Urate Oxidase

ONG-TERM URATE-LOWERING therapy in gout aims to prevent or reverse tissue urate crystal deposits and associated symptoms and signs by maintaining concentrations of uric acid (UA) below the limit of solubility (6.8 mg/dL). 1,2 However, it is common for UA levels to exceed the recommended goal urate range of less than 6.0 mg/dL 2 during oral urate-lowering therapy among the 5 to 6 million US patients with gout, 3,4 a finding documented in a majority of individuals treated with the most frequently prescribed doses of allopurinol (Յ300 mg/d). Although dose titration of available oral uratelowering agents can achieve target UA in most patients, urate-lowering therapy fails for perhaps 3% of patients 9 be-cause of refractoriness, contraindication, or intolerance. Without effective urate lowering, many such patients

Uric acid, a side product of nucleotide metabolism, should be cleared from blood stream since its accumulation can cause cardiovascular diseases and gout. Uricase (urate oxidase) converts uric acid to 5-hydroxyisourate, but it is absent in human and other higher apes. Yet, the recombinant form of uricase, Rasburicase, is now commercially available to cure tumor lysis syndrome by lowering serum uric acid level. Developing new methods to efficiently purify pharmaceutical proteins like uricase has attracted researchers' attention. Self-cleaving intein mediated single column purification is one of these novel approaches. Self-cleaving inteins are modified forms of natural inteins that can excise and join only at one junction site. In this study, the synthetic gene of Aspergillus flavus uricase, a homotetrameric protein, was cloned into pTXB1 vector as a fusion with the N-terminal of Mxe GyrA intein and chitin-binding domain (CBD) for simple purification. Expression was confirmed by ...

Ras is a small GTPase that regulates numerous processes in the cellular development and morphogenesis of many organisms. In this study, we identified and functionally characterized the Clg2p gene of Curvularia lunata, which is homologous with the Ras protein. The Clg2p deletion mutant (ΔClg2p) had altered appressorium formation and conidial morphology and produced fewer, smaller lesions compared with the wild-type strain. When a dominant Clg2p allele was introduced into the mutant, all of these defective phenotypes were completely restored. To further understand the regulation of Clg2p in appressorium formation and conidial morphology, and its role in pathogenicity, seven Clg2p-interacting proteins were screened using a yeast two-hybrid assay. Two of these proteins, Clf, a homologue of Mst11, which corresponds to MAP kinase kinase kinase in Magnaporthe oryzae, and urate oxidase (designated ClUrase) were functionally characterized. Clg2p specifically interacted with Clf through its R...

An all PEDOT:PSS Organic Electrochemical Transistor (OECT) has been developed and used for the selective detection of dopamine (DA) in the presence of interfering compounds (ascorbic acid, AA and uric acid, UA). The selective response has been implemented using a potentiodynamic approach, by varying the operating gate voltage and the scan rate. The trans-conductance curves allow to obtain a linear calibration plot for AA, UA and DA and to separate the redox waves associated to each compound; for this purpose, the scan rate is an important parameter to achieve a good resolution. The sensitivities and limits of detection obtained with the OECT have been compared with those obtained by potential step amperometric techniques (cyclic voltammetry and differential pulse voltammetry), employing a PEDOT:PSS working electrode: our results prove that the all-PEDOT:PSS OECT sensitivities and limits of detection are comparable or even better than those obtained by DPV, a technique that employs a...

This paper describes the immobilization of uricase by entrapment. The entrapping membrane was prepared by the cross-linking of chitosan with polyethylene glycol diglycidyl ether (diepoxy compound). The properties of the immobilized enzyme were investigated and compared with those of the native uricase. The enzyme activity of the immobilized uricase were found to be more dependent on temperature and pH than that of the native enzyme. The uricase immobilized membrane was sufficiently stable for 30 days allowing for the determination of uric acid concentration. The uric acid sensor was constructed using a hydrogen peroxide electrode. 1.

This paper describes the immobilization of uricase by entrapment. The entrapping membrane was prepared by the cross-linking of chitosan with polyethylene glycol diglycidyl ether (diepoxy compound). The properties of the immobilized enzyme were investigated and compared with those of the native uricase. The enzyme activity of the immobilized uricase were found to be more dependent on temperature and pH than that of the native enzyme. The uricase immobilized membrane was sufficiently stable for 30 days allowing for the determination of uric acid concentration. The uric acid sensor was constructed using a hydrogen peroxide electrode. 1.

Nitrogen dioxide (NO2) is an environmental air pollutant and endogenously generated oxidant that contributes to the exacerbation of respiratory disease and can function as an adjuvant to allergically sensitize to an innocuous inhaled Ag. Because uric acid has been implicated as a mediator of adjuvant activity, we sought to determine whether uric acid was elevated and participated in a mouse model of NO2-promoted allergic sensitization. We found that uric acid was increased in the airways of mice exposed to NO2 and that administration of uricase inhibited the development of OVA-driven allergic airway disease subsequent to OVA challenge, as well as the generation of OVA-specific Abs. However, uricase was itself immunogenic, inducing a uricase-specific adaptive immune response that occurred even when the enzymatic activity of uricase had been inactivated. Inhibition of the OVA-specific response was not due to the capacity of uricase to inhibit the early steps of OVA uptake or processin...

