Metabolomic changes in adults with status epilepticus: A human case-control study.

OBJECTIVE: Status epilepticus (SE) is a life-threatening prolonged epileptic seizure that affects ~40 per 100 000 people yearly worldwide. The persistence of seizures may lead to excitotoxic processes, neuronal loss, and neuroinflammation, resulting in long-term neurocognitive and functional disabilities. A better understanding of the pathophysiological mechanisms underlying SE consequences is crucial for improving SE management and preventing secondary neuronal injury. METHODS: We conducted a comprehensive untargeted metabolomic analysis, using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS), on plasma and cerebrospinal fluid (CSF) samples from 78 adult patients with SE and 107 control patients without SE, including 29 with CSF for both groups. The metabolomic fingerprints were compared between patients with SE and controls. Metabolites with differences in relative abundances that could not be attributed to treatment or nutrition provided in the intensive care unit were isolated. Enrichment analysis was performed on these metabolites to identify the most affected pathways. RESULTS: We identified 76 metabolites in the plasma and 37 in the CSF that exhibited differential expression in patients with SE compared to controls. The enrichment analysis revealed that metabolic dysregulations in patients with SE affected primarily amino acid metabolism (including glutamate, alanine, tryptophan, glycine, and serine metabolism), pyrimidine metabolism, and lipid homeostasis. Specifically, patients with SE had elevated levels of pyruvate, quinolinic acid, and keto butyric acid levels, along with lower levels of arginine, N-acetylaspartylglutamate (NAAG), tryptophan, uracil, and uridine. The tryptophan kynurenine pathway was identified as the most significantly altered in SE, resulting in the overproduction of quinolinic acid, an N-methyl-d-aspartate (NMDA) receptor agonist with pro-inflammatory properties. SIGNIFICANCE: This study has identified several pathways that may play pivotal roles in SE consequences, such as the tryptophan kynurenine pathway. These findings offer novel perspectives for the development of neuroprotective therapeutics.

Relation of the kynurenine pathway with normal age: A systematic review.

BACKGROUND: The kynurenine pathway (KP) is gaining more attention as a common pathway involved in age-related conditions. However, which changes in the KP occur due to normal ageing is still largely unclear. The aim of this systematic review was to summarize the available evidence for associations of KP metabolites with age. METHODS: We used an broad search strategy and included studies up to October 2023. RESULTS: Out of 8795 hits, 55 studies were eligible for the systematic review. These studies suggest that blood levels of tryptophan decrease with age, while blood and cerebrospinal fluid levels of kynurenine and its ratio with tryptophan increase. Studies investigating associations between cerebrospinal fluid and blood levels of kynurenic acid and quinolinic acid with age reported either positive or non-significant findings. However, there is a large heterogeneity across studies. Additionally, most studies were cross-sectional, and only few studies investigated associations with other downstream kynurenines. CONCLUSIONS: This systematic review suggests that levels of kynurenines are positively associated with age. Larger and prospective studies are needed that also investigate a more comprehensive panel of KP metabolites and changes during the life-course.

Validation of LC-MS/MS methods for quantitative analysis of kynurenine pathway metabolites in human plasma and cerebrospinal fluid.

Background: Dysregulation of the kynurenine metabolic pathway has been reported in several neurological conditions. Methods & results: Sensitive and selective LC-MS/MS methods have been validated for six kynurenine pathway metabolites in human cerebrospinal fluid and plasma. For each matrix, we validated three methods - one for the simultaneous determination of kynurenine, kynurenic acid, anthranilic acid and 3-hydroxy-kynurenine (four-analyte assay), one for quinolinic acid and one for tryptophan - using stable-isotopically labeled internal standards. The dynamic range and quantitation limits were based on endogenous concentrations for each analyte. Conclusion: The use of validated methods for kynurenine pathway metabolites in human cerebrospinal fluid and plasma will provide definitive information in neurological diseases.

CSF neopterin, quinolinic acid and kynurenine/tryptophan ratio are biomarkers of active neuroinflammation.

