Determination Of Urinary Oxalate Research Articles

Comparative study of oxalate oxidase in three genotypes ofSorghum vulgare

The presence of an oxalate oxidase (EC 1.2.3.4) has been demonstrated in 15,000×g supernatants prepared from 10-day-old seedlings of three genotypes ofSorghum vulgare: grain sorghum hybrid (CSH-5), grain-cum-forage sorghum (PC-6) and forage sorghum (PC-1). The specific activity of the enzyme in the different tissues of seedlings was found to be present in the order leaves > stems > roots in PC-6 and PC-1, but this order was reversed in CSH-5. A comparison of the different properties of the leaf enzyme of these three genotypes of sorghum revealed that the enzyme has maximum activity in the acidic pH range from 4.0 to 5.0 and in the temperature range from 37°C to 40°C. The enzyme was stimulated by Cu2+ and Fe2+. The rate of H2O2 formation in the enzyme reaction was linear up to 5 min and was stoichiometrically related to oxalate consumption. The enzyme is unaffected by Na+ at physiological concentration (0.15 M). The superiority of this enzyme over moss and other plant enzymes for enzymic determination of urinary oxalate is discussed.

Kinetic-enzymatic determination of oxalate in urine by flow-injection analysis with double stopped flow

An enzymatic stopped-flow injection method for the determination of oxalate is proposed. Two sequential haltings of the flow are made, the first while the sample plug is in a reactor packed with controlled-pore glass and immobilized oxalate decarboxylase, and the second on merging of the sample plug with a stream containing an auxiliary enzyme (formate dehydrogenase) as the plug reaches the flow cell, where absorbance-time data are collected. The linear range of the calibration graphs is between 0.1 and 3.0 mM oxalate and their sensitivity depends on the interval over which measurements are made. The proposed method was applied to the determination of oxalate in urine without separation of the analyte by precipitation, with excellent results.

Flow injection determination of oxalate in urine based on an inhibitory effect

Two methods based on the use of the normal and stopped-flow injection modes were developed for the determination of oxalate based on its inhibitory effect on the catalytic action of Fe(III) on the 2,4-diaminophenol/hydrogen peroxide system. The linear determination ranges achieved were between 0.2 and 12.0 μg ml−1 and between 0.2 and 40.0 μg ml−1, the precision was ±5.4%, and ±3.5%, and the sampling rate was 30 and 20 samples h−1 for the normal and stopped-flow method, respectively. Both methods have been applied to the determination of oxalate in urine with excellent results.

Le thiocyanate serait un inhibiteur puissant de l'oxalate oxydase dans le dosage de l'oxalate urinaire

( Urinary thiocyanate appears to be a powerful inhibitor of oxalate oxidase.) The application of an oxalate oxidase electrode to urinary oxalate determination allowed us to observe, after repeated use, an irreversible inhibition of the immobilized enzyme. All known urinary inhibitors previously described do not show such an irreversible phenomenon. Attempts to separate oxalate from the inhibitor by a Sephadex G-10 column were unsuccessful. The inhibitor could be the thiocyanate ion, which is always present in urine as a metabolite of cyanide. Thiocyanate isolation from oxalate is possible by using an anionic-exchange column.

Atomic absorption and UV-VIS absorption spectrophotometric determination of oxalate in urine by ligand exchange extraction

A new method for oxalate determination in urine is described. It neither requires precipitation nor previous extraction of oxalate and is free from the interferences often found with analyes of urine samples. The method is not time consuming and is suited to routine work.

Preventing ascorbate interference in ion-chromatographic determinations of urinary oxalate: four methods compared

In an attempt to decrease ascorbate interference on the ion-chromatographic determination of urinary oxalate, we compared the effectiveness of four different methods for ascorbate elimination by analyzing a representative urine pool supplemented with successive ascorbate additions. Two of the methods--treatment with ferric ions or boric acid--have been described elsewhere; treatments with nitrites or ascorbate oxidase (EC 1.10.3.3) are investigated here as possible alternatives. Consideration of the main features, advantages, and drawbacks of the four procedures leads us to conclude that boric acid dilution is a good routine method and that pre-incubation with ascorbate oxidase reliably prevents ascorbate interference in assays of urinary oxalate.

