Sex and race differences of cerebrospinal fluid metabolites in healthy individuals.

INTRODUCTION: Analyses of cerebrospinal fluid (CSF) metabolites in large, healthy samples have been limited and potential demographic moderators of brain metabolism are largely unknown. OBJECTIVE: Our objective in this study was to examine sex and race differences in 33 CSF metabolites within a sample of 129 healthy individuals (37 African American women, 29 white women, 38 African American men, and 25 white men). METHODS: CSF metabolites were measured with a targeted electrochemistry-based metabolomics platform. Sex and race differences were quantified with both univariate and multivariate analyses. Type I error was controlled for by using a Bonferroni adjustment (0.05/33 = .0015). RESULTS: Multivariate Canonical Variate Analysis (CVA) of the 33 metabolites showed correct classification of sex at an average rate of 80.6% and correct classification of race at an average rate of 88.4%. Univariate analyses revealed that men had significantly higher concentrations of cysteine (p < 0.0001), uric acid (p < 0.0001), and N-acetylserotonin (p = 0.049), while women had significantly higher concentrations of 5-hydroxyindoleacetic acid (5-HIAA) (p = 0.001). African American participants had significantly higher concentrations of 3-hydroxykynurenine (p = 0.018), while white participants had significantly higher concentrations of kynurenine (p < 0.0001), indoleacetic acid (p < 0.0001), xanthine (p = 0.001), alpha-tocopherol (p = 0.007), cysteine (p = 0.029), melatonin (p = 0.036), and 7-methylxanthine (p = 0.037). After the Bonferroni adjustment, the effects for cysteine, uric acid, and 5-HIAA were still significant from the analysis of sex differences and kynurenine and indoleacetic acid were still significant from the analysis of race differences. CONCLUSION: Several of the metabolites assayed in this study have been associated with mental health disorders and neurological diseases. Our data provide some novel information regarding normal variations by sex and race in CSF metabolite levels within the tryptophan, tyrosine and purine pathways, which may help to enhance our understanding of mechanisms underlying sex and race differences and potentially prove useful in the future treatment of disease.

Associations between central nervous system serotonin, fasting glucose, and hostility in African American females.

BACKGROUND: Previous research has shown an association between hostility and fasting glucose in African American women. Central nervous system serotonin activity is implicated both in metabolic processes and in hostility related traits. PURPOSE: The purpose of this study is to determine whether central nervous system serotonin influences the association between hostility and fasting glucose in African American women. METHODS: The study consisted of 119 healthy volunteers (36 African American women, 27 White women, 21 White males, and 35 African American males, mean age 34 ± 8.5 years). Serotonin related compounds were measured in cerebrospinal fluid. Hostility was measured by the Cook-Medley Hostility Scale. RESULTS: Hostility was associated with fasting glucose and central nervous system serotonin related compounds in African American women only. Controlling for the serotonin related compounds significantly reduced the association of hostility to glucose. CONCLUSIONS: The positive correlation between hostility and fasting glucose in African American women can partly be explained by central nervous system serotonin function.

Identification of metabolites from liquid chromatography-coulometric array detection profiling: gas chromatography-mass spectrometry and refractionation provide essential information orthogonal to LC-MS/microNMR.

Liquid chromatography-coulometric array detection (LC-EC) is a sensitive, quantitative, and robust metabolomics profiling tool that complements the commonly used mass spectrometry (MS) and nuclear magnetic resonance (NMR)-based approaches. However, LC-EC provides little structural information. We recently demonstrated a workflow for the structural characterization of metabolites detected by LC-EC profiling combined with LC-electrospray ionization (ESI)-MS and microNMR. This methodology is now extended to include (i) gas chromatography (GC)-electron ionization (EI)-MS analysis to fill structural gaps left by LC-ESI-MS and NMR and (ii) secondary fractionation of LC-collected fractions containing multiple coeluting analytes. GC-EI-MS spectra have more informative fragment ions that are reproducible for database searches. Secondary fractionation provides enhanced metabolite characterization by reducing spectral overlap in NMR and ion suppression in LC-ESI-MS. The need for these additional methods in the analysis of the broad chemical classes and concentration ranges found in plasma is illustrated with discussion of four specific examples: (i) characterization of compounds for which one or more of the detectors is insensitive (e.g., positional isomers in LC-MS, the direct detection of carboxylic groups and sulfonic groups in (1)H NMR, or nonvolatile species in GC-MS), (ii) detection of labile compounds, (iii) resolution of closely eluting and/or coeluting compounds, and (iv) the capability to harness structural similarities common in many biologically related, LC-EC-detectable compounds.

