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Millions of patients suffer from major depressive disorder (MDD), but many do not respond to selective serotonin reuptake inhibitor (SSRI) therapy. We used a pharmacometabolomics-informed pharmacogenomics research strategy to identify genes associated with metabolites that were related to SSRI response. Specifically, 306 MDD patients were treated with citalopram or escitalopram and blood was drawn at baseline, 4 and 8 weeks for blood drug levels, genome-wide single nucleotide polymorphism (SNP) genotyping and metabolomic analyses. SSRI treatment decreased plasma serotonin concentrations (P

Objective: Huntington’s disease (HD) is a rare neurodegenerative disease caused by the expansion of an N-terminal repeat in the huntingtin protein. The protein is expressed in all cells in the body; hence, peripheral tissues, such as blood, may recapitulate processes in the brain. The plasma metabolome may provide a window into active processes that influence brain health and a unique opportunity to noninvasively identify processes that may contribute to neurodegeneration. Alterations in metabolic pathways in brain have been shown to profoundly impact HD. Therefore, identification and quantification of critical metabolomic perturbations could provide novel biomarkers for disease onset and disease progression. Methods: We analyzed the plasma metabolomic profiles from 52 premanifest (PHD), 102 early symptomatic HD, and 140 healthy controls (NC) using liquid chromatography coupled with a highly sensitive electrochemical detection platform. Results: Alterations in tryptophan, tyrosine, purine, and antioxidant pathways were identified, including many related to energetic and oxidative stress and derived from the gut microbiome. Multivariate statistical modeling demonstrated mutually distinct metabolomic profiles, suggesting that the processes that determine onset were likely distinct from those that determine progression. Gut microbiome-derived metabolites particularly differentiated the PHD metabolome, while the symptomatic HD metabolome was increasingly influenced by metabolites that may reflect mutant huntingtin toxicity and neurodegeneration. Interpretation: Understanding the complex changes in the delicate balance of the metabolome and the gut microbiome in HD, and how they relate to disease onset, progression, and phenotypic variability in HD are critical questions for future research.

We measured the levels of mutant huntingtin (mtHtt) and total huntingtin (tHtt) in blood leukocytes from Prospective Huntington At-Risk Observational Study (PHAROS) subjects at 50% risk of carrying the Huntington disease mutation using a homogeneous time-resolved fluorescence (HTRF) assay to assess its potential as a biomarker.Peripheral blood mononuclear cells from consenting PHAROS subjects were analyzed by HTRF using antibodies that simultaneously measured mtHtt and tHtt. mtHtt levels were normalized to tHtt, double-stranded DNA, or protein and analyzed according to cytosine-adenine-guanine repeat length (CAGn), demographics, predicted time to clinical onset or known time since clinical onset, and available clinical measures.From 363 assayed samples, 342 met quality control standards. Levels of mtHtt and mt/tHtt were higher in 114 subjects with expanded CAG repeats (CAG ≥ 37) compared with 228 subjects with nonexpanded CAG repeats (CAG37) (p0.0001). Analysis of relationships to predicted time to onset or to phenoconversion suggested that the HTRF signal could mark changes during the Huntington disease prodrome or after clinical onset.The HTRF assay can effectively measure mtHtt in multicenter sample sets and may be useful in trials of therapies targeting huntingtin.

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.

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.

Mapping metabolic “signatures” can provide new insights into addictive mechanisms and potentially identify biomarkers and therapeutic targets. We examined the differences in metabolites related to the tyrosine, tryptophan, purine, and oxidative stress pathways between cocaine-dependent subjects and healthy controls. Several of these metabolites serve as biological indices underlying the mechanisms of reinforcement, toxicity, and oxidative stress. Metabolomic analysis was performed in 18 DSM-IV-diagnosed cocaine-dependent individuals with at least 2 weeks of abstinence and ten drug-free controls. Plasma concentrations of 37 known metabolites were analyzed and compared using a liquid chromatography electrochemical array platform. Multivariate analyses were used to study the relationship between severity of drug use [Addiction Severity Index (ASI) scores] and biological measures. Cocaine subjects showed significantly higher levels of n-methylserotonin (p

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.

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.

A novel approach to the parallel coupling of normal-bore high-performance liquid chromatography (LC) with electrochemical-array detection (EC-array) and nanoelectrospray mass spectrometry (MS), based on the use of a nanosplitting interface, is described where both detectors are utilized at their optimal detection mode for parallel configuration. The dual detection platform was shown to maintain full chromatographic integrity with retention times and peak widths at half-height between the EC-array and MS displaying high reproducibility with relative standard deviations of

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.

