Urine 5-Hydroxyindoleacetic Acid Negatively Correlates with Migraine Occurrence and Characteristics in the Interictal Phase of Episodic Migraine.

Although migraine belongs to the main causes of disability worldwide, the mechanisms of its pathogenesis are poorly known. As migraine diagnosis is based on the subjective assessment of symptoms, there is a need to establish objective auxiliary markers to support clinical diagnosis. Tryptophan (TRP) metabolism has been associated with the pathogenesis of neurological and psychiatric disorders. In the present work, we investigated an association between migraine and the urine concentration of TRP and its metabolites 5-hydroxyindoleacetic acid (5-HIAA), kynurenine (KYN), kynurenic acid (KYNA) and quinolinic acid (QA) in 21 low-frequency episodic migraine patients and 32 controls. We chose the interictal phase as the episodic migraine patients were recruited from the outpatient clinic and had monthly migraine days as low as 1-2 in many cases. Migraine patients displayed lower urinary levels of 5-HIAA (p < 0.01) and KYNA (p < 0.05), but KYN and QA were enhanced, as compared with the controls (p < 0.05 and 0.001, respectively). Consequently, the patients were characterized by different values of the 5-HIAA/TRP, KYN/TRP, KYNA/KYN, and KYNA/QA ratios (p < 0.001 for all). Furthermore, urinary concentration of 5-HIAA was negatively correlated with Migraine Disability Assessment score and monthly migraine and monthly headache days. There was a negative correlation between Patient Health Questionnaire 9 scores assessing depression. In conclusion, the urinary 5-HIAA level may be further explored to assess its suitability as an easy-to-determine marker of migraine.

Tryptophan-kynurenine ratio as a biomarker of bladder cancer.

OBJECTIVES: To investigate plasma and urinary kynurenine (KYN)-tryptophan (TRP) ratios in bladder cancer, expression of indoleamine 2,3-dioxygenase 1 (IDO1) in relation to tryptophan 2,3-dioxygenase (TDO2) in bladder tumour, and the correlation of KYN-TRP ratio with bladder tumour burden. METHODS: Metabotyping of the TRP-KYN metabolic axis was performed via a clinical case-control study. Expression of IDO1 and TDO2 was measured in human biopsied tissues. Correlational experiments between KYN-TRP ratio and bladder tumour were performed using a murine orthotopic prostate-specific antigen (PSA)-secreting MB49 bladder cancer model. RESULTS: We established for the first time that plasma TRP level was significantly decreased, while both plasma and urinary KYN-TRP ratios were significantly higher in bladder cancer patients, and expression level of IDO1 but not TDO2 was increased in human bladder tumour. We reported the positive correlation between IDO1 expression, KYN-TRP ratio, normalized PSA to creatinine, and bladder tumour burden in the murine model. CONCLUSION: Kynurenine-tryptophan ratio is a promising surveillance biomarker for bladder cancer, but would require further validation before clinical translation.

Urinary metabolomics reveals kynurenine pathway perturbation in newborns with transposition of great arteries after surgical repair.

INTRODUCTION: Transposition of the great arteries (TGA) is a cyanotic congenital heart defect that requires surgical correction, with the use of cardiopulmonary-bypass (CPB), usually within 3 weeks of life. The use of CPB in open heart surgery results in brain hypoperfusion and in a powerful systemic inflammatory response and oxidative stress. OBJECTIVE: We aimed to develop a novel untargeted metabolomics approach to detect early postoperative changes in metabolic profile following neonatal cardiac surgery. METHODS: We studied 14 TGA newborns with intact ventricular septum undergoing arterial switch operation with the use of CPB. Urine samples were collected preoperatively and at the end of the surgery and were analyzed using an untargeted metabolomics approach based on UHPLC-high resolution mass spectrometry. RESULTS: Since post surgery metabolic spectra were heavily contaminated by metabolites derived from administered drugs, we constructed a list of drugs used during surgery and their related metabolites retrieved from urine samples. This library was applied to our samples and 1255 drugs and drug metabolites were excluded from the analysis. Afterward, we detected over 39,000 unique compounds and 371 putatively annotated metabolites were different between pre and post-surgery samples. Among these metabolites, 13 were correctly annotated or identified. Metabolites linked to kynurenine pathway of tryptophan degradation displayed the highest fold change. CONCLUSIONS: This is the first report on metabolic response to cardiac surgery in TGA newborns. We developed an experimental design that allowed the identification of perturbed metabolic pathways and potential biomarkers of brain damage, limiting drugs interference in the analysis.

