Quantitative analysis for estimating injury effects of metal-catalyzed oxidation on human erythrocytes

ObjectiveTo investigate whether human erythrocyte proteins were susceptible to oxidative effects of pharmacological doses of iron and whether resulting damages affect their structure.MethodsConformational changes in hemoglobin were indicated by spectrophotometric analysis from 300 to 650 nm. Carbonyl assay was performed for estimating the protein oxidation in erythrocytes. Oxidative injury in erythrocyte membrane was investigated by evaluation of the structural changes in cytoskeleton proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis in presence of 2-mercaptoethanol and staining with Coomassie briliant blue G-250.ResultsA significant increase in absorbance at 630 nm represented the formation of methemoglobin. Increase in absorbance at 340 nm was indicated by interaction between globin and heme group, which predicted for low oxygen affinity. A decrease in absorbance at 420 nm showed the conversion of oxygen hemoglobin to methemoglobin and significant decrease in oxygen hemoglobin concentration. There was marked elevation in hemichrome compared with control group. Of interest, a positive correlation was observed between iron concentration and hemoglobin absorbance at 340 nm. Elevated levels of carbonyl groups confirmed the oxidative damage to erythrocyte proteins. Analysis of membrane proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis, showed molecular aggregates in the range of 150 to 180 kDa and slight decrease in the intensity of α-spectrin band.ConclusionsIt is possible to predict the situation of everyone who exposed to oxidant agent via a simple blood analysis. In this way, contents of oxidative products in blood samples would be assessed by this method.