Determination Of Kynurenine Research Articles

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.

Improved Voltammetric Determination of Kynurenine at the Nafion Covered Glassy Carbon Electrode - Application in Samples Delivered from Human Cancer Cells.

Nowadays, development of analytical methods responding to a need for rapid and accurate determination of human metabolites is highly desirable. Herein, an electrochemical method employing a Nafion-coated glassy carbon electrode (Nafion/GCE) has been developed for reliable determination of kynurenine (a key tryptophan metabolite) using a differential pulse adsorptive stripping voltammetry. To our knowledge, this is the first analytical method to allow for kynurenine determination at the Nafion-coated electrode. The methodology involves kynurenine pre-concentration in 0.1 M H2SO4 in the Nafion film at the potential of +0.5 V and subsequent stripping from the electrode by differential pulse voltammetry. Under optimal conditions, the sensor can detect 5 nM kynurenine (for the accumulation time of 60 seconds), but the limit of detection can be easily lowered to 0.6 nM by prolonging the accumulation time to 600 seconds. The sensor shows sensitivity of 36.25 μAμM−1cm−2 and 185.50 μAμM−1cm−2 for the accumulation time of 60 and 600 seconds, respectively. The great advantage of the proposed method is easy sensor preparation, employing drop coating method, high sensitivity, short total analysis time, and no need for sample preparation. The method was validated for linearity, precision, accuracy (using a high-performance liquid chromatography), selectivity (towards tryptophan metabolites and different amino acids), and recovery. The comprehensive microscopic and electrochemical characterization of the Nafion/GCE was also conducted with different methods including atomic force microscopy (AFM), optical profilometry, time-of-flight secondary ion mass spectrometry (TOF-SIMS), electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV). The method has been applied with satisfactory results for determination of kynurenine concentration in a culture medium collected from the human ovarian carcinoma cells SK-OV-3 and to measure IDO enzyme activity in the cancer cell extracts.

LC-QTOF/MS determination of tryptophan and kynurenine in infant formulas

A rapid, reliable and sensitive liquid chromatography quadrupole time-of-flight mass spectrometry method for the determination of tryptophan and its metabolite kynurenine in milk formulas for neonates and infants was developed and validated. Two extraction techniques based on EMR Lipid QuEChERS and liquid-liquid extraction with diethyl ether to extract lipids and methanol to precipitate the protein were tested and compared. Four different infant formula products were randomly selected and evaluated for the effect of co-extracted matrix components on the quantitative analysis results. The influence of matrix components on analytical signals was normalized by the use of stable isotope-labeled standards and matrix-matched calibration. The developed method was found to be sensitive and effective for both analytes in all the examined infant formulas with satisfactory linearity (R2 ≥ 0.9995), recovery in the range of 75.7% ± 4.5 – 99.0% ± 1.1, and intra- and inter-day precision with the coefficient of variation below 6.3% and 17.9%, respectively. The limits of detection (LOD) and quantification (LOQ) for both compounds differed significantly between the examined formulas. The LOD and LOQ values were found to be in the range of 2.18–9.85 μg/g and 6.61–29.84 μg/g for the determination of tryptophan and in the range of 0.21–2.71 μg/g and 0.63–8.23 μg/g for the determination of kynurenine, respectively. The method was proved to be suitable for the determination of tryptophan and kynurenine in infant formulas, and it can be used to study the link between tryptophan metabolism via kynurenine pathway and metabolic disorders in infants.

