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

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1 Indian J Med Res 127, April 2008, pp Urinary & serum oxalate determination by oxalate oxidase immobilized on to affixed arylamine glass beads Savina Godara & C.S. Pundir Biochemistry Research Laboratory, Department of Biochemistry & Genetics, MD University Rohtak, India Received January 11, 2007 Background & objectives: 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 40ºC for 15 min. The minimum detection limit of the method was 0.01mM/l in the urine and 2.5 mm/l in the serum. The analytical recovery of added oxalate was per cent in urine and 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 (r 1 = 0.99) and in serum (r 2 = 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 C in reaction buffer. Interpretation & conclusions: 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. Key words Amaranthus - arylamine glass beads- enzyme strip - immobilization - oxalate - oxalate oxidase - serum - urine Oxalate, a metabolic end product in man, can lead to hyperoxaluria due to its enhanced endogenous synthesis or increased absorption from diet. This results in excessive excretion of oxalate in urine, which has also been observed in idiopathic renal calcium oxalate stone formers and a number of ileal diseases. Hence the quantification of urinary oxalate is very important for diagnosis and management of these diseases

2 GODARA & PUNDIR : PREPARATION OF REUSABLE STRIP OF OXALATE OXIDASE 371 Although a number of methods like direct precipitation, solvent extraction, colorimetry, chemiluminescence, isotope dilution, high performance liquid chromatography, ion chromatography, enzymic-uv spectrophotometry are available for oxalate determination in urine, an enzymic colorimetric method employing oxalate oxidase is comparatively more simple, specific, sensitive and rapid, hence suitable for routine use 2. A method for discrete analysis of urinary oxalate has been reported from this laboratory using Amaranthus leaf oxalate oxidase immobilized onto free arylamine glass beads 3. Although the method provided the reuse of enzyme for a period of 90 days and was unaffected by chloride ions (Cl - ) found in biological fluids, the handling of free glass beads was tedious and time consuming and included the risk of their loss during transfer of reaction mixture and washing for reuse. The present study was undertaken to overcome this problem by affixing the glass beads on a plastic strip with a non-reactive fixative before immobilization of enzyme. Material & Methods Chemicals: Zirconia coated arylamine glass beads (pore diameter 55 nm) was a gift from Prof. H.H. Weetall, Corning Glass Works, New York, USA. Horseradish peroxidase (HRP) (RZ=1.0), oxalic acid, bovine serum albumin and 4-aminophenazone were purchased from Sigma Chemical Co. USA. Sodium deoxycholate was from SISCO Research Laboratories Pvt. Ltd. Mumbai. The non-reactive fixative Araldite and totally inert soft, transparent white plastic sheet of about 0.2 mm thickness, used as plastic cover, were purchased from local market. All other chemicals were of analytical reagent grade. Collection of plant material: Healthy Amaranthus spinosus grown at the sides of railway track of north Delhi were identified on the basis of following characters: (i) Erect, armed, glabrous plants up to 60 cm in height (ii) Two axillary, diverticulate sharp spines, (iii) Much branched stem, green or somewhat raddish in colour and glabrous, (iv) Leaves cm in size, ovate or oblong from a slightly decerrent base, gradually narrowed upward, apex, obtuse, rounded and glabrous, and (v) Flowers dense in axillary clusters, racemosely disposed, spikes often branched in lower regions terminal spikes often male flowers only, finally drooping perianth lobes 5, stamens 5, ovary ovoid or oblong, style 3, conical, stigma 3, fimbrillate. The leaves of these plants were collected in ice bath and washed in distilled water, dried in 2 folds of dry filter paper and stored immediately at -20 C until use. Extraction and partial purification of oxalate oxidase: Crude oxalate oxidase from leaves of Amaranthus was prepared 4 with modification. The frozen leaves (100 g) were homogenized in potassium phosphate buffer (0.1M, ph 7.0) containing 0.5 M sucrose in 1:4 ratio (w/v) in a chilled pestle mortar in dark. The homogenate