ONG-TERM URATE-LOWERING therapy in gout aims to prevent or reverse tissue urate crystal deposits and associated symptoms and signs by maintaining concentrations of uric acid (UA) below the limit of solubility (6.8 mg/dL). 1,2 However, it is common for UA levels to exceed the recommended goal urate range of less than 6.0 mg/dL 2 during oral urate-lowering therapy among the 5 to 6 million US patients with gout, 3,4 a finding documented in a majority of individuals treated with the most frequently prescribed doses of allopurinol (Յ300 mg/d). Although dose titration of available oral uratelowering agents can achieve target UA in most patients, urate-lowering therapy fails for perhaps 3% of patients 9 be-cause of refractoriness, contraindication, or intolerance. Without effective urate lowering, many such patients

ObjectiveTo assess the efficacy of pegloticase in achieving and maintaining plasma urate levels of <6 mg/dl in gout patients in whom other treatments have failed, and to assess the pharmacokinetics and safety of pegloticase.MethodsForty‐one patients were randomized to undergo 12–14 weeks of treatment with pegloticase at 1 of 4 dosage levels: 4 mg every 2 weeks, 8 mg every 2 weeks, 8 mg every 4 weeks, or 12 mg every 4 weeks. Plasma uricase activity, plasma urate, and antipegloticase antibodies were measured, pharmacokinetic parameters were assessed, and adverse events were recorded.ResultsThe mean plasma urate level was reduced to ≤6 mg/dl within 6 hours in all dosage groups, and this was sustained throughout the treatment period in the 8 mg and 12 mg dosage groups. The most effective dosage was 8 mg every 2 weeks. Twenty‐six patients received all protocol doses. The percentage of the patients in whom the primary efficacy end point (plasma urate <6 mg/dl for 80% of the study pe...

Objective To define the efficacy and safety of low-dose rasburicase in children from south India with hematologic malignancies. Methods This study is a retrospective analysis of data on 41 children with hematologic malignacies with laboratory evidence of tumor lysis syndrome (TLS) or clinical features indicating high risk for developing TLS. Patients were treated with rasburicase in doses of 0.1-0.15 mg/kg dose, repeated when necessary. Results Male : Female ratio was 32:9. Thirty-six children had laboratory evidence of TLS and 5 were at risk for TLS. Diagnoses were T-cell acute lymphoblastic leukemia (ALL), 19; Pre-B ALL, 17; B-non-Hodgkin lymphoma (NHL), 2; T-NHL, 2; and acute myeloid leukemia (AML), 1. Initial plasma uric acid (PUA): median, 8.5 mg/dl (range, 4.3 to 45.5). Six had creatinine levels of >2 mg/dl on admission; and 10 had peak PO 4 levels of >10 mg/dl. Dose of rasburicase used: median, 0.12 mg/kg (range, 0.08-0.24). Median reduction of PUA at 6 h: 80 % (range 40 to 98 %). Twenty-seven needed only one dose; 12 needed 2 or 3 doses; and two needed 5 doses each. One child required dialysis. None of the children developed anaphylaxis or hemolysis and there were no deaths from TLS. Conclusions Low-dose rasburicase (0.1-0.15 mg/kg) is safe and effective in reducing PUA in Indian children with lymphoid malignancies, and thus it may reduce the risk of renal failure from TLS.

Nineteen strains of microorganisms were screened for uricase production. Gliomastix gueg was recognized to produce high levels of the enzyme. The optimum fermentation conditions for uricase production by Gliomastix gueg were examined. Results showed that uric acid medium was the most favorable one, the optimum temperature was at 30º C, and incubation period required for maximum production was 8 days with aeration level at 150 rpm and at pH 8.0. Sucrose proved to be the best carbon source, uric acid was found to be the best nitrogen source. Both, dipotassium hydrogen phosphate and ferrous chloride as well as some vitamins gave the highest amount of uricase by Gliomastix gueg.

We report on a case of acute kidney injury in a patient with severe volume depletion from high ileostomy output and poor oral intake associated with hyperuricemia and hyperkalemia. The patient presented with similar features on three separate admissions. A conservative management approach was undertaken that included infusion of rasburicase, since the patient refused to undergo hemodialysis on each occasion. Rasburicase is a recombinant urate oxidase enzyme that converts uric acid to allantoin which is five to ten times more soluble than uric acid in urine, thereby enhancing urinary excretion. Rasburicase was administered each time and was followed by a prompt increase in urine output, rapid decrease in serum uric acid level, and recovery of renal function without the need for acute renal replacement therapy. We believe that rasburicase, with a molecular weight of 34 kDa, crossed the glomerular filtration barrier and entered the tubular fluid where it dissolved intra-tubular uric acid, thus relieving obstruction, thereby rapidly enhancing urine excretion and improving renal function. Based on this experience, we propose that rasburicase offers an alternative treatment for acute kidney injury in similar types of settings. To the best of our knowledge this is the first reported case in this situation.

Since the 1940s, purines have been known predominantly for two functions as both nucleic acid building blocks for the genetic material in cells, and as substances which break down to produce urate, and potentially elevate the risk for gout, an arthritis-like ailment or gouty arthritis with an excruciatingly painful presentation in joints There are two extant pathways of purine nucleotide synthesis, the De Novo synthesis pathway and the salvage pathway. The former is the major nucleotide synthesis pathway and the latter is the one essential in brain and bone marrow. The quantity of salvage of certain purines by animals is linked to the related intestinal or bacterial population containing elevate nucleic acid levels. Allopurinol inhibits the enzyme xanthine oxidase and decreases uric acid synthesis, and is broadly applied in the treatment of the excessive production of gout and uric acid.

