Differential Responses of Young and Old Erythrocytes Stored with Vitamin C and Vitamin E in Additive Solution-7

Abstract

Oxidative stress (OS) causes biochemical and morphological alterations in erythrocytes. The primary factors contributing to OS are aging and storage. Antioxidants significantly alleviate OS. Therefore, this study aimed to investigate the response of young and old erythrocytes to vitamin C and vitamin E during storage. Erythrocytes were separated into young and old by the Percoll method. Each erythrocyte subpopulation was categorized into the i) Control (additive solution-7 [AS-7]) and ii) vitamin C and vitamin E in AS-7 (VC+VE) groups and stored for 21 days at 4°C. OS, antioxidant, and aging markers were analyzed on days 1, 14, and 21. The activity of antioxidant enzymes was similar throughout storage in young cells. However, superoxide dismutase activity elevated in old cells (Control and VC+VE) on days 1 and 21. Catalase (CAT) activity increased on days 14 and 21, whereas glutathione peroxidase (GPX) increased on days 1 and 14 in old Controls. However, in old VC+VE, CAT increased on day 21 and GPX increased on day 1. Advanced oxidation protein products, superoxides, glutathione, and uric acid increased in old cells throughout storage. Malondialdehyde decreased in old VC+VE compared with old Control on days 14 and 21. Sialic acids and glutamate oxaloacetate transaminase activity were higher in young cells compared to old cells. Young cells exhibited lower oxidative changes throughout storage. Vitamin C and vitamin E were effective in maintaining the redox balance in old cells. These findings emphasize the need for specific approaches for different subpopulations during erythrocyte banking.

Keywords

young and old erythrocytes vitamin C vitamin E storage oxidative stress

Introduction

Erythrocytes have a finite lifespan in the peripheral circulation for approximately 120 days, after which they are removed in an age-dependent manner.^1,2 However, the shelf-life of stored erythrocytes is limited to 42 days.³ Packed red blood cells have a mixed population of recently matured (young) to senescent (old) cells. The storage of erythrocytes with a higher proportion of old cells may result in a decline in cellular recovery, oxygen supply, and metabolic stability.⁴

Erythrocytes undergo numerous biochemical, morphological, and immunological alterations during aging.^5,6 These include a reduction in cell size, volume, deformability, and hemoglobin content, an increase in cell density, aggregation, binding of autologous immunoglobulin G (IgG),^7–13 disruption of the band 3-mediated anchorage, and phosphatidylserine exposure,^14,15 changes in membrane cation transport,¹⁶ susceptibility to phospholipase A₂,¹⁷ lower sialic acids (SA) content and glutamate oxaloacetate transaminase (GOT) activity,^10–12,18 and the accumulation of lipid peroxidation products.¹⁹

SA regulate the membrane deformability, oxygen-carrying capacity, structure, and distribution of the hemoglobin. As erythrocytes continuously pass through narrow capillaries, their surface SA are gradually sheared off with age.²⁰ GOT activity decreases with erythrocyte age, due to erythrocyte fragmentation, denaturation of the enzyme, and lower levels of the cofactor, pyridoxal phosphate.^21,22 Therefore, SA and GOT or aspartate aminotransferase (AST) are considered as indices of aging.^2,20,23

Erythrocytes undergo changes that affect their viability and functions during storage, collectively known as “storage lesions.” Reactive oxygen species (ROS) overwhelm the antioxidant system, leading to oxidative stress (OS) during storage. However, erythrocytes possess efficient endogenous antioxidant defenses such as enzymatic and nonenzymatic antioxidants to counteract OS.²⁴ Exogenous antioxidants as additives can further enhance the antioxidant capacity, thereby improving the efficacy of stored erythrocytes.

The old erythrocytes during storage exhibit a decline in erythrocyte indices, plasma membrane redox system (PMRS), CD47 expression, accumulation of oxidation products, and reactive species, phosphatidylserine externalization, band 3 clusterization, and microparticle formation compared to young erythrocytes.^15,25–27 Consequently, old erythrocytes encounter higher OS during storage. Therefore, antioxidants as additives have been in focus in recent years as modifying agents in storage solutions.

