Showing papers on "Xanthine published in 2012"

Defects in purine nucleotide metabolism lead to substantial incorporation of xanthine and hypoxanthine into DNA and RNA

Abstract: Deamination of nucleobases in DNA and RNA results in the formation of xanthine (X), hypoxanthine (I), oxanine, and uracil, all of which are miscoding and mutagenic in DNA and can interfere with RNA editing and function. Among many forms of nucleic acid damage, deamination arises from several unrelated mechanisms, including hydrolysis, nitrosative chemistry, and deaminase enzymes. Here we present a fourth mechanism contributing to the burden of nucleobase deamination: incorporation of hypoxanthine and xanthine into DNA and RNA caused by defects in purine nucleotide metabolism. Using Escherichia coli and Saccharomyces cerevisiae with defined mutations in purine metabolism in conjunction with analytical methods for quantifying deaminated nucleobases in DNA and RNA, we observed large increases (up to 600-fold) in hypoxanthine in both DNA and RNA in cells unable to convert IMP to XMP or AMP (IMP dehydrogenase, guaB; adenylosuccinate synthetase, purA, and ADE12), and unable to remove dITP/ITP and dXTP/XTP from the nucleotide pool (dITP/XTP pyrophosphohydrolase, rdgB and HAM1). Conversely, modest changes in xanthine levels were observed in RNA (but not DNA) from E. coli lacking purA and rdgB and the enzyme converting XMP to GMP (GMP synthetase, guaA). These observations suggest that disturbances in purine metabolism caused by known genetic polymorphisms could increase the burden of mutagenic deaminated nucleobases in DNA and interfere with gene expression and RNA function, a situation possibly exacerbated by the nitrosative stress of concurrent inflammation. The results also suggest a mechanistic basis for the pathophysiology of human inborn errors of purine nucleotide metabolism.

Mutations associated with functional disorder of xanthine oxidoreductase and hereditary xanthinuria in humans.

Abstract: Xanthine oxidoreductase (XOR) catalyzes the conversion of hypoxanthine to xanthine and xanthine to uric acid with concomitant reduction of either NAD+ or O2. The enzyme is a target of drugs to treat hyperuricemia, gout and reactive oxygen-related diseases. Human diseases associated with genetically determined dysfunction of XOR are termed xanthinuria, because of the excretion of xanthine in urine. Xanthinuria is classified into two subtypes, type I and type II. Type I xanthinuria involves XOR deficiency due to genetic defect of XOR, whereas type II xanthinuria involves dual deficiency of XOR and aldehyde oxidase (AO, a molybdoflavo enzyme similar to XOR) due to genetic defect in the molybdenum cofactor sulfurase. Molybdenum cofactor deficiency is associated with triple deficiency of XOR, AO and sulfite oxidase, due to defective synthesis of molybdopterin, which is a precursor of molybdenum cofactor for all three enzymes. The present review focuses on mutation or chemical modification studies of mammalian XOR, as well as on XOR mutations identified in humans, aimed at understanding the reaction mechanism of XOR and the relevance of mutated XORs as models to estimate the possible side effects of clinical application of XOR inhibitors.

Novel, Highly Specific N-Demethylases Enable Bacteria To Live on Caffeine and Related Purine Alkaloids

Abstract: The molecular basis for the ability of bacteria to live on caffeine as a sole carbon and nitrogen source is unknown. Pseudomonas putida CBB5, which grows on several purine alkaloids, metabolizes caffeine and related methylxanthines via sequential N-demethylation to xanthine. Metabolism of caffeine by CBB5 was previously attributed to one broad-specificity methylxanthine N-demethylase composed of two subunits, NdmA and NdmB. Here, we report that NdmA and NdmB are actually two independent Rieske nonheme iron monooxygenases with N(1)- and N(3)-specific N-demethylation activity, respectively. Activity for both enzymes is dependent on electron transfer from NADH via a redox-center-dense Rieske reductase, NdmD. NdmD itself is a novel protein with one Rieske [2Fe-2S] cluster, one plant-type [2Fe-2S] cluster, and one flavin mononucleotide (FMN) per enzyme. All ndm genes are located in a 13.2-kb genomic DNA fragment which also contained a formaldehyde dehydrogenase. ndmA, ndmB, and ndmD were cloned as His(6) fusion genes, expressed in Escherichia coli, and purified using a Ni-NTA column. NdmA-His(6) plus His(6)-NdmD catalyzed N(1)-demethylation of caffeine, theophylline, paraxanthine, and 1-methylxanthine to theobromine, 3-methylxanthine, 7-methylxanthine, and xanthine, respectively. NdmB-His(6) plus His(6)-NdmD catalyzed N(3)-demethylation of theobromine, 3-methylxanthine, caffeine, and theophylline to 7-methylxanthine, xanthine, paraxanthine, and 1-methylxanthine, respectively. One formaldehyde was produced from each methyl group removed. Activity of an N(7)-specific N-demethylase, NdmC, has been confirmed biochemically. This is the first report of bacterial N-demethylase genes that enable bacteria to live on caffeine. These genes represent a new class of Rieske oxygenases and have the potential to produce biofuels, animal feed, and pharmaceuticals from coffee and tea waste.