was squeezed though a double layer of cheese cloth and the filtrate was centrifuged at g for 15 min at 4 C in a cooling centrifuge (Remi India, model C- 24) and the pellet was suspended in the same volume of extraction buffer. Sodium deoxycholate (2.5 mg/ml) was added to the suspension and kept at 4 C for 48 h under constant stirring. The suspension was centrifuged at g for 20 min at 4 C and the supernatant (the solubilized enzyme) was collected and subjected to 0-80 per cent (NH 4 ) 2 SO 4 precipitation. The resulting solution was centrifuged at g for 30 min, at 4 C and the pellet obtained was dissolved in extraction buffer and used as partially purified oxalate oxidase. The (NH 4 ) 2 SO 4 precipitation resulted into 13-fold purification of enzyme with 71 per cent yield. The enzyme was stored at -20 C until use. Assay of free oxalate oxidase: The assay of oxalate oxidase was carried out with modification in a 15 ml test tube wrapped with black paper 5. The reaction mixture containing 1.8 ml of 0.05 M sodium citrate buffer (ph 3.5) and 0.1 ml partially purified enzyme was preincubated at 37 C for 5 min. The reaction was started by adding 0.1 ml aqueous oxalate solution (30 mm). After incubation at 37 C for 5 min, 1 ml colour reagent was added and kept at the same temperature in water bath for 20 min to develop the colour. The blank was also run in the same manner except that the enzyme solution was replaced with reaction buffer. A 520 was read in a Spectronic-20 (Milton and Roy, USA) and concentration of H 2 O 2 was extrapolated from standard curve of H 2 O 2.The colour reagent consisted of 50 mg 4-aminophenazone, 100 mg phenol and 1.0 mg HRP per 100 ml of 0.4 M sodium phosphate buffer, ph 7.0 and stored in amber coloured bottle at 4 C and prepared fresh every week. The protein content in oxalate oxidase preparation was measured by Lowry method 6. Unit of enzyme activity: One unit of enzyme is defined as amount of enzyme required to generate 1nmol H 2 O 2 / min. Preparation of reusable strip of oxalate oxidase Affixation of arylamine glass beads on plastic strip - The strip of 15 1 cm size was cut from a plastic

3 372 INDIAN J MED RES, APRIL 2008 sheet. One end of this strip was made round with the help of scissors. A thin layer of 0.2 mm thickness of fixative Araldite was applied uniformly on both side of the round end of strip up to height of 2 cm with the help of a brush. Arylamine glass (50 mg) beads were sprinkled uniformly on the fixative with the help of aluminum foil. The strip was kept in a 15 ml test tube for 24 h at room temperature for affixation of glass beads 7. Immobilization of oxalate oxidase onto affixed arylamine glass beads: This was carried out as described by Lynn with certain modification ml chilled 2N HCl followed by 25 mg solid NaNO 2 were added to the glass beads. The diazotization reaction was allowed to proceed for about 30 min, with occasional shaking. The excess of HNO 2 was decanted and the diazotized beads were washed several times with 0.1 M sodium phosphate buffer (ph 7.0) until the ph of the washing was 7.0.The end of plastic strip containing activated glass beads was dipped in to oxalate oxidase solution (3 ml) and allowed to stand at 4 C for 48 h with occasional shaking. After the immobilization, the strip was taken off and the remaining solution was tested for activity and protein. The strip was dipped in to distilled water 5-6 times to remove the unbound enzyme and tested for enzyme activity. Assay of strip bound oxalate oxidase: It was carried out as described for assay of free oxalate oxidase except that free enzyme was replaced by plastic strip bound oxalate oxidase and reaction buffer was increased by 0.1 ml and the reaction mixture was stirred continuously during incubation. The strip was taken out from the reaction mixture before addition of colour reagent. The strip was stirred in 0.05 M sodium citrate buffer ph 3.5 at 4 C, when not in use. Kinetic properties of immobilized oxalate oxidase: The following kinetic properties of immobilized oxalate oxidase were studied:- optimum ph, incubation temperature, time of incubation, effect of oxalate concentration and calculation of Km and Vmax from Lineweaver Burk plot. Determination of urinary and serum oxalate with Enzyme strip Collection & pretreatment of urine and serum - To collect urine and serum samples, the individuals at Pt BDS PGIMS Hospital, Rohtak were selected under two categories (i) Those who never formed urinary stone as healthy subjects as confirmed by their abdominal X- ray report and (ii) who have already formed urinary stones as stone formers. These two groups were further divided based on sex and age groups. In each group, six individuals were included. 