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The use of detergents when culturing Mycobacterium tuberculosis (M. tuberculosis) are essential to prevent clumping. However, these detergents may influence research outcomes by impacting bacterial morphology and metabolism. This study aimed to assess the metabolome of a M. tuberculosis H37Rv strain cultured with Tyloxapol (H37Rv Tyloxapol ), compared to a control group of H37Rv strain cultured without detergent (H37Rv Control ) to evaluate Tyloxapol's suitability for metabolomic studies. Distinct metabolic alterations were observed in H37Rv Tyloxapol compared to H37Rv Control , primarily associated with fatty acid, sugar and pentose phosphate metabolic pathways. These changes are associated with the surface stress exerted by Tyloxapol on the bacteria, prompting an adaptation of M. tuberculosis metabolism to that usually observed in stress environments. Nevertheless, the effect of Tyloxapol is less pronounced than that of a previous investigation using Tween 80, indicating its potential as the more favourable choice for culturing M. tuberculosis for metabolomic analysis, with due consideration to dosage and result interpretation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: The prevalence of type 2 diabetes has surged to epidemic proportions and despite treatment administration/adherence, some individuals experience poorly controlled diabetes. While existing literature explores metabolic changes in type 2 diabetes, understanding metabolic derangement in poorly controlled cases remains limited., Objective: This investigation aimed to characterize the urine metabolome of poorly controlled type 2 diabetes in a South African cohort., Method: Using an untargeted proton nuclear magnetic resonance metabolomics approach, urine samples from 15 poorly controlled type 2 diabetes patients and 25 healthy controls were analyzed and statistically compared to identify differentiating metabolites., Results: The poorly controlled type 2 diabetes patients were characterized by elevated concentrations of various metabolites associated with changes to the macro-fuel pathways (including carbohydrate metabolism, ketogenesis, proteolysis, and the tricarboxylic acid cycle), autophagy and/or apoptosis, an uncontrolled diet, and kidney and liver damage., Conclusion: These results indicate that inhibited cellular glucose uptake in poorly controlled type 2 diabetes significantly affects energy-producing pathways, leading to apoptosis and/or autophagy, ultimately contributing to kidney and mild liver damage. The study also suggests poor dietary compliance as a cause of the patient's uncontrolled glycemic state. Collectively these findings offer a first-time comprehensive overview of urine metabolic changes in poorly controlled type 2 diabetes and its association with secondary diseases, offering potential insights for more targeted treatment strategies to prevent disease progression, treatment efficacy, and diet/treatment compliance., (© 2024. The Author(s).)

Tuberculous meningitis (TBM) is a severe form of tuberculosis with high neuro-morbidity and mortality, especially among the paediatric population (aged ≤12 years). Little is known of the associated metabolic changes. This study aimed to identify characteristic metabolic markers that differentiate severe cases of paediatric TBM from controls, through non-invasive urine collection. Urine samples selected for this study were from two paediatric groups. Group 1: controls (n = 44): children without meningitis, no neurological symptoms and from the same geographical region as group 2. Group 2: TBM cases (n = 13): collected from paediatric patients that were admitted to Tygerberg Hospital in South Africa on the suspicion of TBM, mostly severely ill; with a later confirmation of TBM. Untargeted 1H NMR-based metabolomics data of urine were generated, followed by statistical analyses via MetaboAnalyst (v5.0), and the identification of important metabolites. Twenty nine urinary metabolites were identified as characteristic of advanced TBM and categorized in terms of six dysregulated metabolic pathways: 1) upregulated tryptophan catabolism linked to an altered vitamin B metabolism; 2) perturbation of amino acid metabolism; 3) increased energy production-metabolic burst; 4) disrupted gut microbiota metabolism; 5) ketoacidosis; 6) increased nitrogen excretion. We also provide original biological insights into this biosignature of urinary metabolites that can be used to characterize paediatric TBM patients in a South African cohort., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Isaiah, Loots, van Reenen, Solomons, van Elsland, Tutu van Furth, van der Kuip and Mason.)

HIV-exposed, uninfected (HEU) children present with suboptimal growth and a greater susceptibility to infection in early life when compared to HIV-unexposed, uninfected (HUU) children. The reasons for these findings are poorly understood. We used a metabolomics approach to investigate the metabolic differences between pregnant women living with HIV (PWLWH) and their HEU infants compared to the uninfected and unexposed controls. Untargeted metabolomic profiling was performed using 1 H-NMR spectroscopy on maternal plasma at 28 weeks' gestation and infant plasma at birth, 6/10 weeks, and 6 months. PWLWH were older but, apart from a larger 28 week mid-upper-arm circumference, anthropometrically similar to the controls. At all the time points, HEU infants had a significantly reduced growth compared to HUU infants. PWLWH had lower plasma 3-hydroxybutyric acid, acetoacetic acid, and acetic acid levels. In infants at birth, threonine and myo-inositol levels were lower in the HEU group while formic acid levels were higher. At 6/10 weeks, betaine and tyrosine levels were lower in the HEU group. Finally, at six months, 3-hydroxyisobutyric acid levels were lower while glycine levels were higher in the HEU infants. The NMR analysis has provided preliminary information indicating differences between HEU and HUU infants' plasma metabolites involved in energy utilization, growth, and protection from infection.

