Kynurenic Acid Concentrations Research Articles

#2194 Targeted metabolomics analysis identifies a specific metabolomic profile in patients with early chronic kidney disease

Abstract Background and Aims The complex interconnections between various metabolites and the kidney play a major role in the pathogenesis of chronic kidney disease (CKD). Hence, by the assessment of the relationship between metabolites and the kidney, more accurate biomarkers could be discovered to prevent and detect CKD at an early stage. The primary goal of this study was to find new serum and urine biomarkers for the diagnosis of early CKD by conducting a metabolomic profiling through targeted metabolomics of both blood and urine in patients with CKD. Method This cross-sectional study comprised 80 patients with CKD who were categorized into six subgroups (G1-G6) based on their eGFR according to the KDIGO Guidelines. Additionally, 20 healthy individuals were included as healthy control participants (group C). Ultra-high-performance liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry was used to perform serum and urine metabolomic profiling. The 6 metabolites evaluated by this study were selected based on the results of a previous untargeted analysis research. Both blood and urine samples underwent multivariate analysis, which was subsequently followed by univariate analysis and quantitative analysis. Initially, the PLSDA score plot and VIP score were utilized. The application of the cross-validation procedure to the initial set of 6 molecules provided outstanding results, including high accuracy, high R² values, and a significant Q2 value. Hence, the model might be deemed as predictive. Random Forest analysis was utilized to conduct biomarker analysis and prediction. The specificity and sensitivity of the compounds identified as possible biomarkers were assessed using the Receiver Operating Characteristic (ROC) curve and the area under the curve (AUC). Results There were strong positive correlations between eGFR and serum levels of Arginine (p < 0.05) and L-Acetylcarnitine (p < 0.05). Of note, the amounts of L-Methionine, L-Phenylalanine, Kynurenic acid, and Indoxyl sulfate in serum showed a progressive increase from group C to subgroups G1-G5. Furthermore, L-Methionine, L-Phenylalanine, Kynurenic acid, and Indoxyl sulfate exhibited a negative correlation with eGFR (p < 0.05). In addition, there was a positive correlation between the levels of urine Arginine, L-Methionine, and L-Phenylalanine and eGFR (p < 0.05). Urine levels of Kynurenic acid, Indoxyl sulfate, and L-Acetylcarnitine were higher in groups G4 and G5, as compared to groups G1-G3b and C, respectively. The urinary concentrations of Indoxyl sulfate, Kynurenic acid, Indoxyl sulfate, and L-Acetylcarnitine exhibited a negative correlation with eGFR (p < 0.05). Conclusion The current study shows that serum and urinary metabolites levels in patients with CKD display an alteration in production, secretion, and excretion in all CKD stages. There was demonstrated a distinct metabolomic profile in the course of CKD which points to novel biomarkers useful for the diagnosis and progression evaluation of CKD from its early stages.

Longitudinal associations of plasma kynurenines and ratios with fatigue and quality of life in colorectal cancer survivors up to 12 months post-treatment.

Fatigue is prevalent in colorectal cancer (CRC) survivors, impacting their health-related quality of life (HRQoL). Inflammation-induced activation of the kynurenine pathway may play a role in cancer-related fatigue and HRQoL, but evidence is scarce. Therefore, we aimed to investigate longitudinal associations of plasma tryptophan, kynurenines, and ratios with fatigue and HRQoL in CRC survivors up to 12 months post-treatment. Repeated measurements at 6 weeks, 6 months, and 12 months post-treatment were performed in 249 stage I-III CRC survivors. Plasma tryptophan and eight kynurenines were analyzed using liquid chromatography-tandem mass spectrometry (LC/MS-MS). Fatigue and HRQoL outcomes were evaluated using validated questionnaires. Confounder-adjusted linear mixed models were conducted to analyze longitudinal associations, with false discovery rate (FDR) correction. Higher tryptophan (Trp), kynurenic acid (KA), and xanthurenic acid (XA) concentrations, as well as a higher kynurenic acid-to-quinolinic acid ratio (KA/QA), were associated with less fatigue and better functioning, while a higher kynurenine-to-tryptophan ratio (KTR) and 3-hydroxykynurenine ratio (HKr) were associated with more fatigue and worse functioning. Finally, higher KA and XA concentrations and a higher KA/QA ratio were associated with a higher overall HRQoL summary score, while a higher HKr was associated with a lower overall HRQoL summary score. In conclusion, we observed that tryptophan and several kynurenines were longitudinally associated with fatigue and HRQoL in CRC survivors up to 12 months post-treatment. Future research is needed to validate our findings and explore the potential of the kynurenine pathway as intervention target for reducing fatigue and enhancing HRQoL after CRC treatment.

The effect of valproate on the amino acids, monoamines, and kynurenic acid concentrations in brain structures involved in epileptogenesis in the pentylenetetrazol-kindled rats.