Modifications to commercial oxalate oxidase based determination of urinary oxalate: A method suitable for routine clinical analysis

A commercially available assay (Sigma) for urinary oxalates based on the oxalate oxidase/peroxidase catalyzed reaction produced, in our hands, recoveries of added oxalate that varied from patient to patient. A modification of the assay is reported that involves the chromatographic separation of interfering substances, such as ascorbate and uric acid, from oxalate prior to analysis. With the modification recoveries of added oxalate were essentially complete and showed less patient-to-patient variability. Correlation with an alternate procedure was greatly improved.

Determination of urinary oxalate by high-performance liquid chromatography monitoring with an ultraviolet detector

High-performance liquid chromatography (HPLC) monitoring with an ultraviolet detector was carried out to measure urinary oxalate levels in urolithiasis. Interfering substances in urine were removed by anion exchange prior to chromatography. This procedure was found excellent with respect to sensitivity, reproducibility, and analytical recovery. The findings were in agreement with colorimetric date. The mean oxalate level in 24-hour urine was 30.5 +/- 15.1 mg in patients with a single episode and 36.3 +/- 9.8 mg in recurrent stone formers. The latter values was significantly higher than the normal control level (27.4 +/- 3.8 mg).

Use of photochemical reactions in flow injection: determination of oxalate in urine

The use of photochemical reactions in flow injection (FI) is reported. The irradiation of an FI reactor with a suitable source facilitates the development of the iron(III)-oxalate reaction, allowing the amperometric determination of the anion in the range 1.0-13.0 micrograms ml-1, with a relative standard deviation of 1.1% and a sampling frequency of 40 h-1. The proposed method was applied successfully to the determination of oxalate in urine samples.

Rapid determination of urinary oxalate by high-performance liquid chromatography

A. MILLANDepartment 01 Chemistry, Faculty 01 Sciences, Universidad de las Islas Baleares,07071 Palma de Mallorca (Spain)J.M. GRASESLaboratoria Agrario, Generalitat de Catalunya (D.A.R.P.), Cabrils, Barcelona (Spain)andF. GRASES*Department 01 Chemistry, Faculty 01 Sciences, Universidad de las Islas Baleares,07071 Palma de Mallorca (Spain)(First received November 14th, 1989; revised manuscript received March 12th, 1990)

Rapid enzymatic determination of urinary oxalate.

This new reagent kit for the quantitative measurement of oxalate in urine is a modification of an earlier Sigma oxalate assay procedure (procedure no. 590), a coupled enzyme assay involving oxalate oxidase and horseradish peroxidase. The new analytical procedure includes methods for processing urine specimens to eliminate interference with oxalate color development at 590 nm by ascorbic acid, divalent cations, and other urinary constituents. The reaction is complete in less than 5 min, and results are linearly related to oxalate concentration up to at least 1 mmol/L. Assay sensitivity and within-run and between-run precision were within the limits acceptable for other urinary oxalate procedures. Analytical recovery of added oxalate was close to 100%. This specific, simple, rapid procedure is suitable for routine clinical use.

Determination of Urinary Oxalate With Disposable Oxalate Oxidase Hembrane Strips

Abstract We describe in this paper a simple and easy method of entrapping oxalate oxidase and peroxidase in acrylamide membrane and demonstrate the use of such enzyme membrane strips in the rapid determination of urinary oxalate. A crude preparation of 45-60% acetone cut obtained from banana fruit peel (Musa paradisiaca; French plantain) homogenate serves as a source of oxalate oxidase, which decomposes oxalate into CO2 and H2O2. the oxalate content of a given urine sample is determined by introducing a small enzyme membrane strip (1 × 1 cm) into an aliquot of buffered urine containing a suitable chromogen for peroxidase and then measuring the colour developed due to the interaction of peroxidase with the newly formed H2O2 and the chromogen. Urine samples are pretreated with sodium nitrite to eliminate the interference of ascorbic acid in the assay. the enzyme membrane assay compares well with that of wet enzyme assay of oxalate in sensitivity and reliability.

Extraction--spectrophotometric determination of oxalate in urine and blood serum.

An extraction--spectrophotometric method is described for the determination of oxalate, based on the formation of a mixed ligand vanadium (V)--mandelohydroxamic acid--oxalate complex. The complex was extracted into a solution of trioctylmethylammonium chloride (Adogen 446) in toluene and the absorbance measured at 535 nm. The experimental variables and interferences in this determination were studied. The detection limit is 0.5 microgram ml-1 and the range of application is between 2 and 8 micrograms ml-1. The method was applied to the determination of oxalate in urine and blood serum.

