Enzymatic Oxidation Of Uric Acid Research Articles

Flow-Through Amperometric Biosensor System Based on Functionalized Aryl Derivative of Phenothiazine and PAMAM-Calix-Dendrimers for the Determination of Uric Acid.

A flow-through biosensor system for the determination of uric acid was developed on the platform of flow-through electrochemical cell manufactured by 3D printing from poly(lactic acid) and equipped with a modified screen-printed graphite electrode (SPE). Uricase was immobilized to the inner surface of a replaceable reactor chamber. Its working volume was reduced to 10 μL against a previously reported similar cell. SPE was modified independently of the enzyme reactor with carbon black, pillar[5]arene, poly(amidoamine) dendrimers based on the p-tert-butylthiacalix[4]arene (PAMAM-calix-dendrimers) platform and electropolymerized 3,7-bis(4-aminophenylamino) phenothiazin-5-ium chloride. Introduction of the PAMAM-calix-dendrimers into the electrode coating led to a fivefold increase in the redox currents of the electroactive polymer. It was found that higher generations of the PAMAM-calix-dendrimers led to a greater increase in the currents measured. Coatings consisted of products of the electropolymerization of the phenothiazine with implemented pillar[5]arene and PAMAM-calix-dendrimers showing high efficiency in the electrochemical reduction of hydrogen peroxide that was formed in the enzymatic oxidation of uric acid. The presence of PAMAM-calix-dendrimer G2 in the coating increased the redox signal related to the uric acid assay by more than 1.5 times. The biosensor system was successfully applied for the enzymatic determination of uric acid in chronoamperometric mode. The following optimal parameters for the chronoamperometric determination of uric acid in flow-through conditions were established: pH 8.0, flow rate 0.2 mL·min-1, 5 U of uricase per reactor. Under these conditions, the biosensor system made it possible to determine from 10 nM to 20 μM of uric acid with the limit of detection (LOD) of 4 nM. Glucose (up to 1 mM), dopamine (up to 0.5 mM), and ascorbic acid (up to 50 μM) did not affect the signal of the biosensor toward uric acid. The biosensor was tested on spiked artificial urine samples, and showed 101% recovery for tenfold diluted samples. The ease of assembly of the flow cell and the low cost of the replacement parts make for a promising future application of the biosensor system in routine clinical analyses.

The Effective Control of Hyperuricemia in Cancer Patients: A New Recombinant Conjugated Variant of Urate Oxidase.

Objective: Management of hyperuricemia is crucial to controlling tumor lysis syndrome (TLS) during cancer therapy. Urate oxidase (UOX) that catalyzes the enzymatic oxidation of uric acid into allantoin, is effective in lowering plasma uric acid levels and controlling hyperuricemia. Recently, we developed a new recombinant conjugate variant of UOX therapeutic drug using PASylation technology. This study was designed to evaluate the stability, plasma half-life and immunogencity of PASylated UOX. Methods:A recombinant variant of PASylated UOX from the Aspergillus flavus was manufactured using bioinformatics and experimental techniques. Ex vivo evaluation of stability of PASylated UOX was done in 50% human serum. For half-life test, recombinant PASylated UOX and rasburicase were administered at 1.5 mg/kg to 10 rats in two different groups and samples were collected after injection Production of antibodies against PASylated drug was also assayed. Results:Residual activity of PASylated UOX in 50% human serum was higher than rasburicase and native UOX. Stability of PASylated UOX at 25°C and 37°C was also higher than rasburicase and native UOX. The PASylated half-life was ~32.1 hours, whereas half-life for rasburicase and native UOX was ~25.1 and ~22.8 hours, respectively. In immunogenicity examination, there is 33% and 36% decrease in the absorbance of native UOX and rasburicase, respectively when compared with that of PASylated UOX.Conclusion:Our data confirmed the efficacy and stability of PASylated UOX in comparison to the rasburicase. In summary, the results indicated that PASylated UOX drug is effective at lowering plasma uric acid levels with prolonged plasma half-life and decreased cost.

