Urinary Metabolites as Predictors of Acute Mountain Sickness Severity.

Individuals sojourning at high altitude (≥2,500m) often develop acute mountain sickness (AMS). However, substantial unexplained inter-individual variability in AMS severity exists. Untargeted metabolomics assays are increasingly used to identify novel biomarkers of susceptibility to illness, and to elucidate biological pathways linking environmental exposures to health outcomes. This study used untargeted nuclear magnetic resonance (NMR)-based metabolomics to identify urine metabolites associated with AMS severity during high altitude sojourn. Following a 21-day stay at sea level (SL; 55m), 17 healthy males were transported to high altitude (HA; 4,300m) for a 22-day sojourn. AMS symptoms measured twice daily during the first 5days at HA were used to dichotomize participants according to AMS severity: moderate/severe AMS (AMS; n=11) or no/mild AMS (NoAMS; n=6). Urine samples collected on SL day 12 and HA days 1 and 18 were analyzed using proton NMR tools and the data were subjected to multivariate analyses. The SL urinary metabolite profiles were significantly different (p≤0.05) between AMS vs. NoAMS individuals prior to high altitude exposure. Differentially expressed metabolites included elevated levels of creatine and acetylcarnitine, and decreased levels of hypoxanthine and taurine in the AMS vs. NoAMS group. In addition, the levels of two amino acid derivatives (4-hydroxyphenylpyruvate and N-methylhistidine) and two unidentified metabolites (doublet peaks at 3.33ppm and a singlet at 8.20ppm) were significantly different between groups at SL. By HA day 18, the differences in urinary metabolites between AMS and NoAMS participants had largely resolved. Pathway analysis of these differentially expressed metabolites indicated that they directly or indirectly play a role in energy metabolism. These observations suggest that alterations in energy metabolism before high altitude exposure may contribute to AMS susceptibility at altitude. If validated in larger cohorts, these markers could inform development of a non-invasive assay to screen individuals for AMS susceptibility prior to high altitude sojourn.

Influence of Myo-inositol Plus Ethanolamine on Plasmalogens and Cell Viability during Oxidative Stress.

Previously, we demonstrated that treatment of rats with myo-inositol plus ethanolamine (ME) elevated brain ethanolamine plasmalogens (PE-Pls) and protected against phosphine-induced oxidative stress. Here we tested the hypothesis that ME treatment elevates PE-Pls in a neuro-2A (N2A) cell culture system and protects against hydrogen peroxide (H2O2)-induced oxidative stress, and we assessed the effects of treatments using myo-inositol with or without (+/-) ethanolamine on ethanolamine phospholipids (PLs) and cell viability following H2O2 exposure. Cells were treated with equimolar amounts (500 µM) of myo-inositol, ethanolamine (Etn), or their combination (ME) for 24 h, followed by an additional 24 h exposure to 650 µM H2O2. NMR analyses evaluated the treatment effects on Etn PLs, while LC-MS/MS analyses assessed the molecular species of Etn PLs preferentially affected by ME and H2O2 treatments, especially PE-Pls and their degradation byproducts-lysophosphatidylethanolamine (LPE) and glycerophosphoethanolamine (GPE). Only ME influenced the cellular levels of PLs. ME yielded a 3-fold increase in PE-Pls and phosphatidylethanolamine (PE) ( p < 0.001) and a preferential 60% increase in PE-Pls containing saturated and monounsaturated fatty acids (SFA+MUFA), while polyunsaturated fatty acid (PUFA) species increased by only 10%. Exposing cells to 650 µM H2O2 caused a significant cell death (56% viability), a 27% decrease in PE-Pls, a 201% increase in PUFA-rich LPE, and a ca. 3-fold increase in GPE. H2O2 had no impact on PE, suggesting that LPE and GPE were primarily the byproducts of PE-Pls (not PE) degradation. Surprisingly, ME pretreatment ameliorated H2O2 effects and significantly increased cell survival to 80% ( p < 0.05). Cellular PE-Pls levels prior to H2O2 treatment were highly correlated ( R2 = 0.95) with cell survival, suggesting a relationship between PE-Pls and cell protection. Data suggest that a preferential increase in PE-Pls containing SFA+MUFA species may protect cells from oxidative stress. Such studies aid in our understanding of the neuroprotective mechanisms that may be associated with plasmalogens and the relevance of these phospholipids to neurodegenerative diseases/disorders.

Lipid, water, and protein composition to facilitate kinetic modeling of the auditory pathway.

