In vivo oxalate degradation by liposome encapsulated oxalate oxidase in rat model of hyperoxaluria.

Background & objectives: High level of urinary oxalate substantially increases the risk of hyperoxaluria, a significant risk factor for urolithiasis. The primary goal of this study was to reduce urinary oxalate excretion employing liposome encapsulated oxalate oxidase in animal model. Methods: A membrane bound oxalate oxidase was purified from Bougainvillea leaves. The enzyme in its native form was less effective at the physiological pH of the recipient animal. To increase its functional viability, the enzyme was immobilized on to ethylene maleic anhydride (EMA). Rats were injected with liposome encapsulated EMA- oxalate oxidase and the effect was observed on degradation of oxalic acid. Results: The enzyme was purified to apparent homogeneity with 60-fold purification and 31 per cent yield. The optimum pH of EMA-derivative enzyme was 6.0 and it showed 70 per cent of its optimal activity at pH 7.0. The EMA-bound enzyme encapsulated into liposome showed greater oxalate degradation in 15 per cent casein vitamin B6 deficient fed rats as compared with 30 per cent casein vitamin B6 deficient fed rats and control rats. Interpretation & conclusions: EMA-oxalate oxidase encapsulated liposome caused oxalate degradation in experimental hyperoxaluria indicating that the enzyme could be used as a therapeutic agent in hyperoxaluria leading to urinary stones.

An amperometric cholesterol biosensor based on epoxy resin membrane bound cholesterol oxidase.

Background & objectives: The use of epoxy resin membrane as a support for immobilization of enzyme has resulted into improved sensitivity and stability of biosensors for uric acid, ascorbic acid and polyphenols. The present work was aimed to prepare an improved amperometric biosensor for determination of serum cholesterol required in the diagnostics and management of certain pathological conditions. Methods: Epoxy resin membrane with immobilized cholesterol oxidase was mounted on the cleaned platinum (Pt) electrode with a parafilm to construct a working electrode. This working electrode along with Ag/AgCl as reference and Ag wire as an auxiliary electrode were connected through a three terminal electrometer to construct a cholesterol biosensor. Results: The sensor showed optimum response within 25 sec at pH 7.0 and 45°C. The linear working range of biosensor was 1.0 to 8.0 mM cholesterol. Km and Imax for cholesterol were 5.0 mM and 9.09 μA, respectively. The biosensor measured serum cholesterol. The minimum detection limit of the sensor was 1.0 mM. The mean analytical recoveries of added cholesterol in serum (2.84 and 4.13 mM) were 91.4±2.8 and 92.3±3.1 per cent (n=6), respectively. Within and between assay coefficient of variation (CV) were <2 and <4 per cent, respectively. Biosensor had a storage life of 6 months at 4°C. Interpretation & conclusions: The use of epoxy resin membrane as a support for immobilization of cholesterol oxidase has resulted into an improved amperometric cholesterol biosensor. The present biosensor had an advantage over the existing biosensors as it worked at comparatively lower potential.

Purification and partial characterization of oxalate oxidase from leaves of forage Sorghum (Sorghum vulgare var. KH-105) seedlings.

An oxalate oxidase was purified to apparent homogeneity from the leaves of 10-days old seedlings of forage Sorghum (Sorghum vulgare var. KH-105). The enzyme had a Mr of 124 kDa with two identical subunits, an optimum pH of 4.5, optimum temperature of 37°C and activation energy (Ea) of 2.0338 Kcal/mol. The rate of reaction was linear up to 7 min. Km value for oxalate was 0.22 mM. The enzyme was stimulated by Cu2+ and inhibited by EDTA, NaCN, diethyldithiocarbamate, na2SO4, but unaffected by NaCl at 0.1 mM concentration. Although the enzyme was stimulated by flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), UV and visible spectra of the enzyme did not match with that of a flavoprotein. The positive reaction of the enzyme with orcinol-H2SO4 reagent indicated its glycoprotein nature. The superiority of the purified enzyme over earlier reported oxalate oxidases for determination of urinary oxalate has been demonstrated.

Urinary & serum oxalate determination by oxalate oxidase immobilized on to affixed arylamine glass beads.