The study aimed to assess the influence of a single valproate (VPA) administration on inhibitory and excitatory neurotransmitter concentrations in the brain structures involved in epileptogenesis in pentylenetetrazol (PTZ)-kindled rats. Adult, male Wistar rats were kindled by repeated intraperitoneal (ip) injections of PTZ at a subconvulsive dose (30mg/kg, three times a week). Due to the different times required to kindle the rats (18-22 injections of PTZ), a booster dose of PTZ was administrated 7days after the last rats were kindled. Then rats were divided into two groups: acute administration of VPA (400mg/kg) or saline given ip. The concentration of amino acids, kynurenic acid (KYNA), monoamines, and their metabolites in the prefrontal cortex, hippocampus, amygdala, and striatum was assessed by high-pressure liquid chromatography (HPLC). It was found that a single administration of VPA increased the gamma-aminobutyric acid (GABA), tryptophan (TRP), 5-hydroxyindoleacetic acid (5-HIAA), and KYNA concentrations and decreased aspartate (ASP) levels in PTZ-kindled rats in the prefrontal cortex, hippocampus, amygdala and striatum. Our results indicate that a single administration of VPA in the PTZ-kindled rats restored proper balance between excitatory (decreasing the level of ASP) and inhibitory neurotransmission (increased concentration GABA, KYNA) and affecting serotoninergic neurotransmission in the prefrontal cortex, hippocampus, amygdala, and striatum.

MM165 - A Small Hybrid Molecule Modulates the Kynurenine Pathway and Attenuates Lipopolysaccharide-Induced Memory Deficits and Inflammation.

Cognitive dysfunctions are now recognized as core symptoms of various psychiatric disorders e.g., major depressive disorder. Sustained immune activation may leads to cognitive dysfunctions. Proinflammatory cytokines shunt the metabolism of tryptophan towards kynurenine and quinolinic acid may accumulate at toxic concentrations. This acid triggers an increase in neuronal nitric oxide synthase function and promotes oxidative stress. The searching for small molecules that can regulate tryptophan metabolites produced in the kynurenic pathway has become an important goal in developing treatments for various central nervous system diseases with an inflammatory component. Previously we have identified a small hybrid molecule - MM165 which significantly reduces depressive-like symptoms caused by inflammation induced by lipopolysaccharide administration. In the present study, we investigated whether this compound would mitigate cognitive deficits induced by lipopolysaccharide administration and whether treatment with it would affect the plasma or brain levels of quinolinic acid and kynurenic acid. Neuroinflammation was induced in rats by administering lipopolysaccharide at a dose of 0.5mg/kg body weight for 10 days. We conducted two tests: novel object recognition and object location, to assess the effect on memory impairment in animals previously treated with lipopolysaccharide. In plasma collected from rats, the concentrations of C-reactive protein and tumor necrosis factor alfa were determined. The concentrations of kynurenic acid and quinolinic acid were determined in plasma and homogenates obtained from the cerebral cortex of rats. Interleukin 6 in the cerebral cortex of rats was determined. Additionally, the body and spleen mass and spontaneous activity were measured in rats. Our study shows that MM165 may mitigate cognitive deficits induced by inflammation after administration of lipopolysaccharide and alter the concentrations of tryptophan metabolites in the brain. Compounds exhibiting a mechanism of action analogous to that of MM165 may serve as foundational structures for the development of a new class of antidepressants.

The Kynurenine Pathway in Attention-Deficit/Hyperactivity Disorder: A Systematic Review and Meta-Analysis of Blood Concentrations of Tryptophan and Its Catabolites.

Preliminary evidence shows that the kynurenine pathway (KP) may be altered in attention-deficit/hyperactivity disorder (ADHD). We thus conducted a systematic review and meta-analysis exploring the peripheral blood concentrations of tryptophan catabolites (TRYCATs) in people with ADHD. We searched the main electronic databases up to 7th December 2023. Standardised mean differences (SMDs) with 95% confidence intervals (95%CIs) were used to compare TRYCAT concentrations between participants with ADHD and healthy controls (HCs). We included eight studies. Random-effects meta-analyses found higher kynurenine (SMD = 0.56; 95%CI: 0.04 to 1.08; p = 0.033; I2 = 90.3%) and lower kynurenic acid (SMD = -0.33; 95%CI: -0.49 to -0.17; p < 0.001; I2 = 0%) concentrations in people with ADHD compared to HCs. Additional analyses on drug-free children with ADHD showed higher tryptophan (SMD = 0.31; 95%CI: 0.11 to 0.50; p = 0.002; I2 = 0%) and kynurenine (SMD = 0.74; 95%CI: 0.30 to 1.17; p < 0.001; I2 = 76.5%), as well as lower kynurenic acid (SMD = -0.37; 95%CI: -0.59 to -0.15; p < 0.001; I2 = 0%) blood levels, as compared to HCs. Despite some limitations, our work provides preliminary evidence on KP alterations in ADHD that may suggest decreased neuroprotection. Further research is needed to clarify the role of the KP in ADHD.

Adult rats sired by obese fathers present learning deficits associated with epigenetic and neurochemical alterations linked to impaired brain glutamatergic signaling.

