Altered interactions of tryptophan metabolites in first-episode neuroleptic-naive patients with schizophrenia.

Schizophrenia is characterized by complex and dynamically interacting perturbations in multiple neurochemical systems. In the past, evidence for these alterations has been collected piecemeal, limiting our understanding of the interactions among relevant biological systems. Earlier, both hyper- and hyposerotonemia were variously associated with the longitudinal course of schizophrenia, suggesting a disturbance in the central serotonin (5-hydroxytryptamine (5-HT)) function. Using a targeted electrochemistry-based metabolomics platform, we compared metabolic signatures consisting of 13 plasma tryptophan (Trp) metabolites simultaneously between first-episode neuroleptic-naive patients with schizophrenia (FENNS, n=25) and healthy controls (HC, n=30). We also compared these metabolites between FENNS at baseline (BL) and 4 weeks (4w) after antipsychotic treatment. N-acetylserotonin was increased in FENNS-BL compared with HC (P=0.0077, which remained nearly significant after Bonferroni correction). N-acetylserotonin/Trp and melatonin (Mel)/serotonin ratios were higher, and Mel/N-acetylserotonin ratio was lower in FENNS-BL (all P-values<0.0029), but not after treatment, compared with HC volunteers. All three groups had highly significant correlations between Trp and its metabolites, Mel, kynurenine, 3-hydroxykynurenine and tryptamine. However, in the HC, but in neither of the FENNS groups, serotonin was highly correlated with Trp, Mel, kynurenine or tryptamine, and 5-hydroxyindoleacetic acid (5HIAA) was highly correlated with Trp, Mel, kynurenine or 3-hydroxykynurenine. A significant difference between HC and FENNS-BL was further shown only for the Trp-5HIAA correlation. Thus, some metabolite interactions within the Trp pathway seem to be altered in the FENNS-BL patients. Conversion of serotonin to N-acetylserotonin by serotonin N-acetyltransferase may be upregulated in FENNS patients, possibly related to the observed alteration in Trp-5HIAA correlation. Considering N-acetylserotonin as a potent antioxidant, such increases in N-acetylserotonin might be a compensatory response to increased oxidative stress, implicated in the pathogenesis of schizophrenia.

Concentration and velocity patterns in a horizontal rotating suspension of non-Brownian settling particles.

We report a systematic experimental study of concentration and velocity patterns formed in a horizontal rotating cylinder filled completely with a monodisperse suspension of non-Brownian settling particles. The system shows a series of concentration and velocity patterns, or phases, with varying rotation rate and solvent viscosity. Individual phases are studied using both side and cross-sectional imaging to examine the detailed flow structures. The overall phase diagram of the system is mapped out as a function of the rotation rate and solvent viscosity. Attempts are made to analyze the functional form of the phase boundaries in order to understand the transition mechanism between different phases.

Pattern formation in a rotating suspension of non-Brownian settling particles.

We report band formation and other pattern formation for a settling suspension of uniform non-Brownian particles in a completely filled horizontal rotating cylinder. The system shows a series of sharp pattern changes that are mapped out as a function of the rotation period and suspension viscosity. The experiment suggests that a large number of patterns and rich dynamics result from the interplay among the viscous drag, and gravitational and centrifugal forces.

N(epsilon)-(gamma-L-glutamyl)-L-lysine (GGEL) is increased in cerebrospinal fluid of patients with Huntington's disease.

Pathological-length polyglutamine (Q(n)) expansions, such as those that occur in the huntingtin protein (htt) in Huntington's disease (HD), are excellent substrates for tissue transglutaminase in vitro, and transglutaminase activity is increased in post-mortem HD brain. However, direct evidence for the participation of tissue transglutaminase (or other transglutaminases) in HD patients in vivo is scarce. We now report that levels of N(epsilon)-(gamma-L-glutamyl)-L-lysine (GGEL)--a 'marker' isodipeptide produced by the transglutaminase reaction--are elevated in the CSF of HD patients (708 +/- 41 pmol/mL, SEM, n = 36) vs. control CSF (228 +/- 36, n = 27); p < 0.0001. These data support the hypothesis that transglutaminase activity is increased in HD brain in vivo.

