Large differences in erythrocyte stability between species reflect different antioxidative defense mechanisms

We have developed a screening assay for erythrocyte stability, which is rapid, easy, inexpensive, robust, and suitable for handling a large number of samples in parallel. Erythrocytes are incubated overnight in 96-well microtiter plates in absence or presence of various oxidants, intact cells are pelleted by centrifugation, and lysis is determined by release of intracellular constituents into the supernatant as either activity of lactate dehydrogenase (LDH) or absorbance of hemoglobin at 406 nm. There is good correlation between the methods. A number of advantages by the present method are that only small amounts of blood is needed, washing is optional, erythrocytes may be stored for at least one day before assay, and large numbers of samples can be handled in parallel. Using this set-up, we have compared erythrocyte stability from several different animal species. We find that erythrocyte susceptibility towards lysis induced by H2O2 and 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) is highly species dependent. The different susceptibility between species is due to cellular components, since swapping of plasma between species has little or no effect. As a novel observation, we find that erythrocytes from chicken are the most sensitive of the species tested towards lysis by H2O2 and are almost four orders of magnitude more sensitive than erythrocytes from man. This is due to a much lower content of catalase in erythrocytes from chicken. A more narrow range is observed for susceptibility towards AAPH and the ranking between the species is different. Thus, chicken erythrocytes are more resistant towards AAPH than some mammals by up to two orders of magnitude. This differential stability towards different oxidative stressors is likely due to evolution/selection of different defense mechanisms.