Showing papers on "Xanthine published in 2016"

Xanthine Oxidoreductase-Derived Reactive Species: Physiological and Pathological Effects

Abstract: Xanthine oxidoreductase (XOR) is the enzyme that catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid and is widely distributed among species. In addition to this housekeeping function, mammalian XOR is a physiological source of superoxide ion, hydrogen peroxide, and nitric oxide, which can function as second messengers in the activation of various pathways. This review intends to address the physiological and pathological roles of XOR-derived oxidant molecules. The cytocidal action of XOR products has been claimed in relation to tissue damage, in particular damage induced by hypoxia and ischemia. Attempts to exploit this activity to eliminate unwanted cells via the construction of conjugates have also been reported. Moreover, different aspects of XOR activity related to phlogosis, endothelial activation, leukocyte activation, and vascular tone regulation, have been taken into consideration. Finally, the positive and negative outcomes concerning cancer pathology have been analyzed because XOR products may induce mutagenesis, cell proliferation, and tumor progression, but they are also associated with apoptosis and cell differentiation. In conclusion, XOR activity generates free radicals and other oxidant reactive species that may result in either harmful or beneficial outcomes.

Hyperuricemia-Related Diseases and Xanthine Oxidoreductase (XOR) Inhibitors: An Overview

Abstract: Uric acid is the final oxidation product of purine metabolism in humans. Xanthine oxidoreductase (XOR) catalyzes oxidative hydroxylation of hypoxanthine to xanthine to uric acid, accompanying the production of reactive oxygen species (ROS). Uric acid usually forms ions and salts known as urates and acid urates in serum. Clinically, overproduction or under-excretion of uric acid results in the elevated level of serum uric acid (SUA), termed hyperuricemia, which has long been established as the major etiologic factor in gout. Accordingly, urate-lowering drugs such as allopurinol, an XOR-inhibitor, are extensively used for the treatment of gout. In recent years, the prevalence of hyperuricemia has significantly increased and more clinical investigations have confirmed that hyperuricemia is an independent risk factor for cardiovascular disease, hypertension, diabetes, and many other diseases. Urate-lowering therapy may also play a critical role in the management of these diseases. However, current XOR-inhibitor drugs such as allopurinol and febuxostat may have significant adverse effects. Therefore, there has been great effort to develop new XOR-inhibitor drugs with less or no toxicity for the long-term treatment or prevention of these hyperuricemia-related diseases. In this review, we discuss the mechanism of uric acid homeostasis and alterations, updated prevalence, therapeutic outcomes, and molecular pathophysiology of hyperuricemia-related diseases. We also summarize current discoveries in the development of new XOR inhibitors.

Novel amperometric xanthine biosensor based on xanthine oxidase immobilized on electrochemically polymerized 10-[4H-dithieno(3,2-b:2′,3′-d)pyrrole-4-yl]decane-1-amine film

Abstract: In this paper, a novel amperometric xanthine (X) biosensor is constructed by xanthine oxidase (XOx) immobilization on the pencil graphite electrode (PGE). Xanthine oxidase is immobilized using glutaraldehyde (GA) on the electrochemically polymerized conducting polymer film. The detection of xanthine is based on its consumed amount due to the enzymatic reaction of xanthine oxidase. The effects of polymer thickness, applied potential, pH, and temperature were investigated and optimum parameters were found to be five cycles, +5 V, +0.5 V and 30 °C, respectively. Storage stability, operation stability of the enzyme electrode, and effect of interferant substances on the amperometric response were also studied. In order to verify the applicability of proposed biosensor, fabricated electrode was used to measure the xanthine concentration in chicken meat samples. The present xanthine biosensor with high selectivity, sensitivity, and stability is promising for practical applications.

Xanthine dehydrogenase: An old enzyme with new knowledge and prospects.

Abstract: Xanthine dehydrogenase (EC 1.17.1.4, XDH) is a typical and complex molybdenum-containing flavoprotein which has been extensively studied for over 110 years. This enzyme catalyzes the oxidation of purines, pterin and aldehydes with NAD+ or NADP+ as electron acceptor, and sometimes can be transformed to xanthine oxidase (EC 1.17.3.2, XOD) capable of utilizing oxygen as the electron acceptor. XDHs are widely distributed in all eukarya, bacteria and archaea domains, and are proposed to play significant roles in various cellular processes, including purine catabolism and production of reactive oxygen species (ROS) and nitric oxide (NO) in both physiological and pathological contexts. The recent applications of XDHs include clinical detections of xanthine and hypoxanthine content in body fluidics, and other diagnostic biomarkers like inorganic phosphorus, 5'-nucleotidase and adenosine deaminase. XDHs can also find applications in environmental degradation of pollutants like aldehydes and industrial application in nucleoside drugs like ribavirin. In this commentary, we would outline the latest knowledge on occurrence, structure, biosynthesis, and recent advances of production and applications of XDH, and highlighted the need to develop XDHs with improved performances by gene prospecting and protein engineering, and protocols for efficient production of active XDHs in response to the increasing demands.

