Xanthurenic Acid Abstracts 1

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Rapid Isocratic Liquid Chromatographic Separation and Quantification of Tryptophan and Six kynurenine Metabolites in Biological Samples with Ultraviolet and Fluorimetric Detection.
            (Badawy and Morgan, 2010) Download
A simple, rapid isocratic liquid chromatographic procedure with ultraviolet and fluorimetric detection is described for the separation and quantification of L-tryptophan (Trp) and six of its kynurenine metabolites (kynurenine, 3-hydroxykynurenine, and 3-hydroxyanthranilic, kynurenic, xanthurenic and anthranilic acids). Using the Perkin Elmer LC 200 system, a reverse phase Synergi 4 μ fusion-RP80 A column (250 × 4.6 mm) (Phenomenex), and a mobile phase of 10 mM sodium dihydrogen phosphate: methanol (73:27, by vol) at pH 2.8 and a flow rate of 1.0-1.2 ml/min at 37 °C, a run took ∼13 min. The run took <7 min at 40 °C and a 1.4 ml/min flow rate. Limits of detection of all 7 analytes were 5-72 nM and their recoveries from human plasma and rat serum and liver varied between 62% and 111%. This simple method is suitable for high throughput work and can be further developed to include quinolinic acid and other Trp metabolites.

Novel spectrophotometric method for the quantitation of urinary xanthurenic acid and its application in identifying individuals with hyperhomocysteinemia associated with Vitamin B₆ deficiency.
            (Chen et al., 2013) Download
A novel spectrophotometric method for the quantification of urinary xanthurenic acid (XA) is described. The direct acid ferric reduction (DAFR) procedure was used to quantify XA after it was purified by a solid-phase extraction column. The linearity of proposed method extends from 2.5 to 100.0 mg/L. The method is precise, yielding day-to-day CVs for two pooled controls of 3.5% and 4.6%, respectively. Correlation studies with an established HPLC method and a fluorometric procedure showed correlation coefficients of 0.98 and 0.98, respectively. Interference from various urinary metabolites was insignificant. In a small-scale screening of elderly conducted at Penghu county in Taiwan (n = 80), we were able to identify a group of twenty individuals having hyperhomocysteinemia (>15  μ mole/L). Three of them were found to be positive for XA as analyzed by the proposed method, which correlated excellently with the results of the activation coefficient method for RBC's AST/B₆ functional test. These data confirm the usefulness of the proposed method for identifying urinary XA as an indicator of vitamin B₆ deficiency-associated hyperhomocysteinemic condition.

Urinary metabolomics of young Italian autistic children supports abnormal tryptophan and purine metabolism.
            (Gevi et al., 2016) Download
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 B6, 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.


 

Tryptophan metabolism, from nutrition to potential therapeutic applications.
            (Le Floc'h et al., 2011) Download
Tryptophan is an indispensable amino acid that should to be supplied by dietary protein. Apart from its incorporation into body proteins, tryptophan is the precursor for serotonin, an important neuromediator, and for kynurenine, an intermediary metabolite of a complex metabolic pathway ending with niacin, CO(2), and kynurenic and xanthurenic acids. Tryptophan metabolism within different tissues is associated with numerous physiological functions. The liver regulates tryptophan homeostasis through degrading tryptophan in excess. Tryptophan degradation into kynurenine by immune cells plays a crucial role in the regulation of immune response during infections, inflammations and pregnancy. Serotonin is synthesized from tryptophan in the gut and also in the brain, where tryptophan availability is known to influence the sensitivity to mood disorders. In the present review, we discuss the major functions of tryptophan and its role in the regulation of growth, mood, behavior and immune responses with regard to the low availability of this amino acid and the competition between tissues and metabolic pathways for tryptophan utilization.

