Associations between circulating interferon and kynurenine/tryptophan pathway metabolites: support for a novel potential mechanism for cognitive dysfunction in SLE.

OBJECTIVE: Quinolinic acid (QA), a kynurenine (KYN)/tryptophan (TRP) pathway metabolite, is an N-methyl-D-aspartate receptor agonist that can produce excitotoxic neuron damage. Type I and II interferons (IFNs) stimulate the KYN/TRP pathway, producing elevated QA/kynurenic acid (KA), a potential neurotoxic imbalance that may contribute to SLE-mediated cognitive dysfunction. We determined whether peripheral blood interferon-stimulated gene (ISG) expression associates with elevated serum KYN:TRP and QA:KA ratios in SLE. METHODS: ISG expression (whole-blood RNA sequencing) and serum metabolite ratios (high-performance liquid chromatography) were measured in 72 subjects with SLE and 73 healthy controls (HCs). ISG were identified from published gene sets and individual IFN scores were derived to analyse associations with metabolite ratios, clinical parameters and neuropsychological assessments. SLE analyses were grouped by level of ISG expression ('IFN high', 'IFN low' and 'IFN similar to HC') and level of monocyte-associated gene expression (using CIBERSORTx). RESULTS: Serum KYN:TRP and QA:KA ratios were higher in SLE than in HC (p<0.01). 933 genes were differentially expressed ≥2-fold in SLE versus HC (p<0.05). 70 of the top 100 most highly variant genes were ISG. Approximately half of overexpressed genes that correlated with KYN:TRP and QA:KA ratios (p<0.05) were ISG. In 36 IFN-high subjects with SLE, IFN scores correlated with KYN:TRP ratios (p<0.01), but not with QA:KA ratios. Of these 36 subjects, 23 had high monocyte-associated gene expression, and in this subgroup, the IFN scores correlated with both KY:NTRP and QA:KA ratios (p<0.05). CONCLUSIONS: High ISG expression correlated with elevated KYN:TRP ratios in subjects with SLE, suggesting IFN-mediated KYN/TRP pathway activation, and with QA:KA ratios in a subset with high monocyte-associated gene expression, suggesting that KYN/TRP pathway activation may be particularly important in monocytes. These results need validation, which may aid in determining which patient subset may benefit from therapeutics directed at the IFN or KYN/TRP pathways to ameliorate a potentially neurotoxic QA/KA imbalance.

Quinolinic acid, a kynurenine/tryptophan pathway metabolite, associates with impaired cognitive test performance in systemic lupus erythematosus.

OBJECTIVES: Interferon-alpha, an important contributor to SLE pathogenesis, induces the enzyme indoleamine 2,3-dioxygenase in the kynurenine/tryptophan (KYN/TRP) pathway. This leads to a potentially neurotoxic imbalance in the KYN/TRP pathway metabolites, quinolinic acid (QA), an N-methyl D-aspartate glutamatergic receptor (NMDAR) agonist, and kynurenic acid (KA), an NMDAR antagonist. We determined whether QA/KA ratios associate with cognitive dysfunction (CD) and depression in SLE. METHODS: This cross-sectional study included 74 subjects with SLE and 74 healthy control (HC) subjects; all without history of neuropsychiatric disorders. Serum metabolite levels (KYN, TRP, QA, KA) were measured concurrently with assessments of cognition (Automated Neuropsychological Assessment Metrics (ANAM), 2×2 array), mood and pain, and compared between SLE and HC. Multivariable modelling in SLE was used to evaluate associations of metabolites with cognitive performance and depression. RESULTS: Serum KYN/TRP and QA/KA ratios were elevated in SLE versus HC (p<0.0001). SLE performed worse than HC on four of five ANAM tests (all p≤0.02) and the 2×2 array (p<0.01), and had higher depression scores (p<0.01). In SLE, elevated QA/KA ratios correlated with poor performance on Match to Sample (MTS), a working memory and visuospatial processing task (p<0.05). Subjects with SLE with elevated QA/KA ratios also had slightly higher odds of depression, but this did not reach significance (p=0.09). Multivariable modelling in SLE confirmed an association between QA/KA ratios and poor MTS performance when considering potentially confounding factors (p<0.05). CONCLUSIONS: Elevated serum KYN/TRP and QA/KA ratios confirm KYN/TRP pathway activation in SLE. The novel association between increased QA/KA ratios and poor cognitive performance supports further study of this pathway as a potential biomarker or therapeutic target for SLE-mediated CD.

Plasma Anthranilic Acid and Leptin Levels Predict HAM-D Scores in Depressed Women.

