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PubMed1990

Human urate oxidase gene: cloning and partial sequence analysis reveal a stop codon within the fifth exon.

Using the cDNA and selected genomic probes of rat urate oxidase, we have screened the human genomic library and isolated seven clones; one clone (clone 13) contained exonic regions which correspond to the exons 5, 6, and 7 of rat urate oxidase gene. The nucleotide sequence was determined for these three exons and exon/intron junctions, and compared with the sequence from the rat gene. A mutation resulting in a stop codon TGA was found in the fifth exon of the human urate oxidase gene. Sequence analysis of the polymerase chain reaction amplified DNA, corresponding to the fifth exon of urate oxidase from DNA samples from four different individuals, confirmed the same TGA stop codon in all. This single stop codon mutation and/or other mutation(s) in this gene may be responsible for the lack of urate oxidase activity in the human.

Amino Acid Sequence↗
PubMed1997

Urate oxidase in prevention and treatment of hyperuricemia associated with lymphoid malignancies.

Standard prophylaxis and treatment of malignancy-associated hyperuricemia in the USA has been allopurinol with vigorous hydration, urinary alkalinization and osmotic diuresis. Urate oxidase, the enzyme that converts uric acid to allantoin (a readily excreted metabolite that has 5- to 10-fold higher solubility than uric acid), is an alternative therapy; however, few published findings support this practice. Between February 1994 and December 1996, we administered non-recombinant urate oxidase (Uricozyme) to 126 children with newly diagnosed non-B cell acute lymphoblastic leukemia (ALL) during the first 5 days of chemotherapy with methotrexate, 6-mercaptopurine or both. Their blood levels of uric acid and other indicators of tumor lysis were measured at diagnosis and during treatment and then compared with findings in 129 similarly treated historical controls who had received allopurinol to control hyperuricemia. Clinical responses to urate oxidase were also determined in eight patients with newly diagnosed B cell ALL or advanced-stage non-Hodgkin lymphoma. Patients treated with urate oxidase had rapid and significantly greater decreases in their blood uric acid levels than did the historical controls (median maximal level during treatment, 2.3 vs 3.9 mg/dl, P < 0.001). They also had lower creatinine (0.6 vs 0.7 mg/dl, P = 0.01) and blood urea nitrogen (11 vs 24 mg/dl, P < 0.001) levels. Similar findings were made in the eight cases of B cell ALL or non-Hodgkin lymphoma. None of the patients required dialysis for acute renal failure. Six (4.5%) of the 134 children given urate oxidase had allergic reactions, manifested primarily by urticaria, bronchospasm and hypoxemia. Thus, non-recombinant urate oxidase is a more effective uricolytic agent than allopurinol but is associated with acute hypersensitivity reactions, even in patients without a history of allergy.

Allopurinol↗
PubMed1988

Localization of urate oxidase in the crystalline cores of rat liver peroxisomes by immunocytochemistry and immunoblotting.

We investigated the immunocytochemical localization of urate oxidase by light and electron microscopy. Rabbits were immunized with urate oxidase prepared from rat liver and the resulting antibody was further purified by affinity chromatography. Immunoblotting of the antigen revealed a single band of Mr 32,500 daltons, consistent with a subunit of uricase. The same band was observed in immunoblots prepared from a total peroxisome fraction and in its subfraction containing the cores, but not in the matrix portion. Immunostaining of 1-micron sections with the antibody against uricase followed by protein A-gold-silver showed fine granules in hepatocytes, which exhibited distinct fluorescence when examined in a microscope equipped with epifluorescence illumination. Incubation of ultra-thin sections of rat liver, embedded in Lowicryl K4M, LR White, or Epon, with the anti-uricase antibody followed by protein A-gold showed prominent labeling of the crystalline cores, with no reaction in the surrounding peroxisomal matrix. In contrast, the core region was spared whereas the matrix was heavily labeled in sections incubated with an antibody against catalase. Direct incubation of cores, isolated by centrifugation, with the anti-uricase antibody followed by protein A-gold revealed gold particles on the surface of isolated cores, with rare particles within the lumen of the polytubular structures that make up the cores. Specificity of the immunolabeling was established in sections incubated with an IgG fraction from pre-immunized rabbits. These observations demonstrate that in normal rat liver urate oxidase is exclusively associated with the crystalline cores in peroxisomes.

Animals↗
PubMed2006

Control of hyperuricemia in subjects with refractory gout, and induction of antibody against poly(ethylene glycol) (PEG), in a phase I trial of subcutaneous PEGylated urate oxidase.

