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Effects of copper/ascorbate-induced oxidative stress on physical properties, Prx2 dimerization, and NADP(H) content of freshly processed RBCs. (A) H 2 O 2 formation by RBCs (5% hematocrit) during 24 hours at 4°C in the absence (Control) or presence of copper/ascorbate (Asc) in fresh blood samples (n = 6). (B) Percent Prx2 dimerization (n = 6) in fresh blood samples exposed to copper/ascorbate for 0 (Control), 1, 4, 24, or 48 hours. (C) Total NADP(H) and (D) NADPH by enzymatic cycling in fresh blood samples exposed to copper/ascorbate for 0, 1, 4, 24, or 48 hours at 4°C (n = 3). (E) Osmotic fragility based on solution osmolality leading to 50% hemolysis (n = 3) and (F) percent lysed erythrocytes (n = 3) in fresh blood samples exposed to copper/ascorbate for 0, 4, 24, or 48 hours at 4°C. Box plots show median, 25th and 75th percentiles (box), and minimum/maximum values (whiskers). Bars show mean values. *P ≤ 0.05, repeated-measures 1-way ANOVA in comparison to control; # P ≤ 0.05, Mann-Whitney nonparametric test.
Source publication
The red blood cell (RBC) storage lesion is a multi-parametric response that occurs during storage at 4°C, but its impact on transfused patients remains unclear. In studies of the RBC storage lesion, the temperature transition from cold storage to normal body temperature that occurs during transfusion has received limited attention. We hypothesized...
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... integrity was only slightly affected after 2-4 weeks' storage at 4°C, but warming to 37°C markedly increased osmotic fragility and decreased deformability after 4 hours, and the effect was even greater after 20 hours ( Figure 1, A and B). Osmotic fragility achieved a plateau by 18 hours and remained stable with incubation longer than 20 hours (Supplemental Figure 2). Temperature sensitivity was acquired during the 4°C storage duration for deformability (weeks 4 and 6), whereas it was independent of storage duration for osmotic fragility (Figure 1, A and B). ...
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... 6 weeks of storage, a 20-hour incubation at 37°C led to 60% dimerization of Prx2 ( Figure 1D) and a decrease in total NADP(H) ( Figure 1E), with a dramatic loss of NADPH ( Figure 1F). The decrease in NADPH was confirmed by both an enzymatic cycling method and HPLC (Supplemental Figure 6, (Figure 2A). Copper/ascorbate treatment of RBCs led to increased Prx2 dimerization within 1 hour ( Figure 2B) and a depletion of total NADP(H) ( Figure 2C) and decreased NADPH/NADP + ratio due to loss of NADPH ( Figure 2D). ...
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... decrease in NADPH was confirmed by both an enzymatic cycling method and HPLC (Supplemental Figure 6, (Figure 2A). Copper/ascorbate treatment of RBCs led to increased Prx2 dimerization within 1 hour ( Figure 2B) and a depletion of total NADP(H) ( Figure 2C) and decreased NADPH/NADP + ratio due to loss of NADPH ( Figure 2D). Increased oxidative stress in RBCs led to deterioration of RBC functional properties, with a progressive increase in osmotic fragility and hemolysis over a 48-hour period (Figure 2, E and F). ...
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... decrease in NADPH was confirmed by both an enzymatic cycling method and HPLC (Supplemental Figure 6, (Figure 2A). Copper/ascorbate treatment of RBCs led to increased Prx2 dimerization within 1 hour ( Figure 2B) and a depletion of total NADP(H) ( Figure 2C) and decreased NADPH/NADP + ratio due to loss of NADPH ( Figure 2D). Increased oxidative stress in RBCs led to deterioration of RBC functional properties, with a progressive increase in osmotic fragility and hemolysis over a 48-hour period (Figure 2, E and F). ...
