Reduced water exchange in sickle cell anemia red cells: a membrane abnormality.

We have measured the diffusional water permeability of sickle cell anemia red blood cells under isotonic conditions using pulsed nuclear magnetic resonance (NMR) techniques. We have found that the equilibrium diffusional permeability for sickle cells is about 1.61.10(-3) cm/s, or about 60% of the value measured for normal cells. This abnormality is not related to the heterogeneity generally found in cell populations in sickle red cells with different mean corpuscular hemoglobin concentrations. We speculate that the abnormality of water exchange under isotonic conditions in sickle cells reflects an alteration of membrane proteins responsible for water exchange, possibly caused by oxidation of Band 3 proteins.

Membrane abnormality of erythrocytes is highly dependent on salt intake and renin profile in essential hypertension: an electron spin resonance study.

The purpose of the present study was to investigate membrane fluidity in essential hypertension using electron spin resonance (ESR) and spin-labelling. Erythrocytes from patients with untreated essential hypertension were examined and compared with age-matched normotensive subjects. The values of outer hyperfine splitting (2T') and order parameter (S) of the ESR spectra for a fatty acid spin label agent (5-nitroxy stearate) were significantly higher in essential hypertension than in the normotensive subjects. However, these values were not changed in secondary hypertension. This finding indicates that the membrane fluidity of erythrocytes was lower in essential hypertension. Further, the abnormality was attenuated with low-salt intake, and, on the contrary, was more prominent with high-salt intake in essential hypertension. Calcium loading to erythrocytes in vitro caused a greater decrease in the membrane fluidity in essential hypertension than in the normotensive controls. This calcium-induced change in the membrane fluidity was significantly inversely correlated with the value of plasma renin activity in essential hypertension. These results suggest that abnormality in the membrane fluidity might be emphasized in the presence of calcium, especially in low-renin essential hypertension, implying enhanced calcium sensitivity in this type of hypertension.

Erythrocyte membrane fluidity decreased in uremic hemodialyzed patients.

Erythrocyte membrane fluidity was studied by means of electron spin resonance in 15 uremic, hemodialyzed patients and 14 normal subjects. Erythrocyte membrane fluidity determined using a 16-nitroxide stearic acid spin label probe was of a significantly lower level in the uremic patients, when compared with normal control subjects. Alterations in molar ratios of membrane free cholesterol to phospholipid are probably not a principal factor contributing to this change in fluidity. Significant decreases of phosphatidylcholine and molar ratios of phosphatidylcholine to sphingomyelin were noted in the erythrocyte membrane of uremic patients, and these alterations may relate to the fluidity change.

Decreased sodium influx and abnormal red cell membrane lipids in a patient with familial plasma lecithin: cholesterol acyltransferase deficiency.

Red cell membrane metabolism in familial lecithin:cholesterol acyltransferase (LCAT) deficiency was investigated. The family presented here is the third case discovered in Japan. An increase of free cholesterol was observed in the red cell membranes, concomitant with increased phosphatidyl choline. Osmotic fragility of the patient's red cells was diminished rather than increased. Red cell survival (51Cr T1/2) was shortened (15 days). Sodium influx was markedly decreased, although sodium efflux, both ouabain-sensitive and ouabain-insensitive, was normal. The activity of acetyl-cholinesterase as a marker of the outer leaflet of the red cell membranes was decreased, while the activity of glyceraldehyde-3-phosphate dehydrogenase as a marker of the inner leaflet was normal. No abnormalities of adenosine triphosphatases in red cell membranes were observed. These results suggest that the alteration of cholesterol metabolism in the plasma of LCAT deficiency increases the red cell membrane cholesterol and affects the functions of the red cell membranes, especially of the outer leaflet, which may result in decreased sodium influx.

Decreased erythrocyte membrane fluidity and altered lipid composition in human liver disease.

Abnormal plasma lipoproteins in patients with liver disease are associated with characteristic changes in erythrocyte membrane lipid composition. The membranes are enriched in cholesterol and phosphatidylcholine and both the cholesterol/phospholipid and phosphatidylcholine/sphingomyelin molar ratios are increased. Phospholipid fatty acid composition is also abnormal; the proportions of arachidonic acid and stearic acid are decreased and that of palmitic acid raised. In this study we have examined the effects of these membrane lipid abnormalities on membrane fluidity. Erythrocyte membrane fluidity was assessed in 30 patients with a variety of liver diseases and in 25 normal subjects using the hydrophobic, fluorescent probe 1,6-diphenylhexa-1,3,5-triene and the values were related to their lipid composition. Membrane fluidity was significantly decreased in the patient erythrocytes (lipid order parameter, S(v)[37 degrees C] = 0.713 +/- 0.018, mean +/- S.D. compared to 0.686 +/- 0.008 in the normal subjects, P < 0.001) and correlated significantly with the cholesterol/phospholipid ratio (r = 0.88, P < 0.001). The fluidity of lipid extracts from the membranes of patient erythrocytes was also decreased, suggesting that decreased membrane fluidity was mainly a consequence of altered lipid composition rather than protein abnormalities. Incubation of patient erythrocytes for 20 hr with normal, heated plasma removed the excess cholesterol without affecting the phosphatidylcholine/sphingomyelin ratio or phospholipid fatty acid composition; following incubation the fluidity of these membranes was similar to that of normal membranes. We conclude that in liver disease changes in the composition of the phospholipid bilayer matrix in the erythrocyte membrane have little influence on its fluidity; the reduced fluidity is predominantly a result of increases in cholesterol relative to phospholipid.-Owen, J. S., K. R. Bruckdorfer, R. C. Day, and N. McIntyre. Decreased erythrocyte membrane fluidity and altered lipid composition in human liver disease.

