Simple and Rapid Method for Determination of Arginase Activity in a Dried Blood Spot on Filter Paper and Hemolysate

Abstract

A semi-automated method for arginase activity in a dried blood spot and hemolysate is described. Urea produced by arginase action was measured with the enzymes of urease and glutamate dehydrogenase, 2-oxoglutarate and NADH. The decrease in absorbance at 340nm at 30°C was measured by a centrifugal analyzer, the Centrifichem 400. The conditions of activation with Mn²⁺, incubation and urea determination are discussed. In dried blood spots, elution of erythrocyte and activation of arginase were performed by incubating discs at 37°C for 30min. We used 25mmol/L MnCI₂ to activate and 150mmol/L arginine as substrate to obtain the maximum velocity of arginase activity. As HCIO₄ was used for the stopper of the enzyme reaction, pH adjustment for the following enzymatic urea determination could be ommited. Linearity and reproducibility were acceptable. The correlation between arginase activities from dried blood disc and hemolysate in the same subjects was r=0.953. The reference values were 41.7±7.75U/g·Hb in dried blood disc and 39.3±9.44 U/g·Hb (mean±SD, n=20) in hemolysate. The activity from dried blood spot is stable at room temperature for one week, which allows to be mailed.