Colorimetric Detection of Uric Acid with High Sensitivity Using Cu2O@Ag Nanocomposites

In this paper, we built the Cu2O@Ag nanocomposites-catalyzed 3,3′,5,5′-tetramethylbenzidine (TMB)–H2O2 system for sensitive, rapid, and low-cost colorimetric sensing of uric acid (UA). Firstly, Ag nanotriangles were synthesized by dynamic method, and then Cu2O was encapsulated on the nanoflakes to synthesize Cu2O@Ag nanocomposites. Characterization of the structure and morphology of nanocomposites material were characterized by transmission electron microscopy, X-ray energy-dispersive spectroscopy, X-ray diffraction and X-ray photoelectron spectroscopy. Cu2O@Ag nanocomposites could catalyze the oxidation of TMB by H2O2 (which is produced by enzymatic oxidation of UA), and the color changes from colorless to blue can be observed directly with the naked eyes or can be detected by UV–Vis spectrophotometer. The colorimetric sensor provided a linear absorbance response in the 0.5–100 μM UA concentration range with a 0.4 μM detection limit (S/N = 3). The recovery of UA in human serum by the proposed sensor is over 95.7%. Thus, these results imply that the UA sensor provides an effective tool in fast clinical analysis of gout without the need for expensive and advanced equipment.

Enhancement of a Novel Extracellular Uricase Production by Media Optimization and Partial Purification by Aqueous Three-Phase System

Uricase (urate: oxygen oxidoreductase, EC 1.7.3.3), an enzyme belonging to the class of oxidoreductases, catalyzes the enzymatic oxidation of uric acid to allantoin and finds a wide variety of application as therapeutic and clinical reagent. In this study, uricase production ability of the bacterial strains isolated from deep litter poultry soil is investigated. The strain with maximum extracellular uricase production capability was identified as Xanthomonas fuscans subsp. aurantifolii based on 16S rRNA sequencing. Effect of various carbon and nitrogen sources on uricase productivity was investigated. The uricase production for this strain was optimized using statistically based experimental designs and resulted in uricase activity of 306 U/L, which is 2 times higher than initial uricase activity. Two-step purification, such as ammonium sulfate precipitation and aqueous two-phase system, was carried out and a twofold increase in yield and specific activity was observed.

ISOLATION, SCREENING AND PRODUCTION STUDIES OF URICASE PRODUCING BACTERIA FROM POULTRY SOURCES

Uricase (urate oxidase EC 1.7.3.3) is a therapeutic enzyme that is widely used to catalyze the enzymatic oxidation of uric acid in the treatment of hyperuricemia and gout diseases. In this study, three bacterial species capable of producing extracellular uricase were isolated from a poultry source and screened based on the size of the clear zone using a uric acid agar plate. The bacterial species capable of producing uricase with the highest uricolytic activity was identified as Bacillus cereus strain DL3 using a 16SrRNA gene sequencing approach. The time-course study of uricase production was performed and the medium was optimized. Carboxymethylcellulose and asparagine were found to be the best carbon and nitrogen sources. Maximum uricolytic activity was observed at pH 7.0 with an inducer concentration of 2.0 g/L. Inoculum size of 5% gave maximum uricolytic activity. The maximum uricolytic activity of 15.43 U/mL was achieved at optimized conditions, which is 1.61 times more than the initial activity. Further, enzymatic stability was determined at different pH and temperature.

