Kynurenine 3-monooxygenase inhibition in blood ameliorates neurodegeneration.

Metabolites in the kynurenine pathway, generated by tryptophan degradation, are thought to play an important role in neurodegenerative disorders, including Alzheimer's and Huntington's diseases. In these disorders, glutamate receptor-mediated excitotoxicity and free radical formation have been correlated with decreased levels of the neuroprotective metabolite kynurenic acid. Here, we describe the synthesis and characterization of JM6, a small-molecule prodrug inhibitor of kynurenine 3-monooxygenase (KMO). Chronic oral administration of JM6 inhibits KMO in the blood, increasing kynurenic acid levels and reducing extracellular glutamate in the brain. In a transgenic mouse model of Alzheimer's disease, JM6 prevents spatial memory deficits, anxiety-related behavior, and synaptic loss. JM6 also extends life span, prevents synaptic loss, and decreases microglial activation in a mouse model of Huntington's disease. These findings support a critical link between tryptophan metabolism in the blood and neurodegeneration, and they provide a foundation for treatment of neurodegenerative diseases.

The Probiotic Lactobacillus reuteri Preferentially Synthesizes Kynurenic Acid from Kynurenine.

The gut-brain axis is increasingly understood to play a role in neuropsychiatric disorders. The probiotic bacterium Lactobacillus (L.) reuteri and products of tryptophan degradation, specifically the neuroactive kynurenine pathway (KP) metabolite kynurenic acid (KYNA), have received special attention in this context. We, therefore, assessed relevant features of KP metabolism, namely, the cellular uptake of the pivotal metabolite kynurenine and its conversion to its primary products KYNA, 3-hydroxykynurenine and anthranilic acid in L. reuteri by incubating the bacteria in Hank's Balanced Salt solution in vitro. Kynurenine readily entered the bacterial cells and was preferentially converted to KYNA, which was promptly released into the extracellular milieu. De novo production of KYNA increased linearly with increasing concentrations of kynurenine (up to 1 mM) and bacteria (107 to 109 CFU/mL) and with incubation time (1-3 h). KYNA neosynthesis was blocked by two selective inhibitors of mammalian kynurenine aminotransferase II (PF-048559989 and BFF-122). In contrast to mammals, however, kynurenine uptake was not influenced by other substrates of the mammalian large neutral amino acid transporter, and KYNA production was not affected by the presumed competitive enzyme substrates (glutamine and α-aminoadipate). Taken together, these results reveal substantive qualitative differences between bacterial and mammalian KP metabolism.

A single prenatal lipopolysaccharide injection has acute, but not long-lasting, effects on cerebral kynurenine pathway metabolism in mice.

In rodents, a single injection of lipopolysaccharide (LPS) during gestation causes chemical and functional abnormalities in the offspring. These effects may involve changes in the kynurenine pathway (KP) of tryptophan degradation and may provide insights into the pathophysiology of psychiatric diseases. Using CD1 mice, we examined acute and long-term effects of prenatal LPS treatment on the levels of kynurenine and its neuroactive downstream products kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK) and quinolinic acid. To this end, LPS (100 µg/kg, i.p.) was administered on gestational day 15, and KP metabolites were measured 4 and 24 h later or in adulthood. After 4 h, kynurenine, KYNA and 3-HK levels were elevated in the fetal brain, 3-HK and KYNA levels were increased in the maternal plasma, and kynurenine was increased in the maternal brain, whereas no changes were seen in the placenta. These effects were less prominent after 24 h, and prenatal LPS did not affect the basal levels of KP metabolites in the forebrain of adult animals. In addition, a second LPS injection (1 mg/kg) in adulthood in the offspring of prenatally saline- and LPS-treated mice caused a similar elevation in 3-HK levels in both groups after 24 h, but the effect was significantly more pronounced in male mice. Thus, acute immune activation during pregnancy has only short-lasting effects on KP metabolism and does not cause cerebral KP metabolites to be disproportionally affected by a second immune challenge in adulthood. However, prenatal KYNA elevations still contribute to functional abnormalities in the offspring.