Transcriptional modulator H2A histone family, member Y (H2AFY) marks Huntington disease activity in man and mouse.

Huntington disease (HD) is a progressive neurodegenerative disease that affects 30,000 individuals in North America. Treatments that slow its relentless course are not yet available, and biomarkers that can reliably measure disease activity and therapeutic response are urgently needed to facilitate their development. Here, we interrogated 119 human blood samples for transcripts associated with HD. We found that the dynamic regulator of chromatin plasticity H2A histone family, member Y (H2AFY) is specifically overexpressed in the blood and frontal cortex of patients with HD compared with controls. This association precedes the onset of clinical symptoms, was confirmed in two mouse models, and was independently replicated in cross-sectional and longitudinal clinical studies comprising 142 participants. A histone deacetylase inhibitor that suppresses neurodegeneration in animal models reduces H2AFY levels in a randomized phase II clinical trial. This study identifies the chromatin regulator H2AFY as a potential biomarker associated with disease activity and pharmacodynamic response that may become useful for enabling disease-modifying therapeutics for HD.

8OHdG as a marker for Huntington disease progression.

Leukocyte 8-hydroxydeoxyguanosine (8OHdG) is an indicator of oxidative stress, impaired metabolism, and mitochondrial dysfunction, features that have been implicated in Huntington disease (HD). Increased levels of 8OHdG have been reported in the caudate, parietal cortex, and peripherally in the serum and leukocytes, in patients diagnosed with HD. However, little is known about levels in prodromal patients and changes that might occur as the disease progresses. To address these issues, 8OHdG was tracked over time for a subset of participants enrolled in the PREDICT-HD study. Participants were stratified into four groups based on proximity to HD diagnosis at study entry: Controls (gene-negative individuals), Low (low probability of near-future diagnosis), Medium, and High. Blood samples were analyzed using Liquid Chromatography Electrochemical Array, and for comparison purposes, a separate cross-sectional sample was analyzed using liquid chromatography coupled with multiple-reaction-monitoring mass spectrometry. Longitudinal data analysis showed that initial status (at study entry) and annual rate of change varied as a function of proximity group, adjusting for sex, education, age at study entry, and site effects. Overall levels were lowest for the Control group and highest for the High group, and the rate of increase varied in a similar manner. The finding that 8OHdG concentrations increased as a function of proximity to projected disease diagnosis and duration indicates support for the continued assessment of 8OHdG as a robust clinical HD biomarker.

Structural characterization of plasma metabolites detected via LC-electrochemical coulometric array using LC-UV fractionation, MS, and NMR.

Liquid chromatography (LC) separation combined with electrochemical coulometric array detection (EC) is a sensitive, reproducible, and robust technique that can detect hundreds of redox-active metabolites down to the level of femtograms on column, making it ideal for metabolomics profiling. EC detection cannot, however, structurally characterize unknown metabolites that comprise these profiles. Several aspects of LC-EC methods prevent a direct transfer to other structurally informative analytical methods, such as LC-MS and NMR. These include system limits of detection, buffer requirements, and detection mechanisms. To address these limitations, we developed a workflow based on the concentration of plasma, metabolite extraction, and offline LC-UV fractionation. Pooled human plasma was used to provide sufficient material necessary for multiple sample concentrations and platform analyses. Offline parallel LC-EC and LC-MS methods were established that correlated standard metabolites between the LC-EC profiling method and the mass spectrometer. Peak retention times (RT) from the LC-MS and LC-EC system were linearly related (r(2) = 0.99); thus, LC-MS RTs could be directly predicted from the LC-EC signals. Subsequent offline microcoil-NMR analysis of these collected fractions was used to confirm LC-MS characterizations by providing complementary, structural data. This work provides a validated workflow that is transferrable across multiple platforms and provides the unambiguous structural identifications necessary to move primary mathematically driven LC-EC biomarker discovery into biological and clinical utility.

Reduced creatine kinase as a central and peripheral biomarker in Huntington's disease.

