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Prostate cancer (PCa) is a common male malignancy and early diagnosis is crucial for successful treatment. The current study aims to validate results from a pilot study that demonstrated an inverse association between urine tyrosine and tryptophan levels and the severity of PCa. This study comprised a cohort of 97 patients with benign prostatic hyperplasia, 93 patients diagnosed with localized PCa, 75 patients diagnosed with locally advanced PCa, and 68 patients diagnosed with metastatic PCa. The tyrosine and tryptophan levels in the samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and electrochemical sensors in accordance with the pilot to maintain uniformity for accurately evaluating the data. One-way ANOVA with post Tukey test as well as the Wilcoxon Rank Sum Test were performed. Analyzing 333 patients across PCa stages with consistent methods, we observed no significant differences in tyrosine and tryptophan levels between PCa patients and controls, finally rejecting the use of tyrosine and tryptophan as PCa biomarkers. We did, however, verify the strong correlation between the urinary concentrations of tyrosine and tryptophan found in the pilot study., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Alatraktchi owns PreDiagnose (supplier of screen-printed sensors). This had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Tryptophan(Trp) is being explored as a potential biomarker for various diseases associated with decreased tryptophan levels; however, metabolomic methods are expensive and time-consuming and require extensive sample analysis, making them urgently needed for trace detection. To exploit the properties of Ti 3 C 2 MXenes a rational porous methyl orange (MO)-delaminated Ti 3 C 2 MXene was prepared via a facile mixing process for the electrocatalytic oxidation of Trp. The hollow-like 3D structure with a more open structure and the synergistic effect of MO and conductive Ti 3 C 2 MXene enhanced its electrochemical catalytic capability toward Trp biosensing. More importantly, MO can stabilize Ti 3 C 2 MXene nanosheets through noncovalent π-π interactions and hydrogen bonding. Compared with covalent attachment, these non-covalent interactions preserve the electronic conductivity of the Ti 3 C 2 MXene nanosheets. Finally, the addition of MO-derived nitrogen (N) and sulfur (S) atoms to Ti 3 C 2 MXene enhanced the electronegativity and improved its affinity for specific molecules, resulting in high-performance electrocatalytic activity. The proposed biosensor exhibited a wide linear response in concentration ranges of 0.01-0.3 µM and 0.5-120 µM, with a low detection limit of 15 nM for tryptophan detection, and high anti-interference ability in complex media of human urine and egg white matrices. The exceptional abilities of the MO/Ti 3 C 2 nanocatalyst make it a promising electrode material for the detection of important biomolecules., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

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.

Herein, we introduce a novel method for tryptophan detection via a reduction reaction facilitated by its interaction with a copper(II) phthalocyanine (CuPc) electrocatalytic electrode. This method addresses challenges associated with the susceptibility of the oxidation response to interference from various species when measuring tryptophan in bodily fluids. The reduction currents exhibit a linear increase with tryptophan concentrations in two ranges: 0.0013-0.10 mM and 0.10-1.20 mM, with the sensitivities of 14.7 ± 0.5 μA mM -1 and 3.5 ± 0.1 μA mM -1 , respectively. The limit of detection (LOD, 3 S B / m ) is determined to be 0.39 μM. The sensor exhibits excellent reproducibility, with the relative standard deviation of <5%. Application of the sensor to authentic urine samples yields a % recovery of 101 ± 4%.

The current study presents a steadfast, simple, and efficient approach for the non-invasive determination of glycosuria of diabetes mellitus using fluorescence spectroscopy. A Xenon arc lamp emitting light in the range of 200-950 nm was used as an excitation source for recording the fluorescent spectra from the urine samples. A consistent fluorescence emission peak of glucose at 450 nm was found in all samples for an excitation wavelength of 370 nm. For confirmation and comparison, the fluorescence spectra of non-diabetic (healthy controls) were also acquired in the same spectral range. It was found that fluorescence emission intensity at 450 nm increases with increasing glucose concentration in urine. In addition, optimized synchronous fluorescence emission at 357 nm was used for simultaneously determining a potential diabetes biomarker, Tryptophan (Trp) in urine. It was also found that the level of tryptophan decreases with the increase in urinary glucose concentration. The quantitative estimation of urinary glucose can be demonstrated based on the intensity of emission light carried by fluorescence light. Moreover, the dissimilarities were further emphasized using the hierarchical cluster analysis (HCA) algorithm. HCA gives an obvious separation in terms of dendrogram between the two data sets based on characteristic peaks acquired from their fluorescence emission signatures. These results recommend that urinary glucose and tryptophan fluorescence emission can be used as potential biomarkers for the non-invasive analysis of diabetes., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The biosynthetic routes leading to de novo nicotinamide adenine dinucleotide (NAD+) production are involved in acute kidney injury (AKI), with a critical role for quinolinate phosphoribosyl transferase (QPRT), a bottleneck enzyme of de novo NAD+ biosynthesis. The molecular mechanisms determining reduced QPRT in AKI, and the role of impaired NAD+ biosynthesis in the progression to chronic kidney disease (CKD), are unknown. We demonstrate that a high urinary quinolinate-to-tryptophan ratio, an indirect indicator of impaired QPRT activity and reduced de novo NAD+ biosynthesis in the kidney, is a clinically applicable early marker of AKI after cardiac surgery and is predictive of progression to CKD in kidney transplant recipients. We also provide evidence that the endoplasmic reticulum (ER) stress response may impair de novo NAD+ biosynthesis by repressing QPRT transcription. In conclusion, NAD+ biosynthesis impairment is an early event in AKI embedded with the ER stress response, and persistent reduction of QPRT expression is associated with AKI to CKD progression. This finding may lead to identification of noninvasive metabolic biomarkers of kidney injury with prognostic and therapeutic implications.

The identification of metabolites in biological samples is challenging due to their chemical and structural diversity. Ion mobility spectrometry (IMS) separates ionized molecules based on their mobility in a carrier buffer gas giving information about the ionic shape by measuring the rotationally averaged collision cross-section (CCS) value. This orthogonal descriptor, in combination with the m / z , isotopic pattern distribution, and MS/MS spectrum, has the potential to improve the identification of molecular molecules in complex mixtures. Urine metabolomics can reveal metabolic differences, which arise as a result of a specific disease or in response to therapeutic intervention. It is, however, complicated by the presence of metabolic breakdown products derived from a wide range of lifestyle and diet-related byproducts, many of which are poorly characterized. In this study, we explore the use of trapped ion mobility spectrometry (TIMS) via LC parallel accumulation with serial fragmentation (PASEF) for urine metabolomics. A total of 362 urine metabolites were characterized from 80 urine samples collected from healthy volunteers using untargeted metabolomics employing HILIC and RP chromatography. Additionally, three analytes (Trp, Phe, and Tyr) were selected for targeted quantification. Both the untargeted and targeted data was highly reproducible and reported CCS measurements for identified metabolites were robust in the presence of the urine matrix. A comparison of CCS values among different laboratories was also conducted, showing less than 1.3% ΔCCS values across different platforms. This is the first report of a human urine metabolite database compiled with CCS values experimentally acquired using an LC-PASEF TIMS-qTOF platform.