Identification of a chemoreceptor that specifically mediates chemotaxis toward metabolizable purine derivatives.

Abstract: Chemotaxis is an essential mechanism that enables bacteria to move toward favorable ecological niches. Escherichia coli, the historical model organism for studying chemotaxis, has five well-studied chemoreceptors. However, many bacteria with different lifestyle have more chemoreceptors, most of unknown function. Using a high throughput screening approach, we identified a chemoreceptor from Pseudomonas putida KT2440, named McpH, which specifically recognizes purine and its derivatives, adenine, guanine, xanthine, hypoxanthine and uric acid. The latter five compounds form part of the purine degradation pathway, permitting their use as sole nitrogen sources. Isothermal titration calorimetry studies show that these six compounds bind McpH-Ligand Binding Domain (LBD) with very similar affinity. In contrast, non-metabolizable purine derivatives (caffeine, theophylline, theobromine), nucleotides, nucleosides or pyrimidines are unable to bind McpH-LBD. Mutation of mcpH abolished chemotaxis toward the McpH ligands identified - a phenotype that is restored by complementation. This is the first report on bacterial chemotaxis to purine derivatives and McpH the first chemoreceptor described that responds exclusively to intermediates of a catabolic pathway, illustrating a clear link between metabolism and chemotaxis. The evolution of McpH may reflect a saprophytic lifestyle, which would have exposed the studied bacterium to high concentrations of purines produced by nucleic acid degradation.

Chemical Evidence for Potent Xanthine Oxidase Inhibitory Activity of Ethyl Acetate Extract of Citrus aurantium L. Dried Immature Fruits

Abstract: Xanthine oxidase is a key enzyme which can catalyze hypoxanthine and xanthine to uric acid causing hyperuricemia in humans. Xanthine oxidase inhibitory activities of 24 organic extracts of four species belonging to Citrus genus of the family Rutaceae were assayed in vitro. Since the ethyl acetate extract of C. aurantium dried immature fruits showed the highest xanthine oxidase inhibitory activity, chemical evidence for the potent inhibitory activity was clarified on the basis of structure identification of the active constituents. Five flavanones and two polymethoxyflavones were isolated and evaluated for inhibitory activity against xanthine oxidase in vitro. Of the compounds, hesperetin showed more potent inhibitory activity with an IC50 value of 16.48 μM. For the first time, this study provides a rational basis for the use of C. aurantium dried immature fruits against hyperuricemia.

The effects of caffeine on wound healing

Abstract: The purine alkaloid caffeine is a major component of many beverages such as coffee and tea. Caffeine and its metabolites theobromine and xanthine have been shown to have antioxidant properties. Caffeine can also act as adenosine-receptor antagonist. Although it has been shown that adenosine and antioxidants promote wound healing, the effect of caffeine on wound healing is currently unknown. To investigate the effects of caffeine on processes involved in epithelialisation, we used primary human keratinocytes, HaCaT cell line and ex vivo model of human skin. First, we tested the effects of caffeine on cell proliferation, differentiation, adhesion and migration, processes essential for normal wound epithelialisation and closure. We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) proliferation assay to test the effects of seven different caffeine doses ranging from 0·1 to 5 mM. We found that caffeine restricted cell proliferation of keratinocytes in a dose-dependent manner. Furthermore, scratch wound assays performed on keratinocyte monolayers indicated dose-dependent delays in cell migration. Interestingly, adhesion and differentiation remained unaffected in monolayer cultures treated with various doses of caffeine. Using a human ex vivo wound healing model, we tested topical application of caffeine and found that it impedes epithelialisation, confirming in vitro data. We conclude that caffeine, which is known to have antioxidant properties, impedes keratinocyte proliferation and migration, suggesting that it may have an inhibitory effect on wound healing and epithelialisation. Therefore, our findings are more in support of a role for caffeine as adenosine-receptor antagonist that would negate the effect of adenosine in promoting wound healing.

