Measurement of urinary oxalate by grain sorghum leaf oxalate oxidase immobilized to affixed alkylamine glass beads

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1 Indian Journal of Biochemistry & Biophysics Vol. 41, April-June 2004, pp Measurement of urinary oxalate by grain sorghum leaf oxalate oxidase immobilized to affixed alkylamine glass beads M Kumari and C S Pundir* Biochemistry Research Laboratory, Department of BioSciences, Maharshi Dayanand University, Rohtak , Haryana, India Received 19 June 2003; revised 1 April 2004 Oxalate in urine was measured by grain Sorghum leaf oxalate oxidase conjugated to alkyl amine glass beads affixed in a beaker. The minimum detection limit was 0.05 mm/l in urine. Recovery of added oxalate in urine was 80.5% and within and between assay, coefficients of variation (CV) were <4% and <5.5%, respectively. Urinary oxalate values obtained by the present method showed a good correlation (r = 0.947) with those by Sigma kit method. The method is not only free from tedious handling of free glass beads and Cl - interference, but also has longer stability and reusability of immobilized enzyme compared to that of barley root and forage Sorghum leaf. Keywords: Oxalate, oxalate oxidase, urine, grain Sorghum, alkylamine glass, immobilized enzyme Determination of oxalate in urine is required in the diagnosis and medical management of urinary stones and intestinal disease 1. Among the various methods available for urinary oxalate determination, enzymic colourimetric method, employing oxalate oxidase (OXO) is simple, sensitive, specific and rapid and thus most suitable for routine testing 2. In fact, enzymic colorimetric method, using barley oxalate OXO has become popular and its commercial kit is available 3. However, the method becomes expensive, when used for large number of clinical samples, as it requires bulk quantity of enzyme. Immobilization of OXO onto free alkylamine glass beads allows the reuse of enzyme and thus reduces the cost 4, but the handling of free glass beads is tedious and timeconsuming. The problem was overcome in our laboratory by affixing the glass beads in a glass beaker with fixatives, such as Araldite and Lakme nail enamel before immobilization of enzyme prepared from barley roots and leaves of forage Sorghum seedlings, respectively 5,6. However, this modified method suffers from low stability and reusability of immobilized enzymes, as the enzyme from barley roots could be reused for 100 times during 15 days 5, while forage Sorghum leaf for 70 *Corresponding author Tel: (91)(1262) pundircs@rediffmail.com Fax: (91)(1262) times over 12 days only 6. Further, barley enzyme is sensitive to Cl - normally present in urine 7. Earlier, oxalate oxidase was purified from leaves of grain Sorghum seedlings in this laboratory, which was insensitive towards physiological concentration of Cl - - and NO 3 and thus was more suitable than barley enzyme for oxalate determination 8. In the present study, we have used various fixatives for affixation of glass beads before immobilization of this enzyme to improve its stability and reusability. The immobilized enzyme was used for the measurement of urinary oxalate. Materials and Methods Alkylamine glass beads (pore diameter=55 nm, Corning Glass Works, New York), horseradish peroxidase (RZ 1.0), oxalic acid, 4-aminophenazone (Sigma Chemicals Co., USA), DEAE-Sephacel, Sephadex G-200 (Pharmacia, Sweden), glutaraldehyde (BDH, UK), the fixatives Araldite (Ciba Speciality Chemicals (India) Ltd., Mumbai), Fevicol and Feviquick (PIDILITE Ind. Ltd., Regent Chambers, Mumbai), Wax of candle, Agar-Agar (Solgel) (Qualigens, Mumbai), Nailpaint (Elle-18, Fiora Cosmetics Ltd., Kindaim, Goa.), Paint{White} (Asian Paint India Ltd., Mumbai.), Quickfix (Wembley Laboratories Ltd., Bahadurgarh, Haryana), DPX and Balsam Canada (LOBA CHEMIE Pvt. Ltd., Mumbai) were used. All other chemicals were of AR