Characterization of diet-dependent metabolic serotypes: analytical and biological variability issues in rats.

This report, the first in a series on diet-dependent changes in the serum metabolome (metabolic serotype), describes validation of the use of high performance liquid chromatography (HPLC) separations coupled with Coulometric array detectors to characterize changes in the metabolome. The long-term aim of these studies is to improve understanding of the effects of significant variation in nutritive status on physiology and on disease processes. Initial studies focus on identifying the effects of dietary (or caloric) restriction on the redox-active components of rat serum. Identification of compounds of interest is being carried out using HPLC separations coupled with coulometric array analysis, an approach allowing simultaneous examination of nearly 1200 serum compounds. The technical and practical issues discussed in this report are related to both analytical validity (HPLC running conditions, computer-automated peak identification, mathematical compensation for chromatographic drift, etc.) and biological variability (individual variability, cohort-cohort variability, outliers). Attention to these issues suggests approximately 250 compounds in serum are sufficiently reliable, both analytically and biologically, for potential use in building mathematical models of serotype.

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.

8-Hydroxy-2'-deoxyguanosine (8OH2'dG) is a principal stable marker of hydroxyl radical damage to DNA. It has been related to a wide variety of disorders and environmental insults, and has been proposed as a useful systematic marker of oxidative stress. Analytic procedures for 8OH2'dG in DNA digests are well established; however, routine measurement of free 8OH2'dG in other body fluids such as urine or plasma has been problematic. This has hindered its evaluation as a general clinical, therapeutic monitoring, or environmental assessment tool. Therefore, we developed a liquid chromatography electrochemical column-switching system based on the use of the unique purine selectivity of porous carbon columns that allows routine accurate measurement of 8OH2'dG in a variety of biologic matrices. This paper describes the rationale of the system design and the protocols developed for 8OH2'dG in urine, plasma, cerebrospinal fluid, tissue, DNA, saliva, sweat, kidney dialysis fluid, foods, feces, culture matrix, and microdialysates. Concentrations in both human and animal body fluids and tissues are reported. The system performance is discussed in the context of a 1-year evaluation of the methods applied to approximately 3600 samples, using internal quality control and external blind testing to determine long-term accuracy. The methods are reliable and accurate, and therefore should prove useful in assessing the role and utility of oxidative DNA damage in aging and human illness.

Purine catabolism: links to mitochondrial respiration and antioxidant defenses?

Type I diabetes in rodents is associated with a spectrum of liver mitochondrial abnormalities ranging from evidence of oxidative stress and altered antioxidant defenses to frank defects in respiration rates and respiratory control ratios. To better address the myriad changes in redox metabolism in these mitochondria, we have applied new chromatographic techniques that enable simultaneous analysis of multiple components of pathways of interest (e.g., purine catabolites and oxidation by-products). We report here a portion of these results, which, in conjunction with other reported data, suggest that purine catabolism may contribute to mitochondrial antioxidant defenses by producing the antioxidant urate. In liver mitochondria from diabetic rats, increases in uric acid (threefold) and its direct precursor xanthine (sixfold) were observed in moderate diabetes, but levels fell essentially to normal in severe disease. Failure to maintain elevated xanthine and uric acid occurred contemporaneously with progressive mitochondrial dysfunction. Regression analysis revealed altered precursor-product relationships between xanthine, its precursors, and uric acid. An independent set of studies in isolated rat liver mitochondria showed that mitochondrial respiration was associated with essentially uniform decreases (approximately 30%) in all purine catabolites measured (urate, xanthine, hypoxanthine, guanine, guanosine, and xanthosine). That result suggests the potential for steady production of urate. Taken together, the two studies raise the possibility that purine catabolism may be a previously unappreciated component of the homeostatic response of mitochondria to oxidant stress and may play a critical role in slowing progressive mitochondrial dysfunction in certain disease states.