Assessment of Prenatal Kynurenine Metabolism Using Tissue Slices: Focus on the Neosynthesis of Kynurenic Acid in Mice.

Several lines of evidence support the hypothesis that abnormally elevated brain levels of kynurenic acid (KYNA), a metabolite of the kynurenine pathway (KP) of tryptophan degradation, play a pathophysiologically significant role in schizophrenia and other major neurodevelopmental disorders. Studies in experimental animal models suggest that KP impairments in these diseases may originate already in utero since prenatal administration of KYNA's bioprecursor, kynurenine, leads to biochemical and structural abnormalities as well as distinct cognitive impairments in adulthood. As KP metabolism during pregnancy is still insufficiently understood, we designed this study to examine the de novo synthesis of KYNA and 3-hydroxykynurenine (3-HK), an alternative biologically active product of kynurenine degradation, in tissue slices obtained from pregnant mice on gestational day (GD) 18. Fetal brain and liver, placenta, and maternal brain and liver were collected, and the tissues were incubated in vitroin the absence or presence of micromolar concentrations of kynurenine. KYNA and 3-HK were measured in the extracellular milieu. Basal and newly produced KYNA was detected in all cases. As KYNA formation exceeded 3-HK production by 2-3 orders of magnitude in the placenta and maternal brain, and as very little 3-HK neosynthesis was detectable in fetal brain tissue, detailed follow-up experiments focused on KYNA only. The fetal brain produced 3-4 times more KYNA than the maternal brain and placenta, though less than the maternal and fetal liver. No significant differences were observed when using tissues obtained on GD 14 and GD 18. Pharmacological inhibition of KYNA's main biosynthetic enzymes, kynurenine aminotransferase (KAT) I and KAT II, revealed qualitative and quantitative differences between the tissues, with a preferential role of KAT I in the fetal and maternal brain and of KAT II in the fetal and maternal liver. Findings using tissue slices from KAT II knockout mice confirmed these conclusions. Together, these results clarify the dynamics of KP metabolism during pregnancy and provide the basis for the conceptualization of interventions aimed at manipulating cerebral KP function in the prenatal period.

Tryptophan-2,3-dioxygenase (TDO) inhibition ameliorates neurodegeneration by modulation of kynurenine pathway metabolites.

Metabolites of the kynurenine pathway (KP) of tryptophan (TRP) degradation have been closely linked to the pathogenesis of several neurodegenerative disorders. Recent work has highlighted the therapeutic potential of inhibiting two critical regulatory enzymes in this pathway-kynurenine-3-monooxygenase (KMO) and tryptophan-2,3-dioxygenase (TDO). Much evidence indicates that the efficacy of KMO inhibition arises from normalizing an imbalance between neurotoxic [3-hydroxykynurenine (3-HK); quinolinic acid (QUIN)] and neuroprotective [kynurenic acid (KYNA)] KP metabolites. However, it is not clear if TDO inhibition is protective via a similar mechanism or if this is instead due to increased levels of TRP-the substrate of TDO. Here, we find that increased levels of KYNA relative to 3-HK are likely central to the protection conferred by TDO inhibition in a fruit fly model of Huntington's disease and that TRP treatment strongly reduces neurodegeneration by shifting KP flux toward KYNA synthesis. In fly models of Alzheimer's and Parkinson's disease, we provide genetic evidence that inhibition of TDO or KMO improves locomotor performance and ameliorates shortened life span, as well as reducing neurodegeneration in Alzheimer's model flies. Critically, we find that treatment with a chemical TDO inhibitor is robustly protective in these models. Consequently, our work strongly supports targeting of the KP as a potential treatment strategy for several major neurodegenerative disorders and suggests that alterations in the levels of neuroactive KP metabolites could underlie several therapeutic benefits.

Key Enabling Technologies for Point-of-Care Diagnostics.