A highly sensitive assay for xanthine oxidoreductase activity using a combination of [(13) C2 ,(15) N2 ]xanthine and liquid chromatography/triple quadrupole mass spectrometry.

Abstract: In this study, we developed a highly sensitive assay for xanthine oxidoreductase (XOR) activity utilizing a combination of [(13) C2 ,(15) N2 ]xanthine and liquid chromatography (LC)/triple quadrupole mass spectrometry (TQMS). In this assay, the amount of [(13) C2 ,(15) N2 ]uric acid (UA) produced by XOR was determined by using LC/TQMS. For this assay, we synthesized [(13) C2 ,(15) N2 ]xanthine as a substrate, [(13) C2 ,(15) N2 ]UA as an analytical standard, and [(13) C3 ,(15) N3 ]UA as an internal standard. The [(13) C2 ,(15) N2 ]UA calibration curve obtained using LC/TQMS under the selected reaction monitoring mode was evaluated, and the results indicated good linearity (R(2) = 0.998, weighting of 1/x(2) ) in the range of 20 to 4000 nM. As a model reaction of less active samples, the XOR activity of serial-diluted mouse plasma was measured. Thereby, the XOR activity of the 1024-fold-diluted mouse plasma was 4.49 ± 0.44 pmol/100 μL/h (mean ± standard deviation, n = 3). This value is comparable to the predicted XOR activity value of healthy human plasma. Hence, this combination method may be used to obtain high-sensitivity measurements required for XOR activity analysis on various organs or human plasma.

Xanthine oxidoreductase and its inhibitors: relevance for gout

Abstract: Xanthine oxidoreductase (XOR) is the rate-limiting enzyme in purine catabolism and converts hypoxanthine to xanthine, and xanthine into uric acid When concentrations of uric acid exceed its biochemical saturation point, crystals of uric acid, in the form of monosodium urate, emerge and can predispose an individual to gout, the commonest form of inflammatory arthritis in men aged over 40 years XOR inhibitors are primarily used in the treatment of gout, reducing the formation of uric acid and thereby, preventing the formation of monosodium urate crystals Allopurinol is established as first-line therapy for gout; a newer alternative, febuxostat, is used in patients unable to tolerate allopurinol This review provides an overview of gout, a detailed analysis of the structure and function of XOR, discussion on the pharmacokinetics and pharmacodynamics of XOR inhibitors-allopurinol and febuxostat, and the relevance of XOR in common comorbidities of gout

Preparation, Characterization and Electrochemical Application of Graphene‐metallic Nanocomposites

Abstract: Three different Graphene-Metallic (Graphene-Me) nanocomposites – Graphene-Silver (Graphene-Ag), Graphene-Gold (Graphene-Au) and Graphene-Platinum (Graphene-Pt) nanocomposites – were prepared and characterized. The electrochemical performances of these nanocomposites were tested by incorporating them with glassy carbon paste electrode (GCPE) and used them in biofuel cells (BFC) and as amperometric xanthine biosensor transducers. Present work contains the first application of Graphene-Au and Graphene-Ag nanocomposite in BFCs and also first application of these Graphene-Me nanocomposites in xanthine biosensors. Considering BFC, power and current densities were calculated as 2.03 µW cm−2 and 167.46 µA cm−2 for the plain BFC, 3.39 µW cm−2 and 182.53 µA cm−2 for Graphene-Ag, 4.43 µW cm−2 and 230.15 µA cm−2 for Grapehene-Au and 6.23 µW cm−2 and 295.23 µA cm−2 for Graphene-Pt nanocomposite included BFCs respectively. For the amperometric xanthine biosensor linear ranges were obtained in the concentration range between 5 µM and 50 µM with the RSD (n=3 for 30 µM xanthine) value of 4.28 % for plain xanthine biosensor, 3 µM and 50 µM with the RSD (n=3 for 30 µM xanthine) value of 9.37 % for Graphene-Ag, 5 µM to 20 µM with the RSD (n=3 for 5 µM xanthine) value of 9.00 % and 30 µM to 70 µM with the RSD (n=3 for 30 µM xanthine) value of 8.80 % for Grapehene-Au and 1 µM and 70 with the the RSD (n=3 for 30 µM xanthine) value of 2.59 % for Grapehene-Pt based xanthine biosensors respectively.

Optimization of High Performance Liquid Chromatography Method for Simultaneous Determination of Some Purine and Pyrimidine Bases.

