Red blood cell subpopulations in freshly drawn blood: application of proteomics and metabolomics to a decades-long biological issue

Abstract

Background: It has long been known that red blood cells comprise various subpopulations, which can be separated through Percoll density gradients.

Materials and methods: In this study, we performed integrated flow cytometry, proteomic and metabolomic analyses on five distinct red blood cell subpopulations obtained by Percoll density gradient separation of freshly drawn leucocyte-depleted erythrocyte concentrates. The relation of density gradient fractions to cell age was confirmed through band 4.1a/4.1b assays.

Results: We observed a decrease in size and increase in cell rugosity in older (denser) populations. Metabolomic analysis of fraction 5 (the oldest population) showed a decrease of glycolytic metabolism and of anti-oxidant defence-related mechanisms, resulting in decreased activation of the pentose phosphate pathway, less accumulation of NADPH and reduced glutathione and increased levels of oxidized glutathione. These observations strengthen conclusions about the role of oxidative stress in erythrocyte ageing in vivo, in analogy with results of recent in vitro studies. On the other hand, no substantial proteomic differences were observed among fractions. This result was partly explained by intrinsic technical limitations of the two-dimensional gel electrophoresis approach and the probable clearance from the bloodstream of erythrocytes with membrane protein alterations. Since this clearance effect is not present in vitro (in blood bank conditions), proteomic studies have shown substantial membrane lesions in ageing red blood cells in vitro.

Conclusion: This analysis shows that the three main red blood cell subpopulations, accounting for over 92% of the total RBC, are rather homogeneous soon after withdrawal. Major age-related alterations in vivo probably affect enzyme activities through post-translational mechanisms rather than through changes in the overall proteomic profile of RBC.

Figure 1

Percoll density gradient of freshly drawn, leucocyte-filtered, RBC concentrates. Five distinct subpopulations are visible, which are numbered from top to bottom. The gradient was prepared by stacking layers of different densities, in agreement with Bosch et al.: 1.096 g/mL, 1.087 g/mL, 1.083 g/mL, 1.080 g/mL and 1.060 g/mL. Percentages of cell recovery are reported for each fractions as means+SD (total=100%). In the right panel, the graph reports densitometric analysis for the band 4.1a/4.1b ratio from the 1D-GE runs for each distinct subpopulation.

Figure 2

Flow cytometry analysis representing forward scattering (FS) and side scattering (SS) on the x and y axis, respectively, for the total red blood cell population (upper left frame) and for each one of the five fractions, as labelled. Each subpopulation has been delimited into a shape enclosing >95% of the counted events, and then superimposed in the frame labelled Total (upper left corner). Fractions 2 to 5 showed greater SS in comparison to fraction 1. The core of events is counted with a homogeneous distribution for fraction 1 as far as FS is concerned. For the other fractions, FS events are mainly shifted leftwards from the main axis (dotted line).

Figure 3

Two-dimensional gel electrophoresis of freshly drawn RBC after separation into five distinct subpopulations through a Percoll density gradient. First dimension isoelectric focusing pI values linearly span between 3 and 10, while molecular weights are indicated on the left.

Figure 4

Time-course metabolomic analyses of leucocyte-filtered RBC subpopulations after separation through a Percoll density gradient. Internal normalisation was performed against the average values for each metabolite among the five distinct subpopulations for all the tested individuals (results are plotted as means+SD). Abbreviations: F6P/G6P=fructose/glucose 6-phosphate; FBP=fructose 1,6 biphosphate; G3P=glyceraldehyde 3-phosphate; PEP=phosphoenolpyruvate; LH=lactate; ATP=adenosine triphosphate; NADH=reduced nicotinamide adenine dinucleotide; NADPH=nicotinamide adenine dinucleotide phosphate; PG=6-phosphogluconate; GSH=reduced glutathione; GSSG=oxidized glutathione; GLTM=glutamine; GLUT=glutamate.