Offspring of obese mothers are at high risk of developing metabolic syndrome and cognitive disabilities. Impaired metabolism has also been reported in the offspring of obese fathers. However, whether brain function can also be affected by paternal obesity has barely been examined. This study aimed to characterize the learning deficits resulting from paternal obesity versus those induced by maternal obesity and to identify the underlying mechanisms. Founder control and obese female and male Wistar rats were mated to constitute three first-generation (F1) experimental groups: control mother/control father, obese mother/control father, and obese father/control mother. All F1 animals were weaned onto standard chow and underwent a learning test at 4 months of age, after which several markers of glutamate-mediated synaptic plasticity together with the expression of miRNAs targeting glutamate receptors and the concentration of kynurenic and quinolinic acids were quantified in the hippocampus and frontal cortex. Maternal obesity induced a severe learning deficit by impairing memory encoding and memory consolidation. The offspring of obese fathers also showed reduced memory encoding but not impaired long-term memory formation. Memory deficits in offspring of obese fathers and obese mothers were associated with a down-regulation of genes encoding NMDA glutamate receptors subunits and several learning-related genes along with impaired expression of miR-296 and miR-146b and increased concentration of kynurenic acid. Paternal and maternal obesity impair offspring's learning abilities by affecting different processes of memory formation. These cognitive deficits are associated with epigenetic and neurochemical alterations leading to impaired glutamate-mediated synaptic plasticity.

Decreased Plasma Levels of Kynurenine and Kynurenic Acid in Previously Treated and First-Episode Antipsychotic-Naive Schizophrenia Patients.

Tryptophan (TRP) catabolites exert neuroactive effects, with the plethora of evidence suggesting that kynurenic acid (KYNA), a catabolite of the kynurenine pathway (KP), acts as the regulator of glutamate and acetylcholine in the brain, contributing to the schizophrenia pathophysiology. Newer evidence regarding measures of KP metabolites in the blood of schizophrenia patients and from the central nervous system suggest that blood levels of these metabolites by no means could reflect pathological changes of TRP degradation in the brain. The aim of this study was to investigate plasma concentrations of TRP, kynurenine (KYN) and KYNA at the acute phase and remission of schizophrenia in a prospective, case-control study of highly selected and matched schizophrenia patients and healthy individuals. Our study revealed significantly decreased KYN and KYNA in schizophrenia patients (p < 0.001), irrespective of illness state, type of antipsychotic treatment, number of episodes or illness duration and no differences in the KYN/TRP ratio between schizophrenia patients and healthy individuals. These findings could be interpreted as indices that kynurenine pathway might not be dysregulated in the periphery and that other factors contribute to observed disturbances in concentrations, but as our study had certain limitations, we cannot draw definite conclusions. Further studies, especially those exploring other body compartments that participate in kynurenine pathway, are needed.

Abstract 17256: Perturbations of Tryptophan Catabolism Through the Kynurenine Pathway Are Associated With Prolonged Mechanical Ventilation and Length of Stay After Stage 2 Palliation for Single Ventricle Heart Disease

Background: Infants with single ventricle heart disease (SVHD) have perturbed kynurenine pathway (KP) function prior to stage 2 palliation. Buildup of kynurenic acid—a byproduct of this pathway—is linked to worse outcomes post stage 2. To date, perturbations along the entire KP have not been delineated in SVHD infants at the time of stage 2 operation. Methods: This is a single center prospective cohort study of 75 infants with SVHD undergoing stage 2 palliation and 50 controls. Targeted analysis of KP metabolites was performed on serum samples via tandem mass spectroscopy prior to stage 2 (baseline) and post-operatively at 2, 24, and 48 hours. Results: Of the 9 KP metabolites, 7 were different between controls and SVHD at baseline (Figure 1A). SVHD infants had lower tryptophan and serotonin; and higher kynurenic acid, 3-hydroxykynurenine, anthranilic acid, picolinic acid, and 3-hydroxyanthranilic acid. After stage 2, there were time related changes in all KP metabolites except tryptophan (Figure 1B). Metabolite concentrations at baseline and 2 hours post-op were poorly correlated with outcomes. At 24 hours, decreases in tryptophan and serotonin along with elevations in kynurenine, kynurenic acid, 3-hydroxykynurine, and quinolinic acid concentrations correlated with outcomes (Figure 1C). At 48 hours, kynurenic acid remained positively correlated with all 3 outcomes (length of stay, hypoxia burden, and intubation length). Conclusions: We demonstrated numerous perturbations in the KP pathway at baseline in SVHD infants compared to controls. We also profiled the trajectory of each metabolite over the 48 hours post stage 2. The link between KP intermediates and poor outcomes post-operatively—but not at baseline—demonstrates that the acute, peri-operative changes in tryptophan catabolism in response to cardiopulmonary bypass are more important to tolerating new stage 2 physiology than chronic changes during the interstage period.