BACKGROUND: Defining the presence of acute and chronic brain inflammation remains a challenge to clinicians due to the heterogeneity of clinical presentations and aetiologies. However, defining the presence of neuroinflammation, and monitoring the effects of therapy is important given its reversible and potentially damaging nature. We investigated the utility of CSF metabolites in the diagnosis of primary neuroinflammatory disorders such as encephalitis and explored the potential pathogenic role of inflammation in epilepsy. METHODS: Cerebrospinal fluid (CSF) collected from 341 paediatric patients (169 males, median age 5.8 years, range 0.1-17.1) were examined. The patients were separated into a primary inflammatory disorder group (n = 90) and epilepsy group (n = 80), who were compared with three control groups including neurogenetic and structural (n = 76), neurodevelopmental disorders, psychiatric and functional neurological disorders (n = 63), and headache (n = 32). FINDINGS: There were statistically significant increases of CSF neopterin, kynurenine, quinolinic acid and kynurenine/tryptophan ratio (KYN/TRP) in the inflammation group compared to all control groups (all p < 0.0003). As biomarkers, at thresholds with 95% specificity, CSF neopterin had the best sensitivity for defining neuroinflammation (82%, CI 73-89), then quinolinic acid (57%, CI 47-67), KYN/TRP ratio (47%, CI 36-56) and kynurenine (37%, CI 28-48). CSF pleocytosis had sensitivity of 53%, CI 42-64). The area under the receiver operating characteristic curve (ROC AUC) of CSF neopterin (94.4% CI 91.0-97.7%) was superior to that of CSF pleocytosis (84.9% CI 79.5-90.4%) (p = 0.005). CSF kynurenic acid/kynurenine ratio (KYNA/KYN) was statistically decreased in the epilepsy group compared to all control groups (all p ≤ 0.0003), which was evident in most epilepsy subgroups. INTERPRETATION: Here we show that CSF neopterin, kynurenine, quinolinic acid and KYN/TRP are useful diagnostic and monitoring biomarkers of neuroinflammation. These findings provide biological insights into the role of inflammatory metabolism in neurological disorders and provide diagnostic and therapeutic opportunities for improved management of neurological diseases. FUNDING: Financial support for the study was granted by Dale NHMRC Investigator grant APP1193648, University of Sydney, Petre Foundation, Cerebral Palsy Alliance and Department of Biochemistry at the Children's Hospital at Westmead. Prof Guillemin is funded by NHMRC Investigator grant APP 1176660 and Macquarie University.

Cerebrospinal fluid quinolinic acid is strongly associated with delirium and mortality in hip-fracture patients.

BACKGROUNDThe kynurenine pathway (KP) has been identified as a potential mediator linking acute illness to cognitive dysfunction by generating neuroactive metabolites in response to inflammation. Delirium (acute confusion) is a common complication of acute illness and is associated with increased risk of dementia and mortality. However, the molecular mechanisms underlying delirium, particularly in relation to the KP, remain elusive.METHODSWe undertook a multicenter observational study with 586 hospitalized patients (248 with delirium) and investigated associations between delirium and KP metabolites measured in cerebrospinal fluid (CSF) and serum by targeted metabolomics. We also explored associations between KP metabolites and markers of neuronal damage and 1-year mortality.RESULTSIn delirium, we found concentrations of the neurotoxic metabolite quinolinic acid in CSF (CSF-QA) (OR 2.26 [1.78, 2.87], P < 0.001) to be increased and also found increases in several other KP metabolites in serum and CSF. In addition, CSF-QA was associated with the neuronal damage marker neurofilament light chain (NfL) (ß 0.43, P < 0.001) and was a strong predictor of 1-year mortality (HR 4.35 [2.93, 6.45] for CSF-QA ≥ 100 nmol/L, P < 0.001). The associations between CSF-QA and delirium, neuronal damage, and mortality remained highly significant following adjustment for confounders and multiple comparisons.CONCLUSIONOur data identified how systemic inflammation, neurotoxicity, and delirium are strongly linked via the KP and should inform future delirium prevention and treatment clinical trials that target enzymes of the KP.FUNDINGNorwegian Health Association and South-Eastern Norway Regional Health Authorities.

