Supplementation of anti-oxidants in leucofiltered erythrocyte concentrates: assessment of morphological changes through scanning electron microscopy. (2014)

Red blood cell (RBC) storage in the blood bank is associated with a sequence of biochemical, metabolic and mechanical changes leading to the progressive loss of cell viability and impairment of RBC survival upon transfusion into the recipient1–6. In detail, in erythrocytes stored for long periods, progressive haemolysis, alterations of permeability and flexibility, along with increased antigenicity are accompanied by an exacerbation of oxidative stress parameters, targeting both the lipid fraction (resulting in the accumulation of malonyldialdehyde and isoprostanes)3 and the protein fraction (with increases in the levels of carbonylated species, protein fragmentation and non-enzymatically glycated haemoglobin)3,5–11. The storage-dependent accumulation of reactive oxygen species (ROS) is mainly related to the progressive loss of metabolic modulation3,12, resulting in the impairment of anti-oxidant defences6. Indeed, in humans, each day about 3% of the body’s haemoglobin undergoes spontaneous autoxidation to methaemoglobin because of the high concentrations of iron in RBC6, whereby Fenton and Haber-Weiss reactions ultimately promote the accumulation of superoxyl and hydroxyl radicals, the latter being considered as the primary mechanism of injury in stored RBC6.

It is worth noting that exacerbated vesiculation during the storage of packed red cells can also be regarded as a self-protective mechanism to eliminate irreversibly altered components, such as carbonylated proteins13. In this view, preventing the accumulation of oxidative stress has been long been regarded as a viable strategy to improve the quality of stored RBC. Over the years, several approaches have been proposed to achieve this aim: (i) supplementation of metal chelators, such as deferoxamine, diethylenetriaminepentaacetic acid or ethylenediaminetetraacetic acid, which bind iron released from ageing erythrocytes, thus protecting RBC from the Fenton and Haber-Weiss reactions14; (ii) oxygen depletion15, to remove the main substrate of ROS-generating reactions and, in parallel, to improve the preservation of high energy phosphate compounds (adenosine triphosphate and diphosphoglycerate), at the expense of anti-oxidant defences16, by relying on the oxygen-dependent modulation of metabolic fluxes according to the model proposed by the groups of Low and Giardina17,18; (iii) supplementation of anti-oxidants in the additive solutions, using compounds containing thiol groups (glutathione loading)19 and vitamins (especially C in the form of ascorbate/dehydroascorbate and vitamin E)20–24.

This last strategy has attracted a great deal of interest during recent decades, especially in the light of the central role of glutathione (GSH) in intracellular anti-oxidant systems. Indeed, glutathione is the substrate for three key RBC anti-oxidant enzymes: glutathione peroxidase, glutathione reductase and glutathione S-transferase. Glutathione peroxidase-catalysed reactions inactivate H2O2 to neutral substances, water and oxidized glutathione (GSSG). Therefore, ROS are not generated in glutathione reactions, as is the case for superoxide dismutase (SOD)-catalysed reactions, from which it can be inferred that glutathione-related enzymatic mechanisms, rather than other enzymatic mechanisms, are primarily committed to protection against the auto-oxidation of erythrocytes25.

Oxidized glutathione is constantly being reduced by glutathione reductase through its utilization of NADPH, which is mainly generated via the pentose phosphate pathway. However, it has been recently confirmed that the progressive loss of metabolic modulation over storage proportionally impairs the capacity of RBC to replenish NADPH reservoirs via the pentose phosphate pathway, especially after the second week of storage2,12. In the light of this, alternative strategies could be pursued:


- to preserve GSH levels by promoting de novo synthesis of this tripeptide by fuelling a key limiting substrate precursor, cysteine, in the form of N-acetylcysteine (NAC)26;



or


- to reduce GSSG back to GSH, via reactions involving dehydroascorbate/ascorbate (vitamin C) 27,28.



Recently, we performed an extensive mass spectrometry-based metabolomics analysis to understand whether vitamin C and NAC supplementation improved storage quality, especially in terms of anti-oxidant potential and glutathione homeostasis and energy metabolism (Pallotta et al., unpublished data). Encouraging results suggested that loading of anti-oxidants boosted GSH levels and helped to prevent malondialdehyde accumulation and reduce haemolysis. However, we could not conclude whether anti-oxidants also preserved RBC morphology better during storage. In the light of the foregoing considerations, we investigated whether supplementation of ascorbate and NAC in the SAGM additive solution helped to preserve RBC morphology better throughout a 42-day storage period.