Spectrophotometric determination of oxalate in urine and plasma with oxalate oxidase.

Abstract

In order to establish a standard procedure for the spectrophotometric determination of urinary and plasma oxalate with oxalate oxidase (Laker, M.F., et al. (1980) Clin. Chem. 26, 827-830; Sugiura, M., et al. (1980) Clin. Chim. Acta 105, 393-399) and to define the limitations of the method, the procedures and reactions involved in the assay have been examined. Among the chromogenic hydrogen donors for peroxidase tested, a combination of 3-methyl-2-benzothiazolinone hydrazone (MBTH) and sodium N-sulfopropylaniline (HALPS) was found to be best for the oxalate determination under the conditions used. Urine contained substance(s) which were inhibitory to the measurement of hydrogen peroxide by the peroxidase-catalyzed oxidative condensation of MBTH and HALPS, but they were largely removed by charcoal treatment at pH 5.6 without significant loss of oxalate. Deproteinization of plasma was carried out by ultrafiltration through a membrane cone (Centriflo CF-25) at neutral pH. The plasma oxalate ultrafiltrability under the conditions employed was calculated to be approximately 95%. A standard assay system for oxalate in these urine and plasma samples was then set up based on a series of studies on the reactions involved in the assay. In the case of normal plasma, however, the absorbance change was very small due to the low concentration of oxalate, and in addition, pretreatment of plasma with excess oxalate decarboxylase followed by the ultrafiltration and oxalate determination did not abolish completely the oxalate oxidase-dependent absorbance increase. It was concluded that the enzymic method was useful for the assay of urinary oxalate and in detecting elevated levels of plasma oxalate such as those in hemodialysis patients but was not sensitive enough to determine accurately the normal or decreased level of oxalate in plasma. The apparent concentration of oxalate in normal human plasma was measured in this work as 3.5 +/- 0.8 microM (mean +/- S.D., n = 8), and this result was interpreted to mean that the concentration of plasma oxalate was less than approximately 3.5 microM, as estimated by the present method.