Decreased cerebrospinal fluid kynurenic acid in epileptic spasms: A biomarker of response to corticosteroids.

BACKGROUND: Epileptic (previously infantile) spasms is the most common epileptic encephalopathy occurring during infancy and is frequently associated with abnormal neurodevelopmental outcomes. Epileptic spasms have a diverse range of known (genetic, structural) and unknown aetiologies. High dose corticosteroid treatment for 4 weeks often induces remission of spasms, although the mechanism of action of corticosteroid is unclear. Animal models of epileptic spasms have shown decreased brain kynurenic acid, which is increased after treatment with the ketogenic diet. We quantified kynurenine pathway metabolites in the cerebrospinal fluid (CSF) of infants with epileptic spasms and explored clinical correlations. METHODS: A panel of nine metabolites in the kynurenine pathway (tryptophan, kynurenine, kynurenic acid, 3-hydroxykynurenine, xanthurenic acid, anthranilic acid, 3-hydroxyanthranilic acid, quinolinic acid, and picolinic acid) were measured using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). CSF collected from paediatric patients less than 3 years of age with epileptic spasms (n=34, 19 males, mean age 0.85, median 0.6, range 0.3-3 yrs) were compared with other epilepsy syndromes (n=26, 9 males, mean age 1.44, median 1.45, range 0.3-3 yrs), other non-inflammatory neurological diseases (OND) (n=29, 18 males, mean age 1.47, median 1.6, range 0.1-2.9 yrs) and inflammatory neurological controls (n=12, 4 males, mean age 1.80, median 1.80, range 0.8-2.5 yrs). FINDINGS: There was a statistically significant decrease of CSF kynurenic acid in patients with epileptic spasms compared to OND (p<0.0001). In addition, the kynurenic acid/kynurenine (KYNA/KYN) ratio was lower in the epileptic spasms subgroup compared to OND (p<0.0001). Epileptic spasms patients who were steroid responders or partial steroid responders had lower KYNA/KYN ratio compared to patients who were refractory to steroids (p<0.005, p<0.05 respectively). INTERPRETATION: This study demonstrates decreased CSF kynurenic acid and KYNA/KYN in epileptic spasms, which may also represent a biomarker for steroid responsiveness. Given the anti-inflammatory and neuroprotective properties of kynurenic acid, further therapeutics able to increase kynurenic acid should be explored. FUNDING: Financial support for the study was granted by Dale NHMRC Investigator grant APP1193648, Petre Foundation, Cerebral Palsy Alliance and Department of Biochemistry at the Children's Hospital at Westmead. Prof Guillemin is funded by NHMRC Investigator grant APP1176660 and Macquarie University.

The Kynurenine Pathway in Traumatic Brain Injury: Implications for Psychiatric Outcomes.

Traumatic brain injury (TBI) is an established risk factor for the development of psychiatric disorders, especially depression and anxiety. However, the mechanistic pathways underlying this risk remain unclear, limiting treatment options and hindering the identification of clinically useful biomarkers. One salient pathophysiological process implicated in both primary psychiatric disorders and TBI is inflammation. An important consequence of inflammation is the increased breakdown of tryptophan to kynurenine and, subsequently, the metabolism of kynurenine into several neuroactive metabolites, including the neurotoxic NMDA receptor agonist quinolinic acid and the neuroprotective NMDA receptor antagonist kynurenic acid. Here, we review studies of the kynurenine pathway (KP) in TBI and examine their potential clinical implications. The weight of the literature suggests that there is increased production of neurotoxic kynurenines such as quinolinic acid in TBI of all severities and that elevated quinolinic acid concentrations in both the cerebrospinal fluid and blood are a negative prognostic indicator, being associated with death, magnetic resonance imaging abnormalities, increased depressive and anxiety symptoms, and prolonged recovery. We hypothesize that an imbalance in KP metabolism is also one molecular pathway through which the TBI-induced neurometabolic cascade may predispose to the development of psychiatric sequelae. If this model is correct, KP metabolites could serve to predict who is likely to develop psychiatric illness while drugs that target the KP could help to prevent or treat depression and anxiety arising in the context of TBI.

