The unaided recovery of marathon-induced serum metabolome alterations.

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 GC-MS metabolomics signature in patients with fibromyalgia syndrome directs to dysbiosis as an aspect contributing factor of FMS pathophysiology.

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.

The 1H-NMR-based metabolite profile of acute alcohol consumption: A metabolomics intervention study.

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.

GC-MS-based urinary organic acid profiling reveals multiple dysregulated metabolic pathways following experimental acute alcohol consumption.

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.

The cross-tissue metabolic response of abalone (Haliotis midae) to functional hypoxia.

Functional hypoxia is a stress condition caused by the abalone itself as a result of increased muscle activity, which generally necessitates the employment of anaerobic metabolism if the activity is sustained for prolonged periods. With that being said, abalone are highly reliant on anaerobic metabolism to provide partial compensation for energy production during oxygen-deprived episodes. However, current knowledge on the holistic metabolic response for energy metabolism during functional hypoxia, and the contribution of different metabolic pathways and various abalone tissues towards the overall accumulation of anaerobic end-products in abalone are scarce. Metabolomics analysis of adductor muscle, foot muscle, left gill, right gill, haemolymph and epipodial tissue samples indicated that South African abalone (Haliotis midae) subjected to functional hypoxia utilises predominantly anaerobic metabolism, and depends on all of the main metabolite classes (proteins, carbohydrates and lipids) for energy supply. Functional hypoxia caused increased levels of anaerobic end-products: lactate, alanopine, tauropine, succinate and alanine. Also, elevation in arginine levels was detected, confirming that abalone use phosphoarginine to generate energy during functional hypoxia. Different tissues showed varied metabolic responses to hypoxia, with functional hypoxia showing excessive changes in the adductor muscle and gills. From this metabolomics investigation, it becomes evident that abalone are metabolically able to produce sufficient amounts of energy when functional hypoxia is experienced. Also, tissue interplay enables the adjustment of H. midae energy requirements as their metabolism shifts from aerobic to anaerobic respiration during functional hypoxia.This article has an associated First Person interview with the first author of the paper.

The altered human serum metabolome induced by a marathon.

INTRODUCTION: Endurance races have been associated with a substantial amount of adverse effects which could lead to chronic disease and long-term performance impairment. However, little is known about the holistic metabolic changes occurring within the serum metabolome of athletes after the completion of a marathon. OBJECTIVES: Considering this, the aim of this study was to better characterize the acute metabolic changes induced by a marathon. METHODS: Using an untargeted two dimensional gas chromatography time-of-flight mass spectrometry metabolomics approach, pre- and post-marathon serum samples of 31 athletes were analyzed and compared to identify those metabolites varying the most after the marathon perturbation. RESULTS: Principle component analysis of the comparative groups indicated natural differentiation due to variation in the total metabolite profiles. Elevated concentrations of carbohydrates, fatty acids, tricarboxylic acid cycle intermediates, ketones and reduced concentrations of amino acids indicated a metabolic shift between various fuel substrate systems. Additionally, elevated odd-chain fatty acids and α-hydroxy acids indicated the utilization of α-oxidation and autophagy as alternative energy-producing mechanisms. Adaptations in gut microbe-associated markers were also observed and correlated with the metabolic flexibility of the athlete. CONCLUSION: From these results it is evident that a marathon places immense strain on the energy-producing pathways of the athlete, leading to extensive protein degradation, oxidative stress, mammalian target of rapamycin complex 1 inhibition and autophagy. A better understanding of this metabolic shift could provide new insights for optimizing athletic performance, developing more efficient nutrition regimens and identify strategies to improve recovery.

Comparative Serum Fatty Acid Profiles of Captive and Free-Ranging Cheetahs (Acinonyx jubatus) in Namibia.

