Efeitos do glicerol sobre a dependência térmica da estabilidade de eritrócitos humanos

Abstract

The incubation of human erythrocytes with ethanol in saline physiologic solution, by 30 minutes, can produce lysis of the expanded morphological state (R), which presents a lower stability, or formation of the contracted morphological state (T), which has a higher stability. The R state occurs in physiological conditions and suffers lysis under less elevated concentrations of ethanol. The lysis of the R state by ethanol can be characterized by the ethanol concentration at the mid-transition point (D50R) of the lysis curve of the erythrocytes by ethanol. The lysis of the R state by hypotonicity can be characterized by the saline concentration at the mid-transition point (H50) of the lysis curve of the erythrocytes by hypotonic stress. The T state occurs in hyperosmolar conditions and suffers lysis at more elevated ethanol concentrations. Depending on its concentration, the proper ethanol can produce lysis or osmostabilization of the human erythrocytes. The objective of this work was to study the effects of glycerol concentration and temperature on the stability (D50R) of the R state of the human erythrocytes in function of the chaotropic actions of ethanol and hypotonicity. The blood samples were collected from 12 healthy female volunteers (24 ± 3 years). The percentage of hemolysis, after incubation by 30 minutes, in function of the ethanol concentration (0-36%) in saline physiologic solution, was studied by spectrophotometry at 540 nm in the absence and in the presence of 1.5 mol.L-1 glycerol at different temperatures (7, 12, 17, 22, 27, 32, 37, 42 and 47°C) and in the presence of different glycerol concentrations (0, 0.5, 1.0, 1.5 and 2.0 mol.L-1) at 7 °C. The percentage of hemolysis, after incubation by 30 minutes, was also evaluated in function of the saline concentration at the absence and in the presence of 1.5 mol.L-1 glycerol at 7 °C. The percentage of hemolysis in function of the ethanol concentration in saline physiologic solution or in function of the saline concentration, were fitted to sigmoidal regression curves. The fittings to the regression curves and the existence of difference among groups were considered significant only when P values were smaller than 0.05. The values of D50R decreased significantly, according decaying sigmoidal curves, with the increase in the temperature in the absence and in the presence of 1.5 mol.L-1 glycerol. This means that the temperature increase induces a transition between two states, one with a lower stability (R) and the other with a higher stability (T). The values of D50R also have declined significantly, according a sigmoidal decaying curve, with the increase in the glycerol concentration. This means that the glycerol concentration also induces a states transition in the erythrocytes, from a lower stability state (R), present at the lower glycerol concentrations, to a higher stability state (T), present at the higher glycerol concentrations. The presence of 1.5 mol.L-1 glycerol decreases progressively the erythrocytes stability with the decrease in the temperature from 32 to 7 °C, but it increases the erythrocytes stability above 37 °C. This behaviour was attributed to the existence of a critical value of osmotic pressure generated by a combination of the ethanol, glycerol and temperature actions. Below this critical value of osmotic pressure there would be predominance of the R state and above that value there would be a predominance of the T state of the erythrocytes. Although the presence of 1.5 mol.L-1 glycerol diminishes the stability of erythrocytes against the ethanol action, it increases the erythrocytes stability against lysis by hypotonicity, at 7 °C, since the H50 values were significantly lower in the presence of the osmolyte than in its absence. At this temperature, the conjunction of the ethanol, temperature and glycerol actions would determine the predominance of R state of the erythrocytes, which has a lower stability and suffers more easily the chaotropic action of the ethanol. However, in the absence of ethanol, the presence of glycerol would determine a lower water entrance in the erythrocytes, making more difficult its lysis by hypotonicity.