Introduction: Various studies have identified TB-induced metabolome variations. However, in most of these studies, a large degree of variation exists between individual patients., Objectives: To identify differential metabolites for TB, independent of patients' sex or HIV status., Methods: Untargeted GCxGC/TOF-MS analyses were applied to the sputum of 31 TB + and 197 TB- individuals. Univariate statistics were used to identify metabolites which are significantly different between TB + and TB- individuals (a) irrespective of HIV status, and (b) with a HIV + status. Comparisons a and b were repeated for (i) all participants, (ii) males only and (iii) females only., Results: Twenty-one compounds were significantly different between the TB + and TB- individuals within the female subgroup (11% lipids; 10% carbohydrates; 1% amino acids, 5% other and 73% unannotated), and 6 within the male subgroup (20% lipids; 40% carbohydrates; 6% amino acids, 7% other and 27% unannotated). For the HIV + patients (TB + vs. TB-), a total of 125 compounds were significant within the female subgroup (16% lipids; 8% carbohydrates; 12% amino acids, 6% organic acids, 8% other and 50% unannotated), and 44 within the male subgroup (17% lipids; 2% carbohydrates; 14% amino acids related, 8% organic acids, 9% other and 50% unannotated). Only one annotated compound, 1-oleoyl lysophosphaditic acid, was consistently identified as a differential metabolite for TB, irrespective of sex or HIV status. The potential clinical application of this compound should be evaluated further., Conclusions: Our findings highlight the importance of considering confounders in metabolomics studies in order to identify unambiguous disease biomarkers., (© 2023. The Author(s).)

Various metabolomics studies have reported increased phenylalanine serum concentrations in SARS-CoV-2 positive cases and have correlated increased phenylalanine with COVID-19 severity. In this study, we report similar results based upon metabolomics analysis of serum collected from a South African cohort of adults with confirmed COVID-19. The novelty of this study is the inclusion of HIV positive cases in the African context. We found that pre-existing HIV co-infection exacerbates the disruption of phenylalanine metabolism in COVID-19. What is lacking in literature is biological context and deeper understanding of perturbed phenylalanine metabolism in COVID-19. We delve deep into the metabolism of phenylalanine in COVID-19 and posit new insights for COVID-19 cases co-infected with HIV; namely, HIV-COVID-19 co-infected individuals do not have sufficient bioavailability of tetrahydrobiopterin (BH 4 ). Hence, we identify BH 4 as a potential supplement to alleviate/lessen COVID-19 symptoms., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors. Published by Elsevier Ltd.)

Introduction: Technological advancements enabled the analyses of limited sample volumes on 1 H NMR. Manual spectral profiling of the data is, however, complex, and timely., Objective: To evaluate the performance of BAYESIL for automated identification and quantification of 1 H NMR spectra of limited volume samples., Method: Aliquots of a pooled African elephant serum sample were analyzed using standard and reduced volumes. Performance was evaluated on confidence scores, non-detects and laboratory CV., Results: Of the 47 compounds detected, 28 had favorable performances. The approach could differentiate samples based on biological variation., Conclusions: BAYESIL is valuable for limited sample 1 H NMR data analyses., (© 2023. The Author(s).)

Background and Aims: The association between the metabolic syndrome (MetS) and plasminogen activator inhibitor-1 (PAI-1) has been well established in cross-sectional studies. It is less clear whether this translates into decreased clot lysis rates and very little information is available on non-European populations. Little is known regarding prospective associations and whether clot lysis progressively worsens in MetS individuals over time. We determined the prospective association of MetS with PAI-1 activity (PAI-1 act ) and clot lysis time (CLT) over a 10-year period., Methods and Results: As many as 2010 African men and women aged ≥30 years were stratified according to MetS status and number of MetS criteria (0-5). We also determined the contribution of the PAI-1 4G/5G polymorphism to these associations and identified which MetS criteria had the strongest associations with PAI-1 act and CLT. Both PAI-1 act and CLT remained consistently elevated in individuals with MetS throughout the 10-year period. PAI-1 act and CLT did not increase more over time in MetS individuals than in controls. The 4G/5G genotype did not influence the association of PAI-1 act or clot lysis with MetS. Increased waist circumference, increased triglycerides and decreased HDL-C were the main predictors of PAI-1 act and CLT., Conclusions: Black South Africans with MetS had increased PAI-1 act and longer CLTs than individuals without MetS. The inhibited clot lysis in MetS did, however, not deteriorate over time compared to controls. Of the MetS criteria, obesity and altered lipids were the main predictors of PAI-1 act and CLT and are thus potential targets for prevention strategies to decrease thrombotic risk., Competing Interests: Declaration of competing interest The authors have nothing to disclose., (Copyright © 2022 The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