Longitudinal associations of macronutrient and micronutrient intake with plasma kynurenines in colorectal cancer survivors up to 12 months posttreatment

BackgroundThe tryptophan-kynurenine pathway is increasingly recognized to play a role in health-related quality of life (HRQoL) after cancer. Because tryptophan is an essential amino acid, and vitamins and minerals act as enzymatic cofactors in the tryptophan-kynurenine pathway, a link between diet and kynurenines is plausible. ObjectivesThis study aimed to investigate the longitudinal associations of macronutrient and micronutrient intake with metabolites of the kynurenine pathway in colorectal cancer (CRC) survivors up to 12 mo posttreatment. MethodsIn a prospective cohort of stage I–III CRC survivors (n = 247), repeated measurements were performed at 6 wk, 6 mo, and 12 mo posttreatment. Macronutrient and micronutrient intake was measured by 7-d dietary records. Plasma concentrations of tryptophan and kynurenines were analyzed using liquid chromatography tandem mass spectrometry (LC/MS-MS). Longitudinal associations were analyzed using linear mixed models adjusted for sociodemographic, clinical, and lifestyle factors. ResultsAfter adjustment for multiple testing, higher total protein intake was positively associated with kynurenic acid (KA) (β as standard deviation [SD] change in KA concentration per 1 SD increase in total protein intake: 0.12; 95% CI: 0.04, 0.20), xanthurenic acid (XA) (standardized β: 0.22; 95% CI: 0.11, 0.33), 3-hydroxyanthranilic acid (HAA) (standardized β: 0.15; 95% CI: 0.04, 0.27) concentrations, and the kynurenic acid-to-quinolinic acid ratio (KA/QA) (standardized β: 0.12; 95% CI: 0.02,0.22). In contrast, higher total carbohydrate intake was associated with lower XA concentrations (standardized β: −0.18; 95% CI: −0.30, −0.07), a lower KA/QA (standardized β: −0.23; 95% CI: −0.34, −0.13), and a higher kynurenine-to-tryptophan ratio (KTR) (standardized β: 0.20; 95% CI: 0.10, 0.30). Higher fiber intake was associated with a higher KA/QA (standardized β: 0.11; 95% CI: 0.02, 0.21) and a lower KTR (standardized β: −0.12; 95% CI: −0.20, −0.03). Higher total fat intake was also associated with higher tryptophan (Trp) concentrations (standardized β: 0.18; 95% CI: 0.06, 0.30) and a lower KTR (standardized β: −0.13; 95% CI: −0.22, −0.03). For micronutrients, positive associations were observed for zinc with XA (standardized β: 0.13; 95% CI: 0.04, 0.21) and 3-hydroxykynurenine (HK) (standardized β: 0.12; 95% CI: 0.03, 0.20) concentrations and for magnesium with KA/QA (standardized β: 0.24; 95% CI: 0.13, 0.36). ConclusionsOur findings show that intake of several macronutrients and micronutrients is associated with some metabolites of the kynurenine pathway in CRC survivors up to 12 mo posttreatment. These results may be relevant for enhancing HRQoL after cancer through potential diet-induced changes in kynurenines. Further studies are necessary to confirm our findings.

Plasma Tryptophan-Kynurenine Pathway Metabolites and Risk for Progression to End-Stage Kidney Disease in Patients With Type 2 Diabetes.

We sought to study the associations between plasma metabolites in the tryptophan-kynurenine pathway and the risk of progression to end-stage kidney disease (ESKD) in patients with type 2 diabetes. Plasma tryptophan, kynurenine, 3-hydroxykynurenine, kynurenic acid, and xanthurenic acid concentrations were measured in discovery (n = 1,915) and replication (n = 346) cohorts. External validation was performed in Chronic Renal Insufficiency Cohort (CRIC) participants with diabetes (n = 1,312). The primary outcome was a composite of incident ESKD (progression to estimated glomerular filtration rate [eGFR] <15 mL/min/1.73 m2, sustained dialysis, or renal death). The secondary outcome was annual eGFR decline. In the discovery cohort, tryptophan was inversely associated with risk for ESKD, and kynurenine-to-tryptophan ratio (KTR) was positively associated with risk for ESKD after adjustment for clinical risk factors, including baseline eGFR and albuminuria (adjusted hazard ratios [HRs] 0.62 [95% CI 0.51, 0.75] and 1.48 [1.20, 1.84] per 1 SD). High levels of kynurenic acid and xanthurenic acid were associated with low risks of ESKD (0.74 [0.60, 0.91] and 0.74 [0.60, 0.91]). Consistently, high levels of tryptophan, kynurenic acid, and xanthurenic acid were independently associated with a slower eGFR decline, while a high KTR was predictive of a faster eGFR decline. Similar outcomes were obtained in the replication cohort. Furthermore, the inverse association between kynurenic acid and risk of ESKD was externally validated in CRIC participants with diabetes (adjusted HR 0.78 [0.65, 0.93]). Accelerated catabolism of tryptophan in the kynurenine pathway may be involved in progressive loss of kidney function. However, shunting the kynurenine pathway toward the kynurenic acid branch may potentially slow renal progression.

Effect of Yarrowia lipolytica yeast biomass with increased kynurenic acid content on selected metabolic indicators in mice.

