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Letters |
Biochemistry Research Laboratory, Department of Bio-Sciences, Maharshi Dayanand University, Rohtak-124 001, Haryana, India
a Author for correspondence. Fax (91) (01262)41132;
To the Editor:
Methods for determination of urinary oxalate, which use oxalate oxidase from mosses, barley, banana peel, and beet stems, may suffer interference from physiological concentrations of Cl- and NO-3, usually found in urine (1)(2)(3)(4)(5)(6). The usual daily excretion of Cl- and NO-3 are 10–15 g/day and 0.5 g/day, respectively. The potential interference of these anions is removed either by passing the urine through an ion-exchange column or by precipitation of oxalate from urine and its redissolution prior to oxalate assay (1)(7). This pretreatment complicates the procedure; consequently, the sensitivity and reproducibility of the determination often suffer. In our laboratory, we have purified an oxalate oxidase from leaves of 10-day-old seedling plants of grain sorghum (CSH-5), which is insensitive to physiological concentrations of Cl- and NO-3 (8). In the present report, we describe a new method of oxalate determination using grain sorghum leaf enzyme, which does not suffer from Cl- and NO-3 interference.
The seeds of grain sorghum (Sorghum vulgare var CSH-5) were
a gift from M/s Nath Seeds Ltd. (Aurangabad, India). The 10-day-old
seedling plants were raised from these seeds in the laboratory
according to our method published previously (8). The leaves
were collected and homogenized with chilled distilled H2O
in a 1:3 ratio and centrifuged at 15 000g for 30 min at
4 °C. Oxalate oxidase activity present in 15 000g
supernatant was purified to apparent homogeneity by using the
combination of 0–80% (NH4)2SO4
precipitation, ion-exchange chromatography on DEAE Sephacel, and gel
filtration on Sephadex G-200 as described (8). The purified
enzyme gave 0.386 U/mg. One enzyme unit is defined as the amount of
enzyme required to produce 1 µmol of
H2O2/min/mL at pH 5.0 and 40 °C. The
purified enzyme was stable for over a period of 1 y, when stored
at -20 °C in 0.05 mol/L sodium succinate buffer, pH 5.0. Unlike
moss, barley, banana peel, beet stem, and Bougainvillea leaf enzyme,
sorghum leaf enzyme was unaffected by Cl- and
NO-3 in their physiological
concentration range (Table 1
).
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To determine urinary oxalate content, 24-h urine samples were collected from apparently healthy individuals in 2-L plastic bottles. Acidified urine (1.0 mL) was diluted with potassium phosphate buffer (0.1 mol, pH 7.0) in a 1:1 ratio, and its final pH was adjusted between 5.0 and 7.0 by HCl or NaOH. To avoid possible ascorbate interference, 200 mg of activated charcoal was added to 2.0 mL of neutralized urine sealed with Parafilm and vortex-mixed for 5 min in a 15-mL test tube. The sample was filtered through Whatman No. 1 filter paper, and filtrate was used for oxalate assay. The assay mixture, in a 15-mL test tube wrapped with black carbon paper, contained 1.7 mL of 0.05 mol/L sodium succinate buffer, pH 5.0, 0.1 mL of 0.01 mol/CuSO4 solution, and 0.1 mL enzyme. This mixture was preincubated at 40 °C for 2 min. The reaction was started by adding 0.1 mL of urine (filtrate). After incubation at 40 °C for 2 min, 1.0 mL of color reagent was added. The tubes were shaken and kept at room temperature (30 ± 5 °C) for 30 min to develop the color. The A520 of the color was read in a Spectronic 20 (Milton & Roy), and the oxalate in the urine sample was calculated from the standard curve between oxalate concentration vs A520. The color reagent consisted of 50 mg of 4-aminophenazone, 100 mg of solid phenol, and 1.0 mg of horseradish peroxidase (RZ = 1.0) per 100 mL of 0.4 mol/L sodium phosphate buffer, pH 7.0 (10) and was stored in an amber-colored bottle at 4 °C and prepared fresh every week.
A linear relationship was obtained between oxalate concentration in the reaction mixture, ranging from 0.1 to 1.2 mmol/L, with color intensity at 520 nm up to an absorbance of 0.11. The analytical recovery of added oxalate (20 and 40 nmol/sample) was 97 ± 2%. The within- and between-day CV for urinary oxalate values (n = 5) were <3.0% and <6.0%, respectively. The oxalate values in 24-h urine from apparently healthy individuals (male, n = 100) were 89–258 µmol/day, with a mean of 185.5. To evaluate the accuracy of the method, we determined the oxalate values in urine samples (n = 20) by the Sigma kit method (x) with modification (11) and by the present method (y). The modified Sigma kit method included the precipitation of urinary oxalate by CaCl2 and ethanol and its redissolution in 0.1 mol/L HCl for removal of ion interference (7), treatment with activated charcoal for removal of ascorbate interference, and measurement of H2O2 generated from urinary oxalate by barley oxalate oxidase in sodium succinate buffer, pH 5.0, by a color reaction using 3-methyl-3-benzothiozolinone and 3-(dimethylamine)benzoic acid and horseradish peroxidase as chromogen (11). The oxalate values obtained by the two methods showed a good correlation (r = 0.9632) with the following regression equation: y = 0.839x - 0.0025, for a mean value of 27 ± 44 mg/L. To test the possible interference by various salts and organic substances found in urine, the following compounds were added in the reaction mixture each at two final concentrations of 0.5 and 1.0 mmol/L: NaCl, KCl, CuSO4, FeSO4, MgSO4, MnCl2, PbCl2, CdCl2, CH3COOK, CH3COONa, NaNO3, Na2HPO4, Na2CO3, urea, glycine, pyruvate, glutamate, citrate, glucose, fructose, creatinine, glycollate, glyoxylate, NADH, and ascorbate. Of these compounds, only ascorbate and NADH caused 91% and 80% inhibition of the color reaction, respectively, at 0.5 mmol/L concentration, which increased to 100% at 1.0 mmol/L concentration in both the cases. Other compounds had practically no effect. NaCl had no effect, even at higher concentrations such as 50, 100, and 200 mmol/L. However, many of these substances may be present in urine at even higher concentrations in various disease states and, therefore, may need to be tested more thoroughly.
Acknowledgments
This work was supported by a grant to C.S.P from Indian Council of Medical Research, N. Delhi, India. The free supply of grain sorghum seeds of M/s Nath Seeds Pvt. Ltd. (Aurangabad, India) is gratefully acknowledged.
References
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