Introduction: PKCδ is ubiquitously expressed in mammalian cells and its dysregulation plays a key role in the onset of several incurable diseases and metabolic disorders. However, much remains unknown about the metabolic pathways and disturbances induced by PKC deficiency, as well as the metabolic mechanisms involved., Objectives: This study aims to use metabolomics to further characterize the function of PKC from a metabolomics standpoint, by comparing the full serum metabolic profiles of PKC deficient mice to those of wild-type mice., Methods: The serum metabolomes of PKCδ knock-out mice were compared to that of a wild-type strain using a GCxGC-TOFMS metabolomics research approach and various univariate and multivariate statistical analyses., Results: Thirty-seven serum metabolite markers best describing the difference between PKCδ knock-out and wild-type mice were identified based on a PCA power value > 0.9, a t-test p-value < 0.05, or an effect size > 1. XERp prediction was also done to accurately select the metabolite markers within the 2 sample groups. Of the metabolite markers identified, 78.4% (29/37) were elevated and 48.65% of these markers were fatty acids (18/37). It is clear that a total loss of PKCδ functionality results in an inhibition of glycolysis, the TCA cycle, and steroid synthesis, accompanied by upregulation of the pentose phosphate pathway, fatty acids oxidation, cholesterol transport/storage, single carbon and sulphur-containing amino acid synthesis, branched-chain amino acids (BCAA), ketogenesis, and an increased cell signalling via N-acetylglucosamine., Conclusion: The charaterization of the dysregulated serum metabolites in this study, may represent an additional tool for the early detection and screening of PKCδ-deficiencies or abnormalities., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Mycobacterium tuberculosis (M. tuberculosis) curli pili (MTP) is a surface located adhesin, which is involved in the initial point-of-contact between the pathogen and the host. Host-pathogen interaction is essential for establishing infection. M. tuberculosis has the ability to infect various host lung cell types, which includes both the epithelial cells and macrophages, and subsequent differences in their cellular function will be evident in their metabolic profiles. Understanding the differences between these cell types and their individual metabolic response to M. tuberculosis infection, with and without the presence of the MTP, will aid to better elucidate the role of this adhesin in modulating metabolic pathways during infection. This may further contribute to the development of improved diagnostic and therapeutic interventions, much needed at present in order to improve control the global tuberculosis (TB) epidemic. This study used a two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC×GC-TOFMS) metabolomics approach to compare the metabolite profiles of A549 epithelial cells to that of THP-1 macrophages, infected with M. tuberculosis, in the presence and absence of MTP. Significant metabolites were identified using various univariate and multivariate statistical analysis. A total of 44, 40, 50 and 34 metabolites were differentially detected when comparing the (a) uninfected A549 epithelial cells to uninfected THP-1 macrophages, (b) wild-type infected A549 epithelial cells to wild-type infected THP-1 macrophages, (c) ∆mtp-infected A549 epithelial cells to ∆mtp-infected THP-1 macrophages (d) complement-infected A549 epithelial cells to complement-infected THP-1 macrophages, respectively. These included metabolites that were involved in amino acid metabolism, fatty acid metabolism, general central carbon metabolism, and nucleic acid metabolism. In the absence of the M. tuberculosis MTP adhesin, the THP-1 macrophages predominantly displayed higher concentrations of amino acids and their metabolic intermediates, than the A549 epithelial cells. The deletion of MTP from M. tuberculosis in the host infection models potentially elicited a pro-inflammatory phenotype, particularly in the macrophage model. In the presence of MTP, the metabolite profile changes indicate potential regulation of host defence mechanisms, accompanied by a reduction in microbicidal abilities of host cells. Hence MTP can be considered a virulence factor of M. tuberculosis. Therefore, blocking MTP interaction with the host may facilitate a faster pathogen clearance during the initial stages of infection, and potentially enhance current therapeutic interventions., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Red beetroot (Beta vulgaris L.) is a multifunctional functional food that reportedly exhibits potent anti-inflammatory, antioxidant, vasodilation, and cellular regulatory properties. This vegetable has gained a fair amount of scientific attention as a possible cost-effective supplement to enhance performance and expedite recovery after physical exercise. To date, no study has investigated the effects of incremental beetroot juice ingestion on the metabolic recovery of athletes after an endurance race. Considering this, as well as the beneficial glucose and insulin regulatory roles of beetroot, this study investigated the effects of beetroot juice supplementation on the metabolic recovery trend of athletes within 48 h after completing a marathon., Methods: By employing an untargeted two-dimensional gas chromatography time-of-flight mass spectrometry approach, serum samples (collected pre-, post-, 24 h post-, and 48 h post-marathon) of 31 marathon athletes that ingested a series (n = 7; 250 ml) of either beetroot juice (n = 15 athletes) or isocaloric placebo (n = 16 athletes) supplements within 48 h post-marathon, were analysed and statistically compared., Results: The metabolic profiles of the beetroot-ingesting cohort recovered to a pre-marathon-related state within 48 h post-marathon, mimicking the metabolic recovery trend observed in the placebo cohort. Since random inter-individual variation was observed immediately post-marathon, only metabolites with large practical significance (p-value ≤0.05 and d-value ≥0.5) within 24 h and 48 h post-marathon were considered representative of the effects of beetroot juice on metabolic recovery. These (n = 4) mainly included carbohydrates (arabitol and xylose) and odd-chain fatty acids (nonanoate and undecanoate). The majority of these were attributed to beetroot content and possible microbial fermentation thereof., Conclusion: Apart from the global metabolic recovery trends of the two opposing cohorts, it appears that beetroot ingestion did not expedite metabolic recovery in athletes within 48 h post-marathon., (© 2021. The Author(s).)