Abstract: Nine purine and pyrimidine bases were separated and determined simultaneously using reversed phase (RP) high performance liquid chromatography (HPLC) in some food samples and biological fluids. Chromatographic behavior of these ionizable compounds highly depends on the interactions with the solvent as confirmed experimentally and by calculation of distribution of this species as a function of pH. Chromatograms show the optimal separation of five purine (uric acid, hypoxanthine, xanthine, adenine, and guanosine), and four pyrimidine (cytosine, uracil, cytidine and tymine) bases at pH around four. Accordingly, acetate buffer was selected due to high buffer capacity in this region. By variation of pH, concentration of buffer and volume ratio between buffer and methanol, we found that a mixture of 50 mM acetate buffer of pH 4.0 ± 0.1 with 3 % of methanol ensures reproducibility, complete separation in less than 15 minutes and compatibility with MS detection. Developed method was validated and applied for the analysis of complex clinical and beverage samples.

Response Surface Optimization for Decaffeination and Theophylline Production by Fusarium solani

Abstract: Coffee processing industries generate caffeine-containing waste that needs to be treated and decaffeinated before being disposed. Five fungal isolates obtained on caffeine-containing mineral media were tested for their ability to utilize caffeine at high concentrations. An isolate identified as Fusarium solani could utilize caffeine as a sole source of carbon and nitrogen up to 5 g/l and could degrade it to an extent of 30-53 % in 120 h. Sucrose that was added as an auxiliary substrate (5 g/l) enhanced the biodecaffeination of caffeine to 88 % in 96 h. The addition of co- substrate (sucrose) not only resulted in higher biodecaffeination efficiency, but also reduced the incubation period from the initial 120 to 96 h. Theophylline and 3-methyl xanthine were obtained as the major metabolites of decaffeination at 96 and 120 h, respectively. Response surface methodology used to optimize the process parameters for maximum biodecaffeination as well as theophylline production showed that a pH of 5.8, temperature of 24 °C and inoculum size of 4.8 × 10(5) spores/ml have resulted in a complete biodecaffeination of caffeine as well as the production of theophylline with a yield of 33 % (w/w). Results thus show that a viable and sustainable process can be developed for the detoxification of caffeine along with the recovery of theophylline, a commercially important chemical.

The inhibitory kinetics and mechanism of dietary vitamins D3 and B2 on xanthine oxidase.

Abstract: Dietary guidelines to promote health are usually based on the patterns' prediction on disease risk of foods and nutrients. Overactivity of xanthine oxidase (XO) is the underlying cause of gout. Herein, the inhibitory kinetics and mechanism of dietary vitamins D3 and B2 on XO were investigated by multispectroscopic methods and a molecular modeling technique. The results showed that vitamin D3 competitively inhibited XO with an inhibition constant of 26.93 ± 0.42 μM by inserting into the active cavity of XO interacting with the surrounding amino acid residues through hydrogen bond and van der Waals forces. Vitamin D3 bound to XO thereby induced the structural compactness of XO which in turn hindered the binding of substrate xanthine to cause the inhibition on XO. Vitamin B2 exhibited a mixed-type inhibition by binding to the vicinity of the active cavity with an inhibition constant of 37.76 ± 0.87 μM through hydrophobic interactions and a feeble hydrogen bond, and it induced the unfolding of the XO structure and an increase of the flexible loops (β-turns and random coils) which might move to cover the active pocket and reduce the binding of the substrate xanthine, and then lead to a lower catalytic activity of the enzyme. In addition, vitamins D3 and B2 showed a synergistic effect on inhibiting the activity of XO in a certain range of concentration. These findings may provide new insights into the inhibitory mechanism of vitamins D3 and B2 on XO and functional research of the vitamins in the supplementary treatment of gout.

Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines

Abstract: Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements.

Streptomyces coelicolor XdhR is a direct target of (p)ppGpp that controls expression of genes encoding xanthine dehydrogenase to promote purine salvage.

