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PubMed2004

Polyaniline-uricase biosensor prepared with template process.

Polyaniline (PANI) uricase biosensor prepared with template process is reported first in this paper. The fabrication process is as follows. Firstly, a PANI-uricase electrode is obtained using one-step process. Secondly, the electrode is hydrolyzed in 6.0 mol/dm(3) hydrochloric acid solution to remove the uricase that may be affected by aniline monomer from PANI film. Finally, active uricase is immobilized into the PANI film based on the principle of the doping and undoping of the conducting polymer and a PANI-uricase biosensor is obtained. Some factors that affect response current are studied, such as temperature, pH, potential and substrate concentration. The determination of biosensors indicates that the response current of the biosensor prepared by template process decreases only by about 18% for 60 days, but that prepared by two-step process decreases by approximately 39% for 40 h. The uricase in PANI-uricase biosensor prepared by template process mainly interacts with the nitrogen linked to the quinoid ring. The biosensor is characterized with FTIR, UV-Vis and SEM for the first time.

Adsorption↗
PubMed2004

Urea potentiometric biosensor based on modified electrodes with urease immobilized on polyethylenimine films.

The development of a new electrochemical sensor consisting in a glass-sealed metal microelectrode coated by a polyethylenimine film is described. The use of polymers as the entrapping matrix for enzymes fulfils all the requirements expected for these materials without damaging the biological material. Since enzyme immobilization plays a fundamental role in the performance characteristics of enzymatic biosensors, we have tested four different protocols for enzyme immobilization to determine the most reliable one. Thus the characteristics of the potentiometric biosensors assembled were studied and compared and it appeared that the immobilization method leading to the most efficient biosensors was the one consisting in a physical adsorption followed by reticulation with dilute aqueous glutaraldehyde solutions. Indeed, the glutaraldehyde immobilized urease sensor provides many advantages, compared to the other types of sensors, since this type of urea biosensor exhibits short response times (15-30s), sigmoidal responses for the urea concentration working range from 1 x 10(-2.5) to 1 x 10(-1.5) M and a lifetime of 4 weeks.

Adsorption↗
PubMed2006

Development and analytical application of an uric acid biosensor using an uricase-immobilized eggshell membrane.

An uric acid biosensor fabricated from a uricase-immobilized eggshell membrane and an oxygen electrode was presented. The detection schemes involve the enzymatic reactions of the uricase leading to the depletion of dissolved oxygen level upon exposure to uric acid solution. The decrease in oxygen level was monitored and related to the uric acid concentration. The scanning electron micrographs show the microstructure of the eggshell membrane within which the uricase is successfully immobilized. The effects of enzyme loading, pH, temperature, and phosphate buffer concentration on the response of the biosensor were investigated in detail. The uric acid biosensor has a linear response range of 4.0-640 microM with a detection limit of 2.0 microM (S/N=3). The response time was less than 100 s. The biosensor exhibited good repeatable response to a 0.10mM uric acid solution with a relative standard deviation of 3.1% (n=7). The reproducibility of fabrication of the biosensors using four different membranes was good with a R.S.D. of 3.2%. The biosensor showed extremely good stability with a shelf-life of at least 3 months. Some common potential interferents in samples such as glucose, urea, ascorbic acid, lactic acid, glycine, DL-alpha-alanine, DL-cysteine, KCl, NaCl, CaCl2, MgSO4, and NH4Cl showed no interferences on the response of the uric acid biosensor. The biosensor was successfully applied to determine the uric acid level in some human serum and urine samples, and the results agreed well with those obtained by a commercial colorimetric assay kit.

Animals↗
PubMed2005

An evaluation of clinical accuracy of the EasyTouch blood uric acid self-monitoring system.