Tumor lysis syndrome is a life-threatening condition for humans due to the lack of urate oxidase. In this study, several variants of PASylated uricase from the Aspergillus flavus species were analyzed computationally to find the appropriate fusions to solve short half-life and stability concern. The Ab initio method was performed using Rosetta software to structurally characterize the PAS sequences. The 3D structures of fusions were predicted for fused C- or N-terminally PAS sequences in different length to the uricase. The refinement and energy minimization steps revealed that physicochemical and conformational properties of fusions improved while the structures possessed prolonged PAS sequences. Molecular docking results showed that the highest binding affinity to uric acid belonged to uricase-PAS1-100 by the formation of six hydrogen and four non-hydrogen bonds. Altogether, the results indicated that the PASylation process would be promising upon the production of urate oxidase w...

We report on a 16-day-old male with metabolic acidosis, hyperuricemia, hyperuricosuria, and nephrocalcinosis caused by Lesch-Nyhan syndrome. Activity of the hypoxanthine-guanine phosphoribosyl transferase (HPRT) enzyme in lysed erythrocytes was undetectable, and molecular DNA analysis confirmed the presence of a 4 -base pair deletion at the 5= end of intervening sequence 8 in the HPRT1 gene, a change that affects a 5' splice site consensus sequence. Rasburicase, a urate oxidase enzyme, was administered on day 26 of life, with an endovenous dose of 0.20 mg/kg/d for 3 days. Plasma urate concentrations normalized (2.96 mg/dL) at 38 days of life. Kidney function was preserved in our patient. In summary, rasburicase proved to be a safe and effective treatment in a patient with Lesch-Nyhan syndrome with uric acid nephropathy in the neonatal period.

CASE REPORT A 47-years old male was seen in the emergency department of a tertiary care hospital in Kolkata India with respiratory distress and diminishing oxygen saturation.The patient had a history of Plasmodium vivax malaria for which he was admitted in the same hospital for 3 days and treated with Tab Lumithar Forte(Artemether and Lumefantrine) and consumed Tab Malirid DS (Primaquine) 15 mg daily for 10 days as home medication.

A new direct colorimetric procedure for uric acid assay in serum or urine is described, utilizing a 3,5-dichloro-2-hydroxybenzene sulfonic acid/4-aminophenazone chromogenic system in the presence of horseradish peroxidase and uricase from Aspergillus flavus. This chromogen system has a high absorptivity, affording useful results with sample/reagent volume ratios as low as 0.025. The procedure is applicable to serum, plasma, or diluted urine. A single working reagent is used; the reaction is complete in less than 15 min at room temperature. The red dye formed is measured at 520 nm; a blank sample measurement is not needed. The standard curve for the method is linear for uric acid concentrations up to 1500 mumol/L. Average analytical recovery of uric acid in human sera and urine exceeded 99%; within-run and between-run precision studies showed CV's of less than or equal to 1.2 and less than or equal to 2.2%, respectively. The new procedure correlated well with the uricase/catalase...

In the present work zinc oxide nanoflakes (ZnO-NF) structures with a wall thickness around 50 to 100 nm were synthesized on a gold coated glass substrate using a low temperature hydrothermal method. The enzyme uricase was electrostatically immobilized in conjunction with Nafion membrane on the surface of well oriented ZnO-NFs, resulting in a sensitive, selective, stable and reproducible uric acid sensor. The electrochemical response of the ZnO-NF-based sensor vs. a Ag/AgCl reference electrode was found to be linear over a relatively wide logarithmic concentration range (500 nM to 1.5 mM). In addition, the ZnO-NF structures demonstrate vast surface area that allow high enzyme loading which results provided a higher sensitivity. The proposed ZnO-NF array-based sensor exhibited a high sensitivity of ~66 mV/ decade in test electrolyte solutions of uric acid, with fast response time. The sensor response was unaffected by normal concentrations of common interferents such as ascorbic acid, glucose, and urea.

ObjectiveTo assess the efficacy of pegloticase in achieving and maintaining plasma urate levels of <6 mg/dl in gout patients in whom other treatments have failed, and to assess the pharmacokinetics and safety of pegloticase.MethodsForty‐one patients were randomized to undergo 12–14 weeks of treatment with pegloticase at 1 of 4 dosage levels: 4 mg every 2 weeks, 8 mg every 2 weeks, 8 mg every 4 weeks, or 12 mg every 4 weeks. Plasma uricase activity, plasma urate, and antipegloticase antibodies were measured, pharmacokinetic parameters were assessed, and adverse events were recorded.ResultsThe mean plasma urate level was reduced to ≤6 mg/dl within 6 hours in all dosage groups, and this was sustained throughout the treatment period in the 8 mg and 12 mg dosage groups. The most effective dosage was 8 mg every 2 weeks. Twenty‐six patients received all protocol doses. The percentage of the patients in whom the primary efficacy end point (plasma urate <6 mg/dl for 80% of the study pe...