Abstract: The gene encoding Streptomyces coelicolor xanthine dehydrogenase regulator (XdhR) is divergently oriented from xdhABC, which encodes xanthine dehydrogenase (Xdh). Xdh is required for purine salvage pathways. XdhR was previously shown to repress xdhABC expression. We show that XdhR binds the xdhABC-xdhR intergenic region with high affinity (Kd ∼ 0.5 nM). DNaseI footprinting reveals that this complex formation corresponds to XdhR binding the xdhR gene promoter at two adjacent sites; at higher protein concentrations, protection expands to a region that overlaps the transcriptional and translational start sites of xdhABC. While substrates for Xdh have little effect on DNA binding, GTP and ppGpp dissociate the DNA-XdhR complex. Progression of cells to stationary phase, a condition associated with increased (p)ppGpp production, leads to elevated xdhB expression; in contrast, inhibition of Xdh by allopurinol results in xdhB repression. We propose that XdhR is a direct target of (p)ppGpp, and that expression of xdhABC is upregulated during the stringent response to promote purine salvage pathways, maintain GTP homeostasis and ensure continued (p)ppGpp synthesis. During exponential phase growth, basal levels of xdhABC expression may be achieved by GTP serving as a lower-affinity XdhR ligand.

Purine metabolism in response to hypoxic conditions associated with breath-hold diving and exercise in erythrocytes and plasma from bottlenose dolphins (Tursiops truncatus)

Abstract: In mammalian tissues under hypoxic conditions, ATP degradation results in accumulation of purine metabolites. During exercise, muscle energetic demand increases and oxygen consumption can exceed its supply. During breath-hold diving, oxygen supply is reduced and, although oxygen utilization is regulated by bradycardia (low heart rate) and peripheral vasoconstriction, tissues with low blood flow (ischemia) may become hypoxic. The goal of this study was to evaluate potential differences in the circulating levels of purine metabolism components between diving and exercise in bottlenose dolphins (Tursiops truncatus). Blood samples were taken from captive dolphins following a swimming routine (n=8) and after a 2min dive (n=8). Activity of enzymes involved in purine metabolism (hypoxanthine guanine phosphoribosyl transferase (HGPRT), inosine monophosphate deshydrogenase (IMPDH), xanthine oxidase (XO), purine nucleoside phosphorylase (PNP)), and purine metabolite (hypoxanthine (HX), xanthine (X), uric acid (UA), inosine monophosphate (IMP), inosine, nicotinamide adenine dinucleotide (NAD(+)), adenosine, adenosine monophosphate (AMP), adenosine diphosphate (ADP), ATP, guanosine diphosphate (GDP), guanosine triphosphate (GTP)) concentrations were quantified in erythrocyte and plasma samples. Enzymatic activity and purine metabolite concentrations involved in purine synthesis and degradation, were not significantly different between diving and exercise. Plasma adenosine concentration was higher after diving than exercise (p=0.03); this may be related to dive-induced ischemia. In erythrocytes, HGPRT activity was higher after diving than exercise (p=0.007), suggesting an increased capacity for purine recycling and ATP synthesis from IMP in ischemic tissues of bottlenose dolphins during diving. Purine recycling and physiological adaptations may maintain the ATP concentrations in bottlenose dolphins after diving and exercise.

Inhibition of Xanthine Oxidase Activity by Ethanolic Extract of Piperomia pellucida L., Acacypha indica L. and Momordica charantia L.

Abstract: BACKGROUND: Uric acid is a final result of purine catabolism, the enzymatic reactions in the body cells from amino acids or ribonucleotide dinucleotide. Peperomia pellucida L. (P. pellucida), Acalypha indica L. (A. indica) and Momordica charantia L. (M. charantia) are plants which have efficacy to reduce levels of uric acid excess. The aim of this research is to find out the effect of ethanol extract of P. pellucida, A. indica and M. charantia in preventing the formation of uric acid excess by inhibiting the action of the enzyme xanthine oxidase and comparing the inhibition activity of xanthine oxidase on treatments. METHODS: The study design is experimental and conducted using the enzyme xanthine oxidase, xanthine (substrate), pH 7.5 phosphate buffer, samples (P. pellucida, A. indica and M. charantia ethanol extracts) and HCL as reaction breaker. Inhibition of xanthine oxidase was determined enzymatically and unreacted xanthine was measured by UV spectrophotometer at 290 nm. The data were expressed as percent inhibition and the inhibitory concentration (IC)50 were determined using linear regresion of inhibition activity vs. concentration. RESULTS: The IC50 of P. pellucida, A. indica and M. charantia ethanol extracts in inhibiting xanthine oxidase were 19.5 ppm, 77.6 ppm and 17.8 ppm, respectively. IC50 of allopurinol was 1.99 μg/ml, and negative control (combination of enzyme and substrate) has absorbance value of 0.75026. CONCLUSION: Ethanol extract of M. charantia showed the most potent inhibition toward xanthine oxidase compared to the other two extracts. KEYWORDS: xanthine oxidase, Peperomia pellucida L., Acalypha indica L., Momordica charantia L.