Nondegradative Extraction and Measurement of Uric Acid from Poultry Litter

Due to limited solubility of uric acid from poultry litter (PL), alkaline solutions are frequently used to extract uric acid from PL for measurement, but the high pH can cause oxidation of the analyte. Our objective was to compare 0.1 M sodium acetate, 0.025 M Li2CO3, and a NaOH/Na2PO4 buffer as a means of extracting uric acid from PL for analysis by high‐pressure liquid chromatography (HPLC). In laboratory experiments, extractant‐induced degradation of reagent uric acid proceeded at a rate of 80 g kg−1 d−1 in 0.025 M of Li2CO3 (pH 11.2) and a NaOH/Na2PO4 buffer (pH 12.4) compared with no degradation in sodium acetate over a 2‐d period. In extracted PL samples, the alkaline extractants were found to enhance the enzymatic oxidation of uric acid to completion in Li2CO3 and by 800 g kg−1 in a NaOH/Na2PO4 buffer over the same 2‐d period. Sodium acetate extraction resulted in minimal loss of analyte (30 g kg−1), possibly due to thermal inactivation of the responsible enzyme uricase. Stabilization of the dissolved uric acid extracted from PL samples allowed for the development of a very accurate measurement procedure by HPLC with ultraviolet (UV)–visible light spectrometer (VIS) detection at 290 nm. Our results suggest a reporting limit of 8.5 mg uric acid–N kg−1 of litter. Of the 118 PL samples that were analyzed in this study, uric acid‐, ammonium‐, and nitrate‐N concentrations accounted for an average of 9.4, 17.4, and 1.2%, respectively, of the total N present in PL.

Distinct mechanisms for rasburicase induced hemolytic anemia and methemoglobinemia in tumor lysis syndrome

To the Editor: I read with great interest the paper by Bauters et al. [1] titled ‘‘Methemoglobinemia and hemolytic anemia after rasburicase administration in a child with leukemia.’’ in which the authors describe the third case in the literature of simultaneous occurrence of these two entities after the use of recombinant form of urate oxidase and also manifestations of acute pancreatitis. It is well recognized that production of hydrogen peroxide as a byproduct of the enzymatic oxidation of uric acid into the more water-soluble allantoin by rasburicase may trigger hemolytic anemia and methemoglobinemia in a genetically susceptible host with glucose-6-phosphate dehydrogenase enzyme deficiency and therefore this drug is contraindicated in this type of situation [2]. However there are several points that should be highlighted in relation to the pathogenesis of these two entities that are not explainable simply because of rasburicase induced hydrogen peroxide production. First of all, tumor lysis syndrome may itself elicit a systemic inflammatory response that leads to activation of endothelial cells and neutrophils which in turn release free radicals and reactive oxygen species (ROS). Besides, pro-inflammatory cytokines released by cell lysis induces nitric oxide production (which is one of the substances that can cause methemoglobinemia) and may combine synergistically with hydrogen peroxide to promote acute hemolysis too [3]. Secondly, serum uric acid plays an important role in protection from oxidative damage since it is a potent free-radical scavenger and increases in response to oxidative stress. So, rapid reduction in uric acid levels by rasburicase can compromise an important anti-oxidant mechanism and facilitates the emergence of acute hemolysis and methemoglobinemia [4]. Thirdly, under normal circumstances, cells are able to defend themselves against ROS damage with enzymes such as superoxide dismutases, catalases, lactoperoxidases, glutathione peroxidases and peroxiredoxins. Small molecules such as ascorbic acid, alpha-tocopherol, beta-carotene, methionine and glutathione also play important roles as cellular antioxidants. Decreased enzymatic activity and low antioxidant levels lead to an increased concentration of ROS which may facilitate rasburicase induced hemolysis and formation of methemoglobin [5]. Fourthly, urate oxidase is an endogenous enzyme found in most mammals but not in humans. Since humans do not express urate oxidase, rasburicase potentially can instigate an immune response that can include anaphylaxis or drug-mediated immune hemolytic anemia [6]. And last but not least, the symptoms of pancreatitis presented by the patient probably should be related to increased oxidative stress. It is known that pancreatic cells are highly susceptible to the effects of ROS. The use of rasburicase leads to a rapid reduction in uric acid and consequently there is an increase of enzymatic activity of xanthine oxidase to convert xanthine into uric acid. It is known that xanthine oxidase generates ROS and this coupled to the production of hydrogen peroxide by rasburicase may potentiate pancreatic inflammation [7, 8]. In summary, various host factors (impaired antioxidative mechanisms), disease characteristics (tumoral burden, cytokine release and ROS generation) and drug-related hydrogen peroxide production combined together create an environment of oxidative stress that ultimately triggers methemoglobinemia, hemolytic anemia and even pancreatic injury. R. Lopes da Silva (&) Hospital Santo Antonio dos Capuchos—CHLC, Alameda Santo Antonio dos Capuchos, 1169-050 Lisbon, Portugal e-mail: ronolosi@gmail.com