Objective To define the efficacy and safety of low-dose rasburicase in children from south India with hematologic malignancies. Methods This study is a retrospective analysis of data on 41 children with hematologic malignacies with laboratory evidence of tumor lysis syndrome (TLS) or clinical features indicating high risk for developing TLS. Patients were treated with rasburicase in doses of 0.1-0.15 mg/kg dose, repeated when necessary. Results Male : Female ratio was 32:9. Thirty-six children had laboratory evidence of TLS and 5 were at risk for TLS. Diagnoses were T-cell acute lymphoblastic leukemia (ALL), 19; Pre-B ALL, 17; B-non-Hodgkin lymphoma (NHL), 2; T-NHL, 2; and acute myeloid leukemia (AML), 1. Initial plasma uric acid (PUA): median, 8.5 mg/dl (range, 4.3 to 45.5). Six had creatinine levels of >2 mg/dl on admission; and 10 had peak PO 4 levels of >10 mg/dl. Dose of rasburicase used: median, 0.12 mg/kg (range, 0.08-0.24). Median reduction of PUA at 6 h: 80 % (range 40 to 98 %). Twenty-seven needed only one dose; 12 needed 2 or 3 doses; and two needed 5 doses each. One child required dialysis. None of the children developed anaphylaxis or hemolysis and there were no deaths from TLS. Conclusions Low-dose rasburicase (0.1-0.15 mg/kg) is safe and effective in reducing PUA in Indian children with lymphoid malignancies, and thus it may reduce the risk of renal failure from TLS.

Rasburicase was administered at a fixed dose of 3 mg to treat 287 episodes of elevated serum uric acid levels (47 mg/dL) in 247 adult patients with hematological malignancies. The median total dose of 36 lg/kg (range: 18-65) was a fraction of the recommended total pediatric dose of 0.75-1.0 mg/kg. The median change in uric acid levels at 24 h was À4.1 mg/dL (range: À12 to þ 1) and À45% (range: À95 to þ 9). Uric acid levels normalized at 24 h in 72% of patients. There was no relationship between the weight-based dose and uric acid decline. The only predictor of success was the baseline uric acid; the failure rate was 84% with baseline level 412 mg/dL and 18% if it was p12. Uric acid levels continued to decline beyond 24 h in most patients without additional treatment. Serum creatinine remained stable over 24 h, and declined over 48 h and 7 days. There was no relationship between the extent of reduction in uric acid levels and serum creatinine. We conclude that a single 3-mg dose of rasburicase, used with close monitoring, is sufficient to treat most adults with uric acid levels up to 12 mg/dL.

Background-Rasburicase, a recombinant urate oxidase, is used to rapidly metabolize uric acid in patients with hyperuricaemia. Rasburicase is an immunogenic therapeutic protein, which has been shown to elicit antibody response in 64 % of healthy volunteers within 1-6 weeks after the initial course, with persistent antibodies for over 1 year. Drug labelling indicates that anaphylaxis rarely occurs (in <1 % of patients) after a single course of therapy with rasburicase, but there are no data available on the incidence of anaphylaxis in patients receiving a subsequent rasburicase course. Objective-To determine the incidence of anaphylaxis after multiple treatment courses of rasburicase. Methods-A retrospective chart review was performed on 97 consecutively treated patients who received repeated courses of rasburicase for hyperuricaemia, to determine whether the risk of anaphylaxis is increased with repeated exposure to rasburicase. Results-None of the 97 patients who were reviewed experienced anaphylaxis during the first rasburicase course; however, six patients (6.2 %) experienced anaphylaxis during a subsequent rasburicase treatment course (p = 0.03).

Urate oxidase is a peroxisomal enzyme with four equal subunits that convert uric acid to allantoin, a more soluble metabolite for excretion. The usage of uricase as a drug in medicine is to treat hyperuricemia. Many microorganisms have been used for uricase production such as Streptomyces exfoliates, Pseudomonas aeruginosa, and Aspergillus flavus. In this study, soil samples were collected and then cultured in a screening medium including uric acid as the sole carbon source. Samples with the higher ability of uricase production were selected for enzyme assay. Enzyme activity was measured by spectrophotometry and the sample with the maximal uricase activity was identified as Aspergillus niger and selected for further studies. According to the results of experiments, the optimized temperature for enzyme production by Aspergillus niger was determined to be 35±2°C. 47963 The best carbon and nitrogen source was glucose and NH4NO3, and the highest enzyme activity was observed in the presence of Cu 2+ ion.

Background: Tumor lysis syndrome (TLS) is a life-threatening disorder characterized by hyperuricemia and metabolic derangements. The efficacy of rasburicase, administered daily for 5 days, has been well established. However, the optimal duration of therapy is unknown in adults. Patients and methods: We evaluated the efficacy of rasburicase (0.15 mg/kg) administered as single dose followed by as needed dosing (maximum five doses) versus daily dosing for 5 days in adult patients at risk for TLS. Results: Eighty of the 82 patients enrolled received rasburicase; 40 high risk [median uric acid (UA) 8.5 mg/dl; range, 1.5-19.7] and 40 potential risk (UA = 5.6 mg/dl; range, 2.4-7.4). Seventy-nine patients (99%) experienced normalization in their UA within 4 h after the first dose; 84% to an undetectable level (<0.7 mg/dl). Thirty-nine of 40 (98%) patients in the daily-dose arm and 34 of 40 (85%) patients in single-dose arm showed sustained UA response. Six high-risk patients within the single-dose arm required second dose for UA >7.5 mg/dl. Rasburicase was well tolerated; one patient with glucose-6-phosphate dehydrogenase deficiency developed methemoglobinemia and hemolysis. Conclusions: Rasburicase is highly effective for prevention and management of hyperuricemia in adults at risk for TLS. Single-dose rasburicase was effective in most patients; only a subset of high-risk patients required a second dose.

