Investigação da associação entre cinética de lise em eritrócitos humanos, parâmetros bioquímicos e hematológicos

Abstract

The analysis of the stability of the erythrocyte membrane is done under incubation at a given temperature for a fixed time, and is based on the amount of existing lysis a result of diminishing in the salt concentration or increased concentration of a denaturing agent. The lysis constants derived from this analysis are of a thermodynamic nature, since they represent an equilibrium situation after a sufficiently long time, although lysis is a very rapid process. These stability constants are valid for the analysis of modulating agents that influence on this equilibrium, but not those who have influence on the kinetics of lysis. This work has developed experimental conditions for the kinetic analysis of the lysis of erythrocytes not by monitoring in real time, but by spectrophotometric monitoring at 540 nm after an interruption of the lysis by hypertonicity at predetermined intervals of time. The kinetics of lysis was studied in the intermediate region of salt concentration (from 0.3 to 0.5 g/dL NaCl) of the hemolysis curve in conditions of fixed time incubation. The concentration of 0.4 g/dL NaCl led to a better definition of the lysis kinetics and was then used to evaluate the kinetics of lysis of erythrocytes from 17 donors that participated of the study. The lysis curve as a function of time was statiscally fitted to a hyperbola, using the analytical routine of the integrated kinetic model (which has the same equation as the Michaelis-Menten model in non-integrated form), allowing the determination of the time required to promote lysis of half of the population of erythrocytes (t1/2) and the maximum absorbance (Amax) reached in the test. The results showed good variance among donors. The association between these constants and hematologic variables, blood glucose and blood lipid was analyzed by Spearman correlation. The constant t1/2 was negatively correlated with total cholesterol (t-C) and LDL-C. The variable Amax, as it was expected, showed significant associations with hematologic variables that are under the influence of hemoglobin levels. Stratification of the study population into two age groups (18-30 and 40-90 y) showed that the values of t1/2 were significantly lower in the older population. Although the population studied has been too small, this study showed that using a kinetic focus in the physico- 4 chemical approach of the RBC lysis is very promising for analyzing the myriad of variables which have influence on the erythrocyte membrane.