Metabolic Response in Rabbit Urine to Occurrence and Relief of Unilateral Ureteral Obstruction.

Ureteral obstruction will lead clinically to hydronephrosis, which may further develop into partial or complete loss of kidney function and even cause permanent histological damage. However, there is little knowledge of metabolic responses during the obstructed process and its recoverability. In this study, a complete unilateral ureteral obstruction (CUUO) model was established in the rabbit, and 1H NMR-based metabolomic analysis of urine was used to reveal the metabolic perturbations in rabbits caused by CUUO and the metabolic recovery after the CUUO was relieved. Univariate and multivariate statistical analyses were used to identify metabolic characteristics. The gradually decreased levels of 3-hydroxykynurenine, 3-methylhistidine, creatinine, guanidoacetate, meta- and para-hydroxyphenylacetate, and phenylacetylglycine and the gradually increased levels of acetate, alanine, citrate, glycine, lactate, and methionine in urine could be regarded as potential biomarkers for the occurrence and severity of ureteral obstruction. And the reduced levels of 3-methylhistidine, creatinine, guanidoacetate, hippurate, meta-hydroxyphenylacetate, and methylguanidine and the elevated levels of 2-aminoisobutyrate, acetylcholine, citrate, lactate, lysine, valine, and α-ketoglutarate in urine compared with the obstructed level could characterize the metabolic recovery of ureteral obstruction. Our results depicted the disturbed biochemical pathways involved in ureteral obstruction and demonstrated the practicability of recovering renal functions for the patients with severe hydronephrosis in clinical practice by removing causes for obstruction.

Urinary l-kynurenine quantification and selective extraction through a molecularly imprinted solid-phase extraction device.

l-Kynurenine is an endogenous metabolite generated by the catabolic pathway of l-tryptophan and it could be a potential biomarker to test the efficacy of several checkpoint inhibitors that have already reached the clinical trials in the antitumor therapy. Thus, a molecularly imprinted polymer specific for the recognition of this metabolite was synthesized and used as innovative system in solid-phase extraction technique for the specific extraction and quantification of l-kynurenine in human urine. The off-line system was firstly tested on l-kynurenine standard solutions, allowing recoveries up to 97.7% (relative standard deviation = 2.2%) and then applied to fortified and deproteinated human urine samples, where a recovery of 84.1% (relative standard deviation = 3.1%) was obtained. The method was validated and it revealed a good linearity in the range of 0.157-20 µg/mL (r2  = 0.9992). The optimized procedure demonstrated a good feasibility on biological samples, allowing a ready quantification of l-kynurenine in the human urine, where the metabolite was found at a very low concentration (0.80 µg/mL). The extraction system developed could attract attention of pharmaceutical industries for l-kynurenine production as potential drug in the treatment of autoimmune disorders through its extraction and purification from biological matrixes.

The kynurenine:tryptophan ratio as a predictor of incident type 2 diabetes mellitus in individuals with coronary artery disease.