A major goal of current clinical research in Huntington's disease (HD) has been to identify preclinical and manifest disease biomarkers, as these may improve both diagnosis and the power for therapeutic trials. Although the underlying biochemical alterations and the mechanisms of neuronal degeneration remain unknown, energy metabolism defects in HD have been chronicled for many years. We report that the brain isoenzyme of creatine kinase (CK-BB), an enzyme important in buffering energy stores, was significantly reduced in presymptomatic and manifest disease in brain and blood buffy coat specimens in HD mice and HD patients. Brain CK-BB levels were significantly reduced in R6/2 mice by approximately 18% to approximately 68% from 21 to 91 days of age, while blood CK-BB levels were decreased by approximately 14% to approximately 44% during the same disease duration. Similar findings in CK-BB levels were observed in the 140 CAG mice from 4 to 12 months of age, but not at the earliest time point, 2 months of age. Consistent with the HD mice, there was a grade-dependent loss of brain CK-BB that worsened with disease severity in HD patients from approximately 28% to approximately 63%, as compared to non-diseased control patients. In addition, CK-BB blood buffy coat levels were significantly reduced in both premanifest and symptomatic HD patients by approximately 23% and approximately 39%, respectively. The correlation of CK-BB as a disease biomarker in both CNS and peripheral tissues from HD mice and HD patients may provide a powerful means to assess disease progression and to predict the potential magnitude of therapeutic benefit in this disorder.

3-Hydroxykynurenine and clinical symptoms in first-episode neuroleptic-naive patients with schizophrenia.

One branch of the tryptophan catabolic cascade is the kynurenine pathway, which produces neurotoxic [3-hydroxykynurenine (3-OHKY), quinolinic acid] and neuroinhibitory (kynurenic acid) compounds. Kynurenic acid acts as a competitive antagonist at the glycine site of N-methyl-d-asparate receptors at high concentrations and as a non-competitive antagonist on the α7-nicotinic acetylcholine receptor at low concentrations. Kynurenine compounds also influence cognitive functions known to be disrupted in schizophrenia. Alterations in tryptophan metabolism are therefore of potential significance for the pathophysiology of this disorder. In this paper, tryptophan metabolites were measured from plasma using high-pressure liquid chromatography coupled with electrochemical coulometric array detection, and relationships were tested between these metabolic signatures and clinical symptoms for 25 first-episode neuroleptic-naive schizophrenia patients. Blood samples were collected and clinical and neurological symptoms were rated at baseline and again at 4 wk following initiation of treatment. Level of 3-OHKY and total clinical symptom scores were correlated when patients were unmedicated and neuroleptic-naive, and this relationship differed significantly from the correlation observed for patients 4 wk after beginning treatment. Baseline psychosis symptoms were predicted only by neurological symptoms. Moreover, baseline 3-OHKY predicted clinical change at 4 wk, with the lowest concentrations of 3-OHKY being associated with the greatest improvement in symptoms. Taken together, our findings suggest a neurotoxic product of tryptophan metabolism, 3-OHKY, predicts severity of clinical symptoms during the early phase of illness and before exposure to antipsychotic drugs. Baseline level of 3-OHKY may also predict the degree of clinical improvement following brief treatment with antipsychotics.

Identification of phenylbutyrate-generated metabolites in Huntington disease patients using parallel liquid chromatography/electrochemical array/mass spectrometry and off-line tandem mass spectrometry.

Oral sodium phenylbutyrate (SPB) is currently under investigation as a histone deacetylation (HDAC) inhibitor in Huntington disease (HD). Ongoing studies indicate that symptoms related to HD genetic abnormalities decrease with SPB therapy. In a recently reported safety and tolerability study of SPB in HD, we analyzed overall chromatographic patterns from a method that employs gradient liquid chromatography with series electrochemical array, ultraviolet (UV), and fluorescence (LCECA/UV/F) for measuring SPB and its metabolite phenylacetate (PA). We found that plasma and urine from SPB-treated patients yielded individual-specific patterns of approximately 20 metabolites that may provide a means for the selection of subjects for extended trials of SPB. The structural identification of these metabolites is of critical importance because their characterization will facilitate understanding the mechanisms of drug action and possible side effects. We have now developed an iterative process with LCECA, parallel LCECA/LCMS, and high-performance tandem MS for metabolite characterization. Here we report the details of this method and its use for identification of 10 plasma and urinary metabolites in treated subjects, including indole species in urine that are not themselves metabolites of SPB. Thus, this approach contributes to understanding metabolic pathways that differ among HD patients being treated with SPB.

