Shogo Otsuji

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.

A new enzymatic approach for estimating total and direct bilirubin

We designed an enzymatic assay for total (TBil) and direct bilirubin (DBil), the principle of which involves measuring the decrease in absorbance at 450 nm produced by bilirubin oxidase from Myrothecium verrucaria. Since TBil and DBil are oxidized at pH 7.2 and 3.7, respectively, the degree of bilirubin oxidation is measurable in each case. An analysis of bilirubin by high-performance liquid chromatography, before and after the enzymatic reaction with bilirubin oxidase, verified the specificity of the enzyme. The results obtained using this method varied linearly with TBil and DBil concentrations up to at least 250 mg/L and 150 mg/L, respectively. Reducing substances, commonly used anticoagulants and hemoglobin showed no apparent interference. The degree of day-to-day precision (CV) for TBil and DBil ranged from 1.2% (206.2 mg/L) to 10.6% (3.5 mg/L) and from 1.8% (84.3 mg/L) to 12.4% (2.1 mg/L), respectively. Values measured using this new method correlated well with those obtained by Malloy-Evelyns method and the slide method employing the Kodak Ektachem analyzer.

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.

Albumin prevents erythrocyte crenation in patients undergoing extracorporeal circulation

Free fatty acid-induced massive erythrocyte crenation has been reported in patients undergoing extracorporeal circulation (EC), suggesting that the crenated cells impair microcirculatory flow and tissue oxygenation during and after the operation. Effect of albumin administration on erythrocyte crenation was examined in 18 patients undergoing coronary bypass operation: 9 patients were given 25 g and another 9 patients were given 50 g of human albumin as a part of the priming solution. Erythrocyte crenation was almost completely prevented in the patients given 50 g albumin. Crenated erythrocytes during EC were 3.6 +/- 2.3% (mean +/- SD) of all erythrocytes and 2.8 +/- 3.7% after EC. This was significantly lower than in patients without albumin administration (63.4 +/- 34.0% during EC and 28.6 +/- 33.3% after EC, n = 20). But the effect was less striking in the patients given 25 g albumin, 32.4 +/- 39.1% during and 28.3 +/- 40.8% after EC. Maintaining an adequate level of plasma albumin is important in preventing erythrocyte crenation during EC, improving microcirculatory flow in patients undergoing open heart surgery.

A rapid enzymatic assay for total blood polyamines

We have designed a rapid, reliable enzymatic assay for total blood polyamines, as based on the combination of substrate specificity of polyamine oxidase (PAO) from Aspergillus terreus and putrescine oxidase (PUO) from Micrococcus rubens. Quinone dye, derived from hydrogen peroxide generated in the oxidation reaction, is measured spectrophotometrically at 555 nm, and total amounts of putrescine (Put), cadaverine (Cad), spermidine (Spd), and spermine (Spm) can be readily determined. The minimum detection limit is about 0.4 mumol/L whole blood. Bilirubin, hemoglobin, reducing substances, and anticoagulants show no apparent interference. Analytical recovery averaged 98.5%. Within-run and between-day precisions ranged from 1.02% (61.04 mumol/L) to 2.84% (13.07) and 1.54% (50.67) to 3.27% (14.74), respectively. Results obtained with this method and those by high-performance liquid chromatography (HPLC) (r = 0.955) or the enzymatic differential method (r = 0.944) correlated well. In our opinion, this method is superior to other assays being used to determine blood polyamines.