The unconventional yeast species Yarrowia lipolytica is a valuable source of protein and many other nutrients. It can be used to produce hydrolytic enzymes and metabolites, including kynurenic acid (KYNA), an endogenous metabolite of tryptophan with a multidirectional effect on the body. The administration of Y. lipolytica with an increased content of KYNA in the diet may have a beneficial effect on metabolism, which was evaluated in a nutritional experiment on mice. In the dry biomass of Y. lipolytica S12 enriched in KYNA (high-KYNA yeast) and low-KYNA (control) yeast, the content of KYNA was determined by high-performance liquid chromatography. Then, proximate and amino acid composition and selected indicators of antioxidant status were compared. The effect of 5% high-KYNA yeast content in the diet on the growth, hematological and biochemical indices of blood and the redox status of the liver was determined in a 7-week experiment on adult male mice from an outbred colony derived from A/St, BALB/c, BN/a and C57BL/6J inbred strains. High-KYNA yeast was characterized by a greater concentration of KYNA than low-KYNA yeast (0.80 ± 0.08 vs. 0.29 ± 0.01 g/kg dry matter), lower content of crude protein with a less favorable amino acid composition and minerals, higher level of crude fiber and fat and lower ferric-reducing antioxidant power, concentration of phenols and glutathione. Consumption of the high-KYNA yeast diet did not affect the cumulative body weight gain per cage, cumulative food intake per cage and protein efficiency ratio compared to the control diet. A trend towards lower mean corpuscular volume and hematocrit, higher mean corpuscular hemoglobin concentration and lower serum total protein and globulins was observed, increased serum total cholesterol and urea were noted. Its ingestion resulted in a trend towards greater ferric-reducing antioxidant power in the liver and did not affect the degree of liver lipid and protein oxidation. The improvement of the quality of Y. lipolytica yeast biomass with increased content of KYNA, including its antioxidant potential, would be affected by the preserved level of protein and unchanged amino acid profile. It will be worth investigating the effect of such optimized yeast on model animals, including animals with metabolic diseases.

Differential Effects of Hypothermia and SZR72 on Cerebral Kynurenine and Kynurenic Acid in a Piglet Model of Hypoxic-Ischemic Encephalopathy.

Kynurenic acid (KYNA), an endogenous neuroprotectant with antiexcitotoxic, antioxidant, and anti-inflammatory effects, is synthesized through the tryptophan-kynurenine (KYN) pathway. We investigated whether brain KYN or KYNA levels were affected by asphyxia in a translational piglet model of hypoxic-ischemic encephalopathy (HIE). We also studied brain levels of the putative blood-brain barrier (BBB) permeable neuroprotective KYNA analogue SZR72, and whether SZR72 or therapeutic hypothermia (TH) modified KYN or KYNA levels. KYN, KYNA, and SZR72 levels were determined using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry in five brain regions 24 h after 20 min of asphyxia in vehicle-, SZR72- and TH-treated newborn piglets (n = 6-6-6) and naive controls (n = 4). Endogenous brain KYN levels (median range 311.2-965.6 pmol/g) exceeded KYNA concentrations (4.5-6.0 pmol/g) ~100-fold. Asphyxia significantly increased cerebral KYN and KYNA levels in all regions (1512.0-3273.9 and 16.9-21.2 pmol/g, respectively), increasing the KYN/Tryptophan-, but retaining the KYNA/KYN ratio. SZR72 treatment resulted in very high cerebral SZR72 levels (13.2-33.2 nmol/g); however, KYN and KYNA levels remained similar to those of the vehicle-treated animals. However, TH virtually ameliorated asphyxia-induced elevations in brain KYN and KYNA levels. The present study reports for the first time that the KYN pathway is altered during HIE development in the piglet. SZR72 readily crosses the BBB in piglets but fails to affect cerebral KYNA levels. Beneficial effects of TH may include restoration of the tryptophan metabolism to pre-asphyxia levels.

Kynurenic Acid Levels and Kynurenine Aminotransferase I, II and III Activities in Ganglia, Heart and Liver of Snail Helix Pomatia.