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... decrease in NADPH was confirmed by both an enzymatic cycling method and HPLC (Supplemental Figure 6, (Figure 2A). Copper/ascorbate treatment of RBCs led to increased Prx2 dimerization within 1 hour ( Figure 2B) and a depletion of total NADP(H) ( Figure 2C) and decreased NADPH/NADP + ratio due to loss of NADPH ( Figure 2D). Increased oxidative stress in RBCs led to deterioration of RBC functional properties, with a progressive increase in osmotic fragility and hemolysis over a 48-hour period (Figure 2, E and F). ...
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... treatment of RBCs led to increased Prx2 dimerization within 1 hour ( Figure 2B) and a depletion of total NADP(H) ( Figure 2C) and decreased NADPH/NADP + ratio due to loss of NADPH ( Figure 2D). Increased oxidative stress in RBCs led to deterioration of RBC functional properties, with a progressive increase in osmotic fragility and hemolysis over a 48-hour period (Figure 2, E and F). No effect on deformability was observed (data not shown). ...
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... reducing equivalents are used for most antioxidant systems in RBCs. The nucleotide salvage metabolism recycles adenine and inosine, allowing the regeneration of NAD + and NADP + , and NADP + is subsequently reduced to NADPH by the PPP (22, 40). A decline in adenine and subsequent accumulation of its deaminated form, hypoxanthine, has been frequently noted as a marker ...
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... integrity was only slightly affected after 2-4 weeks' storage at 4°C, but warming to 37°C markedly increased osmotic fragility and decreased deformability after 4 hours, and the effect was even greater after 20 hours ( Figure 1, A and B). Osmotic fragility achieved a plateau by 18 hours and remained stable with incubation longer than 20 hours (Supplemental Figure 2). Temperature sensitivity was acquired during the 4°C storage duration for deformability (weeks 4 and 6), whereas it was independent of storage duration for osmotic fragility (Figure 1, A and B). ...
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... 6 weeks of storage, a 20-hour incubation at 37°C led to 60% dimerization of Prx2 ( Figure 1D) and a decrease in total NADP(H) ( Figure 1E), with a dramatic loss of NADPH ( Figure 1F). The decrease in NADPH was confirmed by both an enzymatic cycling method and HPLC (Supplemental Figure 6, (Figure 2A). Copper/ascorbate treatment of RBCs led to increased Prx2 dimerization within 1 hour ( Figure 2B) and a depletion of total NADP(H) ( Figure 2C) and decreased NADPH/NADP + ratio due to loss of NADPH ( Figure 2D). ...
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... decrease in NADPH was confirmed by both an enzymatic cycling method and HPLC (Supplemental Figure 6, (Figure 2A). Copper/ascorbate treatment of RBCs led to increased Prx2 dimerization within 1 hour ( Figure 2B) and a depletion of total NADP(H) ( Figure 2C) and decreased NADPH/NADP + ratio due to loss of NADPH ( Figure 2D). Increased oxidative stress in RBCs led to deterioration of RBC functional properties, with a progressive increase in osmotic fragility and hemolysis over a 48-hour period (Figure 2, E and F). ...
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... decrease in NADPH was confirmed by both an enzymatic cycling method and HPLC (Supplemental Figure 6, (Figure 2A). Copper/ascorbate treatment of RBCs led to increased Prx2 dimerization within 1 hour ( Figure 2B) and a depletion of total NADP(H) ( Figure 2C) and decreased NADPH/NADP + ratio due to loss of NADPH ( Figure 2D). Increased oxidative stress in RBCs led to deterioration of RBC functional properties, with a progressive increase in osmotic fragility and hemolysis over a 48-hour period (Figure 2, E and F). ...
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... decrease in NADPH was confirmed by both an enzymatic cycling method and HPLC (Supplemental Figure 6, (Figure 2A). Copper/ascorbate treatment of RBCs led to increased Prx2 dimerization within 1 hour ( Figure 2B) and a depletion of total NADP(H) ( Figure 2C) and decreased NADPH/NADP + ratio due to loss of NADPH ( Figure 2D). Increased oxidative stress in RBCs led to deterioration of RBC functional properties, with a progressive increase in osmotic fragility and hemolysis over a 48-hour period (Figure 2, E and F). ...