2 KUMARI & PUNDIR: URINARY OXALATE DETERMINATION BY IMMOBILIZED OXALATE OXIDASE 103 grade. Seeds of grain Sorghum var. Amarnath were gift from Nath Seeds Pvt. Ltd, Aurangabad, India. Purification of oxalate oxidase (OXO) The 10-days old seedlings of grain Sorghum vulgare var. Amarnath were raised 9 and the purified enzyme was obtained as described earlier 10. The enzyme was purified to 134-fold with 14.6% yield and had specific activity of 0.95 Units mg -1. It was stored at 0-4 C until use. The purified enzyme was concentrated by lyophilization and its homogeneity was confirmed in native PAGE carried at ph 8.8, using silver staining. Immobilization of OXO on affixed alkylamine glass beads A thin layer of 1 mm thickness of various fixatives was applied separately onto inner base of a glass beaker (34 45 mm, O.D height). Alkylamine glass beads (100 mg) were sprinkled uniformly and the beaker was kept overnight at room temperature (30 C) for affixation of glass beads. An OXO purified from Sorghum leaves was immobilized through glutaraldehyde coupling onto alkylamine glass beads affixed on inner base of a glass beaker with different fixatives 11. The affixed glass beads were activated by adding 2.5% glutaraldehyde in 0.1 M potassium phosphate buffer (ph 7.0) with occasional stirring for 2 hr, at room temperature. The excess of glutaraldehyde was poured off and activated beads were washed with 0.05 M potassium phosphate buffer (ph 7.0) until the ph of left out washing buffer was 7.0. The purified enzyme (0.5 U ml -1 ) was added to the activated beads and kept at 4 C for 48 hr with occasional stirring. The affixed glass beads were then washed with 0.1 M sodium phosphate buffer (ph 6.8) until no activity was detected in the washing. The enzyme bound to glass beads was measured by determining the protein in enzyme preparation before and after immobilization. Assay of immobilized OXO The assay was carried out, as described 10 in same beaker (termed as reaction beaker ) containing affixed alkylamine glass beads bound to OXO. The reaction beaker was wrapped with black paper to protect H 2 O 2 generated in the assay from the light. The reaction mixture containing 1.8 ml 0.1 M sodium phosphate buffer (ph 6.8) and 0.1 ml CuSO 4 (10-2 M) was preincubated in the reaction beaker at 38 C for 5 min. The reaction was started by adding 0.1 ml of aqueous oxalate solution (10 mm) and beaker was covered with aluminium foil. After incubation at 38 C for 8 min under continuous stirring, 1.0 ml colour reagent (consisting of 50 mg 4-aminophenazone, 100 mg solid phenol and 1 mg horseradish peroxidase per 100 ml 400 mm sodium phosphate buffer, ph 7) was added and kept at room temperature for 15 min in dark. The colour reagent was stored in amber colored bottle at 4 C and prepared fresh every week The reaction mixture was transferred to a cuvette and absorbance at 520 nm was read in Spectronic-20 (Milton & Roy, USA). The content of H 2 O 2 generated in the reaction was extrapolated from standard curve between absorbance at 520 nm and H 2 O 2 concentration. Determination of urinary oxalate Urine (24 hr) from apparently healthy persons and urinary stone patients of different age groups was collected in 2 L bottles containing 15 ml conc. HCl and diluted with distilled water in 1:2 (v/v) and ph was adjusted to 7 by adding NaOH. To avoid possible interference by ascorbate, 0.1 ml buffered NaNO 2 (3.5 mg ml M sodium phosphate buffer, ph 7) was added per ml diluted urine 11. The urinary oxalate assay was carried out in the same manner as described above except that oxalate solution was replaced by pre-treated urine. The oxalate concentration in the sample was calculated from standard curve between oxalate concentration (0.05 mm to 10 mm) vs. A 520 under standard assay conditions (Fig. 1). Reuse and storage of immobilized enzyme To reuse the immobilized enzyme, beaker was rinsed with 0.1 M sodium phosphate buffer (ph 6.8) 3-4 times, prior to its use in next assay. The reaction beaker containing sufficient distilled water to cover affixed glass beads was stored at 4 C, when not in use. Results and Discussion Immobilization of OXO onto alkylamine glass beads affixed by different fixative inside a glass beaker The immobilized enzyme showed different activities with different fixatives. The retention of OXO activity on to glass beads was maximum in case of quickfix (47%) with a conjugation yield of 8.4 mg/g support, followed by nailpaint (40.7%) with a conjugation yield of 8.2 mg/g support and was minimum in Balsam Canada (Table 1). However, the storage stability of immobilized OXO in distilled

