Human erythrocytes were exposed to gamma-rays and alpha-particles to assess radiation-induced membrane damage and hemoglobin oxidation and denaturation. With all parameters measured, the alpha-particles proved to be less efficient than the gamma-rays. The time-dependence of hemolysis showed also clear differences: with the gamma-rays the process was faster, reaching saturation after 40-90 min (depending on dose), but with the alpha-particles the final level was attained only after about 3-7 h. Hemoglobin oxidation and denaturation could be measured only after gamma-exposure, but they were negligible with the alpha-particles when comparable doses were applied. These results are interpreted by proposing that OH-radicals, whose yields are smaller with densely ionizing radiation, play a crucial role in the induction of the processes for radiation-induced erythrocyte damage.

The effectiveness of radiation-generated HO* radicals in initiating erythrocyte hemolysis in the presence of oxygen and under anaerobic conditions and prehemolytic structural changes in the plasma-erythrocyte membrane were studied. Under anaerobic conditions the efficacy of HO* radicals in induction of hemolysis was 16-fold lower than under air. In both conditions, hemolysis was the final consequence of changes of the erythrocyte membrane. Preceding hemolysis, the dominating process under anaerobic conditions was the aggregation of membrane proteins. The aggregates were principally formed by -S-S- bridges. A decrease in spectrin and protein of band 3 content suggests their participation in the formation of the aggregates. These processes were accompanied by changes in protein conformation determined by means of 4-maleimido-2,2,6,6-tetramethylpiperidine-N-oxyl (MSL) spin label attached to membrane proteins. Under anaerobic conditions, in the range of prehemolytical doses, the reaction of HO* with lipids caused a slight (10-16%) increase in fluidity of the lipid bilayer in its hydrophobic region with a lack of lipid peroxidation. However, in the presence of oxygen, hemolysis was preceded by intense lipid peroxidation and by profound changes in the conformation of membrane proteins. At the radiation dose that normally initiates hemolysis a slight aggregation of proteins was observed. Changes were not observed in particular protein fractions. It can be suggested the cross-linking induced by HO* radicals under anaerobic conditions and a lack of lipid peroxidation are the cause of a decrease in erythrocyte sensitivity to hemolysis. Contrary, under aerobic conditions, molecular oxygen suppresses cross-linking, catalysing further steps of protein and lipid oxidation, which accelerate hemolysis.

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Human erythrocyte suspensions in an isotonic Na-phosphate buffer, pH 7.4, of hematocrit of 2% were exposed under air to gamma radiation at a dose rate of 2.2 kGy. Erythrocytes were irradiated with single doses, and identical doses split into two fractions with an interval time of 3.5 h between following exposures. The obtained results indicated that the irradiation of enucleated human erythrocytes with split doses caused a reduction of hemolysis (2.4 times), a decrease in the level of damage to membrane lipids and the contents of MetHb, compared with identical single doses. However, the splitting of radiation doses did not change the level of damage to the membrane proteins, as was estimated with a maleimide spin label. The obtained results suggest that a decrease in the level of damage to lipids was related to a decrease in hemolysis.

In this study destruction of human deoxyhemoglobin (deoxy-Hb) induced by the action of primary products of water radiolysis, mainly .OH, eaq-, H, and of secondary ethanol radicals was investigated. The extent of destruction was estimated on the basis of absorbance ratio A505/A563 after conversion of hemoglobin into methemoglobin (MetHb) and the changes in the parameters of hemoglobin oxygenation. Deoxy-Hb solutions were irradiated under the atmosphere of argon and N2O, in the absence and presence of ethanol. In each case the radiation-chemical yield for hemoglobin destruction Gd = sigma i fi gi was determined (where: fi--fractional efficiency of destruction by the individual radicals, gi-fraction of radicals reacting with deoxy-Hb). Results were elaborated statistically estimating, by the method of least squares, the parameters of a multidimensional regression function which for each experiment with deoxy-Hb conc. 5 mg ml-1 was of the form: Gd = 0.09 gOH + 0.018 geaq- -0.099 gH + 0.03 g R.Et. The destruction efficiencies f.OH and fR.Et were statistically significant while feaq- and fH were not.

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Structural changes of human deoxyhemoglobin (deoxyHb) induced by water and ethanol radicals were investigated in this study using absorption spectroscopy. Spectra of deoxyHb samples irradiated under various conditions (the atmosphere of argon or N2O in the absence or presence of ethanol) indicate their conversion into methemoglobin (metHb), hemichrome- and cholehemichrome-like products. The absorbance at the characteristic maxima of these derivatives and also of the oxidized or reduced samples following irradiation depends on the dose of radiation and the conditions employed.