Kynurenic acid (KYNA), a tryptophan metabolite along the kynurenine pathway, is an endogenous antagonist of glutamate ionotropic excitatory amino acid (EAA) receptors and the α7 nicotinic acetylcholine receptor (nAChR). The involvement of KYNA in various pathological conditions and during the aging process is significant. KYNA synthesis from L-kynurenine (L-KYN), through the action of several kynurenine aminotransferases (KATs), is present in the central nervous system (CNS) and periphery of mammals. We were interested in investigating the ability of the brain and peripheral organs of Helix pomatia snails to synthesize KYNA, in an in vitro study. In comparative studies between rat and snail, we looked for the synthesis of KYNA in the liver. We then looked for an effect of age on KYNA synthesis. Ten shell parameters of the Helix pomatia snail were used to establish an Age Rating Scale (ARS), i.e. body weight, shell weight, shell length, width and height, shell opening length and width, lip width, number of shell turns and external shell growth rings. An age of the snails was determined according to the ARS and the snails were divided into three groups, i.e. young, middle and old age. Homogenates of dissected regions, i.e. cerebral ganglia (CG), subpharyngeal ganglia (SG) consisting of pedal, visceral and pleural ganglia, heart and liver, were examined. KYNA was measured by high performance liquid chromatography (HPLC) and KAT activities were measured by an enzymatic method. With respect to ARS, an evaluation of the age of the snails between young (1-2 years), middle (5-7 years) and old (9-13 years) showed significant differences (p<0.001). Analysis of KYNA levels in different snail tissues, i.e. CG, SG, heart and liver, showed an occurrence in the low femtomolar range. Marked and significant increases of KYNA were found in the liver of middle and old age groups. In the SG, KYNA decreased significantly with age. There were no differences in KYNA levels between groups in CG and heart. The lowest KAT activity was found in CG and SG (5 pmol/mg/h), while in heart and liver the values were visibly higher (between 8 and 80 pmol/mg/h). Only in the liver, and exceptionally only for KAT I, the activity increased significantly with age, i.e. up to 14 years. No age-related changes in KAT I, II and III activities were found in CG and SG. Snail liver shows a different pattern of KAT activities compared to the rat liver. Regions of the CNS and periphery of the snail Helix pomatia are able to synthesize KYNA due to KAT activities. In the snail liver, KAT I activity increased with age. Notably, there was no age-related increase in KAT activities in the heart and especially in the CNS of Helix pomatia, indicating significant differences from mammals. A moderate KYNA metabolism in the Helix pomatia snail in the periods studied, up to 14 years, could be a physiological phenomenon that protects organs from possible functional insufficiency due to high KYNA levels, as has been suggested. It is reasonable to search for the factor(s) that could regulate the concentration of KYNA in the body of the snail.

LC–MS/MS-based quantification of tryptophan, kynurenine, and kynurenic acid in human placental, fetal membranes, and umbilical cord samples

Tryptophan breakdown metabolites formed along the kynurenine pathway play a significant role in pregnancy and fetal development. To understand their involvement, it is crucial to quantify the levels of tryptophan (TRP), kynurenine (KYN), and kynurenic acid (KYNA) in relevant biological samples such as the placenta, fetal membranes, and umbilical cord. This study used liquid chromatography-tandem mass spectrometry (LC–MS/MS) to determine TRP, KYN, and KYNA levels. The LC–MS/MS method was optimized for high sensitivity and specificity, demonstrating good reproducibility with a precision of < 10% CV and an accuracy of 85–115%. The lower limit of quantification for both TRP and KYN was 0.5 µg/ml, while for KYNA, it was 0.5 ng/mL. The method exhibited linearity within the examined range of concentrations in the homogenate, ranging from 0.5 to 30 µg/ml for TRP and KYN and from 0.5 to 25 ng/ml for KYNA. Using this method, we found significant differences in the concentrations of these substances in investigated maternal–fetal compartments. Placenta samples exhibited higher KYN and lower KYNA concentrations than the umbilical cord and fetal membrane, indicating a potentially important role for kynurenines in late pregnancy. Collectively, this finding may facilitate further research and provide inside into the involvement of the kynurenine pathway of TRP metabolism in fetal development.

#4592 A HIGH PROTEIN DIET INDUCES DEPRESSION-LIKE BEHAVIOR IN CKD RODENT MODELS

Abstract Background and Aims The prevalence of depression among patients with Chronic Kidney Disease (CKD) is higher than the general population. Around 21-27% of CKD patients is estimated to be affected by depression. Several studies hypothesized that mood disorders may be due to reduced family support, older age, low educational status and low quality of life. However, adults with CKD are also known to exhibit altered tryptophan metabolism causing reduced plasma levels of tryptophan and elevated kynurenine and kynurenic acid. Moreover, CKD patients show increased levels of indoxyl sulfate and indole-3-acetic acid, two protein-bound uremic toxins deriving from the gut microbiome metabolism of tryptophan. We postulate that depression is not just due to reduced quality of life but also to a biological reason. In addition, we hypothesize that a high protein diet may influence tryptophan metabolism and therefore affect depression symptoms. Method Eighteen Sprague-Dawley male rats (Janvier, Le Genest- St Isle, France), 7-8 weeks old, were induced with CKD utilizing 5/6 nephrectomy and were randomly assigned to a low protein (n = 10) or a high protein (n = 8) diet. A sham-operated control group for each diet was used (n = 7 respectively). The splash test was adopted to assess depressive symptoms in rats. In short, the splash test is a behavioral test consisting in sprinkling a 10% sucrose solution on the dorsal coat of a rat. Latency (time before the initiation of grooming) and duration of grooming were recorded for 5 min and used as an indication of self-care behavior. Delayed and reduced grooming is considered as a depression-like behavior of rats. Blood and urine creatinine were determined with standard laboratory techniques. Total plasmatic concentrations of tryptophan, kynurenine, kynurenic acid and indoxyl sulfate were measured using LC-MS/MS. Results The latency before the starting of the grooming behavior was significantly higher (p = .0499) in CKD rats on a high protein diet compared to sham rats on the same diet while no difference was seen between the low protein groups. CKD rats reported significantly higher plasma levels of indoxyl sulfate and kynurenic acid and reduced tryptophan. Interestingly, kynurenic acid plasma levels were significantly reduced when administering a low protein diet to CKD rats (p = .0025). Among CKD rats, the latency to the grooming behavior was correlated with plasmatic levels of creatinine clearance (Spearman r = -.57, p value = .019), indoxyl sulfate (Spearman r = .64, p value = .007), and kynurenic acid (Spearman r = .63, p value = .009). The total grooming time was shorter in both CKD groups compared to the sham rats, but not significantly (p = .4881 and p = .4312, respectively). Conclusion These results suggest that biological factors contribute to depression onset and that the decline of kidney function and the accumulation of uremic toxins are potential risk factors for depression. Dietary interventions such as a low protein diet, are potential strategies to lower the plasmatic levels of uremic toxins which may alleviate depressive-like symptoms.