Despite the arguable success of the standardized tuberculosis (TB) treatment regime, a significant number of patients still present with treatment failure. To improve on current TB treatment strategies, we sought to gain a better understanding of the hosts' response to TB therapy. A targeted metabolomics approach was used to compare the urinary acylcarnitine and amino acid profiles of eventually cured TB patients with those of patients presenting with a failed treatment outcome, comparing these patient groups at the time of diagnosis and at weeks 1, 2, and 4 of treatment. Among the significant metabolites identified were histidine, isoleucine, leucine, methionine, valine, proline, tyrosine, alanine, serine, and γ-aminobutyric acid. In general, metabolite fluctuations in time followed a similar pattern for both groups for most compounds but with a delayed onset or shift of the pattern in the successfully treated patient group. These time-trends detected in both groups could potentially be ascribed to a vitamin B6 deficiency and fluctuations in the oxidative stress levels and urea cycle intermediates, linked to the drug-induced inhibition and stimulation of various enzymes. The earlier onset of observed trends in the failed patients is proposed to relate to genotypic and phenotypic variations in drug metabolizing enzymes, subsequently leading to a poor treatment efficiency either due to the rise of adverse drug reactions or to insufficient concentrations of the active drug metabolites. This study emphasizes the need for a more personalized TB treatment approach, by including enzyme phenotyping and the monitoring of oxidative stress and vitamin B6 levels, for example.

The initial host-pathogen interaction is crucial for the establishment of infection. An improved understanding of the pathophysiology of Mycobacterium tuberculosis (M. tuberculosis) during macrophage infection can aid the development of intervention therapeutics against tuberculosis. M. tuberculosis curli pili (MTP) is a surface located adhesin, involved in the first point-of-contact between pathogen and host. This study aimed to better understand the role of MTP in modulating the intertwined metabolic pathways of M. tuberculosis and its THP-1 macrophage host. Metabolites were extracted from pelleted wet cell mass of THP-1 macrophages infected with M. tuberculosis wild-type V9124 (WT), Δmtp-deletion mutant and the mtp-complemented strains, respectively, via a whole metabolome extraction method using a 1:3:1 ratio of chloroform:methanol:water. Metabolites were detected by two-dimensional gas chromatography time-of-flight mass spectrometry. Significant metabolites were determined through univariate and multivariate statistical tests and online pathway databases. Relative to the WT, a total of nine and ten metabolites were significantly different in the Δmtp and complement strains, respectively. All nine significant metabolites were found in elevated levels in the Δmtp relative to the WT. Additionally, of the ten significant metabolites, eight were detected in lower levels and two were detected in higher levels in the complement relative to the WT. The absence of the MTP adhesin resulted in reduced virulence of M. tuberculosis leading to alterations in metabolites involved in carbon, fatty acid and amino acid metabolism during macrophage infection, suggesting that MTP plays an important role in the modulation of host metabolic activity. These findings support the prominent role of the MTP adhesin as a virulence factor as well as a promising biomarker for possible diagnostic and therapeutic intervention., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Mitochondrial diseases (MD), such as Leigh syndrome (LS), present with severe neurological and muscular phenotypes in patients, but have no known cure and limited treatment options. Based on their neuroprotective effects against other neurodegenerative diseases in vivo and their positive impact as an antioxidant against complex I deficiency in vitro, we investigated the potential protective effect of metallothioneins (MTs) in an Ndufs4 knockout mouse model (with a very similar phenotype to LS) crossed with an Mt1 overexpressing mouse model (TgMt1). Despite subtle reductions in the expression of neuroinflammatory markers GFAP and IBA1 in the vestibular nucleus and hippocampus, we found no improvement in survival, growth, locomotor activity, balance, or motor coordination in the Mt1 overexpressing Ndufs4 -/- mice. Furthermore, at a cellular level, no differences were detected in the metabolomics profile or gene expression of selected one-carbon metabolism and oxidative stress genes, performed in the brain and quadriceps, nor in the ROS levels of macrophages derived from these mice. Considering these outcomes, we conclude that MT1, in general, does not protect against the impaired motor activity or improve survival in these complex I-deficient mice. The unexpected absence of increased oxidative stress and metabolic redox imbalance in this MD model may explain these observations. However, tissue-specific observations such as the mildly reduced inflammation in the hippocampus and vestibular nucleus, as well as differential MT1 expression in these tissues, may yet reveal a tissue- or cell-specific role for MTs in these mice.

Background: Several multi-attribute utility instruments (MAUIs) are available from which utilities can be derived for use in cost-utility analysis (CUA). This study provides a review of recommendations from national health technology assessment (HTA) agencies regarding the choice of MAUIs., Methods: A list was compiled of HTA agencies that provide or refer to published official pharmacoeconomic (PE) guidelines for pricing, reimbursement or market access. The guidelines were reviewed for recommendations on the indirect calculation of utilities and categorized as: a preference for a specific MAUI; providing no MAUI preference, but providing examples of suitable MAUIs and/or recommending the use of national value sets; and recommending CUA, but not providing examples of MAUIs., Results: Thirty-four PE guidelines were included for review. MAUIs named for use in CUA: EQ-5D (n = 29 guidelines), the SF-6D (n = 11), HUI (n = 10), QWB (n = 3), AQoL (n = 2), CHU9D (n = 1). EQ-5D was a preferred MAUI in 15 guidelines. Alongside the EQ-5D, the HUI was a preferred MAUI in one guideline, with DALY disability weights mentioned in another. Fourteen guidelines expressed no preference for a specific MAUI, but provided examples: EQ-5D (n = 14), SF-6D (n = 11), HUI (n = 9), QWB (n = 3), AQoL (n = 2), CHU9D (n = 1). Of those that did not specify a particular MAUI, 12 preferred calculating utilities using national preference weights., Conclusions: The EQ-5D, HUI, and SF-6D were the three MAUIs most frequently mentioned in guidelines. The most commonly cited MAUI (in 85% of PE guidelines) was EQ-5D, either as a preferred MAUI or as an example of a suitable MAUI for use in CUA in HTA.