A major trend in biomedical engineering is the development of reliable, self-contained point-of-care (POC) devices for diagnostics and in-field assays. The new generation of such platforms increasingly addresses the clinical and environmental needs. Moreover, they are becoming more and more integrated with everyday objects, such as smartphones, and their spread among unskilled common people, has the power to improve the quality of life, both in the developed world and in low-resource settings. The future success of these tools will depend on the integration of the relevant key enabling technologies on an industrial scale (microfluidics with microelectronics, highly sensitive detection methods and low-cost materials for easy-to-use tools). Here, recent advances and perspectives will be reviewed across the large spectrum of their applications.

Tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase 1 make separate, tissue-specific contributions to basal and inflammation-induced kynurenine pathway metabolism in mice.

BACKGROUND: In mammals, the majority of the essential amino acid tryptophan is degraded via the kynurenine pathway (KP). Several KP metabolites play distinct physiological roles, often linked to immune system functions, and may also be causally involved in human diseases including neurodegenerative disorders, schizophrenia and cancer. Pharmacological manipulation of the KP has therefore become an active area of drug development. To target the pathway effectively, it is important to understand how specific KP enzymes control levels of the bioactive metabolites in vivo. METHODS: Here, we conducted a comprehensive biochemical characterization of mice with a targeted deletion of either tryptophan 2,3-dioxygenase (TDO) or indoleamine 2,3-dioxygenase (IDO), the two initial rate-limiting enzymes of the KP. These enzymes catalyze the same reaction, but differ in biochemical characteristics and expression patterns. We measured KP metabolite levels and enzyme activities and expression in several tissues in basal and immune-stimulated conditions. RESULTS AND CONCLUSIONS: Although our study revealed several unexpected downstream effects on KP metabolism in both knockout mice, the results were essentially consistent with TDO-mediated control of basal KP metabolism and a role of IDO in phenomena involving stimulation of the immune system.

Prenatal Dynamics of Kynurenine Pathway Metabolism in Mice: Focus on Kynurenic Acid.

The kynurenine pathway (KP), the major catabolic route of tryptophan in mammals, contains several neuroactive metabolites, including kynurenic acid (KYNA) and 3-hydroxykynurenine (3-HK). KP metabolism, and especially the fate of KYNA, during pregnancy is poorly understood, yet it may play a significant role in the development of psychiatric disorders later in life. The present study was designed to investigate the prenatal features of KP metabolism in vivo, with special focus on KYNA. To this end, pregnant CD-1 mice were treated systemically with kynurenine (100 mg/kg), KYNA (10 mg/kg), or saline on embryonic day 18. As expected, administration of either kynurenine or KYNA increased KYNA levels in the maternal plasma and placenta. Maternal kynurenine treatment also raised kynurenine levels in the fetal plasma and brain, demonstrating the ability of this pivotal KP metabolite to cross the placenta and increase the levels of both KYNA and 3-HK in the fetal brain. In contrast, maternal administration of KYNA caused only a small, nonsignificant elevation in KYNA levels in fetal plasma and brain. Complementary experiments using an ex vivo placental perfusion procedure confirmed the significant transplacental transfer of kynurenine and demonstrated that only a very small fraction of maternal kynurenine is converted to KYNA in the placenta and released into the fetal compartment under physiological conditions. Jointly, these results help to clarify the contributions of the maternal circulation and the placenta to fetal KYNA in the late prenatal period.

Role of d-amino acid oxidase in the production of kynurenine pathway metabolites from d-tryptophan in mice.