Fluorometric determination of uric acid in bovine milk

The primary objective of this study is to validate a new fast method for determination of uric acid in milk. The method is based on an enzymatic-fluorometric technique that requires minimal pre-treatment of milk samples. The present determination of uric acid is based on the enzymatic oxidation of uric acid to 5-hydroxyisourate via uricase where the liberated hydrogen peroxide reacts with 10-acetyl-3,7-dihydroxyphenoxazine via peroxidase and the fluorescent product, resorufin, is measured fluorometrically. Fresh composite milk samples (n=1,072) were collected from both Jersey (n=38) and Danish Holstein (n=106) cows from one local herd. The average inter- and intra-assay variations were 7·1% and 3·0%, respectively. Percent recovery averaged 103·4, 107·0 and 107·5% for samples spiked with 20, 40 or 60 μm of standard, respectively, with a correlation (r=0·98; P<0·001) observed between the observed and expected uric acid concentrations. A positive correlation (r=0·96; P<0·001) was observed between uric acid concentrations using the present method and a reference assay. Storage at 4°C for 24 h resulted in lower (P<0·01) uric acid concentrations in milk when compared with no storage or samples stored at -18°C for 24 h. Addition of either allopurinol (a xanthine oxidase inhibitor) or dimethylsulfoxide (a solvent for allopurinol) did not affect milk uric acid concentrations (P=0·96) and may indicate that heat treatment before storage and analysis was sufficient to degrade xanthine oxidase activity in milk. No relationship was observed between milk uric acid and milk yield and milk components. Authors recommend a single heat treatment (82°C for 10 min) followed by either an immediate analysis of fresh milk samples or storage at -18°C until further analysis.

Urate oxidase (rasburicase) for treatment of severe tophaceous gout

Gout is a clinical disorder caused by deposition of urate crystals in a joint leading to acute inflammatory response with acute pain. In severe and longstanding gout, the crystals accumulate in soft tissues such as cartilage, subcutaneous tissue or even veins leading to the development of tophi responsible for very large deposit formations and disability. Most cases of gout present with the sudden onset of severe acute arthritis in a peripheral joint in the leg. Urate oxidase, or uricase (EC 1.7.3.3), is a peroxisomal liver enzyme that catalyses the enzymatic oxidation of uric acid into the more water-soluble allantoin (Figure 1). Urate oxidase is an endogenous enzyme found in most mammals but not in humans. During primate evolution, the inactivation of the hominoid urate oxidase gene was caused by independent nonsense or frameshift mutations and has taken a two-step deterioration process, first in the promotor and second in the coding region [1]. Two nonsense mutations were found in the human urate oxidase gene, which confirms, at the molecular level, that the urate oxidase gene in humans is non-functional [2–3]. Because uric acid is a powerful scavenger of free radicals, it has been proposed that uric acid plays an important role in protection hominoids from oxidative damage and the prolonged live span [1,4]. Urate oxidase is used in humans for the control of increased serum uric acid in patients with acute tumour lysis syndrome after receiving chemotherapy. Rasburicase (SR 29142), a recombinant urate oxidase expressed in Saccharomyces cerevisiae, has been demonstrated to be superior to allopurinol in the control of uric acid in a randomized trial of paediatric and adult patients at risk of acute tumour lysis syndrome [5,6]. However, only few case reports address the potential role of urate oxidase for treatment of severe tophaceous gout. A French group treated three heart transplant patients with uncontrollable gout with non-recombinant urate oxidase and observed shrinking of tophi and improved mobility of the fingers in all three patients [7]. Phillips and co-workers used rasburicase for treatment of severe tophaceous gout refractory to high-dose allopurinol in a patient with end-stage renal disease and observed a regression of gout tophi [8]. The treatment was well tolerated in all reported patients and produced no adverse effects. We analysed efficacy and safety of rasburicase in the long-term control of hyperuricaemia in an adult kidney transplant patient with severe tophaceous gout.