This study presents an electrochemical biosensor developed for uric acid (UA) determination using carbon paste electrode (CPE) modified with copper (II) oxide (CuO) particles and urate oxidase (UOx) enzyme. Base CPE is prepared using a multi-walled carbon nanotube (MWCNT) and a polydimethylsiloxane (PDMS) binder. The main sensing process is based on the oxidation of UA into 5-hydroxyisourate (HIU) as catalyzed by UOx, forming H 2 O 2 as byproduct, and then the H 2 O 2 reduction-oxidation (redox) reaction converts CuO to form Cu 2 O; the amount of H 2 O 2 and hence UA in the sample is measured by the oxidative current measured for the conversion of Cu 2 O back to CuO. Cyclic Voltammetry (CV) measurements revealed that the activity of UOx was retained with an apparent Michaelis constant (K m app) to be equal to 41.46 µM. Differential Pulse Voltammetry (DPV) measurements of UA using UOx-CuO-CPE showed a linear response ranging from 10 µM to 79.4 µM UA with a limit of detection (LOD) determined to be equal to 8.82 µM. UOx-CuO-CPE was shown to be selective towards UA even in the presence of creatinine, xanthine, and glucose. Furthermore, UOx-CuO-CPE was shown to be reusable (3.28% RSD), and its fabrication is repeatable using single factor Analysis of Variance (ANOVA) [F(1.396) < F critical (5.143)]. UOx-CuO-CPE was also shown to be stable even after five weeks of storage using the two-sample t-test [t(0.156) < t critical (4.303)]. Based on a recovery test using synthetic urine sample, this study showed the applicability of UOx-CuO-CPE in the detection of UA in human urine with 90.27%-102.03% recovery (n = 3).

ONE OF THE COMPLICATIONS of the initial phase of acute leukemia therapy is uric acid-induced nephropathy caused by massive cell lysis. Prophylactic therapy has included the use of allopurinol, a xanthine-oxidase inhibitor, and of fluids, bicarbonate, and diuretics (acetazolamide).' Urate-oxidase, an enzyme which converts uric acid into allantoin, is claimed to be particularly useful in the prevention of this complication, but there are few published data regarding its use. We have studied the efficacy of urate-oxidase therapy for the prevention of uric acid induced nephropathy in the initial phase of antineoplastic treatment in a homogeneous group of pediatric patients with acute leukemia. MATERIALS AND METHODS Urate-oxidase Uricozyme (Clin-Midy, Paris). Physicochemical profile. The highly purified enzyme is obtained from cultures of Aspergillus flavus. The molecular weight is 93,000 _+ 3,000 with an isoelectric point of 6.3, a Michaelis constant of 6. l0 s M, and a pH range of action of 6-11 (optimum 8.5)2-Mechanism of action. Urate-oxidase catalyzes the oxi

Uric acid in urine was analyzed using porcine liver uricase and horseradish peroxidase immobilized on alkylamine and arylamine glass beads (pore diameter 55nm) respectively. The minimum detection limit was 5.0mg/0.1 ml urine. The recovery of added uric acid was 92%. Within and between assay CVs were <1.3% and <5.3% respectively. A good correlation (r=0.93) was found between urinary urate values obtained by a commercial kit method and the present method. The uric acid in 24hr urine of apparently healthy adults and person suffering from various diseases was found to be 450 to 900mg/24hr, 659mg/24hr (range, mean) and 910-1400mg/24hr, 1145mg/24hr (range, mean) respectively with the present method.

Commercially available uricase and peroxidase have been immobilized onto alkylamine glass and arylamine glass beads respectively. A discrete method has been developed to determine uric acid in serum using immobilized uricase and peroxidase. The method is based on generation of H O from serum uric acid by immobilized uricase and its measurement by a colour reaction 2 2 catalyzed by immobilized peroxidase. The minimum detection limit of the method was 8 mg / 0.1 ml sample. The mean analytical recovery of added uric acid in serum was 87.5%. The within and between assay coefficient of variation (C. V.) were , 6.58% and , 10.77% respectively. The serum uric acid in apparently healthy adults and persons suffering from different disease was found to be 25-55 mg / ml, 3262.25 (range, mean6S.D.) and 55-200 mg / ml; 5266.4 (range, mean6S.D.) respectively by our method. A good correlation (r 5 0.8170) was obtained between the serum urate values by this method and with those obtained by commercial Enzo-kit method.

Commercially available uricase and peroxidase have been immobilized onto alkylamine glass and arylamine glass beads respectively. A discrete method has been developed to determine uric acid in serum using immobilized uricase and peroxidase. The method is based on generation of H O from serum uric acid by immobilized uricase and its measurement by a colour reaction 2 2 catalyzed by immobilized peroxidase. The minimum detection limit of the method was 8 mg / 0.1 ml sample. The mean analytical recovery of added uric acid in serum was 87.5%. The within and between assay coefficient of variation (C. V.) were , 6.58% and , 10.77% respectively. The serum uric acid in apparently healthy adults and persons suffering from different disease was found to be 25-55 mg / ml, 3262.25 (range, mean6S.D.) and 55-200 mg / ml; 5266.4 (range, mean6S.D.) respectively by our method. A good correlation (r 5 0.8170) was obtained between the serum urate values by this method and with those obtained by commercial Enzo-kit method.