Objective: To better characterize the cerebrospinal fluid (CSF) metabolic profile of tuberculous meningitis (TBM) cases using a South African paediatric cohort., Methods: 1 H NMR metabolomics was used to analyse the CSF of a South African paediatric cohort. Univariate and multivariate statistical analyses were performed to compare a homogeneous control group with a well-defined TBM group., Results: Twenty metabolites were identified to discriminate TBM cases from controls. As expected, reduced glucose and elevated lactate were the dominating discriminators. A closer investigation of the CSF metabolic profile yielded 18 metabolites of statistical significance. Ten metabolites (acetate, alanine, choline, citrate, creatinine, isoleucine, lysine, myo-inositol, pyruvate and valine) overlapped with two other prior investigations. Eight metabolites (2-hydroxybutyrate, carnitine, creatine, creatine phosphate, glutamate, glutamine, guanidinoacetate and proline) were unique to our paediatric TBM cohort., Conclusions: Through strict exclusion criteria, quality control checks and data filtering, eight unique CSF metabolites associated with TBM were identified for the first time and linked to: uncontrolled glucose metabolism, upregulated proline and creatine metabolism, detoxification and disrupted glutamate-glutamine cycle in the TBM samples. Associated with oxidative stress and chronic neuroinflammation, our findings collectively imply destabilization, and hence increased permeability, of the blood-brain barrier in the TBM cases., (Copyright © 2020. Published by Elsevier Ltd.)

Endurance athlete performance is greatly dependent on sufficient post-race system recovery, as endurance races have substantial physiological, immunological and metabolic effects on these athletes. To date, the effects of numerous recovery modalities have been investigated, however, very limited literature exists pertaining to metabolic recovery of athletes after endurance races without the utilisation of recovery modalities. As such, this investigation is aimed at identifying the metabolic recovery trend of athletes within 48 h after a marathon. Serum samples of 16 athletes collected 24 h before, immediately after, as well as 24 h and 48 h post-marathon were analysed using an untargeted two-dimensional gas chromatography time-of-flight mass spectrometry metabolomics approach. The metabolic profiles of these comparative time-points indicated a metabolic shift from the overall post-marathon perturbed state back to the pre-marathon metabolic state during the recovery period. Statistical analyses of the data identified 61 significantly altered metabolites including amino acids, fatty acids, tricarboxylic acid cycle, carbohydrates and associated intermediates. These intermediates recovered to pre-marathon related concentrations within 24 h post-marathon, except for xylose which only recovered within 48 h. Furthermore, fluctuations in cholesterol and pyrimidine intermediates indicated the activation of alternative recovery mechanisms. Metabolic recovery of the athletes was attained within 48 h post-marathon, most likely due to reduced need for fuel substrate catabolism. This may result in the activation of glycogenesis, uridine-dependent nucleotide synthesis, protein synthesis, and the inactivation of cellular autophagy. These results may be beneficial in identifying more efficient, targeted recovery approaches to improve athletic performance.

The association between hypercoagulability and use of drospirenone (DRSP) and ethinylestradiol (EE) containing combined oral contraceptives (COCs) is an important clinical concern. We have previously reported that the two formulations of DRSP combined with EE (namely, DRSP/20EE and DRSP/30EE) bring about a prothrombotic state in hemostatic traits of female users. We report here the serum metabolomic changes in the same study cohort in relation to the attendant prothrombotic state induced by COC use, thus offering new insights on the underlying biochemical mechanisms contributing to the altered coagulatory profile with COC use. A total of 78 healthy women participated in this study and were grouped as follows: control group not using oral contraceptives ( n  = 25), DRSP/20EE group ( n  = 27), and DRSP/30EE group ( n  = 26). Untargeted metabolomics revealed changes in amino acid concentrations, particularly a decrease in glycine and an increase in both cysteine and lanthionine in the serum, accompanied by variations in oxidative stress markers in the COC users compared with the controls. Of importance, this study is the first to link specific amino acid variations, serum metabolites, and the oxidative metabolic profile with DRSP/EE use. These molecular changes could be linked to specific biophysical coagulatory alterations observed in the same individuals. These new findings lend evidence on the metabolomic substrates of the prothrombotic state associated with COC use in women and informs future personalized/precision medicine research. Moreover, we underscore the importance of an interdisciplinary approach to evaluate venous thrombotic risk associated with COC use.