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... treatment of RBCs led to increased Prx2 dimerization within 1 hour ( Figure 2B) and a depletion of total NADP(H) ( Figure 2C) and decreased NADPH/NADP + ratio due to loss of NADPH ( Figure 2D). Increased oxidative stress in RBCs led to deterioration of RBC functional properties, with a progressive increase in osmotic fragility and hemolysis over a 48-hour period (Figure 2, E and F). No effect on deformability was observed (data not shown). ...
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... reducing equivalents are used for most antioxidant systems in RBCs. The nucleotide salvage metabolism recycles adenine and inosine, allowing the regeneration of NAD + and NADP + , and NADP + is subsequently reduced to NADPH by the PPP (22, 40). A decline in adenine and subsequent accumulation of its deaminated form, hypoxanthine, has been frequently noted as a marker ...
Citations
... Thus, erythrocytes must be well equipped with an efficient antioxidant and chaperone machinery [13,14]. The main non-enzymatic erythrocyte antioxidant in concentration terms is glutathione (GSH), which is present at a concentration of about 2 mM [15,16], much higher than uric acid/urate (50-500 μM) [17,18], ascorbate (about 50 μM) [19][20][21], NADH (≤50 μM), NADPH (5-40 μM) [22][23][24], and thioredoxin (Trx) (3-5 μM) [25,26] (Figure 1). The primary role in the removal of ROS is played by antioxidant enzymes. ...
... In contrast to fresh cells, oxidation was not reversed by incubation of stored blood with glucose [183]. In another study, the extent of Prdx2 oxidation was found to increase slightly during incubation at 4 • C for up to 6 weeks and much more during subsequent incubation at 37 • C [24]. Surprisingly, no differences in the Trx levels, NADPH concentrations, or TrxR activity were found between fresh and stored erythrocytes [185]. ...
Peroxiredoxin 2 (Prdx2) is the third most abundant erythrocyte protein. It was known previously as calpromotin since its binding to the membrane stimulates the calcium-dependent potassium channel. Prdx2 is present mostly in cytosol in the form of non-covalent dimers but may associate into doughnut-like decamers and other oligomers. Prdx2 reacts rapidly with hydrogen peroxide (k > 107 M−1 s−1). It is the main erythrocyte antioxidant that removes hydrogen peroxide formed endogenously by hemoglobin autoxidation. Prdx2 also reduces other peroxides including lipid, urate, amino acid, and protein hydroperoxides and peroxynitrite. Oxidized Prdx2 can be reduced at the expense of thioredoxin but also of other thiols, especially glutathione. Further reactions of Prdx2 with oxidants lead to hyperoxidation (formation of sulfinyl or sulfonyl derivatives of the peroxidative cysteine). The sulfinyl derivative can be reduced by sulfiredoxin. Circadian oscillations in the level of hyperoxidation of erythrocyte Prdx2 were reported. The protein can be subject to post-translational modifications; some of them, such as phosphorylation, nitration, and acetylation, increase its activity. Prdx2 can also act as a chaperone for hemoglobin and erythrocyte membrane proteins, especially during the maturation of erythrocyte precursors. The extent of Prdx2 oxidation is increased in various diseases and can be an index of oxidative stress.
... To study the physiology of transfused RBCs, in vitro conditions that mimic some aspects of the recipient's environment have been used. Stored RBCs have been exposed to body temperature (Roch et al., 2019) and healthy, diseased or pro-inflammatory plasma (Mittag et al., 2015;Tzounakas et al., 2016;Anastasiadi et al., 2021;Längst et al., 2021) to examine the impact of temperature transition and plasma components upon these cells. There are also in vitro models that aim to unravel immunologic derangements, by exposing stored RBCs to human T and B cells (Long et al., 2014). ...
