Electrochemical Determination of Xanthine Oxidase-Catalyzed Oxidation of Xanthine

Abstract

A rapid electrochemical (chronoamperometric) method for the determination of xanthine oxidase catalyzed oxidation of xanthine and hypoxanthine is described. The assay is based on the anodic oxidation of the product, uric acid, at a stationary carbon paste electrode. Metabolism was monitored as reaction proceeded by direct insertion of a three-electrode assembly into incubation mixtures, applying a potential and measuring current after a 7 sec controlled electrolysis. The method requires no sample preparation, nor utilization of external reagents, and is compared with the on-line spectrophotonetric analysis based on monitoring the appearance of uric acid detected as an increase in absorbance at 290 nm.     

A Multiple Enzyme Method for Maasurement of K,Na ATPase

Abstract

A multiple enzyme method for spectrophotometrical measurement of K, Na ATPase activity is described. The method includes three enzymatic steps: Hydrolyses of ATP by ATPase, conversion of inosine to hypoxanthine and ribose -1- phosphate by purine nucleoside phosphorylase and finally xanthine oxidase mediated oxidation of hypoxanthine to xanthine with consequent formation of formazan from a tetrazolium salt. The phospholytic cleavage of inosine in the medium is dependent of an ATPase- phosphate complex.     

Amperometric Biosensor for Hypoxanthine Based on Immobilized Xanthine Oxidase on Iron (III) Meso‐tetraphenylporphyrin Nanoparticles Modified Glassy Carbon Electrode

Abstract

The preparation and performance of hypoxanthine (Hx) electrochemical biosensor, which was based on iron (III) meso‐tetraphenylporphyrin (FeTPP) nanoparticles (NPs), is reported in this work. FeTPP NPs prepared by mixing solvent techniques with diameters ca. 25~45 nm and were used as a mediator. The XOD/FeTPPNP/GC electrode exhibited good amperometric signal for Hx. Based on the consumption of dissolved oxygen during the oxidation process of Hx catalyzed by the immobilized XOD, the biosensor could detect the concentration of Hx up to 0.34 mM with a detection limit of 1.0 µM. The usefulness of this biosensor for the analysis of real sample was also demonstrated by determining Hx in rat brain dialysate coupled with microdialysis.     

A Selective Determination of Hypoxanthine,Xanthine and Inosine by Reversed-Phase Liquid Chromatography Coupled with Enzyme Reactors

Abstract

A Selective and sensitive assay of hypoxyanthine, xanthine and inosine by reversed-phase liquid chromatography coupled with immobilized enzyme reactors is described. The flourometric detection of hydrogen peroxide using immobilized peroxidase and p-hydroxyphenylacetic acid was applied to the assay of hypoxanthine, xanthine and inosine, which were oxidized to hydrogen peroxide in the presence of the immobilized enzymes (purine nucleoside phosphorylase and/or xanthine oxidase. The enzymes were immobilized the the intermolecular cross-linking method on controlled pore-glass. The method established was applied to serum and urine samples. The detection limits of hypoxanthine, xanthine and inosine were approximately 130, 300 and 650 pg per injection, respectively.     

Immobilization of xanthine oxidase and its use in the quantitation of hypoxanthine in fish muscle tissue extracts using a flow injection method

Fish muscle extracts (Scomberomorus— brasiliensis- carite) were analyzed for their hypoxanthine content using a flow injection system incorporating an immobilized xanthine oxidase bioreactor. The xanthine oxidase was immobilized under mild conditions to a 2-fluoro-1-methylpyridinium Fractogel support. The uric acid produced from the oxidation of hypoxanthine by the immobilized xanthine oxidase at pH 7.0 and 35‡C was monitored at 290 ran. Hypoxanthine concentrations as low as 4.4 Μmol/L can be detected. Up to 30 samples per hour can be analyzed at a flow rate of 1 mL/min, using 150 ΜL sample volumes and a bioreactor dimension of 1.0 cm x 2.0 mm id. Recovery yields were between 92 and 99%. Both within day and between day precisions gave CVs < 5.00% (n = 30). Good correlation (r = 0.998) is obtained when 78 fish samples were analyzed for their hypoxanthine content both by this FI method and a reference HPLC method.     