The kynurenine pathway (KP), the major catabolic route of the essential amino acid l-tryptophan (l-TRP), contains several neuroactive compounds, including kynurenic acid, 3-hydroxykynurenine (3-HK), and quinolinic acid (QUIN). The role of the d-enantiomer (d-TRP) in KP metabolism has received little attention so far. d-TRP can be converted to l-TRP by d-amino acid oxidase, and the same enzyme can produce d-kynurenine, a known bioprecursor of KYNA. To analyze these complex metabolic events systematically in vivo, we injected mice with d-TRP (300 mg/kg, i.p.) and examined KP metabolism in the absence or presence of the d-amino acid oxidase inhibitor 3-methylpyrazole-5-carboxylic acid (MPC; 100 mg/kg, i.p.,). After 90 min, newly formed l-TRP was recovered in plasma, liver, forebrain, and cerebellum, and MPC prevented its neosynthesis in all tissues. In the same animals, de novo production of d-kynurenine from d-TRP was also observed, but was much higher in the periphery than in the brain. d-TRP administration raised KYNA, 3-HK, and QUIN levels in all tissues examined, and KYNA production from d-TRP was significantly reduced after pre-treatment with MPC. These results indicate that catabolic routes other than those classically ascribed to l-TRP and l-kynurenine can account for the synthesis of KYNA, 3-HK and QUINin vivo. The essential amino acid l-tryptophan is catabolized via the kynurenine pathway (KP). We explored the role of the d-enantiomer in KP metabolism in mice in vivo. We report that d-tryptophan is metabolized in both brain and periphery and converted to KP metabolites, including d-kynurenine and l-kynurenine, kynurenic acid, 3-hydroxykynurenine, and quinolinic acid. Pharmacological experiments confirm the involvement of d-amino acid oxidase in these processes. Our results indicate that this enzyme participates in the synthesis of KP metabolites from d-tryptophan.

Restraint Stress during Pregnancy Rapidly Raises Kynurenic Acid Levels in Mouse Placenta and Fetal Brain.

Stressful events during pregnancy adversely affect brain development and may increase the risk of psychiatric disorders later in life. Early changes in the kynurenine (KYN) pathway (KP) of tryptophan (TRP) degradation, which contains several neuroactive metabolites, including kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), and quinolinic acid (QUIN), may constitute a molecular link between prenatal stress and delayed pathological consequences. To begin testing this hypothesis experimentally, we examined the effects of a 2-h restraint stress on KP metabolism in pregnant FVB/N mice on gestational day 17. TRP, KYN, KYNA, 3-HK, and QUIN levels were measured in maternal and fetal plasma and brain, as well as in the placenta, immediately after stress termination and 2 h later. In the same animals, we determined the activity of TRP 2,3-dioxygenase (TDO) in the maternal liver and in the placenta. Compared to unstressed controls, mostly transient changes in KP metabolism were observed in all of the tissues examined. Specifically, stress caused significant elevations of KYNA levels in the maternal plasma, placenta, and fetal brain, and also resulted in increased levels of TRP and KYN in the placenta, fetal plasma, and fetal brain. In contrast, 3-HK and QUIN levels remained unchanged from control values in all tissues at any time point. In the maternal liver, TDO activity was increased 2 h after stress cessation. Taken together, these findings indicate that an acute stress during the late gestational period preferentially affects the KYNA branch of KP metabolism in the fetal brain. Possible long-term consequences for postnatal brain development and pathology remain to be examined.

Reduced kynurenine pathway metabolism and cytokine expression in the prefrontal cortex of depressed individuals.

BACKGROUND: Neuroinflammatory processes are increasingly believed to participate in the pathophysiology of a number of major psychiatric diseases, including depression. Immune activation stimulates the conversion of the amino acid tryptophan to kynurenine, leading to the formation of neuroactive metabolites, such as quinolinic acid and kynurenic acid. These compounds affect glutamatergic neurotransmission, which plays a prominent role in depressive pathology. Increased tryptophan degradation along the kynurenine pathway (KP) has been proposed to contribute to disease etiology. METHODS: We used postmortem brain tissue from the ventrolateral prefrontal cortex (VLPFC) to assess tissue levels of tryptophan and KP metabolites, the expression of several KP enzymes and a series of cytokines as well as tissue pathology, including microglial activation. Tissue samples came from nonpsychiatric controls (n = 36) and individuals with depressive disorder not otherwise specified (DD-NOS, n = 45) who died of natural causes, homicide, accident, or suicide. RESULTS: We found a reduction in the enzymatic conversion of tryptophan to kynurenine, determined using the kynurenine:tryptophan ratio, and reduced messenger RNA expression of the enzymes indoleamine-2,3-dioxygenase 1 and 2 and tryptophan-2,3-dioxygenase in depressed individuals irrespective of the cause of death. These findings correlated with reductions in the expression of several cytokines, including interferon-γ and tumour necrosis factor-α. Notably, quinolinic acid levels were also lower in depressed individuals than controls. LIMITATIONS: Information on the use of antidepressants and other psychotropic medications was insufficient for statistical comparisons. CONCLUSION: Contrary to expectations, the present results indicate that depression, in the absence of medical illness or an overt inflammatory process, is associated with compromised, rather than increased, KP metabolism in the VLPFC.