A randomized comparison between rasburicase and allopurinol in children with lymphoma or leukemia at high risk for tumor lysis: Presented in part at the American Society of Hematology Conference in Miami Beach, FL, December 1998.

AbstractStandard therapy in the United States for malignancy-associated hyperuricemia consists of hydration, alkalinization, and allopurinol. Urate oxidase catalyzes the enzymatic oxidation of uric acid to a 5 times increased urine soluble product, allantoin. Rasburicase is a new recombinant form of urate oxidase available for clinical evaluation. This multicenter randomized trial compared allopurinol to rasburicase in pediatric patients with leukemia or lymphoma at high risk for tumor lysis. Patients received the assigned uric acid-lowering agent for 5 to 7 days during induction chemotherapy. The primary efficacy end point was to compare the area under the serial plasma uric acid concentration curves during the first 96 hours of therapy (AUC0-96). Fifty-two patients were randomized at 6 sites. In an intent-to-treat analysis, the mean uric acid AUC0-96 was 128 ± 70 mg/dL.hour for the rasburicase group and 329 ± 129 mg/dL.hour for the allopurinol group (P < .0001). The rasburicase versus allopurinol group experienced a 2.6-fold (95% CI: 2.0-3.4) less exposure to uric acid. Four hours after the first dose, patients randomized to rasburicase compared to allopurinol achieved an 86% versus 12% reduction (P < .0001) of initial plasma uric acid levels. No antirasburicase antibodies were detected at day 14. This randomized study demonstrated more rapid control and lower levels of plasma uric acid in patients at high risk for tumor lysis who received rasburicase compared to allopurinol. For pediatric patients with advanced stage lymphoma or high tumor burden leukemia, rasburicase is a safe and effective alternative to allopurinol during initial chemotherapy.

Uricase-catalyzed oxidation of uric acid using an artificial electron acceptor and fabrication of amperometric uric acid sensors with use of a redox ladder polymer.

Electrochemical oxidation of uric acid catalyzed by uricase (uric acid oxidase, UOx; EC 1.7.3.3) was studied using several redox compounds including 5-methylphenazinium (MP) and 1-methoxy-5-methylphenazinium (MMP) as electron acceptors for UOx, which does not contain any redox cofactor. It was found that MP and MMP were useful to mediate electrons from UOx to an electrode in the enzymatic oxidation of uric acid. A novel redox polymer, poly(N-methyl-o-phenylenediamine)(poly-MPD), containing the MP units was also found to possess the mediation ability for UOx, and poly-MPD was immobilized together with UOx onto an electrode substrate covered with a self-assembled monolayer of 2-aminoethanethiolate with use of glutaraldehyde as a binding agent. The resulting electrode (poly-MPD/UOx/Au) exhibited amperometric responses to uric acid with very fast response of approximately 30 s, allowing reagentless amperometric determination in a concentration range covering that in the blood of a healthy human being. Kinetic parameters of the apparent Michaelis constant and the maximum current response obtained at the poly-MPD/UOx/Au suggested that electrochemical oxidation of uric acid was controlled by diffusion of uric acid into the enzyme film and that the redox polymer worked well in mediating between active sites of UOx molecules and the electrode substrate.

Nonlinear dynamics of the distributed biochemical systems functioning in the dissipative structure formation mode

Nonlinear dynamical biomolecular systems can evidently be considered as prototypes of information processing devices at molecular level capable to solve problems of high computational complexity. Keeping in mind this goal the dynamics of biochemical system based on enzymatic oxidation of uric acid was considered. The system was studied in the version of distributed biomolecular structure having predetermined geometry of enzyme distribution on a porous planar medium. Being in the regime of stepwise dissipative structure formation this system demonstrated complicated modes of behaviour.