An inexpensive method for sensitive enzymatic determination of oxalate in urine and plasma.

In this simple, sensitive, and rapid enzymatic method for the determination of oxalate in urine or plasma, oxalate oxidase (EC 1.2.3.4) prepared from barley seedlings is used to convert oxalate to carbon dioxide and hydrogen peroxide, which is determined photometrically at 600 nm, with use of horseradish peroxidase, by oxidative coupling of 3-methyl-2-benzothiazoline hydrazine with N,N-dimethylaniline. Plasma is pre-treated by ultrafiltration and co-precipitation of oxalate with calcium sulfate and ethanol, urine by dilution and reversed-phase chromatography on C18 columns. Analytical recovery for urine is 99 +/- 2%, for plasma 92 +/- 3%. The normal range for urinary excretion is 0.10 to 0.56 mmol/24 h, and for the concentration in plasma 0.4 to 3.7 mumol/L. There were no significant sex-related differences in urinary excretion or plasma concentration. Our within- and between-assay coefficients of variation were, respectively, less than 3.4% and less than 6.0% for urine, and less than 1.5% and less than 4.3% for plasma.

Determination of oxalic acid in urine: A review

Available methods for determination of oxalate in urine are reviewed and classified into seven groups, namely: (1) direct precipitation; (2) solvent liquid-liquid extraction; (3) isotope dilution; (4) chromatographic (gas and HPLC); (5) enzymatic (involving enzymes dissolved or immobilized on reactors or electrodes); (6) flow methods (segmented and non-segmented); (7) other methods. In every case the advantages and disadvantages of the different methods are discussed.

Approaches to the liquid chromatographic determination of carboxylic acids using potentiometric detection with a metallic copper electrode

A metallic copper electrode is evaluated as a potentiometric detector for carboxylic acids. The application of this device to ion-exchange chromatography is illustrated by the determination of oxalate in urine. Oxalate was selectively detected in the presence of a 100-fold excess of sulphate after separation on a low-capacity methacrylate anion-exchange column using 0.7mM potassium hydrogen phthalate at pH 7.1 as eluent. Calibration plots were linear up to 50ppm of oxalate. Potentiometric detection has also been applied to ion-exclusion chromatography using 0.005% phosphoric acid as eluent. With this method detection limits of 0.2, 2.1, 5.0 and 5.3µg were obtained for formic, acetic, propionic and iso-butyric acids, respectively.

Critical evaluation of a commercial enzyme kit (Sigma) for determining oxalate concentrations in urine.

The Sigma reagent kit for urinary oxalate determination is reportedly simple, rapid, and specific for oxalate. We evaluated the kit and identified a number of shortcomings. Our investigations suggest that the recommended time for chromophore development is too short and should be doubled. Oxalate recovery during the extraction procedure depends strongly on urine pH. For complete extraction, urine should be acidified to pH 1.8-2.4. We also observed positive interference from ascorbate in urine. This interference was substantial, absorbances produced from ascorbate standards being approximately 80% of those obtained from oxalate standards of similar concentration. Our investigations also indicate the presence of a substance on the Sigma adsorbent that is eluted during the extraction procedure and interferes in the color reaction. These interferences represent potentially major sources of imprecision in the assay.

Obviating interferences in the assay of urinary oxalate.

Evaluation of the Sigma Kit for determination of urinary oxalate highlighted two major limitations of which users must be aware: the pH of urine samples before extraction is critical, and ascorbic acid interferes positively. I give details, and describe simple modifications to the manufacturer's protocol that overcome these problems.

Determination of oxalate in urine by flow injection analysis

A method is described for the determination of oxalate in urine using flow injection analysis and fluorimetry. Oxalate is precipitated with calcium chloride at pH 4.5, redissolved in H 2SO 4 and measured by flow injection analysis. The minimum detection limit is 6 μmol/l. The coefficient of variation is 7%. Results are in good accordance with normal values found with traditional oxalate analysis.

Immobilized enzyme electrode for the determination of oxalate in urine.

Immobilized enzyme electrode for the determination of oxalate in urine.