Department of Ecological and Biological Sciences, University of Tuscia, Viterbo, Italy.
Blood Transfus. 2013 Jan;11(1):75-87. doi: 10.2450/2012.0164-11. Epub 2012 Jul 11.
It has long been known that red blood cells comprise various subpopulations, which can be separated through Percoll density gradients.
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.
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.
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.
长期以来，人们一直知道红细胞包含各种亚群，可以通过 Percoll 密度梯度分离。
在这项研究中，我们对通过 Percoll 密度梯度分离新鲜白细胞去除的红细胞浓缩物获得的五个不同的红细胞亚群进行了综合流式细胞术、蛋白质组学和代谢组学分析。通过带 4.1a/4.1b 测定法证实了密度梯度级分与细胞年龄的关系。
我们观察到较老（较密）群体的细胞大小减小，细胞粗糙度增加。对第 5 级分（最古老的群体）的代谢组学分析显示糖酵解代谢和抗氧化防御相关机制减少，导致戊糖磷酸途径的激活减少，NADPH 和还原型谷胱甘肽的积累减少，氧化型谷胱甘肽水平增加。这些观察结果加强了关于氧化应激在体内红细胞衰老中的作用的结论，与最近的体外研究结果相似。另一方面，各级分之间没有明显的蛋白质组学差异。这一结果部分解释了二维凝胶电泳方法的内在技术限制以及可能从血液中清除具有膜蛋白改变的红细胞。由于这种清除效应在体外不存在（在血库条件下），蛋白质组学研究表明体外衰老红细胞的膜损伤严重。
该分析表明，占总 RBC 超过 92%的三个主要红细胞亚群在刚抽出后就相当均匀。体内与年龄相关的主要变化可能通过翻译后机制而不是通过 RBC 整体蛋白质组学谱的变化来影响酶活性。