Influence of repeated levodopa administration on rabbit striatal serotonin metabolism, and comparison between striatal and CSF alterations.

Parkinson's disease (PD) is characterized by decreased striatal dopamine, but serotonin (5-HT) is also reduced. Because 5-HT decreases following a single levodopa injection, levodopa has been suggested to contribute to PD's serotonergic deficits. However, in a recent study, rat striatal serotonin levels were reported to increase following 15-day levodopa administration. To address this issue, we administered levodopa (50 mg/kg) to rabbits for 5 days, then measured serotonin, its precursors tryptophan and 5-hydroxytryptophan (5-HTP), and its major metabolite 5-hydroxyindole-acetic acid (5-HIAA) in striatum and CSF. Striatal serotonin and tryptophan were unchanged, while 5-HTP and 5-HIAA increased 4- and 7-fold, respectively. CSF 5-HTP and 5-HIAA were also significantly increased. In levodopa-treated animals, 5-HTP concentrations were moderately correlated (r = 0.679) between striatum and CSF, while weak correlations were present between striatal and CSF concentrations of both serotonin and 5-HIAA. These results suggest that repeated levodopa treatment increases striatal serotonin turnover without changing serotonin content. However, levodopa-induced alterations in striatal serotonin metabolism may not be accurately reflected by measurement of serotonin and 5-HIAA in CSF.

Simultaneous analysis of the majority of low-molecular-weight, redox-active compounds from mitochondria.

Studies of the interaction between oxidative stress and mitochondrial dysfunction are complicated by analytical limitations, especially the need to assess multiple parameters in relatively small samples. We have addressed this problem by developing a methodology for the simultaneous analysis of the majority of low-molecular-weight, redox-active compounds from mitochondria using HPLC separations followed by coulometric array detection. The method described should also be applicable for the study of redox-active compounds in other subcellular organelles as well as in intact cells and tissues. The protocol described enables simultaneous measurement of antioxidants (e.g., tocopherols, ascorbate, lipoates, uric acid, and glutathione), markers of oxidative stress (e.g., o-tyrosine, m-tyrosine, nitrotyrosine, dityrosine, glutathione disulfide, and 8-hydroxydeoxyguanosine) as well as other metabolites (e.g., purines and indoles). In all, ca. 600 redox active compounds can be detected, most with a limit of detection of approximately 5 pg on column. Results, including analytical parameters, from a study of liver mitochondria from control and diabetic rats are presented to demonstrate utility of this methodology.

Altered guanosine and guanine concentrations in rabbit striatum following increased dopamine turnover.

The significance of guanine nucleotides and nucleosides in neurodegenerative disorders is suggested by recent reports that these molecules enhance neurite branching and astrocyte proliferation. The objective of this study was to investigate the influence of increased dopamine metabolism, produced by 5-day treatment of rabbits with reserpine (2 mg/kg) or levodopa (LD) (50 mg/kg), on striatal concentrations of guanosine, guanine, and their metabolites. Reserpine treatment decreased striatal guanosine by 41% and increased guanine by 50%, while LD decreased guanosine by 48% (all p < 0.01 vs. vehicle-treated controls). LD also increased guanine by 22% (not statistically significant). Xanthine and uric acid concentrations were unchanged. Because of the neurotrophic properties of guanosine and guanine, changes in striatal concentrations of these purines secondary to increased dopamine (DA) turnover may have relevance for survival of remaining dopaminergic neurons in Parkinson's disease (PD).

A comparative bear model for immobility-induced osteopenia.