AIMS/HYPOTHESIS: The tryptophan metabolite kynurenine has potent immune modulatory and vasoactive properties. Experimental data implicate kynurenine in obesity-related morbidities. Epidemiological studies are, however, sparse. We evaluated associations of the plasma and urine kynurenine:tryptophan ratio (KTR) to incident type 2 diabetes. METHODS: We followed 2519 individuals with coronary artery disease (CAD; 73.1% men) without diabetes at baseline for a median of 7.6 years, during which 173 (6.9%) new incidences of type 2 diabetes were identified. Multivariate Cox regression analyses were applied to investigate the prospective relationships of plasma and urine KTR with new onset type 2 diabetes. RESULTS: At inclusion, mean (SD) age was 61.3 (10.4) years, BMI was 25.9 (3.71) kg/m2 and median (interquartile range) HbA1c was 5.6% (5.0%-6.0%) (38 [31-42] mmol/mol). Plasma KTR was not significantly related to type 2 diabetes risk. By contrast, urine KTR showed a strong positive association. Comparing quartile 4 with quartile 1, the HRs (95% CIs) were 2.59 (1.56, 4.30) and 2.35 (1.39, 3.96) in the age- and sex-adjusted and multivariate models, respectively. CONCLUSIONS/INTERPRETATION: Urine KTR is a strong predictor of incident type 2 diabetes in individuals with CAD. Potential clinical implications and possible pathogenic roles of renal kynurenine excretion in type 2 diabetes development should be further elucidated.

The tryptophan/kynurenine pathway, systemic inflammation, and long-term outcome after kidney transplantation.

Tryptophan is metabolized along the kynurenine pathway, initially to kynurenine, and subsequently to cytotoxic 3-hydroxykynurenine. There is increasing interest in this pathway because of its proinflammatory nature, and drugs interfering in it have received increasing attention. We aimed to investigate whether serum and urinary parameters of the tryptophan/kynurenine pathway, and particularly cytotoxic 3-hydroxykynurenine, are associated with systemic inflammation and long-term outcome in renal transplant recipients (RTR). Data were collected in outpatient RTR with a functioning graft for >1 yr. Tryptophan, kynurenine, and 3-hydroxykynurenine in serum and urine were measured using LC-MS/MS. A total of 561 RTR (age: 51 ± 12 yr; 56% male) were included at a median of 6.0 (2.6-11.6) yr posttransplantation. Baseline median serum tryptophan was 40.0 (34.5-46.0) µmol/l, serum kynurenine was 1.8 (1.4-2.2) µmol/l, and serum 3-hydroxykynurenine was 42.2 (31.0-61.7) nmol/l. Serum kynurenine and 3-hydroxykynurenine were strongly associated with parameters of systemic inflammation. During follow-up for 7.0 (6.2-7.5) yr, 51 RTR (9%) developed graft failure and 120 RTR (21%) died. Both serum kynurenine and 3-hydroxykynurenine were independently associated with graft failure [HR 1.72 (1.23-2.41), P = 0.002; and HR 2.03 (1.42-2.90), P < 0.001]. Serum 3-hydroxykynurenine was also independently associated with mortality [HR 1.37 (1.08-1.73), P = 0.01], whereas serum kynurenine was not. Urinary tryptophan/kynurenine pathway parameters were not associated with outcome. Of tryptophan metabolites, serum 3-hydroxykynurenine is cross-sectionally most strongly and consistently associated with systemic inflammation and prospectively with adverse long-term outcome after kidney transplantation. Serum 3-hydroxykynurenine may be an interesting biomarker and target for the evaluation of drugs interfering in the tryptophan/kynurenine pathway.

Urine 3-hydroxykynurenine is higher during the postovulatory phase than in the preovulatory phase indicating a higher vitamin B6 requirement.

The relationship between l-tryptophan to nicotinamide metabolism and the menstrual cycle of Japanese women was investigated. Nine metabolism intermediates from urine samples collected during the preovulatory and postovulatory phases were measured. Only urine 3-hydroxykynurenine was higher in the postovulatory phase than in the preovulatory phase. This increase in 3-hydroxykynurenine suggests a decreased reaction of 3-hydroxykynurenine → 3-hydroxyanthranilic acid catalyzed by kynureninase, a vitamin B6 enzyme.

Urinary excretion of kynurenine and tryptophan, cardiovascular events, and mortality after elective coronary angiography.