Graphitized mesoporous carbon modified glassy carbon electrode for selective sensing of xanthine, hypoxanthine and uric acid

Abstract: An efficient electrochemical sensor for simultaneous electrochemical sensing of three purine compounds, uric acid (UA), xanthine (X) and hypoxanthine (HX), using a graphitized mesoporous carbon (GMC) modified glassy carbon electrode (GCE/GMC) has been demonstrated in pH 7 phosphate buffer solution without any enzyme, prior to electrode activation, surfactant and sample pre-concentration step. Electrochemical investigation of the GCE/GMC with [Fe(CN)6]3− indicates metallic conductor like surface features of the modified electrode. A diffusion controlled reaction mechanism was identified for the electro-oxidation of the three purine compounds with an electrocatalytic pathway, except for the UA, where it shows a surface area effect with a mixed-diffusion and adsorption controlled mechanism at higher scan rates (>70 mV s−1). Calculated full-width of the half maximum values for the simultaneous detection of the three purine compounds are 42, 53 and 64 mV respectively and these are the lowest values ever reported in the literature, suggesting effective electron-transfer behaviour of the modified electrode for the purine oxidations. Calibration plots for the simultaneous detection of the purine compounds were linear in the concentration range of 20–400 μM, 20–320 μM and 20–240 μM for UA, X and HX with detection limit values of 110 nM, 388 nM, and 351 nM respectively. Selective sensing of the purine compounds in human blood-plasma, urine and fish samples was successfully demonstrated with recovery values ∼100%.

Benzothiazinones: a novel class of adenosine receptor antagonists structurally unrelated to xanthine and adenine derivatives.

Abstract: 2-(Acyl)amino-4H-3,1-benzothiazin-4-ones and related thienothiazinones were identified as structurally novel antagonists at adenosine receptors (ARs). 6-Methyl-2-benzoylamino-4H-3,1-benzothiazin-4-...

Amperometric detection of hypoxanthine and xanthine by enzymatic amplification using a gold nanoparticles-carbon nanohorn hybrid as the carrier.

Abstract: A novel gold nanoparticles–single-walled carbon nanohorn (GNPs–SWCNH) hybrid was synthesized for the construction of an amperometric biosensing platform. The GNPs–SWCNH hybrid was stable in aqueous solution for at least two weeks, and was characterized with scanning electron microscopy, transmission electron microscopy, and electrochemical impedance spectroscopy. The average diameter of GNPs in situ synthesized on the SWCNH was 5–8 nm, and the good interaction between GNPs and SWCNH was confirmed by ultraviolet-visible absorption spectroscopy. The GNPs–SWCNH immobilized on a platinum electrode showed high electrochemical activity toward the oxidation of hydrogen peroxide and uric acid with low applied potentials. Combining with the enzymatic reaction of xanthine oxidase (XOx), a biosensor for hypoxanthine and xanthine was constructed. The XOx–GNPs–SWCNH-based biosensor exhibited good responses to hypoxanthine and xanthine with the linear ranges of 1.5 to 35.4 and 2.0 to 37.3 μM, and the detection limits of 0.61 and 0.72 μM, respectively. The recovery test showed acceptable results. The gold nanoparticles functionalized carbon nanohorns provided a promising way to construct an electrochemical platform for sensitive biosensing.

Low-potential amperometric enzyme biosensor for xanthine and hypoxanthine.

