The Elevation of Adenosine-Triphosphate Levels in Human Erythrocytes

It has previously been possible to double the level of ATP in human erythrocytes by incubation of the cells at 37° for 10 hr with glucose and adenine. The present study describes a further increase in the ATP level and some of the possible mechanisms involved. Addition of 5 mM pyruvate to a medium containing 32 mM inorganic phosphate, glucose, and adenine elevated the level of ATP threefold during a 10-hr incubation. Pyruvate could be replaced by inosine but the presence of both limited the elevation of ATP to twice that of fresh cells. This limitation may be overcome by the use of 96 mM phosphate in the incubation medium, in which case the intracellular level of ATP is tripled within 2 hr. The conditions which limit the accumulation of ATP are associated with low intracellular phosphate concentrations and the accumulation of organic phosphates, especially, in the presence of inosine, 2,3-diphosphoglycerate. Utilizing ¹⁴C-glucose labeled in carbons 1, 2, or 6, it has been shown that when ATP is being rapidly elevated, the pentose moiety of the adenine nucleotides is mainly supplied (about 80%) by oxidation of carbon 1 of glucose, catalyzed by the dehydrogenases of the hexosemonophosphate shunt. In the presence of pyruvate this activity is doubled. Pyruvate reoxidizes NADPH formed by this pathway, since lactic dehydrogenase has some specificity towards the NADPH. The involvement of the dehydrogenases of the hexosemonophosphate shunt is illustrated by the use of erythrocytes deficient in glucose-6-phosphate dehydrogenase. Incubation of these cells for 5 hr with glucose and adenine results in only a slight increase in ATP formation, and pyruvate has no additional effect. Addition of inosine, however, leads to the same increment in ATP levels seen in normal cells. The ATP and 2,3-diphosphoglycerate levels in 6-wk preserved blood can also be increased to three times that of fresh cells by incubation with glucose, adenine, pyruvate, and inosine in a medium high in inorganic phosphate.