PEG-modified recombinant mammalian urate oxidase (PEG-uricase) is being developed as a treatment for patients with chronic gout who are intolerant of, or refractory to, available therapy for controlling hyperuricemia. In an open-label phase I trial, single subcutaneous injections of PEG-uricase (4 to 24 mg) were administered to 13 such subjects (11 had tophaceous gout), whose plasma uric acid concentration (pUAc) was 11.3 +/- 2.1 mg/dl (mean +/- SD). By day seven after injection of PEG-uricase, pUAc had declined by an average of 7.9 mg/dl and had normalized in 11 subjects, whose mean pUAc decreased to 2.8 +/- 2.2 mg/dl. At doses of 8, 12, and 24 mg, the mean pUAc at 21 days after injection remained no more than 6 mg/dl. In eight subjects, plasma uricase activity was still measurable at 21 days after injection (half-life 10.5 to 19.9 days). In the other five subjects, plasma uricase activity could not be detected beyond ten days after injection; this was associated with the appearance of relatively low-titer IgM and IgG antibodies against PEG-uricase. Unexpectedly, these antibodies were directed against PEG itself rather than the uricase protein. Three PEG antibody-positive subjects had injection-site reactions at 8 to 9 days after injection. Gout flares in six subjects were the only other significant adverse reactions, and PEG-uricase was otherwise well tolerated. A prolonged circulating life and the ability to normalize plasma uric acid in markedly hyperuricemic subjects suggest that PEG-uricase could be effective in depleting expanded tissue stores of uric acid in subjects with chronic or tophaceous gout. The development of anti-PEG antibodies, which may limit efficacy in some patients, is contrary to the general assumption that PEG is non-immunogenic. PEG immunogenicity deserves further investigation, because it has potential implications for other PEGylated therapeutic agents in clinical use.

Adult↗
PubMed2002

Urate-oxidase in the prevention and treatment of metabolic complications in patients with B-cell lymphoma and leukemia, treated in the Société Française d'Oncologie Pédiatrique LMB89 protocol.

PURPOSE: To evaluate the frequency of metabolic complications and dialysis due to tumor lysis syndrome in patients with B-cell advanced-stage non-Hodgkin's lymphoma (NHL) and L3 leukemia at initiation of chemotherapy including the use of urate-oxidase. PATIENTS AND METHODS: Retrospective review of the clinical records of 410 patients with stage III and IV B-cell NHL and L3 leukemia treated in France and prospectively registered in the LMB89 protocol. RESULTS: During the first week of chemotherapy, only 34 of 410 patients recorded metabolic problems that included hypocalcemia (< 70 mg/dl) in 24 patients, hyperphosphatemia (> 6.5 mg/dl) in 28 and elevation of creatinine > or = 2 SD in 16. Six patients underwent dialysis for life-threatening problems and a seventh as a preventive measure. In the other 27 cases, metabolic problems were successfully resolved using urate-oxidase in combination with alkaline hyperhydration. Among the 410 patients, one case of hemolysis was reported and there was no severe allergic reaction to urate-oxidase. CONCLUSIONS: Only 1.7% of patients in our study receiving urate-oxidase during their induction chemotherapy needed renal dialysis. Urate-oxidase was well tolerated, and used as prophylaxis and/or treatment of hyperuricemia and tumor lysis syndrome consistently gave a lower rate of renal and metabolic complications than in other series of similar patients.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2005

Protective effect of urate oxidase on uric acid induced-monocyte apoptosis.