AIMS: Kynurenine is a potent endothelium-derived vasodilator. Its synthesis from tryptophan is stimulated by interferon γ and may represent an important compensatory pathway for the regulation of vascular function in inflammatory conditions. We assessed associations of urine kynurenine to tryptophan ratio (KTR) levels to incident major coronary events (MCEs), acute myocardial infarction (AMI), and ischaemic stroke and mortality in patients with suspected stable coronary artery disease (CAD). METHODS AND RESULTS: A total of 3224 patients (mean age 62 years, 69% men) underwent urine and blood sampling prior to elective coronary angiography and were subsequently followed up for median 55 months. A total of 8.4% experienced an MCE, 7.8% suffered an AMI, and 7.6% died. In age- and gender-adjusted analyses, the hazard ratios [HRs; 95% confidence intervals (CI)] of MCE, AMI, and all-cause mortality were 1.43 (1.29-1.59), 1.44 (1.29-1.59), and 1.38 (1.23-1.54) per standard deviation increment of the (log-transformed) urinary KTR, respectively. These estimates were only minimally attenuated after adjustment for potential confounders. The addition of the urine KTR to a model of conventional risk factors significantly improved goodness of fit, discrimination, and risk classification for these clinical endpoints. No association was seen between the urine KTR and the risk of incident ischaemic stroke. CONCLUSION: A novel urinary inflammation marker, KTR, is strongly associated with adverse prognosis in patients with suspected stable CAD. Underlying pathomechanisms should be further elucidated.

Urinary metabolomics reveals potential biomarkers for early detection of pregnancy in Mithun (Bos frontalis) cows.

The present study investigated the urinary metabolic profiles of early pregnant and non-pregnant Mithun to identify potential pregnancy detection biomarkers. Urine samples were collected on days 0, 10, 18, 35 and 45 of gestation from pregnant (n = 6) and on days 0, 10 and 18 from non-pregnant (n = 6) Mithun. Urinary metabolites were assessed using proton nuclear magnetic resonance (1H NMR) spectroscopy and identified 270 metabolites. Statistical analyses demonstrated pronounced distinctions in metabolite profiles between pregnant and non-pregnant samples. Twenty-five metabolites that could discriminate between pregnant and non-pregnant Mithun based on Variable Importance in Projection (VIP) scores >1 were identified. Upon further examination of six metabolites (kynurenine, kynurenate, 3-hydroxykynurenine, quinolinate, tyrosine and leucine) identified with high VIP scores, ROC curve analyses demonstrated their significant predictive potential, with AUC values ranging between 0.50 and 0.85. Additionally, a combined panel of top 25 metabolites yielded an AUC value of 0.85. Pathway analysis identified seven potential metabolic pathway modulations during early gestation, with particular emphasis on phenylalanine, tyrosine and tryptophan biosynthesis, tryptophan pathway and pathways involved in the metabolism of various amino acids. In conclusion, kynurenine, kynurenate, 3-hydroxykynurenine, quinolinate, tyrosine, and leucine show promise as non-invasive urinary biomarkers for early pregnancy detection in Mithun. SIGNIFICANCE: This study presents the first report on the metabolic profile of urine from early pregnant and non-pregnant Mithun (Bos frontalis). The metabolites like kynurenine and its derivatives (kynurenate, 3-hydroxykynurenine and quinolinate), tyrosine and leucine were documented signature urinary metabolites associated with early pregnancy in Mithun. The identified combination of metabolites holds promise as predictive biomarkers for non-invasive urinary-based early pregnancy diagnostics in Mithun. In addition, this study identified changes in metabolic pathways that involve phenylalanine, tyrosine, tryptophan and related amino acids and biomarkers identified were either precursors or products within these metabolic pathways.

Tryptophan catabolism is associated with acute GVHD after human allogeneic stem cell transplantation and indicates activation of indoleamine 2,3-dioxygenase.

