The Elevation of Adenosine-Triphosphate Levels in Human Erythrocytes

It has previously been possible to doublethe level of ATP in human erythrocytes byincubation of the cells at 37° for 10 hr withglucose and adenine. The present study describes a further increase in the ATP leveland some of the possible mechanisms involved. Addition of 5 mM pyruvate to amedium containing 32 mM inorganic phosphate, glucose, and adenine elevated thelevel of ATP threefold during a 10-hr incubation. Pyruvate could be replaced byinosine but the presence of both limitedthe elevation of ATP to twice that of freshcells. This limitation may be overcome bythe use of 96 mM phosphate in the incubation medium, in which case the intracellular level of ATP is tripled within 2 hr. Theconditions which limit the accumulation ofATP are associated with low intracellularphosphate concentrations and the accumulation of organic phosphates, especially, inthe presence of inosine, 2,3-diphosphoglycerate. Utilizing ¹⁴C-glucose labeled incarbons 1, 2, or 6, it has been shown thatwhen ATP is being rapidly elevated, thepentose moiety of the adenine nucleotidesis mainly supplied (about 80%) by oxidationof carbon 1 of glucose, catalyzed by thedehydrogenases of the hexosemonophosphate shunt. In the presence of pyruvatethis activity is doubled. Pyruvate reoxidizesNADPH formed by this pathway, sincelactic dehydrogenase has some specificitytowards the NADPH. The involvement ofthe dehydrogenases of the hexosemonophosphate shunt is illustrated by the useof erythrocytes deficient in glucose-6-phosphate dehydrogenase. Incubation ofthese cells for 5 hr with glucose andadenine results in only a slight increase inATP formation, and pyruvate has no additional effect. Addition of inosine, however,leads to the same increment in ATP levelsseen in normal cells. The ATP and 2,3-diphosphoglycerate levels in 6-wk preserved blood can also be increased to threetimes that of fresh cells by incubation withglucose, adenine, pyruvate, and inosine ina medium high in inorganic phosphate.

Submitted on January 26, 1973 Revised on March 27, 1973 Accepted on March 28, 1973