Immobilized Oxalate Oxidase Research Articles

Determination of Urinary Oxalate with Arylamine Glass-Bound Sorghum Oxalate Oxidase and Horseradish Peroxidase

We describe an enzymic colourimetric method for determination of oxalate level in urine using arylamine glass-bound sorghum leaf oxalate oxidase and horseradish peroxidase. The method is based on quantification of H2O2 generated from oxidation of urinary oxalate by immobilized oxalate oxidase, by a colour reaction consisting of 4-aminophnazone, phenol and immobilized peroxidase as chromogen. Minimum detection limit of the method was 0.05 mmol/l. Analytical recovery of added oxalate in urine was 96.8± 3.0% (mean ±S.D.). Within and between day coefficient of variation (CV) for urinary oxalate in urine were < 3.5% and <6.46 % respectively. The urinary oxalate values in apparently healthy and urinary stone formers as measured by the present method were correlated with those by modified Sigma Kit method (r= 0.929). The method has the advantages that it provides ca 200 times reuse of oxalate oxidase and peroxidase and free from interferences by Cl- and NO3- normally found in urine. Int J Appl Sci Biotechnol, Vol 4(3): 346-351

Chemical activation of egg shell membrane for covalent immobilization of enzymes and its evaluation as inert support in urinary oxalate determination

Egg shell membrane is a novel, robust, microporous, cost effective, easily available organic support material. In recent studies egg shell membranes were utilized as organic support for enzyme immobilization. But low conjugation yield limits its application as good support for biotechnological industries. In present study egg shell membrane was chemically treated to introduce free functional groups for covalent linkage of proteins to increase its conjugation yield and stability of conjugate complex. Many enzymes were tested for immobilization on modified egg shell membrane like oxalate oxidase, glucose oxidase, peroxidase and lipase. A fifteen to sixteen fold increase in conjugation yield was observed when immobilization was performed after chemical treatment in comparison to immobilization on native membrane with slight change in specific activity of immobilized enzyme which ranges from 5% to 15%. Egg shell membrane bound enzymes showed slight changes in their kinetic properties after immobilization. Egg shell membrane bound oxalate oxidase shows detection limit of 1.5 μM when used for urinary oxalate determination. Egg shell membrane support shows no interference to enzyme activity and a good correlation of 0.99 was observed with the values estimated using commercially available Sigma kit. The immobilized oxalate oxidase, glucose oxidase, peroxidase and lipase were stable up to duration of 180 days and there is respective loss of 10%, 13%, 24%, and 33% of initial activity. Overall result strengthens our view of using chemically modified egg shell membrane as solid support for better immobilization of enzymes and can be used in various biotechnological applications.

Preparation of biomolecule-ligated polystyrene cuvets and their applications in diagnostics

Activated polystyrene cuvet is used for the determination of oxalate in urine sample and immunoglobulin in human sera. Polystyrene cuvet was activated by introducing active functional group onto its surface by coating the surface with a heterobifunctional photolinker, 1-fluoro-2-nitro-4-azidobenzene and exposing to UV light at 365 nm. Modified cuvet was active enough to covalently link oxalate oxidase in just 45 min at 50 °C without any additional catalyst or reagent. Remarkable reduction of time was achieved when immobilization was carried out by microwave irradiation. It took only 50 s to immobilize oxalate oxidase in the activated cuvet when exposed to microwaves in a domestic microwave oven. Immobilized-oxalate oxidase was stable, reusable and conveniently used for determination of urinary oxalate for diagnosis of primary hyperoxaluria and calcium oxalate nephrolithaiasis. Detection of human IgG involving ELISA in antihuman IgG-immobilized cuvet showed the feasibility of the cuvet in other immunoassays.