Simultaneous analysis of neopterin, kynurenine and tryptophan by amine-HPLC shows minor oxidative stress from short-term exhaustion exercise

Abstract Introduction: Neopterin, kynurenine and tryptophan can be used to measure activation of monocytes and macrophages during immunological events such as exercise inducing inflammation. Endurance exercise and high-impact sports have shown significant increases in these biomarkers. Measurement is typically conducted by high-performance liquid chromatography (HPLC) using C18 or SCX columns. However, kynurenine and tryptophan are not measured simultaneously to neopterin using these separation systems. Here we have used an amine column for separation and simultaneous determination of neopterin, kynurenine and tryptophan. Methods: Optimization and validation for the amine-HPLC method was conducted using plasma from 43 participants subjected to a short maximal exercise bicycling regime or rest period. The order of exercise and rest was randomized and separated by a 3-5 week washout period. Results: Using an amine column developed with ammonium acetate formic acid (33%) and acetonitrile (72%) provided optimal separation and run time for analysis. Neopterin increased significantly post-exercise and subsided to baseline by 30 minutes. Total neopterin remained elevated until 60 minutes following exercise. Conclusion: Amine-HPLC can be used for simultaneous determination of kynurenine, tryptophan and neopterin in plasma. Short intense exercise causes a significant increase in plasma neopterin suggesting a prolonged activation of monocytes and macrophages.

Simultaneous Determination of Kynurenine and Kynurenic Acid by High-Performance Liquid Chromatography Photoirradiation System Using a Mobile Phase Containing 18-crown-6.

A high-performance liquid chromatography (HPLC) system has been developed for the fluorometric determination of kynurenine (KYN) and kynurenic acid (KYNA) in human serum using a mobile phase containing 18-crown-6. A retention time of KYNA was adjusted with pH of phosphate buffer in 18-crown-6. KYN and KYNA were separated on a CAPCELLPAK C18 (250 × 4.6 mm i.d.). The mobile phase consisted of 35 mmol/L phosphate buffer (pH 8.0)/methanol (85/15, v/v) containing 35 mmol/L hydrogen peroxide and 10 mmol/L 18-crown-6. The retention times of KYN and KYNA were 18and 24 minutes, respectively. The calibration graphs of KYN and KYNA were linear over the range 180 to 2900 and 1 to 84 nmol/L by injecting a 50-μL volume of KYN and KYNA, respectively. Pretreatment of serum was achieved by deproteinization only. The mean recoveries of KYN and KYNA from serum were more than 97%.

Simultaneous Determination of Kynurenine, Fumaric acid and Pyroglutamic acid as Degradation Products in Amino acids Formulation

The aim of the work was to develop and validate a HPLC method to quantify the Kynurenine (Kyn), Fumaric acid (Fum) and Pyroglutamic acid (Pyr) in amino acids (AAs) formulation as degradation products. Kyn, Fum and Pyr are the degradation products of Tryptophan (Trp), Aspartic acid (Asp) and Glutamic acid (Glu) respectively. Protein plays a crucial role in almost all biological processes and AAs are the building blocks of proteins. Trp is an essential amino acid, whereas Asp and Glu are non-essential AAs. Separation was main challenge due to non-significant absorption of UV radiation and zwitterion nature of AAs. However, separation was achieved using YMC C18AQ (4.6×250mm) 5µm column at 45°C with Waters Alliance e2695 HPLC equipped with PDA detector at 210 nm with gradient mobile phase consisting of phosphate buffer (pH 2.1) and acetonitrile as mobile phase A (98:2) and Acetonitrile as mobile phase B at a flow rate of 0.8 mL/min. This method shows excellent linear response with correlation coefficient (R > 0.9995) for all the three degradation products. The percentage recovery of three degradation products (Kyn, Fum and Pyr) in Amino acid formulation were found to be within the acceptance limit of 97 to 103 %. The precision of the method was less than the maximum allowable limit of percentage of relative standard deviation (% RSD) was = 2. Forced degradation of the drug product was carried out as per ICH guidelines with a view of stability indicating property of the method. The validated dosing range covered from 0.2 mg/L to 4 mg/L of Kyn; 0.1 mg/L to 1.5 mg/L of Fum; and 1.0 mg/L to 80 mg/L of Pyr. The developed method could be presented as a suitable method for separation and quantification of these degradation products in AAs formulations. The method specificity, accuracy, precision, linearity and robustness were proved in validation. The developed and validated HPLC method is simple, realistic and suitable for monitoring of Kyn, Fum, and Pyr in AAs formulations.