Abstract: The bacterial xanthine dehydrogenase (XDH) from Rhodobacter capsulatus was immobilized on an edge-plane pyrolytic graphite (EPG) electrode to construct a hypoxanthine/xanthine biosensor that functions at physiological pH. Phenazine methosulfate (PMS) was used as a mediator which acts as an artificial electron-transfer partner for XDH. The enzyme catalyzes the oxidation of hypoxanthine to xanthine and also xanthine to uric acid by an oxidative hydroxylation mechanism. The present electrochemical biosensor was optimized in terms of applied potential and pH. The electrocatalytic oxidation response showed a linear dependence on the xanthine concentration ranging from 1.0 × 10–5 to 1.8 × 10–3 M with a correlation coefficient of 0.994. The modified electrode shows a very low detection limit for xanthine of 0.25 nM (signal-to-noise ratio = 3) using controlled potential amperometry.

The Role of Aldehyde Oxidase and Xanthine Oxidase in the Biotransformation of a Novel Negative Allosteric Modulator of Metabotropic Glutamate Receptor Subtype 5

Abstract: Negative allosteric modulation (NAM) of metabotropic glutamate receptor subtype 5 (mGlu5) represents a therapeutic strategy for the treatment of childhood developmental disorders, such as fragile X syndrome and autism. VU0409106 emerged as a lead compound within a biaryl ether series, displaying potent and selective inhibition of mGlu5. Despite its high clearance and short half-life, VU0409106 demonstrated efficacy in rodent models of anxiety after extravascular administration. However, lack of a consistent correlation in rat between in vitro hepatic clearance and in vivo plasma clearance for the biaryl ether series prompted an investigation into the biotransformation of VU0409106 using hepatic subcellular fractions. An in vitro appraisal in rat, monkey, and human liver S9 fractions indicated that the principal pathway was NADPH-independent oxidation to metabolite M1 (+16 Da). Both raloxifene (aldehyde oxidase inhibitor) and allopurinol (xanthine oxidase inhibitor) attenuated the formation of M1, thus implicating the contribution of both molybdenum hydroxylases in the biotransformation of VU0409106. The use of 18O-labeled water in the S9 experiments confirmed the hydroxylase mechanism proposed, because 18O was incorporated into M1 (+18 Da) as well as in a secondary metabolite (M2; +36 Da), the formation of which was exclusively xanthine oxidase-mediated. This unusual dual and sequential hydroxylase metabolism was confirmed in liver S9 and hepatocytes of multiple species and correlated with in vivo data because M1 and M2 were the principal metabolites detected in rats administered VU0409106. An in vitro-in vivo correlation of predicted hepatic and plasma clearance was subsequently established for VU0409106 in rats and nonhuman primates.

Supramolecular immobilization of redox enzymes on cyclodextrin-coated magnetic nanoparticles for biosensing applications

Abstract: Mono-6-formyl-β-cyclodextrin moieties were attached to (3-aminopropyl)triethoxysilane-coated superparamagnetic Fe 3 O 4 nanoparticles by reductive alkylation with NaBH 3 CN. The oligosaccharide-capped core–shell nanoparticles were employed as support for the supramolecular immobilization of two different adamantane-modified enzymes, tyrosinase and xanthine oxidase, through host–guest interactions. The enzyme-modified nanomaterial was further used to magnetically modify carbon paste electrodes for constructing amperometric biosensors toward cathecol and xanthine. The tyrosinase and xanthine oxidase based biosensors showed excellent electroanalytical behaviours, with linear ranges of 100 nM–12 μM cathecol and 5.0–120 μM xanthine, sensitivities of 12 mA/M and 130 mA/M, and low detection limits of 22 nM and 2.0 μM, respectively. The supramolecular nature of the immobilization approach was confirmed by electroanalytical methods.