Higher concentrations of kynurenic acid in CSF are associated with the slower clinical progression of Alzheimer's disease.

The kynurenine pathway's (KP) malfunction is closely related to Alzheimer's disease (AD), for antagonistic kynurenic acid (KA) and agonistic quinolinic acid act on the N-methyl-D-aspartate receptor, a possible therapeutic target in treating AD. In our longitudinal case-control study, KP metabolites in the cerebrospinal fluid were analyzed in 311 patients with AD and 105 cognitively unimpaired controls. Patients with AD exhibited higher concentrations of KA (β=0.18, P<0.01) and picolinic acid (β=0.20, P<0.01) than the controls. KA was positively associated with tau pathology (β=0.29, P<0.01), and a higher concentration of KA was associated with the slower progression of dementia. The higher concentrations of neuroprotective metabolites KA and picolinic acid suggest that the activation of the KP's neuroprotective branch is an adaptive response in AD and may be a promising target for intervention and treatment. Highlights Patients with Alzheimer's disease (AD) exhibited higher concentrations of kynurenic acid and picolinic acid than controls. Higher concentrations of kynurenic acid were associated with slower progression of AD. Potential neurotoxic kynurenines were not increased among patients with AD. Activation of the kynurenine pathway's neuroprotective branch may be an adaptive response in AD.

The neurotoxic branch of the kynurenine pathway is highly activated in the central nervous system of patients with pneumococcal meningitis

BackgroundAcute bacterial meningitis (ABM) causes excessive activation of N-methyl-D-aspartate receptors (NMDAr), leading to cortical and hippocampal neuron death. As opposite, enteroviral meningitis is more frequently benign. The kynurenine (KYN) pathway is the major catabolic route of tryptophan (TRP) and some of its metabolites are agonists or antagonists of NMDAr. MethodsIn order to investigate the pathogen-specific patterns of KYN pathway modulation in the central nervous system of children with acute meningococcal (MM), pneumococcal (PM) or enteroviral (VM) meningitis, the cerebrospinal fluid (CSF) concentrations of TRP, KYN, kynurenic acid (KYNA) and quinolinic acid (QUINA) were evaluated by ultra-high performance liquid chromatography (uHPLC) coupled to mass spectrometry. In addition, CSF levels of IL-6, IL-10 and TNF-α were quantified by multi-analyte flow assay. The data was mined and integrated using statistical and machine learning methods. ResultsThe three forms of meningitis investigated herein up-regulated the neurotoxic branch of the KYN pathway within the intrathecal space. However, this response, represented by the concentration of QUINA, was six and nine times higher in PM patients compared to MM or VM, respectively. CSF levels of IL-6, TNF-α, and IL-10 were increased in MM and PM patients when compared to controls. In VM, CSF IL-6 and IL-10, but not TNF-α were increased compared to controls, although not reaching the high levels found in bacterial meningitis. No correlation was found between the concentrations or the ratios of any pair of KYN metabolites and any cytokine or standard cytochemical parameter tested. ConclusionsCNS infection with meningococci, pneumococci, and enteroviruses intrathecally activate the KYN pathway, favoring its neurotoxic branch. However, in PM, higher CSF levels of QUINA, compared to MM and VM, may contribute to its poorer neurologic outcome.

Albumin-bound kynurenic acid is an appropriate endogenous biomarker for assessment of the renal tubular OATs-MRP4 channel

Renal tubular secretion mediated by organic anion transporters (OATs) and the multidrug resistance-associated protein 4 (MRP4) is an important means of drug and toxin excretion. Unfortunately, there are no biomarkers to evaluate their function. The aim of this study was to identify and characterize an endogenous biomarker of the renal tubular OATs-MRP4 channel. Twenty-six uremic toxins were selected as candidate compounds, of which kynurenic acid was identified as a potential biomarker by assessing the protein-binding ratio and the uptake in OAT1-, OAT3-, and MRP4-overexpressing cell lines. OAT1/3 and MRP4 mediated the transcellular vectorial transport of kynurenic acid in vitro. Serum kynurenic acid concentration was dramatically increased in rats treated with a rat OAT1/3 (rOAT1/3) inhibitor and in rOAT1/3 double knockout (rOAT1/3−/−) rats, and the renal concentrations were markedly elevated by the rat MRP4 (rMRP4) inhibitor. Kynurenic acid was not filtered at the glomerulus (99% of albumin binding), and was specifically secreted in renal tubules through the OAT1/3-MRP4 channel with an appropriate affinity (Km) (496.7 μM and 382.2 μM for OAT1 and OAT3, respectively) and renal clearance half-life (t1/2) in vivo (3.7 ± 0.7 h). There is a strong correlation in area under the plasma drug concentration-time curve (AUC0–t) between cefmetazole and kynurenic acid, but not with creatinine, after inhibition of rOATs. In addition, the phase of increased kynurenic acid level is earlier than that of creatinine in acute kidney injury process. These results suggest that albumin-bound kynurenic acid is an appropriate endogenous biomarker for adjusting the dosage of drugs secreted by this channel or predicting kidney injury.