3 104 INDIAN J. BIOCHEM. BIOPHYS., VOL. 41, APRIL-JUNE 2004 Table 1 Immobilization of Sorghum leaf oxalate oxidase onto alkylamine glass beads (pore diameter 55 nm) affixed on inner base of a glass beaker by various fixatives [In a glass beaker (34 45mm) containing a layer of 1 mm thickness of various fixatives on its inner base, 100 mg of alkylamine glass beads were sprinkled uniformly and affixed beads were activated by 2.5% glutaraldehyde in 0.05 M sodium phosphate buffer (ph 7.0). Purified enzyme (1.2 mg containing 0.56 U) was added to the activated beads and kept at 4ºC for 48 hr. Unbound enzyme was removed. One unit of oxalate oxidase is defined as the amount of enzyme required to generate 1 μmol H 2 O 2 /min /ml under standard assay condition] Fixative Oxalate oxidase coupled to 100 mg glass beads Conjugation yield (mg/g) Retention of specific activity (%) Control Fevicol Quick fix Nailpaint Paint Wax Feviquick Araldite Agar-agar DPX Balsam Canada Table 2 Storage stability of oxalate oxidase onto affixed alkylamine glass beads with various fixatives [The beaker containing affixed alkylamine glass beads bound Sorghum leaf oxalate oxidase was stored at 4 C with a layer of distilled water on immobilized enzyme, when not in use. Unit activity of immobilized enzyme is defined as 1 μmol H 2 O 2 produced per min. under standard assay conditions] Fixative Initial activity of immobilized enzyme (nmol H 2 O 2 /min) Days at which immobilized enzyme lost its 50% activity Control Fevicol QuickFix Nailpaint Paint Wax Feviquick Araldite Agar-agar DPX Balsam Canada water at 4 C was highest in the case of nailpaint, compared to other fixatives (Table 2). The half-life of immobilized enzyme bound to glass beads affixed by various fixatives was found in the following order: Nailpaint> Wax> Araldite>Feviquick> Paint> Quickfix> Agar> DPX> Balsam Canada> Fevicol (Table 2). As the OXO bound to glass beads affixed with nailpaint showed highest half-life, it was studied further. Kinetic properties of nailpaint affixed glass bead bound OXO The kinetic properties of Sorghum OXO immobilized to alkylamine glass beads affixed on inner base of a glass beaker by nailpaint were studied and compared with the native/free enzyme 10 and enzyme bound to free alkylamine glass beads 14 (Table 3). An increase in optimum ph of the enzyme after immobilization on affixed beads could be due to loss of NH 2 groups from surface of enzyme upon conjugation 15. An increase in activation energy (E a ) of affixed glass bound enzyme, compared to that of free enzyme might be due to the change in conformation of the enzyme, unfavourable electron flow between reactant and support and unfavourable change of polarity or ionic strength in the vicinity of active site of enzyme 15. An increase in K m for oxalate, but decrease in V max after immobilization on affixed beads might be due to change in the enzyme conformation, steric hindrance, micro-environmental effect and bulk and diffusional effects after immobilization 15. Determination of oxalate in urine by affixed glass beads bound OXO A method was developed for determination of oxalate in urine employing Sorghum OXO immobilized to glass beads affixed by nailpaint inside a 25 ml glass beaker. The method has the advantage that it not only allows the reuse of enzyme with an ease and free from Cl - interference but also has longer stability and higher reusability of immobilized enzyme than that employing barley root and forage Sorghum leaf

4 KUMARI & PUNDIR: URINARY OXALATE DETERMINATION BY IMMOBILIZED OXALATE OXIDASE 105 Table 3 Kinetic parameters of native, free alkylamine and nailpaint affixed alkylamine conjugated Sorghum oxalate oxidase (OXO) on the inner base of glass beaker Parameters Free OXO 9 OXO immobilized to free glass beads 12 OXO immobilized to affixed glass beads (Present method) Optimum ph Temp. for maximum activity ( C) E a (Kcal/mol) Time for linearity (min) K m for oxalate mol/l mol/l mol/l V max μmol/min 0.25 μmol/min 0.14 μmol/min Stability at 4 C 50% loss in 15 days 10% loss in 6 months 50% loss in 6 months No inhibition detected by NaCl (up to 100 mm concentration) in the activity of free enzyme and enzyme bound to free and affixed alkylamine glass beads Fig 1 Standard curve for A 520 vs. oxalic acid concentrations using affixed alkylamine glass bound grain Sorghum leaf oxalate oxidase [A glass beaker containing Sorghum leaf oxalate oxidase bound to affixed glass beads on its inner base, 1.8 ml 0.05 M sodium phosphate buffer (ph 6.8), 0.1 ml 0.01 M CuSO 4 and 0.1 ml oxalate solu-tion of different concentrations was incubated at 37ºC for 8 min under continuous stirring. 