As a means to increase the growth rate and reduce aggregation, Tween 80 is routinely added to growth media during mycobacterial culturing. This detergent has, however, been associated with causing alterations to the morphology, pathogenicity and virulence of these bacteria. In an attempt to better understand the underlying mechanism of these alterations, we investigated the effect of Tween 80 on the metabolomes of a M. tuberculosis lab strain (H37Rv) and multidrug-resistant clinical strain (R179), using GC-GCxTOF-MS metabolomics. The metabolite markers identified indicated Tween 80-induced disparities in the central carbon metabolism of both strains, with an upregulation in the glyoxylate cycle, glucogenogenesis and the pentose phosphate pathway. The results also signified an increased production of mycobacterial biosynthetic precursors such as triacylglycerols, proteinogenic amino acids and nucleotide precursors, in the presence of the detergent. Collectively, these metabolome variations mimic the phenotypic changes observed when M. tuberculosis is grown in vivo, in a lipid rich environment. However, in addition to the increased availability of oleic acid as a carbon source from Tween 80, the observed variations, and the morphological changes associated with the detergent, could also be a result of an overall stress response in these bacteria. This study is the first to identify specific metabolome variations related to the addition of Tween 80 to the growth media during M. tuberculosis culturing. The consideration of these results during the method development and data interpretation phases of future metabolomics investigations will improve the quality of the analyses as well as the credibility of potential research outcomes. These results will also assist in the interpretation of research questions specifically aimed at aspects of mycobacterial metabolism, even when using other methodologies such as transcriptomics or fluxomics., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Introduction: Manifestations of fatigue range from chronic fatigue up to a severe syndrome and myalgic encephalomyelitis. Fatigue grossly affects the functional status and quality of life of affected individuals, prompting the World Health Organization to recognize it as a chronic non-communicable condition., Objectives: Here, we explore the potential of urinary metabolite information to complement clinical criteria of fatigue, providing an avenue towards an objective measure of fatigue in patients presenting with the full spectrum of fatigue levels., Methods: The experimental group consisted of 578 chronic fatigue female patients. The measurement design was composed of (1) existing clinical fatigue scales, (2) a hepatic detoxification challenge test, and (3) untargeted proton nuclear magnetic resonance ( 1 H-NMR) procedure to generate metabolomics data. Data analysed via an in-house Matlab script that combines functions from a Statistics and a PLS Toolbox., Results: Multivariate analysis of the original 459 profiled 1 H-NMR bins for the low (control) and high (patient) fatigue groups indicated complete separation following the detoxification experimental challenge. Important bins identified from the 1 H-NMR spectra provided quantitative metabolite information on the detoxification challenge for the fatigue groups., Conclusions: Untargeted 1 H-NMR metabolomics proved its applicability as a global profiling tool to reveal the impact of toxicological interventions in chronic fatigue patients. No clear potential biomarker emerged from this study, but the quantitative profile of the phase II biotransformation products provide a practical visible effect directing to up-regulation of crucial phase II enzyme systems in the high fatigue group in response to a high xenobiotic-load.

Chronic fatigue, in its various manifestations, frequently co-occur with pain, sleep disturbances and depression and is a non-communicable condition which is rapidly becoming endemic worldwide. However, it is handicapped by a lack of objective definitions and diagnostic measures. This has prompted the World Health Organization to develop an international instrument whose intended purpose is to improve quality of life (QOL), with energy and fatigue as one domain of focus. To complement this objective, the interface between detoxification, the exposome, and xenobiotic-sensing by nuclear receptors that mediate induction of biotransformation-linked genes, is stimulating renewed attention to a rational development of strategies to identify the metabolic profiles in complex multifactorial conditions like fatigue. Here we present results from a seven-year study of a cohort of 576 female patients suffering from low to high levels of chronic fatigue, in which phase I and phase II biotransformation was assessed. The biotransformation profiles used were based on hepatic detoxification challenge tests through oral caffeine, acetaminophen and acetylsalicylic acid ingestion coupled with oxidative stress analyses. The interventions indicated normal phase I but increased phase II glucuronidation and glycination conjugation. Complementarity was indicated between a fatigue scale, medical symptoms and associated energy-related parameters by application of Chi-square Automatic Interaction Detector (CHAID) analysis. The presented study provides a cluster of data from which we propose that multidisciplinary inputs from the combination of a fatigue scale, medical symptoms and biotransformation profiles provide the rationale for the development of a comprehensive laboratory instrument for improved diagnostics and personalized interventions in patients with chronic fatigue with a view to improving their QOL., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Fibromyalgia syndrome (FMS) is a chronic pain syndrome. Previous analyses of untargeted metabolomics data indicated altered metabolic profile in FMS patients., Objectives: We report a semi-targeted explorative metabolomics study on the urinary metabolite profile of FMS patients; exploring the potential of urinary metabolite information to augment existing medical diagnosis., Methods: All cases were females. Patients had a medical history of persistent FMS (n = 18). Control groups were first-generation family members of the patients (n = 11), age-related individuals without indications of FMS (n = 10), and healthy, young (18-22 years) individuals (n = 41). The biofluid investigated was early morning urine samples. Data generation was done through gas chromatography-mass spectrometry (GC-MS) analysis and data processing and analyses were performed using Matlab, R, SPSS and SAS software., Results: Quantitative analysis revealed the presence of 196 metabolites. Unsupervised and supervised multivariate analyses distinguished all three control groups and the FMS patients, which could be related to 14 significantly increased metabolites. These metabolites are associated with energy metabolism, digestion and metabolism of carbohydrates and other host and gut metabolites., Conclusions: Overall, urinary metabolite profiles in the FMS patients suggest: (1) energy utilization is a central aspect of this pain disorder, (2) dysbiosis seems to prevail in FMS patients, indicated by disrupted microbiota metabolites, supporting the model that microbiota may alter brain function through the gut-brain axis, with the gut being a gateway to generalized pain, and (3) screening of urine from FMS is an avenue to explore for adding non-invasive clinical information for diagnosis and treatment of FMS.