A ketogenic diet substantially reshapes the human metabolome

Western dietary habits (WD) have been shown to promote chronic inflammation, which favors the development of many of today's non-communicable diseases. Recently, ketogenic diets (KD) have emerged as an immune-regulating countermeasure for WD-induced metaflammation. To date, beneficial effects of KD have been solely attributed to the production and metabolism of ketone bodies. Given the drastic change in nutrient composition during KD, it is reasonable to assume that there are widespread changes in the human metabolome also contributing to the impact of KD on human immunity. The current study was conducted to gain insight into the changes of the human metabolic fingerprint associated with KD. This could allow to identify metabolites that may contribute to the overall positive effects on human immunity, but also help to recognize potential health risks of KD. We conducted a prospective nutritional intervention study enrolling 40 healthy volunteers to perform a three-week ad-libitum KD. Prior to the start and at the end of the nutritional intervention serum metabolites were quantified, untargeted mass spectrometric metabolome analyses and urine analyses of the tryptophan pathway were performed. KD led to a marked reduction of insulin (-21.45%±6.44%, p=0.0038) and c-peptide levels (-19.29%±5.45%, p=0.0002) without compromising fasting blood glucose. Serum triglyceride concentration decreased accordingly (-13.67%±5.77%, p=0.0247), whereas cholesterol parameters remained unchanged. LC-MS/MS-based untargeted metabolomic analyses revealed a profound shift of the human metabolism towards mitochondrial fatty acid oxidation, comprising highly elevated levels of free fatty acids and acylcarnitines. The serum amino acid (AA) composition was rearranged with lower abundance of glucogenic AA and an increase of BCAA. Furthermore, an increase of anti-inflammatory fatty acids eicosatetraenoic acid (p<0.0001) and docosahexaenoic acid (p=0.0002) was detected. Urine analyses confirmed higher utilization of carnitines, indicated by lower carnitine excretion (-62.61%±18.11%, p=0.0047) and revealed changes to the tryptophan pathway depicting reduced quinolinic acid (-13.46%±6.12%, p=0.0478) and elevated kynurenic acid concentrations (+10.70%±4.25%, p=0.0269). A KD fundamentally changes the human metabolome even after a short period of only three weeks. Besides a rapid metabolic switch to ketone body production and utilization, improved insulin and triglyceride levels and an increase in metabolites that mediate anti-inflammation and mitochondrial protection occurred. Importantly, no metabolic risk factors were identified. Thus, a ketogenic diet could be considered as a safe preventive and therapeutic immunometabolic tool in modern medicine. German Clinical Trials Register; DRKS-ID: DRKS00027992 (www.drks.de).

Measurement of kynurenine pathway metabolites by tandem mass spectrometry

ObjectivesRecent studies have shown that derangements in kynurenine pathway metabolite levels are associated with various pathologies such as neurodegenerative diseases, schizophrenia, depression, bipolar disorder, rheumatoid arthritis, and cancer. Therefore, reliable, accurate, fast, and multiplex measurement methods for kynurenines have become increasingly important. This study aimed to validate a new mass spectrometric method for analyzing tryptophan metabolites. MethodsA tandem mass spectrometric method, including protein precipitation and evaporation steps, was developed to measure serum levels of tryptophan, kynurenine, kynurenic acid, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid. Samples were separated using a Phenomenex Luna C18 reversed-phase column. The kynurenine pathway metabolites were detected by tandem mass spectrometry. The developed method was validated according to Clinical & Laboratory Standards Institute (CLSI) guidelines and applied to hemodialysis samples. ResultsThe developed method was linear at the concentrations of 48.8 – 25,000, 0.98 – 500, 1.2–5000, 1.2–5000, and 0.98–250 ng/mL for tryptophan, kynurenic acid, kynurenine, 3-hydroxyanthranilic acid, and 3-hydroxykynurenine, respectively. The imprecisions were less than 12 %. The median serum concentrations of tryptophan, kynurenine, kynurenic acid, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid were 10530, 1100, 218, 17.6, and 25.4 ng/mL in pre-dialysis blood samples, respectively. They were 4560, 664, 135, 7.4, and 12.8 ng/mL in post-dialysis blood samples, respectively. ConclusionsA fast, simple, cost-effective, accurate, robust, and validated tandem mass spectrometric method was developed, and the method was successfully used for the quantitation of kynurenine pathway metabolite concentrations in hemodialysis patients.