... These changes seem to render the cells more susceptible to alterations in their intracellular oxidative burden when exposed to a "closer to normal" environment. In vitro studies on the transition of stored RBCs from cold to body temperature (without the addition of plasma) revealed a decrease in NADPH levels as well as dimerization of Prdx2 from middle storage onwards (Roch et al., 2019). The only exogenous oxidative stimuli that induced a distinct ROS generation profile post-reconstitution (namely rapid accumulation in both shortly-and medium-stored RBCs) was tBHP, a general reagent that hits numerous cellular targets. ...
The 24-hour (24 h) post-transfusion survival of donor red blood cells (RBCs) is an important marker of transfusion efficacy. Nonetheless, within that period, donated RBCs may encounter challenges able to evoke rapid stress-responses. The aim of the present study was to assess the effect of exposure to plasma and body temperature upon stored RBCs under recipient-mimicking conditions in vitro from the first hours "post-transfusion" up to 24 h. For this purpose, packed RBCs from seven leukoreduced CPD/ SAGM units were reconstituted with plasma of twenty-seven healthy individuals and incubated for 24 h at 37 °C. Three units were additionally used to examine stress-responses in 3-hour intervals post mixing with plasma (n = 5) until 24 h. All experiments were performed in shortly-, medium-, and long-stored RBCs. Hemolysis, redox, morphology, membrane protein binding and vesiculation parameters were assessed. Even though spontaneous hemolysis was minimal post-reconstitution, it presented a time-dependent increase. A similar time-course profile was evident for the concentration of procoagulant extracellular vesicles and the osmotic fragility (shortly-stored RBCs). On the contrary, mechanical fragility and reactive oxygen species accumulation were characterized by increases in medium-stored RBCs, evident even from the first hours in the recipient-mimicking environment. Finally, exposure to plasma resulted in rapid improvement of morphology, especially in medium-stored RBCs. Overall, some RBC properties vary significantly during the first 24 h post-mixing, at levels different from both the storage ones and the standard end-of-24 h. Such findings may be useful for understanding the performance of RBCs and their possible clinical effects −especially on susceptible recipients− during the first hours post-transfusion.
... One of the basic requirements of biochemistry laboratories is to maintain a constant temperature at predetermined levels during the working hours. For example, the gel-based agglutination test, which is extensively used for blood typing, produces the most accurate results when stabilized at the body temperature [5]. The test is based on the gel technique for detecting red blood cell agglutination reactions, where the gel acts as a trap [4]. ...
... with the set parameters. The control action branches moved away from the P-action line as decreasing 5 . When parallel to the P line, they are converted into a symmetrical sigmoid function. ...
A gel card incubator is an important clinical interface device that maintains gel cards at body temperature, completing the initial step in determining blood grouping. Achieving a much faster closed-loop response to shorten the warm-up period and maintaining a uniform temperature inside the incubator are crucial requirements for medical incubation devices. To accomplish incubator control requirements and deal with deadtime, this study proposes a novel nonlinear plus integral control (NPI) scheme coupled with a time-delay compensator using an incubator dynamic model. The NPI successfully shaped the control structure by utilizing a few nonlinear parameters. The control strategy, which promises a significant performance combining linear and nonlinear control actions, is easy to realize, simple to tune, and allows a smooth transition between control actions. Experiments were performed in a microprocessor-controlled gel card incubator using a computer interface to demonstrate the superiority of the proposed control technique. The comparative results prove that the proposed control scheme exhibits significantly faster and superior responses for transient and steady states. It also achieves successful disturbance rejection and less energy consumption than the Smith predictor-based PI control.
... 6 Thus, unsurprisingly present-day preventive medicine and intensive care both rely on monitoring the optical properties of blood to report on health. [7][8][9][10][11] It is generally accepted that the optical absorption behaviour of blood in the visible spectral region (450 to 700 nm) should relate to the oxidation state of the Fe centre found in the centre of the protoporphyrin ring. Why is this important? ...