Improved fluorometric quantification of urinary xanthurenic acid.
            (Liu et al., 1996) Download
Measurement of urinary xanthurenic acid (XA) has been used clinically to study a variety of disorders caused by vitamin B6 deficiency. To obviate some cumbersome steps of current methods for measuring XA in human urine, we have developed a simple fluorometric method. We apply the urine sample to a solid-phase extraction column containing trimethylaminopropyl group bound to silica, which enables us to purify and concentrate the XA from the urine without contamination from various tryptophan metabolites. The XA in the acidic eluate can then be quantified fluorometrically. The linearity of the proposed method extends from 0.2 to 10.0 mg/L. The method is precise, yielding day-to-day CVs for two pooled control specimens (1.08 and 1.90 mg/L) of 1.2% and 2.6%, respectively. Correlation studies with an established HPLC method and with a spectrophotometric procedure showed correlation coefficients of 0.99 and 0.98, respectively. Interference from vitamin C, uric acid, salicylate, acetaminophen, vanillylmandelic acid, and homovanillic acid was insignificant. The proposed method for urinary XA is rapid, simple, and suitable for routine use in the clinical laboratory.


 

Development of an LC-MS/MS method for the analysis of serotonin and related compounds in urine and the identification of a potential biomarker for attention deficit hyperactivity/hyperkinetic disorder.
            (Moriarty et al., 2011) Download
Serotonin is a major neurotransmitter and affects various functions both in the brain and in the rest of the body. It has been demonstrated that altered serotinergic function is implicated in various psychiatric disorders including depression and schizophrenia. Serotonin has also been implicated along with dopamine in attention deficit-hyperkinetic disorder (AD-HKD). This study provides a versatile validated method for the analysis of serotonin, hydroxyindole acetic acid and dopamine in urine using LC-MS/MS. This method was then used to quantify these analytes in a test group of 17 children diagnosed with severe AD-HKD. This group was compared to a matched control group to investigate the possibility that one of these compounds may be a potential biomarker for this condition. The developed method provided good linear calibration curves for the multiplex assay of analytes in urine (0.05-3.27 nmol/L; R(2) ≥ 0.9977). Acceptable inter-day repeatability was achieved for all analytes with RSD values (n = 9) ranging from 1.1% to 9.3% over a concentration range of 0.11-3.27 μmol/L in urine. Excellent limits of detection (LOD) and limits of quantitation (LOQ) were achieved with LODs of 8.8-18.2 nmol/L and the LOQs of 29.4-55.7 nmol/L for analytes in urine. Recoveries were in the ranges of 98-104%, 100-106% and 91-107% for serotonin, 5-HIAA and dopamine, respectively. An appropriate sample clean-up procedure for urine was developed to ensure efficient recovery and reproducibility on analysis. Evaluation of matrix effects was also carried out and the influence of ion suppression on analytical results reported. Confirmatory analysis was carried out on a linear trap quadrupole-Orbitrap mass spectrometer to obtain high mass accuracy data of the target analytes in the clinical samples.

Testing the functional capacity of the tryptophan-niacin pathway in man by analysis of urinary metabolites.
            (Price et al., 1965)  Download1  Download2  Download3

Urinary metabolites as noninvasive biomarkers of gastrointestinal diseases: A clinical review.
            (Sarosiek et al., 2016) Download
The diagnosis of gastrointestinal (GI) disorders is usually based on invasive techniques such as endoscopy. A key important factor in GI cancer is early diagnosis which warrants development of non- or less-invasive diagnostic techniques. In addition, monitoring and surveillance are other important parts in the management of GI diseases. Metabolomics studies with nuclear magnetic resonance and mass spectrometry can measure the concentration of more than 3000 chemical compounds in the urine providing possible chemical signature in different diseases and during health. In this review, we discuss the urinary metabolomics signature of different GI diseases including GI cancer and elaborate on how these biomarkers could be used for the classification, early diagnosis and the monitoring of the patients. Moreover, we discuss future directions of this still evolving field of research.