Environments combining JP-8 jet fuel exposure with heightened ambient noise may accelerate hearing loss induced by noise. To reduce animal use and facilitate kinetic modeling of this military aviation fuel, tissue-specific parameters are required, including water, protein, and lipid content. However, tissues involved in hearing, including cochlea, brainstem, frontal, and temporal lobe, have not been characterized before. Therefore, water content was determined by lyophilization of rat auditory tissues and the protein of the freeze dried remainder was quantified using a bicinchoninic acid assay. Lipids were extracted from fresh-frozen rat auditory tissues and separated into neutral lipids, free fatty acids, neutral phospholipids, and acidic phospholipids using solid phase extraction. Phospholipid fractions were confirmed by 31 P nuclear magnetic resonance analysis showing distinct phospholipid profiles. Lipid content in reference tissues, such as kidney and adipose, confirmed literature values. For the first time, lipid content in the rat auditory pathway was determined showing that total lipid content was lowest in cochlea and highest in brainstem compared with frontal and temporal lobes. Auditory tissues displayed distinct lipid fraction profiles. The information on water, protein, and lipid composition is necessary to validate algorithms used in mathematical models and predict partitioning of chemicals of future interest into these tissues. This research may reduce the use of animals to measure partition coefficients for prospective physiological models.

Intestinal Epithelial Cells Regulate Gut Eotaxin Responses and Severity of Allergy.

Intestinal epithelial cells (IECs) are known to regulate allergic sensitization. We addressed the role of the intrinsic IKKß signaling in IECs in the effector phase of allergy following oral allergen challenge and its impact on the severity of responses is poorly. Upon orally sensitization by co-administration of ovalbumin with cholera toxin as adjuvant, wild-type and mice lacking IKKß in IECs (IKKßΔIEC mice) developed similar levels of serum IgE and allergen-specific secretory IgA in the gut. However, subsequent allergen challenges in the gut promoted allergic lower responses in KKßΔIEC mice. Analysis of cytokines and chemokines in serum and gut tissues after oral allergen challenge revealed impaired eotaxin responses in IKKßΔIEC mice, which correlated with lower frequencies of eosinophils in the gut lamina propria. We also determined that IECs were a major source of eotaxin and that impaired eotaxin production was due to the lack of IKKß signaling in IECs. Oral administration of CCL11 to IKKßΔIEC mice during oral allergen challenge enhanced allergic responses to levels in wild-type mice, confirming the role of IEC-derived eotaxin as regulator of the effector phase of allergy following allergen challenge. Our results identified targeting IEC-derived eotaxin as potential strategy to limit the severity of allergic responses to food antigens.

Simultaneous fecal microbial and metabolite profiling enables accurate classification of pediatric irritable bowel syndrome.

BACKGROUND: We previously showed that stool samples of pre-adolescent and adolescent US children diagnosed with diarrhea-predominant IBS (IBS-D) had different compositions of microbiota and metabolites compared to healthy age-matched controls. Here we explored whether observed fecal microbiota and metabolite differences between these two adolescent populations can be used to discriminate between IBS and health. FINDINGS: We constructed individual microbiota- and metabolite-based sample classification models based on the partial least squares multivariate analysis and then applied a Bayesian approach to integrate individual models into a single classifier. The resulting combined classification achieved 84 % accuracy of correct sample group assignment and 86 % prediction for IBS-D in cross-validation tests. The performance of the cumulative classification model was further validated by the de novo analysis of stool samples from a small independent IBS-D cohort. CONCLUSION: High-throughput microbial and metabolite profiling of subject stool samples can be used to facilitate IBS diagnosis.

Altered gut microbial energy and metabolism in children with non-alcoholic fatty liver disease.

Obesity is becoming the new pediatric epidemic. Non-alcoholic fatty liver disease (NAFLD) is frequently associated with obesity and has become the most common cause of pediatric liver disease. The gut microbiome is the major metabolic organ and determines how calories are processed, serving as a caloric gate and contributing towards the pathogenesis of NAFLD. The goal of this study is to examine gut microbial profiles in children with NAFLD using phylogenetic, metabolomic, metagenomic and proteomic approaches. Fecal samples were obtained from obese children with or without NAFLD and healthy lean children. Stool specimens were subjected to 16S rRNA gene microarray, shotgun sequencing, mass spectroscopy for proteomics and NMR spectroscopy for metabolite analysis. Children with NAFLD had more abundant Gammaproteobacteria and Prevotella and significantly higher levels of ethanol, with differential effects on short chain fatty acids. This group also had increased genomic and protein abundance for energy production with a reduction in carbohydrate and amino acid metabolism and urea cycle and urea transport systems. The metaproteome and metagenome showed similar findings. The gut microbiome in pediatric NAFLD is distinct from lean healthy children with more alcohol production and pathways allocated to energy metabolism over carbohydrate and amino acid metabolism, which would contribute to development of disease.