Uremic patients have a higher risk of infection and malignancy than normal subjects. Previous studies have deomonstrated that monocytes isolated from uremic patients display an increased apoptosis rate compared to normal subjects; furthermore uremic plasma can increase apoptosis rates on U937, a human monocytic cell line. In several pathological conditions, precipitation of uric acid crystals can lead to renal insufficiency or acute renal failure by different mechanisms. In recent studies uric acid has been shown to induce inflammatory response from monocytes and it has been suggested to be involved in cell dysfunction. Rasburicase is a new recombinant urate oxidase developed to prevent and treat hyperuricaemia in patients with cancer or renal failure; it degrades uric acid to allantoin, a less toxic and more soluble product. In the present study, we aimed at determining whether uric acid may be a factor affecting U937 apoptosis, and whether urate oxidase may reduces or even prevent uric acid induced cell apoptosis. Hoechst staining and internucleosome ledder fragmentation of DNA showed that uric acid increased the percentage of apoptotic cells comparing to the control and that when the U937 cells were incubated with uric acid and urate oxidase the percentage of apoptosis significantly decreased (from 43+/-7% to 19+/- 3%, p<0.05). Also, the activity of caspase-8 and caspase-3 showed the same trend (caspase 3: from 2.7+/-0.53 to 1.6+/-0.42; caspase-8: from 2.2+/-0.43 to 1.3+/-0.57). A reduction of intracellular reduced glutathione (GSH) concentration was found in uric acid treated cells while the addition of urate oxidase in the uric acid incubated cells decreased the GSH extrusion. The concentration of TNF-alpha was increased in the sample incubated with uric acid comparing to the control. Uric acid is an inducer of apoptosis on U937 cell line, and therefore it may be a component of the mosaic of uremic toxins both in acute and chronic renal disease. We can hypothesize that uric acid might be directly involved in the apoptotic process trough the activation of both death receptor and mitochondrial-mediated pathways. We have, also, demonstrated that urate oxidase is able to prevent at least in part, the effect of uric acid on U937 apoptosis. This effect might be a result of different mechanisms of action.

Apoptosis↗
PubMed1999

Hyperuricemia and renal insufficiency associated with malignant disease: urate oxidase as an efficient therapy?

Hyperuricemia is a common finding in patients with malignant diseases. Chemotherapy can induce life-threatening tumor lysis syndrome with severe hyperuricemia, other metabolic abnormalities, and acute renal failure. Intrarenal precipitation of uric acid contributes to renal insufficiency in this situation. Allopurinol, by preventing the conversion of hypoxanthine and xanthine to uric acid, has been long considered the standard pharmacological approach to hyperuricemia and prevention of tumor lysis syndrome. However, allopurinol itself may facilitate precipitation of xanthine crystals and has little influence on already-formed uric acid crystals deposited in the kidney. Urate oxidase further oxidizes uric acid to the highly water-soluble allantoin in mammals, except humans, who lack this enzyme. We report four cases of hyperuricemia (initial serum uric acid concentrations, 14.0 to 25.0 mg/dL) associated with malignant diseases treated with exogenous urate oxidase. Two of the patients showed full-blown tumor lysis syndrome. A single urate oxidase infusion (1,000 U) readily reduced serum uric acid levels in all patients. Furthermore, renal insufficiency, determined by serum creatinine concentrations, improved in three of the four patients. No adverse effects were observed. Currently, a recombinant urate oxidase is undergoing clinical testing and may make this efficient therapy more widely available. We believe that treatment with urate oxidase is a safe and efficient therapy for patients with cancer-associated hyperuricemia and may be effective even in individuals with only moderately elevated serum uric acid concentrations.

Journal Article↗
PubMed1995

Urate-oxidase for the treatment of tophaceous gout in heart transplant recipients. A report of three cases.

Gout in heart transplant recipients is common and poses a significant therapeutic challenge. Concomitant administration of azathioprine and allopurinol therapy carries a high risk of leukopenia. Uricosuric agents can cause renal lithiasis and/or acute renal failure in patients with renal failure and/or high urinary levels of uric acid. We report our experience with urate-oxidase in three heart transplant recipients with severe polyarticular and tophaceous gout, a history of leukopenia under allopurinol and unresponsiveness or contraindications to uricosuric agents. Urate-oxidase was given parenterally in a dosage of 1000 units per day, seven days a month. The injections were done intramuscularly in one patient and intravenously in the other two, who were under anticoagulant therapy. Patients 1 and 2 received 12 and 6 courses, respectively. The third patient had had four courses and was still under treatment at the time of this writing. Shrinking of the tophi and improved mobility of the fingers were seen in all three patients after the second course. No adverse effects were recorded. Our experience suggests that urate-oxidase therapy may decrease the urate burden in patients with severe tophaceous gout. Urate-oxidase therapy should be viewed as a phase in the treatment of gout, which must be followed by administration of another agent.

Adult↗
PubMed2002

Investigations into the uricolytic properties of urate oxidase in a granivorous (Columba livia domestica) and in a carnivorous (Buteo jamaicensis) avian species.