24 h urine sample was collected in each case in a 2 litre plastic bottle containing 15 ml conc HCl. The final ph of acidified urine was adjusted between 5.0 to 7.0 by addition of NaOH or HCl. The urine was diluted in 1:1 with 0.1M potassium phosphate buffer (ph 7.0) 9. To avoid possible interference by ascorbate, 0.1ml buffered sodium nitrite (3.5 mg/ml prepared in 0.1 M sodium phosphate buffer, ph 7.0) was added per ml of urine 10. Blood samples (2 ml) from the same individuals was collected and serum separated. To avoid possible ascorbate interference, 0.1 ml 5 mm sodium nitrite (in 0.5 M sodium phosphate buffer ph 6.0) was added to 0.1 ml acidified serum 10. Determination of serum and urinary oxalate: This was carried out in the same manner as described for assay of immobilized oxalate oxidase except that oxalate solution was replaced by pretreated urine or serum and optimal assay conditions were maintained. The concentration of oxalate in urine or serum was extrapolated from standard curve drawn from oxalate concentration ranging from to 0.4 mm and A 520. Reuse of enzyme strip: The enzyme strip was dipped in the 0.05 M sodium citrate buffer, ph 3.5, five to six times before its use in the next assay. The strip was stored in the same buffer at 4 C, when not in use. Statistical analysis: The urinary and serum oxalate values of stone formers were compared with those in healthy individuals in each group using the student s t test. Results & Discussion An oxalate oxidase partially purified from mature leaves of Amaranthus spinosus plants has been immobilized covalently onto arylamine glass beads affixed on one end of a plastic strip with 56 per cent retention of initial activity of free enzyme and conjugation yield of 40 mg/g. A comparison of immobilization of oxalate oxidase from different plant sources onto free and affixed arylamine and alkylamine glass beads shows better conjugation yield of Amaranthus enzyme immobilized onto affixed arylamine glass beads and higher retention of activity of Amaranthus enzyme immobilized onto free alkylamine glass beads (Table I). Kinetic properties of plastic strip bound oxalate oxidase: Compared to free enzyme, the strip bound

4 GODARA & PUNDIR : PREPARATION OF REUSABLE STRIP OF OXALATE OXIDASE 373 Table I. A comparison of immobilization of oxalate oxidase from different plant sources on to free & affixed arylamine & alkylamine glass beads Parameter Support Barley Sorghum leaf Amaranthus leaf Conjugation Free arylamine yield Affixed arylamine (Present) (mg/g support) Free alkylamine Affixed alkylamine % Retention Free arylamine Affixed arylamine (Present) Free alkylamine Affixed alkylamine Superscript numerals represent reference numbers Amaranthus enzyme showed no change in its optimum ph i.e., 3.5, in contrast to barley enzyme whose ph was decreased from 3.2 to 3.0 and sorghum enzyme, whose ph was increased from 5.0 to 5.5 after immobilization 11,15. The time of incubation for maximum activity of Amaranthus enzyme was increased from 5 to 15 min after immobilization, in contrast to barley enzyme, where time of incubation decreased from 10 to 5 min after immobilization but similar to Sorghum enzyme where it increased from 2 to 5 min. Apparent Km value for oxalate was increased from to M, after immobilization, similar to that for barley 11 enzyme ( to M) and Sorghum 15 enzyme ( to M). Incubation temperature for maximum activity of Amaranthus enzyme was increased slightly from 37 to 40 C after immobilization, while it has been shown to decrease for barley 11 from 35 to 30 C and increase for Sorghum 15 from 40 to 45 C. Urinary and serum oxalate determination with oxalate oxidase strip: The method using reusable strip of oxalate oxidase was simple, sensitive and specific method for discrete analysis of oxalate in urine and serum. The method has the advantage as it provides enormous ease in reuse of enzyme and unaffected by Cl - normally found in biological fluids. Further, the method avoids accumulation of dye (product of colour reaction) in the vicinity