Analytical methods to determine the potential misuse of the ghrelin mimetics capromorelin (CP-424,391), macimorelin (macrilen, EP-01572) and tabimorelin (NN703) in sports were developed. Therefore, different extraction strategies, i.e. solid-phase extraction, protein precipitation, as well as a "dilute-and-inject" approach, from urine and EDTA-plasma were assessed and comprehensive in vitro/in vivo experiments were conducted, enabling the identification of reliable target analytes by means of high resolution mass spectrometry. The drugs' biotransformation led to the preliminary identification of 51 metabolites of capromorelin, 12 metabolites of macimorelin and 13 metabolites of tabimorelin. Seven major metabolites detected in rat urine samples collected post-administration of 0.5-1.0 mg of a single oral dose underwent in-depth characterization, facilitating their implementation into future confirmatory test methods. In particular, two macimorelin metabolites exhibiting considerable abundances in post-administration rat urine samples were detected, which might contribute to an improved sensitivity, specificity, and detection window in case of human sports drug testing programs. Further, the intact drugs were implemented into World Anti-Doping Agency-compliant initial testing (limits of detection 0.02-0.60 ng/ml) and confirmation procedures (limits of identification 0.18-0.89 ng/ml) for human urine and blood matrices. The obtained results allow extension of the test spectrum of doping agents in multitarget screening assays for growth hormone-releasing factors from human urine., (© 2021 The Authors. Biomedical Chromatography published by John Wiley & Sons Ltd.)

Tryptophan (Trp), also known as α-amino β -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 C 18 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.

A facile chiral composite (3D-NGMWCNT@(S,S)-CIL) was prepared by integrating three-dimensional N-doped graphene oxide multi-walled carbon nanotubes (3D-NGMWCNT) and chiral ionic liquid ((S,S)-CIL) via electrodeposition. SEM, XRD, XPS, and electrochemical methods were used to characterize this composite and it revealed that the integrated 3D-NGMWCNT@(S,S)-CIL composite showed excellent electrochemical performance. Therefore, a 3D-NGMWCNT@(S,S)-CIL/GCE electrochemical sensor was constructed for enantioselective recognition of Trp enantiomers. The coefficient (I L /I D ) of the 3D-NGMWCNT@(S,S)-CIL/GCE chiral sensor was 2.26 by differential pulse voltammograms (DPV), revealing that the synthesized 3D-NGMWCNT@(S,S)-CIL had a higher affinity for L-Trp than D-Trp. Moreover, UV-V is spectroscopy and a water contact angle test also proved this result. The 3D-NGMWCNT@(S,S)-CIL/GCE sensor had a detection limit of 0.024 μM and 0.055 μM, and sensitivity of 62.35 μA·mM -1 ·cm -2 and 30.40 μA·mM -1 ·cm -2 for L-Trp and D-Trp, respectively, with a linear response range of 0.01 to 5 mM. In addition, the 3D-NGMWCNT@(S,S)-CIL/GCE chiral sensor showed excellent stability, and good reproducibility and was applied to detect L-Trp or D-Trp in real samples. The novel 3D-NGMWCNT@(S,S)-CIL/GCE chiral sensor provides an efficient and convenient strategy for chiral enantioselective recognition. Schematic construction of the 3D-NGMWCNT@(S,S)-CIL/GCE chiral electrochemical sensors.

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., (© 2020 The Authors BJU International © 2020 BJU International Published by John Wiley & Sons Ltd.)

Urine autofluorescence at 295 nm is significantly higher in patients with malignant melanoma at each clinical stage compared to the healthy group. The largest difference is in the early-stages and without metastases. With increasing stage, the autofluorescence at 295 nm decreases. There is also a significant negative correlation between autofluorescence and Clark classification. Based on our results, it is assumed that the way malignant melanoma grows also affects urinary autofluorescence.

Metabolic syndrome (MetS) is a multifactor condition predisposing for diabetes, cardiovascular diseases and other degenerative disorders. Although several diagnostic criteria have been established, none of them is specific and there is a call for better pathophysiological explanation of MetS and for the discovery of molecular biomarkers. Phenotype characterization at metabolome level might be useful for both purposes. To this end, our aim was to perform comparative untargeted metabolomics of urines from MetS patients and from the control group. The study participants included 52 diagnosticated and 50 healthy individuals from Leon city in central Mexico; 23 anthropometric and clinical parameters were measured and submitted to Principal Component Analysis (PCA). The obtained PCA model allowed us for selection of 11 MetS patients and 13 control subjects, correspondingly representative for each of the two groups (clearly separated in PCA). The first morning urines from these subjects were ambulatory collected and, after methanol extraction and acidification, were submitted to capillary liquid chromatography-high resolution mass spectrometry (LC-HRMS). The obtained data were analyzed on MetaboScape® platform (Bruker Daltonics). Specifically, t -test applied to LC-HRMS data revealed several ions presenting at least 3-fold higher intensities in MetS with respect to the control samples (p < 0.05). Data analysis and complementary experiments yielded the identification of the following metabolites: indole-3-acetic acid, indole-3-acetic acid-O-glucuronide, N-(indol-3-ylacetyl) glutamine, indole-3-carbaldehyde and hydroxyhexanoycarnitine. Additionally, indole-3-carboxylic acid was annotated with 2.13-fold higher abundance in MetS patients. To assess the contribution of individual metabolites in the difference between two groups of subjects, partial least square discriminant analysis was performed for LC-HRMS data and the obtained values of variable importance in projection (VIP), confirmed the association of six above mentioned compounds with MetS. Overall, this study provides direct evidence on the disturbed catabolism of tryptophan in metabolic syndrome.

Higher serum 6-bromotryptophan has been associated with lower risk of chronic kidney disease (CKD) progression, implicating mechanisms beyond renal clearance. We studied genetic determinants of urine 6-bromotryptophan and its association with CKD risk factors and incident end-stage kidney disease (ESKD) in 4,843 participants of the German Chronic Kidney Disease (GCKD) study. 6-bromotryptophan was measured from urine samples using mass spectrometry. Patients with higher levels of urine 6-bromotryptophan had higher baseline estimated glomerular filtration rate (eGFR, p < 0.001). A genome-wide association study of urine 6-bromotryptophan identified two significant loci possibly related to its tubular reabsorption, SLC6A19, and its production, ERO1A, which was also associated with serum 6-bromotryptophan in an independent study. The association between urine 6-bromotryptophan and time to ESKD was assessed using Cox regression. There were 216 ESKD events after four years of follow-up. Compared with patients with undetectable levels, higher 6-bromotryptophan levels were associated with lower risk of ESKD in models unadjusted and adjusted for ESKD risk factors other than eGFR (

Introduction: The design of training programs for football players is not straightforward due to intra- and inter-individual variability that leads to different physiological responses under similar training loads., Objective: To study the association between the external load, defined by variables obtained using electronic performance tracking systems (EPTS), and the urinary metabolome as a surrogate of the metabolic adaptation to training., Methods: Urine metabolic and EPTS data from 80 professional football players collected in an observational longitudinal study were analyzed by ultra-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry and assessed by partial least squares (PLS) regression., Results: PLS models identified steroid hormone metabolites, hypoxanthine metabolites, acetylated amino acids, intermediates in phenylalanine metabolism, tyrosine, tryptophan metabolites, and riboflavin among the most relevant variables associated with external load. Metabolic network analysis identified enriched pathways including steroid hormone biosynthesis and metabolism of tyrosine and tryptophan. The ratio of players showing a deviation from the PLS model of adaptation to exercise was higher among those who suffered a muscular lesion compared to those who did not., Conclusions: There was a significant association between the external load and the urinary metabolic profile, with alteration of biochemical pathways associated with long-term adaptation to training. Future studies should focus on the validation of these findings and the development of metabolic models to identify professional football players at risk of developing muscular injuries.

Transcription factor (TF)-based metabolite detection mainly depends on TF-regulated gene expression in cells. From TF activation to gene transcription and translation, the signal travels a relatively long way before it is received. Here, we propose a TF-splinting duplex DNA nanoswitch to detect metabolites. We show its feasibility using tryptophan repressor (TrpR) to detect l-tryptophan as a model. The assay has been optimized and characterized after obtaining a proof of concept, and the detection of l-tryptophan in complex biological samples is feasible. Unlike an equivalent gene expression approach, the whole process is a single-step, enzyme-free, and signal-on method. It can be completed within 20 min. This proposed TF-splinting duplex has the potential to be applied to the quick and convenient detection of other metabolites or even TFs.