Kuruma shrimp Marsupenaeus japonicus Bate, under the stress of 0.36 and 1.39 mM nitrite at 30 % (parts per thousand, g kg À1) for 48 h, were examined for nucleotide-related compounds, specific activities of xanthine dehydrogenase (XDH), xanthine oxidase (XOD), and uricase. The levels of total nucleotide-related compounds, including xanthine and hypoxanthine, in the gill increased directly with ambient nitrite, whereas the levels of total nucleotide-related compounds, including xanthine and hypoxanthine, in the hepatopancreas were inversely related to ambient nitrite. Specific activity of XOD in the hepatopancreas increased directly with ambient nitrite, whereas no significant difference in uricase activity in the hepatopancreas was observed among three treatments. In another experiment, M. japonicus, following 48 h exposure to 0.36 and 1.39 mM nitrite, were examined for ammonia, urea, and urate levels in tissues. Hemolymph urea and exoskeleton urate levels increased directly with ambient nitrite, whereas hemolymph urate and exoskeleton urea levels were inversely related to ambient nitrite. It is concluded that M. japonicus exhibited uricogenesis and uricolysis, and an increase of uricogenesis occurred for the shrimp under nitrite stress. Urate produced in the hepatopancreas was transported and accumulated in the epidermis, and removed along with the exoskeleton at the time of molting.

Available recombinant uricases (rasburicase, pegloticase) are potent hypouricaemic agents for tophaceous gout, but their long-term use is in question. We have performed a literature review on uricases, using Scirus, PubMed, Science Direct, and several other search engines. We have also consulted the records of drug regulatory authorities and patents on uricases. Rasburicase (Fasturtec ®) was approved in Europe for tumour lysis syndrome induced by chemotherapy, in a single daily infusion dose for a maximum of 7 days. A retrospective study (n = 10) conducted in patients with gout and three clinical cases have shown that infusions spaced over time, over several months, ensure the control of serum uric acid and help to eliminate or significantly reduce the size of tophi. However, repeated gout attacks (despite colchicine) and hypersensitivity reactions (despite corticosteroids) have dampened enthusiasm for its use in gout. Pegloticase was recently approved by the Food and Drug Administration (FDA) for patients with chronic gout, refractory or intolerant to conventional hypouricaemic therapy. A 6 month study versus placebo showed that pegloticase (infused at 8 mg every 2 weeks), induced a significant decrease in plasma uric acid in about 40% of patients (associated with a tendency for tophi dissolution). However, the remaining patients were non-responders, which correlated with the formation of pegloticase antibodies and infusion reactions. Research efforts are needed to develop less immunogenic uricases. In conclusion, some uricases could have an important role in the treatment of gout, for instance as a first-line treatment and over a short period of several months in patients with severe and tophaceous gout to allow rapid tophi dissolution.

We report the presence of a new subcompartment in rat liver peroxisomal matrix in which only D-amino acid oxidase is localized and other matrix enzymes are absent. By electron microscopic observation, the rat liver peroxisome has generally been considered to consist of a single limiting membrane, an electron-dense crystalline core, and a homogeneous matrix. Immunohistochemical staining for D-amino acid oxidase by the protein A-gold technique revealed the presence of a small area in the matrix that was immunoreactive for the enzyme and was less electron-dense than the surrounding matrix. The localization of D-amino acid oxidase in this small area of the peroxisomal matrix was confirmed by immunoelectron microscopy on freeze-substituted tissues processed without chemical fixation. To analyze the characteristics of the electron-lucent area, immunoreactivity for various peroxisomal enzymes, including catalase, acyl-CoA oxidase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunct...

Rasburicase is currently approved at a dosage of 0.15-0.2 mg/kg once/day for 5 days in pediatric patients with cancer to lower plasma uric acid concentrations and manage tumor lysis syndrome (TLS). Information on rasburicase dosing in adults is limited, with some data on using rasburicase as a single dose instead of multiple daily doses. Therefore, we evaluated the efficacy of a single dose of rasburicase for preventing or managing TLS in adults. We collected retrospective data for 11 adults with hematologic malignancies who received a single 6-mg dose of rasburicase. All patients received intravenous hydration with urinary alkalinization and allopurinol; however, due to adverse reactions, two patients received short courses of allopurinol. Only patients at high risk for TLS (e.g., large tumor burden, increasing uric acid concentration) or those with TLS received rasburicase. The single dose of rasburicase 6 mg resulted in a median 0.0773-mg/kg dose (range 0.0232-0.1361 mg/kg). The single 6-mg dose rapidly lowered uric acid concentrations in 10 of the 11 patients. The median uric acid concentration of 11.7 mg/dl (range 7.4-17.4 mg/dl) declined to 2.0 mg/dl (range 0.5-15.4 mg/dl) within a day after rasburicase administration (p=0.022). In these 10 patients, uric acid concentrations remained low despite subsequent chemotherapy, and none required additional rasburicase doses. The only patient who did not respond to the single 6-mg rasburicase dose was a morbidly obese man (259 kg, body mass index 87 kg/m 2) who subsequently responded to an additional dose of rasburicase 12 mg. These results warrant further investigation of a single 6-mg dose of rasburicase in adults with TLS or at high-risk for developing TLS.