Inhibitory activity of xanthine oxidase by fractions Crateva adansonii

Abstract: Objective To study the inhibitory effect of various extracts from Crateva adansonii ( C. adansonii ) used traditionally against several inflammatory diseases such as rheumatism, arthritis, and gout, was investigated on purified bovine milk xanthine oxidase (XO) activity. Methods Xanthine oxidase inhibitory activity was assayed spectrophotometrically and the degree of enzyme inhibition was determined by measuring the increase in absorbance at 295 nm associated with uric acid formation. Enzyme kinetics was carried out using Lineweaver-Burk plots using xanthine as the substrate. Results Among the fractions tested, the chloroform fraction exhibited highest potency (IC 50 20.2±1.6 μg/mL) followed by the petroleum ether (IC 50 30.1±2.2 μg/mL), ethyl acetate (IC 50 43.9±1.4 μg/mL) and residual (IC 50 98.0±3.3 μg/mL) fractions. The IC 50 value of allopurinol used, as the standard was 5.7±0.3 μg/mL. Conclusions Enzyme inhibition mechanism indicated that the mode of inhibition was of a mixed type. Our findings suggest that the therapeutic use of these plants may be due to the observed Xanthine oxidase inhibition, thereby supporting their use in traditional folk medicine against inflammatory-related diseases, in particular, gout.

Fabrication of an amperometric xanthine biosensor based on polyvinylchloride membrane

Abstract: A method is described for fabrication of an amperometric xanthine biosensor based on polyvinylchloride (PVC) membrane bound xanthine oxidase (XOD). The membrane bound enzyme oxidizes xanthine into uric acid and H2O2, which is split into 2H++O2+2e- at high potential (0.4V) and measured as current (mA).Sensor showed optimum response within 30 s, at pH 7.0 and 35C. A linear relationship was observed between current and xanthine concentration ranging from 0.025 to 0.4 × 10-6 M, Km for xanthine and Imax were 0.45 × 10-6 M and 0.002 mA, respectively. Minimum detection limit of the biosensor was 2.5 × 10-8 M. Biosensor was utilized for determination of xanthine in fish meat cow and buffalos milk. Biosensor was used 100 times over a period of 45 days with only 30% loss of initial activity, when stored at 4C. PRACTICAL APPLICATIONS Evaluation of fish meat freshness is of great significance in food industries for the quality control of fish products. After the death of a fish, xanthine is a major metabolite in the degradation of adenosine triphosphate in the fish meat. Thus, xanthine attracts much attention as an indicator for estimating fish freshness. Moreover, the content of xanthine in human body fluids can also provide useful index for certain pathologic state, especially for xanthinuria.

Prolonged fasting increases purine recycling in post-weaned northern elephant seals.

Abstract: SUMMARY Northern elephant seals are naturally adapted to prolonged periods (1–2 months) of absolute food and water deprivation (fasting). In terrestrial mammals, food deprivation stimulates ATP degradation and decreases ATP synthesis, resulting in the accumulation of purines (ATP degradation byproducts). Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) salvages ATP by recycling the purine degradation products derived from xanthine oxidase (XO) metabolism, which also promotes oxidant production. The contributions of HGPRT to purine recycling during prolonged food deprivation in marine mammals are not well defined. In the present study we cloned and characterized the complete and partial cDNA sequences that encode for HGPRT and xanthine oxidoreductase (XOR) in northern elephant seals. We also measured XO protein expression and circulating activity, along with xanthine and hypoxanthine plasma content in fasting northern elephant seal pups. Blood, adipose and muscle tissue samples were collected from animals after 1, 3, 5 and 7 weeks of their natural post-weaning fast. The complete HGPRT and partial XOR cDNA sequences are 771 and 345 bp long and encode proteins of 218 and 115 amino acids, respectively, with conserved domains important for their function and regulation. XOR mRNA and XO protein expression increased 3-fold and 1.7-fold with fasting, respectively, whereas HGPRT mRNA (4-fold) and protein (2-fold) expression increased after 7 weeks in adipose tissue and muscle. Plasma xanthine (3-fold) and hypoxanthine (2.5-fold) levels, and XO (1.7- to 20-fold) and HGPRT (1.5- to 1.7-fold) activities increased during the last 2 weeks of fasting. Results suggest that prolonged fasting in elephant seal pups is associated with increased capacity to recycle purines, which may contribute to ameliorating oxidant production and enhancing the supply of ATP, both of which would be beneficial during prolonged food deprivation and appear to be adaptive in this species.

X-ray spectroscopy of heterocyclic biochemicals: xanthine, hypoxanthine, and caffeine.