OBJECTIVES: Self-monitoring blood uric acid device is an important tool for patients to efficiently monitor their blood uric acid concentrations. The objective of the present study was to evaluate the accuracy of EasyTouch uric acid monitoring system. DESIGN AND METHODS: Capillary blood uric acid concentrations measured using EasyTouch and the reference values obtained from COBAS MIRA were performed in the Department of Laboratory Medicine, Wei-Gong Memorial Hospital. Results were evaluated using (1) linear regression analysis, (2) percentage of readings within a defined range of deviation from the reference value, and (3) coefficients of variation (CVs) calculated from 60 measurements in series. RESULTS: The window of the 177 EasyTouch readings covered a wide range from 0.1785 to 0.6367 mmol/L (3-10.7 mg/dL). Linear regression analysis yielded a regression slope of 0.975, an intercept of 0.0118 mmol/L and an R2 of 0.8966. Of the EasyTouch readings, 64 (36.2%), 61 (34.5%), 34 (19.2%), 9 (5.08%), and 9 (5.08%) were within the intervals of <5%, 5-10%, 10-15%, 15-17%, and >17%, respectively, of the reference values. Further analysis for the performance of each lot of strips showed that both coefficients of correlation and the percentages of readings within the CLIA's criterion (+/-17%), respectively, were in a narrow range from 0.8777 to 0.9541 and from 92.1% to 100%. The CVs for the seven lots of strips (lot 1 to lot 7) ranged from 2.93% to 6.33%, 3.2% to 5.9%, 3.64% to 7.0%, 2.84% to 7.6%, 2.68% to 5.42%, 3.03% to 6.93%, and 3.18% to 5.17%, respectively. CONCLUSION: EasyTouch is an acceptable diagnostic device which provides accurate uric acid measurements.

Adult↗
PubMed2005

Quantification of urinary allantoin by capillary zone electrophoresis during recombinant urate oxydase (rasburicase) therapy.

OBJECTIVES: Rasburicase (Fasturtec) is used to prevent or treat hyperuricemia associated with chemotherapy. We developed a capillary zone electrophoresis method to measure urinary allantoin, the degradation product of uric acid by rasburicase. DESIGN AND METHODS: Electrophoresis was performed using a P/ACE 5500 system (Beckman) with a fused silica capillary tube and a UV-visible detector set at 214 nm. Urine samples from 10 patients with non-Hodgkin's lymphoma were analyzed to validate the technique. RESULTS: Using a sodium tetraborate running buffer, urinary allantoin was separated from related compounds and internal standard in less than 30 min. The method was linear up to 1.25 g/L (quantification limit: 30 mg/L); precision was below 10%. The total amount of allantoin excreted in patients treated by rasburicase ranged from 1.5 g to 7.9 g/4 days. CONCLUSION: This CZE assay is a simple, rapid and reproducible method to measure allantoin in urine. Different elimination profiles have been found in patients treated with rasburicase.

Allantoin↗
PubMed2005

Allopurinol/uricase and ibuprofen enhance engraftment of cardiomyocyte-enriched human embryonic stem cells and improve cardiac function following myocardial injury.