Background and Aims: Studies have linked several metabolites to the risk of coronary heart disease (CHD) among Western populations, but prospective studies among Asian populations on the metabolite-CHD association remain limited., Methods and Results: We evaluated the association of urinary metabolites with CHD risk among Chinese adults in a nested case-control study of 275 incident cases and 275 matched controls (127 pairs of men and 148 pairs of women). Fifty metabolites were measured by a predefined metabolomics panel and adjusted using urinary creatinine. Conditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs). After adjusting for traditional CHD risk factors, urinary tryptophan showed a positive association with incident CHD: OR (95% CI) for the highest vs. lowest quartiles was 2.02 (1.15-3.56) among all study participants (p-trend = 0.02). The tryptophan-CHD association was more evident among individuals with dyslipidemia than among those without the condition (OR [95% CI] for the highest vs. lowest quartiles = 3.90 [1.86-8.19] and 0.74 [0.26-2.06], respectively; p-interaction<0.01). Other metabolites did not show significant associations with CHD risk among all study participants. However, a positive association of methionine with CHD risk was observed only among women (OR [95% CI] for the highest vs. lowest quartiles = 2.77 [1.17-6.58]; p-interaction = 0.03), and an inverse association of inosine with CHD risk was observed only among men (OR [95% CI] for the highest vs. lowest quartiles = 0.29 [0.11-0.81]; p-interaction = 0.04)., Conclusion: Elevated urinary tryptophan may be related to CHD risk among Chinese adults, especially for those with dyslipidemia., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest. The sponsors had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, and approval of the manuscript; and decision to submit the manuscript for publication. The contents of this article are solely the responsibility of the authors and do not represent the official views of the Tennessee Department of Health., (Copyright © 2019 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

Tryptophan is an essential dietary amino acid that originates uremic toxins that contribute to end-stage kidney disease (ESKD) patient outcomes. We evaluated serum levels and removal during haemodialysis and haemodiafiltration of tryptophan and tryptophan-derived uremic toxins, indoxyl sulfate (IS) and indole acetic acid (IAA), in ESKD patients in different dialysis treatment settings. This prospective multicentre study in four European dialysis centres enrolled 78 patients with ESKD. Blood and spent dialysate samples obtained during dialysis were analysed with high-performance liquid chromatography to assess uremic solutes, their reduction ratio (RR) and total removed solute (TRS). Mean free serum tryptophan and IS concentrations increased, and concentration of IAA decreased over pre-dialysis levels (67%, 49%, -0.8%, respectively) during the first hour of dialysis. While mean serum total urea, IS and IAA concentrations decreased during dialysis (-72%, -39%, -43%, respectively), serum tryptophan levels increased, resulting in negative RR (-8%) towards the end of the dialysis session ( p < 0.001), despite remarkable Trp losses in dialysate. RR and TRS values based on serum (total, free) and dialysate solute concentrations were lower for conventional low-flux dialysis ( p < 0.001). High-efficiency haemodiafiltration resulted in 80% higher Trp losses than conventional low-flux dialysis, despite similar neutral Trp RR values. In conclusion, serum Trp concentrations and RR behave differently from uremic solutes IS, IAA and urea and Trp RR did not reflect dialysis Trp losses. Conventional low-flux dialysis may not adequately clear Trp-related uremic toxins while high efficiency haemodiafiltration increased Trp losses.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) that is associated with demyelination and neuronal loss. Over recent years, the immunological and neuronal effects of tryptophan (Trp) metabolites have been largely investigated, leading to the hypothesis that these compounds and the related enzymes are possibly involved in the pathophysiology of MS. Specifically, the kynurenine pathway of Trp metabolism is responsible for the synthesis of intermediate products with potential immunological and neuronal effects. More recently, Trp metabolites, originating also from the host microbiome, have been identified in MS, and it has been shown that they are differently regulated in MS patients. Here, we sought to discuss whether, in MS patients, a specific urinary signature of host/microbiome Trp metabolism can be potentially identified so as to select novel biomarkers and guide toward the identification of specific metabolic pathways as drug targets in MS., (Copyright © 2020 Gaetani, Boscaro, Pieraccini, Calabresi, Romani, Di Filippo and Zelante.)

Background: Concentrated cherry juice reportedly contains melatonin which, in turn, has been highlighted as an important regulator in initiating sleep., Aim: The present investigation aims to clarify whether Night Time Recharge (NTR), a marketed sleep aid containing cherry extract, improves key sleep parameters in young, active adults with mildly poor sleep., Methods: A double-blind, randomized, placebo-controlled, cross-over study design was employed. Twenty participants (nine female) consumed either NTR or a placebo for seven days. Accelerometers were used to assess sleep quality and physical activity levels. Urinary levels of 6-sulphatoxymelatonin (6-SMT), a marker of melatonin synthesis, was assessed via enzyme-linked immunosorbent assay., Results: 6-SMT levels increased following NTR treatment (28.95 ng/ml) compared with placebo (4.0 ng/ml) ( p < 0.001). There was also a significant difference ( p = 0.047) in dietary tryptophan consumption during the NTR treatment (1236 mg) versus placebo (1149 mg). No trace of melatonin was detected from our analysis of the supplement. NTR had no significant effect on any sleep parameters with the exception of sleep latency ( p = 0.001)., Conclusions: As chemical analysis of NTR by liquid-chromatography mass-spectrometry identified no detectable melatonin, the tryptophan content of the supplement is a likely reason for improvement in sleep latency. These results are in contrast to previous studies which have found a positive effect on sleep following cherry supplementation. Future work should focus on sleep latency and investigating whether cherry juice is effective in participants with problems in initiating sleep.

Purposes: Indoleamine-2,3-dioxygenase-1 (IDO1) is a key enzyme of tryptophan metabolism which regulates T cell function in immune cells and little is known about the role of IDO1 expression in bladder cancer cells. The study is aimed to evaluate the clinical relevance of IDO1 expression in human bladder urothelial carcinoma (UC)., Materials and Methods: One hundred and sixty paraffin-embedded UC tissues (130 bladder, 30 upper urinary tract) and 47 adjacent normal tissues were retrieved for IDO1 immunostaining. Urine samples from UC and non-UC patients were collected before surgery for measuring the concentration of tryptophan and its metabolites. Clinicopathological correlates of IDO1 expression and the prognostic values in human bladder cancer were explored. External validation was performed with 4 published bladder cancer datasets, as well as in vitro studies., Results: As compared with normal adjacent tissues, UC exhibited a higher frequency of IDO1 expression (chi-square, P = 0.0005). IDO1 expression is an independent poor prognostic factor for disease progression [hazard ratio and 95% confidence interval, 3.80 (1.46-9.86), P = 0.006], which is associated with decreased number of intratumoral infiltrating CD8+ lymphocyte (unpaired t test, P = 0.026). External validation showed that patients with higher IDO1 expression exhibit decreased disease-specific survival than those with lower IDO1 expression. Furthermore, IDO1 expression correlated positively with the expression of several EMT markers, including ZEB2, fibronectin and vimentin. The in vitro T24 cell subline demonstrated that IDO1 expression can up-regulate ZEB2 expression probably through miR-200c signaling., Conclusion: IDO1 expression predicts poorer survival and up-regulates ZEB2 expression in human bladder cancer., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Chemometrics multivariate calibration coupled with high performance liquid chromatography-diode array detection (HPLC-DAD) analytical strategy was applied for fast and sensitive quantification of the eight small molecules (uric acid, creatinine, tyrosine, homovanillic acid, hippuric acid, indole-3-acetic acid, tryptophan and 2-methylhippuric acid) in human urine. The objective of this work was to get the successful resolution of the complex matrix with minimum experimental time in the presence of highly overlapping peaks, of distortions in the time and baseline aspects among chromatograms, and of the presence of unknown and background interferences. All the analysis were based on a short C18 column with the chromatographic system operating in isocratic mode and all analytes can be successfully quantified within 6 min. The second-order HPLC-DAD data acquired were handled intelligently by two typical chemometrics tools including alternating trilinear decomposition (ATLD) and multivariate curve resolution-alternating least squares (MCR-ALS). Reasonable resolution and satisfactory quantification results were obtained regardless of the complex matrix interferences from the urine samples and the second-order advantage was fully exploited. With the validation by classic HPLC method, the proposed strategy could take extra advantages such as increased selectivity and sensitivity, shorter analysis time, undemanding elution conditions and sufficiency of lower limit of quantification benefit from multivariate calibration. The method was shown as a promising means for fast and sensitive determination of small molecules in human urine and also for fast diagnosis or surveillance in related diseases., (Copyright © 2019. Published by Elsevier B.V.)