A novel, robust method for quantification of multiple kynurenine pathway metabolites in the cerebrospinal fluid.

Aim: Kynurenine metabolites are potential modulators of psychiatric disease. We aimed to develop a highly sensitive biochemical analysis of cerebrospinal fluid (CSF) tryptophan (TRP) metabolites, to investigate the stability of metabolites and to confirm our previous findings of aberrant CSF quinolinic acid (QUIN) and picolinic acid (PIC) in suicide attempters using this method. Methodology & results: Ten CSF TRP metabolites were analyzed with ultraperformance LC-MS/MS. The method showed small intra- and interassay variation. Metabolites were stable following freeze-thaw cycles. A decreased CSF PIC/QUIN ratio was found in suicide attempters. Conclusion: The feasibility of reliably determining CSF TRP metabolites were demonstrated, including separation of the two isomers PIC and nicotinic acid (NA) and the finding of a reduced PIC/QUIN ratio replicated in suicide attempters.

Twin study shows association between monocyte chemoattractant protein-1 and kynurenic acid in cerebrospinal fluid.

Preclinical studies indicate a link between the kynurenine pathway and monocyte chemoattractant protein-1 (MCP-1), but there is a lack of clinical studies examining this further. We here perform a secondary analysis of kynurenine metabolites and MCP-1 in cerebrospinal fluid of 23 twins affected from schizophrenia, bipolar disorder or unaffected. We show an association between MCP-1 and kynurenic acid (KYNA), driven by unique environmental influences and a less pronounced association between MCP-1 and tryptophan. No association was detected between MCP-1 and quinolinic acid. Further studies on the mechanism behind the putative relationship between KYNA and MCP-1 are needed.

Elevated quinolinic acid levels in cerebrospinal fluid in subacute sclerosing panencephalitis.

Subacute sclerosing panencephalitis (SSPE) is a rare neurodegenerative disorder caused by a persistent infection with aberrant measles virus. Indoleamine-2, 3-dioxygenase (IDO) initiates the increased production of kynurenine pathway (KP) metabolites quinolinic acid (QUIN), which has an excitotoxic effect for neurons. We measured serum IDO activity and cerebrospinal fluid (CSF) levels of QUIN. The CSF QUIN levels were significantly higher in SSPE patients than in controls, and increased according as neurological disability in a patient studied. Elevation of CSF QUIN and progression of SSPE indicate a pathological role of KP metabolism in the inflammatory neurodestruction.

HIV, prospective memory, and cerebrospinal fluid concentrations of quinolinic acid and phosphorylated Tau.

There is mounting evidence that prospective memory (PM) is impaired during HIV infection despite treatment. In this prospective study, 66 adults (43 HIV+ and 23 HIV negative) underwent PM assessment and cerebrospinal fluid (CSF) examination. HIV+ participants had significantly lower PM but significantly higher CSF concentrations of CXCL10 and quinolinic acid (QUIN). Higher CSF phosphorylated Tau (pTau) was associated with worse PM. In a secondary analysis excluding outliers, higher QUIN correlated with higher pTau. CSF QUIN is thus elevated during HIV infection despite antiretroviral therapy and could indirectly contribute to impaired PM by influencing the formation of pTau.

Kynurenic acid and psychotic symptoms and personality traits in twins with psychiatric morbidity.