of immobilized enzyme and thus eliminates its possible interference in the assay. A linear relationship was found between A 520 vs. oxalate concentration ranging from to 0.4 mm in reaction mixture. The minimum detection limit of the method in urine was 0.01 mm oxalate/l, which was lower than that for the method employing barley 11 enzyme (0.02 mm) and Sorghum 12 enzyme (0.1 mm). The minimum detection limit of the method in the serum was 2.5 mm/l, which was similar to sorghum 16 enzyme. Table II. Analytical recovery of added oxalic acid in urine by oxalate oxidase immobilized onto arylamine glass beads affixed on a plastic strip Oxalate Mean oxalate found % Recovery by immobilized added (mg\l), n=6 oxalate oxidase (mean ± SD) (mg\l) Nil ± 0.51 Nil ± 1.2 Table III. Analytical recovery of added oxalic acid in serum by oxalate oxidase immobilized onto arylamine glass beads affixed on a plastic strip Oxalate Mean oxalate found % Recovery by immobilized added (mm/l), n=6 oxalate oxidase (mean ± SD) (mm/l) Nil ± 2.4 Nil ± 1.8 For analytical recovery, solid oxalate was added into urine (20 and 30 mg/l) and serum (1.5 and 2.5mM/ l). The oxalate content was measured before and after addition of oxalate. The analytical recoveries of added oxalate were per cent (mean + SD) in urine which was higher than that using barley enzyme 11 (88.9%) and Sorghum enzyme 12 (82%) and per cent (mean + SD) in serum, which was higher than that using Sorghum 16 enzyme (89.5 %) (Tables II, III). The oxalate content of six urine and six serum samples was determined six times in one run (within batch) on the first day and after one week storage at -20 C (between batch). The oxalate values in urine and serum samples agreed with each other and within batch and between batch coefficients of variation (CV) were 4.6 and 5.2 per cent in urine

5 374 INDIAN J MED RES, APRIL 2008 and 7.4 and 5.8 per cent in serum respectively, showing the high reproducibility and reliability of the method (Table IV). To test the accuracy of method, the oxalate value was determined in 12 urine and 12 serum samples of apparentaly healthy persons by both the present method (y) and standard enzymatic colorimetric kit method (x). The values obtained by both methods showed a good correlation for oxalate in urine (r 1 = 0.99) with regression equation being y = 1.023x (Fig. 1) and oxalate in serum (r 2 = 0.92) with regression equation being y = x (Fig. 2). Fig. 1. Correlation between urinary oxalate values determined by Sigma Kit Method (X-axis) and present method (Y axis) with regression equation being y = x & r 1 = Table IV. Within and between assay coefficient of variation (CV) for determination of urinary and serum oxalate by oxalate oxidase immobilized onto arylamine glass beads affixed on a plastic strip Type of assay Mean oxalate % CV ± SD (mg/l) Urinary: Within assay a ± 0.67 Between assay b ± 0.91 Serum: Within assay a ± 0.27 Between assay b ± 0.23 a Sample assayed on the same day b Same Samples were assayed after 1 wk storage at -20 C Serum oxalate by present method (mm/l) Serum oxalate by Sigma Kit Method (mm/l) Fig. 2. Correlation between serum oxalate values determined by Sigma Kit Method (X-axis) & present method (Y-axis) with regression equation being y = x & r 2 = Table V. Urinary oxalate values of apparently healthy persons and stone formers as determined by Amaranthus leaf oxalate oxidase immobilized onto arylamine glass beads affixed on a plastic strip Age group Sex Urinary oxalate (yr) (mg/h) Healthy individuals Stone formers Male 14.35± ±3.6 Female 12.5± ± Male 15.4± ±3.4 Female 14.35± ± Male 14.75± ±4.3 Female 16.63± ± Male 18.18± ±8.7 Female 17.4± ±4.9 Above 51 Male 27.5± ±5.2 Female 25.63± ±4.7 Values are mean ± SD (n = 6) for each group and significant (P<0.001) in all stone formers compared to healthy individuals in their respective group