Activation of polyvinyl chloride sheet surface for covalent immobilization of oxalate oxidase and its evaluation as inert support in urinary oxalate determination

Polyvinyl chloride (PVC) sheets are a promising material for enzyme immobilization owing to the PVC’s properties such as being chemically inert, corrosion free, weather resistant, tough, lightweight, and maintenance free and having a high strength-to-weight ratio. In this study, this attractive material surface was chemically modified and exploited for covalent immobilization of oxalate oxidase using glutaraldehyde as a coupling agent. The enzyme was immobilized on activated PVC surface with a conjugation yield of 360 μg/cm 2. The scanning electron micrographs showed the microstructures on the PVC sheet surface revealing the successful immobilization of oxalate oxidase. A colorimetric method was adopted in evaluating enzymatic activity of immobilized and native oxalate oxidase. The immobilized enzyme retained 65% of specific activity of free enzyme. Slight changes were observed in the optimal pH, incubation temperature, and time for maximum activity of immobilized oxalate oxidase. PVC support showed no interference when immobilized oxalate oxidase was used for estimation of oxalic acid concentration in urine samples and showed a correlation of 0.998 with the values estimated with a commercially available Sigma kit. The overall results strengthen our view that PVC sheet can be used as a solid support for immobilization of enzymes and in the field of clinical diagnostics, environmental monitoring and remediation.

Interference-free sample preparation for the determination of plasma oxalate analyzed by HPLC-ER: preliminary results from calcium oxalate stone-formers and non-stone-formers

Background: Oxalate generation at pH-values above 5.0 and an oxalate–protein binding in acidified plasma would appear to complicate the determination of oxalate in plasma. Methods: To avoid complex sample preparation we used a high-performance liquid chromatographic system with an inline enzyme reactor (HPLC-ER) containing immobilised oxalate oxidase. The detection limit was 0.68 μmol/l. Blood was drawn in lithium–heparin vessels and immediately centrifuged at 4 °C. The yielded plasma was ultrafiltered using a Centrisart-I-tube. To inhibit oxalate generation by ascorbic acid, the ultrafiltrate was acidified with 1 mol/l hydrochloric acid during ultrafiltration at 4 °C. The liquid thus yielded was used for HPLC-ER analysis. Blood samples were obtained from 133 healthy adults (63 men, 70 women, aged 20–94 years) with no history of renal disorder and from 79 patients (53 men, 26 women, aged 19–77 years) with a history of calcium oxalate stone formation. Results: Mean plasma oxalate was 2.65±2.31 μmol/l for healthy subjects and 4.21±0.56 μmol/l for stone formers. Conclusions: Analysis yielded no significant differences between males and females. A correlation between age and plasma oxalate was found for the healthy adults ( p<0.001).

Use of a Ruthenium(III), Iron(II), and Nickel(II) Hexacyanometallate-Modified Graphite Electrode with Immobilized Oxalate Oxidase for the Determination of Urinary Oxalate

This paper describes the performance of a biosensor with an Ru(III), Ni(II), and Fe(II) hexacyanometallate-modified graphite electrode and immobilized oxalate oxidase for the determination of urinary oxalate. The addition of ruthenium enhances the electrochemical reversibility and chemical stability of the electrocrystallized layer and improves the sensitivity of the biosensor. Hydrogen peroxide, produced by the enzyme-catalyzed oxidation of oxalate, was measured at -50 mV vs an Hg Hg2CI2 3M KCl electrode in a solution of pH 3.6 succinic buffer, 0.1 M KCl, and 5.4mM ethylenediaminetetraacetic acid. The linear concentration range for the determination of oxalate was 0.18-280 microM. The recoveries of added oxalate (10-35 microM) from aqueous solution ranged from 99.5 to 101.7%, whereas from urine samples without oxalate (or with a concentration of oxalate below the detection limit) the recoveries of added oxalate ranged from 91.4 to 106.6%. The oxalate in 24 h urine samples, taken during their daily routine from 35 infants and children, was measured and found to range from 0.6 to 121.7 mg/L. There were no interferences from uric acid, acetylsalicylic acid, and urea in the concentration range investigated, but paracetamol and ascorbic acid did interfere. A good correlation (R2 = 0.9242) was found between values obtained for oxalate in real urine samples by 2 laboratories, with the proposed biosensor and ion chromatography, respectively.