BACKGROUND & OBJECTIVE: Although the measurement of oxalate in urine and serum by Amaranthus leaf oxalate oxidase immobilized on free arylamine glass beads is highly sensitive and specific, the handling of glass beads is tedious and cumbersome. The present study was undertaken to overcome this problem. METHODS: Partially purified Amaranthus spinosus leaf oxalate oxidase was immobilized through diazotization onto arylamine glass beads affixed on the surface of a plastic strip by a non reactive fixative and employed for oxalate determination in urine and serum samples collected from healthy individuals and urinary stone formers. RESULTS: The immobilized enzyme retained 56 per cent of its initial activity with a conjugation yield of 40 mg/g support. The strip bound enzyme showed maximum activity at pH 3.5 when incubated at 40 degrees C for 15 min. The minimum detection limit of the method was 0.01 mM/l in the urine and 2.5 microM/l in the serum. The analytical recovery of added oxalate was 97.7+/-1.2 per cent in urine and 92.0+/-2.4 per cent in serum. Within and between assay coefficient of variation (CV) were 4.6 and 5.2 per cent in urine and 7.4 and 5.8 per cent in serum respectively. A good correlation for oxalate in urine (r1= 0.99) and in serum (r2= 0.92) was obtained between Sigma kit method and the present method. The strip could be reused 150 times over a period of 2 months, when stored at 4 degrees C in reaction buffer. INTERPRETATION & CONCLUSION: Immobilization of Amaranthus leaf oxalate oxidase on to affixed glass beads provided enormous ease in its reuse for determination of oxalate in urinary and serum samples.

Discrete analysis of bile acid in serum and bile with 3 alpha-hydroxysteroid dehydrogenase and diaphorase immobilized onto alkylamine glass beads.

3alpha-Hydroxysteroid dehydrogenase (3alpha-HSD) from Pseudomonas testosteronei and diaphorase (lipoyl dehydrogenase) from Clostridium spp were immobilized individually onto alkylamine glass beads through glutaraldehyde coupling. A cost-effective enzymic colorimetric method for determination of bile acid in the serum and bile was developed employing mixture of the immobilized enzymes. The method was based upon measurement of NADH generated from NAD+ during oxidation of bile acid by immobilized 3alpha-HSD with a color reagent consisting of nitrobluetetrazolium (NBT) chloride salt and immobilized diaphorase in 0.065 M sodium phosphate buffer (pH 7.0). The minimum detection limit of the method was 4.8 pmol/L in the serum and 19.5 micromol/L in bile. The per cent recovery of added bile acid in the serum and bile was 89.1 and 95.0, respectively. Within and between batch coefficients of variation (CV) for bile acid determination were <1.0% and <0.2% in the serum and <0.2% and <0.6% in bile, respectively. A good correlation for bile acid in the serum (r1= 0.95) and in bile (r2 = 0.93) was obtained by a standard chemical method (a commonly used method in India) and the present method. The mixture of immobilized 3alpha-HSD and diaphorase lost 30% of its initial activity after 4 months of regular use. The cost of bile acid determination for 100 the serum and bile samples by the present method was found to be lower than by a commercially available method (Sigma kit 450-A).

Determination of serum glucose using co-immobilized glucose oxidase and peroxidase onto arylamine glass beads affixed on a plastic strip.

Glucose oxidase (GOD) from Aspergillus niger and horseradish peroxidase (POD) were co-immobilized onto arylamine glass beads affixed on a plastic strip with a conjugation yield of 28.2 mg/g and 43% retention of their initial specific activity. The coimmobilized enzymes showed maximum activity at pH 7.5 when incubated at 37 degrees C for 15 min. A simple, specific and sensitive method for discrete analysis of the serum glucose was developed employing this strip. The minimum detection limit of the method was 5 mg/dl. Within and between assay coefficient of variations for the serum were <5.6% and <10.6% (n = 6) respondely. A good correlation (r = 0.943) was found between the glucose values obtained by the enzyme colorimetric method employing free GOD and POD and the present method. The strip lost 50% of its initial activity after its 150 regular uses for a period of one month, when stored in reaction buffer at 4 degrees C. The method is cost-effective than the enzymic colorimetric method, as the enzyme strip is reusable.

Correlation between chemical composition of biliary calculi and sera of stone formers.