OBJECTIVE: A major limitation of stem cell transfer is early donor-cell death. Here, we seek to enhance myocardial repair following injury through transplantation of cardiomyocyte-enriched human embryonic stem cells (hESC) and recipient treatment with cytoprotective (allopurinol+uricase) and anti-inflammatory (ibuprofen) agents. METHODS: We injected 10(6) (15% hESC-derived cardiomyocytes) green fluorescent protein (GFP+) hESC in the infarcted area following left anterior descending artery (LAD)-ligation in SCID-beige mice. In Group I, 1.6 mg allopurinol and 0.2 mg of uricase were injected i.p. for 3 days prior to cell transplantation. In Group II, 0.35 mg/ml of ibuprofen were added to the drinking water before and after cell implantation. In Group III, the LAD was ligated and allopurinol/uricase was administered without cell treatment. In Group IV, ibuprofen was added to the drinking water and the LAD was ligated without additional cell treatment. In Group V, only cells were transplanted. Group VI involved infarcted controls and Group VII involved sham-operated mice (all groups: n=5). We evaluated heart function (ejection fraction (EF)) by MRI (4.7 T) 3 weeks later. The hearts were harvested for histology. RESULTS: Differentiated hESC formed clusters and expressed alpha-sarcomeric actin and Connexin 43. Cell treatment improved heart function, which was best in the ibuprofen- and allopurinol-treated groups (+cell transfer), compared to the infarcted controls [EF: Group I: 76.6+/-8.6%, Group II: 78.6+/-7.3%, Group III: 58.1+/-5.7%, Group IV: 53.9+/-5.2%, Group V: 57.7+/-7.5%, Group VI: 43.5+/-4.3%, and Group VII: 66.3+/-7.8%]. We did not observe tumors in any group. CONCLUSIONS: Allopurinol/uricase and ibuprofen enhance differentiated hESC-engraftment and myocardial restoration following transplantation into the injured heart.

Acute Disease↗
PubMed2005

Transthyretin-related proteins function to facilitate the hydrolysis of 5-hydroxyisourate, the end product of the uricase reaction.

Purine catabolic pathway in Bacillus subtilis is consisted of more than 14 genes. Among these genes, pucL and pucM are required for uricase activity. While PucL is known to encode the uricase itself, the function of PucM is still unclear although this protein is also indispensable for uric acid decomposition. Here, we provide evidence that PucM, a transthyretin-related protein, functions to facilitate the hydrolysis of 5-hydroxyisourate, the end product of the uricase reaction. Based on these results, we propose that transthyretin-related proteins present in diverse organisms are not functionally related to transthyretin but actually function as a hydroxyisourate hydrolase.

Amino Acid Sequence↗
PubMed2005

The comparison of the estimation of enzyme kinetic parameters by fitting reaction curve to the integrated Michaelis-Menten rate equations of different predictor variables.

The estimation of enzyme kinetic parameters by nonlinear fitting reaction curve to the integrated Michaelis-Menten rate equation ln(S(0)/S)+(S(0)-S)/K(m)=(V(m)/K(m))xt was investigated and compared to that by fitting to (S(0)-S)/t=V(m)-K(m)x[ln(S(0)/S)/t] (Atkins GL, Nimmo IA. The reliability of Michaelis-Menten constants and maximum velocities estimated by using the integrated Michaelis-Menten equation. Biochem J 1973;135:779-84) with uricase as the model. Uricase reaction curve was simulated with random absorbance error of 0.001 at 0.075 mmol/l uric acid. Experimental reaction curve was monitored by absorbance at 293 nm. For both CV and deviation <20% by simulation, K(m) from 5 to 100 micromol/l was estimated with Eq. (1) while K(m) from 5 to 50 micromol/l was estimated with Eq. (2). The background absorbance and the error in the lag time of steady-state reaction resulted in negative K(m) with Eq. (2), but did not affect K(m) estimated with Eq. (1). Both equations gave better estimation of V(m). The computation time and the goodness of fit with Eq. (1) were 40-fold greater than those with Eq. (2). By experimentation, Eq. (1) yielded K(m) consistent with the Lineweaver-Burk plot analysis, but Eq. (2) gave many negative parameters. Apparent K(m) by Eq. (1) linearly increased, while V(m) were constant, vs. xanthine concentrations, and the inhibition constant was consistent with the Lineweaver-Burk plot analysis. These results suggested that the integrated rate equation that uses the predictor variable of reaction time was reliable for the estimation of enzyme kinetic parameters and applicable for the characterization of enzyme inhibitors.

Algorithms↗
PubMed2006

Uric acid recycling in the shield bug, Parastrachia japonensis (Hemiptera: Parastrachiidae), during diapause.