Discrete analysis of plasma oxalate with alkylamine glass bound sorghum oxalate oxidase and horseradish peroxidase

We have reported a simple method of determination of plasma oxalate using a Cl − and NO 3 − insensitive oxalate oxidase purified from grain sorghum leaf and commercially available peroxidase from horseradish [Pundir et al., Ind. J. Biochem. Biophys., 35 (1998) 120–122]. The present report describes the immobilization of both the enzymes onto alkylamine glass, their kinetic properties and application for discrete analysis of plasma oxalate. In the analytic method, H 2O 2 generated from plasma oxalate by immobilized oxalate oxidase is measured colorimetrically at 520 nm by oxidative coupling with 4-aminophenazone, and phenol catalyzed by immobilized peroxidase. The minimum detection limit of the method is 2.5 μmol/l. Analytic recovery of added oxalate in plasma was 89.5±4.1% (mean±S.D.). The within and between day CV for plasma oxalate measurement were <9.37 and <11.0%, respectively. The normal range of plasma oxalate as measured by the present method was 3.6 to 5.7 μmol/l. The method is not only free from interference by plasma Cl − and NO 3 − but also provides the reuse of glass beads and thus reduces the cost of analysis for routine.

Quantification of Urinary Oxalate with Alkylamine Glass Bound Amaranthus Leaf Oxalate Oxidase

ABSTRACT A method has been developed for discrete analysis of urinary oxalate employing alkylamine glass bound oxalate oxidase partially purified from mature leaves of Amaranthus spinosus. In the method H2O2 is generated from urinary oxalate by immobilized oxalate oxidase, which is measured by a colour reagent containing 4-aminophenazone, phenol and horseradish peroxidase in phosphate buffer, pH 7.0. The chromogen formed is read at 520nm. The minimum detection limit of the method is 0.9mg/L. The analytical recovery of added oxalate in urine was 98.0±2.7% (mean±SD). Within batch and between batch CV's were less than 4% and 3%, respectively, for urinary oxalate determination. The mean value of urinary oxalate measured by the present method is comparable to that obtained by the Sigma kit method (r=0.98).

Determination of urinary oxalate with oxalate oxidase and peroxidase immobilized on to glass beads.

We have reported the immobilization of barley oxalate oxidase on to alkylamine glass beads through glutaraldehyde coupling (Pundir, C. S., Satyapal and Kuchhal, N. K. (1993) Clinical Chemistry 39, 1750-1751). The present report describes the immobilization of commercially available horseradish peroxidase on to zirconia-coated arylamine glass beads through diazotization and a new method for the discrete assay of urinary oxalate using both immobilized oxalate oxidase and peroxidase. In the method, urinary oxalate is precipitated with CaCl2, redissolved in HCl and then assayed using immobilized enzymes. The oxalate in 24 h urine samples from apparently healthy male adults was measured by this method and found to be in the range of 12.2-28.0 mg with a mean of 19.8 mg. The percentage recovery of added oxalate (17.5 mg/l) was 96.7 +/- 3.4 (mean +/- SD). The mean value of urinary oxalate by our method is comparable with those obtained by the Sigma kit method. The cost of oxalate determination in 100 urine samples by the present method has been compared with that of the Sigma kit method.

An improved HPLC-enzyme-reactor method for the determination of oxalic acid in complex matrices

In this paper we present an improved method for the selective and sensitive determination of oxalate in different matrices such as urine, plasma, and food. The method uses ion chromatography for the separation of anions. To overcome problems with interfering matrix-anions, colourings, and macromolecules, we used an inline enzyme-reactor (ER) containing immobilised oxalate oxidase, which converts oxalate to hydrogen peroxide. Hydrogen peroxide was analysed with high sensitivity by amperometric detection. The determination limit for the HPLC-ER method was 1.5 μol/l, the mean recovery in urine was 102%. The evaluation in a urinary matrix achieved CV values from 2.2% to 6.7% for the within-run precision and CV values from 3.7% to 8.6% for the between-batch precision. The results of the new method were statistically equivalent to those obtained by enzymatic kits. We present first results of the HPLC-ER method, when applied to body fluids and food analysis.