Cheetahs (Acinonyx jubatus) are highly specialised large felids, currently listed as vulnerable on the IUCN red data list. In captivity, they are known to suffer from a range of chronic non-infectious diseases. Although low heterozygosity and the stress of captivity have been suggested as possible causal factors, recent studies have started to focus on the contribution of potential dietary factors in the pathogenesis of these diseases. Fatty acids are an important component of the diet, not only providing a source of metabolisable energy, but serving other important functions in hormone production, cellular signalling as well as providing structural components in biological membranes. To develop a better understanding of lipid metabolism in cheetahs, we compared the total serum fatty acid profiles of 35 captive cheetahs to those of 43 free-ranging individuals in Namibia using gas chromatography-mass spectrometry. The unsaturated fatty acid concentrations differed most remarkably between the groups, with all of the polyunsaturated and monounsaturated fatty acids, except arachidonic acid and hypogeic acid, detected at significantly lower concentrations in the serum of the free-ranging animals. The influence of age and sex on the individual fatty acid concentrations was less notable. This study represents the first evaluation of the serum fatty acids of free-ranging cheetahs, providing critical information on the normal fatty acid profiles of free-living, healthy individuals of this species. The results raise several important questions about the potential impact of dietary fatty acid composition on the health of cheetahs in captivity.

Contribution towards a Metabolite Profile of the Detoxification of Benzoic Acid through Glycine Conjugation: An Intervention Study.

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.

A hypothetical astrocyte-microglia lactate shuttle derived from a 1H NMR metabolomics analysis of cerebrospinal fluid from a cohort of South African children with tuberculous meningitis.

Tuberculosis meningitis (TBM) is the most severe form of extra-pulmonary tuberculosis and is particularly intense in small children; there is no universally accepted algorithm for the diagnosis and substantiation of TB infection, which can lead to delayed intervention, a high risk factor for morbidity and mortality. In this study a proton magnetic resonance (1H NMR)-based metabolomics analysis and several chemometric methods were applied to data generated from lumber cerebrospinal fluid (CSF) samples from three experimental groups: (1) South African infants and children with confirmed TBM, (2) non-meningitis South African infants and children as controls, and (3) neurological controls from the Netherlands. A total of 16 NMR-derived CSF metabolites were identified, which clearly differentiated between the controls and TBM cases under investigation. The defining metabolites were the combination of perturbed glucose and highly elevated lactate, common to some other neurological disorders. The remaining 14 metabolites of the host's response to TBM were likewise mainly energy-associated indicators. We subsequently generated a hypothesis expressed as an "astrocyte-microglia lactate shuttle" (AMLS) based on the host's response, which emerged from the NMR-metabolomics information. Activation of microglia, as implied by the AMLS hypothesis, does not, however, present a uniform process and involves intricate interactions and feedback loops between the microglia, astrocytes and neurons that hamper attempts to construct basic and linear cascades of cause and effect; TBM involves a complex integration of the responses from the various cell types present within the CNS, with microglia and the astrocytes as main players.

Paracetamol prevents hyperglycinemia in vervet monkeys treated with valproate.

Valproate administration increases the level of the inhibitory transmitter, glycine, in the urine and plasma of patients and experimental animals. Nonketotic hyperglycinemia (NKH), an autosomal recessive disorder of glycine metabolism, causes increased glycine concentrations in blood, urine, and cerebrospinal fluid (CSF), most likely due to a defect in the glycine cleavage enzyme or possibly deficits in glycine transport across cell membranes. We investigated the relationship between the hyperglycinemic effect of valproate and induced pyroglutamic aciduria via paracetamol in the vervet monkey. Firstly it was determined if valproate could induce hyperglycinemia in the monkey. The second aim was to increase glutamic acid (oxoproline) urine excretion using paracetamol as a pre-treatment and to assess whether valproate has an influence on the γ-glutamyl cycle. Hyperglycinemia was induced in healthy vervet monkeys when treated with a single oral dose of 50 mg/kg valproate. An acute dose of 50 mg/kg paracetamol increased oxoproline in the urine. Pre-treatment with paracetamol opposed the hyperglycinemic effect of valproate. However, the CSF:serum glycine ratio in a nonketotic monkey increased markedly after paracetamol treatment and remained high following valproate treatment. These results indicate that the γ-glutamyl cycle does indeed play a role in the hyperglycinemic effect of valproate treatment, and that paracetamol may have value in preventing and/or treating valproate-induced NKH.

Treatment of an adrenomyeloneuropathy patient with Lorenzo's oil and supplementation with docosahexaenoic acid--a case report.