Background and objectives: Melanin, which has a confirmed role in melanoma cell behaviour, is formed in the process of melanogenesis and is synthesized from tryptophan, L-tyrosine and their metabolites. All these metabolites are easily detectable by chromatography in urine. Materials and Methods: Urine samples of 133 individuals (82 malignant melanoma patients and 51 healthy controls) were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC). The diagnosis of malignant melanoma was confirmed histologically. Results: Chromatograms of melanoma patients showed increased levels of 5,6-dihydroxyindole-2-carboxylic acid, vanilmandelic acid, homovanilic acid, tryptophan, 5-hydroxyindole-3-acetic acid, and indoxyl sulphate compared to healthy controls. Concentration of indoxyl sulphate, homovanilic acid and tryptophan were significantly increased even in the low clinical stage 0 of the disease (indoxyl sulphate, homovanilic acid and tryptophan in patients with clinical stage 0 vs. controls expressed as medium/ interquartile range in µmol/mmol creatinine: 28.37/15.30 vs. 5.00/6.91; 47.97/33.08 vs. 7.33/21.25; and 16.38/15.98 vs. 3.46/6.22, respectively). Conclusions: HPLC detection of metabolites of L-tyrosine and tryptophan in the urine of melanoma patients may play a significant role in diagnostics as well as a therapeutic strategy of melanoma cancer.

A composite was prepared from graphene functionalized with 3,4,9,10-perylene tetracarboxylic acid and chitosan (rGO-PTCA-chitosan) by a chemical method. It involves non-covalent functionalization of rGO with PTCA followed by amidation reaction with chitosan. Scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy and electrochemical methods were used to characterize the composites. By combining the chiral features of chitosan and the excellent electrochemical behaviors of rGO-PTCA, a graphene-based material with enantioselectivity was constructed for electrochemical chiral recognition of tryptophan (Trp) enantiomers. A glassy carbon electrode (GCE) modified with rGO-PTCA-chitosan had a higher recognition capability for L-Trp than for D-Trp. Best operated at a working voltage near 0.78 V (vs. SCE), the enantioselectivity coefficient is 3.0. The sensor has a linear response in the 1 mM to 10 mM Trp concentration range and a 1.2 μM detection limit (at S/N = 3) for L-Trp, and of 3.0 μM to D-Trp. The sensor was successfully used to detect Trp enantiomers in real samples, and a recognition mechanism is presented. Graphical abstract Schematic presentation of a composoie prepared by graphene functionalized with 3,4,9,10-perylene tetracarboxylic acid and chitosan (rGO-PTCA-chitosan) via a chemical method. It involves non-covalent functionalization of rGO with PTCA followed by amidation reaction with chitosan and voltammetric determination of tryptophan enantiomers.

Gestational diabetes mellitus (GDM) is a disorder in pregnancy with highest impact in the future life of both mother and newborn. Increasing incidence, economic impact, and potential for severe GDM-related pregnancy complications are some factors that have motivated the deep study of physiopathology, risk factors for developing GDM, and potential biomarkers for its diagnosis. In the present pilot study, we analyzed the urinary metabolome profile of GDM patients in the 3rd trimester of pregnancy, when GDM is already established and the patients are under dietary and pharmacological control. An untargeted metabolomics method based on liquid chromatography⁻mass spectrometry analysis was developed to identify differentially expressed metabolites in the GDM group. We identified 14 metabolites that are significantly upregulated in the urine of GDM patients, and, more importantly, we identified those related with the steroid hormone biosynthesis and tryptophan (TRP) metabolism pathways, which are associated with GDM pathophysiology. Thus, these metabolites could be screened as potential prognostic biomarkers of type two diabetes mellitus, coronary artery disease and chronic renal failure in future follow-up studies with GDM patients.

Introduction: Metabolic syndrome (MetS) is a disorder defined by having three of five features: increased waist circumference (WC), hypertriglyceridemia, decreased high-density lipoprotein-cholesterol, hypertension and an elevated blood glucose (BG). Metabolic Syndrome ( MetS) affects 35% of American adults and significantly increases risk for Atherosclerotic cardiovascular disease (ASCVD) and type-2 diabetes (T2DM). An understanding of the metabolome will help elucidate the pathogenesis of MetS and lead to better management. We hypothesize that the metabolites, gamma-aminobutyric acid (GABA), d-pyroglutamic acid (PGA) and N-acetyl-d-tryptophan (NAT) will be altered in nascent MetS patients without the confounding of ASCVD or T2DM. We also correlated these metabolites with biomarkers of inflammation., Patients and Methods: This was an exploratory study of 30 patients with nascent MetS and 20 matched controls undertaken in 2018. Metabolites were evaluated from patient's frozen early morning urine samples and were correlated with biomarkers of inflammation and adipokines. They were assayed by the NIH Western Metabolomics Center using liquid chromatography/mass spectrometry and standardized to urinary creatinine. All patients had normal hepatic and renal function., Results: GABA and PGA levels were significantly increased in MetS patients compared to controls: 2.8-fold and 2.9-fold median increases respectively with p < 0.0001 and p = 0.004, possibly deriving from glutamate. NAT was significantly decreased by 90% in MetS patients compared to controls, p < 0.001. GABA correlates significantly with cardio-metabolic (CM) features including WC, blood pressure systolic (BP-S) while NAT correlated inversely with WC, BP-S, blood glucose (BG) and triglycerides (TG). GABA correlated positively with chemerin, leptin, Fetuin A and endotoxin. NAT correlated inversely with WC, BP-S, BG, TG, high sensitivity C - reactive protein (hsCRP), toll-like receptor-4 (TLR-4), lipopolysaccharide binding protein (LBP), chemerin and retinol binding protein-4 (RBP-4)., Conclusions: We make the novel observation of increased GABA and PGA with decreased NAT in patients with MetS. While GABA and PGA correlates positively with CM features and biomediators of inflammation, the metabolite NAT correlated inversely. Thus, GABA and PGA could contribute to the pro-inflammatory state of MetS while NAT could mitigate this pro-inflammatory response., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Modified nucleosides (mNS) in urine are shown to be encouraging markers in cancer, mostly in patients presenting with high tumor mass such is breast and lung cancer. To our knowledge, mNS have not been investigated in head and neck squamous cell carcinoma (HNSCC). HNSCC is characterized by early metastasis into locoregional lymph nodes and slow infiltrating growth, but even in the advanced stage exhibits only a relatively low cancer volume. Therefore, reliable distinction between HNSCC and healthy controls by urinary mNS might pose substantial analytical problems and even more as patients with HNSCC mostly have an increased exposure to tobacco smoke and excessive alcohol consumption which affect the renal mNS pattern., Materials and Methods: Urinary mNS in samples of 93 therapy-naive patients with HNSCC and 242 healthy controls were quantified by reversed-phase high-performance liquid chromatography. Considering that the circadian rhythm causes diuresis-induced variations in concentration, the mNS-to-creatinine ratio was chosen to compare patients and controls. For sensitivity and specificity in discriminating between patients and controls, the corresponding curve was plotted. Additionally, logistic regression was carried out and a multilayer perceptron neuronal network (NN) was created., Results: Fifteen mNS were detectable in cases and controls; concentrations of 11 were found to be significantly different. The sensitivity and specificity depend on the total volume of the lesion; HNSCC with volume <20 ml was reliably detected, but those with a volume of 20 ml or greater produced amounts of mNS which led to the most accurate detection of HNSCC based on HNSCC-specific mNS patterns., Conclusion: Analysis of urinary mNS allows for detection of small-volume HNSCC, with acceptable specificity and sensitivity if the tumor volume exceeds 20 ml., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Nicotinamide adenine dinucleotide (NAD + ) extends longevity in experimental organisms, raising interest in its impact on human health. De novo NAD + biosynthesis from tryptophan is evolutionarily conserved yet considered supplanted among higher species by biosynthesis from nicotinamide (NAM). Here we show that a bottleneck enzyme in de novo biosynthesis, quinolinate phosphoribosyltransferase (QPRT), defends renal NAD + and mediates resistance to acute kidney injury (AKI). Following murine AKI, renal NAD + fell, quinolinate rose, and QPRT declined. QPRT +/- mice exhibited higher quinolinate, lower NAD + , and higher AKI susceptibility. Metabolomics suggested an elevated urinary quinolinate/tryptophan ratio (uQ/T) as an indicator of reduced QPRT. Elevated uQ/T predicted AKI and other adverse outcomes in critically ill patients. A phase 1 placebo-controlled study of oral NAM demonstrated a dose-related increase in circulating NAD + metabolites. NAM was well tolerated and was associated with less AKI. Therefore, impaired NAD + biosynthesis may be a feature of high-risk hospitalizations for which NAD + augmentation could be beneficial.