Metabolomics studies of disease conditions related to chronic alcohol consumption provide compelling evidence of several perturbed metabolic pathways underlying the pathophysiology of alcoholism. The objective of the present study was to utilize proton nuclear magnetic resonance (1H-NMR) spectroscopy metabolomics to study the holistic metabolic consequences of acute alcohol consumption in humans. The experimental design was a cross-over intervention study which included a number of substances to be consumed-alcohol, a nicotinamide adenine dinucleotide (NAD) supplement, and a benzoic acid-containing flavoured water vehicle. The experimental subjects-24 healthy, moderate-drinking young men-each provided six hourly-collected urine samples for analysis. Complete data sets were obtained from 20 of the subjects and used for data generation, analysis and interpretation. The results from the NMR approach produced complex spectral data, which could be resolved sufficiently through the application of a combination of univariate and multivariate methods of statistical analysis. The metabolite profiles resulting from acute alcohol consumption indicated that alcohol-induced NAD+ depletion, and the production of an excessive amount of reducing equivalents, greatly perturbed the hepatocyte redox homeostasis, resulting in essentially three major metabolic disturbances-up-regulated lactic acid metabolism, down-regulated purine catabolism and osmoregulation. Of these, the urinary excretion of the osmolyte sorbitol proved to be novel, and suggests hepatocyte swelling due to ethanol influx following acute alcohol consumption. Time-dependent metabolomics investigations, using designed interventions, provide a way of interpreting the variation induced by the different factors of a designed experiment, thereby also giving methodological significance to this study. The outcomes of this approach have the potential to significantly advance our understanding of the serious impact of the pathophysiological perturbations which arise from the consumption of a single, large dose of alcohol-a simulation of a widespread, and mostly naive, social practice.

Metabolomics studies of diseases associated with chronic alcohol consumption provide compelling evidence of several perturbed metabolic pathways. Moreover, the holistic approach of such studies gives insights into the pathophysiological risk factors associated with chronic alcohol-induced disability, morbidity and mortality. Here, we report on a GC-MS-based organic acid profiling study on acute alcohol consumption. Our investigation - involving 12 healthy, moderate-drinking young men - simulated a single binge drinking event, and indicated its metabolic consequences. We generated time-dependent data that predicted the metabolic pathophysiology of the alcohol intervention. Multivariate statistical modelling was applied to the longitudinal data of 120 biologically relevant organic acids, of which 13 provided statistical evidence of the alcohol effect. The known alcohol-induced increased NADH:NAD + ratio in the cytosol of hepatocytes contributed to the global dysregulation of several metabolic reactions of glycolysis, ketogenesis, the Krebs cycle and gluconeogenesis. The significant presence of 2-hydroxyisobutyric acid supports the emerging paradigm that this compound is an important endogenous metabolite. Its metabolic origin remains elusive, but recent evidence indicated 2-hydroxyisobutyrylation as a novel regulatory modifier of histones. Metabolomics has thus opened an avenue for further research on the reprogramming of metabolic pathways and epigenetic networks in relation to the severe effects of alcohol consumption.

Background: Fibromyalgia syndrome (FMS) is a chronic pain syndrome. A plausible pathogenesis of the disease is uncertain and the pursuit of measurable biomarkers for objective identification of affected individuals is a continuing endeavour in FMS research. Our objective was to perform an explorative metabolomics study (1) to elucidate the global urinary metabolite profile of patients suffering from FMS, and (2) to explore the potential of this metabolite information to augment existing medical practice in diagnosing the disease., Methods: We selected patients with a medical history of persistent FMS (n = 18), who described their recent state of the disease through the Fibromyalgia Impact Questionnaire (FIQR) and an in-house clinical questionnaire (IHCQ). Three control groups were used: first-generation family members of the patients (n = 11), age-related individuals without any indications of FMS or related conditions (n = 10), and healthy young (18-22 years) individuals (n = 20). All subjects were female and the biofluid under investigation was urine. Correlation analysis of the FIQR showed the FMS patients represented a well-defined disease group for this metabolomics study. Spectral analyses of urine were conducted using a 500 MHz 1 H nuclear magnetic resonance (NMR) spectrometer; data processing and analyses were performed using Matlab, R, SPSS and SAS software., Results and Discussion: Unsupervised and supervised multivariate analyses distinguished all three control groups and the FMS patients, and significant increases in metabolites related to the gut microbiome (hippuric, succinic and lactic acids) were observed. We have developed an algorithm for the diagnosis of FMS consisting of three metabolites - succinic acid, taurine and creatine - that have a good level of diagnostic accuracy (Receiver Operating Characteristic (ROC) analysis - area under the curve 90%) and on the pain and fatigue symptoms for the selected FMS patient group., Conclusion: Our data and comparative analyses indicated an altered metabolic profile of patients with FMS, analytically detectable within their urine. Validation studies may substantiate urinary metabolites to supplement information from medical assessment, tender-point measurements and FIQR questionnaires for an improved objective diagnosis of FMS.