This is a case report of adrenomyeloneuropathy (AMN), the adult variant of adrenoleukodystryphy (ALD). The diagnoses in the patient, aged 34, was confirmed via increased serum very long chain fatty acid concentration (VLCFA). Treatment started with the cholesterol lowering drug, atorvastatin, followed by add-on therapy with Lorenzo's oil (LO) and finally supplementation with docosahexaenoic acid (DHA). The magnetic resonance imaging (MRI) scan of the AMN patient before DHA treatment, already showed abnormal white matter in the brain. Although the MRI showed no neurological improvement after 6 months of DHA treatment, no selective progression of demyelination was detected in the AMN patient. Contrary to what was expected, LO failed to sustain or normalize the VLCFA levels or improve clinical symptoms. It was however, shown that DHA supplementation in addition to LO, increased DHA levels in both plasma and red blood cells (RBC). Additionally, the study showed evidence that the elongase activity in the elongation of eicosapentaenoic acid (EPA) to docosapentaenoic acid (DPA) might have been significantly compromised, due to the increased DHA levels.

The styrene metabolite, phenylglyoxylic acid, induces striatal-motor toxicity in the rat: influence of dose escalation/reduction over time.

Exposure to the industrial solvent, styrene, induces locomotor and cognitive dysfunction in rats, and parkinsonian-like manifestations in man. The antipsychotic, haloperidol (HP), well known to induce striatal toxicity in man and animals, and styrene share a common metabolic pathway yielding p-fluoro phenylglyoxylic acid and phenylglyoxylic acid (PGA), respectively. Using an exposure period of 30 days and the vacous chewing movement (VCM) model as an expression of striatal-motor toxicity, we found that incremental PGA dosing (220-400 mg/kg) significantly increased VCMs up to day 25, but decreased to control levels shortly after reaching maximum dose. However, a diminishing dose of PGA (400-200 mg/kg) did not evoke an immediate worsening of VCMs but precipitated a significant increase in VCMs following dosage reduction to 200 mg/kg on day 22. PGA exposure, therefore, compromises striatal-motor function that is especially sensitive to changes in exposure dose. Longer alternating dose exposure studies are needed to establish whether motor dysfunction is progressive in severity or longevity. These findings are of significance for the environmental toxicology of styrene in the chemical industry.

An overview of a cohort of South African patients with mitochondrial disorders.

Mitochondrial disorders are frequently encountered inherited diseases characterized by unexplained multisystem involvement with a chronic, intermittent, or progressive nature. The objective of this paper is to describe the profile of patients with mitochondrial disorders in South Africa. Patients with possible mitochondrial disorders were accessed over 10 years. Analyses for respiratory chain and pyruvate dehydrogenase complex enzymes were performed on muscle. A diagnosis of a mitochondrial disorder was accepted only if an enzyme activity was deficient. Sixty-three patients were diagnosed with a mitochondrial disorder, including 40 African, 20 Caucasian, one mixed ancestry, and two Indian patients. The most important findings were the difference between African patients and other ethnicities: respiratory chain enzyme complexes CI+III or CII+III deficiencies were found in 52.5% of African patients, being of statistical significance (p value = 0.0061). They also presented predominantly with myopathy (p value = 0.0018); the male:female ratio was 1:1.2. Twenty-five (62.5%) African patients presented with varying degrees of a myopathy accompanied by a myopathic face, high palate, and scoliosis. Fourteen of these 25 also had ptosis and/or progressive external ophthalmoplegia. No patients of other ethnicities presented with this specific myopathic phenotype. Caucasian patients (16/20) presented predominantly with central nervous system involvement. Of the South African pediatric neurology patients, Africans are more likely to present with myopathy and CII+III deficiency, and Caucasian patients are more likely to present with encephalopathy or encephalomyopathy.

Melatonin prevents the free radical and MADD metabolic profiles induced by antituberculosis drugs in an animal model.

