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.

Determination of acid base balance and oil water partition coefficient of an atopy patch test of levofloxacin.

Levofloxacin, a quinolone used in the treatment of an upper respiratory tract infection is involved in skin reactions. Patch tests are helpful for the aetiological diagnosis. Unfortunately, they are known for their lack of sensibility. A weak transcutaneous penetration due to the physicochemical characteristics of tested drugs is a well-known cause of false negative results but these characteristics are usually unknown. The aim of the present study was to evaluate physicochemical parameters by potentiometric method in order to optimize the transcutaneous penetration of a preparation for patch test. This method is applicable to a medium such as the preparation for patch test. In order to value the stability of the preparation, qualitative and quantitative analysis of the level of levofloxacin was effectuated. In our study, spectrophotometric and potentiometric data at T0 and six months later with the preparation showed the stability of the preparation. The proportions of non-ionized (45%) and ionized levofloxacin (55%) were not modified. Further experiments are needed to choose the minimal efficient dose of the suspected molecule able to detect a positive reaction in cases of allergic reaction. In addition, this method could be useful in qualitative and quantitative control of preparations made at hospital.

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.

In vitro and in vivo antimalarial activity of derivatives of 1,10-phenanthroline framework.

A series of trisubstituted 1,10-phenanthrolines was prepared. These compounds exhibited mild to high biological activities in vitro both toward chloroquino-resistant FcB1-Columbia and FcM29-Cameron strains and Nigerian chloroquino-sensitive strain of Plasmodium falciparum. Cytotoxicity of the most active compounds was estimated showing that one compound (10) exhibited a selective activity against malaria parasite (selectivity indexes of 52 and 144). Antiplasmodial activity of this derivative was optimized by N-10 alkylation and the phenanthrolinium salt (15) submitted to an in vivo study using mice infected by P. vinckei petteri showing an ED50 of 7.86 mg/kg/day.

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.

The protective effects of lidocaine on human erythrocytes stored for seven days at 04 degrees C.

Erythrocyte storage may result in cell damage due to an alteration of membrane integrity, which results in potassium efflux and hemolysis. Lidocaine has been shown to protect erythrocytes from oxidative stress by a possible membrane effect. We conducted this study to examine the effects of lidocaine on human erythrocyte storage. Erythrocytes were kept for seven days at 04 degrees C in the absence or in presence of plasma, and of lidocaine at 36.9 and 221.6 microM. Cell damage was assessed by measuring potassium efflux in the supernatant after seven days, and studying potassium efflux and hemolysis induced by oxidative stress. As expected, erythrocyte storage in the presence of plasma was less deleterious. Lidocaine decreased potassium efflux after 7 days' storage. Resistance toward oxidative stress was greater when the erythrocytes had been kept in the presence of plasma. Considering that lidocaine is widely used in various clinical situations, this data may be of clinical relevance.