In this study, we compared different types of biomolecular markers in kidney cancer patients and in normal healthy controls, using fluorescence emission spectra and synchronous fluorescence excitation spectra. We were able to provide an accurate classification of the spectral features of kidney cancer patients relative to that of normal controls, in terms of the concentration ratios of biomolecules (viz., tryptophan, NADH, FAD, basic porphyrin, and acidic porphyrin) based on the intensity of their spectral peaks. The specificity and sensitivity of the method were 90%. The rationale of our current approach is to evolve an innovative protocol for the spectral characterization of in vitro optical analyses suitable for both small clinics and hospitals., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A glassy carbon electrode (GCE) was modified with poly(L-arginine) (P-Arg), reduced graphene oxide (rGO) and gold nanoparticle (AuNP) to obtain an electrode for simultaneous determination of dopamine (DA), serotonin (5-HT) and L-tryptophan (L-Trp) in the presence of ascorbic acid (AA). The modified GCE was prepared via subsequent 'layer-by-layer' deposition using an electrochemical technique. The surface morphology of the modified electrode was studied by scanning electron microscopy, and electrochemical characterizations were carried out via cyclic voltammetry and electrochemical impedance spectroscopy. The modified electrode showed excellent electrocatalytic activity toward DA, 5-HT and L-Trp at pH 7.0. Figures of merit for the differential pulse voltammetric reponse are as follows: (a) Response to DA is linear in two intervals, viz. 1.0-50 nM and 1.0-50 μM DA concentration range, the typical working voltage is 202 mV (vs. Ag/AgCl), and the detection limit is 1 nM (at an S/N ratio of 3). For 5-HT, the respective data are 10 to 500 nM and 1.0 to 10 μM, 381 mV, and 30 nM. For L-Trp, the respective data are 10-70 nM and 10-100 μM, 719 mV, and 0.1 μM. The modified GCE is fairly selective. It was successfully applied to the simultaneous determination of DA, 5-HT, and L-Trp in spiked urine samples, and high recovery rates were found. Graphical abstract Schematic presentation of the voltammetric sensor based on a glassy carbon electrode modified with poly(L-arginine), reduced graphene oxide (rGO) and gold nanoparticle (GCE/P-Arg/ErGO/AuNP) for simultaneous determination of dopamine (DA), serotonin (5-HT) and L-tryptophan (L-Trp).

Autism spectrum disorder (ASD) is a range of neurodevelopmental problems without certain causes. Conventional diagnostic or screening tools for ASD rely on the observation of children's behavioral presentations. Novel methods are focused on the alterations of some important biochemical matters in ASD patients, which are applicable in the screening for ASD. This study investigated and compared amino acids in the first morning urine from age and sex matched ASD and non-ASD children using high performance liquid chromatography. Significantly lower urinary free methionine, phenylalanine, valine, tryptophan, and leucine plus isoleucine were observed in ASD children. The effects of using urinary free amino acids (UFAAs) singly or conjointly to classify participants into ASD or control group were analyzed and compared. ROC curves on these UFAAs singly in classification performed the sensitivity of 0.593-0.889 and the specificity of 0.704-0.963. Binary-logistic regression analysis of these UFAAs obtained a final regression model comprised of urinary free valine and tryptophan. The ROC curve established by the linear combination of the two amino acids achieved a sensitivity of 0.926 and a specificity of 0.889, which showed superiority to single UFAA and comparability to existing diagnostic or screening tools. It was suggested that the multivariate model based on UFAAs was possibly applicable in screening for children at higher risk of ASD., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Indoleamine-2,3-dioxygenase (IDO) catalyzes the first step of tryptophan (Trp) catabolism, yielding kynurenine (Kyn) metabolites. The kynurenine-to-tryptophan (K/T) ratio is used as a surrogate for biological IDO enzyme activity. IDO expression is increased during Escherichia coli urinary tract infection (UTI). Thus, our objective was to develop a method for measurement of Kyn/Trp ratio in human blood and urine and evaluate its use as a biomarker of UTI., Methods: A mass spectrometric method was developed to measure Trp and Kyn in serum and urine specimens. The method was applied to clinical urine specimens from symptomatic pediatric patients with laboratory-confirmed UTI or other acute conditions and from healthy controls., Results: The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was linear to 500 μmol/L for both Trp and Kyn. Imprecision ranged from 5 to 15% for Trp and 6-20% for Kyn. Analytical recoveries of Trp and Kyn ranged from 96 to 119% in serum and 90-97% in urine. No correlation was found between the K/T ratio and circulating IDO mass (r = 0.110) in serum. Urinary Kyn and Trp in the pediatric test cohort demonstrated elevations in the K/T ratio in symptomatic patients with UTI (median 13.08) and without UTI (median 14.38) compared to healthy controls (median 4.93; p < 0.001 for both comparisons). No significant difference in K/T ratio was noted between symptomatic patients with and without UTI (p = 0.84)., Conclusions: Measurement of Trp and Kyn by LC-MS/MS is accurate and precise in serum and urine specimens. While urinary K/T ratio is not a specific biomarker for UTI, it may represent a general indicator of a systemic inflammatory process., (Copyright © 2018 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: Bright light (BL) has been shown to be effective in enhancing both cognitive and physical performances. Alterations in nighttime melatonin levels have also been observed. However, evaluations of light-induced changes in the preceding biochemical processes are absent. Therefore, the impact of a single morning BL exposure on sensorimotor and visuomotor performance, as well as tryptophan (trp) and trp metabolites, was evaluated in this study., Methods: In a crossover design, 33 healthy volunteers were randomly exposed to 30 min of < 150 lx at eye level (office light, OL) and 5000 lx at eye level (bright light, BL) of 6500 K in the morning hours. Trp, sulfatoxymelatonin (aMT6s), and kynurenine (kyn) courses over the morning hours were analyzed, and changes in sensori- and visuomotor measures were examined., Results: Motoric performance increased in both setups, independent of light intensity. aMT6s and kyn decreased equally under both lighting conditions. Trp levels decreased from a mean (95% confidence interval) of 82.0 (77.2-86.9) to 66.5 (62.5-70.1) in the OL setup only., Conclusion: These data suggest that BL in the morning hours has a limited effect on visuo- and sensorimotor performance. Nevertheless, trp degradation pathways in the morning show diverse courses after OL and BL exposure. This suggests that trp courses can potentially be altered by BL exposure.