Increased cytokines and kynurenic acid (KYNA) levels in cerebrospinal fluid (CSF) have been reported in patients with schizophrenia and bipolar disorder. The aim of the present study was to investigate cytokines and kynurenines in the CSF of twin pairs discordant for schizophrenia or bipolar disorder and to study these CSF markers in relation to psychotic symptoms and personality traits. CSF levels of tryptophan (TRP), KYNA, quinolinic acid (QUIN), interleukin (IL)-6, IL-8 and tumor necrosis factor-alpha (TNF-α) were analyzed in 23 twins with schizophrenia or bipolar disorder, and in their not affected co-twins. Ratings of psychotic symptoms and personality traits were made using the Scales for Assessment of Negative and Positive symptoms, the Structured Clinical Interview for DSM-IV - Axis II Disorders, and the Schizotypal Personality Questionnaire - Brief. A total score for psychotic symptoms and personality traits was constructed for analysis. CSF KYNA was associated with the score for psychotic symptom and personality traits. TNF-α and IL-8 were associated, and the intra-pair differences scores of TNF-α and IL-8 were highly correlated. Intraclass correlations indicated genetic influences on CSF KYNA, TRP, IL-8 and TNF-α. The association between KYNA and psychotic symptoms further supports a role of KYNA in psychotic disorders.

An enzyme in the kynurenine pathway that governs vulnerability to suicidal behavior by regulating excitotoxicity and neuroinflammation.

Emerging evidence suggests that inflammation has a key role in depression and suicidal behavior. The kynurenine pathway is involved in neuroinflammation and regulates glutamate neurotransmission. In the cerebrospinal fluid (CSF) of suicidal patients, levels of inflammatory cytokines and the kynurenine metabolite quinolinic acid (QUIN), an N-methyl-d-aspartate receptor agonist, are increased. The enzyme amino-ß-carboxymuconate-semialdehyde-decarboxylase (ACMSD) limits QUIN formation by competitive production of the neuroprotective metabolite picolinic acid (PIC). Therefore, decreased ACMSD activity can lead to excess QUIN. We tested the hypothesis that deficient ACMSD activity underlies suicidal behavior. We measured PIC and QUIN in CSF and plasma samples from 137 patients exhibiting suicidal behavior and 71 healthy controls. We used DSM-IV and the Montgomery-Åsberg Depression Rating Scale and Suicide Assessment Scale to assess behavioral changes. Finally, we genotyped ACMSD tag single-nucleotide polymorphisms (SNPs) in 77 of the patients and 150 population-based controls. Suicide attempters had reduced PIC and a decreased PIC/QUIN ratio in both CSF (P<0.001) and blood (P=0.001 and P<0.01, respectively). The reductions of PIC in CSF were sustained over 2 years after the suicide attempt based on repeated measures. The minor C allele of the ACMSD SNP rs2121337 was more prevalent in suicide attempters and associated with increased CSF QUIN. Taken together, our data suggest that increased QUIN levels may result from reduced activity of ACMSD in suicidal subjects. We conclude that measures of kynurenine metabolites can be explored as biomarkers of suicide risk, and that ACMSD is a potential therapeutic target in suicidal behavior.

LC-MS/MS-based quantification of kynurenine metabolites, tryptophan, monoamines and neopterin in plasma, cerebrospinal fluid and brain.

AIM: The kynurenine (KYN) pathway is implicated in diseases such as cancer, psychiatric, neurodegenerative and autoimmune disorders. Measurement of KYN metabolite levels will help elucidating the involvement of the KYN pathway in the disease pathology and inform drug development. METHODOLOGY: Samples of plasma, cerebrospinal fluid or brain tissue were spiked with deuterated internal standards, processed and analyzed by LC-MS/MS; analytes were chromatographically separated by gradient elution on a C18 reversed phase analytical column without derivatization. CONCLUSION: We established an LC-MS/MS method to measure 11 molecules, namely tryptophan, KYN, 3-OH-KYN, 3-OH-anthranilic acid, quinolinic acid, picolinic acid, kynurenic acid, xanthurenic acid, serotonin, dopamine and neopterin within 5.5 min, with sufficient sensitivity to quantify these molecules in small sample volumes of plasma, cerebrospinal fluid and brain tissue.

Activation of the kynurenine pathway and increased production of the excitotoxin quinolinic acid following traumatic brain injury in humans.