Simultaneous Determination of Tryptophan, Kynurenine and Niacin in Serum of Periparturient Dairy Cows by High-Performance Liquid Chromatography with Diode Array Detection

Simultaneous Determination of Tryptophan, Kynurenine and Niacin in Serum of Periparturient Dairy Cows by High-Performance Liquid Chromatography with Diode Array Detection Tryptophan is an essential amino acid and substrate for important biochemical pathways, e.g. generating the neurotransmitter serotonin or building kynurenine and nicotinamide via the kynurenine pathway which can be induced by the enzyme tryptophan 2,3-dioxygenase (TDO) via glucocorticoid hormones or by indoleamine 2,3-dioxygenase (IDO) after immune activation. As the kynurenine to tryptophan ratio is regarded as indicator for an activated immune system, we developed an analytical method for the simultaneous determination of kynurenine, tryptophan and nicotinamide in serum via high-performance liquid chromatography (HPLC) und assessed their suitability as inflammatory markers in dairy cows. Validation parameters have shown that the HPLC method meets the requirements for routine analysis of tryptophan and its metabolites kynurenine and nicotinamide in serum with high linearity within the respective working ranges and limits of quantifications of 0.41 µmol L-1 for nicotinamide, 0.43 µmol L-1 for kynurenine and 3.40 µmol L-1 for tryptophan. The intra-day and inter-day variations were 2.3 and 3.6% (nicotinamide), 3.1 and 6.3% (kynurenine) and 1.9 and 5.2% (tryptophan), respectively. Tryptophan degradation via the kynurenine pathway was investigated in 10 periparturient dairy cows out of two dietary groups during the transition period. The course of tryptophan concentrations in cow serum around calving was similar to those found in pregnant women with a decrease until birth, followed by increased and normalized postpartum tryptophan concentrations. However, rising kynurenine concentrations and a simultaneous incline of the kynurenine to tryptophan ratio during gestation could not be confirmed in our study, apart from a peak of the kynurenine to tryptophan ratio at parturition which might be due to stress hormone-induced activation of hepatic TDO and extrahepatic IDO. Additional measurements of interferon-γ and glucocorticoids as putative inductors of TDO and IDO would be useful to verify the suitability of the kynurenine to tryptophan ratio as an immune marker in the bovine.

Determination of Kynurenine and Tryptophan in Human Plasma by Stacking-Micellar Electrokinetic Chromatography

Kynurenine and tryptophan are important biomarkers for disorders in nervous and immune systems, while the low content of kynurenine in human plasma makes the determination by routine approaches difficult. In this work, an on-line preconcentration method for capillary electrophoresis was developed for the quantification of kynurenine and tryptophan. A concomitant sweeping phenomenon was observed due to the combination of micellar electrokinetic chromatography and stacking. The poor sensitivity in capillary electrophoresis was improved so that the determination of low concentrations of kynurenine became possible. Parameters that may affect the efficiency of the on-line concentration were systematically investigated and optimized. A large volume sample injection was employed for sampling at 100 mbar for 112 sec. A voltage of −20 kV was applied, and the ionic sample matrix was removed with stacking while analytes were kept in the capillary. The voltage was switched to +18 kV and micellar electrokinetic chromatography was performed for separation and determination of kynurenine and tryptophan. Based on peak heights, the concentration factors for kynurenine and tryptophan were 33 and 31, respectively. The analytical performance of the established method was studied; good linearity of kynurenine was observed in the range of 0.3–30 µM (R2 = 0.9988), and 0.3–60 µM (R2 = 0.9963) was obtained for tryptophan. LODs of 0.15 µM for kynurenine and 0.30 µM for tryptophan were obtained. The optimum method was successfully tested in human plasma; kynurenine and tryptophan were well identified, which suggests that this method has potential for the analysis of biological samples.

