A Comparison of Normal Red Cell ATP Levels as Measured by the Firefly System and the Hexokinase System

By ERNEST BEUTLER AND CLARAMMA K. MATHAI T HE ADENOSINE TRIPHOSPHATE (ATP) content of erythrocytes is of considerable interest from the viewpoint of red cell preservation, hereditaly abnormalities of red cell metabolism, such as pyruvate kinase deficiency, and certain genetic disorders characterized by increased or decreased red cell ATP levels. Several internally consistent methods are available for the measurement of red cell ATP. These methods include: ( 1 ) paper and column chromatography; ( 2 ) measuring glucose-6-phosphate formation in the hexokinase reaction with glucose-6-phosphate dehydrogenase ( G-6-PD ) and TPN; (3 ) employing the “backwards” glyceraldehyde phosphate dehydrogenase reaction to measure the oxidation of DPNH; and ( 4 ) the firefly technic. Each of these technics has been reported to give reproducible results with human blood, and adequate recoveries of ATP added to blood have been achieved. The simplest and most reproducible of these methods is the firefly luciferase method; yet, this system yields results which are considerably higher than those reported using other technics (Table 1). The firefly system differs from all other methods in use in that the blood is added directly to a buffer and boiled without washing of red cells or preparation of a deproteinized filtrate. The discrepancy between results obtained with the firefly technic and other methods could thus be due to lack of specificity of the firefly reaction, to destruction of ATP during processing by the other methods, or to incomplete extraction of ATP from red cells. It has also been suggested that the presence of hemoglobin may result in falsely high ATP levels when the firefly technic is employed. It is the purpose of this report to investigate some of the possible causes of discrepancies between red cell ATP levels as determined by the firefly technic and red cell ATP level as estimated by other methods. The hexokinase technic has been employed for comparative purposes.