The effect of alcohols on red blood cell mechanical properties and membrane fluidity depends on their molecular size

Abstract

The role of membrane fluidity in determining red blood cell (RBC) deformability has been suggested by a number of studies. The present investigation evaluated alterations of RBC membrane fluidity, deformability and stability in the presence of four linear alcohols (methanol, ethanol, propanol and butanol) using ektacytometry and electron paramagnetic resonance (EPR) spectroscopy. All alcohols had a biphasic effect on deformability such that it increased then decreased with increasing concentration; the critical concentration for reversal was an inverse function of molecular size. EPR results showed biphasic changes of near-surface fluidity (i.e., increase then decrease) and a decreased fluidity of the lipid core; rank order of effectiveness was butanol > propanol > ethanol > methanol, with a significant correlation between near-surface fluidity and deformability (r = 0.697; p<0.01). The presence of alcohol enhanced the impairment of RBC deformability caused by subjecting cells to 100 Pa shear stress for 300 s, with significant differences from control being observed at higher concentrations of all four alcohols. The level of hemolysis was dependent on molecular size and concentration, whereas echinocytic shape transformation (i.e., biconcave disc to crenated morphology) was observed only for ethanol and propanol. These results are in accordance with available data obtained on model membranes. They document the presence of mechanical links between RBC deformability and near-surface membrane fluidity, chain length-dependence of the ability of alcohols to alter RBC mechanical behavior, and the biphasic response of RBC deformability and near-surface membrane fluidity to increasing alcohol concentrations.

Figure 1. Elongation index (EI)-shear stress (SS) curves for RBC in suspending medium containing the four alcohols at different concentrations.

(A) Methanol. (B) Ethanol. (C) Propanol. (D) Butanol. The data are means ± SE from 10 experiments on blood samples from different donors. Note that error bars often lie within the symbols and therefore are not visible. The curves representing the EI-SS relations for RBC suspensions containing different concentrations of alcohols were significantly different from each other for all for alcohols as determined by two-way ANOVA (P<0.001).

Figure 2. Half-maximal shear stress (SS_1/2) and maximum elongation index (EI_max) calculated by Lineweaver-Burk approach.

The curves presented in Figure 1 were used in calculus. (A) SS_1/2 for RBC exposed to ethanol; (B) EI_max for RBC exposed to ethanol; (C) SS_1/2 for RBC exposed to propanol; (D) EI_max for RBC exposed to propanol. The data are means ± SE from 10 experiments on blood samples from different donors. Difference from control without alcohol were tested by one-way ANOVA: * P<0.05; ** P<0.01.

Figure 3. Effect of alcohol concentration on SS_1/2/EI_max parameters.

The effects were determined using the EI-SS curves presented in Figure 1. SS_1/2/EI_max is inversely proportional to RBC deformability. The data are means ± SE from 10 experiments on blood samples from different donors. Statistical significance of the differences in comparison to control (no alcohol) are: * P<0.05 and ** P<0.01, as tested by one-way ANOVA.

Figure 4. Alcohol concentrations at which the SS_1/2/EI_max parameter reached a minimum (i.e., maximal RBC deformability).

The minimal concentrations were determined using the fitted curves presented in Figure 3.

Figure 5. Effect of alcohol concentration on elongation indexes (EI) at 100 Pa SS during a 300 s period of shearing.

(A) Methanol. (B) Ethanol. (C) Propanol. (D) Butanol. EI values are presented as % of the initial value. Each curve represents the mean from 10 experiments. Standard Error (SE) varied between 0.05 and 2.12, and showed increase with the duration of SS (error bars not shown for clarity). The curves were compared to control using two-way ANOVA, with significant differences (P<0.0001) observed for all four concentrations of methanol, ethanol at 0.687 and 1.202 M, both concentrations of propanol, and the single concentration of butanol.

Figure 6. Changes of SS_1/2/EI_max following the application of 100 Pa SS for 300 s.

(A) Methanol. (B) Ethanol. (C) Propanol. (D) Butanol. Data are means + SE from 10 experiments on blood samples from different donors. Differences between values that were obtained before (white bars) and after (black bars) the exposure to 100 Pa SS, as determined by the means of two-way ANOVA: * P<0.05; ** P<0.01; *** P<0.001.

Figure 7. The effects of alcohols on RBC membrane fluidity at various concentrations as determined by EPR spectroscopy.

(A) The order parameter (S) of 5-DS-labeled RBC. S for 2 M methanol was 0.729 ± 0.007 (not shown). (B) Rotational correlation time (τ) of 16-DS-labeled RBC. τ for 2 M methanol was 18.20 ± 0.88^** (10^-10 s) (not shown). Data are mean ± SD from 4 experiments on blood samples from different donors. Statistically significant differences compared to control (no alcohol) using one-way ANOVA: * P<0.05, ** P<0.01.

Figure 8. Hemolysis (%) plotted against alcohol concentration for four alcohols with different molecular sizes.

Data are mean ± SE from 7 experiments on blood samples from different donors. Statistical significance from control (no alcohol) tested by one-way ANOVA: * P<0.05; ** P<0.01.

Figure 9. Light microscopy images of RBC in the presence of alcohols.

The cells were observed in dilute (1:200), fresh, wet-mount, unstained preparations in which cell shape was unaffected by the "glass slide" effect (i.e., crenation due to alkaline conditions at glass surface).