Tetsuro Kamada

Dietary Sardine Oil Increases Erythrocyte Membrane Fluidity in Diabetic Patients

The effects of dietary sardine oil rich in eicosapentaenoic acid, C20:5 (EPA), on erythrocyte membrane fluidity and membrane and plasma lipids were investigated in diabetic and control subjects. Before consumption of this oil, the levels of erythrocyte membrane fluidity were lower in the diabetic subjects, as noted in our previous work (Diabetes 1983; 32:585–91). Decreased membrane polyunsaturated fatty acid contents were evident. Daily consumption of 2700 mg of sardine oil for 8 wk increased erythrocyte membrane fluidity, as determined by electron spin resonance using the 12- or 16- stearic acid label. This increase was seen after 4 wk, and the level remained elevated for 8 wk. Membrane EPA of phospholipid acyl-chains significantly increased after 4 wk and was even more apparent after 8 wk. Membrane-free cholesterol to phospholipid molar ratios significantly decreased after 8 wk. Both the diabetic and normal subjects responded to the sardine oil in the same way. After feeding with sardine oil, there no longer were differences in erythrocyte membrane fluidity between, the normal and diabetic subjects. We propose that improvement in membrane fluidity may contribute to the amelioration of altered cell membrane functions in diabetic patients.

Higher Levels of Erythrocyte Membrane Microviscosity in Diabetes

Significantly higher levels of erythrocyte membrane microviscosity (MV) [η̅: 5.22 ± 0.17 (4.70–5.92), mean ± SD (range), poise, N = 67, P < 0.005] measured by fluorescence depolarization using 1,6-diphenyl-1,3,5-hexatriene as a fluorescent probe were found in diabetic patients when compared with normal controls [5.05 ± 0.15 (4.70–5.29), N = 22]. No significant differences in MV existed between males and females, nor was MV significantly correlated with diabetic age, duration of diabetes, plasma cholesterol, cholesterol/phospholipid ratios, and plasma lecithin :cholesterol acyltransferase activities. No significant difference in MV was observed between groups with or without diabetic retinopathy. There was, however, significantly higher MV [5.29 ± 0.19 (5.00–5.92), N = 20, P < 0.05] in the group with fasting blood glucose (FBG) > 140 mg/dl than that [5.19 ± 0.15 (4.70–5.46), N = 47] in the group with FBG < 140 mg/dl. The changes in erythrocyte membrane MV presented in this study appear to be related to the current metabolic control of diabetic patients and are considered to be one of the factors responsible for the reduced erythrocyte deformability in diabetes.

Lower Levels of Erythrocyte Membrane Fluidity in Diabetic Patients: A Spin Label Study

The dynamic properties of intact erythrocyte membrane in diabetic patients were investigated by means of electron spin resonance using three stearic acid spin labels (SAL): 5-, 12-, and 16-SAL. Significantly lower levels of erythrocyte membrane fluidity were revealed with 16-SAL as a probe in diabetic patients when compared with normal controls. However, there were no significant differences in fluidity values using 5- or 12-SAL between the two groups. Therefore, it became obvious that the decrease in fluidity was located in deeper sites (hydrophobic region) of the erythrocyte double membrane in diabetic patients. It was strongly suggested that changes in the membrane cholesterol to phospholipid molar ratios are not a principal factor contributing to the fluidity change. A significant increase of sphingomyelin and decrease of phosphatidylethanolamine were found in the erythrocyte membrane of diabetic patients and an alteration in membrane phospholipidclasses and their acyl-chains could conceivably be related to the fluidity change. There were no significant correlations betweenchange in membrane fluidity and most plasma lipids, plasma lecithin-cholesterol acyl-transferase activities erythrocyte glycosylated hemoglobin, erythrocyte adenosine triphosphate, fasting blood glucose or duration of the disease. Plasma cholesterol of high-density lipoprotein showed a significant negative correlation with the membrane fluidity values. Some of the possible factors contributing to and the significance of the lower levels of erythrocyte membrane fluidity were discussed in conjunction with both metabolic and clinical aspects in diabetic patients.

Lowered membrane fluidity of younger erythrocytes in diabetes

In vivo age-related changes in membrane fluidity of erythrocytes were investigated by a spin label method after fractionation of the cells by discontinuous density gradient centrifugation. Membrane fluidity was lower in older than in younger erythrocytes in both the normal and diabetic subjects. Cells from diabetic subjects showed a significantly lower level of membrane fluidity for all three age groups (younger, middle and older) than the corresponding cells from normal subjects. The magnitude of progression in the decrease in membrane fluidity in erythrocytes did not differ significantly between both groups of subjects. Both erythrocyte ATP and acetylcholinesterase activity declined, while glycosylated hemoglobin (HbA1c) increased with cell age in both groups of subjects. The HbA1c level in each corresponding fraction was higher in diabetic subjects than normal subjects, but was not correlated with membrane fluidity in either group. Neither the ATP level nor acetylcholinesterase activity in each corresponding fraction differed between groups. Membrane fluidity was significantly correlated with acetylcholinesterase activity in both normal and diabetic subjects. Our results indicate that decreased erythrocyte membrane fluidity in diabetic patients does not form gradually during their life span but develops soon after the cells enter the circulation or during their maturation in the bone marrow.