A quantitative chemical analysis of total cholesterol, bilirubin, calcium, inorganic phosphate and iron of three types of biliary calculi (cholesterol, pigment and mixed) of 40 gall stone former was carried out and correlated to with those of there sera. A moderately positive correlation for inorganic phosphate, Ca2+ and Fe2+ content of sera and calculi of cholesterol stone patient was found. A Good positive correlation for total cholesterol, a moderately positive correlation for bilirubin and iron but no correlation for inorganic phosphate and calcium content of sera and calculi of pigment stone patient was observed. A good moderately positive correlation for iron but no correlation for total cholesterol, bilirubin, inorganic phosphate and Ca2+ content of sera and calculi of stone patient was found.

Immobilization of Amaranthus leaf oxalate oxidase on alkylamine glass.

A membrane bound oxalate oxidase from leaves of Amaranthus spionsus has been partially purified and immobilized on alkylamine glass with a yield of 9.2 mg protein/g support. The enzyme retained 99.4% of initial activity of free enzyme after immobilization. There was no change in the optimum pH (3.5) and Vmax but the temperature for maximum activity was slightly decreased (35 degrees C) and energy of activation (Ea) and Km for oxalate were increased after immobilization. The immobilized enzyme preparation was stable for 6 months, when stored in distilled water at 4 degrees C. Presence of Cl- did not affect the activity of immobilized enzyme.

Determination of plasma oxalate with chloride ion insensitive oxalate oxidase.

A simple colorimetric method has been developed for determination of oxalate in plasma using a C1-insensitive oxalate oxidase purified from grain sorghum leaves. The ultrafiltered plasma collected in 3.5 N HC1 was pretreated with NaNO2 to avoid possible interference by ascorbate. The minimum detection limit of the method is 0.225 mg/l. The % recovery of the added oxalate was 91.5 +/- 5.0 (mean +/- S.D., n = 15). The coefficient of variation within and between batch were < 3 and < 5 respectively. The mean plasma oxalate concentration in healthy subjects was 0.29 mg/l. The method has the advantage over other enzymic methods that it doesn't require the removal of C1- prior to oxalate assay.

Chemical analysis of urinary calculi in Haryana.

Quantitative chemical analysis of 225 urinary calculi (128 Renal, 57 bladder and 40 ureter) collected from hospitals of different districts of Haryana was carried out. CaOxM was found to be present as a major component in all the stones analysed, while MAP, HA, CA and UA were found in 83.7%, 83.6%, 13% and 78.9% cases, respectively. The content of CaOxM was found to be higher in renal stones as compared to ureter and bladder stones, while the concentration of MAP was higher in bladder stones as compared to kidney and ureter stones. HA content was higher in ureter and UA was higher in bladder stones as compared to other urinary stones.

Barley oxalate oxidase immobilized on zirconia-coated alkylamine glass using glutaraldehyde.

A method for immobilizing barley oxalate oxidase to zirconia coated alkylamine glass through the process of glutaraldehyde coupling has been described. The immobilized enzyme retained 97.2% of the specific activity, with a conjugation yield of 6.63 mg/g support and showed an increase in optimum pH. The Km value of immobilized enzyme was unaltered but Vmax was decreased compared to free enzyme. The conjugated enzyme was stable at 4 degrees C for 2 years. A number of inorganic ions and metabolic substances did not denature the immobilized enzyme. The clinical importance of this work is demonstrated.

Isolation, purification, immobilization of oxalate oxidase and its clinical applications.

Oxalate oxidase known to catalyse the aerobic oxidation of oxalic acid into CO2 and H2O2, has been found in bacteria, fungi, mosses and some higher plants. So far, a membrane bound oxalate oxidase from Pseudomonas sp. OX-53 and a soluble oxalate oxidase from seedling plants of barley and grain sorghum has been purified to homogeneity by conventional purification methods. The enzyme has been immobilized onto insoluble support such as nylon tubing, zirconia coated alkylamine glass, polyamide membrane, CO2 gas sensing electrode, H2O2 sensor probe and polyanionic electrolyte such as ethylaminemaleic anhydride (EMA). Compared to free enzyme the immobilized enzyme showed an increase in optimum pH, decrease in Vmax and time for maximum activity, higher resistance to inhibition by NaCl but no change in Km value. The immobilized enzyme has been used in both continuous flow system and discrete assays and in enzyme electrode for determination of oxalate in urine, blood and food stuff, which is essentially required for the diagnosis and treatment of hyperoxaluria and calcium oxalate urinary stones. The degradation of endogenous oxalate in rat by immobilized oxalate oxidase has opened a new vistas in enzyme therapy of hyperoxaluria.