The National Institutes of Health (NIH) and the National Aeronautics and Space Administration (NASA) are seeking solutions to the human problem of osteopenia, or immobility-induced bone loss. Bears, during winter dormancy, appear uniquely exempted from the debilitating effects of immobility osteopenia. NIH and ESA, Inc. are creating a large database of metabolic information on human ambulatory and bedrest plasma samples for comparison with metabolic data obtained from bear plasma samples collected in different seasons. The database generated from NASA's HR113 human bedrest study showed a clear difference between plasma samples of ambulatory and immobile subjects through cluster analysis using compounds determined by high performance liquid chromatography with coulometric electrochemical array detection (HPLC-EC). We collected plasma samples from black bears (Ursus americanus) across 4 seasons and from 3 areas and subjected them to similar analysis, with particular attention to compounds that changed significantly in the NASA human study. We found seasonal differences in 28 known compounds and 33 unknown compounds. A final database contained 40 known and 120 unknown peaks that were reliably assayed in all bear and human samples; these were the primary data set for interspecies comparison. Six unidentified compounds changed significantly but differentially in wintering bears and immobile humans. The data are discussed in light of current theories regarding dormancy, starvation, and anabolic metabolism. Work is in progress by ESA Laboratories on a larger database to confirm these findings prior to a chemical isolation and identification effort. This research could lead to new pharmaceuticals or dietary interventions for the treatment of immobility osteopenia.

Markers of dopamine depletion and compensatory response in striatum and cerebrospinal fluid.

Though depletion of CSF homovanillic acid (HVA) concentration has often been regarded as a direct indicator of dopamine (DA) deficiency in Parkinson's Disease (PD), CSF HVA is normal in mildly affected patients. To explore why, we measured DA and its metabolites in striatum and CSF in rabbits receiving reserpine for 5 days. Reserpine, which depletes striatal DA by disrupting vesicular storage of the neurotransmitter, results in a compensatory increase of DA turnover. In response to a 96% depletion of striatal DA, its catabolic intermediates 3,4-dihydroxyphenylacetic acid (DOPAC) and 3-methoxytyramine (3-MT) decreased 64% and 92% in striatum, although the endproduct, HVA, was unchanged. In contrast, CSF concentrations of HVA and DOPAC increased significantly, though 3-MT and levodopa (LD) were unaltered. A 5-fold rise in striatal LD concentration after reserpine-induced DA depletion provided evidence for enhanced DA synthesis. As in PD, the compensatory increase of DA synthesis after reserpine administration confounds the ability of CSF HVA to reflect DA depletion.

Kynurenine pathway abnormalities in Parkinson's disease.

We measured metabolites of tyrosine and tryptophan (TRP) in the frontal cortex, putamen (PT), and pars compacta of the substantia nigra (SN) of control and Parkinson's disease (PD) brain tissues. Dopamine concentrations were significantly decreased in the PT and SN of PD tissue, regardless of L-dopa therapy. However, 3-O-methyldopa (3OMD) concentration showed a significant increase in each region of the PD group treated with L-dopa (PD[+]) as compared with both the control group and the PD group without L-dopa therapy (PD[-]). Therefore, 3OMD concentration appears to be a reliable marker of L-dopa therapy. Serotonin concentration was lower in each region of the PD groups than in the control group. Although the magnitude of decrease was greater in the PD(+) group, there was no statistical significance between the two PD groups. The same patterns of decrease were present in kynurenine (KYN) and kynurenic acid (KYA) concentrations, but the molar ratios of TRP to KYN and KYN to KYA were unchanged among three groups. In contrast, 3-hydroxykynurenine (3OHKY) concentration was increased in the PT PD(-) group and in three regions of the PD(+) group. Since the KYN pathway leads to formation of nicotinamide-adenine dinucleotide (NADH), the present results may be a further indication of a defect in NADH:ubiquinone oxidoreductase (complex I) in mitochondria in PD.

Rett syndrome: stimulation of endogenous biogenic amines.

Transient hypercapnic hyperoxemia was induced in two Rett syndrome children by the administration of a gaseous mixture of 80% O2 and 20% CO2. Time course studies of neurotransmitters and their metabolites showed an immediate and marked increase in central biogenic amine turnover following inhalation of the gas mixture. The increased turnover of biogenic amines was associated with improved clinical changes. This suggests a coupled relationship and provides further support for an etiological role of neurotransmitter dysfunction in Rett syndrome. In a complementary study, elevation of pulmonary CO2 by application of a simple rebreathing device resulted in improvement of abnormal blood gases and elimination of the Cheyne-Stokes-like respiratory pattern of the Rett syndrome. Near normalization of the EEG occurred when a normal respiratory pattern was imposed by means of a respirator. Taken together, these results lead to the preliminary conclusion that cerebral hypoxemia secondary to abnormal respiratory function may contribute to diminished production of biogenic amines in Rett syndrome.