A Simple LC–MS/MS Method for Determination of Kynurenine and Tryptophan Concentrations in Human Plasma from Hiv-Infected Patients

Indoleamine 2,3-dioxygenase, catalyzing tryptophan (Trp) metabolism through the kynurenine (Kyn) metabolic pathway, plays important roles in immune suppression and the CNS. In this article, we report a simple, rapid and specific LC-MS/MS method for accurate determination of Kyn and Trp concentrations in human plasma from HIV-infected patients. The human plasma sample (100 µl) was mixed with Kyn-d4 and Trp-d5 internal standards and then precipitated with trifluoroacetic acid. The supernatant was directly analyzed by LC-MS/MS. The assay using surrogate matrix calibrators was validated for precision, accuracy, matrix effect, extraction efficiency and stability. Some assay validation issues for endogenous substance bioanalysis using an LC-MS/MS method are discussed. A simple, specific and reproducible LC-MS/MS method has been developed and validated for measuring Kyn and Trp in human plasma samples.

Determination of tryptophan and kynurenine in human plasma by liquid chromatography–electrochemical detection with multi‐wall carbon nanotube‐modified glassy carbon electrode

A novel method was developed for the simultaneous determination of kynurenine and tryptophan by high-performance liquid chromatography with electrochemical detection at multi-wall carbon nanotube (MWCNT)-modified glassy carbon electrode. The separation and detection conditions were optimized. The typical HPLC experiments were conducted by using a reversed-phase ODS column with a mobile phase consisting of stock acetate buffer (pH 5)-methanol (4:1, v/v) using an isocratic elution at the flow rate of 1.0 mL/min. The obtained LODs for kynurenine and tryptophane were 0.5 and 0.4 µmol/L, respectively. The analytical method for human plasma samples was validated and confirmed by LC-UV and LC-MS. The recoveries were in the range of 84.8-110%, and the precision was lower than 5.9%.

Optimization of Zn 2+-containing mobile phase for simultaneous determination of kynurenine, kynurenic acid and tryptophan in human plasma by high performance liquid chromatography

In the present work we have developed a standard-addition HPLC method using a mobile phase containing low concentration of ZnAc 2 to determine physiological level of kynurenine (KYN), kynurenic acid (KYNA) and tryptophan (TRP) in human plasma simultaneously. The method greatly improved the sensitivity of KYNA, the resolution of KYNA and TRP, and avoided clotting risk caused by high concentration of ZnAc 2 in mobile phase. Samples were deproteinized by addition of equal volume of 0.6 mol/L HClO 4. Analytes in supernatants were separated by an Agilent HC-C18 (2) analytical column; an aqueous mobile phase containing 20 mmol/L NaAc, 3 mmol/L ZnAc 2 and 7% acetonitrile at flow rate of 1.0 mL/min. Detections were performed by a variable wavelength detector at wavelength 365 nm for KYN and a fluorescence detector at wavelengths excitation 344 nm and emission 398 nm for KYNA and TRP. Good linear responses were found with r 2 > 0.999 for all analytes within the concentration range of physiological levels. The limit of detection of the developed method was 0.03 μmol/L, 0.9 nmol/L and 0.4 μmol/L for KYN, KYNA and TRP respectively. Recoveries from spiked human plasma were 95.4–99.7% for KYN, 98.9–104% for KYNA and 96.5–100.2% for TRP. All CVs for the repeatability and intermediate precision were less than 5%. We conclude that the developed method is helpful for the research investigations in KYN pathway of TRP metabolism.

