.

A new limit for blood metabolite analysis using 1H NMR spectroscopy.

Human blood is the most widely used biospecimen in the clinic and the metabolomics field. While both mass spectrometry and NMR spectroscopy are the two premier analytical platforms in the metabolomics field, NMR exhibits several unsurpassed characteristics for blood metabolite analysis, the most important of which are its ability to identify unknown metabolites and its quantitative nature. However, the relatively small number of metabolites accessible by NMR has restricted the scope of its applications. Enhancing the limit of identified metabolites in blood will therefore greatly impact NMR-based metabolomics. Continuing our efforts to address this major issue, our current study describes the identification of 12 metabolites, which expands the number of quantifiable blood metabolites by ~15%. These results, in combination with our earlier efforts, now provide access to nearly 90 metabolites, which is the highest to date for a simple 1D 1H NMR experiment that is widely used in the metabolomics field. Metabolites were identified based on the comprehensive investigation of human blood and plasma using 1D/2D NMR techniques. The newly identified metabolites were validated based on chemical shift databases, spectra of authentic compounds obtained under conditions identical to blood/plasma, and, finally, spiking experiments using authentic compounds. Considering the high reproducibility of NMR and the sensitivity of chemical shifts to altered sample conditions, experimental protocols and peak annotations are provided for the newly identified metabolites, which serve as a template for identification of blood metabolites for routine applications. Separately, the identified metabolites were evaluated for their sensitivity to preanalytical conditions. The results reveal that among the newly identified metabolites, inosine monophosphate (IMP) and nicotinamide are associated with labile coenzymes and their levels are sensitive to preanalytical conditions. The study demonstrates the expansion of quantifiable blood metabolites using NMR to a new height and is expected to greatly impact blood metabolomics.

Hydrogen-Deuterium Addition and Exchange in N-Ethylmaleimide Reaction with Glutathione Detected by NMR Spectroscopy.

Glutathione (GSH) is an important and ubiquitous thiol compound abundantly present in virtually every living cell. It is a powerful antioxidant critically required to protect cells from oxidative damage and free radical injury. Its quantification in ex vivo analysis remains a major challenge because it spontaneously oxidizes to form glutathione disulfide. N-Ethylmaleimide (NEM) is a well-known Michael acceptor, which reacts rapidly and irreversibly with thiol and prevents disulfide bond formation. Based on thiol conjugation to NEM, recently, the concentration of GSH was determined in human blood using NMR spectroscopy [Anal. Chem, 2021, 93(44): 14844-14850]. It was found that hydrogen-deuterium addition and exchange occur during the thiol-maleimide reaction as well as NMR analysis, generating a series of poorly explored diastereomers/isotopomers. Here, we establish a general NMR approach to identify the thiosuccinimide diastereomers/isotopomers derived from the thiol-maleimide reaction. The thiol-Michael addition reaction was conducted for GSH and another thiol compound, cysteine, separately, using D2O and H2O. The conjugates were characterized by 1H/13C 1D/2D NMR under different solvent, buffer, and pH conditions. The Michael addition combined with the H/D exchange formed twelve unique diastereomers/isotopomers. NMR measurements allowed the distinct assignment of all structures in solutions and quantification of H/D addition and exchange. Interestingly, the deuterium exchange rate was dependent on structure, pH, and buffer. The elucidation of the thiol-maleimide reaction and H/D exchange mechanism can potentially impact areas including metabolomics, small molecule synthesis, and bioconjugation chemistry.

A Combination of Nicotinamide and D-Ribose (RiaGev) Is Safe and Effective to Increase NAD+ Metabolome in Healthy Middle-Aged Adults: A Randomized, Triple-Blind, Placebo-Controlled, Cross-Over Pilot Clinical Trial.

Nicotinamide adenine dinucleotide (NAD+) is an essential cofactor required for proper functioning of all cells and its decline is correlated with advancing age and disease. This randomized, triple-blind, placebo-controlled, crossover pilot study assessed the efficacy and safety of a combination of nicotinamide with D-ribose (RiaGev) for NAD metabolome enhancement and related benefits in healthy middle-aged adults. Supplementing with 1520 mg RiaGev twice daily for 7 days significantly increased the NAD+ metabolome in blood, especially NADP+ by 27% compared to the placebo group (p = 0.033) and over the baseline (p = 0.007). Increases in glutathione and high energy phosphates were also observed in the blood. Seven-day supplementation with RiaGev significantly (p = 0.013) reduced overall blood glucose without significant changes in insulin secretion (p = 0.796), suggesting an improved insulin sensitivity and glucose tolerance. The waking salivary cortisol of the subjects steadily and significantly decreased (p = 0.026) in the RiaGev group in contrast to the placebo. Subjects in the RiaGev group showed less fatigue, improved mental concentration and motivation over the baseline (p = 0.015, 0.018, and 0.012, respectively) as observed through the Checklist Individual Strength (CIS) questionnaire. There were no clinically relevant adverse events, or alterations in hematology, electrolytes, liver, and kidney markers pre- and post-supplementation. RiaGev appears to be safe and efficacious in increasing NAD+ metabolome in healthy middle-aged adults, as shown by this study.