Monitoring tryptophan metabolism in chronic immune activation.
            (Schröcksnadel et al., 2006) Download
The essential amino acid tryptophan is a constituent of proteins and is also a substrate for two important biosynthetic pathways: the generation of neurotransmitter 5-hydroxytryptamine (serotonin) by tryptophan 5-hydroxylase, and the formation of kynurenine derivatives and nicotinamide adenine dinucleotides. The latter pathway is initiated by the enzymes tryptophan pyrrolase (tryptophan 2,3-dioxygenase, TDO) and indoleamine 2,3-dioxygenase (IDO). TDO is located in liver cells, whereas IDO is expressed in a variety of cells including monocyte-derived macrophages and dendritic cells and is preferentially induced by Th1-type cytokine interferon-gamma. Tryptophan depletion via IDO is part of the cytostatic and antiproliferative activity mediated by interferon-gamma in cells. In vivo tryptophan concentration can be measured by HPLC by monitoring its natural fluorescence (285 nm excitation and 365 nm emission wavelength). IDO activity is characterized best by the kynurenine to tryptophan ratio which correlates with concentrations of immune activation markers such as neopterin. Low serum/plasma tryptophan concentration is observed in infectious, autoimmune, and malignant diseases and disorders that involve cellular (Th1-type) immune activation as well as during pregnancy due to accelerated tryptophan conversion. Thus, in states of persistent immune activation, low tryptophan concentration may contribute to immunodeficiency. Decreased serum tryptophan can also effect serotonin biosynthesis and thus contribute to impaired quality of life and depressive mood. As such, monitoring tryptophan metabolism in chronic immunopathology provides a better understanding of the association between immune activation and IDO and its role in the development of immunodeficiency, anemia and mood disorders.

Organ Correlation with Tryptophan Metabolism Obtained by Analyses of TDO-KO and QPRT-KO Mice.
            (Shibata and Fukuwatari, 2016) Download
The aim of this article is to report the organ-specific correlation with tryptophan (Trp) metabolism obtained by analyses of tryptophan 2,3-dioxygenase knockout (TDO-KO) and quinolinic acid phosphoribosyltransferase knockout (QPRT-KO) mice models. We found that TDO-KO mice could biosynthesize the necessary amount of nicotinamide (Nam) from Trp, resulting in the production of key intermediate, 3-hydroxyanthranilic acid. Upstream metabolites, such as kynurenic acid and xanthurenic acid, in the urine were originated from nonhepatic tissues, and not from the liver. In QPRT-KO mice, the Trp to quinolinic acid conversion ratio was 6%; this value was higher than expected. Furthermore, we found that QPRT activity in hetero mice was half of that in wild-type (WT) mice. Urine quinolinic acid levels remain unchanged in both hetero and WT mice, and the conversion ratio of Trp to Nam was also unaffected. Collectively, these findings show that QPRT was not the rate-limiting enzyme in the conversion. In conclusion, the limiting factors in the conversion of Trp to Nam are the substrate amounts of 3-hydroxyanthranilic acid and activity of 3-hydroxyanthranilic acid 3,4-dioxygenase in the liver.

Urinary excretion ratio of xanthurenic acid/kynurenic acid as a functional biomarker of niacin nutritional status.
            (Shibata et al., 2016) Download
The present study was conducted to survey functional biomarkers for evaluation of niacin nutritional status. Over 500 enzymes require niacin as a coenzyme. Of these, we chose the tryptophan degradation pathway. To create niacin-deficient animals, quinolinic acid phosphoribosyltransferase-knock out mice were used in the present study because wild type mice can synthesize nicotinamide from tryptophan. When the mice were made niacin-deficient, the urinary excretion of xanthurenic acid (XA) was extremely low compared with control mice; however, it increased according to the recovery of niacin nutritional status. The urinary excretion of kynurenic acid (KA) was the reverse of XA. Kynurenine 3-monooxygenase, which needs NADPH, was thought to be suppressed by niacin deficiency. Thus, we calculated the urinary excretion ratio of XA:KA as a functional biomarker of niacin nutrition. The ratio increased according to recovering niacin nutritional status. Low values equate with low niacin nutritional status.

Measurement of urinary tryptophan metabolites by reverse-phase high-pressure liquid chromatography.
            (Tarr, 1981) Download
Rapid separation a n d determination of tryptophan, N^-formylkynurenine, kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, nicotinic acid, nicotinamide, trigonelline, kynurenic acid and xanthurenic acid from pea seedling using reverse-phase high-pressure liquid chromatography is described. Accuracy of the determinations was better than 5 % and less than 50 pmol of each metabolite could be measured reliably.