There are limited studies that examined the effect of antioxidants on young and old erythrocytes during storage. Arduini et al.²⁸ observed that L-carnitine could protect against the loss of potassium ions in old erythrocytes and reduced osmotic swelling in both young and old erythrocytes. Vitamin C and vitamin E were effective during storage in erythrocytes.²⁹

Therefore, the objectives were to investigate i) the intermittent changes occurring during storage in young and old erythrocytes and, ii) the influence of vitamin C and vitamin E on young and old erythrocytes during storage in terms of oxidative modulations. Thus, the OS markers and antioxidant defenses were analyzed.

Materials and Methods

Ethical committee approval was obtained for this study (841/b/04/CPCSEA) (IAEC/NCP/117/2022).

Blood sampling

Wistar rats (male, 4 months old, n = 10) were lightly anesthetized with ketamine hydrochloride and restrained in dorsal recumbency. In brief, the syringe needle was inserted just below the xiphoid cartilage and slightly to the left of the midline. Blood (4–5 mL) was carefully aspirated from the heart into plastic collecting tubes containing anticoagulant, citrate, phosphate, dextrose, and adenine (CPDA)-1²⁹ (sodium citrate, 89 mM/L; citric acid, 17 mM/L; sodium dihydrogen phosphate, 5.8 mM/L; adenine, 2 mM/L; glucose, 177 mM/L; pH, 5.6). Whole blood and CPDA-1 were mixed in a 1:7 ratio.

Erythrocyte separation

The blood was centrifuged at 2000 rpm at 4°C for 20 minutes. The erythrocyte pellet was washed with isotonic phosphate buffer and resuspended in isotonic phosphate buffer ((a) 0.155 M/L NaH₂PO₄ [monobasic sodium phosphate], (b) 0.103M/L Na₂HPO₄ [dibasic sodium phosphate], pH 7.4, 310 mOsm) to a final hematocrit of 50%.³⁰ The young and old erythrocytes were separated using a Percoll density gradient.

Isolation of young and old erythrocyte subpopulations by Percoll density gradient centrifugation

Young and old erythrocytes were isolated using a modified method by Rennie et al.³¹ In brief, two solutions of bovine serum albumin (BSA) were prepared, solution 1: 4.8% BSA in water and solution 2: 4.8% BSA in Percoll (1.13 g/mL). Solutions A and B were prepared by mixing 19 parts of solutions 1 and 2 respectively with 1 part of HEPES buffer (4- (2-hydroxyethyl)-1-piperazineethanesulfonic acid) (pH 7.4). The two solutions were mixed at 80%, 71%, 67%, 64%, and 40% (v/v) to form discontinuous Percoll-BSA gradients. The erythrocyte suspension (50% hematocrit) was then layered carefully over the gradient and centrifuged at 3500 rpm for 20 minutes at 4°C. The erythrocytes fractioned in the 71% layer were “young cells,” and cells found in the 80% layer were “old cells.” These separated young and old erythrocytes were washed and resuspended in isotonic phosphate buffer (pH 7.4) to a final hematocrit of 50%.

Experimental design

The young and old erythrocytes were categorized into i) Control (additive solution-7 [AS-7]) and ii) VC+VE (vitamin C [10 mM] and vitamin E [2 mM] in AS-7) groups. The AS-7 and erythrocyte suspension were mixed at a ratio of 1.1: 4.5.³² The samples were treated on day 1 and stored for 21 days at 4°C. The aliquots were taken on days 1, 14, and 21 of storage and the OS, antioxidant, and aging markers were analyzed. A hemolysate was prepared by diluting the erythrocyte suspension in a hypotonic phosphate buffer ((a) 0.01 M/L NaH₂PO₄, (b) 6.6 mM/L Na₂HPO₄, pH 7.4) (1:14 ratio)³⁰ and stored at −20°C for further analysis.

Antioxidant enzymes

Superoxide dismutase

Superoxide dismutase (SOD) activity was measured by the method of Misra and Fridovich.³³ Hemolysate was added to the carbonate buffer (0.05 M) [(A) Na₂CO₃ (5.3 g/L), (B) NaHCO₃ (4.2 g/L), pH 10.2]. Epinephrine (30 mM) was added to the above mixture and the absorbance was read at 480 nm. SOD activity (units/mg protein) was expressed as the amount of enzyme that inhibits the oxidation of epinephrine by 50%.