Urate oxidase (Uox) catalyses the first reaction of oxidative uricolysis, a three-step enzymatic pathway that allows some animals to eliminate purine nitrogen through a water-soluble compound. Inactivation of the pathway in hominoids leads to elevated levels of sparingly soluble urate and puts humans at risk of hyperuricemia and gout. The uricolytic activities lost during evolution can be replaced by enzyme therapy. Here we report on the functional and structural characterization of Uox from zebrafish and the effects on the enzyme of the missense mutation (F216S) that preceded Uox pseudogenization in hominoids. Using a kinetic assay based on the enzymatic suppression of the spectroscopic interference of the Uox reaction product, we found that the F216S mutant has the same turnover number of the wild-type enzyme but a much-reduced affinity for the urate substrate and xanthine inhibitor. Our results indicate that the last functioning Uox in hominoid evolution had an increased Michaeli...

Reactive oxygen species (ROS) produced by neutrophils are crucial for defense against infectious diseases, and the adequate measurement of ROS levels is an important way to evaluate the possibility of infections. The fluorescent probe dihydrorhodamine 123 has been applied exclusively to the measurement of ROS thus far. We developed a novel method for detecting ROS, which utilizes the chemiluminescent probes Luminol and Diogenes. The new method quantitatively detects ROS produced by as few as 10 to 10 4 neutrophils. Furthermore, this method can detect ROS levels in one microliter of whole blood or ROS produced by Epstein-Barr immortalized B lymphocytes. This method will be valuable for prompt diagnosis of neonatal chronic granulomatous diseases in which neutrophils aberrantly produce superoxide.

Background: The optimal rasburicase dose for adult patients has not been determined. Objective: To retrospectively examine use of rasburicase in our centre and to evaluate the effect of a single dose of rasburicase on urate and serum creatinine levels in our adult patients. Method: A retrospective chart review was conducted of all adult patients who received rasburicase for treatment of tumour lysis syndrome-associated hyperuricaemia at our academic, urban medical centre from July 2002 to October 2006. Result: Twenty-one patients received rasburicase with an average first dose of 0AE15 ± 0AE03 mg/kg. The drug dosing was calculated based on the patients' ideal body weight (IBW) or adjusted body weight (aBW) for those who were more than 30% above their IBW. Patients experienced a mean serum urate reduction of 89AE7 ± 9AE0% from the baseline through the first 24 h after a single rasburicase dose (11AE4 ± 4AE5 mg/dL vs. 1AE4 ± 1AE4 mg/dL, respectively, P < 0AE001). The urate levels remained within normal limits (<8 mg/dL) in all the patients for 48 h after a single dose of rasburicase. The major limitation of our study is that in 18 of 21 patients we lacked adequate documentation to ascertain that the blood samples sent for urate analysis after drug administration were handled according to the manufacturer's recommendations. However, in this small group of patients, we observed that the effect of rasburicase on serum urate was similar to the total study population. The effect was sustained for 48 h after a single dose. Serum creatinine levels at 24-72 h after the single rasburicase dose were not significantly different from baseline (1AE8 mg/dL vs. 2AE3 mg/dL, respectively, P = 0AE14). Conclusion: Rasburicase is an effective treatment for patients with hyperuricaemia and may aid in the prevention of hyperuricaemia-associated nephrotoxicity. From our experience, a single dose of 0AE15 mg/kg (IBW or aBW) of rasburicase appears to effectively decrease and maintain urate levels within normal limits for 48 h.

Assessment of Nurses’ Behavior in Prevention of overstimulation of Preterm Infants in NICU Wards of Tehran Universities of Medical Sciences Premature Interruption of intrauterine growth results in vulnerability of the preterm infants. Preterm infants are exposed to various stimulants in hospitals which have significant effects on their growth and development. Due to the presence of factors such as high technology, light, noise and various activities, NICU environment is quite different from that of the intrauterine environment. In the NICU environment, the preterm infant is continuously exposed to a stressful environment which is in sharp contrast to the physiological needs of their developing nervous system. Over and above the job of tending to the infant, an important job of the nurse is to provide proper conditions for the development of the nervous system of the preterm infant. This is done only through the control of NICU environment. Sometimes, nurses who are the sole responsi...

In the present work zinc oxide nanoflakes (ZnO-NF) structures with a wall thickness around 50 to 100 nm were synthesized on a gold coated glass substrate using a low temperature hydrothermal method. The enzyme uricase was electrostatically immobilized in conjunction with Nafion membrane on the surface of well oriented ZnO-NFs, resulting in a sensitive, selective, stable and reproducible uric acid sensor. The electrochemical response of the ZnO-NF-based sensor vs. a Ag/AgCl reference electrode was found to be linear over a relatively wide logarithmic concentration range (500 nM to 1.5 mM). In addition, the ZnO-NF structures demonstrate vast surface area that allow high enzyme loading which results provided a higher sensitivity. The proposed ZnO-NF array-based sensor exhibited a high sensitivity of ~66 mV/ decade in test electrolyte solutions of uric acid, with fast response time. The sensor response was unaffected by normal concentrations of common interferents such as ascorbic acid, glucose, and urea.