UNLABELLED: During inflammation, the kynurenine pathway (KP) metabolises the essential amino acid tryptophan (TRP) potentially contributing to excitotoxicity via the release of quinolinic acid (QUIN) and 3-hydroxykynurenine (3HK). Despite the importance of excitotoxicity in the development of secondary brain damage, investigations on the KP in TBI are scarce. In this study, we comprehensively characterised changes in KP activation by measuring numerous metabolites in cerebrospinal fluid (CSF) from TBI patients and assessing the expression of key KP enzymes in brain tissue from TBI victims. Acute QUIN levels were further correlated with outcome scores to explore its prognostic value in TBI recovery. METHODS: Twenty-eight patients with severe TBI (GCS ≤ 8, three patients had initial GCS = 9-10, but rapidly deteriorated to ≤8) were recruited. CSF was collected from admission to day 5 post-injury. TRP, kynurenine (KYN), kynurenic acid (KYNA), QUIN, anthranilic acid (AA) and 3-hydroxyanthranilic acid (3HAA) were measured in CSF. The Glasgow Outcome Scale Extended (GOSE) score was assessed at 6 months post-TBI. Post-mortem brains were obtained from the Australian Neurotrauma Tissue and Fluid Bank and used in qPCR for quantitating expression of KP enzymes (indoleamine 2,3-dioxygenase-1 (IDO1), kynurenase (KYNase), kynurenine amino transferase-II (KAT-II), kynurenine 3-monooxygenase (KMO), 3-hydroxyanthranilic acid oxygenase (3HAO) and quinolinic acid phosphoribosyl transferase (QPRTase) and IDO1 immunohistochemistry. RESULTS: In CSF, KYN, KYNA and QUIN were elevated whereas TRP, AA and 3HAA remained unchanged. The ratios of QUIN:KYN, QUIN:KYNA, KYNA:KYN and 3HAA:AA revealed that QUIN levels were significantly higher than KYN and KYNA, supporting increased neurotoxicity. Amplified IDO1 and KYNase mRNA expression was demonstrated on post-mortem brains, and enhanced IDO1 protein coincided with overt tissue damage. QUIN levels in CSF were significantly higher in patients with unfavourable outcome and inversely correlated with GOSE scores. CONCLUSION: TBI induced a striking activation of the KP pathway with sustained increase of QUIN. The exceeding production of QUIN together with increased IDO1 activation and mRNA expression in brain-injured areas suggests that TBI selectively induces a robust stimulation of the neurotoxic branch of the KP pathway. QUIN's detrimental roles are supported by its association to adverse outcome potentially becoming an early prognostic factor post-TBI.

A role for inflammatory metabolites as modulators of the glutamate N-methyl-D-aspartate receptor in depression and suicidality.

BACKGROUND: Patients with depression and suicidality suffer from low-grade neuroinflammation. Pro-inflammatory cytokines activate indoleamine 2,3-dioxygenase, an initial enzyme of the kynurenine pathway. This pathway produces neuroactive metabolites, including quinolinic- and kynurenic acid, binding to the glutamate N-methyl-d-aspartate-receptor, which is hypothesized to be part of the neural mechanisms underlying symptoms of depression. We therefore hypothesized that symptoms of depression and suicidality would fluctuate over time in patients prone to suicidal behavior, depending on the degree of inflammation and kynurenine metabolite levels in the cerebrospinal fluid (CSF). METHODS: We measured cytokines and kynurenine metabolites in CSF, collected from suicide attempters at repeated occasions over 2 years (total patient samples n=143, individuals n=30) and healthy controls (n=36). The association between the markers and psychiatric symptoms was assessed using the Montgomery Asberg Depression Rating Scale and the Suicide Assessment Scale. RESULTS: Quinolinic acid was increased and kynurenic acid decreased over time in suicidal patients versus healthy controls. Furthermore, we found a significant association between low kynurenic acid and severe depressive symptoms, as well as between high interleukin-6 levels and more severe suicidal symptoms. CONCLUSIONS: We demonstrate a long-term dysregulation of the kynurenine pathway in the central nervous system of suicide attempters. An increased load of inflammatory cytokines was coupled to more severe symptoms. We therefore suggest that patients with a dysregulated kynurenine pathway are vulnerable to develop depressive symptoms upon inflammatory conditions, as a result the excess production of the NMDA-receptor agonist quinolinic acid. This study provides a neurobiological framework supporting the use of NMDA-receptor antagonists in the treatment of suicidality and depression.