Altered physicochemical characteristics of polyethylene glycol linked beet stem oxalate oxidase

Oxalate oxidase (EC 1.2.3.4), obtained from the beet stem, was covalently linked to polyethylene glycol (PEG). Compared with native enzyme, the modified oxalate oxidase exhibited decreased electrophoretic mobility, increased storage stability, higher thermal stability, and resistance to heavy metal inactivation and proteolytic digestion. The chemical modification of oxalate oxidase with PEG also brought about a marked shift in its optimal pH, from pH 4.5 to 6.5, without altering its Michaelis constant (K(m)) significantly. These acquired properties of the immobilized oxalate oxidase render it suitable for possible applications in clinical, nutritional, and medical fields. (c) 1995 John Wiley & Sons, Inc.

Flow Injection Analysis of Oxalate in Foods Using Titanium(IV)-Porphyrin Reagent

A rapid and sensitive flow injection analysis (FIA) with spectrophotometric detection by a Ti(IV)-porphyrin reagent is described for the determination of oxalate in foods using an immobilized oxalate oxidase reactor to yield hydrogen peroxide. The reagent is an aqueous solution of oxo[5, 10, 15, 20-tetra(4-pyridyl)porphyrinato]titanium(IV), TiO(tpypH4)4+, which reacts with hydrogen peroxide to form a monoperoxo complex, TiO2(tpypH4)4+, having an absorption peak at 450nm. The absorbance was proportional to the concentration of hydrogen peroxide. The flow-injection manifold comprised a two-channel system with an immobilized oxalate oxidase column. A linear relation was observed between the absorbance and the oxalate concentration ranging from 5×10-7to 2.5×10-4M (r=0.999), and 25 samples could be analyzed in one hour. The relative standard deviation for ten replicate measurements was 0.48% at 2.5×10-5M (500pmol/injection). The application of this system for the determination of oxalate in foods gave satisfactory results. A good correlation (r=0.996) was seen between the results obtained by the proposed method and by the conventional method using a commercial F-kit.

France — Comparative performance and application of immobilized oxalate oxidase sensors : In ANAL. CHIM. ACTA (299/1 (75–79) 1994) M.A. Saka Amini & J.J. Vallon of Hospital Edouard Herriot report on “Comparison of performances and analytical applications of two immobilized oxalate oxidase sensors”

France — Comparative performance and application of immobilized oxalate oxidase sensors : In ANAL. CHIM. ACTA (299/1 (75–79) 1994) M.A. Saka Amini & J.J. Vallon of Hospital Edouard Herriot report on “Comparison of performances and analytical applications of two immobilized oxalate oxidase sensors”

Comparison of performances and analytical applications of two immobilized oxalate oxidase sensors

The enzyme oxalate oxidase was immobilized on a pre-activated Nylon membrane or on a collagen membrane after activation of the carboxyl groups with the aid of an acyl-azide reaction. Oxalate was quantitated amperometrically via electrooxidation of the hydrogen peroxide generated in the reaction between oxalic acid and oxygen. The specific activities for the collagen and Nylon membranes were 0.13 and 0.74 U cm 2 , respectively. The enzyme activities for the two membranes were determined as a function of pH and ionic strength. The detection limits of oxalate were 1 × 10 −5 M for the collagen sensor and 4 × 10 −7 M for the Nylon sensor.

Amperometric determination of oxalate in plasma and urine by liquid chromatography with immobilized oxalate oxidase.

A detection system for plasma and urinary oxalate involving a postcolumn enzymatic reaction and electrochemical detection is described. Oxalate oxidase was immobilized on AF-Tresyl Toyopearl 650 gel. The immobilized oxalate oxidase was packed into a stainless-steel column (10 x 4 mm I.D.) and used on-line as an immobilized enzyme reactor (IMER). The hydrogen peroxide produced by enzymatic reaction was detected amperometrically at a platinum electrode maintained at +0.5 V vs. Ag/AgCl. The HPLC separation of oxalate was carried out using a Capcell Pak C8 column and an isocratic mobile phase containing 80 mM KH2PO4 and 5 mM tetra-n-butylmammonium phosphate as an ion-pair reagent. The IMER was active and stable in the mobile phase employed. The plot of peak height against concentration of oxalate was linear in the range 0.1-1.6 mumol/ml with a correlation coefficient of 0.9989. The detection limit was 10 nmol/ml at a signal-to-noise ratio of 3. The within-day and between-day coefficients of variation were 5.3 and 7.9%, respectively.