Quinolinic acid/tryptophan ratios predict neurological disease in SIV-infected macaques and remain elevated in the brain under cART.

Activation of the kynurenine pathway (KP) of tryptophan catabolism likely contributes to HIV-associated neurological disorders. However, KP activation in brain tissue during HIV infection has been understudied, and the effect of combination antiretroviral therapy (cART) on KP induction in the brain is unknown. To examine these questions, tryptophan, kynurenine, 3-hydroxykynurenine, quinolinic acid, and serotonin levels were measured longitudinally during SIV infection in the striatum and CSF from untreated and cART-treated pigtailed macaques. Messenger RNA (mRNA) levels of KP enzymes also were measured in the striatum. In untreated macaques, elevations in KP metabolites coincided with transcriptional induction of upstream enzymes in the KP. Striatal KP induction was also temporally associated-but did not directly correlate-with serotonin losses in the brain. CSF quinolinic acid/tryptophan ratios were found to be the earliest predictor of neurological disease in untreated SIV-infected macaques, outperforming other KP metabolites as well as the putative biomarkers interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1). Finally, cART did not restore KP metabolites to control levels in the striatum despite the control of the virus, though CSF metabolite levels were normalized in most animals. Overall, these results demonstrate that cerebral KP activation is only partially resolved with cART and that CSF QUIN/TRP ratios are an early, predictive biomarker of CNS disease.

Targeted deletion of kynurenine 3-monooxygenase in mice: a new tool for studying kynurenine pathway metabolism in periphery and brain.

Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the kynurenine pathway (KP) of tryptophan degradation, has been suggested to play a major role in physiological and pathological events involving bioactive KP metabolites. To explore this role in greater detail, we generated mice with a targeted genetic disruption of Kmo and present here the first biochemical and neurochemical characterization of these mutant animals. Kmo(-/-) mice lacked KMO activity but showed no obvious abnormalities in the activity of four additional KP enzymes tested. As expected, Kmo(-/-) mice showed substantial reductions in the levels of its enzymatic product, 3-hydroxykynurenine, in liver, brain, and plasma. Compared with wild-type animals, the levels of the downstream metabolite quinolinic acid were also greatly decreased in liver and plasma of the mutant mice but surprisingly were only slightly reduced (by ∼20%) in the brain. The levels of three other KP metabolites: kynurenine, kynurenic acid, and anthranilic acid, were substantially, but differentially, elevated in the liver, brain, and plasma of Kmo(-/-) mice, whereas the liver and brain content of the major end product of the enzymatic cascade, NAD(+), did not differ between Kmo(-/-) and wild-type animals. When assessed by in vivo microdialysis, extracellular kynurenic acid levels were found to be significantly elevated in the brains of Kmo(-/-) mice. Taken together, these results provide further evidence that KMO plays a key regulatory role in the KP and indicate that Kmo(-/-) mice will be useful for studying tissue-specific functions of individual KP metabolites in health and disease.

A method for the determination of D-kynurenine in biological tissues.

D-kynurenine (D-KYN), a metabolite of D-tryptophan, can serve as the bioprecursor of kynurenic acid (KYNA) and 3-hydroxykynurenine, two neuroactive compounds that are believed to play a role in the pathophysiology of several neurological and psychiatric diseases. In order to investigate the possible presence of D-KYN in biological tissues, we developed a novel assay based on the conversion of D-KYN to KYNA by purified D-amino acid oxidase (D-AAO). Samples were incubated with D-AAO under optimal conditions for measuring D-AAO activity (100 mM borate buffer, pH 9.0), and newly produced KYNA was detected by high-performance liquid chromatography (HPLC) with fluorimetric detection. The detection limit for D-KYN was 300 fmol, and linearity of the assay was ascertained up to 300 pmol. No assay interference was noted when other D-amino acids, including D-serine and D-aspartate, were present in the incubation mixture at 50-fold higher concentrations than D-KYN. Using this new method, D-KYN was readily detected in the brain, liver, and plasma of mice treated systemically with D-KYN (300 mg/kg). In these experiments, enantioselectivity was confirmed by determining total kynurenine levels in the same samples using a conventional HPLC assay. Availability of a sensitive, specific, and simple method for D-KYN measurement will be instrumental for evaluating whether D-KYN should be considered for a role in physiology and pathology.