Blue diaper syndrome (BDS) (Online Mendelian Inheritance in Man number 211000) is an extremely rare disorder that was first described in 1964. The characteristic finding is a bluish discoloration of urine spots in the diapers of affected infants. Additional clinical features of the first described patients included diarrhea, inadequate weight gain, hypercalcemia, and nephrocalcinosis. An intestinal defect of tryptophan absorption was postulated as the underlying pathology. However, functional evidence for this theory is lacking. No genetic cause has been identified so far. Here, we report on a boy who presented with neonatal-onset diarrhea, metabolic acidosis, transient hepatopathy, recurrent hypoglycemia, and blue-stained urine spots in his diapers. An ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis of urine samples at different time points demonstrated the constant presence of indigo derivatives, thereby confirming the diagnosis of BDS. Of note, the visibility of indigo derivatives in the urine was highly dependent on the urine's pH. To identify the underlying genetic cause of the disease, whole-exome sequencing was performed, leading to the identification of a homozygous frameshift mutation in proprotein convertase subtilisin/kexin type 1 ( PCSK1 ; NM&#95;000439.4: c.679del, p.[Val227Leufs*12]). PCSK1 encodes prohormone convertase 1/3, and mutations within this gene have been reported as a rare cause of early-onset malabsorptive diarrhea and multiple endocrine dysfunction. In our report, we suggest that BDS can be caused by PCSK1 mutations., Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2018 by the American Academy of Pediatrics.)

Using a non-targeted metabolomics platform, we recently identified C-mannosyltryptophan and pseudouridine as non-traditional kidney function markers. The aims of this study were to obtain absolute concentrations of both metabolites in blood and urine from individuals with and without CKD to provide reference ranges and to assess their fractional excretions (FE), and to assess the agreement with their non-targeted counterparts. In individuals without/with CKD, mean plasma and urine concentrations for C-mannosyltryptophan were 0.26/0.72 µmol/L and 3.39/4.30 µmol/mmol creatinine, respectively. The respective concentrations for pseudouridine were 2.89/5.67 µmol/L and 39.7/33.9 µmol/mmol creatinine. Median (25 th , 75 th percentiles) FEs were 70.8% (65.6%, 77.8%) for C-mannosyltryptophan and 76.0% (68.6%, 82.4%) for pseudouridine, indicating partial net reabsorption. Association analyses validated reported associations between single metabolites and eGFR. Targeted measurements of both metabolites agreed well with the non-targeted measurements, especially in urine. Agreement for composite nephrological measures FE and urinary metabolite-to-creatinine ratio was lower, but could be improved by replacing non-targeted creatinine measurements with a standard clinical creatinine test. In summary, targeted quantification and additional characterization in relevant populations are necessary steps in the translation of non-traditional biomarkers in nephrology from non-targeted discovery to clinical application.

Recent reports show that the worldwide incidence of autism spectrum disorder (ASD) is dramatically increasing, although ASD etiology and pathogenesis are still far to be fully elucidated. Some dietary-derived essential compounds, such as the amino acid tryptophan, appear to be impaired in patients with ASD. Tryptophan (Trp) plays a significant role in the human organism and serves as a precursor for a wide range of bioactive compounds, including major neurotransmitters. Research indicates that tryptophan might be deficient in subjects with ASD. Deficiency in the tryptophan level can be retrieved by investigating Trp levels or its major metabolite kynurenine in urines. The purpose of the present study is to quantify tryptophan content in urine samples (n = 236) of ASD patients, who underwent a supplemented dietary panel with B vitamins and magnesium, compared to controls (without this diet regimen). The samples were analyzed with gas chromatography-mass spectrometry. Additionally, the correlation between body mass index (BMI) and the level of this amino acid in urine was accomplished. Basic parameters of urine samples were also evaluated. Statistical evaluations in the concentration of tryptophan in ASD patients with different severity of symptoms were reported. A significant difference in tryptophan levels in all groups was observed. Supplementation with B vitamins and magnesium has an influence on the Trp concentration. Furthermore, no correlation between BMI and tryptophan levels was found. These results assess that the Trp level in ASD subjects is critical and that intake of B vitamins and magnesium with diet might influence its metabolic homeostasis.

While over 10% of the human metabolome is directly associated with the gut microbial metabolism, specific metabolites are largely uncharacterized. Therefore, methods for the identification and quantification of microbiota-associated metabolites in biological fluids such as urine or plasma are necessary in order to elucidate the molecular basis of host-microbiota interaction. In this study, we focused on the tryptophan metabolism, employing quantitative assays by ultra-high performance liquid chromatography (UHPLC) and tandem mass spectrometry, specifically selected reaction monitoring (SRM). Metabolite standards were utilized to generate SRM library for 16 intermediates of the tryptophan metabolism which were human endogenous as well as microbiota-associated based on the HMDB classification. Next, the SRM assays were utilized for screening in maternal urine samples and in dried urine specimens from neonates. The approach resulted in the discovery of microbiota-associated metabolites (methyl indole-3-acetate and methyl indol-3-propionate) previously unreported in urine samples and additionally in quantification of 8 intermediates of the tryptophan metabolism. To the best of our knowledge, this study represents the first attempt to explore previously unreported microbial metabolites in urine by UHPLC-SRM and novel methodology for simultaneous determination of microbiota-modulated component of Trp metabolism., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Alzheimer's disease (AD), a neurodegenerative disorder, is the major form of dementia. As AD is an irreversible disease, it is necessary to focus on earlier intervention. However, the potential biomarkers of preclinical AD are still not clear. In this study, urinary metabolomics based on ultra-high-performance liquid chromatography coupled with quadruple time-of-flight mass spectrometry was performed for delineating the metabolic changes and potential early biomarkers in APPswe/PS1dE9 (APP/PS1) transgenic mice. A total of 24 differentially regulated metabolites were identified when comparing transgenic mice to wild-type mice using multivariate statistical analysis. Among them, 10 metabolites were significantly upregulated and 14 metabolites were downregulated. On the basis of these potential biomarkers, metabolic pathway analysis found that pentose and glucuronate interconversions, glyoxylate and dicarboxylate metabolism, starch and sucrose metabolism, the citrate cycle, tryptophan metabolism, and arginine and proline metabolism were disturbed in APP/PS1 mice. Our study revealed that levels of endogenous metabolites in the urine of APP/PS1 mice changed prior to the emergence of learning and cognitive impairment, which may be associated with abnormal nitric oxide production pathways and metabolic disorders of monoaminergic neurotransmitters. In conclusion, this study showed that metabolomics provides an early indicator of disease occurrence for AD.

Tryptophan (Trp) is an essential amino acid that plays an important role in protein synthesis and is a precursor of various substances related to diverse biological functions. An imbalance in Trp metabolites is associated with inflammatory diseases. The accurate and precise measurement of these compounds in biological specimens would provide meaningful information for understanding the biochemical states of various metabolic syndrome-related diseases, such as hyperlipidemia, hypertension, diabetes, and obesity. In this study, we developed a rapid, accurate, and sensitive liquid chromatography-tandem mass spectrometry-based method for the simultaneous targeted analysis of Trp and its related metabolites of the kynurenine (Kyn), serotonin, and tryptamine pathways in urine. The application of the developed method was tested using urine samples after protein precipitation. The detection limits of Trp and its metabolites were in the range of 0.01 to 0.1 μg/mL. The method was successfully validated and applied to urine samples from controls and patients with metabolic syndrome. Our results revealed high concentrations of Kyn, kynurenic acid, xanthurenic acid, and quinolinic acid as well as a high Kyn-to-Trp ratio (KTR) in patients with metabolic syndromes. The levels of urine Kyn and KTR were significantly increased in patients under 60 years old. The profiling of urinary Trp metabolites could be a useful indicator for age-related diseases including metabolic syndrome. ᅟ.