Improved separation and detection of picolinic acid and quinolinic acid by capillary electrophoresis-mass spectrometry: application to analysis of human cerebrospinal fluid.

"Quinolinic acid (QA)", a metabolite of the kynurenine pathway (KP), is implicated as a major neurological biomarker, which causes inflammatory disorders, whereas there is an increase evidence of the role of picolinic acid (PA) in neuroinflammation. Therefore, there is an urgent need to develop new clinical test for early diagnosis of neuroinflammatory disorders. A comparison is made between three different platforms such as high performance liquid chromatography-electrospray mass spectrometry (HPLC-ESI-MS/MS), nano LC-Chip/ESI-MS/MS, as well as the use of cationic (quaternary ammonium) and anionic (sulfonated) coated capillaries in capillary electrophoresis (CE)-ESI-MS/MS. The comparison revealed that CE-ESI-MS/MS method using a quaternary ammonium coated capillary is the best method for analysis of PA and QA. A simple stacking procedure by the inclusion of acetonitrile in the artificial cerebrospinal fluid (CSF) sample was employed to improve the peak shape and sensitivity of KP metabolites in CE-ESI-MS/MS. The developed CE-ESI-MS/MS assay provided high resolution, high specificity and high sensitivity with a total analysis time including sample preparation of nearly 12 min. In addition, excellent intra-day and inter-day repeatability of migration times and peak areas of the metabolites were observed with respective relative standard deviation (RSD) of less than 2.0% and 2.5%. Somewhat broader variations in repeatability for a 3 independently prepared coated capillary (total 35 runs each) with % RSD up to 3.8% and 5.8% was observed for migration time and peak areas, respectively. Artificial CSF was used as a surrogate matrix to simultaneously generate calibration curves over a concentration range of 0.02-10 µM for PA and 0.4-40 µM for QA. The method was then successfully applied to analyze PA and QA in human CSF, demonstrating the potential of this CE-ESI-MS/MS method to accurately quantitate with high specificity and sensitivity.

Microglia/macrophage-derived inflammatory mediators galectin-3 and quinolinic acid are elevated in cerebrospinal fluid from newborn infants after birth asphyxia.

Activation of microglia/macrophages is important in neonatal hypoxic-ischemic (HI) brain injury. Based on experimental studies, we identified macrophage/microglia-derived mediators with potential neurotoxic effects after neonatal HI and examined them in cerebrospinal fluid (CSF) from newborn infants after birth asphyxia. Galectin-3 is a novel inflammatory mediator produced by microglia/macrophages. Galectin-3 is chemotactic for inflammatory cells and activates nicotinamide adenine dinucleotide phosphate (NADPH) oxidase resulting in production and release of reactive oxygen species (ROS). Matrix metalloproteinase-9 (MMP-9) is a tissue-degrading protease expressed by activated microglia in the immature brain after HI. Both galectin-3 and MMP-9 contribute to brain injury in animal models for neonatal HI. Quinolinic acid (QUIN) is a neurotoxic N-methyl-D-aspartate (NMDA) receptor agonist also produced by activated microglia/macrophages. Galectin-3 and MMP-9 were measured by ELISA and QUIN by mass spectrometry. Asphyxiated infants (n=20) had higher levels of galectin-3 (mean (SEM) 2.64 (0.43) ng/mL) and QUIN (335.42 (58.9) nM) than controls (n=15) (1.36 (0.46) ng/mL and 116.56 (16.46) nM, respectively), p<0.05 and p<0.01. Infants with septic infections (n=10) did not differ from controls. Asphyxiated infants with abnormal outcome had higher levels of galectin-3 (3.96 (0.67) ng/mL) than those with normal outcome (1.76 (0.32) ng/mL), p=0.02, and the difference remained significant in the clinically relevant group of infants with moderate encephalopathy. MMP-9 was detected in few infants with no difference between groups. The potentially neurotoxic macrophage/microglia-derived mediators galectin-3 and QUIN are increased in CSF after birth asphyxia and could serve as markers and may contribute to injury.