Therapeutic attenuation of mitochondrial dysfunction and oxidative stress in neurotoxin models of Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder for which there is no current therapy preventing cumulative neuronal loss. There is substantial evidence that mitochondrial dysfunction, oxidative stress, and associated caspase activity underlie the neurodegeneration observed. One potential drug therapy is the potent free radical scavenger and antioxidant cystamine, which has demonstrated significant clinical potential in models of neurodegenerative disorders and human neurological disease. This study examined the oral efficacy of cystamine in the MPTP and 6-hydroxydopamine neurotoxin models of PD. The neuroprotective effects of cystamine treatment significantly ameliorated nigral neuronal loss, preserved striatal dopaminergic projections, and improved striatal dopamine and metabolite levels, as compared to MPTP alone. Cystamine normalized striatal 8-hydroxy-2'-deoxyguanosine levels and ATP concentrations, consistent with reduced oxidative stress and improved mitochondrial function. Cystamine also protected against MPTP-induced mitochondrial loss, as identified by mitochondrial heat shock protein 70 and superoxide dismutase 2, with concomitant reductions in cytochrome c and caspase-3 activities. The neuroprotective value of cystamine was confirmed in the 6-hydroxydopamine model. Together these findings show cystamine's therapeutic benefit to reduce neuronal loss through attenuation of oxidative stress and mitochondrial dysfunction, providing the rationale for human clinical trials in PD patients.

Phase 2 study of sodium phenylbutyrate in ALS.

The objective of the study was to establish the safety and pharmacodynamics of escalating dosages of sodium phenylbutyrate (NaPB) in participants with ALS. Transcription dysregulation may play a role in the pathogenesis of ALS. Sodium phenylbutyrate, a histone deacetylase inhibitor, improves transcription and post-transcriptional pathways, promoting cell survival in a mouse model of motor neuron disease. Forty research participants at eight sites enrolled in an open-label study. Study medication was increased from 9 to 21 g/day. The primary outcome measure was tolerability. Secondary outcome measures included adverse events, blood histone acetylation levels, and NaPB blood levels at each dosage. Twenty-six participants completed the 20-week treatment phase. NaPB was safe and tolerable. No study deaths or clinically relevant laboratory changes occurred with NaPB treatment. Histone acetylation was decreased by approximately 50% in blood buffy-coat specimens at screening and was significantly increased after NaPB administration. Blood levels of NaPB and the primary metabolite, phenylacetate, increased with dosage. While the majority of subjects tolerated higher dosages of NaPB, the lowest dose (9 g/day), was therapeutically efficient in improving histone acetylation levels.

Metabolomic profiling to develop blood biomarkers for Parkinson's disease.

The development of biomarkers for the diagnosis and monitoring disease progression in Parkinson's disease (PD) is of great importance since diagnosis based on clinical parameters has a considerable error rate. In this study, we utilized metabolomic profiling using high performance liquid chromatography coupled with electrochemical coulometric array detection (LCECA) to look for biomarkers in plasma useful for the diagnosis of PD. We examined 25 controls and 66 PD patients. We also measured 8-hydroxy-2-deoxyguanosine (8-OHdG) levels as a marker of oxidative damage to DNA. We initially examined the profiles of unmedicated PD subjects compared to controls to rule out confounding effects of symptomatic medications. We found a complete separation of the two groups. We then determined the variables, which played the greatest role in separating the two groups and applied them to PD subjects taking dopaminergic medications. Using these parameters, we achieved a complete separation of the PD patients from controls. 8-OHdG levels were significantly increased in PD patients, but overlapped controls. Two other markers of oxidative damage were measured in our LCECA profiles. Uric acid was significantly reduced while glutathione was significantly increased in PD patients. These findings show that metabolomic profiling with LCECA coulometric array has great promise for developing biomarkers for both the diagnosis, as well as monitoring disease progression in PD.

Neuroprotective effects of synaptic modulation in Huntington's disease R6/2 mice.

Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disorder in which the neostriatum degenerates early and most severely, with involvement of other brain regions. There is significant evidence that excitotoxicity may play a role in striatal degeneration through altered afferent corticostriatal and nigrostriatal projections that may modulate synaptically released striatal glutamate. Glutamate is a central tenant in provoking excitotoxic cell death in striatal neurons already weakened by the collective molecular events occurring in HD. In addition, transcriptional suppression of trophic factors occurs in human and transgenic mouse models of HD, suggesting that a loss of trophic support might contribute to degeneration. Since anti-glutamate approaches have been effective in improving disease phenotype in HD mice, we examined whether deafferentation of the corticostriatal and nigrostriatal pathways may mitigate striatal stress and neurodegeneration. Both surgical and chemical lesions of the corticostriatal and nigrostriatal pathways, respectively, improved the behavioral, neuropathological, and biochemical phenotype in R6/2 transgenic mice and extended survival. Decortication ameliorated hindlimb clasping, striatal neuron atrophy, and huntingtin-positive aggregates, improved N-acetyl aspartate/creatine levels, reduced oxidative stress, and significantly lowered striatal glutamate levels. In addition, 6-hydroxydopamine lesioned mice showed extended survival along with a significant reduction in striatal glutamate. These results suggest that synaptic stress is likely to contribute to neurodegeneration in HD, whereas transsynaptic trophic influences may not be as salient. Thus, modulation of synaptic influences continues to have therapeutic potential in HD.

Increased levels of gamma-glutamylamines in Huntington disease CSF.

Transglutaminases (TGases) catalyze several reactions with protein substrates, including formation of gamma-glutamyl-epsilon-lysine cross-links and gamma-glutamylpolyamine residues. The resulting gamma-glutamylamines are excised intact during proteolysis. TGase activity is altered in several diseases, highlighting the importance of in situ enzymatic determinations. Previous work showed that TGase activity (as measured by an in vitro assay) and free gamma-glutamyl-epsilon-lysine levels are elevated in Huntington disease (HD) and that gamma-glutamyl-epsilon-lysine is increased in HD CSF. Although free gamma-glutamyl-epsilon-lysine was used in these studies as an index of in situ TGase activity, gamma-glutamylpolyamines may also be diagnostic. We have devised methods for the simultaneous determination of four gamma-glutamylamines in CSF: gamma-glutamyl-epsilon-lysine, gamma-glutamylspermidine, gamma-glutamylputrescine, and bis-gamma-glutamylputrescine and showed that all are present in normal human CSF at concentrations of approximately 150, 670, 40, and 240 nM, respectively. The high gamma-glutamylspermidine/gamma-glutamylputrescine and gamma-glutamylspermidine/bis-gamma-glutamylputrescine ratios presumably reflect in part the large spermidine to putrescine mole ratio in human brain. We also showed that all four gamma-glutamylamines are elevated in HD CSF. Our findings support the hypotheses that (i) gamma-glutamylpolyamines are reflective of TGase activity in human brain, (ii) polyamination is an important post-translational modification of brain proteins, and (iii) TGase-catalyzed modification of proteins is increased in HD brain.

Beta-defensin 1, aryl hydrocarbon receptor and plasma kynurenine in major depressive disorder: metabolomics-informed genomics.

Major depressive disorder (MDD) is a heterogeneous disease. Efforts to identify biomarkers for sub-classifying MDD and antidepressant therapy by genome-wide association studies (GWAS) alone have generally yielded disappointing results. We applied a metabolomics-informed genomic research strategy to study the contribution of genetic variation to MDD pathophysiology by assaying 31 metabolites, including compounds from the tryptophan, tyrosine, and purine pathways, in plasma samples from 290 MDD patients. Associations of metabolite concentrations with depressive symptoms were determined, followed by GWAS for selected metabolites and functional validation studies of the genes identified. Kynurenine (KYN), the baseline plasma metabolite that was most highly associated with depressive symptoms, was negatively correlated with severity of those symptoms. GWAS for baseline plasma KYN concentrations identified SNPs across the beta-defensin 1 (DEFB1) and aryl hydrocarbon receptor (AHR) genes that were cis-expression quantitative trait loci (eQTLs) for DEFB1 and AHR mRNA expression, respectively. Furthermore, the DEFB1 locus was associated with severity of MDD symptoms in a larger cohort of 803 MDD patients. Functional studies demonstrated that DEFB1 could neutralize lipopolysaccharide-stimulated expression of KYN-biosynthesizing enzymes in monocytic cells, resulting in altered KYN concentrations in the culture media. In addition, we demonstrated that AHR was involved in regulating the expression of enzymes in the KYN pathway and altered KYN biosynthesis in cell lines of hepatocyte and astrocyte origin. In conclusion, these studies identified SNPs that were cis-eQTLs for DEFB1 and AHR and, which were associated with variation in plasma KYN concentrations that were related to severity of MDD symptoms.