To study the uricolytic properties of urate oxidase in granivorous and carnivorous birds, experiments were conducted in pigeons (Columba livia domestica) and Red-tailed Hawks (Buteo jamaicensis). Plasma concentrations of allantoin and uric acid were determined at various times in experimental groups before and after receiving 100, 200, and 600 U/kg urate oxidase once daily and were compared with controls. All regimens caused a significant decrease in plasma uric acid concentrations within 2 days after the first administration, when compared with controls. Furthermore, both doses used in Red-tailed Hawks (200 and 100 U/kg) caused a significant postprandial suppression of plasma uric acid concentrations. Plasma allantoin concentrations were significantly higher when compared with controls. The ability of urate oxidase to prevent the physiological postprandial hyperuricaemia in Red-tailed Hawks and to reduce plasma uric acid concentrations to undetectable levels, shows the great potential of this drug for treating avian hyperuricaemia.

Allantoin↗
PubMed2002

Urate oxidase from the rust Puccinia recondita is a heterotetramer with two different-sized monomers.

Uricase (urate: oxygen oxidoreductase; EC 1.7.3.3) from the rust Puccinia recondita was purified to electrophoretic homogeneity. Preparations with a specific activity of 8.4 U/mg were used for characterization of the enzyme, which showed a strong similarity to other plant and fungal urate oxidases. The enzyme had a pH optimum of 9.0, a K(m) of 35 microM for urate, and it was inhibited only by oxonate and xanthine. A molecular mass of 152 kDa was estimated for the native protein. SDS-PAGE analysis revealed a striking difference to most urate oxidases, since two different-sized subunits were detected. These results suggest that P. recondita uricase is a tetramer with two types of subunits.

Basidiomycota↗
PubMed2006

A preliminary neutron diffraction study of rasburicase, a recombinant urate oxidase enzyme, complexed with 8-azaxanthin.

Crystallization and preliminary neutron diffraction measurements of rasburicase, a recombinant urate oxidase enzyme expressed by a genetically modified Saccharomyces cerevisiae strain, complexed with a purine-type inhibitor (8-azaxanthin) are reported. Neutron Laue diffraction data were collected to 2.1 A resolution using the LADI instrument from a crystal (grown in D2O) with volume 1.8 mm3. The aim of this neutron diffraction study is to determine the protonation states of the inhibitor and residues within the active site. This will lead to improved comprehension of the enzymatic mechanism of this important enzyme, which is used as a protein drug to reduce toxic uric acid accumulation during chemotherapy. This paper illustrates the high quality of the neutron diffraction data collected, which are suitable for high-resolution structural analysis. In comparison with other neutron protein crystallography studies to date in which a hydrogenated protein has been used, the volume of the crystal was relatively small and yet the data still extend to high resolution. Furthermore, urate oxidase has one of the largest primitive unit-cell volumes (space group I222, unit-cell parameters a = 80, b = 96, c = 106 A) and molecular weights (135 kDa for the homotetramer) so far successfully studied with neutrons.

Binding Sites↗
PubMed2005

Exclusion of urate oxidase as a candidate gene for hyperuricosuria in the Dalmatian dog using an interbreed backcross.

Hyperuricosuria, an autosomal recessive disorder, is characterized by high levels of uric acid in the urine of Dalmatian dogs. Whereas high levels of uric acid are known to be caused by the silencing of the urate oxidase (uox) gene in humans and higher primates, the molecular basis for the Dalmatian defect is unknown. Transplantation studies show that the organ responsible for the Dalmatian phenotype is the liver, which is where urate oxidase is exclusively expressed and uric acid is converted into allantoin. We cloned and sequenced the canine uox cDNA and compared the sequence between a Dalmatian and non-Dalmatian dog. No change in cDNA sequence was identified. A Dalmatian x pointer backcross family was used to track the segregation of microsatellite markers surrounding the urate oxidase locus. The uox gene was excluded for Dalmatian hyperuricosuria based on the cDNA sequence identity and negative LOD scores.

Animals↗
PubMed1975

Mitochondria and peroxisomes from the cellular slime mould Dictyostelium discoideum. Isolation techniques and urate oxidase association with peroxisomes.