Catalase

Hemolysate was treated with absolute alcohol and incubated at 0°C for 30 minutes. Hydrogen peroxide (H₂O₂; 6.6 mM) and phosphate buffer (0.1 M) were added to the above mixture and a decrease in absorbance was detected at 240 nm. Catalase (CAT) activity was estimated using the extinction coefficient of 43.6 M cm⁻¹.³⁴ One unit of CAT activity is equivalent to one mole of H₂O₂ degraded/minute/mg protein.

Glutathione peroxidase

Glutathione peroxidase (GPX) was analyzed by the method of Flohe and Gunzler.³⁵ Phosphate buffer (0.1 M), glutathione (GSH) reductase (0.24 units), and 10 mM GSH were added to hemolysate and preincubated for 10 minutes at 37°C, followed by the addition of 1.5 mM nicotinamide adenine dinucleotide phosphate (NADPH) in 0.1% NaHCO₃. The overall reaction was started by adding prewarmed H₂O₂ and a decrease in absorption was monitored at 340 nm. GPX activity was expressed as units/mg protein, where one unit corresponds to the oxidation of 1 mM of NADPH per minute.

Protein oxidation

Advanced oxidation protein products

Advanced oxidation protein products (AOPP) levels were estimated by Witko’s method.³⁶ Hemolysate was diluted in 0.1 M phosphate buffer saline and 1.16 M L⁻¹ potassium iodide was added. Then acetic acid was added and immediately read at 340 nm. AOPP levels were calculated by using the extinction coefficient of 26 mM⁻¹ cm⁻¹.

Protein sulfhydryl

Protein sulfhydryl (P-SH) was measured as described by Habeeb.³⁷ Hemolysate was treated with 0.08 M sodium phosphate buffer containing 0.5 mg/mL of Na₂-ethylenediaminetetraacetic acid (EDTA), and 2% sodium dodecyl sulfate (SDS). Subsequently, 0.1 mL of 5,5′-dithiobis-2-nitrobenzoic acid was added, vortexed, and absorbance was measured at 412 nm. P-SH was calculated by using the extinction coefficient of 13,600 M⁻¹ L⁻¹ cm⁻¹.

Lipid peroxidation

Malondialdehyde

Hemolysate samples were treated with 8.1% SDS, and incubated at room temperature for 10 minutes. 20% acetic acid was added followed by 0.6% thiobarbituric acid, and the samples were boiled until a pale yellow or pink color developed. Butanol-pyridine (15:1) was added, and centrifuged at 1000 rpm for 5 minutes, and the absorbance of the colored supernatant was measured at 532 nm using standard (1,1,3,3-tetra methoxy propane).³⁸

Conjugate dienes

Conjugate dienes (CD) were assessed by the method of Olas and Wachowicz.³⁹ Hemolyasate samples were diluted with ether: ethanol (1:3 v/v), vortexed, and centrifuged at 8000 rpm for 20 minutes. The absorbance of the supernatant was measured at 234 nm and calculated using the molar extinction coefficient of 29,500 M⁻¹ cm⁻¹.

Thiobarbituric acid reactive substances

Thiobarbituric acid reactive substances (TBARS) were determined by the method of Bar-Or et al.⁴⁰ Hemolysate was treated with 0.9% NaCl and incubated at 37°C for 20 minutes. Next, 0.8 M HCl containing 12.5% TCA and 1% thiobarbituric acid (TBA) was added and kept in a boiling water bath for 20 minutes and cooled at 4°C. Centrifugation was carried out at 4660 rpm and absorbance was measured at 532 nm. TBARS were calculated by using the extinction coefficient of 15,6000 M⁻¹ cm⁻¹.

Reactive species

Superoxides

Superoxide levels were estimated by the method of Olas and Wachowicz.³⁹ Hemolysate was treated with cytochrome C (160 μM), incubated at 37°C and the absorbance was measured at 550 nm. The extinction coefficient of 18700 M⁻¹cm⁻¹ was used to calculate superoxide levels.

Nitrites

Nitrite levels were determined by the method of Yegin et al.⁴¹ Hemolysate was treated with distilled water followed by a coupling reagent (Griess reagent [1% sulfanilamide, 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride and 2.5% phosphoric acid]), vortexed, and incubated for 10 minutes. The absorbance was measured at 520 nm. Sodium nitrite was used as a standard to determine the amount of nitrites.