In captivity, cheetahs (Acinonyx jubatus) frequently suffer from several unusual chronic diseases that rarely occur in their free-ranging counterparts. In order to develop a better understanding of their metabolism and health we documented the urine organic acids of 41 apparently healthy captive cheetahs, in an untargeted metabolomic study, using gas chromatography-mass spectrometry. A total of 339 organic acids were detected and annotated. Phenolic compounds, thought to be produced by the anaerobic fermentation of aromatic amino acids in the distal colon, as well as their corresponding glycine conjugates, were present in high concentrations. The most abundant organic acids in the cheetahs' urine were an as yet unidentified compound and a novel cadaverine metabolite, tentatively identified as N 1 ,N 5 -dimethylpentane-1,5-diamine. Pantothenic acid and citramalic acid concentrations correlated negatively with age, while glutaric acid concentrations correlated positively with age, suggesting possible dysregulation of coenzyme A metabolism in older cheetahs. This study provides a baseline of urine organic acid reference values in captive cheetahs and suggests important avenues for future research in this species., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: ERp is a variable selection and classification method for metabolomics data. ERp uses minimized classification error rates, based on data from a control and experimental group, to test the null hypothesis of no difference between the distributions of variables over the two groups. If the associated p-values are significant they indicate discriminatory variables (i.e. informative metabolites). The p-values are calculated assuming a common continuous strictly increasing cumulative distribution under the null hypothesis. This assumption is violated when zero-valued observations can occur with positive probability, a characteristic of GC-MS metabolomics data, disqualifying ERp in this context. This paper extends ERp to address two sources of zero-valued observations: (i) zeros reflecting the complete absence of a metabolite from a sample (true zeros); and (ii) zeros reflecting a measurement below the detection limit. This is achieved by allowing the null cumulative distribution function to take the form of a mixture between a jump at zero and a continuous strictly increasing function. The extended ERp approach is referred to as XERp., Results: XERp is no longer non-parametric, but its null distributions depend only on one parameter, the true proportion of zeros. Under the null hypothesis this parameter can be estimated by the proportion of zeros in the available data. XERp is shown to perform well with regard to bias and power. To demonstrate the utility of XERp, it is applied to GC-MS data from a metabolomics study on tuberculosis meningitis in infants and children. We find that XERp is able to provide an informative shortlist of discriminatory variables, while attaining satisfactory classification accuracy for new subjects in a leave-one-out cross-validation context., Conclusion: XERp takes into account the distributional structure of data with a probability mass at zero without requiring any knowledge of the detection limit of the metabolomics platform. XERp is able to identify variables that discriminate between two groups by simultaneously extracting information from the difference in the proportion of zeros and shifts in the distributions of the non-zero observations. XERp uses simple rules to classify new subjects and a weight pair to adjust for unequal sample sizes or sensitivity and specificity requirements.

Benzoic acid is widely used as a preservative in food products and is detoxified in humans through glycine conjugation. Different viewpoints prevail on the physiological significance of the glycine conjugation reaction and concerns have been raised on potential public health consequences following uncontrolled benzoic acid ingestion. We performed a metabolomics study which used commercial benzoic acid containing flavored water as vehicle for designed interventions, and report here on the controlled consumption of the benzoic acid by 21 cases across 6 time points for a total of 126 time points. Metabolomics data from urinary samples analyzed by nuclear magnetic resonance spectroscopy were generated in a time-dependent cross-over study. We used ANOVA-simultaneous component analysis (ASCA), repeated measures analysis of variance (RM-ANOVA) and unfolded principal component analysis (unfolded PCA) to supplement conventional statistical methods to uncover fully the metabolic perturbations due to the xenobiotic intervention, encapsulated in the metabolomics tensor (three-dimensional matrices having cases, spectral areas and time as axes). Identification of the biologically important metabolites by the novel combination of statistical methods proved the power of this approach for metabolomics studies having complex data structures in general. The study disclosed a high degree of inter-individual variation in detoxification of the xenobiotic and revealed metabolic information, indicating that detoxification of benzoic acid through glycine conjugation to hippuric acid does not indicate glycine depletion, but is supplemented by ample glycine regeneration. The observations lend support to the view of maintenance of glycine homeostasis during detoxification. The study indicates also that time-dependent metabolomics investigations, using designed interventions, provide a way of interpreting the variation induced by the different factors of a designed experiment-an approach with potential to advance significantly our understanding of normal and pathophysiological perturbations of endogenous or exogenous origin., Competing Interests: The authors have declared that no competing interests exist.

Background: Metabolomics datasets are often high-dimensional though only a limited number of variables are expected to be informative given a specific research question. The important task of selecting informative variables can therefore become complex. In this paper we look at discriminating between two groups. Two tasks need to be performed: (i) finding variables which differ between the two groups; and (ii) determining how the selected variables can be used to classify new subjects. We introduce an approach using minimum classification error rates as test statistics to find discriminatory and therefore informative variables. The thresholds resulting in the minimum error rates can be used to classify new subjects. This approach transforms error rates into p-values and is referred to as ERp., Results: We show that non-parametric hypothesis testing, based on minimum classification error rates as test statistics, can find statistically significantly shifted variables. The discriminatory ability of variables becomes more apparent when error rates are evaluated based on their corresponding p-values, as relatively high error rates can still be statistically significant. ERp can handle unequal and small group sizes, as well as account for the cost of misclassification. ERp retains (if known) or reveals (if unknown) the shift direction, aiding in biological interpretation. The threshold resulting in the minimum error rate can immediately be used to classify new subjects. We use NMR generated metabolomics data to illustrate how ERp is able to discriminate subjects diagnosed with Mycobacterium tuberculosis infected meningitis from a control group. The list of discriminatory variables produced by ERp contains all biologically relevant variables with appropriate shift directions discussed in the original paper from which this data is taken., Conclusions: ERp performs variable selection and classification, is non-parametric and aids biological interpretation while handling unequal group sizes and misclassification costs. All this is achieved by a single approach which is easy to perform and interpret. ERp has the potential to address many other characteristics of metabolomics data. Future research aims to extend ERp to account for a large proportion of observations below the detection limit, as well as expand on interactions between variables.