Abstract: The electronic structures of the purine derivatives xanthine, hypoxanthine and caffeine have been investigated in the gas phase using C, N, and O 1s X-ray photoemission (XPS) and near edge X-ray absorption fine structure (NEXAFS) spectroscopy. The results have been interpreted by means of ab initio calculations using the third-order algebraic-diagrammatic construction (ADC(3)) method for the one-particle Green’s function and the second-order ADC method (ADC(2)) for the polarization propagator. The carbon, nitrogen and oxygen K-edge NEXAFS spectra of xanthine and caffeine are very similar, since the molecules differ only by substitution of three hydrogen atoms by methyl groups. For hypoxanthine, the electronic structure and spectra differ considerably from xanthine as the purine ring is more highly conjugated, and there is one less oxo group. Effects due to oxo-hydroxy tautomerism were not observed. However, the two oxo tautomeric forms of hypoxanthine oxo-N(9)-H and oxo-N(7)-H are populated in the gas pha...

Exposure time to caffeine affects heartbeat and cell damage‐related gene expression of zebrafish Danio rerio embryos at early developmental stages

Abstract: Caffeine is white crystalline xanthine alkaloid that is naturally found in some plants and can be produced synthetically. It has various biological effects, especially during pregnancy and lactation. We studied the effect of caffeine on heartbeat, survival and the expression of cell damage related genes, including oxidative stress (HSP70), mitochondrial metabolism (Cyclin G1) and apoptosis (Bax and Bcl2), at early developmental stages of zebrafish embryos. We used 100 µm concentration based on the absence of locomotor effects. Neither significant mortality nor morphological changes were detected. We monitored hatching at 48 h post-fertilization (hpf) to 96 hpf. At 60 and 72 hpf, hatching decreased significantly (P 0.05). Heartbeats per minute were 110, 110 and 112 in control at 48, 72 and 96 hpf, respectively. Caffeine significantly increased heartbeat - 122 and 136 at 72 and 96 hpf, respectively. Quantitative RT-PCR showed significant up-regulation after caffeine exposure in HSP70 at 72 hpf; in Cyclin G1 at 24, 48 and 72 hpf; and in Bax at 48 and 72 hpf. Significant down-regulation was found in Bcl2 at 48 and 72 hpf. The Bax/Bcl2 ratio increased significantly at 48 and 72 hpf. We conclude that increasing exposure time to caffeine stimulates oxidative stress and may trigger apoptosis via a mitochondrial-dependent pathway. Also caffeine increases heartbeat from early phases of development without affecting the morphology and survival but delays hatching. Use of caffeine during pregnancy and lactation may harm the fetus by affecting the expression of cell-damage related genes.

Analytical Techniques Used for Determination of Methylxanthines and their Analogues—Recent Advances

Abstract: Methylxanthines (caffeine, theophylline, theobromine) are a popular group of natural purine alkaloids, which are components of many commonly used drugs, parapharmaceuticals and a wide range of food products (e.g. coffee, tea, energy drinks, etc.). Caffeine metabolites (theophylline, paraxanthine, theobromine and other 1-, 3-, 7-methyl tri-, di- and mono-derivatives of xanthine) and hypoxanthine metabolites (xanthine, uric acid) play an important role in the biochemical processes of mammalian organisms. That is why they are the markers of many diseases and are in the focus of the clinicians, pharmaceutical industry and ecologists. In addition to the natural methylxanthines there are structurally related synthetic derivatives: pentoxifylline, dyphylline, xantinol nicotinate and 8-chlorotheophylline that are still valuable pharmaceuticals needing to be monitored. A review of the modern techniques used for determination of methylxanthines for the last 10 years (2000 – March 2010) is presented.

Identification and Mode of Action of 5-Hydroxymethyl-2-furfural (5-HMF) and 1-Methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic Acid (MTCA) as Potent Xanthine Oxidase Inhibitors in Vinegars

Abstract: Vinegars have been used as an alternative remedy for treating gout, but the scientific basis remains to be elucidated. In this study, seven commercial vinegars and one laboratory-prepared red-koji vinegar were evaluated for the inhibitory activity of xanthine oxidase (XO), a critical enzyme catalyzing uric acid formation. Red-koji vinegar exhibited potent xanthine oxidase inhibitory (XOI) activity and was used for isolating active compounds. The substances under two peaks with XOI activity from HPLC were identified as 5-hydroxymethyl-2-furfural (5-HMF) and 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (MTCA), by LC-MS-MS and NMR. The XO half-maximal inhibitory concentrations (IC(50)) of 5-HMF and MTCA were 168 and 860 μg/mL, respectively. In further mode-of-action analysis, the inhibitory mechanism of each compound was elucidated at the IC(50) level in the presence of various concentrations of xanthine as the substrate. The following Michaelis-Menten kinetics analysis of XO inhibition revealed uncompetitive and competitive patterns for 5-HMF and MTCA, respectively.