Total antioxidant capacity: cupric ion-reducing antioxidant capacity-BCS assay

Cupric ion-reducing antioxidant capacity (CUPRAC) was measured using the method described by Campos et al.⁴² Briefly, the hemolysate was treated with 0.25 mM bathocuproinedisulfonic acid disodium salt (BCS) in 10 mM phosphate buffer (pH 7.4) and an initial absorbance was read at 490 nm. CuSO₄ (0.5 mM) was added, and incubated at room temperature followed by 0.01 M EDTA to arrest the reaction. The absorbance was read at 490 nm, compared with a standard uric acid and expressed in mM uric acid equivalents/L.

PMRS

The PMRS was estimated by a modified method of Avron and Shavit.⁴³ The hemolysate was mixed with phosphate buffer saline (PBS) (containing 5 mM glucose and 1 mM potassium ferricyanide), incubated for 30 minutes at 37°C, centrifuged at 3500 rpm and the supernatant was treated with sodium acetate (3 M), citric acid (0.2 M), and FeCl₃ (3.3 M). The amount of ferrocyanide was measured using 4,7-diphenyl-1,10-phenanthrolinedisulfonic acid disodium salt (6.2 mM) and the absorbance was read at 535 nm. PMRS activity was calculated using the extinction coefficient of 20,500 M⁻¹cm⁻¹ and expressed as μM ferrocyanide/mL hemolysate/30 minutes.

Nonenzymatic antioxidants

GSH

GSH was measured using the method described by Beutler et al.⁴⁴ Briefly, hemolysate was treated with 4% sulphosalicylic acid, vortexed, and centrifuged at 8000 rpm for 15 minutes. The supernatant was treated with 10 mM DTNB and the absorbance was read at 412 nm. GSH was calculated using the extinction coefficient 13600 M⁻¹cm⁻¹.

Uric acid

Uric acid levels were determined using the Uricase/POD method kit provided by Aspen Laboratories.⁴⁵ The hemolysate was treated with the reagent, mixed well, and incubated at 37°C. The absorbance was measured at 492 nm.

Aging markers

SA

SA was estimated using the method of Warren.⁴⁶ Briefly, the sample was treated with 0.2 M sodium periodate solution and incubated at room temperature for 20 minutes. Sodium arsenite solution (10%) was added and vortexed until the brown color disappeared. A solution of 0.6% TBA in 0.5 M sodium sulfate was added and incubated in a boiling water bath for 15 minutes. The reaction mixture was cooled and equal volumes of cyclohexanone were added to the top layer. The mixture was then centrifuged at 2000 rpm for 5 minutes at room temperature and the absorbance of the supernatant was read at 549 nm. The molar extinction coefficient, 57,000 M⁻¹cm⁻¹ was used to determine the SA content.

GOT or AST

The levels of GOT/AST in hemolysate were assessed utilizing the modified International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) method at 340 nm as described in the Aspen Laboratories kit.²³ Reagents 1 and 2 were mixed (4:1 ratio), added to the hemolysate, and incubated at 37°C. The change in absorbance per minute was measured at 340 nm for 3 minutes, which is proportional to the GOT activity in the sample.

Protein determination

Protein in the samples was determined using the standard BSA following the method of Lowry et al.⁴⁷

Statistical analyses

The results obtained were expressed as mean ± SE. Two-way ANOVA and a Bonferroni post hoc test were performed using GraphPad Prism 6 software, and p < 0.05 was considered statistically significant.

Results

The results are represented as comparisons between i) Day 1 versus other storage days in their respective groups, ii) Control group versus Antioxidant group on a particular storage day, and iii) young versus old erythrocytes on a particular day of storage.

Antioxidant enzymes

SOD

SOD activity remained constant throughout storage in both the Control and VC+VE groups.

SOD activity increased by 3-fold in old cells compared with young cells, on days 1 (p < 0.05), 14, and 21 (p < 0.01) (Fig. 1A).

FIG. 1.

(A) Superoxide dismutase activity in stored erythrocytes. (B) Catalase activity in stored erythrocytes. (C) Glutathione peroxidase activity in stored erythrocytes. Values are expressed as mean ± SE from 10 samples. Significance between the groups and subgroups was analyzed by two-way ANOVA followed by Bonferroni post hoc test, using GraphPad Prism 6 software. p-Value < 0.05 was considered significant. (@) represents significant changes during storage with respect to day 1 and (#) represents significant changes between young and old erythrocytes on a particular day of storage.

