Assay for Hexokinase Activity in Intact Red Cells and Its Alterations on Storage.

To determine hexokinase activity in intact red cells, one may either measure the disappearance of glucose or the appearance of glucose 6-phosphate. The former is not absolutely specific for the hexokinase reaction. The latter can only be done after the cells are broken because phosphorylated compounds are reluctant to move across red cell membranes. Alternatively, studies of hexokinase activity can and have been made on hemolysates, but such studies violate intracellular environmental conditions including natural inhibitors, activators, and cofactors. For example, when red cells are hemolyeed, their adenosine triphosphate is rapidly destroyed (I), and adenosine triphosphate is as necessary for the phosphorylation of glucose as the enzyme, hexokinase. Our objective was to assay continuously the rate of formation of glucose 6-phosphate in intact red cells. The assay described in the present work depends on the continuing presence in the intact human red cell of adequate amounts of glucose-6-P dehydrogenase and triphosphopyridine nucleotide such that when methylene blue is added, oxygen uptake will become dependent on the rate of production of glucose-6-P. Oxygen uptake will then measure hexokinase activity. The use of methylene blue to enhance oxygen uptake via shunt pathways has been well established (2). Inasmuch as some standard assays for hexokinase activity in hemolysates are based on the spectrophotometric determination of reduced triphosphopyridine nucleotide in a suitably arranged system (3), it seemed reasonable to extend the principle to an assay based on following oxygen uptake of a suspension of intact red cells to which dye had been added. However, it remained to be established that adequate amounts of glucose-6-P dehydrogenase and triphosphopyridine nucleotide were already present inside the red cells, since adding them extracellularly would be of no use. Fortunately, inosine could be substituted for glucose as a glucose-6-Pgenerating source, and when this was done, the oxygen uptake with methylene blue proved the adequacy of supply of glucose6-P dehydrogenase and triphosphopyridine nucleotide. In subsequent utilization of this assay system, an aliquot was always run with inosine to insure adequacy of glucose-6-P dehydrogenase and triphosphopyridine nucleotide stores. Inasmuch as the hexokinase reaction is the first step in the conversion of glucose to lactate in the mammalian red cell, it is an attractive step to incriminate in the gradual failure of glycolysis that occurs in stored (banked) red cells. Both Denstedt and