Simultaneous determination of tryptophan and kynurenine in plasma samples of children patients with Kawasaki disease by high-performance liquid chromatography with programmed wavelength ultraviolet detection

A simple, fast, sensitive and specific high-performance liquid chromatography (HPLC) method is developed for simultaneous determination of kynurenine (Kyn) and tryptophan (Trp) with ultraviolet (UV) detection setting programmed wavelength. The separation was carried out on an Agilent Hypersil ODS column (125 mm × 4.0 mm, 5 μm) in less than 6 min and the eluate was monitored by the programmed wavelength detection setting at 360 nm from 0 min to 4 min for Kyn, and at 278 nm from 4 min to 6 min for Trp in a single run with UV detector. The linearities of the method were from 0.20 μmol/L to 21.2 μmol/L for Kyn and 2.25–678.0 μmol/L for Trp, and the detection limits were 0.028 μmol/L for Kyn and 0.053 μmol/L for Trp, respectively. Satisfactory precisions and recoveries were obtained by this method. The assay was employed to analyze plasma samples of children patients with Kawasaki disease (KD). The result showed great difference between Kawasaki disease and control group.

Determination of kynurenine in serum by high‐performance liquid with on‐column fluorescence derivatization

Determination of kynurenine in serum by high‐performance liquid with on‐column fluorescence derivatization

Simultaneous Determination of Kynurenine and Tryptophan in Serum by High Performance Liquid Chromatography

A method was established for the simultaneous determination of kynurenine (Kyn) and tryptophan (Trp) in serum by high performance liquid chromatography-ultraviolet detection (HPLC-UV). It employed a Symmetry Shield RP-C18 column (150 mm x 3.9 mm i.d., 5 microm) and a mobile phase of 15 mmol/L sodium acetate-acetic acid solution containing 2.7% (v/v) acetonitrile (pH 3.6) at a flow rate of 1.0 mL/min. The ultraviolet detector was operated at 225 nm. Serum samples were first precipitated with a 5.0% perchloric acid solution, then centrifuged to remove protein residue and finally analyzed by HPLC. The retention time of Kyn was 3.5 min, the linear range of the method was from 0.098 to 49 micromol/L, and the detection limit was 0.02 micromol/L. The recoveries of Kyn were from 90.82% to 93.45%, the intraday and interday variations were 2.37% and 3.66%, respectively. The retention time of Trp was 8.1 min, the linear range of the method was from 4.9 to 490 micromol/L, and the detection limit was 0.20 micromol/L. The recoveries of Trp were from 95.51% to 98.67%, the intraday and interday variations were 1.50% and 2.65%, respectively. The method is simple, fast, accurate, and suitable for routine analysis.

A new, simple, bioassay for human IFN-γ

IFN-γ induces the production of N-formyl-kynurenine from L-tryptophan in various cell types by the induction of the enzyme indoleamine 2,3-dioxygenase (IDO). The IFN-γ induced IDO activity in the glioblastoma cell line 86HG39 and cells of clone 2D9 derived from this cell line was found to be greater than that in Hela cells and U373MG cells. Consequently 2D9 cells were used in all subsequent experiments. The determination of kynurenine in the supernatant of IFN-γ activated cells was performed photometrically using a microplate reader. It was found that the amount of kynurenine produced was directly proportional to the amount of IFN-γ used to activate cells. The detection limit for IFN-γ of this assay was 20 U/ml. The induction of L-tryptophan degradation was specific for IFN-γ since neither IFN-α, IFN-β, IL-1, IL-2, IL-6, GM-CSF nor TNFα induced the production of detectable amounts of kynurenine by 86HG39 and 2D9 cells. Furthermore, a mab directed against IFN-γ was able to completely block the IFN-γ induced IDO activation. This bioassay was used to determine the IFN-γ content of supernatants harvested from toxoplasma antigen specific human T cell lines and clones. This assay gave reproducible results which correlated well with the IFN-γ content detected in the same samples using a commercially available ELISA kit. Furthermore in the case of T cell supernatant stimulated 2D9 cells a mab directed against IFN-γ was able to completely block IDO induction. We conclude that the measurement of kynurenine production induced by IFN-γ can be used to determinate IFN-γ content. This is a simple bioassay which can be performed with standard laboratory equipment.