Ultratrace Xanthine Determination in Diluted Alkaline Electrolyte by Adsorptive Stripping Voltammetry at the Mercury Film Electrode

Abstract

A stripping method for the determination of xanthine at the submicromolar concentration level is described. The method is based on controlled adsorptive accumulation of xanthine at a thin-film mercury electrode followed by a linear scan voltammetry measurement of the surface species. Optimum experimental conditions were found to be the use of a 5.0 × 10^?3 M NaOH solution, an accumulation potential of 0.00 V, and a scan rate of 20 mV s^?1. The response of xanthine is linear over the concentration range 20–140 ppb. For an accumulation time of 30 min, the detection limit was found to be 36 ppt (2.3 × 10^?10 M). The more convenient relations for measuring xanthine in the presence of the metals, hypoxanthine, amino acids, and other nitrogenated bases were also investigated. The utility of the method is demonstrated by the presence of xanthine in adenosine-5′-triphosphate or DNA.     

The Application of UV-Radioactivity High Performance Liquid Chromatography to the Study of Hypoxanthine Transport in Human Erythrocytes

Abstract

A procedure is presented for the simultaneous measurement of concentrations of labeled and non labeled hypoxanthine by HPLC in order to study hypoxanthine transport in erythrocytes. A radioactivity detector connected on-line to the high performance liquid chromatograph in series with a UV detector provides on-line quantitative monitoring of hypoxanthine in erythrocytes or incubation medium. The procedure provides a rapid, sensitive and convenient means for the study of hypoxanthine transport.     

Quantitation of Purines and Pyrimidines in Human Serum by High-Performance Liquid Chromatography

Abstract

A high-performance liquid chromatography assay was developed for the purpose of examining xanthine, hypoxanthine, uridine, thymidine, allopurinol, and oxypurinol in serum of patients with advanced carcinoma treated with methorexate and 5-fluorouracil. After the addition of an internal standard, serum samples were extracted of purines and pyrimidines with trichloroacetic acid and neutralized with tert-amine. Aliquots of the extracted serum were injected onto a C18 reverse-phase column and purines and pyrimidines were eluted with a gradient of MeOH/H₂O and KH₂PO₄ solutions. Absorbance was detected with a variable-wavelength UV spectrophotometer at 254 nm and 280 nm. This assay can be readily applied to quantitate baseline and treatment-induced variations in serum purine and pyrimidine levels which may correlate with clinical response and/or toxicity in patients with cancer.     

An Improved Method for Micro-Fluorimetric Determination of 6-Mercaptopurine

Abstract

A sensitive and specific fluorimetric assay for 6-mercaptopurine (6-MP) is described. The method consists of enzymatic (xanthine oxidase) conversion of 6- MP to the oxypurine derivatives, followed by oxidation with acidic chromate to the corresponding 6-sulfonate. The fluorescent product has excitation and emission maxima at 330 and 400 nm, respectively. The good recovery and reproducibility were obtained. The sensitivity (the smallest amount detectables) was about 22 pg/ml for 6- MP in water. Different spiked serum samples containing 6-MP and azathioprine (AZA) were also analyzed and the sensitivity for 6-MP was about 2.2 ng/ml.     

Pheophytin analogues from the medicinal lichen Usnea diffracta

A new pheophytin, (13²S, 17S, 18S)-13²-hydroxy-20-chloro-ethylpheophorbide a (3), along with two known analogues (1–2) were isolated from the lichen Usnea diffracta Vainio (Parmeliaceae). Among them, compound 3 was a rare C-20-chloro type pheophytin obtained from lichens. Their structures were elucidated by extensive spectroscopic analysis, and all the compounds were obtained for the first time from U. diffracta. Compounds (1–3) were evaluated for their xanthine oxidase (XO) inhibitory activities in vitro, and the results showed that 1–3 possessed significant enzyme inhibitory actions with IC₅₀ values of 46.9 ± 3.8, 75.9 ± 7.4 and 42.1 ± 1.7 μg/mL, respectively.     

