Quantification of urinary oxalate with a chloride- and nitrate-insensitive oxalate oxidase

Abstract

Methods for the determination of urinary oxalate employing oxalate oxidase extracted from mosses, barley, banana peel and beet stems may suffer from interference by physiological concentrations of chloride and nitrate normally found in urine. 1±4 The normal daily excretion of chloride and nitrate into urine is 250 mmol/L and 8 mmol/L, respectively. This interference can be removed either by passing the urine through an ion-exchange column or by precipitating the oxalate from the urine and redissolving it prior to assay. 1,5 This pretreatment reduces the sensitivity and reproducibility of the determination of oxalate. In our laboratory, we have puri®ed an oxalate oxidase from roots of 10-day-old seedling plants of a grain sorghum hybrid (CSH-5), which is insensitive to physiological concentrations of chloride (250 mmol/L) and nitrate (8 mmol/L) and therefore more suitable for the enzymic determination of urinary oxalate than enzymes from other sources. 6 METHODS Preparation and puri®cation of oxalate oxidaseApart from Sephadex and Sephacel (which were obtained from Pharmacia LKB, Uppsala, Sweden) all reagents were obtained from Sigma Chemical Co (St Louis, MO, USA). Ten-day-old seedling plants of grain sorghum hybrid (CSH-5) were raised in the laboratory. The roots were collected and oxalate oxidase was extracted and partially puri®ed as described previously. 6 The enzyme was further puri®ed by gel ®ltration on a Sephadex G-200 column (1´52 45 cm) using 20 mmol/L potassium phosphate buffer (pH 7´0), followed by passage of the active fractions through a DEAE-Sephacel column (2´52 29 cm) using a linear gradient of KCl (0±0´6 mol/L) in 20 mmol/L potassium phosphate buffer pH 7´0. Active DEAE-Sephacel fractions were precipitated with 0±70% (NH 4 ) 2 SO 4 and then further puri®ed on a Sephadex G-200 column, (1´52 30 cm) using 20 mmol/L potassium phosphate buffer (pH 7´0). The active fractions of the last step were pooled and treated as puri®ed enzyme. The puri®ed enzyme exhibited a single band in polyacrylamide gel disc electrophoresis using Coomassie blue as protein stain and had a speci®c activity of 1´9 units/mg. One enzyme unit is de®ned as the amount of enzyme required to produce 1 mmol of H 2 O 2 /min/mL at pH 5´0 and 40 Ê C. The puri®ed enzyme was stable over a period of 1 year when stored at 2 20 Ê C in 20 mmol/L sodium phosphate buffer pH 7´0. Oxalate assayTo determine urinary oxalate content, 24-h urine samples were collected from apparently healthy individuals in 2-L plastic bottles containing 15 mL of concentrated HCl. The ®nal pH of the urine was maintained below pH 1´5 during and after the collection. Acidi®ed urine (1´0 mL) was diluted with potassium phosphate buffer (100 mmol/L pH 7´0) in 1:1 ratio and its ®nal pH was adjusted to between 5´0 and 7´0 by adding 5 mol/L NaOH dropwise with constant mixing. To prevent oxalogenesis from ascorbate present in the urine, 0´1 mL of buffered NaNO 2 (35 mg/10 mL in 10 mmol/L sodium phosphate buffer pH 7´0) was added to 2´0 mL of diluted urine.The colour reagent f...