On-line mass spectrometric investigation of the peroxidase-catalysed oxidation of uric acid

The enzymatic and electrochemical oxidation pathways of uric acid were determined on-line with thermospray-tandem mass spectrometry. Products and intermediates formed as a result of electrooxidation were monitored as the electrode potential was varied. Electrochemical results served as a model for the enzymatic studies. In fact, electrochemical studies were essential for elucidating the structures of intermediates because of the high conversion efficiencies in electrooxidation. Products and intermediates formed as a result of enzymatic oxidation of uric acid were monitored as the reaction time was varied. When the enzymatic oxidation of uric acid with peroxidase and H2O2 was studied, the same intermediates and products were observed as in the electrochemical oxidation. The tandem mass spectrometric results provide convincing evidence that the primary intermediate produced during both the enzymatic and electrochemical oxidation of uric acid has a quinonoid diimine structure. The primary intermediate can follow three distinct reaction pathways to produce the identified final products. The final enzymatic and electrochemical oxidation products observed in these studies were urea, CO2, alloxan, alloxan monohydrate, allantoin, 5-hydroxyhydantoin-5-carboxamide and parabanic acid.

Enzymatic and electrochemical oxidation of uric acid Mechanism for the peroxidase catalyzed oxidation uric acid

The oxidation of uric acid has been studied by thin-layer spectroelectrochemistry using an optically transparent gold minigrid electrode and enzymatically using peroxidase. Under both enzymatic and electrochemical conditions a u.v.-absorbing intermediate (λ max =302–304 nm between pH 7–9.3) is observed. This intermediate is proposed to be an imine-alcohol species formed by rapid hydration of the primary reaction product which is a diimine. Under the conditions employed in both the enzymatic electrochemical studies the imine-alcohol intermediate is hydrated in a first order reaction with an observed rate constant of 3.5×10 −3 s −1 between pH 7–9.3. This study establishes the similarity between the electrochemical and enzymatic oxidation of uric acid and indicates that electrochemical investigations of the redox behavior of purines can provide unique insights into their biological redox properties.

Enzymatic and electrochemical oxidation of uric acid: A mechanism for the peroxidase catalyzed oxidation of uric acid

The oxidation of uric acid has been studied by thin-layer spectroelectrochemistry using an optically transparent gold minigrid electrode and enzymatically using peroxidase. Under both enzymatic and electrochemical conditions a u.v.-absorbing intermediate (λ max=302–304 nm between pH 7–9.3) is observed. This intermediate is proposed to be an imine-alcohol species formed by rapid hydration of the primary reaction product which is a diimine. Under the conditions employed in both the enzymatic electrochemical studies the imine-alcohol intermediate is hydrated in a first order reaction with an observed rate constant of 3.5×10 −3s −1 between pH 7–9.3. This study establishes the similarity between the electrochemical and enzymatic oxidation of uric acid and indicates that electrochemical investigations of the redox behavior of purines can provide unique insights into their biological redox properties.

The fluorometric determination of uric acid by use of the uricase-peroxidase system and 3,5-diacetyl-1,4-dihydrolutidine as secondary substrate

During the enzymatic oxidation of uric acid by means of uricase (urate: oxygen oxidoreductase, E.C. 1.7.3.3) hydrogen peroxide is generated which in the presence of peroxidase 3,5-diacetyl-1,4-dihydrolutidine (DDL) oxidizes to substituted pyridine. Connected with this is a proportional decrease of fluorescence of the DDL. Maintaining the specificity of the uricase reaction, with the fluorescence measurement a higher sensitivity of uric acid determination is achieved than by applying spectrophotometric methods. Thus as little as 25 · 10 −12 moles of uric acid can still be determined. The application of the method to serum will be described.