ERYTHROCYTE CRENATION INDUCED BY FREE FATTY ACIDS IN PATIENTS UNDERGOING EXTRACORPOREAL CIRCULATION

Normal erythrocyte morphology is necessary for proper distribution of blood-flow in the microcirculation. Erythrocyte shape was studied in 20 patients undergoing extracorporeal circulation (EC) during coronary bypass surgery. Crenated erythrocytes comprised a mean 64% of all erythrocytes during and 29% after EC. Free fatty acid (FFA) content of the erythrocyte membranes was significantly increased at both times, and FFA content correlated with the proportion of crenated erythrocytes. Washing crenated cells with defatted albumin solution transformed them back to discocytes, simultaneously removing more FFA than that removed from pre-EC discocytes. The plasma FFA to albumin ratio became disproportionately increased during EC; the increased level correlated with severity of erythrocyte crenation. Maintaining a higher level of albumin during EC by adding 50 g human albumin to the extracorporeal system prevented erythrocyte crenation. Entry of plasma FFA not bound to albumin into erythrocyte membranes during EC causes massive erythrocyte crenation.

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.

Metabolic dependence of the fluidity of intact erythrocyte membrane

The motion of spin labeled phosphatidylcholine embedded in the lipid bilayer of erythrocyte membrane increased in association with metabolic ATP-depletion. The fluidity change was reversed by subsequent ATP-replenishment. However, levels of cellular 1,2-diacylglycerol, GSH or intracellular Ca2+ contributed little to the fluidity change. Temperature dependence of the fluidity indicated the disappearance of inflection points in ATP-depleted cells, thereby suggesting alterations in protein-lipid interactions. The fluidity change was also reproduced with oxidizing agents which cross-link spectrin. We suggest that the lipid phase state of membrane is maintained by protein-lipid interactions which depend on the metabolic state of the cells, particularly ATP levels.

Qualitative and quantitative analysis of erythrocyte surface membrane sialyl residues using affinity cytochemistry with special reference to diabetic patients

SummaryErythrocyte surface membrane sialyl residues were investigated by means of affinity cytochemistry using the avidin-biotin complex technique. Mild oxidation with the periodate (MO)-biotin hydrazide (BHZ)-ferritin avidin conjugate (FAv) sequence revealed numerous ferritin particles on erythrocytes from healthy donors. The ferritin particles attached on the perpendicularly sectioned membrane were seen at an average distance of 10 to 12 nm from the outer dense leaflet of the cell membrane. Pretreatment with neuraminidase followed by the MO-BHZ-FAv sequence almost eliminated erythrocyte ferritin labeling. Erythrocytes from diabetic patients showed less dense ferritin labeling compared with those from healthy donors. Quantiative analysis of sialyl residues demonstrated a marked reduction in ferritin labeling of erythrocytes from diabetic patients which was significantly less (p< 0.01) than that of erythrocytes from healthy donors. This observation supports previous biochemical data demonstrating lower levels of surface membrane negative charge and sialyl residues on erythrocytes from patients with diabetes mellitus.

Changes in polyphosphoinositides and phosphatidic acid of erythrocyte membranes in diabetes

We studied metabolic pool size of polyphosphoinositides and phosphatidate of erythrocyte membranes from normal and diabetic subjects using 32P for 20-h incubation, a sufficiently long period to reach isotopic equilibrium between monoesterphosphate bond and gamma-phosphate of ATP. Phosphatidylinositol 4-monophosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and phosphatidate were the phospholipids labelled. Metabolic pools of individual phospholipids were estimated, based on their proportionate and absolute radioactivity. A significant decline in radioactivity of phosphatidate and PtdIns(4,5)P2 was seen in erythrocytes from the diabetic subjects, indicating suppression of the metabolically labile pool of these two phospholipids. There was no significant change in PtdIns4P radioactivity between the groups. The direct effect of insulin on phosphorylation of polyphosphoinositides and phosphatidate was also evaluated by a short incubation period of erythrocyte membranes with [gamma-32P]-ATP. Added insulin increased the incorporation of 32P into phosphatidate in a dose-dependent manner that reached a steady state at 2 nM. We conclude that the metabolically labile pool size of phosphatidate is decreased and that of polyphosphoinositides is altered in erythrocyte membranes from diabetic patients.

Changes in erythrocyte membrane fluidity by endotoxin in rats.

The effect of endotoxin on fluidity and lipid composition of the erythrocyte membrane was studied in rats following the intraperitoneal administration of endotoxin (30 mg-kg−1 body weight). Erythrocyte membrane fluidity measured with 16-stearic acid spin label (16-SAL) was significantly decreased in the endotoxin-treated rats as compared with control. A decrease of lysophosphatidylcholine in the membrane lipid was evident in the endotoxin-treated rats. The cholesterol to phospholipid molar ratios and other phospholipid fractions did not differ significantly in the two groups.The levels of plasmaβ-glucuronidase activity and lipoperoxide were significantly increased in the endotoxin-treated rats when compared to controls. There were significant correlations between the parameter of 16-SAL in erythrocytes and plasmaβ-glucuronidase activities or lipoperoxide from both endotoxin-treated and control groups,P<0.005 orP<0.02 respectively.In conclusion endotoxin decreased rat erythrocyte membrane fluidityin vivo. Since membrane fluidity is closely related to the vital functions of the membranes, the change described could be related to the abnormality of cell membrane functions in endotoxin shock state.