6 GODARA & PUNDIR : PREPARATION OF REUSABLE STRIP OF OXALATE OXIDASE 375 Table VI. Serum oxalate values of apparently healthy persons and stone formers as determined by Amaranthus leaf oxalate oxidase immobilized onto arylamine glass beads affixed on a plastic strip Age group Sex Serum oxalate (yr) (mm/l) Healthy individuals Stone formers Male 4.1± ±0.6 Female 3.5± ± Male 4.2± ±0.7 Female 3.8± ± Male 4.9± ±0.69 Female 3.6± ± Male 4.4± ±0.64 Female 4.6± ±0.67 Above 51 Male 5.4± ±0.78 Female 4.8± ±0.87 Values are mean ± SD (n=6) for each group and significant (P<0.001) in all the stone formers compared to healthy individuals in their respective group The intra-class correlation coefficient was calculated to study the agreement between the two methods 17, but the interpretation was the same. The oxalate values in 24 h urine samples of apparently healthy individuals and stone formers were in the range 12.5 to 27.5 mg/l and 28.9 to 47.5 mg/24 h respectively, which were significantly higher in stone formers compared to healthy individuals (P<0.001) (Table V). The oxalate value in serum of apparently healthy individuals and stone formers were in the range of 3.5 to 5.4 mm/l and 5.75 to 11.6 mm/l respectively (Table VI). These observation revealed that both urinary and serum oxalate values were significantly higher in stone formers compared to healthy individuals (P<0.001). Fig. 3. Storage stability of Amaranthus leaf oxalate oxidase immobilized onto arylamine glass beads affixed on a plastic strip, in reaction buffer at 4 C. The strip bound enzyme did not show any noticeable change in its activity up to 60 days during its regular use (150 times) when stored at 4 C in reaction buffer (0.05M sodium citrate buffer ph 3.5). The strip lost 50 per cent of its initial activity after four months (Fig. 3). Conclusion A reusable enzyme strip of Amaranthus leaf oxalate oxidase was prepared for determination of serum and urinary oxalate. The method has the advantage as it provides enormous ease in reuse of enzyme and unaffected by CI normally found in biological fluids. Acknowledgment The authors thank HOD Biochemistry, Pt BDS PGIMS, Rohtak for providing urine and serum samples for this study. References 1. Galosy R, Clark L, Ward DI, Pak CYC. Renal oxalate excretion in calcium urolithiasis. J Urol 1979; 123 : Decastro MDL. Determination of oxalic acid in urine-a review. J Pharm Biomed 1988; 6 : Goyal L, Singh S, Pundir CS. Immobilization of Amaranthus leaf oxalate oxidase on arylamine glass. Indian J Chem Technol 2000; 7 : Goyal L, Thakur M, Pundir CS. Purification and properties of a membrane bound oxalate oxidase from Amaranthus leaves. Plant Sci 1999; 142 : Pundir CS, Chauhan DN. Detection and solubilization of a membrane bound oxalate oxidase from leaves of Amaranthus spinosus. Physiol Mol Biol Plant 1996; 2 : Lowry OH, Rosebrough NJ, Farr AL, Randall R. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193 :

7 376 INDIAN J MED RES, APRIL Tank N, Suman, Pundir CS. Determination of serum glucose using coimmobilized glucose oxidase & peroxidase on to arylamine glass beads affixed on a plastic strip. Indian J Biochem Biophys 2005; 42 : Lynn M. Inorganic support Intermediate: Covalent coupling of enzymes on Inorganic supports. In: Weetall HH, editor. Immobilized enzyme, antigen antibodies and peptides. New York: Marcel Dekker Inc.; p Kohlbecker G, Butz M. Direct spectrophotometric determination of serum and urinary oxalate with oxalate oxidase. J Clin Chem Clin Biochem 1981; 19 : Kasidas GP, Rose GA. Measurement of plasma oxalate in healthy subjects and in patients with chronic renal failure using immobilized oxalate oxidase. Clin Chim Acta 1985; 22 : Chandran P, Thakur M, Pundir CS. Improved determination of urinary oxalate with alkylamine glass bound barley oxalate oxidase. J Biotechnol 2001; 85 : Kumari M, Pundir CS. Measurement of urinary oxalate by grain sorghum leaf oxalate oxidase immobilized to affixed alkylamine glass beads. Indian J Biochem Biophys 2004; 41 : Pundir CS, Kuchhal NK, Satyapal. Barley oxalate oxidase immobilized on zirconia-coated alkylamine glass using glutaraldehyde. Indian J Biochem Biophys 1993; 30 : Madanpotra S, Chaudhary R, Singh S, Pundir CS. Preparation of a reusable strip of barley oxalate oxidase for determination of urinary oxalate. Indian J Chem Technol 2003; 11 : Pundir CS, Thakur M, Goyal L, Bhargava AK. Immobilization of Sorghum leaf oxalate oxidase onto alkylamine and arylamine glass. Chin J Biotechnol 1999; 15 : Thakur M, Goyal L, Pundir CS. Discrete analysis of plasma oxalate with alkylamine glass bound sorghum oxalate oxidase and horseradish peroxidase. J Biochem Biophys Methods 2000; 44 : Bland MJ, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lacent 1986; 8 : Reprint requests: Prof. C.S. Pundir, Department of Biochemistry & Genetics, Dean Faculty of Life Sciences M.D. University, Rohtak , India pundircs@rediffmail.com