The objective was to determine the effect of combined antituberculosis (anti-TB) drug therapy and an antioxidant, melatonin, on the free radical and organic acid profiles in an experimental rat model. A combined anti-TB drug, Rifater, consisting of 12.0 mg rifampicin, 0.8 mg isoniazid, and 23.0 mg pyrazinamide and 18.56 microg melatonin/kg body weight per day (corresponding to average physiological human intake) were orally administered to Sprague-Dawley rats. Hydroxyl radical production was monitored by quantifying 2,3-dihydroxybenzoic acid produced after intraperitonial sodium salicylate injections. Organic acid extractions and gas chromatography-coupled mass spectrometry analyses were performed on collected urine samples. The results show hydroxyl radicals (P = 0.0019) and organic acids (P-value range: 0.037 to <0.001), characteristic of a multiple acyl-CoA dehydrogenase defect (MADD), were elevated with Rifater treatment and these elevations were significantly lowered with melatonin pretreatment (P-value range: 0.031 to <0.001), probably because of its inherent antioxidant activity. We conclude that hydroxyl radical production and an increased organic acid profile induced by anti-TB medication indicates inhibition of the electron transport chain. We also conclude that free radicals leading to clinical symptoms associated with an MADD metabolic profile induced by anti-TB treatment could be alleviated by melatonin intervention.

Measurement of mitochondrial respiration in permeabilized murine neuroblastoma (N-2alpha) cells, a simple and rapid in situ assay to investigate mitochondrial toxins.

Most mitochondria-based methods used to investigate toxins require the use of relatively large amounts of material and hence compromised sensitivity in assay. We adopted procedures from methods initially developed to diagnose mitochondrial encephalomyopathies and unified these into a single assay. Eukaryotic cell membranes are selectively permeabilized with digitonin to render a system in which mitochondrial respiration can be measured rapidly and with considerable sensitivity. Mitochondria remain intact, uninjured, and in their natural environment where mitochondrial respiration can be measured in situ under physiologically relevant conditions. This approach furthermore allows measurement of toxin effects on individual mitochondrial complexes. Numerous compounds at varying concentrations can be screened for mitochondrial toxicity, while the site of mitochondrial inhibition can be determined simultaneously. We used this assay to investigate, in murine neuroblastoma (N-2alpha) cells, the mitochondrial inhibitory properties of the parkinsonian-inducing proneurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and its neurotoxic monoamine oxidase-B (MAO-B)-generated metabolite, the 1-methyl-4-phenylpyridinium species (MPP(+)). Within the time frame of each measurement (15 min), MPTP (< or = 1 mM) did not interfere with in situ mitochondrial respiration. As expected, MPP(+) was found to be a potent Complex I inhibitor but surprisingly also found to inhibit Complex IV. Optimized conditions for performing this assay are provided.

Acetyl-L-carnitine prevents total body hydroxyl free radical and uric acid production induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the rat.

Acetyl-L-carnitine (ALCAR) is intimately involved in the transport of long chain fatty acids across the inner mitochondrial membrane during oxidative phosphorylation. ALCAR also has been reported to attenuate the occurrence of parkinsonian symptoms associated with 1-methyl-1,2,3,6-tetrahydropyridine (MPTP) in vivo, and protects in vitro against the toxicity of the neurotoxic 1-methyl-4-phenylpyridinium (MPP+) metabolite of MPTP. The mechanism for these protective effects remains unclear. ALCAR may attenuate hydroxyl (HO*) free radical production in the MPTP/MPP+ neurotoxic pathway through several mechanisms. Most studies on MPTP/MPP+ toxicity and protection by ALCAR have focused on in vivo brain chemistry and in vitro neuronal culture studies. The present study investigates the attenuative effects of ALCAR on whole body oxidative stress markers in the urine of rats treated with MPTP. In a first study, ALCAR totally prevented the MPTP-induced formation of HO* measured by salicylate radical trapping. In a second study, the production of uric acid after MPTP administration-a measure of oxidative stress mediated through xanthine oxidase-was also prevented by ALCAR. Because ALCAR is unlikely to be a potent radical scavenger, these studies suggest that ALCAR protects against MPTP/MPP+-mediated oxidative stress through other mechanisms. We speculate that ALCAR may operate through interference with organic cation transporters such as OCTN2 and/or carnitine-acylcarnitine translocase (CACT), based partly on the above findings and on semi-empirical electronic similarity calculations on ALCAR, MPP+, and two other substrates for these transporters.