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Uric acid estimation: a comparison of the manual uricase-UV and the phosphotungstate auto-analyzer methods.

1. Uric acid estimations by the uricase-UV and the autoanalyzer colorimetric procedures were compared. 2. At all levels of serum uric acid concentration measured, results with the uricase-UV method were significantly lower than those with the phosphotungstate method. 3. The data indicate that the critical uric acid level (measured by the autoanalyzer) at which allopurinol, uricosuric, and other agents are instituted should be decreased approximately 10 per cent when determinations are done by the uricase method.

Autoanalysis↗

A precise semi-automated uricase method for determining uric acid in serum or urine.

1. A semi-automated uricase method is described for measuring uric acid in serum or urine directly at 292 nm at reduced cost. 2. The coefficient of variation within and between batch assays, is estimated to be 2.5% and 4.3% respectively, which is less than the estimated daily intrapersonal variation in uric acid levels. 3. Recovery of uric acid added to samples ranges from 98.5% to 101.2% with a mean of 99.6%. 4. The cost of reagents per sample determination is estimated at $0.05, and in batch analyses the mean time required per sample determination is 2-3 minutes.

Analysis of Variance↗

Inhibition of uricase by pyrimidine and purine drugs.

A number of barbiturates inhibit uricase from porcine liver competitively, the Ki, values being in the range 1 to 13 X 10(-3) mol/L. In general the Ki increases as the size of the substituents at C-5 increases. Theophylline also inhibits the enzyme competitively (Ki = 1 X 10(-3) mol/L) but theobromine and caffeine have no inhibitory action at 0.01 mol/L. A number of sulphonamides and some other heterocyclic compounds, at 10(-3) mol/L, do not inhibit uricase. The possible effects of such inhibition in clinical assays for plasma/serum urate are discussed but it is concluded that the compounds which were studied are unlikely to cause significant interference in the assay.

Animals↗

Definitive characterization of uric acid as an interferent in peroxidase indicator reactions and a proposed mechanism of action.

We have characterized and identified uric acid as an interferent to peroxidase catalyzed reactions where hydrogen peroxide is generated at relatively low concentrations. The implications of these findings are important for those utilizing peroxidase as an indicator reaction where low primary substrate concentrations require their preliminary extraction or chemical modification. We have shown that the elimination of uric acid as an interferent from biologic fluid obviates the necessity for such treatment. In amniotic fluid, our data suggests that uric acid represents the only interference to peroxidase-catalyzed reactions, especially when using p-substituted phenols as proton donors. The removal of uric acid has been shown to eliminate hydrogen peroxide reduction and should allow for an increase in sensitivity and specificity for measurements incorporating a peroxidase-coupled indicator reaction, hence, more effective use of these reaction sequences. To our knowledge, this is the first report of a mechanism to eliminate hydrogen peroxide reduction in amniotic fluid.

Amniotic Fluid↗

Therapeutic proteins: a comparison of chemical and biological properties of uricase conjugated to linear or branched poly(ethylene glycol) and poly(N-acryloylmorpholine).

Uricase from Bacillus fastidiosus (UC) was covalently linked to linear PEG (PEG-1) (Mw 5 kDa), branched PEG (PEG-2) (Mw 10 kDa) and to poly(N-acryloylmorpholine) (PAcM) (Mw 6 kDa). The conjugation of UC with linear PEG and PAcM was accompanied by complete loss of enzymatic activity but, if uric acid as site protecting agent was included in the reaction mixture, the conjugate protein retained enzymatic activity. On the other hand, the modification with PEG-2 gave a conjugate that also maintained enzymatic activity in the absence of any active site protection. This behaviour must be related to hindrance of the branched polymer in reaching the enzyme active site. The UC conjugates exhibited increased resistance to proteolytic digestion while minor variations in the inhibitory constant, optimal pH, heat stability, affinity for substrate, were observed. Pharmacokinetic investigations in mice demonstrated increased residence time in blood for all the conjugates as compared with native uricase. Uricase conjugated with linear PEG was longer lasting in blood UC derivative, followed by branched PEG and the PAcM conjugates. Unconjugated uricase was rapidly removed from circulation. All these data are in favour of the use of the less known amphiphilic polymer PAcM as an alternative to PEGs in modification of enzymes devised for therapeutic applications.