OBJECTIVES: Major depressive disorder (MDD) is associated with dysregulations of leptin and tryptophan-kynurenine (Trp-Kyn) (TKP) pathways. Leptin, a pro-inflammatory cytokine, activates Trp conversion into Kyn. However, leptin association with down-stream Kyn metabolites in MDD is unknown. METHODS: Fasting plasma samples from 29 acutely ill drug-naïve (n = 16) or currently non-medicated (⩾6 weeks; n = 13) MDD patients were analyzed for leptin, Trp, Kyn, its down-stream metabolites (anthranilic [AA], kynurenic [KYNA], xanthurenic [XA] acids and 3-hydroxykynurenine [3HK]), C-reactive protein (CRP), neopterin, body mass index (BMI), and insulin resistance (HOMA-IR). Depression severity was assessed by HAM-D-21. RESULTS: In female (n = 14) (but not in male) patients HAM-D-21 scores correlated with plasma levels of AA (but not other Kyn metabolites) (rho = -0.644, P = .009) and leptin (Spearman's rho = -0.775, P = .001). Inclusion of AA into regression analysis improved leptin prediction of HAM-D from 48.5% to 65.9%. Actual HAM-D scores highly correlated with that calculated by formula: HAM-D = 34.8518-(0.5660 × leptin [ng/ml] + 0.4159 × AA [nmol/l]) (Rho = 0.84, P = .00015). In male (n = 15) (but not in female) patients leptin correlated with BMI, waist circumference/hip ratio, CRP, and HOMA-IR. CONCLUSIONS: Present findings of gender specific AA/Leptin correlations with HAM-D are important considering that AA and leptin are transported from plasma into brain, and that AA formation is catalyzed by kynureninase-the only TKP gene associated with depression according to genome-wide analysis. High correlation between predicted and actual HAM-D warrants further evaluation of plasma AA and leptin as an objective laboratory test for the assessment of depression severity in female MDD patients.

Cerebrospinal fluid monoamine levels in central disorders of hypersomnolence.

STUDY OBJECTIVES: Whether the cause of daytime sleepiness in narcolepsy type 1 (NT1) is a direct consequence of the loss of orexin (ORX) neurons or whether low orexin reduces the efficacy of the monoaminergic systems to promote wakefulness is unclear. The neurobiology underlying sleepiness in other central hypersomnolence disorders, narcolepsy type 2 (NT2), and idiopathic hypersomnia (IH), is currently unknown. METHODS: Eleven biogenic amines including the monoaminergic neurotransmitters and their metabolites and five trace amines were measured in the cerebrospinal fluid (CSF) of 94 drug-free subjects evaluated at the French National Reference Center for Narcolepsy: 39 NT1(orexin-deficient) patients, 31 patients with objective sleepiness non orexin-deficient (NT2 and IH), and 24 patients without objective sleepiness. RESULTS: Three trace amines were undetectable in the sample: tryptamine, octopamine, and 3-iodothyronamine. No significant differences were found among the three groups for quantified monoamines and their metabolites in crude and adjusted models; however, CSF 5-hydroxyindoleacetic acid (5-HIAA) levels tended to increase in NT1 compared to other patients after adjustment. Most of the biomarkers were not associated with ORX-A levels, clinical or neurophysiological parameters, but a few biomarkers (e.g. 3-methoxy-4-hydroxyphenylglycol and norepinephrine) correlated with daytime sleepiness and high rapid eye movement (REM) sleep propensity. CONCLUSIONS: We found no striking differences among CSF monoamines, their metabolites and trace amine levels, and few associations between them and key clinical or neurophysiological parameters in NT1, NT2/IH, and patients without objective sleepiness. Although mostly negative, these findings are a significant contribution to our understanding of the neurobiology of hypersomnolence in these disorders that remain mysterious and deserve further exploration.

Peripheral kynurenine-3-monooxygenase deficiency as a potential risk factor for metabolic syndrome in schizophrenia patients.

Increased predisposition of schizophrenia patients (SP) to development of obesity and insulin resistance suggested common signaling pathway between metabolic syndrome (MetS) and schizophrenia. Deficiency of kynurenine-3-monooxygenase (KMO), enzyme catalyzing formation of 3-hydroxykynurenine (3-HK) from kynurenine (Kyn), a tryptophan (Trp) metabolite, might contribute to development of MetS as suggested by non-expression of KMO genes in human fat tissue and elevated serum concentrations of Kyn and its metabolites, kynurenic (KYNA) and anthranilic (ANA) acids, in diabetic patients and Zucker fatty rats (ZFR). Markers of KMO deficiency: decreased 3-HK and elevated Kyn, KYNA and ANA, were observed in brains and spinal fluids of SP, and in brains and serum of experimental animals with genetically- or pharmacologically-induced KMO deficiency. However, elevated concentrations of ANA and decreased 3-HK were reported in serum of SP without concurrent increase of Kyn and KYNA. Present study aimed to re-assess serum Kyn metabolites (HPLC-MS) in a sub-group of SP with elevated KYNA. We found increased Kyn concentrations (by 30%) and Kyn:Trp ratio (by 20%) in serum of SP with elevated KYNA concentrations (by 40%). Obtained results and our previous data suggest that peripheral KMO deficiency might be manifested by, at least, two different patterns: elevated ANA with decreased 3-HK; and elevated KYNA and KYN. The latter pattern was previously described in type 2 diabetes patients and might underline increased predisposition of SP to development of MetS. Assessment of peripheral KMO deficiency might identify SP predisposed to MetS. Attenuation of the consequences of peripheral KMO deficiency might be a new target for prevention/treatment of obesity and diabetes in SP.