Induction of indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme in tryptophan degradation along the kynurenine pathway, acts as a potent immunoregulatory loop. To address its role in human allogeneic stem cell transplantation, we measured major tryptophan metabolites, such as quinolinic acid and kynurenine, in serial urine specimens from 51 patients by liquid chromatography-tandem mass spectrometry. Samples were collected between admission and day 90 after transplantation, and metabolite levels were correlated with early clinical events and outcome. In selected patients, IDO gene expression was assessed by quantitative RT-PCR in intestinal biopsies. Surviving patients had significantly lower metabolite levels on days 28, 42, and 90, respectively, compared with patients dying of GVHD and associated complications (n = 10). Kynurenine levels were directly correlated with severity and clinical course of GVHD: Mean urinary quinolinic acid levels were 4.5 ± 0.3 µmol/mmol creatinine in the absence of acute GVHD, 8.0 ± 1.1 µmol/mmol creatinine for GVHD grade 1 or 2, and 13.5 ± 2.7 µmol/mmol creatinine for GVHD grade 3 or 4 (P < .001), respectively. GVHD-dependent induction of IDO was further suggested by increased expression of IDO mRNA in intestinal biopsies from patients with severe GVHD. Our data indicate reactive release of kynurenines in GVHD-associated inflammation.

The niacin required for optimum growth can be synthesized from L-tryptophan in growing mice lacking tryptophan-2,3-dioxygenase.

In mammals, nicotinamide (Nam) is biosynthesized from l-tryptophan (l-Trp). The enzymes involved in the initial step of the l-Trp→Nam pathway are l-Trp-2,3-dioxygenase (TDO) and indoleamine-2,3-dioxygenase (IDO). We aimed to determine whether tdo-knockout (tdo(-/-)) mice fed a diet without preformed niacin can synthesize enough Nam to sustain optimum growth. Wild-type (WT) and tdo(-/-) mice were fed a chemically defined 20% casein diet with or without preformed niacin (30 mg nicotinic acid/kg) for 28 d. Body weight, food intake, and liver NAD concentrations did not differ among the groups. In the groups of mice fed the niacin-free diet, urinary concentrations of the upstream metabolites kynurenine (320% increase, P < 0.0001), kynurenic acid (270% increase, P < 0.0001), xanthurenic acid (770% increase, P < 0.0001), and 3-hydroxyanthranilic acid (3-HA; 450% increase, P < 0.0001) were higher in the tdo(-/-) mice than in the WT mice, while urinary concentrations of the downstream metabolite quinolinic acid (QA; 50% less, P = 0.0010) and the sum of Nam and its catabolites (10% less, P < 0.0001) were lower in the tdo(-/-) mice than in the WT mice. These findings show that the kynurenine formed in extrahepatic tissues by IDO and subsequent enzymes can be metabolized up to 3-HA, but not into QA. However, the tdo(-/-) mice sustained optimum growth even when fed the niacin-free diet for 1 mo, suggesting they can synthesize the minimum necessary amount of Nam from l-Trp, because the liver can import blood kynurenine formed in extrahepatic tissues and metabolize it into Nam via NAD and the resulting Nam is then distributed back into extrahepatic tissues.

Supplementing healthy women with up to 5.0 g/d of L-tryptophan has no adverse effects.

Because of the frequent use of L-tryptophan (L-Trp) in dietary supplements, determination of the no-observed-adverse-effect-level is desirable for public health purposes. We therefore assessed the no-observed-adverse-effect-level for L-Trp and attempted to identify a surrogate biomarker for excess L-Trp in healthy humans. A randomized, double-blind, placebo-controlled, crossover intervention study was performed in 17 apparently healthy Japanese women aged 18-26 y with a BMI of ≈ 20 kg/m(2). The participants were randomly assigned to receive placebo (0 g/d) or 1.0, 2.0, 3.0, 4.0, or 5.0 g/d of L-Trp for 21 d each with a 5-wk washout period between trials. Food intake, body weight, general biomarkers in blood and urine, and amino acid composition in blood and urine were not affected by any dose of L-Trp. Administration of up to 5.0 g/d L-Trp had no effect on a profile of mood states category measurement. The urinary excretion of nicotinamide and its catabolites increased in proportion to the ingested amounts of L-Trp, indicating that participants could normally metabolize this amino acid. The urinary excretion of L-tryptophan metabolites, including kynurenine (Kyn), anthranilic acid, kynurenic acid, 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid, and quinolinic acid (QA), all of which are intermediates of the L-TRP→Kyn→QA pathway, was in proportion to L-Trp loading. The response of 3-HK was the most characteristic of these L-Trp metabolites. This finding suggests that the urinary excretion of 3-HK is a good surrogate biomarker for excess L-Trp ingestion.

