Sensitive Spectrophotometric Assay for Plasma Oxalate

Abstract

Precise measurement of plasma oxalate is difficult because the concentration in healthy humans is fairly low (1–3 μmol/L) (1)(2). A colorimetric enzymatic assay that uses oxalate oxidase is commonly used for oxalate detection (2)(3). This assay is fairly straightforward for detecting urinary oxalate, which occurs at concentrations in the millimolar range. Plasma oxalate, however, occurs at concentrations in the micromolar range, and signals generated by oxidase degradation are difficult to detect. Our reference laboratory previously used an oxalate oxidase–based assay that required enzyme immobilization on a nylon coil and uses an HPLC with spectrophotometric detection (1). This method has several disadvantages, including limited automation potential, and requires subjective estimation of peak size. We therefore took advantage of the enhanced sensitivity of currently available spectrophotometers (absorbance measured down to a sensitivity of 0.0001) to develop a plasma assay that uses soluble oxalate oxidase (1)(2)(3)(4). The reagents used were as follows: oxalate reagents A and B and the Oxalate Urine Control Elevated, purchased from Trinity Biotech; hydrochloric acid (0.01 and 12 mol/L), sodium hydroxide (10 mol/L), potassium citrate monohydrate (crystalline; formula weight 324.22), EDTA disodium salt dihydrate (crystalline powder; formula weight 372.24), citric acid monohydrate (granular; formula weight 210.14), and sodium nitrite (crystalline; formula weight 69.00) from Fisher; and oxalic acid dihydrate (formula weight 126.07) and Triton from Sigma. Disposable polystyrene semimicro (1.5 mL) cuvettes were purchased through Fisher. Absorbance was measured with the Beckman Coulter DU800 UV/Visible Spectrophotometer. Stock citrate buffer (0.33 mol/L) was prepared by dissolving 30.5 g of potassium citrate, 50 g of citric acid, and 2 g of EDTA disodium salt in 1 L of distilled, deionized H2O, with a mean (SD) resulting pH of 3.3 (0.2). Working citrate buffer (0.066 mol/L) was prepared from …