The networks of human gut microbe-metabolite associations are different between health and irritable bowel syndrome.

The goal of this study was to determine if fecal metabolite and microbiota profiles can serve as biomarkers of human intestinal diseases, and to uncover possible gut microbe-metabolite associations. We employed proton nuclear magnetic resonance to measure fecal metabolites of healthy children and those diagnosed with diarrhea-predominant irritable bowel syndrome (IBS-D). Metabolite levels were associated with fecal microbial abundances. Using several ordination techniques, healthy and irritable bowel syndrome (IBS) samples could be distinguished based on the metabolite profiles of fecal samples, and such partitioning was congruent with the microbiota-based sample separation. Measurements of individual metabolites indicated that the intestinal environment in IBS-D was characterized by increased proteolysis, incomplete anaerobic fermentation and possible change in methane production. By correlating metabolite levels with abundances of microbial genera, a number of statistically significant metabolite-genus associations were detected in stools of healthy children. No such associations were evident for IBS children. This finding complemented the previously observed reduction in the number of microbe-microbe associations in the distal gut of the same cohort of IBS-D children.

Identification of serum analytes and metabolites associated with aerobic capacity.

Studies aimed at identifying serum markers of cellular metabolism (biomarkers) that are associated at baseline with aerobic capacity (VO2max) in young, healthy individuals have yet to be reported. Therefore, the goal of the present study was to use the standard chemistry screen and untargeted mass spectrometry (MS)-based metabolomic profiling to identify significant associations between baseline levels of serum analytes or metabolites with VO2max (77 subjects, age range 18-35 years). Use of multivariable linear regression identified three analytes (standard chemistry screen) and twenty-three metabolites (MS-based metabolomics) containing significant, sex-adjusted associations with VO2max. In addition, fourteen metabolites were found to contain sex-specific associations with aerobic capacity. Subsequent stepwise multivariable linear regression identified the combination of SGOT, 4-ethylphenylsulfate, tryptophan, γ-tocopherol, and α-hydroxyisovalerate as overall, sex-adjusted baseline predictors of VO2max (adjusted R(2) = 0.66). However, the results of the stepwise model were found to be sensitive to outliers; therefore, random forest (RF) regression was performed. Use of RF regression identified a combination of seven covariates that explained 57.6 % of the variability inherent in VO2max. Furthermore, inclusion of significant analytes, metabolites and sex-specific metabolites into a stepwise regression model identified the combination of five metabolites in males and seven metabolites in females as being able to explain 80 and 58 % of the variability inherent in VO2max, respectively. In conclusion, the evidence presented in the current report is the first attempt to identify baseline serum biomarkers that are significantly associated with VO2max in young, healthy adult humans.

Comparative metabolomic and genomic analyses of TCDD-elicited metabolic disruption in mouse and rat liver.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) elicits a broad spectrum of species-specific effects that have not yet been fully characterized. This study compares the temporal effects of TCDD on hepatic aqueous and lipid metabolite extracts from immature ovariectomized C57BL/6 mice and Sprague-Dawley rats using gas chromatography-mass spectrometry and nuclear magnetic resonance-based metabolomic approaches and integrates published gene expression data to identify species-specific pathways affected by treatment. TCDD elicited metabolite and gene expression changes associated with lipid metabolism and transport, choline metabolism, bile acid metabolism, glycolysis, and glycerophospholipid metabolism. Lipid metabolism is altered in mice resulting in increased hepatic triacylglycerol as well as mono- and polyunsaturated fatty acid (FA) levels. Mouse-specific changes included the induction of CD36 and other cell surface receptors as well as lipases- and FA-binding proteins consistent with hepatic triglyceride and FA accumulation. In contrast, there was minimal hepatic fat accumulation in rats and decreased CD36 expression. However, choline metabolism was altered in rats, as indicated by decreases in betaine and increases in phosphocholine with the concomitant induction of betaine-homocysteine methyltransferase and choline kinase gene expression. Results from these studies show that aryl hydrocarbon receptor-mediated differential gene expression could be linked to metabolite changes and species-specific alterations of biochemical pathways.

Characterization of 1H NMR spectroscopic data and the generation of synthetic validation sets.