Deletion of quinolinate phosphoribosyltransferase gene accelerates frailty phenotypes and neuromuscular decline with aging in a sex-specific pattern.

Decline in neuromuscular function with aging is known to be a major determinant of disability and all-cause mortality in late life. Despite the importance of the problem, the neurobiology of age-associated muscle weakness is poorly understood. In a previous report, we performed untargeted metabolomics on frail older adults and discovered prominent alteration in the kynurenine pathway, the major route of dietary tryptophan degradation that produces neurotoxic intermediate metabolites. We also showed that neurotoxic kynurenine pathway metabolites are correlated with increased frailty score. For the present study, we sought to further examine the neurobiology of these neurotoxic intermediates by utilizing a mouse model that has a deletion of the quinolinate phosphoribosyltransferase (QPRT) gene, a rate-limiting step of the kynurenine pathway. QPRT-/- mice have elevated neurotoxic quinolinic acid level in the nervous system throughout their lifespan. We found that QPRT-/- mice have accelerated declines in neuromuscular function in an age- and sex-specific manner compared to control strains. In addition, the QPRT-/- mice show premature signs of frailty and body composition changes that are typical for metabolic syndrome. Our findings suggest that the kynurenine pathway may play an important role in frailty and age-associated muscle weakness.

Tryptophan challenge in individuals with schizophrenia and healthy controls: acute effects on circulating kynurenine and kynurenic acid, cognition and cerebral blood flow.

Cognitive impairments predict poor functional outcomes in people with schizophrenia. These impairments may be causally related to increased levels of kynurenic acid (KYNA), a major metabolic product of tryptophan (TRYP). In the brain, KYNA acts as an antagonist of the of α7-nicotinic acetylcholine and NMDA receptors, both of which are involved in cognitive processes. To examine whether KYNA plays a role in the pathophysiology of schizophrenia, we compared the acute effects of a single oral dose of TRYP (6 g) in 32 healthy controls (HC) and 37 people with either schizophrenia (Sz), schizoaffective or schizophreniform disorder, in a placebo-controlled, randomized crossover study. We examined plasma levels of KYNA and its precursor kynurenine; selected cognitive measures from the MATRICS Consensus Cognitive Battery; and resting cerebral blood flow (CBF) using arterial spin labeling imaging. In both cohorts, the TRYP challenge produced significant, time-dependent elevations in plasma kynurenine and KYNA. The resting CBF signal (averaged across all gray matter) was affected differentially, such that TRYP was associated with higher CBF in HC, but not in participants with a Sz-related disorder. While TRYP did not significantly impair cognitive test performance, there was a trend for TRYP to worsen visuospatial memory task performance in HC. Our results demonstrate that oral TRYP challenge substantially increases plasma levels of kynurenine and KYNA in both groups, but exerts differential group effects on CBF. Future studies are required to investigate the mechanisms underlying these CBF findings, and to evaluate the impact of KYNA fluctuations on brain function and behavior. (Clinicaltrials.gov: NCT02067975).

Enzymatic transamination of D-kynurenine generates kynurenic acid in rat and human brain.