Objective interpretation of metabolomics data requires understanding both analytical and biological measurement errors. Here we address analytical measurement errors, the sources of these errors, and how this variability can impact metabolomic profiles. Sources considered include room temperature exposure (which could affect sample stability), spiking with authentic standards, the number of study replicates, the overall temporal design of the experimental series, and the complexity of the biological matrix of the samples (individual or pooled sera). The study focused on the analysis of ∼80 rat sera metabolites by HPLC coupled with coulometric array detectors. Time delay and room temperature exposure had minimal effects on the total relative metabolite concentrations and variability (mean: ∼94–98% of control, CVmedian: ±5–7%), but the concentrations of some specific metabolites were significantly altered. Changes observed in the concentrations of specific metabolites ranged as high as ±7-fold, with changes in variability ranging from 0.3% to 68%. Spiked samples demonstrated more complex behavior when allowed to decay over time than did control samples. The spiking of sera and standards with 43 known metabolites increased variability of the apparent concentrations of metabolites up to ∼24% as opposed to ∼3% in pure sera. Increased variability was metabolite-specific. In both pure and spiked sera, ∼80–95% of metabolites had CVs equivalent to standard analytical CVs for these metabolites. Experimental design, number of replicates, and complexity of the biological matrix had comparable effects. These results suggest that, under carefully controlled conditions, these analytical issues are not significant sources of variability relative to biological variation for most metabolites.

Motor neuron diseases (MND) are a heterogeneous group of disorders that includes amyotrophic lateral sclerosis (ALS) and result in death of motor neurons. These diseases may produce characteristic perturbations of the metabolome, the collection of small-molecules (metabolites) present in a cell, tissue, or organism. To test this hypothesis, we used high performance liquid chromatography followed by electrochemical detection to profile blood plasma from 28 patients with MND and 30 healthy controls. Of 317 metabolites, 50 were elevated in MNDpatients and more than 70 were decreased (p < 0.05). Among the compounds elevated, 12 were associated with the drug Riluzole. In a subsequent study of 19 subjects with MND who were not taking Riluzole and 33 healthy control subjects, six compounds were significantly elevated in MND, while the number of compounds with decreased concentration was similar to study 1. Our data also revealed a distinctive signature of highly correlated metabolites in a set of four patients, three of whom had lower motor neuron (LMN) disease. In both datasets we were able to separate MND patients from controls using multivariate regression techniques. These results suggest that metabolomic studies can be used to ascertain metabolic signatures of disease in a non-invasive fashion. Elucidation of the structures of signature molecules in ALS and other forms of MND should provide insight into aberrant biochemical pathways and may provide diagnostic markers and targets for drug design.

Metabolic serotypes sensitive to caloric intake may enable sera metabolomic profiles to validate epidemiological parameters and predict disease risk in humans. This long-range goal is complicated by the lack of known state markers and the requirement for simultaneous monitoring of multiple small changes. Therefore, analytical precision for appropriate high data density studies using HPLC separations coupled with coulometric array detectors was evaluated over a two month period in pooled rat sera samples (previously collected and stored at −80 °C), and in authentic biochemical standards. In sera, mean coefficients of variation (CV) of retention time and ratio accuracy within the established metabolic serotype varied within ±1% and ±3%, respectively. In sets of purified standards, the same parameters fluctuated, correspondently, in ranges of ±0.1% and ±1%. Median CV of the metabolite concentrations were ~13% in standards and ~11–19% in sera, and varied non-monotonically with the analytical system status and experimental design. These parameters were shown to be sufficiently controlled so as not to dominate intra-group biological variability in serum metabolomics studies. Continuation of experimental runs across an analytical breakpoint (column replacement) was associated with disproportionate changes in metabolite concentrations, independent of maintained analytical precision. These changes were sufficient to shift overall profile localization in megavariate projection analyses. We developed a mathematical approach to normalize this break and use partial least squares projection to latent structure discriminant analysis to confirm validity of this normalization approach. This generally applicable mathematical correction helps enable longer term high data density studies by removing a critical source of systemic variation.