Nymphs of the univoltine shield bug, Parastrachia japonensis grow by feeding on the drupes of their sole food plant, which are available for only 2 weeks a year. The new adults soon enter a reproductive diapause and survive without feeding for at least 10 months up to 2 years. Uric acid was found to be the predominant component among four waste nitrogenous compounds, i.e., uric acid, allantoin, allantoic acid and urea in the body of both nymphs and adults in all stages, and to be predominantly excreted by the nymphs and reproductive adults. However, adults in diapause excreted negligible amounts of these compounds. Erwinia-like bacteria were found exclusively in the cecum of midgut, in which three uricolytic enzymes, i.e., uricase, allantoinase and allantoicase were detected. Ninety % of adults in diapause could survive on water for 9 months, but those given 0.02% rifampicin aqueous solution all died within this period, with significant reduction of the bacteria and uricase activity in the cecum. Rifampicin treatment resulted in a considerable reduction of free amino acids, especially proline in the hemolymph. These results suggest that uric acid is recycled as an amino acid source with the aid of Erwinia-like bacteria, and uricase functions as a key enzyme for this process.

Animals↗
PubMed2006

The crystal structure of the transthyretin-like protein from Salmonella dublin, a prokaryote 5-hydroxyisourate hydrolase.

The mechanism of binding of thyroid hormones by the transport protein transthyretin (TTR) in vertebrates is structurally well characterised. However, a homologous family of transthyretin-like proteins (TLPs) present in bacteria as well as eukaryotes do not bind thyroid hormones, instead they are postulated to perform a role in the purine degradation pathway and function as 5-hydroxyisourate hydrolases. Here we describe the 2.5 Angstroms X-ray crystal structure of the TLP from the Gram-negative bacterium Salmonella dublin, and compare and contrast its structure with vertebrate TTRs. The overall architecture of the homotetramer is conserved and, despite low sequence homology with vertebrate TTRs, structural differences within the monomer are restricted to flexible loop regions. However, sequence variation at the dimer-dimer interface has profound consequences for the ligand binding site and provides a structural rationalisation for the absence of thyroid hormone binding affinity in bacterial TLPs: the deep, negatively charged thyroxine-binding pocket that characterises vertebrate TTR contrasts with a shallow and elongated, positively charged cleft in S. dublin TLP. We have demonstrated that Sdu_TLP is a 5-hydroxyisourate hydrolase. Furthermore, using site-directed mutagenesis, we have identified three conserved residues located in this cleft that are critical to the enzyme activity. Together our data reveal that the active site of Sdu_TLP corresponds to the thyroxine binding site in TTRs.

Amidohydrolases↗
PubMed2004

A single dose of rasburicase is sufficient for the treatment of hyperuricemia in patients receiving chemotherapy.

Hydration, urinary alkalization, and allopurinol are the standard of care in the treatment and prevention of hyperuricemia. Rasburicase is a new alternative for the management of hyperuricemia in cancer patients. Criteria for the use of rasburicase were developed by the Hematology/Oncology Pharmacy and Therapeutics Subcommittee of the Detroit Medical Center and implemented in 2003. The guidelines limit rasburicase use to one dose, with additional doses as needed, compared to the five doses recommended by the manufacturer, in cancer patients with hyperuricemia and bulky tumor who require immediate chemotherapy. During the period of March to September 2003, eight patients received rasburicase, according to the guidelines, for the management of hyperuricemia. One dose of rasburicase produced a rapid and sustained therapeutic effect of lowering the plasma uric acid levels in all patients. The levels remained below 4 mg/dL throughout the administration of chemotherapy for up to 96 h. Utilizing the guidelines resulted in a significant cost savings of 100,000 US dollars.

Adult↗
PubMed2005

[Acute tumor lysis syndrome: update on therapy].

