Biochemical characterization of size-separated human red blood cells Vaysse J , Gattegno L, Pilardeau P. Biochemical characterization of size-separated human red blood cells. Eur J Haematol 1992: 48: 83-86. Abstract: Human red blood cells (RBC) are heterogeneous with respect to their size; the physiological significance of this heterogeneity has not yet been fully elucidated. To further investigate this problem, some characteristics of human RBC fractionated according to their mean corpuscular volume (MCV) by counterflow centrifugation were determined. Larger RBC are more prone to hypotonic lysis. The membrane protein content per cell increases with the MCV, but no obvious difference in the distribution of the major proteins can be demonstrated. The lipid content per cell also rises with the RBC size, while the percentages of the main lipid components d o not significantly vary. However, the variations of sialic acid content per RBC according to MCV are more important than those of protein or lipid; thus, the sialic acid-to-protein ratio gradually increases with the MCV. This indicates that, in spite of the lack of major changes in the membrane composition, some qualitative differences exist between large and small cells.
Jenny Vaysse’, Liliane Gattegno’ and Paul Pilardeau
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Laboratoire de Biochimie, HBpital Jean Verdier, Bondy, * Laboratoire de Biologie Cellulaire, Faculte de MBdecine, Bobigny France
Key words: red blood cell - size - elutriation - osmotic fragility - sialic acid - lipid - protein - membrane
Correspondence: Jenny Vaysse, Laboratoire de Biochimie, HBpital Jean Verdier, 93 140 Bondy, France Accepted for publication 13 November 1991
Introduction
Size-separated RBC characteristics
Human RBC are heterogeneous with respect to density, size, osmotic fragility, rheological behavior, metabolism and function. The causes and the physiological importance of this heterogeneity remain controversial. To investigate this problem, many fractionation procedures based on differences in some physical properties have been described; they allow the collection of more homogeneous RBC populations used for biochemical, physiological and hematological studies. Fractionation according to RBC density (1) or to RBC volume (2) has been shown to result in separation according to cell age; thus, comparison of the characteristics of density- or size-fractionated RBC may help to understand the mechanism of the in viva RBC ageing process. While properties of density-separated RBC have been extensively studied (for review, 3), little is known about those of size-separated RBC (4, 5); the present study was thus designed to further investigate this topic.
Ghosts were prepared from the RBC subpopulations according to Dodge ( 6 ) . The total protein content was determined by the Lowry method. Proteins were fractionated by electrophoresis (7) on 4-30 % SDS-polyacrylamide gels stained with Coomassie blue or periodate-Schiff and scanned by densitometry. Total sialic acid was measured according to Warren (8). Cholesterol and choline-containing phospholipids were estimated on ghosts by enzymatic procedures (Boehringer Mannheim France, 38420 Meylan, France). Lipids were extracted from ghosts and analyzed by thin-layer chromatography (9). RBC osmotic fragility was studied according to Dacie and Lewis (10). Mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC) and mean corpuscular hemoglobin content (MCH) were determined with a Coulter Counter Model S (Coultronics, 95 Margency, France).
Material and methods
RBC fractionation according to size
Human RBC were fractionated according to size by elutriation as previously described (5).
Statistics
Significance of the variations was assessed by variance analysis. Linear regression and the Spearman rank correlation test were used to evaluate the relationships between the different parameters. 83
Vaysse et al. Results
The membrane protein content expressed as mg/ RBC (Fig. 1) linearly increases with the MCV (p
Jenny Vaysse’, Liliane Gattegno’ and Paul Pilardeau
’
’
Laboratoire de Biochimie, HBpital Jean Verdier, Bondy, * Laboratoire de Biologie Cellulaire, Faculte de MBdecine, Bobigny France
Key words: red blood cell - size - elutriation - osmotic fragility - sialic acid - lipid - protein - membrane
Correspondence: Jenny Vaysse, Laboratoire de Biochimie, HBpital Jean Verdier, 93 140 Bondy, France Accepted for publication 13 November 1991
Introduction
Size-separated RBC characteristics
Human RBC are heterogeneous with respect to density, size, osmotic fragility, rheological behavior, metabolism and function. The causes and the physiological importance of this heterogeneity remain controversial. To investigate this problem, many fractionation procedures based on differences in some physical properties have been described; they allow the collection of more homogeneous RBC populations used for biochemical, physiological and hematological studies. Fractionation according to RBC density (1) or to RBC volume (2) has been shown to result in separation according to cell age; thus, comparison of the characteristics of density- or size-fractionated RBC may help to understand the mechanism of the in viva RBC ageing process. While properties of density-separated RBC have been extensively studied (for review, 3), little is known about those of size-separated RBC (4, 5); the present study was thus designed to further investigate this topic.
Ghosts were prepared from the RBC subpopulations according to Dodge ( 6 ) . The total protein content was determined by the Lowry method. Proteins were fractionated by electrophoresis (7) on 4-30 % SDS-polyacrylamide gels stained with Coomassie blue or periodate-Schiff and scanned by densitometry. Total sialic acid was measured according to Warren (8). Cholesterol and choline-containing phospholipids were estimated on ghosts by enzymatic procedures (Boehringer Mannheim France, 38420 Meylan, France). Lipids were extracted from ghosts and analyzed by thin-layer chromatography (9). RBC osmotic fragility was studied according to Dacie and Lewis (10). Mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC) and mean corpuscular hemoglobin content (MCH) were determined with a Coulter Counter Model S (Coultronics, 95 Margency, France).
Material and methods
RBC fractionation according to size
Human RBC were fractionated according to size by elutriation as previously described (5).
Statistics
Significance of the variations was assessed by variance analysis. Linear regression and the Spearman rank correlation test were used to evaluate the relationships between the different parameters. 83
Vaysse et al. Results
The membrane protein content expressed as mg/ RBC (Fig. 1) linearly increases with the MCV (p