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/m 2 and median (interquartile range) HbA 1c 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.

Background: Acute intermittent porphyria (AIP) is a rare metabolic disorder due to a deficiency of porphobilinogen deaminase, the third enzyme of the heme biosynthetic pathway. This low enzymatic activity may predispose to the appearance of acute neurological attacks. Seminal studies suggested that AIP was associated with changes in tryptophan homeostasis with inconclusive results. Therefore, the aim of this study was to analyze the urinary metabolome of AIP patients focusing on tryptophan metabolism using state-of-the-art technology., Methods: This was a case-control study including a group of 25 AIP patients with active biochemical disease and increased excretion of heme-precursors and 25 healthy controls. Tryptophan and related compounds and metabolites including: large neutral amino acids (LNAAs), serotonin, kynurenine, kynurenic acid and anthranilic acid were quantified in urine by liquid chromatography tandem-mass spectrometry (LC-MS/MS). Twenty-nine biological markers (including metabolic ratios and absolute concentrations) were compared between patients and controls., Results: Significant differences were found in the tryptophan-kynurenine metabolic pathway. Compared to controls, AIP patients showed: (a) increased urinary excretion of kynurenine and anthranilic acid (P<0.005); (b): elevation of the kynurenine/tryptophan ratio (P<0.001) and (c): decrease of the kynurenic acid/kynurenine ratio (P=0.001). In contrast, no differences were found in the serotonin metabolic pathway independently of the markers and ratios used., Conclusions: The results of the study demonstrate that there is an imbalance in the kynurenine metabolic pathway in AIP patients, with an increase of the kynurenine/tryptophan ratio in urine and a reduction of the kynurenic acid/kynurenine ratio. The modified ratios suggest induction of indoleamine 2,3-deoxygenase and decreased activity of kynurenine aminotransferase in the liver. The results confirm that LC-MS/MS is useful for the characterization of the urinary metabolome of hepatic porphyrias., (Copyright © 2017. Published by Elsevier B.V.)

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., (Copyright © 2017 the American Physiological Society.)

Antibiotics excreted into the intestinal tract may disrupt the microbiota that provide colonization resistance against enteric pathogens and alter normal metabolic functions of the microbiota. Many of the bacterial metabolites produced in the intestinal tract are absorbed systemically and excreted in urine. Here, we used a mouse model to test the hypothesis that alterations in levels of targeted bacterial metabolites in urine specimens could provide useful biomarkers indicating disrupted or intact colonization resistance. To assess in vivo colonization resistance, mice were challenged with Clostridium difficile spores orally 3, 6, and 11 days after the completion of 2 days of treatment with piperacillin-tazobactam, aztreonam, or saline. For concurrent groups of antibiotic-treated mice, urine samples were analyzed by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify the concentrations of 11 compounds targeted as potential biomarkers of colonization resistance. Aztreonam did not affect colonization resistance, whereas piperacillin-tazobactam disrupted colonization resistance 3 days after piperacillin-tazobactam treatment, with complete recovery by 11 days after treatment. Three of the 11 compounds exhibited a statistically significant and >10-fold increase (the tryptophan metabolite N -acetyltryptophan) or decrease (the plant polyphenyl derivatives cinnamoylglycine and enterodiol) in concentrations in urine 3 days after piperacillin-tazobactam treatment, followed by recovery to baseline that coincided with the restoration of in vivo colonization resistance. These urinary metabolites could provide useful and easily accessible biomarkers indicating intact or disrupted colonization resistance during and after antibiotic treatment., (Copyright © 2017 American Society for Microbiology.)

In this present investigation, we introduced a novel electrochemical sensor for the detection of tryptophan (TRP) based on green pompoms flower-like cerium vanadate (CeVO 4 ). The flower-like CeVO 4 microstructure was prepared by the simple hydrothermal treatment with the assistance of urea for the first time. The as-prepared flower-like CeVO 4 microstructure was characterized by various analytical and spectroscopic techniques such as X-ray diffraction, Raman spectroscopy fourier transform infrared spectroscopy, scanning electron microscopy and energy-dispersive X-ray spectroscopy studies. The electrochemical properties are evaluated by the cyclic voltammetry (CV) and differential pulse voltammetry (DPV). As an electrochemical sensor, the green pompoms flower-like CeVO 4 modified glassy carbon electrode (GCE) displayed an excellent electrocatalytic activity for the detection of TRP. The obtained electrochemical results revealed that the oxidation of TRP, exhibited a lower potential and higher anodic peak current when compared to unmodified GCE. These results were suggested that the flower-like CeVO 4 /GCE have a good electrocatalytic activity towards the TRP oxidation. The flower-like CeVO 4 sensor exhibited the wide linear concentration range and low detection limit of 0.1-94µM and 0.024µM respectively. Finally, the proposed sensor was successfully applied to the determination of TRP in real sample analysis such as food and biological samples with satisfied recoveries., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The tyrosine, tryptophan, and glutamate metabolic pathways play key roles on pathological state of neuronal functions and the change of their levels in biological systems reflects the progress degree of neuronal diseases. Comprehensive profiling of these metabolites is important to find new biomarkers for diagnosis or prognosis of various neuronal diseases. However, the overall profiling analysis of various neurochemicals in biological sample is confronted with several limitations due to their low concentration and physicochemical properties and the coexistence of matrices. We developed an efficient and feasible method using gas chromatography-tandem mass spectrometry (GC-MS/MS). Wide-bore mixed cation exchange (MCX) SPE process enables a rapid and effective cleanup of 20 neurochemicals even including acidic and basic neurochemicals in a single SPE cartridge by using different composition of eluents. Selective derivatization of various types of metabolites was applied to achieve highly chromatographic separation and sensitive mass detection. Appropriate selection of precursor and product transition ions used in multiple reaction-monitoring (MRM) mode based on the MS/MS fragmentations of the derivatized neurochemicals could be significantly minimized the matrix effects and enhanced the reliability of quantification results. The developed method was validated in terms of linearity, limits of detection, precision, accuracy, and matrix effects. The intra- and inter-assay analytical variations were less than 10%. The overall linearity for all of the targets was excellent (R 2 ≥0.996). The detection limits ranged between 0.38 and 8.13ng/mL for the acidic neurochemicals and between 0.02 and 11.1ng/mL for the basic neurochemicals. The developed protocol will be expected to be a promising tool for the understanding of the pathological state and diagnosis of various neuronal diseases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Autism spectrum disorder (ASD) is still diagnosed through behavioral observation, due to a lack of laboratory biomarkers, which could greatly aid clinicians in providing earlier and more reliable diagnoses. Metabolomics on human biofluids provides a sensitive tool to identify metabolite profiles potentially usable as biomarkers for ASD. Initial metabolomic studies, analyzing urines and plasma of ASD and control individuals, suggested that autistic patients may share some metabolic abnormalities, despite several inconsistencies stemming from differences in technology, ethnicity, age range, and definition of "control" status., Methods: ASD-specific urinary metabolomic patterns were explored at an early age in 30 ASD children and 30 matched controls (age range 2-7, M:F = 22:8) using hydrophilic interaction chromatography (HILIC)-UHPLC and mass spectrometry, a highly sensitive, accurate, and unbiased approach. Metabolites were then subjected to multivariate statistical analysis and grouped by metabolic pathway., Results: Urinary metabolites displaying the largest differences between young ASD and control children belonged to the tryptophan and purine metabolic pathways. Also, vitamin B 6 , riboflavin, phenylalanine-tyrosine-tryptophan biosynthesis, pantothenate and CoA, and pyrimidine metabolism differed significantly. ASD children preferentially transform tryptophan into xanthurenic acid and quinolinic acid (two catabolites of the kynurenine pathway), at the expense of kynurenic acid and especially of melatonin. Also, the gut microbiome contributes to altered tryptophan metabolism, yielding increased levels of indolyl 3-acetic acid and indolyl lactate., Conclusions: The metabolic pathways most distinctive of young Italian autistic children largely overlap with those found in rodent models of ASD following maternal immune activation or genetic manipulations. These results are consistent with the proposal of a purine-driven cell danger response, accompanied by overproduction of epileptogenic and excitotoxic quinolinic acid, large reductions in melatonin synthesis, and gut dysbiosis. These metabolic abnormalities could underlie several comorbidities frequently associated to ASD, such as seizures, sleep disorders, and gastrointestinal symptoms, and could contribute to autism severity. Their diagnostic sensitivity, disease-specificity, and interethnic variability will merit further investigation.