Mitochondrial toxicity of nucleoside analogues has been proposed to be the etiology of a range of side-effects from antiretroviral therapy of HIV infection. In this study, urinary 8-hydroxy-2'-deoxyguanosine (8OH2'dG), a metabolite of oxidized DNA, was measured to determine if antiretroviral therapy leads to oxidative damage to DNA. A cross-sectional study was carried out measuring urinary 8OH2'dG in three groups of HIV-infected patients: (1) antiretroviral medication naïve, (2) patients on antiretroviral medications without lipodystrophy and (3) patients on antiretroviral medications with lipodystrophy. Twenty-five patients were enrolled in each group. The mean spot urinary 8OH2'dG measurements per mg creatinine for the three groups were: antiretroviral naïve 4.27 +/- 0.61 (ng 8OH2'dG/mg creatinine +/- SEM), on antiretroviral medications without lipodystrophy 2.88 +/- 0.26, and on antiretroviral medications with lipodystrophy 3.27 +/- 0.30. The differences between the means of the three groups is not statistically significant (p = 0.055), and these results are not significantly different from reported values for healthy controls [A carbon column-based liquid chromatography electrochemical approach to routine 8-hydroxy-2-deoxyguanosine measurements in urine and other biologic matrices: a one-year evaluation of methods. Free Radical Biology and Medicine 27 (1999) 647-666].

Our research seeks to identify a serum profile, or serotype, that reflects substantial changes in food intake. Earlier studies demonstrated that a number of low-molecular-weight, redox-active compounds of metabolome were sufficiently stable analytically and biologically to identify biomarkers of dietary restriction (DR, restriction of total food intake) in rats. A second initial requirement is to demonstrate feasibility, i.e., that concentration changes in selected serum metabolites can contain sufficient information to classify rats by diet. The current study distinguished 101 (female) and 112 (male) chromatographically identifiable compounds that differ between ad libitum (AL) consumption and DR 6-mo-old rats. In a cohort of female rats, both hierarchical cluster analysis (HCA) and principal component analyses (PCA) could distinguish dietary groups with 100% efficiency (101 metabolites). Repeating the classification studies using the 63 biologically and analytically most robust metabolites decreased noise without affecting categorical separation. In a cohort of male rats, PCA, but not HCA, distinguished the original dietary groups with 100% accuracy (112 metabolites). A subset of 52 of the 112 metabolites enabled both HCA and PCA to group the male rats with 100% accuracy. These data demonstrate that quantitative analysis of selected serum metabolites can yield sufficient information by which to classify the dietary intake of a group of rats, identify such markers chromatographically and set the stage for validation of these metabolic serotypes in independent datasets.

Schizophrenia (SZ) is a biochemically complex disorder characterized by widespread defects in multiple metabolic pathways whose dynamic interactions, until recently, have been difficult to examine. Rather, evidence for these alterations has been collected piecemeal, limiting the potential to inform our understanding of the interactions amongst relevant biochemical pathways. We herein review perturbations in purine and neurotransmitter metabolism observed in early SZ using a metabolomic approach. Purine catabolism is an underappreciated, but important component of the homeostatic response of mitochondria to oxidant stress. We have observed a homeostatic imbalance of purine catabolism in first-episode neuroleptic-naïve patients with SZ (FENNS). Precursor and product relationships within purine pathways are tightly correlated. Although some of these correlations persist across disease or medication status, others appear to be lost among FENNS suggesting that steady formation of the antioxidant uric acid (UA) via purine catabolism is altered early in the course of illness. As is the case for within-pathway correlations, there are also significant cross-pathway correlations between respective purine and tryptophan (TRP) pathway metabolites. By contrast, purine metabolites show significant cross-pathway correlation only with tyrosine, and not with its metabolites. Furthermore, several purine metabolites (UA, guanosine, or xanthine) are each significantly correlated with 5-hydroxyindoleacetic acid (5-HIAA) in healthy controls, but not in FENNS at baseline or 4-week after antipsychotic treatment. Taken together, the above findings suggest that purine catabolism strongly associates with the TRP pathways leading to serotonin (5-hydroxytryptamine, 5-HT) and kynurenine metabolites. The lack of a significant correlation between purine metabolites and 5-HIAA, suggests alterations in key 5-HT pathways that may both be modified by and contribute to oxidative stress via purine catabolism in FENNS.

Objective More must be learned about metabolic and biochemical alterations that contribute to the development and expression of drug dependence. Experimental opioid administration influences mechanisms and indices of oxidative stress, such as antioxidant compounds and purine metabolism. We examined perturbations of neurotransmitter-related pathways in opioid dependence (OD). Methods In this preliminary study, we used a targeted metabolomics platform to explore whether biochemical changes were associated with OD by comparing OD individuals (n = 14) and non-drug users (n = 10). Results OD patients undergoing short-term methadone detoxification showed altered oxidation–reduction activity, as confirmed by higher plasma levels of α- and γ- tocopherol and increased GSH/GSSG ratio. OD individuals had also altered purine metabolism, showing increased concentration of guanine and xanthosine, with decreased guanosine, hypoxanthine and hypoxanthine/xanthine and xanthine/xanthosine ratios. Other drug use in addition to opioids was associated with partly different biochemical changes. Conclusions This is a preliminary investigation using metabolomics and showing multiple peripheral alterations of metabolic pathways in OD. Further studies should explore the metabolic profile of conditions of opioid abuse, withdrawal and long-term abstinence in relation to agonist and antagonist treatment and investigate biochemical signatures of opioid substances and medications. Copyright © 2009 John Wiley & Sons, Ltd.