Connecting inflammation with glutamate agonism in suicidality.

The NMDA-receptor antagonist ketamine has proven efficient in reducing symptoms of suicidality, although the mechanisms explaining this effect have not been detailed in psychiatric patients. Recent evidence points towards a low-grade inflammation in brains of suicide victims. Inflammation leads to production of quinolinic acid (QUIN) and kynurenic acid (KYNA), an agonist and antagonist of the glutamatergic N-methyl-D-aspartate (NMDA) receptor, respectively. We here measured QUIN and KYNA in the cerebrospinal fluid (CSF) of 64 medication-free suicide attempters and 36 controls, using gas chromatography mass spectrometry and high-performance liquid chromatography. We assessed the patients clinically using the Suicide Intent Scale and the Montgomery-Asberg Depression Rating Scale (MADRS). We found that QUIN, but not KYNA, was significantly elevated in the CSF of suicide attempters (P<0.001). As predicted, the increase in QUIN was associated with higher levels of CSF interleukin-6. Moreover, QUIN levels correlated with the total scores on Suicide Intent Scale. There was a significant decrease of QUIN in patients who came for follow-up lumbar punctures within 6 months after the suicide attempt. In summary, we here present clinical evidence of increased QUIN in the CSF of suicide attempters. An increased QUIN/KYNA quotient speaks in favor of an overall NMDA-receptor stimulation. The correlation between QUIN and the Suicide Intent Scale indicates that changes in glutamatergic neurotransmission could be specifically linked to suicidality. Our findings have important implications for the detection and specific treatment of suicidal patients, and might explain the observed remedial effects of ketamine.

CSF concentrations of brain tryptophan and kynurenines during immune stimulation with IFN-alpha: relationship to CNS immune responses and depression.

Cytokine-induced activation of indoleamine 2,3-dioxygenase (IDO) catabolizes L-tryptophan (TRP) into L-kynurenine (KYN), which is metabolized to quinolinic acid (QUIN) and kynurenic acid (KA). QUIN and KA are neuroactive and may contribute to the behavioral changes experienced by some patients during exposure to inflammatory stimuli such as interferon (IFN)-alpha. A relationship between depressive symptoms and peripheral blood TRP, KYN and KA during treatment with IFN-alpha has been described. However, whether peripheral blood changes in these IDO catabolites are manifest in the brain and whether they are related to central nervous system cytokine responses and/or behavior is unknown. Accordingly, TRP, KYN, QUIN and KA were measured in cerebrospinal fluid (CSF) and blood along with CSF concentrations of relevant cytokines, chemokines and soluble cytokine receptors in 27 patients with hepatitis C after approximately 12 weeks of either treatment with IFN-alpha (n=16) or no treatment (n=11). Depressive symptoms were assessed using the Montgomery-Asberg Depression Rating Scale. IFN-alpha significantly increased peripheral blood KYN, which was accompanied by marked increases in CSF KYN. Increased CSF KYN was in turn associated with significant increases in CSF QUIN and KA. Despite significant decreases in peripheral blood TRP, IFN-alpha had no effect on CSF TRP concentrations. Increases in CSF KYN and QUIN were correlated with increased CSF IFN-alpha, soluble tumor necrosis factor-alpha receptor 2 and monocyte chemoattractant protein-1 as well as increased depressive symptoms. In conclusion, peripheral administration of IFN-alpha activated IDO in concert with central cytokine responses, resulting in increased brain KYN and QUIN, which correlated with depressive symptoms.