丝印电极法体外筛选黄嘌呤氧化酶抑制剂方法研究

建立丝印电极快速筛选黄嘌呤氧化酶(XO)抑制剂的新方法. 利用丝网印刷技术制作一次性使用的丝印碳糊电极, 采用吸附法将羧基化的多壁碳纳米管(MWNTs)修饰在电极表面, 建立测定尿酸(UA)的简单快捷的计时电流分析方法, 将该法应用于XO抑制剂的体外筛选. 在0.3 V (vs. Ag/AgCl)的工作电位下, UA在MWNTs修饰的丝印电极上产生灵敏的响应电流, 响应时间30 s, 测定UA的线性范围为2～300 μmol•L^－1, 线性方程为Y (μmol•L^－1)＝43.8240X (μA)－0.1592, r＝0.9998, 最低检出限为1 μmol•L^－1. 用该法对4种中药水提物进行了筛选, 相对空白对照组, 桑寄生500 μg•mL^－1浓度组有显著性差异. 该法简单、快速, 电极制作成本低, 所需仪器简单, 适用于体外大量筛选XO抑制剂.     

Isolation,characterization, and xanthine oxidase inhibitory activities of flavonoids from the leaves of Perilla frutescens

Abstract

Phytochemical investigation of the water extract from the leaves of Perilla frutescens (Lamiaceae) led to the isolation of a new flavanone, a new chalcone, and a new aurone, namely, (2S)-5,7-dimethoxy-8,4'-dihydroxyflavanone (1), 2',4'-dimethoxy-4,5',6'-trihydroxychalcone (2), and (Z)-4,6-dimethoxy-7,4'-dihydroxyaurone (3), respectively. The structures were unambiguously elucidated on the basis of spectroscopic data. And the absolute configuration of 1 was determined by analysis of electronic circular dichroism spectrum. The isolated compounds were evaluated for their inhibitory effects on xanthine oxidase in vitro. Among them, 2 showed more potent activity than the positive control allopurinol, a well-known XO inhibitor clinically used for treatment of gout. Lineweaver-Burk transformation of the inhibition kinetics data demonstrated that it was a mixed-type inhibitor.

     

Chemiluminescent Determination of Xanthine Oxidase Activity Using A Sensitive Low-Light Detection System

Abstract

A highly sensitive and rapid chemiluminescent assay for the determination of the activity of xanthine oxidase (XOD) was developed. The chemiluminescent signal was obtained from the catalyzed oxidation of hypoxanthine, accelerated and amplified using a Fe-EDTA complex and perborate, which acts on luminol. The same luminescent mixture was previously used as detection system for immunoassays. Two different mixtures were used, which differ in their luminol and perborate content, with (CLMrho) or without (CLMb) addition of 0.1 μM rhodamine fluorophor. The response obtained from XOD standard solutions in buffer was linear from 5 to 500 U L^?1 and from 0.7 to 250 U L^?1 for CLMrho and CLMb respectively, at 25°C. 5 and 0.7 U L^?1 were the detection limits at 1 standard deviation level. The intra- and inter-assay relative standard deviations ranged from 6 to 12 % for both CLM. Measurements were made using the high performance, low-light level imaging Berthold luminograph LB-980 which allows simultaneous determination of several samples distributed on a microtiterplate. Various kinds of milk were analyzed for XOD content, which in pasteurized milk depends on the fat content and in the UHT milk disappears owing to the heat treatment.     