CAT

CAT activity increased by 14-fold (p < 0.001) and 20-fold (p < 0.0001) on days 14 and 21, respectively, in old Control, whereas it increased by 11-fold (p < 0.01) in old VC+VE on day 21 in comparison to day 1.

CAT activity showed an increment of 38% (p < 0.01) in old Controls compared to young Controls on day 21 (Fig. 1B)

GPX

GPX activity decreased by 35% (p < 0.05) in old VC+VE on day 21 compared with day 1.

GPX activity increased by 94% (p < 0.01) in old Controls compared to young Controls on days 1 and 14, and it increased by 55% (p < 0.01) in old VC+VE compared to young VC+VE on day 1 (Fig. 1C).

Protein oxidation

AOPP

AOPP levels were similar in both the Control and VC+VE groups during storage.

AOPP levels increased by 3-fold (p < 0.05) in the old cells compared to their respective young cells throughout the storage period (Fig. 2A).

FIG. 2.

(A) Advanced oxidation protein products in stored erythrocytes. (B) Protein sulfhydryl in stored erythrocytes. Values are expressed as mean ± SE from 10 samples. Significance between the groups and subgroups was analyzed by two-way ANOVA followed by Bonferroni post hoc test, using GraphPad Prism 6 software. p-Value < 0.05 was considered significant. (#) represents significant changes between young and old erythrocytes on a particular day of storage.

P-SH

The changes in P-SH levels were similar throughout storage compared to day 1.

There were changes between the Controls and VC+VE groups throughout storage; however, these variations were statistically similar.

P-SH levels were higher by 1-fold (p < 0.01) in old VC+VE compared to Young VC+VE on day 1 (Fig. 2B).

Lipid peroxidation

Malondialdehyde

Malondialdehyde (MDA) levels showed increments of 5-fold (p < 0.0001) and 3-fold (p < 0.0001) on days 14 and 21, respectively, in old Controls and 13-fold (p < 0.0001) and 9-fold (p < 0.001) on days 14 and 21, respectively, in old VC+VE compared with day 1.

MDA levels decreased by 45% (p < 0.0001) and 41% on days 14 and 21, respectively, in old VC+VE compared with old Control.

MDA levels increased by 3-fold (p < 0.0001) and 4-fold (p < 0.0001) on days 14 and 21, respectively, in old Controls versus young Controls. Similarly, MDA levels showed increments of 4-fold (p < 0.0001) in old VC+VE compared to young VC+VE on day 14 (Fig. 3A).

FIG. 3.

(A) Malondialdehyde in stored erythrocytes. (B) Conjugate dienes stored erythrocytes. (C) Thiobarbituric acid reactive substances in stored erythrocytes. Values are expressed as mean ± SE from 10 samples. Significance between the groups and subgroups was analyzed by two-way ANOVA followed by Bonferroni post hoc test, using GraphPad Prism 6 software. p-Value < 0.05 was considered significant. (@) represents significant changes during storage with respect to day 1; (*) represents significant changes between Control and VC+VE on a particular day of storage; (#) represents significant changes between young and old erythrocytes on a particular day of storage.

CD

CD decreased by 46% (p < 0.01) in young Controls on days 14 and 21 and by 30% (p < 0.01) in old Control on day 14 compared with day 1.

CD showed increments of 81% (p < 0.01) and 45% (p < 0.05) in old Controls and VC+VE compared with young Controls and VC+VE respectively, on day 14. Similarly, CD increased by 108% (p < 0.0001) in old Controls compared to young Controls on day 21 (Fig. 3B).

TBARS

TBARS levels were similar throughout storage in all the groups (Fig. 3C).

Reactive species

Superoxides

Superoxides increased by 124% (p < 0.001) and 89% (p < 0.05) in old Controls and old VC+VE, respectively, on day 21 compared with day 1.

Superoxides showed an increment of 285% (p < 0.0001) in old cells compared to their respective young cells, on day 21 (Fig. 4A).

FIG. 4.