An amperometric biosensor for fish freshness detection from xanthine oxidase immobilized in polypyrrole-polyvinylsulphonate film

Abstract: A new amperometric biosensor was developed for determining hypoxanthine in fish meat. Xanthine oxidase with pyrrole and polyvinylsulphonate was immobilized on the surface of a platinum electrode by electropolymerization. The determination of xanthine-hypoxanthine was performed by means of oxidation of uric acid liberated during the enzyme reaction on the surface of the enzyme electrode at + 0.30V (SCE). The effects of pH, substrate concentration, and temperature on the response of the xanthine-hypoxanthine biosensor were investigated. The linear working range of the enzyme electrode was 1.0 × 10−7 –1.0 × 10−3 M of the hypoxanthine concentration, and the detection limit was 1.0 × 10−7M. The apparent Km(app) and Imax of the immobilized xanthine oxidase were found to be 0.0154 mM and 1.203 μA/mM, respectively. The best pH and temperature value for xanthine oxidase were selected as 7.75 and 25°C, respectively. The sensor was used for the determination of hypoxhantine in fish meat. Results show that th...

Amperometric determination of xanthine in tea, coffee, and fish meat with graphite rod bound xanthine oxidase

Abstract: A method is described for construction of an amperometric xanthine biosensor based on graphite rod modified through adsorption of xanthine oxidase. Enzymatically produced H2O2 from xanthine was split into 2H+ + O2 + 2e− at 0.6 V and the current was measured, which was directly proportional to xanthine concentration ranging from 1 ° 10−7 to 6 ° 10−7 M with a detection limit of 1 ° 10−7 M. The biosensor exhibited optimum response within 35 sec at pH 7.0 and 35°C. It was employed for determination of xanthine in tea leaves (0.9 ° 10−5−2.5 ° 10−5 mmol/g), coffee powder (3.2 μmol/g) and fish meat (90 mmol/g). The content of xanthine in fish meat increased 6.5 times with its storage at room temperature during 15 days. The enzyme electrode could be reused 200 times during the span of 30 days, when stored in reaction buffer at 4°C.

Tissue distribution and metabolism of guanosine in rats following intraperitoneal injection.

Abstract: Guanosine has long been known as an endogenous purine nucleoside deeply involved in the modulation of several intracellular processes, especially G-protein activity. More recently, it has been reported to act as an extracellular signaling molecule released from neurons and, more markedly, from astrocytes either in basal conditions or after different kinds of stimulation including hypoxia. Moreover, in vivo studies have shown that guanosine plays an important role as both a neuroprotective and neurotrophic agent in the central nervous system. Specific high-affinity binding sites for this nucleoside have been found on membrane preparations from rat brain. The present study was undertaken to investigate the distribution and metabolic profiles of guanosine after administering the nucleoside to gain a better understanding of the biological effects of this potential drug candidate. Rats were given an intraperitonal (i.p.) injection of 2, 4, 8 or 16 mg/kg of guanosine combined with 0.05% of [3H]guanosine. Plasma samples were collected 7.5, 15, 30, 60 and 90 min after the guanosine-mixture administration and analyzed by either a liquid scintillation counter or by HPLC connected to a UV and to an on-line radiochemical detector to measure the levels of guanosine and its metabolic products guanine, xanthine and uric acid. The levels of guanosine, guanine and xanthine were also measured in brain, lung, heart, kidney and liver tissue homogenates at the defined time points after the injection of 8 mg/kg of the guanosine-mixture. We found that the levels of radioactivity in plasma increased linearly in a dose- and time-dependent manner. Guanosine was widely distributed in all tissues examined in the present study, at almost twice its usual levels. In addition, guanine levels dramatically increased in all the organs. Interestingly, enzymatic analysis of the plasma samples showed the presence of a soluble purine nucleoside phosphorylase, a key enzyme in the purine salvage pathway and nucleoside catabolism. Since guanosine has been shown to be neuroprotective and astrocytes have been reported to play critical roles in mediating neuronal survival and functions in different neurodegenerative disorders, we also performed uptake and release.