Enzymatic Assay of Oxalate in Urine by Flow Injection Analysis Using Immobilized Oxalate Oxidase and Chemiluminescence Detection

Abstract A flow injection analysis (FIA) method for the assay of oxalate in urine samples is described, which utilizes an incorporated column reactor of immobilized oxalate oxidase. The hydrogen peroxide generated is detected by chemiluminescence via reaction with luminol and hexacyanoferrate(III). Special empasis is placed on the role of L-ascorbic acid, which has been claimed to constitute the most serious interference in these types of assays. The possible elimination af this constituent by addition of sodium nitrite, as previously used in batch procedures, did not improve the selectivity of the approach. The FIA approach described, which has a detection limit (3[sgrave]) of 34 μM in undiluted urine samples and allows a sampling frequency of ca. 55 s/h, is shown to offer itself as convenient and rapid means for screening purposes.

Flow-injection amperometric determination of oxalate with immobilized oxalate oxidase

Oxalate is immobilized on controlled-pore glass and is used on-line in a glass minicolumn (2.5×25 mm). The hydrogen peroxide formed is detected amperometrically. Oxalate (6×10 −6−9×10 −4 M) is determined in a flowing stream of pH 3.5 citrate (or succinate) buffer. As little as 20 ng (in 40 μl; 5.7×10 −6 M) of oxalate can be detected. Copper inhibition can be removed either by adding EDTA to the carrier stream or incorporating a chelating-resin minicolumn into the flow system prior to the enzyme column.

Procedure for the measurement of calcium oxalate and phosphate crystals in urine.

A method is described for the chemical measurement of calcium oxalate and phosphate crystal formation in urine. The crystals were centrifuged, washed and the oxalate measured by an immobilised oxalate oxidase technique and the phosphate by a standard centrifugal analyser procedure. The methods proved precise and recoveries were good. Calcium oxalate crystal formation after evaporation is time-dependent and this parameter must therefore be standardised. Values for normal urinary calcium oxalate crystal concentration after concentration are given.

Removal of ascorbate from urine prior to assaying with a commercial oxalate kit

There is an increasing awareness of the importance of urinary oxalate in the pathogenesis of urinary calcium stones. Reliable estimates of urinary oxalate is therefore of prime importance. A commercial kit [l] for the rapid enzymatic determination of oxalate has been introduced and is used by many laboratories. Recently, various laboratories have reported the conversion of ascorbate to oxalate in urine during analysis and post-collection periods [2-61. It has been shown previously [2,3] that ascorbate can be converted to oxalate during the alkaline elution in the sample preparation step used in the commercial (Sigma) kit [l]. The use of acidic ferric chloride was advocated to overcome this problem. A ‘sodium nitrite’ method is used to remove ascorbate in urine prior to assaying for oxalate in an automated enzymatic (immobilised oxalate oxidase) assay [7] used routinely in these laboratories. In this study we have compared the removal of ascorbate in urine with acidic ferric chloride with the sodium nitrite method prior to assaying for oxalate by the Sigma kit.

Degradation of oxalate in rats implanted with immobilized oxalate oxidase

Accumulation of oxalate leads to hyperoxaluria and calcium oxalate nephrolithiasis in man. Since oxalate is a metabolic end product in mammals, the feasibility of its enzymic degradation has been tested in vivo in rats by administering exogenous oxalate oxidase. Oxalate oxidase, isolated from banana fruit peels, in its native form was found to be non-active at the physiological pH of the recipient animal. However, its functional viability in the recipient animal was ensured by its prior binding with ethylenemaleic anhydride, thus shifting its pH activity curve towards the alkaline range. Rats implanted with dialysis membrane capsules containing such immobilized oxalate oxidase in their peritoneal cavities effectively metabolized intraperitoneally injected [ 14C]oxalate as well as its precursor [ 14C]glyoxalate. The implantation of capsules containing coentrapped multienzyme preparations of oxalate oxidase, catalase and peroxidase led to a further degradation of administered [ 14C]oxalate in rats.