The mechanism of decline of age-dependent enzymes in the red blood cell.

Buoyant density centrifugation on discontinuous gradients separates red blood cells (RBCs) according to age, as shown by radiolabelling experiments both in vitro and in vivo. Changes observed in these gradients reflect in vivo rates of decline. A progressive metabolic decline may render the RBC incapable of surviving stresses in the circulation. It was hypothesized that changes only take place at the reticulocyte-mature RBC transition. RBC hexokinase (HK) has two isozymes, one predominant in reticulocytes, the other in mature RBCs. We compared its decline in the density gradient, with that of pyrimidine-5'-nucleotidase (P5N), glutamate-oxaloacetate transaminase (GOT) and pyruvate kinase (PK). The decline of HK and P5N was clearly biphasic; for GOT and PK instead there was a single slope. Thus changes taking place at the reticulocyte-RBC transition are clearly identified by a biphasic slope in the gradient. The view of a progressive metabolic decline in vivo for the RBC therefore remains valid.