Enzymatic Depletion of Mitochondrial Inorganic Polyphosphate (polyP) Increases the Generation of Reactive Oxygen Species (ROS) and the Activity of the Pentose Phosphate Pathway (PPP) in Mammalian Cells.

Inorganic polyphosphate (polyP) is an ancient biopolymer that is well preserved throughout evolution and present in all studied organisms. In mammals, it shows a high co-localization with mitochondria, and it has been demonstrated to be involved in the homeostasis of key processes within the organelle, including mitochondrial bioenergetics. However, the exact extent of the effects of polyP on the regulation of cellular bioenergetics, as well as the mechanisms explaining these effects, still remain poorly understood. Here, using HEK293 mammalian cells under Wild-type (Wt) and MitoPPX (cells enzymatically depleted of mitochondrial polyP) conditions, we show that depletion of polyP within mitochondria increased oxidative stress conditions. This is characterized by enhanced mitochondrial O2- and intracellular H2O2 levels, which may be a consequence of the dysregulation of oxidative phosphorylation (OXPHOS) that we have demonstrated in MitoPPX cells in our previous work. These findings were associated with an increase in basal peroxiredoxin-1 (Prx1), superoxide dismutase-2 (SOD2), and thioredoxin (Trx) antioxidant protein levels. Using 13C-NMR and immunoblotting, we assayed the status of glycolysis and the pentose phosphate pathway (PPP) in Wt and MitoPPX cells. Our results show that MitoPPX cells display a significant increase in the activity of the PPP and an increase in the protein levels of transaldolase (TAL), which is a crucial component of the non-oxidative phase of the PPP and is involved in the regulation of oxidative stress. In addition, we observed a trend towards increased glycolysis in MitoPPX cells, which corroborates our prior work. Here, for the first time, we show the crucial role played by mitochondrial polyP in the regulation of mammalian redox homeostasis. Moreover, we demonstrate a significant effect of mitochondrial polyP on the regulation of global cellular bioenergetics in these cells.

Extending the Scope of 1H NMR-Based Blood Metabolomics for the Analysis of Labile Antioxidants: Reduced and Oxidized Glutathione.

Glutathione is a ubiquitous cellular antioxidant, which is critically required to protect cells from oxidative damage and free radical injury. It is practically impossible to analyze glutathione in its native form after isolation from biological mixtures since the active form (reduced glutathione, GSH) spontaneously gets converted to the oxidized form (oxidized glutathione, GSSG). To address this challenge, numerous highly sensitive detection methods, including mass spectrometry, have been used in conjunction with derivatization to block the oxidation of GSH. Efforts so far to quantitate GSH and GSSG using the nuclear magnetic resonance (NMR) spectroscopy method have remained unsuccessful. With a focus on addressing this challenge, in this study, we describe an extension to our recent whole blood analysis method [ Anal. Chem. 2017, 89, 4620-4627] that includes the important antioxidants GSH and GSSG. Fresh and frozen human whole blood specimens as well as standard GSH and GSSG were comprehensively investigated using NMR without and with derivatization using N-ethylmaleimide (NEM). NMR experiments detect two diastereomers, distinctly, for the derivatized GSH and enable the analysis of both GSH and GSSG in human whole blood with an accuracy of >99%. Interestingly, the excess (unreacted) NEM used for blocking the GSH can be removed from the samples during a drying step after extraction, with no need for additional processing. This is an important characteristic that offers an added advantage for simultaneous analysis of the antioxidants (GSH and GSSG), redox coenzymes (oxidized nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), oxidized nicotinamide adenine dinucleotide phosphate (NADP+), reduced nicotinamide adenine dinucleotide phosphate (NADPH)), energy coenzymes (adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP)), and a large number of other blood metabolites using the same one-dimensional (1D) NMR spectrum. The presented method broadens the scope of global metabolite profiling and adds a new dimension to NMR-based blood metabolomics. Further, the method demonstrated here for human blood can be extended to virtually any biological specimen.

NMR-Based Metabolomics.

Nuclear magnetic resonance (NMR) spectroscopy is a major analytical method used in the growing field of metabolomics. Although NMR is relatively less sensitive than mass spectrometry, this analytical platform has numerous characteristics including its high reproducibility and quantitative abilities, its nonselective and noninvasive nature, and the ability to identify unknown metabolites in complex mixtures and trace the downstream products of isotope labeled substrates ex vivo, in vivo, or in vitro. Metabolomic analysis of highly complex biological mixtures has benefitted from the advances in both NMR data acquisition and analysis methods. Although metabolomics applications span a wide range of disciplines, a majority has focused on understanding, preventing, diagnosing, and managing human diseases. This chapter describes NMR-based methods relevant to the rapidly expanding metabolomics field.