Myotonic muscular dystrophy. Time-dependent alterations in erythrocyte membrane fluidity.

The muscle cell membrane may be the site of the basic molecular defect in myotonic muscular dystrophy. Many laboratories, including our own, have suggested that this defect may also be manifested in membrane of extraneural tissue. In previous studies, we found that electron spin resonance results suggested an increased membrane fluidity in erythrocyte membranes that had aged two days in buffer, but we and others could find no such changes in fresh erythrocyte membranes. To investigate these findings further, the results of an initial study of the time course of the membrane fluidity changes in erythrocytes in myotonic muscular dystrophy are given in the present report. They suggested that increased membrane fluidity changes in erythrocytes in myotonic muscular dystrophy are given in the present report. They suggested that increased membrane fluidity in myotonic dystrophy is manifested after two days of in vivo ageing and confirm our original findings. These results are discussed in relation to possible effects of metabolic deprivation or of protein-lipid alterations in erythrocytes.

Functional study of lipids of PNH red cell membranes: susceptibility of liposomes to reactive lysis.

Lipids extracted with chloroform-methanol from red blood cell membranes of 7 PNH and 13 control subjects were used for the preparation of liposomes, which were then examined with the reactive lysis test. PNH liposomes lysed to a higher extent than control liposomes as indicated by the higher dilution of the limiting complement reagent that was necessary to lyse 50% of the PNH liposomes. A similar finding was also observed with liposomes made of lipids from AET-treated red cells. The enhanced reactive lysis can be attributed to the polar lipid fraction, as indicated by the increased lysis of hybrid liposomes prepared from this polar lipids extracted from PNH erythrocyte membrane and lipids extracted from normal erythrocyte membrane. The increased susceptibility to reactive lysis does not seem to be specific of PNH liposomes, since it was also observed with liposomes prepared from lipids of red cells from beta-thalassemia major and autoimmune hemolytic disease.

Red cell membrane protein changes caused by freezing and the mechanism of cryoprotection by glycerol.

Membranes isolated from frozen-thawed erythrocytes and analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate have significantly decreased band six, which is the glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase. The total membrane protein and sialic acid contents of these membranes are also significantly decreased. The red blood cell membrane protein abnormality is reproduced by suspending membranes in NaCl solutions of increasing molarity. Glycerol prevents the elution of band six in NaCl solutions less than 0.2 M and ameliorates it in solutions of higher ionic strength. When intact cells are suspended in hypertonic salt solution, there is no elution of band six, indicating that exposure of the inner surface of the membrane to toxic concentrations of solutes results in this elution. The data indicate that freezing with its associated hypertonicity induces a specific membrane change which is ameliorated by the addition of glycerol.

Simplified E-UFA test for multiple sclerosis (MS): some sources of "false" results.

A simple modification of the Erythrocyte-Unsaturated Fatty Acid (E-UFA) Test for Multiple Sclerosis is described, whereby well washed erythrocytes (RBC) are allowed to stand in Hanks medium 199, for about 21 days at 4 degrees C. The control-experimental difference in electrophoretic mobility of RBC rises to nearly 20%. Precautions in interpretation and wider implications of the SE-UFA test, recently uncovered, are briefly indicated.

Inherited erythrocyte metabolic and membrane disorders.

Erythrocyte metabolic defects which lead to hemolytic anemia are abnormalities of the glycolytic pathway, of the pentose phosphate pathway, and of nucleotide metabolism. Hemolytic anemias may also be caused by inherited defects involving the membrane and membrane abnormalities secondary to intracellular abnormalities and inclusions. The more common disorders are focused upon, and newer findings impacting on the field are discussed.

Erythrocyte cellular and membrane deformability in hereditary spherocytosis.

In order to determine whether the relative rigidity of the hereditary spherocytosis (HS) red cell is due to membrane rididity or merely to an altered surface/volume ratio, we investigated the deformability of resealed red cell membranes from patients with HS. Whereas the osmotic fragility of intact red cells of HS patients showed the expected increase, the osmotic fragility of resealed HS membranes was normal, thus indicating that their surface/volume ratio was normal. Measurements with an ektacytometer showed that deformability of intact HS cells was markedly diminished, whereas deformability of resealed HS membranes was normal. These findings indicate that the HS red cell membrane is not intrinsically abnormally rigid, as has been suggested, but that the lack of deformability of the erythrocyte is primarily a function of the altered surface/volume ratio.