A mathematical model of tryptophan metabolism via the kynurenine pathway provides insights into the effects of vitamin B-6 deficiency, tryptophan loading, and induction of tryptophan 2,3-dioxygenase on tryptophan metabolites.

Vitamin B-6 deficiency is associated with impaired tryptophan metabolism because of the coenzyme role of pyridoxal 5'-phosphate (PLP) for kynureninase and kynurenine aminotransferase. To investigate the underlying mechanism, we developed a mathematical model of tryptophan metabolism via the kynurenine pathway. The model includes mammalian data on enzyme kinetics and tryptophan transport from the intestinal lumen to liver, muscle, and brain. Regulatory mechanisms and inhibition of relevant enzymes were included. We simulated the effects of graded reduction in cellular PLP concentration, tryptophan loads and induction of tryptophan 2,3-dioxygenase (TDO) on metabolite profiles and urinary excretion. The model predictions matched experimental data and provided clarification of the response of metabolites in various extents of vitamin B-6 deficiency. We found that moderate deficiency yielded increased 3-hydroxykynurenine and a decrease in kynurenic acid and anthranilic acid. More severe deficiency also yielded an increase in kynurenine and xanthurenic acid and more pronounced effects on the other metabolites. Tryptophan load simulations with and without vitamin B-6 deficiency showed altered metabolite concentrations consistent with published data. Induction of TDO caused an increase in all metabolites, and TDO induction together with a simulated vitamin B-6 deficiency, as has been reported in oral contraceptive users, yielded increases in kynurenine, 3-hydroxykynurenine, and xanthurenic acid and decreases in kynurenic acid and anthranilic acid. These results show that the model successfully simulated tryptophan metabolism via the kynurenine pathway and can be used to complement experimental investigations.

[Screening of urinary biomarkers in patients with type 2 diabetes mellitus].

OBJECTIVE: To screen the biomarkers of the middle-aged type 2 diabetes (T2DM) in urine based on metabolomics. METHODS: First, both the metabolite profiles of morning urine sample in T2DM and control group were obtained by liquid chromatography-mass spectrometry (LC-MS). Second, an automated compound (feature) extraction algorithm was employed for processing background subtracted single MS data. Then principal component analysis (PCA) and t-Test analysis was followed after preprocessing on the extractive data. Third, identifying the potential biomarkers that show significant difference between the two groups by searching the database online and comparing the results of MS/MS experiments to those available in literature and those standards. RESULTS: Three endogenous metabolites, among which tryptophan, kynurenic acid, and shown down-regulation while kynurenine shown up-regulation in T2DM, significantly differentiate between T2DM and control group. CONCLUSION: Tryptophan, kynurenic acid and kynurenine may be considered as the potentail biomarkers related to T2DM.

Psychosocial dwarfism: psychopathological aspects and putative neuroendocrine markers.

There exists an extensive terminology for defining the situation of children who, in varying circumstances, suffer from affective deprivation (AD), within an unsatisfactory family situation or in institutions. Nevertheless, the neuroendocrine mechanisms (if they exist) determining it have yet to be identified. Our objective was to determine if specific neuroendocrine markers, all of them previously implicated in affective disorders, could be modified, and in which sense, in affective deprivation syndrome of the child. For this purpose, we studied three separate groups of children: (1) control group (CG); (2) children suffering from AD; and (3) children with non-organic failure to thrive (NOFT). In every case, we studied the serum levels of melatonin, serotonin, ß-endorphins and adrenocorticotropic hormone (ACTH); and kynurenine pathway tryptophan metabolites (both during the day and at night). Significantly, there was a conspicuous reduction in the levels of each of the neuroendocrine markers (melatonin, serotonin, ß-endorphins and ACTH) in the group suffering from affective deficiency, a diminution which was even more noticeable in the group of patients presenting delayed growth. Furthermore, as also occurs in other affective disorders, there were corresponding modifications in the metabolisation of tryptophan. We report the existence of neuroendocrine mechanisms that are associated with the above-mentioned clinical manifestations in these patients, mechanisms that may underlie the close connection existing between AD syndrome and the cause of NOFT. These data suggest that the AD syndrome and NOFT comprise a single process, but one with a different evolutionary continuum of psychosocial dwarfism.

