Measurement of erythrocyte glucose 6 phosphate dehydrogenase activity with a centrifugal analyzer

Abstract

Two improved methods for determination of erythrocyte glucose 6 phosphate dehydrogenase activity are described. One method is an 'enzyme linked' procedure in which an excess of 6 phosphogluconate dehydrogenase is used to produce 2 moles of NADPH for each mole of glucose 6 phosphate oxidized. In the other method 2,3 diphosphoglycerate is used to inhibit the variable contribution of endogenous 6 phosphogluconate dehydrogenase to glucose 6 phosphate dehydrogenase activity in erythrocyte lysates. These assays require 100 μl of blood and are performed on a centrifugal analyzer in a final reaction volume of 410 μl at 37 °C. NADPH formation is monitored at 340 nm. Hemoglobin is measured as oxyhemoglobin in the same reaction mixture used to determine enzyme activity by changing the wavelength to 550 nm. Results are expressed as international units of glucose 6 phosphate dehydrogenase activity per gram of hemoglobin. The coefficient of correlation between 'enzyme linked' assay and 'standard assay' was .992, the slope of the regression line was 1.07, and the intercept was at -0.76. When results of the 'enzyme linked' assay were compared to those of the 'nonlinked' assay with added 2,3 diphosphoglycerate, the slope of the regression line was .994, the intercept 0.109, and the correlation coefficient .994.