Peripheral Tryptophan - Kynurenine Metabolism Associated with Metabolic Syndrome is Different in Parkinson's and Alzheimer's Diseases.

Insulin resistance (IR), obesity and other components of metabolic syndrome [MetS] are highly associated with Alzheimer's (AD) and Parkinson's (PD) diseases. Dysregulation of kynurenine (Kyn) pathway (KP) of tryptophan (Trp) metabolism was suggested as major contributor to pathogenesis of AD and PD and MetS. KP, the major source of NAD+ in humans, occurs in brain and peripheral organs. Considering that some, but not all, peripherally originated derivatives of Kyn penetrate blood brain barrier, dysregulation of central and peripheral KP might have different functional impact. Up-regulated Kyn formation from Trp was discovered in central nervous system of AD and PD while assessments of peripheral KP in these diseases yield controversial results. We were interested to compare peripheral kynurenines in AD and PD with emphasis on MetS-associated kynurenines, i.e., kynurenic (KYNA) and anthranilic (ANA) acids and 3-hydroxykynurenine (3-HK). Serum concentrations of KP metabolites were evaluated (HPLC-MS method). In PD patients Trp concentrations were lower, and Kyn: Trp ratio, Kyn, ANA and KYNA were higher than in controls. 3-HK concentrations of PD patients were below the sensitivity threshold of the method. In AD patients. ANA serum concentrations were approximately 3 fold lower, and KYNA concentrations were approximately 40% higher than in controls. Our data suggest different patterns of KP dysregulation in PD and AD: systemic chronic subclinical inflammation activating central and peripheral KP in PD, and central, rather than peripheral, activation of KP in AD triggered by Aß1-42. Dysregulation of peripheral KP in PD and AD patients might underline association between neurodegenerative diseases and MetS.

Anthranilic Acid: A Potential Biomarker and Treatment Target for Schizophrenia.

Dysregulation of Trp-Kyn pathway is the most recent hypothesis of mechanisms of schizophrenia. In particular, over-production of kynurenic acid (KYNA), one of the three immediate downstream metabolites of kynurenine (Kyn) along tryptophan (Trp): Kyn pathway, has been considered as a new target for therapeutic intervention in schizophrenia. Up-regulation of KYNA formation was suggested to occur at the expense of down-regulated production of 3-hydroxyKyn (3-HK), the second immediate downstream metabolite of Kyn. We were interested to assess the third immediate downstream Kyn metabolite, anthranilic acid (AA). Serum AA concentrations were evaluated in schizophrenia patients and control subjects by HPLC-mass spectrometry method. We found 2-fold increase of AA and 3-fold decrease of 3-HK concentrations in serum of schizophrenia patients. Up regulated formation of AA might contribute to mechanisms of schizophrenia considering experimental evidences of AA augmentation of autoimmune processes in rat and mice; clinical findings of AA elevation in rheumatoid arthritis and type 1 diabetes, autoimmune diseases diametrical to schizophrenia; and involvement of autoimmunity in development of schizophrenia. Present data warrant further studies of AA as biological marker in, at least, a subgroup (associated with autoimmune mechanisms) of schizophrenia patients and as a new target for therapeutic intervention.

Boron-doped diamond electrodes for the electrochemical oxidation and cleavage of peptides.

Electrochemical oxidation of peptides and proteins is traditionally performed on carbon-based electrodes. Adsorption caused by the affinity of hydrophobic and aromatic amino acids toward these surfaces leads to electrode fouling. We compared the performance of boron-doped diamond (BDD) and glassy carbon (GC) electrodes for the electrochemical oxidation and cleavage of peptides. An optimal working potential of 2000 mV was chosen to ensure oxidation of peptides on BDD by electron transfer processes only. Oxidation by electrogenerated OH radicals took place above 2500 mV on BDD, which is undesirable if cleavage of a peptide is to be achieved. BDD showed improved cleavage yield and reduced adsorption for a set of small peptides, some of which had been previously shown to undergo electrochemical cleavage C-terminal to tyrosine (Tyr) and tryptophan (Trp) on porous carbon electrodes. Repeated oxidation with BDD electrodes resulted in progressively lower conversion yields due to a change in surface termination. Cathodic pretreatment of BDD at a negative potential in an acidic environment successfully regenerated the electrode surface and allowed for repeatable reactions over extended periods of time. BDD electrodes are a promising alternative to GC electrodes in terms of reduced adsorption and fouling and the possibility to regenerate them for consistent high-yield electrochemical cleavage of peptides. The fact that OH-radicals can be produced by anodic oxidation of water at elevated positive potentials is an additional advantage as they allow another set of oxidative reactions in analogy to the Fenton reaction, thus widening the scope of electrochemistry in protein and peptide chemistry and analytics.