[Quantitative analysis of tryptophan and its metabolites in urine by ultra performance liquid chromatography-tandem mass spectrometry].

Tryptophan (Trp), also known as α-amino ß-indolepropionic acid, is an essential amino acid, which is involved in various physiological processes. Studies have shown that tumors, infectious diseases, and neurological diseases are accompanied by Trp-related metabolic disorders. Understanding the excretion of Trp and its metabolites in normal individuals is of great significance for treating Trp-related diseases and monitoring the health. A rapid quantitative method was developed based on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Further, this method was applied to the simultaneous determination of Trp and its metabolites, including kynurenine (Kyn), kynurenic acid (KA), 3-hydroxykynurenine (3-OH-Kyn), 3-hydroxyanthranilic acid (3-OH-AA), xanthurenic acid (XA), 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA). The excretion and amount of target compounds in random urine samples collected from healthy participants were studied using this method. Urine samples were collected from healthy male volunteers (between 20-22 years old) without any diet and exercise restrictions. Urine samples were collected between 11∶00-13∶00 daily for 10 d. Thereafter, the urine samples were diluted, centrifuged, and subjected to pre-column derivatization with dansyl chloride (DNS-Cl). Caffeic acid (CA) was used as the internal control. Later, the derivatives were detected using triple quadrupole mass spectrometry with electron pray ionization (ESI) in positive and multi reaction monitoring (MRM) modes. The samples were separated using a Thermo C18 column (50 mm×3 mm, 2.7 µm) with 0.1% aqueous formic acid aqueous solution and methanol as mobile phases at a flow rate of 0.2 mL/min. The three most abundant ions for each derivative were selected for downstream analysis, and the internal control was used for quantification. The polarity and molecular weight of the compounds were found to be altered effectively after DNS-Cl derivatization treatment. The dansyl group effectively altered the polarities of the derivatives, such that their retention behaviors in the reverse elution system were similar and they were well separated. The interference due to impurities was effectively eliminated using the MRM mode. The results showed significant linear correlation, since the correlation coefficients were greater than 0.9740. The recoveries were between 93.24%-107.65%, and the LODs were 0.005-0.5 ng/mL for the eight compounds. Trp prototype and the seven target metabolites, including 3-OH-Kyn, 3-OH-AA, XA, Kyn, KA, 5-HIAA, and 5-HT generated through Trp-5-HT and Trp-Kyn pathways were detected in the urine samples. These results indicated that Trp was excreted in a prototypic form or after being metabolized. The level of the target compounds in random urine samples of individuals were 0.99-3.72 (3-OH-Kyn), 2.51-21.11 (3-OH-AA), 0.25-1.12 (XA), 0.15-1.53 (Kyn), 0.24-2.58 (KA), 0-0.31 (5-HT), and 2.2-17.94 (5-HIAA) µg/mL. For the same individual, in the state of physical health, the fluctuations of Trp and its metabolites in urine were large. Due to these large fluctuations in the absolute content, the difference between individuals was not significant. The data generated using 70 urine samples revealed that the amount of excreted Trp being metabolized was 124%-268% of prototype, which further indicated that the excretion after metabolism was the major underlying mechanism. Upon comparing the levels of metabolites in the Trp-5-HT and Trp-Kyn pathways, the results indicated that the levels of 3-OH-AA and 3-OH-Kyn generated upon Trp degradation through the Kyn pathway was higher than those of the other products. Trp was degraded via Kyn pathway to produce 3-OH-AA, which was the main metabolite of Trp found to be present in the body. This manuscript detected the levels of Trp and its metabolites, as well as summarized the characteristics of excretion using random urine samples, which could provide valuable information for clinical practice.

Improved LC-MS/MS method for the determination of 42 neurologically and metabolically important molecules in urine.