We report how Raman difference imaging provides insight on cellular biochemistry in vivo as a function of sub-cellular dimensions and the cellular environment. We show that this approach offers a sensitive diagnostic to address blood biochemistry at the cellular level. We examine Raman microscopic images of the distribution of the different hemoglobins in both healthy (discocyte) and unhealthy (echinocyte) blood cells and interpret these images using pre-calculated, accurate pre-resonant Raman tensors for scattering intensities specific to hemogolobins. These tensors are developed from theoretical calculations of models of the oxy, deoxy and met forms of heme benchmarked against the experimental visible spectra of the corresponding hemoglobins. The calculations also enable assignments of the electronic transitions responsible for the colour of blood: these are mainly ligand to metal charge transfer transitions.
... Hemoglobin and other proteins with MW greater than 30 kDa were removed from RBC lysates using centrifugal filters with a 30 kDa threshold, and filtrates were heated to 60°C for 30 minutes per the manufacturer's protocol to decompose NADP + and permit measurement of the remaining NADPH. A detection reagent was added, and NADP(H) was monitored by absorbance at 450 nm for a period of 3 hours and quantified using standards [37]. ...
Red blood cells (RBCs) are susceptible to sustained free radical damage during circulation, while the changes of antioxidant capacity and regulatory mechanism of RBCs under different oxygen gradients remain unclear. Here, we investigated the changes of oxidative damage and antioxidant capacity of RBCs in different oxygen gradients and identified the underlying mechanisms using an in vitro model of the hypoxanthine/xanthine oxidase (HX/XO) system. In the present study, we reported that the hypoxic RBCs showed much higher oxidative stress injury and lower antioxidant capacity compared with normoxic RBCs. In addition, we found that the disturbance of the recycling process, but not de novo synthesis of glutathione (GSH), accounted for the significantly decreased antioxidant capacity of hypoxic RBCs compared to normoxic RBCs. We further elucidated the underlying molecular mechanism by which oxidative phosphorylation of Band 3 blocked the hexose monophosphate pathway (HMP) and decreased NADPH production aggravating the dysfunction of GSH synthesis in hypoxic RBCs under oxidative conditions.
... In future in vitro experiments, simulation of transfusion via post-storage incubation at 37 • C in plasma could be used to better reflect the behavior of the cells after transfusion [51,52]. It could also be interesting to test higher concentrations of antioxidants. ...
After blood donation, the red blood cells (RBCs) for transfusion are generally isolated by centrifugation and then filtrated and supplemented with additive solution. The consecutive changes of the extracellular environment participate to the occurrence of storage lesions. In this study, the hypothesis is that restoring physiological levels of uric and ascorbic acids (major plasmatic antioxidants) might correct metabolism defects and protect RBCs from the very beginning of the storage period, to maintain their quality. Leukoreduced CPD-SAGM RBC concentrates were supplemented with 416 µM uric acid and 114 µM ascorbic acid and stored during six weeks at 4 °C. Different markers, i.e., haematological parameters, metabolism, sensitivity to oxidative stress, morphology and haemolysis were analyzed. Quantitative metabolomic analysis of targeted intracellular metabolites demonstrated a direct modification of several metabolite levels following antioxidant supplementation. No significant differences were observed for the other markers. In conclusion, the results obtained show that uric and ascorbic acids supplementation partially prevented the metabolic shift triggered by plasma depletion that occurs during the RBC concentrate preparation. The treatment directly and indirectly sustains the antioxidant protective system of the stored RBCs.
Background:
The quality of red blood cells (RBCs) stored in red cell concentrates (RCCs) is influenced by processing, storage and donor characteristics, and can have a clinical impact on transfused patients. To evaluate RBC properties and their potential impact in a transfusion setting, a simple in vitro-transfusional model has been developed.