The isolation of cell organelles from Dictyostelium discoideum was attempted using a variety of techniques. Cell homogenization (e.g. Potter-Elvehjem, glass beads) gave poor yields of organelles which were, in addition, exceptionally fragile and unstable in density gradients. An isolation method was developed using Triton X-100 in buffered sorbitol/Ficoll solutions at concentrations optimal for plasma membrane rupture. Immediately following cell lysis the solutions were diluted to sub-optimal Triton X-100 concentrations. Sedimentabilities of malate dehydrogenase, citrate synthetase, urate oxidase and catalase of around 55%, 40%, 35% and 55% respectively could be demonstrated using this method. The organelles were more resistant to breakage during resuspension following differential centrifugation and remained largely intact during density gradient centrifugation. The distribution of adenylate kinase activity in gradients showed that at least half the mitochondria retained an intact outer membrane. The mitochondria and peroxisomes could not be clearly separated using conventional sucrose-Ficoll density gradients. Separation was achieved by incubating the cell homogenate with succinate and a tetrazolium dye (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl monotetrazolium chloride). Succinate dehydrogenase activity of mitochondria reduced the tetrazolium dye and the product (formazan) was deposited on the mitochondrial membranes ("heavy-labelling"). The mitochondria then sedimented to denser regions of the gradient while catalase distribution remained unchanged. The treatment left both organelles intact. The mitochondria (1.21 g/ml) were slightly denser than the peroxisomes (1.19 g/ml). The peroxisomes contained catalase and urate oxidase; no other hydrogen-peroxide-producing oxidases were detected. The slime mould urate oxidase resembled the mammalian enzyme. It had an apparent Km value of 12.5 muM, an optimum of activity at pH 8.5 in borate buffer and was competitively inhibited by trichloropurine.

2,6-Dichloroindophenol↗
PubMed2002

A new class of gadolinium complexes employed to obtain high-phasing-power heavy-atom derivatives: results from SAD experiments with hen egg-white lysozyme and urate oxidase from Aspergillus flavus.

Seven gadolinium complexes are shown to be excellent compounds for the preparation of heavy-atom derivatives for macromolecular crystallography projects. De novo phasing has been carried out using single-wavelength anomalous diffraction (SAD) on a series of gadolinium-derivative crystals of two proteins: hen egg-white lysozyme and urate oxidase from Aspergillus flavus. Lysozyme derivative crystals were obtained by co-crystallizing the protein with the corresponding gadolinium complex at a concentration of 100 mM. Diffraction data were collected to a resolution of 1.7 A using Cu K(alpha) radiation from a rotating-anode generator, making use of the high anomalous signal of gadolinium at this wavelength. Urate oxidase derivative crystals were obtained by soaking native crystals in 100 mM gadolinium complex solutions. Diffraction data were collected to a resolution close to 3 A using X-rays at the Gd L(III) absorption edge, taking advantage of the sharp white line on that edge. For all urate oxidase derivative crystals and three of the lysozyme crystals, SAD phasing led to electron-density maps of very high quality, allowing unambiguous chain tracing. From this study, the binding effectiveness of the gadolinium complexes seems to be related to the nature of the precipitant used for crystallization. These gadolinium complexes represent a new class of high-phasing-power heavy-atom derivatives that may be used for high-throughput structure-determination projects.

Animals↗
PubMed2000

Urate oxidase electrode based on dissolved oxygen probe for urine uric acid determination.

A biosensor for the specific determination of uric acid in urine was developed using urate oxidase (EC 1.7.3.3) in combination with a dissolved oxygen probe. Urate oxidase was immobilized with gelatin by means of glutaraldehyde and fixed on a pretreated teflon membrane to serve as enzyme electrode. The electrode response was maximum when 50 mM glycine buffer was used at pH 9.2 and 35 degrees C. The enzyme electrode response depends linearly on uric acid concentration between 5-40 microM with a response time of 5 min. The enzyme electrode is stable for more than 2 weeks and during this period over 35 assays were performed.

Biosensing Techniques↗
PubMed2006

High pressure macromolecular crystallography: the 140-MPa crystal structure at 2.3 A resolution of urate oxidase, a 135-kDa tetrameric assembly.

We report the three-dimensional structure determined by high-pressure macromolecular crystallography (HPMX) of a 135-kDa homo-tetrameric enzyme, urate oxidase from Aspergillus flavus complexed with its potent inhibitor 8-azaxanthin. Urate oxidase crystals are quite sensitive to pressure, as three-dimensional order is lost at about 180 MPa. A highly complete 2.3 A resolution data set was collected at 140 MPa, close to the critical pressure. Crystal structures at atmospheric pressure and at high pressure were refined in the orthorhombic space group I222 with final crystallographic R factors 14.1% and 16.1%, respectively. The effect of pressure on temperature factors, ordered water molecules, hydrogen bond lengths, contacts, buried surface areas as well as cavity volume was investigated. Results suggest that the onset of disruption of the tetrameric assembly by pressure has been captured in the crystalline state.

Aspergillus flavus↗