1.0 ml color reagent was added and A 520 was read after keeping it in dark for 15 min; values are mean of three replications] enzyme 5,6. The following analytic parameters were determined to evaluate the method. Linearity A linear relationship was found between A 520 vs. oxalate concentration ranging from 0.05 mm to 10 mm in reaction mixture up to an absorbance of 0.11 (Fig. 1). Minimum detection limit and recovery The minimum detection limit of the method is 0.05 mm oxalate/l urine (Fig. 1), which is comparable with that of using barley enzyme immobilized to affixed alkylamine glass beads by Araldite (0.04 mm/l) 5. Fig. 2 Correlation between urinary oxalate values determined by the modified SIGMA kit method (X) and by the present method (Y) Analytical recovery of added oxalate in urine samples (20 mg L -1 and 30 mg L -1 ) was 82±4.8% and 78±5% (mean±sd, n=6), which is lower than that using barley enzyme (96.7±3.4%) 4 and Sorghum enzyme (97.5%) bound to free alkylamine glass beads 1. Precision and accuracy To check the reproducibility and reliability of the method, oxalate content of same urine samples in one run (within batch) and after one week storage at -20 C (between batch) were determined. The within batch and between batch coefficients of variation (CV) for urinary oxalate determination were <4% and <5.5%, respectively, and were comparable to earlierreports 4,5,14. Oxalate values as determined by the present method in 24 hr urine were mg L -1 with mean of 32.2 mg L -1 in apparently healthy male adults and mgl -1 with mean of 54.6 mg L -1 in male urinary stone formers. The values of 10 to 32 mg L -1 (mean 22.0 mgl -1 ) and 15 to 67 mg L -1 (mean 43.6

5 106 INDIAN J. BIOCHEM. BIOPHYS., VOL. 41, APRIL-JUNE 2004 mg L -1 ) were found in apparently healthy females adults and female urinary stone formers, respectively. For testing the accuracy of the method, the urinary oxalate values obtained by present method (y) were compared with those obtained by enzymic colorimetric kit method (Sigma). The correlation coefficient (r) was and regression equation being y = 0.869x , indicating the high accuracy of the method (Fig. 2). Interference The effect of urinary substances viz., pyruvate, glucose, citrate, haemoglobin, albumin, cysteine, urea, uric acid, creatinine, hippuric acid, acetone, ascorbic acid, bilirubin, cholesterol and NaCl was studied at their physiological concentration 16. These substances had practically no effect, except ascorbate, which caused 68% inhibition of colour reaction. However, the inhibition by ascorbate was lower than that reported for free and alkylamine conjugated Sorghum enzyme (100% and 82% inhibition, respectively) 17, indicating the protection of enzyme onto affixed alkylamine glass beads by nailpaint. Reusability and storage The immobilized grain Sorghum leaf oxalate oxidase retained 50% of its initial activity after 250 uses for over a period of 6 months, when stored at 4 C in distilled water, which is higher than that for barley root enzyme (100 uses over 15 days) and forage Sorghum leaf enzyme (70 uses over 12 days) 6. In conclusion, the use of grain Sorghum leaf oxalate oxidase immobilized through glutaraldehyde onto affixed alkylamine glass beads by nailpaint in enzymic colorimetric measurement of urinary oxalate allows reuse of enzyme with ease and free from Cl - interference. Further, the affixed glass beads also provide longer stability and higher reusability of the enzyme, compared to barley root and forage Sorghum leaf enzymes. Acknowledgement We are grateful to M/s Nath Seeds Pvt. Ltd., Aurangabad, India for free supply of grain Sorghum seeds and Prof H H Weetall of Corning Glass Work, New York for gifting zirconia-coated alkylamine glass beads. References 1 Decastro M D L (1988) J Pharm Biomed 6, Sharma S R & Thind S K (1993) Scan Micro 7, Crider Q E & Curran D F (1984) Clin Biochem 17, Pundir C S, Satypal & Kuchhal N K (1993) Clin Chem 39, Chandran P, Thakur M & Pundir C S (2001) J Biotechnol 85, Kalra V & Pundir C S (2004) Indian J Biotechnol 3, Potezny N, Bais R, O Loughlin PD, Edwards J B, Rofe A M & Conyers R A J (1983) Clin Chem 29, Pundir C S, Thakur M & Satyapal (1998) Clin Chem 44, Pundir C S & Nath R (1984) Phytochemistry 23, Satyapal & Pundir C S (1993) Biochem Biophys Acta 1161, Lynn M (1975) in Immobilized Enzymes, Antigens Antibodies and Peptides (Weetall H H, ed.), pp. 1-48, Marcel Dekker Inc, New York 12 Lowry O H, Rosenbrough N J, Farr A L & Randall R J (1951) J Biol Chem 193, Kasidas G P & Rose G A (1985) Ann Chem Biochem 22, Thakur M, Pundir C S (1999) Biotechnol Tech 13, Kennedy J F (1985) in Handbook of Enzymes Biotechnology (Wiseman A, ed.), pp , John Wiley & Sons, New York 16 Hawk P B (1985) in Hawk s Physiological Chemistry (Oser B L, ed.), XIVth edn, pp , Tata McGraw Hill Publishing Company Ltd., New Delhi. 17 Pundir C S, Thakur M, Goyal L & Bhargava A K (1999) Chin J Biotechnol 15,