Effects of various osmolarity on human red blood cells in terms of potassium efflux and hemolysis induced by free radicals.

OBJECTIVES: To study in an in vitro model of erythrocytes exposed to free radicals the effects of hyperosmolarity and hypoosmolarity on the induced potassium efflux and hemolysis. DESIGN SETTING: Erythrocytes were separated from plasma and suspended in 3 different phosphate buffer solutions (HYPO, ISO, and HYPER) containing, respectively, 100, 150 and 200 mmol/l of Na. Free radicals were generated from 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH). Potassium efflux (flame photometry) and hemolysis (Drabkin method) were measured. Measurements were expressed in % (versus total) and area under % versus time curves were calculated (% min). An ANOVA was used for statistical analysis. RESULTS: In presence of AAPH, hemolysis was significantly greater in HYPO (732.45+/-40% min) and lower in HYPER (578.97+/-15% min) as compared to ISO (608.30+/-42% min). Potassium efflux was significantly increased in HYPER (7508.04+/-85% min) as compared with HYPO (5308.74+/-62% min) and ISO (6561.39+/-62% min). DISCUSSION: Our data suggest that hyperosmolarity increases the resistance of the red blood cells when exposed to free radicals as shown by the decreased hemolysis. In such conditions, the potassium efflux analysis appears to be inappropriate to evaluate the free radical effects on erythrocytes.

Comparison of three methods for oxidative stress-induced potassium efflux measurement.

The aim of the study was to compare the potassium efflux measurements (flame photometry (FP), specific electrode (SE) and atomic absorption photometry (AAP)) using a model of erythrocytes exposed to an oxidative stress in various conditions of osmolarity. Human erythrocytes were incubated in 3 different values of osmolarity and in the presence of 50mM AAPH, potassium efflux was measured by FP, SE and AAP at t=0 and every 30min for 2h. These methods were similar for the measurement of global potassium efflux. However, SE detected important amounts of potassium at the beginning of the experiment or in absence of AAPH in comparison with AAP and FP. It is noteworthy that these different methods of measurements were not altered by the osmolarity. FP and AAP make it possible to study the potassium efflux during oxidative stress while SE should be used only for global measurements.

Effects of an oxidative stress on human hemoglobin: a multiwavelength visible spectrometry study.

This study was carried out to investigate hemoglobin behavior and the role of cell membrane during oxidative stress of human red blood cells induced by a water-soluble radical initiator, 2,2'-azobis(amidino-propane) hydrochloride (AAPH) and compare the observed data to the one obtained with purified human haemoglobin solution. The different forms of hemoglobin were identified and quantified by multiwavelength visible spectrometry using multiple linear regression analysis. Hemolysis was quantified by the Drabkin method. Oxidative stress on purified hemoglobin solutions induced an early formation of Hb(+). In intact erythrocytes, no modified form of haemoglobin was found. Only the hemoglobin released by hemolysis in the extracellular medium was notified in the same way as purified haemoglobin. Thus, the cell membrane appears to protect intraerythrocytic hemoglobin toward an extracellular oxidative stress. Oxidative stress-induced by hemolysis does not seem to be due to changes in intraerythrocytic hemoglobin forms.