PURPOSE: The tumor lysis syndrome (TLS) is a set of complications that can arise from treatment of high burden, drug sensitive and rapidly proliferating neoplasm particularly of hematological origin. This syndrome can be observed before any treatment because of spontaneous tumoral cellular death, and is generally worsened when chemotherapy is initiated. CURRENT KNOWLEDGE AND KEY POINTS: Although TLS is primarily observed during therapy of acute leukemia, Burkitt's lymphomas and lymphoblastic lymphomas, it can also be observed in other hematological malignancies and during the treatment of rare solid tumors. Important progress has recently been made in the management of TLS. The use of urate oxydase can rapidly control TLS induced hyperuricemia, which help to prevent the risk of calcium phosphate crystal precipitation. FUTURE PROSPECTS AND PROJECTS: A global strategy for the management of SLT, combining adapted hydration, urate oxydase, and a close cooperation between intensive care units and hematology units can control this complication in most of the patients. The early management of TLS can, indeed, have an impact on the global therapy of these patients who need to be treated with high-dose anti-cancer agents with renal elimination.

Acute Disease↗
PubMed2005

Uric acid, evolution and primitive cultures.

Hypertension is epidemic and currently affects 25% of the world's population and is a major cause of stroke, congestive heart failure, and end-stage renal disease. Interestingly, there is evidence that the increased frequency of hypertension is a recent event in human history and correlates with dietary changes associated with Westernization. In this article, we review the evidence that links uric acid to the cause and epidemiology of hypertension. Specifically, we review the evidence that the mutation of uricase that occurred in the Miocene that resulted in a higher serum uric acid in humans compared with most other mammals may have occurred as a means to increase blood pressure in early hominoids in response to a low-sodium and low-purine diet. We then review the evidence that the epidemic of hypertension that evolved with Westernization was associated with an increase in the intake of red meat with a marked increase in serum uric acid levels. Indeed, gout and hyperuricemia should be considered a part of the obesity, type 2 diabetes, and hypertension epidemic that is occurring worldwide. Although other mechanisms certainly contribute to the pathogenesis of hypertension, the possibility that serum uric acid level may have a major role is suggested by these studies.

Animals↗
PubMed1992

Identification of an amino acid residue involved in the substrate-binding site of rat liver uricase by site-directed mutagenesis.

Computer analysis has shown that a conserved amino acid sequence (Leu 160 to Lys 164) of rat liver uricase is also present in other enzymes with purine substrates. The significances of the amino acids in this sequence were studied by site-directed mutagenesis. Replacement of Lys 164 by Glu or Ile resulted in loss of uricase activity and decrease in binding of the competitive inhibitor xanthine. The far ultraviolet circular dichroic spectra of the mutant uricases were identical to that of the wild type protein, indicating that the replacement of Lys 164 by other amino acids did not result in serious modification of the conformation of uricase. These findings suggest that this amino acid is involved in the substrate-binding site of the enzyme.

Amino Acid Sequence↗
PubMed1988

A human genomic sequence highly homologous to the 3'-untranslated region of rat uricase mRNA.

A human genomic sequence was isolated from a library using a rat uricase cDNA probe. Sequence analysis has shown that it is highly homologous to the 3'-untranslated region of rat uricase mRNA. Total loss of uricase activity in human is, therefore, not due to total loss of the gene. Discovery of high degree of conservation of the non-coding region of the gene would be of great interest as we attempt to learn the process of gene evolution.

Animals↗
PubMed1975

Identification of a creatine kinase inhibitor in human serum.

Human skeletal muscle creatine kinase is inhibited by a dialyzable factor in human serum. The inhibitor was fractionated from other serum components by ultrafiltration and chromatography on Sephadex G-10 and was identified as uric acid by spectral, chromatographic, and kinetic methods. Its quantitative kinetic properties are identical to those of uric acid, both as a substrate for uricase and as an inhibitor of creatine kinase. Inhibition of creatine kinase by the natural inhibitor and by known uric acid is completely reversible by addition of thiol compounds.

Chromatography, Gel↗