Cohen N S, Ekholm J E, Luthra M G, Hanahan D J
Biochim Biophys Acta. 1976 Jan 21;419(2):229-42. doi: 10.1016/0005-2736(76)90349-7.
A simple, reproducible method for the separation of human erythrocytes, described recently (Murphy, J. R. (1973) J. Lab. Clin. Med. 82, 334-341) has been utilized for the purpose of obtaining a wide range of biochemical data on these cells. Using phthalate ester density centrifugation of the fractions obtained by Murphy's method, we established that the cells were separated exclusively on the basis of their densities. Data on a wide range of biochemical and hematological parameters, when compared with previously reported density separation procedures showed that this simple technique can be used to fractionate the cells according to their densities (age) in their own plasma. Cells of increasing density consistently and reproducibly exhibited an increase in hemoglobin concentration, a moderate elevation in Na+ and a decrease in the following: K+, acetylcholinesterase, sialic acid, membrane protein, 2,3-diphosphoglycerate, ATP, cholesterol, phospholipid, mean corpuscular volume and critical hemolytic volume, However, no change in mean corpuscular hemoglobin was evident. The observed differences were not artifacts of the centrifugation process. This was determined in recentrifuged top fractions from which new top and bottom cells were obtained. The latter cells resembled the top fraction from which they were obtained, rather than the original bottom fraction. Whereas the parameters mentioned above exhibited consistency and reproducibility, such was not the case with the ATPase values. Depending on the cell density group examined and/or buffer as well as other conditions, significant variability in the activity levels of the ouabain sensitive, as well as the Ca2+ -stimulated ATPase, was observed. Use of these enzyme activities as indicators of cell age must be viewed with caution.
最近描述的一种简单、可重复的人红细胞分离方法（Murphy，J. R.（1973年）《实验室与临床医学杂志》82卷，334 - 341页）已被用于获取这些细胞的广泛生化数据。通过邻苯二甲酸酯密度离心法处理墨菲方法得到的组分，我们确定细胞完全是根据其密度进行分离的。与先前报道的密度分离程序相比，关于广泛的生化和血液学参数的数据表明，这种简单技术可用于根据细胞在自身血浆中的密度（年龄）对细胞进行分级分离。密度增加的细胞始终且可重复地表现出血红蛋白浓度升高、Na⁺适度升高以及以下各项降低：K⁺、乙酰胆碱酯酶、唾液酸、膜蛋白、2,3 - 二磷酸甘油酸、ATP、胆固醇、磷脂、平均红细胞体积和临界溶血体积。然而，平均红细胞血红蛋白未见明显变化。观察到的差异不是离心过程的假象。这是通过对重新离心的上层组分进行测定得出的，从中获得了新的上层和下层细胞。后者细胞类似于从中获得它们的上层组分，而不是原始的下层组分。虽然上述参数表现出一致性和可重复性，但ATP酶值并非如此。根据所检查的细胞密度组和/或缓冲液以及其他条件，观察到哇巴因敏感型以及Ca²⁺刺激型ATP酶的活性水平存在显著变异性。将这些酶活性用作细胞年龄指标时必须谨慎看待。