Liquid chromatography-mass spectrometry-based metabolomic analysis of livers from aged rats.

Abstract: We used UPLC–Q-TOF MS to analyze hepatic metabolites of rats aged 6, 12, 18, and 24 months; the MS data were processed by partial least-squares discriminant analysis (PLS-DA) to investigate the discrimination among sample groups. Rats were significantly separated with increasing age, except those aged between 6 and 12 months. We identified only 25 of 120 metabolites contributing to the separation: lipid metabolites (glycerol-3-phosphate, linolenic acid, lysophosphatidylcholines [lysoPCs]), energy metabolism intermediates (betaine, carnitine, acylcarnitines, creatine, pantothenic acid), nucleic acid metabolites (inosine, xanthosine, uracil, hypoxanthine, xanthine), and tyrosine. Aging accumulated energy metabolism intermediates, hypoxanthine, xanthine, and 2 major lysoPCs (C18:0 and C22:6). The NAD level and NAD/NADH ratio decreased with age. It was indicated that aging might decrease energy production through β-oxidation because of a decrease in NAD despite the accumulation of lipid energy metabolism inte...

Biocompatible Xanthine-Quadruplex Scaffold for Ion-Transporting DNA Channels

Abstract: Molecular dynamics simulations and adaptive biasing force analysis of the quadruplex DNA dynamics in an explicit solvent reveal fundamentally different mechanisms of Na+ transport in xanthine- and guanine-based DNA systems. The barrier to the transport of K+ through the xanthine-based quadruplex is significantly lower than those reported for the guanine-based analogs.

Urine metabolomics reveals novel physiologic functions of human aldehyde oxidase and provides biomarkers for typing xanthinuria

Abstract: Classical xanthinuria is a rare inherited metabolic disorder caused by either isolated xanthine dehydrogenase (XDH) deficiency (type I) or combined XDH and aldehyde oxidase (AO) deficiency (type II). XDH and AO are evolutionary related enzymes that share a sulfurated molybdopterin cofactor. While the role of XDH in purine metabolism is well established, the physiologic functions of AO are mostly unknown. XDH and AO are important drug metabolizing enzymes. Urine metabolomic analysis by high pressure liquid chromatography and mass spectrometry of xanthinuric patients was performed to unveil physiologic functions of XDH and AO and provide biomarkers for typing xanthinuria. Novel endogenous products of AO, hydantoin propionic acid, N1-methyl-8-oxoguanine and N-(3-acetamidopropyl) pyrrolidin-2-one formed in the histidine, nucleic acid and spermidine metabolic pathways, respectively, were identified as being lowered in type II xanthinuria. Also lowered were the known AO products, N1-methyl-2-pyridone-5-carboxamide and N1-methyl-4-pyridone-5-carboxamide in the nicotinamide degradation pathway. In contrast to the KEGG annotations, the results suggest minor role of human AO in the conversion of pyridoxal to pyridoxate and gentisaldehyde to gentisate in the vitamin B6 and tyrosine metabolic pathways, respectively. The perturbations in purine degradation due to XDH deficiency radiated further from the previously known metabolites, uric acid, xanthine and hypoxanthine to guanine, methyl guanine, xanthosine and inosine. Possible pathophysiological implications of the observed metabolic perturbations are discussed. The identified biomarkers have the potential to replace the allopurinol-loading test used in the past to type xanthinuria, thus facilitating appropriate pharmacogenetic counseling and gene directed search for causative mutations.

Different inhibitory potency of febuxostat towards mammalian and bacterial xanthine oxidoreductases: insight from molecular dynamics

Abstract: Febuxostat, a drug recently approved in the US, European Union and Japan for treatment of gout, inhibits xanthine oxidoreductase (XOR)-mediated generation of uric acid during purine catabolism. It inhibits bovine milk XOR with a Ki in the picomolar-order, but we found that it is a much weaker inhibitor of Rhodobacter capsulatus XOR, even though the substrate-binding pockets of mammalian and bacterial XOR are well-conserved as regards to catalytically important residues and three-dimensional structure, and both permit the inhibitor to be accommodated in the active site, as indicated by computational docking studies. To clarify the reason for the difference of inhibitory potency towards the two XORs, we performed molecular dynamics simulations. The results indicate that differences in mobility of hydrophobic residues that do not directly interact with the substrate account for the difference in inhibitory potency.