Electrochemical Determination of Superoxide Based on Cytochrome c Immobilized on DDAB‐Modified Powder Microelectrode

Abstract

Cytochrome c was immobilized at didodecyldimethylammonium bromide (DDAB)‐modified powder microelectrode and presented quasi‐reversible electrochemistry. The apparent surface coverage of cytochrome c is greatly enhanced by using powder microelectrode technique, which is 1.21×10^?8 mol/cm², more than one to three orders of magnitude larger than that obtained with thiol and DNA‐modified Au electrode. The cytochrome c modified powder microelectrode was applied for the amperometric determination of superoxide generated by the reaction of hypoxanthine with xanthine oxidase (XOD) in the presence of dioxygen.

The detection sensitivity of the modified powder microelectrode is 0.74 µA/cm² µM, which is larger than that reported in previous publications. The detection limit of the modified powder microelectrode (PME) is 0.5 µM, and the linear detection range is 0.86～5.93 µM (values of the concentration are all in terms of hypoxanthine concentration in the solution).     

Mediated amperometric biosensor for hypoxanthine based on a hydroxymethylferrocene-modified carbon paste electrode

An amperometric enzyme electrode for the determination of hypoxanthine in fish meat is described. The hypoxanthine sensor was prepared from xanthine oxidase immobilized by covalent binding to cellulose triacetate and a carbon paste electrode containing hydroxymethylferrocene. The xanthine oxidase membrane was retained behind a dialysis membrane at a carbon paste electrode. The sensor showed a current response to hypoxanthine due to the bioelectrocatalytic oxidation of hypoxanthine, in which hydroxymethyiferrocene served as an electron-transfer mediator. The limit of detection is 6 × 10^?7 M, the relative standard deviation is 2.8% (n=28) and the response is linear up to 7 × 10^?4 M. The sensor responded rapidly to a low hypoxanthine concentration (7 × 10^?4 M), the steady-state current response being achieved in less than 1 min, and was stable for more than 30 days at 5 ° C. Results for tuna samples showed good agreement with the value determined by the conventional method.     

Simultaneous determination of three purines in Alysicarpus vaginalis (L.) DC. by hollow fiber‐based liquid‐phase microextraction combined with high‐performance liquid chromatography

In this paper, a three‐phase hollow fiber liquid‐phase microextraction (HF‐LPME) method combined with high‐performance liquid chromatography (HPLC) was developed for the determination of hypoxanthine (HX), xanthine (Xan) and adenine (A) and then for the first time successfully applied to the analysis of HX, Xan and A in Alysicarpus vaginalis (L.) DC. medicinal materials. Different factors affecting the HF‐LPME procedure were investigated and optimized. Under optimal extraction conditions (1‐octanol as organic solvent, pH of the donor and acceptor phase 10.0 and 3.5, respectively, extraction time 40 min, stirring rate 800 rpm and salt addition 10%, w/v), HX, Xan and A could be determined within the test ranges with a good correlation coefficient (r² > 0.9992). The limit of detection for HX, Xan and A was 153, 173 and 97 ng/mL, respectively, and the intra‐ and inter‐day relative standard deviations were no more than 9.8%. The content of HX, Xan and A in Alysicarpus vaginalis (L.) DC. medicinal materials was 120.40, 18.37 and 62.75 µg/g, respectively. This procedure afforded a convenient, sensitive, accurate and inexpensive method with a high extraction efficiency for determination of HX, Xan and A. Copyright © 2013 John Wiley & Sons, Ltd.     

Electrochemical determination of xanthine and hypoxanthine in rat striatum with an acetylene black-dihexadecyl hydrogen phosphate composite film modified electrode by HPLC coupled with in vivo microdialysis

The fabrication and application of a new electrochemical detector for use in HPLC is presented. The detector consists of an electrode modified with a composite film composed of acetylene black and dihexadecyl hydrogen phosphate. The electrochemistry of xanthine and hypoxanthine at this chemically modified electrode (CME) was investigated by cyclic voltammetry. It is found that the CME exhibits efficient electrocatalytic activity towards xanthine and hypoxanthine, with good sensitivity, stability, and lifetime. The linear ranges cover three orders of magnitude, and the detection limits are 6.0 × 10^?8 mol L^?1 for xanthine and 2.5 × 10^?7 mol L^?1 for hypoxanthine (at an S/N ratio of 3). The method was coupled to in-vivo microdialysis sampling and successfully applied to quantify xanthine and hypoxanthine in rat striatal microdialysates of freely moving rats.     