Kynurenic acid concentrations are reduced in Huntington's disease cerebral cortex.

Huntington's disease (HD) is characterized by gradually evolving selective neuronal death. Several lines of evidence suggest that an excitotoxic mechanism may play a role. Tryptophan metabolism leads to production of quinolinic acid, an N-methyl-D-aspartate (NMDA) receptor agonist, and to kynurenic acid, an antagonist at these same receptors. We recently found increased kynurenine to kynurenic acid ratios in HD postmortem putamen and decreased kynurenic acid concentrations in cerebrospinal fluid, consistent with decreased formation of kynurenic acid in HD brain. In the present study we used HPLC with 16 sensor coulometric electrochemical detection to measure kynurenic acid and 18 other electrochemically active compounds in 6 cortical regions, caudate and cerebellum from controls, HD, Alzheimer's disease (AD), and Parkinson's disease (PD) patients. Significant reductions in kynurenic acid concentrations were found in 5 of 6 cortical regions examined. Smaller reductions of kynurenic acid in the caudate, cerebellum and frontal pole were not significant. No significant reductions were found in the AD and PD patients. Both uric acid and glutathionine were significantly reduced in several regions of HD cerebral cortex, which could signify abnormal energy metabolism in HD. Since kynurenic acid is an antagonist of excitatory amino acid receptors, a deficiency could contribute to the pathogenesis of neuronal degeneration in HD.

Neurochemical analysis of focal ischemia in rats.

BACKGROUND AND PURPOSE: Increases in uric acid follow experimental stroke, which may be related to free radical formation by xanthine oxidase. The present study examined the time course of changes in xanthine and uric acid and their relationship to changes in the free radical scavengers glutathione, cysteine, and ascorbic acid. METHODS: Focal ischemia was induced by occluding the middle cerebral artery, followed by transient occlusion of the common carotid arteries for 60 minutes. At varying time points, animals were sacrificed, and ischemic cortex was dissected. Neurochemical measurements were made by high-performance liquid chromatography with 16-sensor electrochemical detection. RESULTS: Marked increases in uric acid were seen at all time points, with a maximal increase at 1 day and a persistent increase lasting up to 21 days. There were smaller reciprocal decreases in xanthine. Glutathione, cysteine, and ascorbic acid showed significant decreases, consistent with the generation of free radicals. Reductions in levels of cysteine and glutathione were significantly correlated with increases in uric acid levels. CONCLUSIONS: These findings confirm marked alterations in purine metabolism following focal ischemia and suggest that xanthine oxidase contributes to the generation of free radicals.

Kynurenine pathway measurements in Huntington's disease striatum: evidence for reduced formation of kynurenic acid.

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.

Simultaneous measurements of tryptophan and its metabolites, kynurenine and serotonin, in the superficial layers of the spinal dorsal horn. A study in normal and arthritic rats.

It has been demonstrated that 5-hydroxytryptamine (5-HT) is not the only neuroactive metabolite of tryptophan (TRP) in the CNS. The presence of kynurenine (KYN) and its metabolites has been reported in the brain of several mammalian species and the neuroactive properties of these compounds are now well established. In the present study, we report the identification of KYN in the superficial layers of the rat spinal dorsal horn. KYN was measured simultaneously with TRP. 5-hydroxytryptophan, 5-HT, 5-hydroxyindoleacetic acid and 5-HT-O-sulfate by means of liquid chromatography with coulometric electrode array detection. The results observed in the normal rat and in an animal model of persistent pain, the arthritic rat, are discussed in view of the hypothesis relating to the involvement of the bulbospinal serotonergic system in pain mechanisms and of the possible participation of KYN and its metabolites in these mechanisms.