Evaluation of Fumaric Acid and Maleic Acid as Internal Standards for NMR Analysis of Protein Precipitated Plasma, Serum, and Whole Blood.

Significant advances have been made in unknown metabolite identification and expansion of the number of quantifiable metabolites in human plasma, serum, and whole blood using NMR spectroscopy. However, reliable quantitation of metabolites is still a challenge. A major bottleneck is the lack of a suitable internal standard that does not interact with the complex blood sample matrix and also does not overlap with metabolite peaks apart from exhibiting other favorable characteristics. With the goal of addressing this challenge, a comprehensive investigation of fumaric and maleic acids as potential internal standards was made along with a comparison with the conventional standards, TSP (trimethylsilylpropionic acid) and DSS (trimethylsilylpropanesulfonic acid). Both fumaric acid and maleic acid exhibited a surprisingly high performance with a quantitation error <1%, while the TSP and DSS caused an average error of up to 35% in plasma, serum, and whole blood. Further, the results indicate that while fumaric acid is a robust standard for all three biospecimens, maleic acid is suitable for only plasma and serum. Maleic acid is not suited for the analysis of whole blood due to its overlap with coenzyme peaks. These findings provide new opportunities for improved and accurate quantitation of metabolites in human plasma, serum, and whole blood using NMR spectroscopy. Moreover, the use of protein precipitation prior to NMR analysis mirrors the sample preparation commonly used for mass spectrometry based metabolomics, such that these findings further strengthen efforts to combine and compare NMR and MS based metabolite data of human plasma, serum, and whole blood for metabolomics based research.

Metabolomics in chronic pain research.

BACKGROUND AND OBJECTIVE: Metabolomics deals with the identification and quantification of small molecules (metabolites) in biological samples. As metabolite levels can reflect normal or altered metabolic pathways, their measurement provides information to improve the understanding, diagnosis and management of diseases. Despite its immense potential, metabolomics applications to pain research have been sparse. This paper describes current metabolomics techniques, reviews published human metabolomics pain research and compares successful metabolomics research in other areas of medicine with the goal of highlighting opportunities offered by metabolomics to advance pain medicine. DATABASES AND DATA TREATMENT: Non-systematic review. RESULTS: Our search identified 19 studies that adopted a metabolomics approach in: fibromyalgia (7), chronic widespread pain (4), other musculoskeletal pain conditions (5), neuropathic pain (1), complex regional pain syndrome (1) and pelvic pain (1). The studies used either mass spectrometry or nuclear magnetic resonance. Most are characterized by small sample sizes. Some consistency has been found for alterations in glutamate and testosterone metabolism, and metabolic imbalances caused by the gut microbiome. CONCLUSIONS: Metabolomics research in chronic pain is in its infancy. Most studies are at the pilot stage. Metabolomics research has been successful in other areas of medicine. These achievements should motivate investigators to expand metabolomics research to improve the understanding of the basic mechanisms of human pain, as well as provide tools to diagnose, predict and monitor chronic pain conditions. Metabolomics research can lead to the identification of biomarkers to support the development and testing of treatments, thereby facilitating personalized pain medicine.

Distinguishing NASH Histological Severity Using a Multiplatform Metabolomics Approach.

Nonalcoholic fatty liver disease (NAFLD) is categorized based on histological severity into nonalcoholic fatty liver (NAFL) or nonalcoholic steatohepatitis (NASH). We used a multiplatform metabolomics approach to identify metabolite markers and metabolic pathways that distinguish NAFL from early NASH and advanced NASH. We analyzed fasting serum samples from 57 prospectively-recruited patients with histologically-proven NAFLD, including 12 with NAFL, 31 with early NASH and 14 with advanced NASH. Metabolite profiling was performed using a combination of liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy analyzed with multivariate statistical and pathway analysis tools. We targeted 237 metabolites of which 158 were quantified. Multivariate analysis uncovered metabolite profile clusters for patients with NAFL, early NASH, and advanced NASH. Also, multiple individual metabolites were associated with histological severity, most notably spermidine which was more than 2-fold lower in advanced fibrosis vs. early fibrosis, in advanced NASH vs. NAFL and in advanced NASH vs. early NASH, suggesting that spermidine exercises a protective effect against development of fibrosing NASH. Furthermore, the results also showed metabolic pathway perturbations between early-NASH and advanced-NASH. In conclusion, using a combination of two reliable analytical platforms (LC-MS and NMR spectroscopy) we identified individual metabolites, metabolite clusters and metabolic pathways that were significantly different between NAFL, early-NASH, and advanced-NASH. These differences provide mechanistic insights as well as potentially important metabolic biomarker candidates that may noninvasively distinguish patients with NAFL, early-NASH, and advanced-NASH. The associations of spermidine levels with less advanced histology merit further assessment of the potential protective effects of spermidine in NAFLD.