Glycolytic activity in human red cell populations separated by a combination of density and counterflow centrifugation. Evidence for an improved separation of red cells according to age

Abstract

Human red blood cells were separated by a discontinuous Percoll density gradient into fractions of increasing density. Red cells comprising the lowest and highest density fractions, respectively, were subsequently separated according to mean cell volume (MCV) by means of counterflow centrifugation. The activities of 4 red cell age-dependent enzymes (hexokinase (HK), pyruvate kinase (PK), glucose-6-phosphate dehydrogenase (G6PD) and aspartate aminotransferase (ASAT)) were highest in the red cell fraction with low density/large MCV, although the difference from red cell enzyme activities in the total low density fraction was not significant. These 4 enzyme activities were lowest in the fraction of red cells with high density/small MCV. The relative activities of the enzymes in the high density/small MCV fraction, as compared to the unseparated cell population, were: HK (58%), PK (49%), G6PD (53%) and ASAT (28%). These activities were all significantly lower than those measured in the total high density red cell fraction. The rates of lactate production in the low density/large MCV cells (0.89 ± 0.15 μmol.min¹.10⁻¹ cells) is approximately 3-fold higher than in high density/small MCV cells (0.33 ± 0.03 μmol.min⁻¹ .10⁻¹¹ cells). This latter value is 1.8-fold lower than the rate of lactate production in the total high density red cell fraction (0.59 ± 0.14 μmol.min⁻¹.10⁻¹¹ cells) and is, in contrast to lactate production in other density/size fractions, insensitive to phosphate as a metabolic stimulus. It is argued that the combination of density gradient and counter-flow centrifugation offers a greater potential for obtaining an old red cell population than classical red cell density centrifugation alone.