Improved method for determining erythrocyte creatine by the diacetyl-alpha-naphthol reaction: elimination of...

We describe a simple, accurate, and reproducible method for determining erythrocyte creatine. The method is free from glutathione inhibition and is adaptable to use with standard spectrophotometers as well as centrifugal analyzers. A clear filtrate, essentially free of protein, hemoglobin, and glutathione, is prepared from 0.1 mL of packed erythrocytes by treatment with Ba(OH)2 and ZnSO4, then reacted with diacetyl-alpha-naphthol. The standard curve for this method is linear from 10 to 500 mg/L. We show that endogenous sulfhydryl species such as erythrocyte glutathione will interfere with the creatine-diacetyl-alpha-naphthol reaction. This observation confirmed a suspicion of underestimation of erythrocyte creatine by the method of Griffiths (Clin. Chim. Acta 9: 210, 1964). Added p-chloromercuribenzoic acid did not completely eliminate this inhibition. In the present method these interfering sulfhydryl species are eliminated from the reaction mixture, thus obviating the need for p-chloromercuribenzoic acid and dialysis. The reference interval for this method is 42-80 mg/L.