Simultaneous determination of kynurenines, neurotransmitters, pterins and steroids linked to various neurological and metabolic diseases have important diagnostic significance for related pathology and drug monitoring. An improved, sensitive and selective ultra-high performance liquid chromatography coupled to electrospray ionization triple quadrupole mass spectrometric (UHPLC-MS/MS) method, based on our earlier publication, has been proposed for the quantitative measurement of 42 metabolites in human urine. The assay covers a larger number of analytes, uses an advanced, Waters Atlantis T3 chromatographic column and similarly meets the guideline of European Medicines Agency (EMA) on bioanalytical method validation. Analytical performance met all the EMA requirements and the assay covered the relevant clinical concentrations. Linear correlation coefficients were all > 0.998. Intra-day and inter-day accuracy and precision were 87-118%, 81-120% and 2-20%, respectively including the lower limit of quantification (LLOQ). The assay is expected to facilitate the diagnosis and allows drug level monitoring from urine.

Metabolomic analysis identifies inflammatory and noninflammatory metabolic effects of genetic modification in a mouse model of Crohn's disease.

Interleukin-10 is an immunosuppressive cytokine involved in the regulation of gastrointestinal mucosal immunity toward intestinal microbiota. Interleukin-10-deficient (IL10(-/-)) mice develop Crohn's disease-like colitis unless raised in germ-free conditions. Previous gas chromatography-mass spectrometry (GC-MS) metabolomic analysis revealed urinary metabolite differences between IL10(-/-) and wildtype C57BL/6 mice. To determine which of these differences were specifically associated with intestinal inflammation arising from IL10-deficiency, urine samples from IL10(-/-) and wildtype mice, housed in either conventional or specific pathogen-free conditions, were subjected to GC-MS metabolomic analysis. Fifteen metabolite differences, including fucose, xanthurenic acid, and 5-aminovaleric acid, were associated with intestinal inflammation. Elevated urinary levels of xanthurenic acid in IL10(-/-) mice were attributed to increased production of kynurenine metabolites that may induce T-cell tolerance toward intestinal microbiota. Liquid chromatography-mass spectrometry analysis confirmed that plasma levels of kynurenine and 3-hydroxykynurenine were elevated in IL10(-/-) mice. Eleven metabolite differences, including glutaric acid, 2-hydroxyglutaric acid, and 2-hydroxyadipic acid, were unaffected by the severity of inflammation. These metabolite differences may be associated with residual genes from the embryonic stem cells of the 129P2 mouse strain that were used to create the IL10(-/-) mouse, or may indicate novel functions of IL10 unrelated to inflammation.

Melatonin and elimination of kynurenines in children with Down's syndrome.

BACKGROUND: Heightened activity of superoxide dimutase is an effect derived from the gene dose in the trisomy of Down's syndrome (DS), and has been related to the increased production of hydrogen peroxide and with greater lipid peroxidation. Many of the degenerative changes observed in patients with DS have been associated with the pathological effects of free radicals, and for this reason it is of interest to determine the levels present in these patients of powerful antioxidant molecules such as melatonin, and of metabolites with important neuroprotector and neurotoxic consequences such as those derived from the kynurenine pathway. PATIENTS AND METHODS: A study was made of 15 children with DS, together with a control group of 15 non-DS children, matched for age and sex, examined at the Hospital Costa del Sol, Marbella, Spain. Serum melatonin and serotonin were analyzed by RIA; urinary tryptophan metabolites (kynurenine pathway) were determined during periods of light and darkness (09.00-21.00 h and 21.00-9.00 h) by thin-layer chromatography. RESULTS: The mean values of serotonin and melatonin were found to be lower in the patients with DS, although the level of nocturnal secretion of melatonin was higher. Urinary excretion of kynurenine was lower in the patients with DS, although greater quantities of kynurenic acid and anthranilic acid were excreted. CONCLUSIONS: Patients with DS present levels of plasma melatonin and urinary kynurenine that are lower than the corresponding levels in the control population, together with higher values of kynurenic acid and anthranilic acid. These circumstances constitute an added risk to these patients of damage by free radicals.