Fluorimetric determination of kynurenine in human serum by high-performance liquid chromatography coupled with post-column photochemical reaction with hydrogen peroxide

A high-performance liquid chromatographic method involving post-column photochemical reaction and fluorimetric detection has been developed for the determination of kynurenine in serum. Kynurenine was separated on a column of Capcell Pak C 18 (resistant to pH 10). The mobile phase consisted of 0.05 M Na 2B 4O 7-0.1 M KH 2PO 4 buffer (pH 8.5)-ethanol (97:3, v/v) containing 60 m M hydrogen peroxide. The post-column reagent, containing 60% (v/v) ethanol, was mixed with the mobile phase, which was irradiated with ultraviolet light to induce fluorescence. The recovery of kynurenine was 95.9 ± 5.0% ( n = 6). The method allows the determination of as little as 2 pmol of kynurenine.

Determination of serum kynurenine and hepatic tryptophan dioxygenase activity by high-performance liquid chromatography

The status of the oxidative metabolism of l-tryptophan is usually evaluated by the determination of tryptophan metabolites in serum or urine and/or the activities of various oxidative enzymes in tissues. I have developed assays for serum kynurenine and hepatic tryptophan dioxygenase (TDO) activity based on the determination of kynurenine (KYN) by isocratic, reverse phase HPLC with spectrophotometric detection at 365 nm. Sample pretreatment prior to HPLC requires little more than perchloric acid precipitation of serum or a TDO incubation mixture. The analytical recovery for the serum assay was 101 ± 2%, while the run-to-run coefficient of variation at normal KYN levels was approximately 8%. Serum KYN levels in 40 apparently healthy fasting humans were normally distributed and ranged from 0.27 to 0.69 μg/ml ( χ ± SD: 0.47 ± 0.1). Serum KYN in predialysis specimens from a group of 20 patients with chronic renal failure demonstrated a highly significant increase ( χ ± SD: 0.83 ± 0.35 μg/ml; P < 0.001) as compared to the reference population. It is possible that such an increase might contribute to the pathophysiology of the uremic state. The analytical recovery of KYN from TDO incubation mixtures was approximately 90%. There was no evidence for the onward metabolism of KYN during the assay of whole liver homogenates. The mean (±SD) TDO activity of rat liver homogenates preincubated with ascorbate and hematin was 2.3 ± 0.8 μmol/h/g wet wt (30°C). The sensitivity, specificity, and convenience of these two methods suggest that they are suitable for routine use in the investigation of the biology and pathology of oxidative tryptophan metabolism.

Determination of kynurenine in human plasma by gas chromatography/negative ion chemical ionization mass spectrometry.

Determination of kynurenine in human plasma by gas chromatography/negative ion chemical ionization mass spectrometry.

Determination of kynurenine in serum by high-performance liquid chromatography after enzymatic conversion to 3-hydroxykynurenine

A method for the determination of the physiological level of kynurenine in human serum based upon conversion of kynurenine to 3-hydroxykynurenine by enzymatic reaction with the mitochondrial fraction and NADPH and analysis by reversed-phase high-performance liquid chromatography with electrochemical detection is described. Tryptophan gave no interference. For one analysis, 0.2 ml of serum was sufficient, compared with the large volume (5.0 ml) required for other methods.

Determination of kynurenine by a simple gas—liquid chromatographic method applicable to urine, plasma, brain and cerebrospinal fluid

A simple, sensitive and specific method for the determination of kynurenine is described. This is based on alkaline cleavage of kynurenine, followed by solvent extraction, trifluoroacetylation and gas—liquid chromatography with electron capture detection. Using this method kynurenine has been determined in urine and plasma, and for the first time in brain and cerebrospinal fluid. Increases in kynurenine in brain, plasma and urine are demonstrated following tryptophan administration to man and rat.