Recent evidence suggests that there may be overactivation of the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptors in Huntington's disease (HD). Tryptophan metabolism by the kynurenine pathway produces both quinolinic acid, an NMDA receptor agonist, and kynurenic acid, an NMDA receptor antagonist. In the present study, multiple components of the tyrosine and tryptophan metabolic pathways were quantified in postmortem putamen of 35 control and 30 HD patients, using HPLC with 16-sensor electrochemical detection. Consistent with previous reports in HD putamen, there were significant increases in 5-hydroxyindoleacetic acid, 5-hydroxytryptophan, and serotonin concentrations. Within the kynurenine pathway, the ratio of kynurenine to kynurenic acid was significantly (p less than 0.01) increased twofold in HD patients as compared with controls, consistent with reduced formation of kynurenic acid in HD. CSF concentrations of kynurenic acid were significantly reduced in HD patients as compared with controls and patients with other neurologic diseases. Because kynurenic acid is an endogenous inhibitor of excitatory neurotransmission and can block excitotoxic degeneration in vivo, a relative deficiency of this compound could directly contribute to neuronal degeneration in HD.

We used two analytic methods (a multichannel coulometric electrode array with high-performance liquid chromatography, and gas chromatography-mass spectrophotometry) to measure CSF dopamine (DA) and its metabolites in mildly affected, unmedicated subjects with Parkinson's disease (PD). The mean (+/- SD) concentration of homovanillic acid (HVA), the most abundant product of DA turnover, was 164.57 +/- 95.05 nM. As sequential aliquots of CSF were collected from the first to 23rd ml, CSF HVA concentration almost doubled. After HVA, 3-O-methyldopa (3-O-MD) was the next most abundant compound. The summed concentrations of 3-O-MD, 3,4-dihydroxyphenylacetic acid, 3-methoxytyramine, DA, DA-3-sulfate, homovanillol, and levodopa (LD) amounted to 12.6% of HVA. Concentrations of the DA metabolites did not correlate to a variety of indices of PD severity. The presence of LD and 3-O-MD may be indicators of DA synthesis and possibly could reflect compensatory processes among surviving dopaminergic neurons of the PD brain.

This paper presents results of a survey of mercury concentrations in coal, ash, water, fly ash, and flue gas discharges from a 5.5 × 106 Ib/hr steam generator serving a 775 MW (net) turbine-generator set. Representative composite or grab samples were obtained for inlet coal and outlet ash and water. Stack samples were obtained for fly ash and mercury vapor emissions while the unit was operated at 660 MW (net) (85% of full load). Samples were analyzed by anodic stripping voltammetry, plasma emission spectroscopy, and neutron activation analysis to determine mercury concentration entering the furnace in the coal and leaving the furnace in the flue gas, fly ash, bottom and hopper ash, and water. Method inter-comparisons are discussed. A material balance for mercury has been calculated from fuel, ash, and stack gas flow rates. About 90% of the mercury in the coal is released and appears as vapor discharged in the stack gas while 10% remains in the residual ash. For a 700 MW (net) unit, about 5 lb/day of mercu...

Aerosol particles in Cambridge, Massachusetts, were collected by cascade impactor and analyzed for chlorine, bromine, and iodine by neutron activation and for lead electrochemically. The Casella four-stage impactor selected particles of radius greater than about 0.2–0.5 μ, and two sampling sites were chosen on the M.I.T. campus—on the roof of the 90-meter-high Green Center for Earth Sciences and at the curb by a busy street. The ranges 1 to 6 μg Cl/m3 STP and 2 to 10 ng I/m3 STP are in good agreement with observations in unpolluted Hawaii air, but the range 20 to 800 ng Br/m3 STP stands an order of magnitude higher and agrees with observations in Washington, D.C., polluted air. Lead concentrations are 0.1 to 2.2 μg Pb/m3 STP and are strongly correlated with high Br concentrations during hazy weather (low visibility) and/or curb-site sampling. The data suggest that the initial exhaust product is a particulate lead halide which suffers loss of Br in the atmosphere.