Acrylic Resins↗

Use of UV scanning techniques in the identification of serum constituents separated by high-performance liquid chromatography.

Serum constituents separated by the reversed-phase partition mode of high-performance liquid chromatography were identified by using a stopped-flow UV scanning technique. This method of peak identification eliminates post-chromatographic sample handling. Results from UV scanning were correlated with those obtained from absorbance ratios of reference compounds and the enzymatic peak-shift technique.

Chromatography, High Pressure Liquid↗

The effect of decreasing the concentration of urinary urate on the crystallization of calcium oxalate in undiluted human urine.

The effect of lowering the urate concentration, using uricase immobilized on to nylon tubing, on the nucleation, growth and aggregation of calcium oxalate crystals in undiluted human urine was examined. The median urate concentration was significantly (p less than 0.01) reduced from 2.8 to 0.55 mmol/l., but this had no reproducible effect on the metastable limits, the volume of crystalline calcium oxalate deposited and the size of the crystals and aggregates produced from the 10 urine samples examined. It was concluded that further studies aimed at testing the effects of a raised urinary urate concentration on the crystallization of calcium oxalate should be made a matter of high priority.

Calcium Oxalate↗

Immunogenicity and antigenicity of soluble cross-linked enzyme/albumin polymers: Advantages for enzyme therapy.

Soluble cross-linked polymers of hog-liver uricase and an excess of either rabbit or dog albumin were injected repeatedly into rabbits in order to determine their antigenicity and immunogenicity. Whereas the enzyme in its free form induces antibody production, homologous albumin polymerised with the enzyme renders the complex non-immunogenic and non-antigenic. Dog-albumin/uricase polymers injected into rabbits induce antibody formation against the dog albumin but not against the uricase. The results suggest an important advantage of these soluble enzyme polymers in enzyme-replacement therapy.

Albumins↗

Discrete analysis of serum uric acid with immobilized uricase and peroxidase.

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 H2O2 from serum uric acid by immobilized uricase and its measurement by a colour reaction catalyzed by immobilized peroxidase. The minimum detection limit of the method was 8 microg/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 microg/ml, 32+/-2.25 (range, mean+/-S.D.) and 55-200 microg/ml; 52+/-6.4 (range, mean+/-S.D.) respectively by our method. A good correlation (r = 0.8170) was obtained between the serum urate values by this method and with those obtained by commercial Enzo-kit method.

Colorimetry↗

Determination of uric acid in serum using isotachophoresis.

An operational system is described for the isotachophoretic determination of uric acid in serum, making use of column coupling. The method has been compared with a standard enzymatic procedure. With the present technique small amounts of serum (ca. 3 microliter) can be applied without any pretreatment. Urate recovery was 99.0-100.5%. Under the non-physiological measuring conditions used, 12-28% of control serum uric acid was bound to macromolecules of molecular weight exceeding 25,000. The day-to-day variations of the isotachophoretic procedure were smaller than those of the enzymatic method, whereas standard deviations were comparable. The isotachophoretic procedure is less influenced by certain metabolites.

Adult↗

A purification method of the diagnostic enzyme Bacillus uricase using magnetic beads and non-specific protease.

A simple purification method of the Bacillus uricase (Uao) was newly developed. The gene coding for Uao with a C-terminal 6-histidine tag (Uao-HT) was constructed and overexpressed. Using the non-specific proteases, such as proteinase K, the tag was easily removed because Uao-HT includes its C-terminal region to be specifically cleaved by them. Such treatment of Uao-HT with the proteases did not affect its enzymatic properties and enabled us to purify it from the crude extract with a single-step protocol; the cell lysate containing Uao-HT was mixed with the Ni ion-chelating magnetic beads and then the adsorbed enzyme was eluted with the proteinase K-containing buffer after untagged proteins were washed out. The isolated enzyme yielded a single band on SDS-PAGE and was fully active. This method is extremely useful for high-throughput purification of mutants because of compatibility with automation.

Amino Acid Sequence↗