The time-course linkage between hemolysis, redox, and metabolic parameters during red blood cell storage with or without uric acid and ascorbic acid supplementation

Abstract

Oxidative phenomena are considered to lie at the root of the accelerated senescence observed in red blood cells (RBCs) stored under standard blood bank conditions. It was recently shown that the addition of uric (UA) and/or ascorbic acid (AA) to the preservative medium beneficially impacts the storability features of RBCs related to the handling of pro-oxidant triggers. This study constitutes the next step, aiming to examine the links between hemolysis, redox, and metabolic parameters in control and supplemented RBC units of different storage times. For this purpose, a paired correlation analysis of physiological and metabolism parameters was performed between early, middle, and late storage in each subgroup. Strong and repeated correlations were observed throughout storage in most hemolysis parameters, as well as in reactive oxygen species (ROS) and lipid peroxidation, suggesting that these features constitute donor-signatures, unaffected by the diverse storage solutions. Moreover, during storage, a general "dialogue" was observed between parameters of the same category (e.g., cell fragilities and hemolysis or lipid peroxidation and ROS), highlighting their interdependence. In all groups, extracellular antioxidant capacity, proteasomal activity, and glutathione precursors of preceding time points anticorrelated with oxidative stress lesions of upcoming ones. In the case of supplemented units, factors responsible for glutathione synthesis varied proportionally to the levels of glutathione itself. The current findings support that UA and AA addition reroutes the metabolism to induce glutathione production, and additionally provide mechanistic insight and footing to examine novel storage optimization strategies.

Keywords: accelerated aging; ascorbic acid; glutathione; oxidative stress; red blood cell; storage lesion; uric acid.

FIGURE 1

Intra-parameter correlations during storage. (A) Correlations between the levels of hemolysis and oxidative parameters at early, middle and late storage. All r values are statistically significant and concern controls, but similar statistically significant values were found in uric acid (UA)-, ascorbic acid (AA)- and mix-enhanced units (see also Figure 2). (B) Scatter plot of the levels of early and middle storage for extracellular antioxidant capacity (bold font p < 0.05). The same pattern with slightly different R ² values was evident for all storage periods examined. (C) Scatter plot regarding the levels of pentose phosphate pathway (PPP) metabolites (for control and UA: D-erythrose 4-phosphate; for AA, sedoheptulose 7-phosphate; for mix: glucose 6-phosphate) between early and middle storage. Hb, hemoglobin; MCF, mean corpuscular fragility; MFI, mechanical fragility index; ROS, reactive oxygen species; MDA, malondialdehyde; AU, arbitrary units.

FIGURE 2

Inter- and intra-parameter correlations during storage. Correlations between the levels of hemolysis (A) and oxidative (B) parameters at early, middle and late storage for control, uric acid (UA)-, ascorbic acid (AA)- and mix-enhanced units. All r values shown exceed the threshold for statistical significance (p < 0.05). Scatter plots presenting correlations between the levels of early and middle or middle and late storage (C) regarding purine metabolites for control and UA-supplemented units (R ² values with bold font: p < 0.05). Xanthosine day 21 control values were ten-fold multiplied to fit the graphical representation. Hb, hemoglobin; MCF, mean corpuscular fragility; MFI, mechanical fragility index; NS, not significant; Ox, oxidative; MDA, malondialdehyde; ROS, reactive oxygen species; AU, arbitrary units.

FIGURE 3

Correlations common across all groups. (A) Correlations between redox, proteostasis and metabolism parameters of early and middle storage with oxidative stress of middle and late storage for control, uric acid (UA)-, ascorbic acid (AA)- and mix-enhanced units. (B) Representative scatter plots regarding some of the correlations graphically presented in panel (A). In the first scatter plot S-adenosyl methionine (SAM) is shown for controls and cystathionine for supplementations. All R ² values shown exceed the threshold for statistical significance (p < 0.05). TCA, tricarboxylic acid; GSSG, glutathione disulfide; Hb, hemoglobin; ROS, reactive oxygen species; AU, arbitrary units; RFU, relative fluorescence units; TR-like, trypsin-like; TBA, thiobarbituric acid.

FIGURE 4

Differential correlation profile in enhanced blood units. (A) Unique correlations between redox, proteostasis and metabolism parameters of early and middle storage with redox parameters of middle and late storage for uric acid (UA)-, ascorbic acid (AA)- and mix-enhanced units. (B) Representative scatter plots regarding some of the correlations graphically presented in panel (A). In the first scatter plot total antioxidant capacity (AC) is shown for mix, UA-dependent AC for UA and UA-independent AC for AA units. All R ² values shown exceed the threshold for statistical significance (p < 0.05). Hb, hemoglobin; GSSG, glutathione disulfide; ROS, reactive oxygen species; TBA, thiobarbituric acid; AU, arbitrary units; tBHP, tert-butyl hydroperoxide; RFU, relative fluorescence units.