Dose ranging and efficacy study of high-dose coenzyme Q10 formulations in Huntington's disease mice.

There is substantial evidence that a bioenergetic defect may play a role in the pathogenesis of Huntington's Disease (HD). A potential therapy for remediating defective energy metabolism is the mitochondrial cofactor, coenzyme Q10 (CoQ10). We have reported that CoQ10 is neuroprotective in the R6/2 transgenic mouse model of HD. Based upon the encouraging results of the CARE-HD trial and recent evidence that high-dose CoQ10 slows the progressive functional decline in Parkinson's disease, we performed a dose ranging study administering high levels of CoQ10 from two commercial sources in R6/2 mice to determine enhanced efficacy. High dose CoQ10 significantly extended survival in R6/2 mice, the degree of which was dose- and source-dependent. CoQ10 resulted in a marked improvement in motor performance and grip strength, with a reduction in weight loss, brain atrophy, and huntingtin inclusions in treated R6/2 mice. Brain levels of CoQ10 and CoQ9 were significantly lower in R6/2 mice, in comparison to wild type littermate control mice. Oral administration of CoQ10 elevated CoQ10 plasma levels and significantly increased brain levels of CoQ9, CoQ10, and ATP in R6/2 mice, while reducing 8-hydroxy-2-deoxyguanosine concentrations, a marker of oxidative damage. We demonstrate that high-dose administration of CoQ10 exerts a greater therapeutic benefit in a dose dependent manner in R6/2 mice than previously reported and suggest that clinical trials using high dose CoQ10 in HD patients are warranted.

High-performance liquid chromatography separations coupled with coulometric electrode array detectors: a unique approach to metabolomics.

Metabolomics is the systematic and theoretically comprehensive study of the small molecules that comprise a biological sample, e.g., sera or plasma. The primary analytical tools used in metabolomics are nuclear magnetic resonance and mass spectroscopy. We here address a different tool, high-performance liquid chromatography (HPLC) separations coupled with coulometric electrode array detection. This system has unique advantages, notably sensitivity and high quantitative precision, but also has unique limitations, such as obtaining little structural information on the metabolites of interest and limited scale-up capacity. The system also only detects redox-active compounds, which can be either a benefit or a detriment, depending on the experimental goals and design. Here, we discuss the characteristics of this HPLC/coulometric electrode array system in the context of metabolomics, and then present the method as practiced in our groups.

Metabolomics in the study of aging and caloric restriction.

Metabolomics is the systematic and theoretically comprehensive study of the small molecules that comprise a biological sample, e.g., sera or plasma. Metabolomics, in conjunction with other "-omics" approaches, offers a new window onto the study of aging and caloric restriction. Here, we present the methodology that we are using, high-performance liquid chromatography separations coupled with coulometric electrode array detection, to probe the metabolome of aging and caloric-restricted animals. This system has unique advantages, notably sensitivity and high quantitative precision, but also has unique disadvantages, such as the ability to obtain little structural information on the metabolites of interest and limited scale-up capacity. The system also only detects redox-active compounds, which can be either a benefit or a detriment, depending on the experimental goals and design.

Biological variability dominates and influences analytical variance in HPLC-ECD studies of the human plasma metabolome.

BACKGROUND: Biomarker-based assessments of biological samples are widespread in clinical, pre-clinical, and epidemiological investigations. We previously developed serum metabolomic profiles assessed by HPLC-separations coupled with coulometric array detection that can accurately identify ad libitum fed and caloric-restricted rats. These profiles are being adapted for human epidemiology studies, given the importance of energy balance in human disease. METHODS: Human plasma samples were biochemically analyzed using HPLC separations coupled with coulometric electrode array detection. RESULTS: We identified these markers/metabolites in human plasma, and then used them to determine which human samples represent blinded duplicates with 100% accuracy (N = 30 of 30). At least 47 of 61 metabolites tested were sufficiently stable for use even after 48 hours of exposure to shipping conditions. Stability of some metabolites differed between individuals (N = 10 at 0, 24, and 48 hours), suggesting the influence of some biological factors on parameters normally considered as analytical. CONCLUSION: Overall analytical precision (mean median CV, ~9%) and total between-person variation (median CV, ~50-70%) appear well suited to enable use of metabolomics markers in human clinical trials and epidemiological studies, including studies of the effect of caloric intake and balance on long-term cancer risk.