Voltammetric determination of hypoxanthine based on the enhancement effect of mesoporous TiO2-modified electrode

A mesoporous TiO₂ was synthesized according to the reported method, and then used to modify the carbon paste electrode (CPE). The electrochemical behavior of hypoxanthine was investigated with great detail. Compared with the unmodified CPE, the mesoporous TiO₂-modified CPE greatly enhances the oxidation signal of hypoxanthine. Due to huge surface area, well-defined and special mesopores, the mesoporous TiO₂-modified CPE shows considerable enhancement effect toward hypoxanthine. Based on this, a sensitive, rapid and convenient electrochemical method was developed for the determination of hypoxanthine. The linear range is over the range from 2.0 × 10⁻⁷ to 5.0 × 10⁻⁵ mol L⁻¹, and the limit of detection is estimated to be 5.0 × 10⁻⁸ mol L⁻¹. The relative standard deviation (RSD) for 10 mesoporous TiO₂-modified CPEs is 5.7%. Finally, this sensing method was successfully used to determine hypoxanthine in human blood serum samples.     

On-line electrochemical measurements of cerebral hypoxanthine of freely moving rats

This study demonstrates an on-line method for continuous measurements of cerebral hypoxanthine in the freely moving rats with integration of selective electrochemical biosensing with in vivo microdialysis sampling. The selective electrochemical biosensing is achieved by using xanthine oxidase (XOD) as the specific sensing element and Prussian blue (PB) as the electrocatalyst for the reduction of H₂O₂ generated from the oxidase-catalyzed reaction. The method is virtually interference-free from the coexisting electroactive species in the brain and exhibits a good stability and reproducibility. Upon integrated with in vivo microdialysis, the on-line method is well suitable for continuous measurements of cerebral hypoxanthine of freely moving rats, which is illustrated by the measurements of the microdialysates after the hypoxanthine standard was externally infused into the rat brain. This study essentially offers a facile on-line electrochemical approach to continuous measurements of cerebral hypoxanthine and could find some interesting applications in physiological and pathological investigations associated with hypoxanthine.     

Anti-hyperuricemia activity and toxicity prediction of a novel xanthine oxidoreductase inhibitor

A potent xanthine oxidoreductase inhibitor (LS087) was recently proved to exhibit a similar hypouricemic potency to febuxostat. A hyperuricemia model induced by potassium oxonate and hypoxanthine was proposed in specific pathogen-free male Kunming mice, and the serum urea nitrogen, creatinine and uric acid levels were measured after oral administration of LS087. Furthermore, renal histopathology was conducted by staining with hematoxylin and eosin, periodic acid–Schiff and Masson's trichrome stains, respectively. The results showed that the levels of serum urea nitrogen and uric acid significantly decreased compared with the model group, but the level of creatinine showed no significant changes. The pathological abnormalities in kidney tubules were improved after LS087 administration. Ten metabolites (M1–M10) of LS087 were identified after a single oral dosing of 10 mg/kg in rats. M6 was the primary LS087 metabolite in vivo with a pathway of methylation. The toxicity and potential risks of LS087 and its metabolites were predicted using the ProTox-II software. LS087 and the major metabolites (M2, M3, M5, M6, M7 and M8) were predicted to have no potential hepatotoxicity, but some metabolites with a total rate of <1% (M1, M4, M9, and M10) showed potential hepatotoxicity. M1 and M8 showed potential carcinogenicity. The LS087 biotransformation pathway in rat was well characterized.