Introduction: The efficacy of pegloticase, a polyethylene glycol (PEG)-conjugated mammalian recombinant uricase, approved for chronic refractory gout, can be limited by the development of antibodies (Ab). Analyses from 2 replicate, 6-month, randomized controlled trials were performed to characterize Ab responses to pegloticase. Methods: Anti-pegloticase, anti-PEG, and anti-uricase Ab were determined by validated enzyme-linked immunosorbent assays. Ab titers were analyzed for possible relationships with serum pegloticase concentrations, serum uric acid (sUA) lowering, and risk of infusion reactions (IRs). Results: Sixty-nine (41%) of 169 patients receiving pegloticase developed high titer anti-pegloticase Ab (> 1:2430) and 40% (67/169) developed anti-PEG Ab; 1 patient receiving placebo developed high titer anti-pegloticase Ab. Only 14% (24/169) of patients developed anti-uricase Ab, usually at low titer. In responders, patients showing sustained UA lowering, mean anti-pegloticase titers at week 25

Urate oxidase is an important enzyme with therapeutic and diagnostic applications. Rasburicase is a recombinant urate oxidase enzyme approved by FDA to use in the treatment of hyperuricemia conditions. Various hosts such as Saccharomyces cerevisiae, Hansenula polymorpha and Escherichia coli have been used to express the enzyme. Today, Pichia pastoris is considered as an important host for heterologous protein expression since it has beneficial characteristics such as strong promoters, simple scale up, post translational modifications, high cell density cultivation and simple genetic manipulation. In this study, Aspergillus flavus urate oxidase gene was cloned in pPICZαA expression vector and expressed in P. pastoris. The recombinant urate oxidase was expressed in secretory form and was confirmed through RT-PCR, SDS-PAGE analysis and western blotting. The enzyme activity was determined using a colorimetric assay. A production yield of 0.43 U/ml of culture supernatant was obtained.

Hyperuricemia is a condition characterized by abnormally elevated levels of uric acid in the blood. It has been a leading morbidity disease. Microbial uricase can be used to oxidize uric acid into allantoin and hydrogen peroxide in the presence of oxygen and therefore has the potential to play an essential role in reducing uric acid in the people suffering from degenerative disease of hyperuricemia. The present study aims to select uric acid oxidizing-Lactobacillus plantarum isolates based on their genetic determinant and uricase kinetics. A collection of Lactobacillus plantarum isolates were grown on a selective differential medium followed by measuring their uricase activity spectrophotometrically. Specific primers for detection of uricase gene were designed. The uricase coding gene (uox) was then detected in all of the selected isolates by using a qPCR method employing the designed specific primers. The uricase kinetics was determined by the Lineweaver-Burk method. Results showed...

Bean leaf urate oxidase (host enzyme) activity increases in both compatible and incompatible interactions once the ®rst haustorium has dierentiated (1 day post-inoculation) after Uromyces phaseoli infection. Progressive development of the parasite in susceptible leaves leads to further enhancement of urate oxidase activity and both host and fungal enzymes are synergistically involved to give a progressive impetus to purine degradation. The necrogenic process associated with hypersensitivity in the incompatible response blocks fungal growth but does not change the pattern of urate oxidase enhancement. A pronounced 32 kDa-subunit accumulation during the late infection stage revealed by SDS±PAGE analysis, greatly exceeds the enzyme activity increase suggesting a relevant drop in urate oxidase speci®c activity. This de novo enzyme synthesis is connected with greater gene expression, but only in susceptible leaves and concomitantly with the presence of the highest amount of fungal enzyme. These data are consistent with the hypothesis that an up-regulation of plant urate oxidase gene expression occurs at the post-transcriptional level rather than an over expression of the urate oxidase gene. We conclude that a general pathogenic eect and host growth reduction takes place as a consequence of an alteration in the catalytic property of host urate oxidase, and purine pool depauperation. The possibility that excess H 2 O 2 , generated by enhanced activity of urate oxidase, may contribute to hypersensitive cell death induction is also discussed.

Limitations in efficacy and/or tolerance of currently available urate-lowering therapies (ULTs), such as oral xanthine oxidase inhibitors, uricosurics, and intravenous uricase agents contribute to the development of refractory gout. Renal excretion is the major route of uric acid elimination, but the intestinal tract plays an increasingly recognized role in urate homeostasis, particularly in chronic kidney disease (CKD) in which the renal elimination of urate is impaired. We targeted intestinal degradation of urate in vivo with ALLN-346, an orally administered, engineered urate oxidase, optimized for proteolytic stability, and activity in the gut. We tested ALLN-346 in uricase/urate oxidase deficient mice (URKO mice) with severe hyperuricemia, hyperuricosuria, and uric acid crystalline obstructive nephropathy. A total of 55 male and female URKO mice were used in the two consecutive studies. These seminal, proof-of-concept studies aimed to explore both short-(7-day) and long-term (19-day) effects of ALLN-346 on the reduction of plasma and urine urate. In both the 7-and 19-day studies, ALLN-346 oral therapy resulted in the normalization of urine uric acid excretion and a significant reduction of hyperuricemia by 44 and 28% when therapy was given with food over 24 h or was limited for up to 6 h, respectively. Fractional excretion of uric acid (FEUA) was normalized with ALLN-346 therapy. Oral enzyme therapy with engineered urate oxidase (ALLN-346) designed to degrade urate in the intestinal tract has the potential to reduce hyperuricemia and the renal burden of filtered urate in patients with hyperuricemia and gout with and without CKD.