Anaerobic storage of red blood cells. (2010)

Oxidative damage – lipid and protein oxidation / peroxidation and cross-linking caused by reactive oxygen species, such as hydroxyl, peroxy and alkoxy radicals – is one of the major factors contributing to the development of storage lesions. RBC contain a highly reactive mixture of iron (in Hb) and oxygen (dissolved in the cytosol and bound to Hb) at very high concentrations. Iron atoms in Hb must be maintained in a ferrous state in order to reversibly bind O2. In vivo, ferrous iron in Hb is protected from oxidation within RBC, but when the cells are removed from the body, and stored under refrigerated conditions, the mechanisms protecting the RBC lose their efficiency and Hb becomes vulnerable to oxidation.

Haemoglobin oxidation and the oxidative damage pathway
Haemoglobin and the products of its denaturation play a central role in the development of oxidative damage to stored RBC by serving as catalysts of this process. Although the importance of denatured Hb in the context of oxidative damage during refrigerated storage was raised back in 1989 by Wolfe109, relatively few studies that address this issue directly have been reported since. The involvement of Hb in the development of storage lesions has been reviewed recently110.

Haemoglobin contains four ferrous ions, one in each of its four subunits coordinated in the porphyrin ring. To fulfil its main physiological function, each deoxyHb molecule binds four molecules of O2 reversibly, without exchanging electrons (Figure 1A). RBC maintain a highly reducing environment in their cytosol to preserve iron in its ferrous form and use enzymes efficiently to reverse Hb oxidation.



Figure 1

Haemoglobin and pathways of oxidative damage in RBC.



A small fraction of oxyHb spontaneously auto-oxidizes to form ferric methaemoglobin (metHb) and a superoxide anion. In circulation, ferric metHb is reduced back to ferrous Hb by NADH-linked cytochrome b5 reductase. In refrigerated storage, however, this reaction is retarded while the formation of metHb is enhanced for stored RBC with partially oxygenated Hb (Figure 1B). Once formed, metHb is inherently unstable; it denatures readily first to reversible hemichromes, then to irreversible hemichromes, and finally to globin and free haem (haemin; Figure 1C). The stability of metHb may be further compromised at storage temperatures similar to a lower thermodynamic stability of met-myoglobin at 4°C (cold denaturation)111.

Ferric iron in hemichromes, free haem and molecular iron released from haem can all function as a Fenton reagent in the Harber-Weiss cycle fed by H2O2 to generate highly reactive hydroxyl radicals (Figure 1D). Hydroxyl radicals (•OH) attack proteins and (in the presence of oxygen) initiate a lipid peroxidation cycle in RBC membrane (Figure 1E). Polyunsaturated fatty acids in lipids are attacked by •OH to form lipid radicals, which then form lipid hydroperoxyl radicals with oxygen, which in turn attack polyunsaturated lipid to complete the cycle. This cycle continues in presence of oxygen until two radicals react to terminate the reaction, resulting in cross-linked lipids112.


Haemoglobin auto-oxidation under conditions of partial oxygen depletion
H2O2 is a substrate for the Harber-Weiss reaction that produces hydroxyl radicals and itself is a product of superoxide dismutase reacting on superoxide anion, a byproduct of Hb auto-oxidation (Figure 1B and D). Because metHb forms when oxyHb auto-oxidizes, the concentration of dissolved O2 is a critical factor in determining the rate of metHb formation. Balagopalakrishna et al. reported that the rate of metHb formation and superoxide production reached a maximum when Hb was only partially occupied by O2 in a hypoxic state (SO2 ∼60%) rather than fully oxygenated at a high pO2113. In this context, the current practice of collecting and processing venous blood, and placing it in storage at a starting SO2 of nearly 60% and then allowing the blood to oxygenate further to nearly 100% during the 6 weeks of storage as oxygen slowly diffuses through the plastic film of the storage bag, likely exacerbates the oxidative damage experienced by stored RBC.


The effect of oxidative damage on stored red blood cells
Obtaining unequivocal direct evidence of oxidative damage to RBC during refrigerated storage has been difficult prior to the application of highly sensitive and specific proteomic techniques114,115. For example, a simple measurement of metHb levels may not accurately reflect the rate of metHb formation or the extent of oxidative reactions during storage due to the transient nature of unstable metHb. A small fraction of RBC that were nearly senescent at the time of blood collection and damaged RBC may cause high readings of free Hb, haem and other by-products of oxidation. Thus, most available evidence regarding the mechanisms of Hb-mediated oxidative damage and their impact on the physiological function of RBC were obtained from in vitro experiments exposing RBC to oxidative stress104,116–121 or from studying RBC from patients with various haemoglobinopathies (such as sickle cell disease or thalassaemia)122–125 and other conditions (such as glucose-6-phosphate dehydrogenase deficiency or myelodysplastic syndrome)126,127.

During conventional (aerobic) storage, a progressive increase in overall protein oxidation has been observed128–130. D’Amici et al. demonstrated a significant reduction in the rate of increase in the number of spots on a two-dimensional gel of RBC proteins (an indicator of protein modification and fragmentation) in the first week of storage for anaerobically stored RBC relative to those stored aerobically (conventionally), suggesting a reduced rate of RBC protein oxidation under anaerobic conditions. By employing standard proteomic techniques, they also identified proteins that were modified early and in an O2-dependent manner, including band 4.2 (prevalent), band 4.1, band 3 and spectrin (minor)114. Messian et al. observed a loss of oxygen-dependent metabolic modulation in RBC stored aerobically131. In addition to these physical and chemical perturbations, a decrease in glutathione (GSH) levels and in activity of GSH-dependent glutathione peroxidase were observed during storage132.

After Hb auto-oxidizes to metHb, it denatures to hemichromes, which then precipitate onto the lipids and the cytoskeleton of RBC membrane. Subsequent clustering of band 3 caused by this precipitation allows the immune system of the recipient to recognise and cull the transfused RBC from the circulation109. Precipitated hemichromes also disrupt the interactions between other proteins of the cytoskeleton (spectrin, actin and band 4.1)109,133. Hemichromes undergo further denaturation into haemin, globin and free molecular iron, all of which readily partition into lipid bilayers. Free haem is a potent haemolytic agent; it causes RBC haemolysis by collapsing the cationic gradient134. Haemin and free molecular iron can also function as Fenton reagents to generate hydroxyl radicals, which in turn initiate lipid and protein peroxidation reactions. Peroxidation of the lipids and proteins of the RBC membrane results in an increased susceptibility to cation leaks, elevated phosphatidyl serine exposure, vesiculation and haemolysis. All of these changes prime RBC for suicidal cell death (eryptosis) in the circulation135. Excessive vesiculation due to oxidative damage leads to a disproportionately higher loss of membrane area than volume; as the surface area-to-volume ratio decreases, stored RBC change shape, ultimately approaching a sphere. This change in the surface area-to-volume ratio and the cross-linking of membrane lipid and proteins of the cytoskeleton caused by the hydroxyl radicals contribute to the significant reduction of deformability of stored RBC93,96,109,118,120,124,125.