 

Potato- an important source of nutritional kynurenic acid.
            (Turski et al., 2012) Download
Kynurenic acid (KYNA) is a metabolite of tryptophan which is formed along the kynurenine pathway. KYNA may possess neuroprotective, anti-inflammatory, antioxidant and antiproliferative properties. This study measured the concentration of KYNA in various varieties of potatoes and products made from potatoes. KYNA content was determined by means of the high-performance liquid chromatography with fluorescence detection. KYNA was found in all 16 studied varieties of potato tubers in amounts varying from 0.239 to 3.240 μg/g dry weight. The content of KYNA in potato tubers declined during long-term storage. The content of KYNA in French fries varied from 0.100 to 0.646 μg/g dry weight. KYNA content in potato crisps was 0.478 and 0.576 μg/g dry weight. Hence, all in all, we concluded that the amount of KYNA potentially delivered to the human body in potatoes and various foods produced from potatoes is high and might be compared to the amount of KYNA present in a maximum daily dose of popular herbs and herbal medicines.

 


References

Badawy, AA and CJ Morgan (2010), ‘Rapid Isocratic Liquid Chromatographic Separation and Quantification of Tryptophan and Six kynurenine Metabolites in Biological Samples with Ultraviolet and Fluorimetric Detection.’, Int J Tryptophan Res, 3 175-86. PubMed: 22084598
Chen, CF, et al. (2013), ‘Novel spectrophotometric method for the quantitation of urinary xanthurenic acid and its application in identifying individuals with hyperhomocysteinemia associated with Vitamin B₆ deficiency.’, Biomed Res Int, 2013 678476. PubMed: 24151616
Gevi, F, et al. (2016), ‘Urinary metabolomics of young Italian autistic children supports abnormal tryptophan and purine metabolism.’, Mol Autism, 7 47. PubMed: 27904735
Le Floc’h, N, W Otten, and E Merlot (2011), ‘Tryptophan metabolism, from nutrition to potential therapeutic applications.’, Amino Acids, 41 (5), 1195-205. PubMed: 20872026
Liu, M, et al. (1996), ‘Improved fluorometric quantification of urinary xanthurenic acid.’, Clin Chem, 42 (3), 397-401. PubMed: 8598102
Moriarty, M, et al. (2011), ‘Development of an LC-MS/MS method for the analysis of serotonin and related compounds in urine and the identification of a potential biomarker for attention deficit hyperactivity/hyperkinetic disorder.’, Anal Bioanal Chem, 401 (8), 2481-93. PubMed: 21866401
Price, JM, RR Brown, and N Yess (1965), ‘Testing the functional capacity of the tryptophan-niacin pathway in man by analysis of urinary metabolites.’, Adv Metab Disord, 2 159-225. PubMed: 4250300
Sarosiek, I, et al. (2016), ‘Urinary metabolites as noninvasive biomarkers of gastrointestinal diseases: A clinical review.’, World J Gastrointest Oncol, 8 (5), 459-65. PubMed: 27190585
Schröcksnadel, K, et al. (2006), ‘Monitoring tryptophan metabolism in chronic immune activation.’, Clin Chim Acta, 364 (1-2), 82-90. PubMed: 16139256
Shibata, K and T Fukuwatari (2016), ‘Organ Correlation with Tryptophan Metabolism Obtained by Analyses of TDO-KO and QPRT-KO Mice.’, Int J Tryptophan Res, 9 1-7. PubMed: 27147825
Shibata, K, M Yamazaki, and Y Matsuyama (2016), ‘Urinary excretion ratio of xanthurenic acid/kynurenic acid as a functional biomarker of niacin nutritional status.’, Biosci Biotechnol Biochem, 1-9. PubMed: 27429300
Tarr, JB (1981), ‘Measurement of urinary tryptophan metabolites by reverse-phase high-pressure liquid chromatography.’, Biochem Med, 26 (3), 330-38. PubMed: 7332544
Turski, MP, et al. (2012), ‘Potato- an important source of nutritional kynurenic acid.’, Plant Foods Hum Nutr, 67 (1), 17-23. PubMed: 22392498