Regeneration of phosphorylated metabolites in stored erythrocytes in an open perfusion system: studies using 31P NMR spectroscopy

Human erythrocytes were maintained at high haematocrit in a metabolically functional state for several hours in a thermodynamically open perfusion apparatus. The concentrations of ATP and 2,3-bisphosphoglycerate (2,3-DPG) and pH were continuously monitored before and after metabolic perturbations by using 31P NMR; the monitoring was achieved with a 31P flow-through probe. Methylphosphonate was added to plasma perfusion medium as a phosphorus concentration standard and as a 31P NMR pH probe molecule. The rates of decline of ATP and 2,3-DPG levels in fresh cells in a glucose-free medium were measured as were the rates of reformation in response to a 'rejuvenation' medium. Also, rates of ATP and 2,3-DPG synthesis during perfusion with Krebs bicarbonate-0.5 mmol/l glucose and perfusion with pooled plasma were measured in cells that had been previously stored at 4 degrees C for 5 weeks.