MOTIVATION: Common contemporary practice within the nuclear magnetic resonance (NMR) metabolomics community is to evaluate and validate novel algorithms on empirical data or simplified simulated data. Empirical data captures the complex characteristics of experimental data, but the optimal or most correct analysis is unknown a priori; therefore, researchers are forced to rely on indirect performance metrics, which are of limited value. In order to achieve fair and complete analysis of competing techniques more exacting metrics are required. Thus, metabolomics researchers often evaluate their algorithms on simplified simulated data with a known answer. Unfortunately, the conclusions obtained on simulated data are only of value if the data sets are complex enough for results to generalize to true experimental data. Ideally, synthetic data should be indistinguishable from empirical data, yet retain a known best analysis. RESULTS: We have developed a technique for creating realistic synthetic metabolomics validation sets based on NMR spectroscopic data. The validation sets are developed by characterizing the salient distributions in sets of empirical spectroscopic data. Using this technique, several validation sets are constructed with a variety of characteristics present in 'real' data. A case study is then presented to compare the relative accuracy of several alignment algorithms using the increased precision afforded by these synthetic data sets. AVAILABILITY: These data sets are available for download at http://birg.cs.wright.edu/nmr_synthetic_data_sets.

Evidence that plasmalogen is protective against oxidative stress in the rat brain.

The antioxidant capabilities of phosphatidylethanolamine plasmalogen (PlsEtn), in vivo, against lipid peroxidation were investigated via acute phosphine (PH(3)) administration in rats. Oxidative stress was assessed from measures of malondialdehyde and various enzyme activities, while NMR analyses of lipid and aqueous tissue extracts provided metabolic information in cerebellum, brainstem, and cortex. Brainstem had the highest basal [PlsEtn], and showed only moderate PH(3)-induced oxidative damage with no loss of ATP. The lowest basal [PlsEtn] was observed in cortex, where PH(3) caused a 51% decrease in [ATP]. The largest oxidative effect occurred in cerebellum, but [ATP] was unaffected. Myo-inositol+ethanolamine pretreatment attenuated all PH(3) effects. Specifically, the pretreatment attenuated the ATP decrease in cortex, and elevated brain [PlsEtn] in the cerebellum, nearly abolishing the cerebellar oxidative effects. Our data suggest a high basal [PlsEtn], or the capacity to synthesize new ethanolamine lipids (particularly PlsEtn) may protect against PH(3) toxicity.

Total body water volume for white children and adolescents and anthropometric prediction equations: the Fels Longitudinal Study.

BACKGROUND: There are few studies of total body water (TBW) volume in children. Such studies are needed, as are new prediction equations for the clinical management of children with renal insufficiency and those receiving dialysis. METHODS: Mixed longitudinal data were from 124 white boys and 116 white girls 8 to 20 years of age. TBW volume was measured by deuterium nuclear magnetic resonance spectroscopy, and random effects models were used to determine patterns of change over time. Sex-specific TBW prediction equations were developed using regression analysis. RESULTS: Boys had significantly greater (P < 0.05) mean TBW volumes than girls at all but 3 ages. TBW was significantly (P < 0.05) associated with age and maturation in the boys and the girls. In boys, mean TBW/WT varied from 0.55 to 0.59, while in the girls the mean declined from 0.53 to 0.49 by 16 years of age. Boys had significantly larger means for TBW/WT than girls, who had a significant, slight negative trend with age. The prediction equations were TBW = -25.87 + 0.23 (stature) + 0.37 (weight) for boys and TBW =-14.77 + 0.18 (stature) + 0.25 (weight) for girls. CONCLUSION: Means are provided for TBW in white children from 8 to 20 years of age, whose average fatness affected the percentage of TBW in body weight. These updated TBW prediction equations perform better than those available from the past.

Administration of myo-inositol plus ethanolamine elevates phosphatidylethanolamine plasmalogen in the rat cerebellum.

Plasmalogens are ether-linked phospholipids highly abundant in nervous tissue. Previously we demonstrated that acute administration of myo-inositol (myo-Ins) + [2-(13)C] ethanolamine ([2-(13)C]Etn) significantly elevated phosphatidylethanolamine plasmalogen (PlsEtn) in rat whole brain. Current experiments investigated the effects of acute myo-Ins+[2-(13)C]Etn administration on [PlsEtn] and the biosynthesis of new Etn lipids using NMR spectroscopy in rat cerebral cortex, hippocampus, brainstem, midbrain and cerebellum. Treated rats received a single dose of myo-Ins + [2-(13)C]Etn and controls received saline rather than myoIns. Data reveal that the cerebellum is the brain region most affected by treatment, which resulted in a 22% increase in [PlsEtn] and 89% increase in newly synthesized Etn lipids relative to controls (P < 0.05). Furthermore, the cerebellar PlsEtn/phosphatidylethanolamine ratio and molar percentage of PlsEtn were significantly elevated by 12% and 8%, respectively (P < 0.05). These data suggest that myo-Ins influences Etn lipid metabolism in brain, particularly in the cerebellum where there is a stimulation in the biosynthesis of new Etn lipids with a preference towards PlsEtn.