Chemical labeling of electrochemically cleaved peptides.

RATIONALE: Cleavage of peptide bonds C-terminal to tyrosine and tryptophan after electrochemical oxidation may become a complementary approach to chemical and enzymatic cleavage. A chemical labeling approach specifically targeting reactive cleavage products is presented here and constitutes a promising first step towards the development of a new proteomics workflow. METHODS: Hexylamine was used to react with the spirolactone moieties generated after electrochemical oxidation and cleavage of tripeptides. The influence of pH and reaction time on the yield was determined and the excess of tagging reagent was optimized. Selective detection of the tagged cleavage products was achieved by precursor ion scanning in a triple quadrupole mass spectrometer. RESULTS: Optimal labeling was reached under aqueous conditions when working at pH 10 with a reaction time of 0.5 min. The excess of hexylamine over spirolactone groups can be significantly decreased by working under non-aqueous conditions in pure acetonitrile to prevent spirolactone hydrolysis. The specific formation of hexylamine-containing y(1) reporter ions generated by collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) allows for selective detection by precursor ion scanning of the cleaved and labeled peptides. CONCLUSIONS: This work presents a method for selective labeling and detection of electrochemically cleaved Tyr- and Trp-containing peptides for which reaction conditions have been optimized with hexylamine as labeling agent. This workflow offers new possibilities for electrochemical oxidation, cleavage and labeling of peptides and proteins.

Electrochemical oxidation and cleavage of tyrosine- and tryptophan-containing tripeptides.

Electrochemical oxidation of peptides and proteins has been shown to lead to specific cleavage next to tyrosine (Tyr) and tryptophan (Trp) residues which makes the coupling of electrochemistry to mass spectrometry (EC-MS) a potential instrumental alternative to chemical and enzymatic cleavage. A set of Tyr and Trp-containing tripeptides has been studied to investigate the mechanistic aspects of electrochemical oxidation and the subsequent chemical reactions including peptide bond cleavage, making this the first detailed study of the electrochemistry of Trp-containing peptides. The effect of adjacent amino acids was studied leading to the conclusion that the ratios of oxidation and cleavage products are peptide-dependent and that the adjacent amino acid can influence the secondary chemical reactions occurring after the initial oxidation step. The effect of parameters such as potential and solvent conditions showed that control of the oxidation potential is crucial to avoid dimer formation for Tyr and an increasing number of oxygen insertions (hydroxylations) for Trp, which occur above 1000 mV (vs Pd/H(2)). While the formation of reactive intermediates after the first oxidation step is not strongly dependent on experimental conditions, an acidic pH is required for good cleavage yields. Working under strongly acidic conditions (pH 1.9-3.1) led to optimal cleavage yields (40-80%), whereas no or little cleavage occurred under basic conditions. Online EC-MS allowed determining the optimal potential for maximum cleavage yields, whereas EC-LC-MS/MS revealed the nature and distribution of the reaction products.

Oxidative protein labeling in mass-spectrometry-based proteomics.

Oxidation of proteins and peptides is a common phenomenon, and can be employed as a labeling technique for mass-spectrometry-based proteomics. Nonspecific oxidative labeling methods can modify almost any amino acid residue in a protein or only surface-exposed regions. Specific agents may label reactive functional groups in amino acids, primarily cysteine, methionine, tyrosine, and tryptophan. Nonspecific radical intermediates (reactive oxygen, nitrogen, or halogen species) can be produced by chemical, photochemical, electrochemical, or enzymatic methods. More targeted oxidation can be achieved by chemical reagents but also by direct electrochemical oxidation, which opens the way to instrumental labeling methods. Oxidative labeling of amino acids in the context of liquid chromatography(LC)-mass spectrometry (MS) based proteomics allows for differential LC separation, improved MS ionization, and label-specific fragmentation and detection. Oxidation of proteins can create new reactive groups which are useful for secondary, more conventional derivatization reactions with, e.g., fluorescent labels. This review summarizes reactions of oxidizing agents with peptides and proteins, the corresponding methodologies and instrumentation, and the major, innovative applications of oxidative protein labeling described in selected literature from the last decade.