In the mammalian brain, the α7 nicotinic and NMDA receptor antagonist kynurenic acid is synthesized by irreversible enzymatic transamination of the tryptophan metabolite l-kynurenine. d-kynurenine, too, serves as a bioprecursor of kynurenic acid in several organs including the brain, but the conversion is reportedly catalyzed through oxidative deamination by d-amino acid oxidase. Using brain and liver tissue homogenates from rats and humans, and conventional incubation conditions for kynurenine aminotransferases, we show here that kynurenic acid production from d-kynurenine, like the more efficient kynurenic acid synthesis from l-kynurenine, is blocked by the aminotransferase inhibitor amino-oxyacetic acid. In vivo, focal application of 100 µM d-kynurenine by reverse microdialysis led to a steady rise in extracellular kynurenic acid in the rat striatum, causing a 4-fold elevation after 2 h. Attesting to functional significance, this increase was accompanied by a 36% reduction in extracellular dopamine. Both of these effects were duplicated by perfusion of 2 µM l-kynurenine. Co-infusion of amino-oxyacetic acid (2 mM) significantly attenuated the in vivo effects of d-kynurenine and essentially eliminated the effects of l-kynurenine. Thus, enzymatic transamination accounts in part for kynurenic acid synthesis from d-kynurenine in the brain. These results are discussed with regard to implications for brain physiology and pathology.

Gas chromatography/tandem mass spectrometry detection of extracellular kynurenine and related metabolites in normal and lesioned rat brain.

We describe here a gas chromatography-tandem mass spectrometry (GC/MS/MS) method for the sensitive and concurrent determination of extracellular tryptophan and the kynurenine pathway metabolites kynurenine, 3-hydroxykynurenine (3-HK), and quinolinic acid (QUIN) in rat brain. This metabolic cascade is increasingly linked to the pathophysiology of several neurological and psychiatric diseases. Methodological refinements, including optimization of MS conditions and the addition of deuterated standards, resulted in assay linearity to the low nanomolar range. Measured in samples obtained by striatal microdialysis in vivo, basal levels of tryptophan, kynurenine, and QUIN were 415, 89, and 8 nM, respectively, but 3-HK levels were below the limit of detection (<2 nM). Systemic injection of kynurenine (100 mg/kg, i.p.) did not affect extracellular tryptophan but produced detectable levels of extracellular 3-HK (peak after 2-3 h: ~50 nM) and raised extracellular QUIN levels (peak after 2h: ~105 nM). The effect of this treatment on QUIN, but not on 3-HK, was potentiated in the N-methyl-D-aspartate (NMDA)-lesioned striatum. Our results indicate that the novel methodology, which allowed the measurement of extracellular kynurenine and 3-HK in the brain in vivo, will facilitate studies of brain kynurenines and of the interplay between peripheral and central kynurenine pathway functions under physiological and pathological conditions.

Tryptophan Challenge in Healthy Controls and People with Schizophrenia: Acute Effects on Plasma Levels of Kynurenine, Kynurenic Acid and 5-Hydroxyindoleacetic Acid.

The pivotal tryptophan (TRP) metabolite kynurenine is converted to several neuroactive compounds, including kynurenic acid (KYNA), which is elevated in the brain and cerebrospinal fluid of people with schizophrenia (SZ) and may contribute to cognitive abnormalities in patients. A small proportion of TRP is metabolized to serotonin and further to 5-hydroxyindoleacetic acid (5-HIAA). Notably, KP metabolism is readily affected by immune stimulation. Here, we assessed the acute effects of an oral TRP challenge (6 g) on peripheral concentrations of kynurenine, KYNA and 5-HIAA, as well as the cytokines interferon-γ, TNF-α and interleukin-6, in 22 participants with SZ and 16 healthy controls (HCs) using a double-blind, placebo-controlled, crossover design. TRP raised the levels of kynurenine, KYNA and 5-HIAA in a time-dependent manner, causing >20-fold, >130-fold and 1.5-fold increases in kynurenine, KYNA and 5-HIAA concentrations, respectively, after 240 min. According to multivariate analyses, neither baseline levels nor the stimulating effects of TRP differed between participants with SZ and HC. Basal cytokine levels did not vary between groups, and remained unaffected by TRP. Although unlikely to be useful diagnostically, measurements of circulating metabolites following an acute TRP challenge may be informative for assessing the in vivo efficacy of drugs that modulate the neosynthesis of KYNA and other products of TRP degradation.