Materials and methods:
Transfusion was simulated by mixing a washed RBC pool from two male-derived RCCs stored at 4°C with a pool of 15 male-derived fresh frozen plasma (FFP) units, representing the recipient, at a hematocrit (HCT) of 30% ("control" setting) or 5% (alternative model). The mixtures were incubated at 37°C, 5% of CO2 up to 48 h. Different metabolites, hemolysis and microvesicles (MVs) were quantified at several incubation times and RBC-morphology changes and deformability after incubation. For each model, biological triplicates have been investigated with RCCs at storage days 2 and 43.
Results:
The 5%-HCT model restored the 2,3-DPG level and maintained the ATP level. Furthermore, glucose consumption and corresponding lactate production were increased in the 5%- vs the 30%-HCT condition. Lower hemolysis was observed with 5%-HCT, but only at day 2. However, morphological analysis by digital holographic microscopy (DHM) revealed a decreased fraction of discocytes at 5% rather than at 30% of HCT at storage day 2 but at day 43, the trend was inverted. Concordantly, RBCs incubated at 5% of HCT were more deformable than at 30% at day 43 (p<0.0001).
Discussion:
Higher metabolic activity of RBCs in the 5%-HCT condition was promoted by a higher glucose availability and limited cell-waste accumulation. The conditions of the new proposed model thus enabled rejuvenation of RBCs and maintained them in a physiological-close state in contrast to the 30%-HCT model. It may be used as a first approach to evaluate e.g., the impact of donor and recipient characteristics on RBC properties.
Band 3 (anion exchanger 1 - AE1) is the most abundant membrane protein in red blood cells (RBCs), the most abundant cell in the human body. A compelling model posits that - at high oxygen saturation - the N-term cytosolic domain of AE1 binds to and inhibits glycolytic enzymes, thus diverting metabolic fluxes to the pentose phosphate pathway to generate reducing equivalents. Dysfunction of this mechanism occurs during RBC aging or storage under blood bank conditions, suggesting a role for AE1 in the regulation of blood storage quality and efficacy of transfusion – a life-saving intervention for millions of recipients worldwide. Here we leverage two murine models carrying genetic ablations of AE1 to provide mechanistic evidence of its role in the regulation of erythrocyte metabolism and storage quality. Metabolic observations in mice recapitulated those in a human subject lacking expression of AE11-11 (band 3 Neapolis), while common polymorphisms in the region coding for AE11-56 correlate with increased susceptibility to osmotic hemolysis in healthy blood donors. Through thermal proteome profiling and cross-linking proteomics, we provide a map of the RBC interactome, with a focus on AE11-56 and validate recombinant AE1 interactions with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). As a proof-of-principle and further mechanistic evidence of the role of AE1 in the regulation of redox homeostasis of stored RBCs, we show that incubation with a cell-penetrating AE11-56 peptide can rescue the metabolic defect in glutathione recycling and boost post-transfusion recoveries of stored RBCs from healthy human donors and genetically ablated mice.
Purpose of review:
Over the last decades, clinical studies have suggested that transfusion of red blood cells (RBCs) might negatively impact patient outcomes. Even though large randomized clinical trials did not show differences in mortality when transfusing fresh versus standard-issue RBC units, data imply that RBCs at the very end of storage could elicit negative effects.
Recent findings:
Certain alterations of RBCs during cold storage -- such as an increase of potassium and lactate in the storage solution -- have been discovered a century ago. In recent years, proteomic and metabolomic studies have shed more light into pathophysiological changes of RBCs during storage and have helped to specify the definition of old blood. These advancements are now utilized to increase the quality of stored RBCs and devise therapeutic strategies (e.g. nitric oxide, haptoglobin, or reduction of the iron load) when transfusing old blood.
Summary:
Further research to improve the quality of RBC units and to study populations potentially at risk is warranted. Until the question whether transfusion of old blood is detrimental for specific patient populations has been answered, a deliberate use of RBC transfusion should be implemented.