A liquid phase microextraction based on hollow fibre followed by liquid chromatographic determination was developed for the extraction and quantitation of the hallucinogenic muscimol from urine samples. Method applicability on polar hallucinogens was also tested on two alkaloids, a psychedelic hallucinogen, tryptamine and a polar amino acid, tryptophan which exists in its charged state in the entire pH range. A multivariate design of experiments was used in which a half fractional factorial approach was applied to screen six factors (donor phase pH, acceptor phase HCl concentration, carrier composition, stirring rate, extraction time and salt content) for their extent of vitality in carrier mediated liquid microextractions. Four factors were deemed essential for the effective extraction of each analyte. The vital factors were further optimized for the extraction of single-spiked analyte solutions using a central composite design. When the simultaneous extraction of analytes was performed under universal factor conditions biased towards maximizing the enrichment of muscimol, a good composite desirability value of 0.687 was obtained. The method was finally applied on spiked urine samples with acceptable enrichments of 4.1, 19.7 and 24.1 obtained for muscimol, tryptophan and tryptamine respectively. Matrix-based calibration curves were used to address matrix effects. The r(2) values of the matrix-based linear regression prediction models ranged from 0.9933 to 0.9986. The linearity of the regression line of the matrix-based calibration curves for each analyte was directly linked to the analyte enrichment repeatability which ranged from an RSD value of 8.3-13.1%. Limits of detection for the developed method were 5.12, 3.10 and 0.21ngmL(-1) for muscimol, tryptophan and tryptamine respectively. The developed method has proven to offer a viable alternative for the quantitation of muscimol in human urine samples., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Nonylphenol (NP) was quantified using liquid chromatography tandem mass spectrometry (LC-MS/MS) in the urine and plasma of rats treated with 0, 50, and 250 mg/kg/day of NP for four consecutive days. A urinary metabolomic strategy was originally implemented by high performance liquid chromatography time of flight mass spectrometry (HPLC-QTOF-MS) to explore the toxicological effects of NP and determine the overall alterations in the metabolite profiles so as to find potential biomarkers. It is essential to point out that from the observation, the metabolic data were clearly clustered and separated for the three groups. To further identify differentiated metabolites, multivariate analysis, including principal component analysis (PCA), orthogonal partial least-squares discriminant analysis (OPLS-DA), high-resolution MS/MS analysis, as well as searches of Metlin and Massbank databases, were conducted on a series of metabolites between the control and dose groups. Finally, five metabolites, including glycine, glycerophosphocholine, 5-hydroxytryptamine, malonaldehyde (showing an upward trend), and tryptophan (showing a downward trend), were identified as the potential urinary biomarkers of NP-induced toxicity. In order to validate the reliability of these potential biomarkers, an independent validation was performed by using the multiple reaction monitoring (MRM)-based targeted approach. The oxidative stress reflected by urinary 8-oxo-deoxyguanosine (8-oxodG) levels was elevated in individuals highly exposed to NP, supporting the hypothesis that mitochondrial dysfunction was a result of xenoestrogen accumulation. This study reveals a promising approach to find biomarkers to assist researchers in monitoring NP.

Analysis of L-tryptophan (Trp) in biological samples has great importance for biomedical studies. Amino acid tyrosine (Tyr) that usually coexist with Trp in biological fluids can significantly interfere with reliable determination of Trp. In the current study, we demonstrate the development of two ultra-sensitive electrochemical sensor and label-free aptasensor for selective analysis of Trp in biological samples (i.e., cow's milk and human plasma, saliva and urine samples). In addition, without using AgCl/KCl, an Ag pseudo-reference screen printed electrode (Ag-PR-SPE) was exploited as a reference electrode. To prepare the engineered Trp sensor/aptasensor, a gold SPE was first modified with multiwall carbon nanotube (MWCNT-AuSPE) and then armed with Trp aptamer molecules (Apt-MWCNT-AuSPE). The prepared sensors were characterized using constant current-potentiometric stripping analysis (CC-PSA) and electrochemical impedance spectroscopy (EIS). The MWCNT-AuSPE and Apt-MWCNT-AuSPE were compared with respect to the linear detection range, limit of detection (LOD), accuracy, precision, repeatability. MWCNT-AuSPE and Apt-MWCNT-AuSPE demonstrate fast near-Nernstian response for PSA of Trp over the concentration ranging from 1.0 × 10(-9) to 2.0 × 10(-4) mol L(-1) and 1.0 × 10(-11) to 1.0 × 10(-4) mol L(-1) with detection limits of 3.6 × 10(-10) mol L(-1) and 4.9 × 10(-12) mol L(-1), respectively. Common interfering species present in the biological fluids (i.e., tyrosine, uric acid, ascorbic acid) showed no effects on the determination of Trp using CC-PSA. MWCNT-AuSPE and Apt-MWCNT-AuSPE represented well reproducibility and great precision with relative standard deviation (RSD) of 2.9% and 5.3% respectively. In comparison with the MWCNT-AuSPE, Apt-MWCNT-AuSPE provided higher sensitivity, selectivity and accuracy of Trp detection in real samples. Based on these findings, we propose the developed Apt-MWCNT-AuSPE as a simple detection method for analysis of Trp in biological samples., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: The aim of the current study was to explore the role of aromatic amino acids (AAAs) in blood in relation to attention-deficit/hyperactivity disorder (ADHD). Given their impact on the synthesis of serotonin and dopamine, decreased concentrations of the AAAs tryptophan, tyrosine and phenylalanine in blood may contribute to the expression of ADHD symptoms. Decreased AAA blood concentrations, in turn, may be related to lowered dietary protein intake or to abnormal AAA catabolism, as evidenced by increased urinary AAA concentrations., Methods: Eighty-three children with ADHD (75% males) and 72 typically developing (TD) children (51% males), aged 6 to 13 years, participated in the study. AAA concentrations were assessed in blood spots and an 18-hour urinary sample. A nutritional diary was filled out by parents to calculate dietary protein intake. Parent and teacher questionnaires assessed symptoms of ADHD, oppositional defiant disorder, conduct disorder, and autism spectrum disorder., Results: Children with ADHD showed normal AAA concentrations in blood spots and urine, as well as normal protein intake compared to controls. No associations between AAA concentrations and symptoms of ADHD or comorbid psychiatric disorders were found., Conclusions: This study is the first to explore AAA metabolism in children with ADHD using a well-defined and relatively large sample. We found that AAA deficiencies are not related to ADHD. The results do not support treatment with AAA supplements in children with ADHD. Future studies regarding the cause of serotonin and dopamine alterations in ADHD should focus on other explanations, such as effects of altered transport of AAAs.