Studies of myo-inositol and plasmalogen metabolism in rat brain.

Plasmalogens are ether-linked phospholipids that are abundant in nervous tissues. Their biological role is unclear, but may involve membrane structure/function and antioxidant activities. This study further investigates a recent report that chronic administration of myo-inositol in rats increased brain phosphatidylethanolamine plasmalogen (PlsEtn). We examined the effects of myo-inositol administration on the incorporation of [2-(13)C]ethanolamine ([2-(13)C]Etn) into rat brain phospholipids using NMR spectroscopy. Rats received either acute myo-inositol (single dose) +/- [2-(13)C]Etn, or chronic myo-inositol (10-day treatment) + [2-(13)C]Etn. Controls received saline rather than myo-inositol. Acute myo-inositol produced a 68% increase in brain [myo-inositol] and an increase in the incorporation of [2-(13)C]Etn into phospholipids (P < .05). The PlsEtn/phosphatidylethanolamine ratio and the [PlsEtn] were increased by 27% and 30%, respectively. The PlsEtn content as a mole percentage of total phospholipids was elevated (P < or = .05). Acute administration of myo-inositol + ethanolamine illustrates a positive correlation between the brain [myo-inositol] and the biosynthesis of ethanolamine phospholipids, with preferential synthesis of PlsEtn.

NMR-based metabolomics.

Similar to genomics and proteomics which yield vast amounts of data about the expression of genes and proteins, metabolomics refers to the whole metabolic profile of the cell. The focus of this report concerns the use of nuclear magnetic resonance (NMR) spectroscopy for metabolic analyses and, in particular, its use in toxicology for examining the metabolic profile of biofluids. Examples from the literature will demonstrate how 1H NMR and pattern recognition methods are used to obtain the urinary metabolic profile, and how this profile is affected by exposure to various toxicants. These particular studies which focus on the metabolic profiles of biofluids, specifically urine, are referred to as metabonomics. NMR-based metabonomics provides a means to categorize organ-specific toxicity, monitor the onset and progression of toxicological effects, and identify biomarkers of toxicity. A future challenge, however, is to describe the cellular metabolome for purposes of understanding cellular functions (i.e., metabolomics). Thus the capabilities and advantages of multinuclear NMR to provide metabolic information in cells and tissues will also be discussed. Such information is essential if metabolomics is to provide a complementary dataset which together with genomics and proteomics can be used to construct computer network models to describe cellular functions.

Kinetic analyses of liver phosphatidylcholine and phosphatidylethanolamine biosynthesis using (13)C NMR spectroscopy.

Choline and ethanolamine are substrates for de novo synthesis of phosphatidylcholine (PtdC) and phosphatidylethanolamine (PtdE) through the CDP-choline and CDP-ethanolamine pathways. In liver, PtdE can also be converted to PtdC by PtdE N-methyltransferase (PEMT). We investigated these kinetics in rat liver during a 60 min infusion with (13)C-labeled choline and ethanolamine. NMR analyses of liver extracts provided concentrations and (13)C enrichments of phosphocholine (Pcho), phosphoethanolamine (Peth), PtdC, and PtdE. Kinetic models showed that the de novo and PEMT pathways are 'channeled' processes. The intermediary metabolites directly derived from exogenous choline and ethanolamine do not completely mix with the intracellular pools, but are preferentially used for phospholipid synthesis. Of the newly synthesized PtdC, about 70% was derived de novo and 30% was by PEMT. PtdC and PtdE de novo syntheses displayed different kinetics. A simple model assuming constant fluxes yielded a modest fit to the data; allowing upregulated fluxes significantly improved the fit. The ethanolamine-to-Peth flux exceeded choline-to-Pcho, and the rate of PtdE synthesis (1.04 micromol/h/g liver) was 2-3 times greater than that of PtdC de novo synthesis. The metabolic pathway information provided by these studies makes the NMR method superior to earlier radioisotope studies.