(A) Superoxides in stored erythrocytes. (B) Nitrites in stored erythrocytes. Values are expressed as mean ± SE from 10 samples. Significance between the groups and subgroups was analyzed by two-way ANOVA followed by Bonferroni post hoc test, using GraphPad Prism 6 software. p-Value < 0.05 was considered significant. (@) represents significant changes during storage with respect to day 1; (#) represents significant changes between young and old erythrocytes on a particular day of storage.

Nitrites

Nitrite levels showed an increment of 122% (p < 0.0001) in old Controls on day 21. Nitrites decreased by 52% (p < 0.001), whereas they increased by 90% (p < 0.0001) in old VC+VE on days 14 and 21. Nitrites also increased in young VC+VE by 65% (p < 0.01) on day 21 compared with day 1.

Nitrites showed increments of 94% (p < 0.0001) and 67% (p < 0.0001) in old Control and VC+VE, respectively, compared with their respective young cells on day 21 (Fig. 4B).

Total antioxidant capacity

Total antioxidant capacity (TAC) was decreased by 57% (p < 0.0001) and 46% (p < 0.001) on days 14 and 21, respectively, in young Controls, and by 50% (p < 0.05) on day 21 in young VC+VE compared with day 1.

TAC was maintained in Controls and VC+VE in both young and old cells throughout storage (Fig. 5A).

FIG. 5.

(A) Total antioxidant capacity in stored erythrocytes. (B) Plasma membrane redox system in stored erythrocytes. (C) Glutathione in stored erythrocytes. (D) Uric acid in stored erythrocytes. Values are expressed as mean ± SE from 10 samples. Significance between the groups and subgroups was analyzed by two-way ANOVA followed by Bonferroni post hoc test, using GraphPad Prism 6 software. p-Value < 0.05 was considered significant. (@) represents significant changes during storage with respect to day 1; (#) represents significant changes between young and old erythrocytes on a particular day of storage.

PMRS

PMRS was similar in all the groups throughout storage.

PMRS increased by 1-fold (p < 0.001) in old VC+VE compared with young VC+VE on day 1 (Fig. 5B).

Nonenzymatic antioxidants

GSH

GSH levels were similar in all the groups during storage.

GSH levels increased by 103% (p < 0.0001) in old Controls and 70% (p < 0.0001) in old VC+VE compared with their respective young cells throughout storage (Fig. 5C).

Uric acid

Uric acid levels were decreased by 32% (p < 0.01) in old VC+VE on day 14 compared with day 1.

Uric acid levels showed increments of 87% (p < 0.0001), 78% (p < 0.05), and 65% in old Controls on days 1, 14, and 21 respectively, compared with young Controls. Uric acid levels also increased by 59% (p < 0.0001) and 74% (p < 0.0001) on days 1 and 21, respectively, in old VC+VE compared with young VC+VE (Fig. 5D).

Aging markers

SA

SA levels were maintained throughout storage in all the groups (Fig. 6A).

FIG. 6.

(A) Sialic acids in stored erythrocytes. (B) Glutamic oxaloacetate transaminase activity in stored erythrocytes. Values are expressed as mean ± SE from 10 samples. Significance between the groups and subgroups was analyzed by two-way ANOVA followed by Bonferroni post hoc test, using GraphPad Prism 6 software. p-Value < 0.05 was considered significant.

GOT or AST

GOT activity was maintained similarly throughout storage. However, GOT activity was higher in young cells compared to old cells throughout storage (Fig. 6B).

Discussion

Oxidative alterations occur during the second week of erythrocyte storage.⁴⁸ Therefore, the initial analysis of samples was confined to intervals of 14 days. The young cells disappear from day 21 of storage.^26,49 Therefore, storage period was limited to 21 days. SA and GOT serve as markers of erythrocyte aging, whereas old erythrocytes have lower levels of SA levels (30%) and GOT activity compared with young cells.^2,20,23 This was confirmed in this study by estimating the GOT activity and SA content.

Erythrocytes are more susceptible to oxidative damage with age,² leading to a decline in their function and viability.⁵ SOD converts superoxide radicals into H₂O₂ and oxygen. CAT and GPX scavenge H₂O₂.²⁴ However, the GSH-dependent activity of GPX has been considered the primary line of defense against H₂O₂.

Young erythrocytes exhibit lower oxidative changes compared to old cells.^27,50 This was evidenced by constant protein oxidation products throughout storage and a decline in primary lipid peroxidation product, CD, on days 14 and 21, indicating a cessation in lipid propagation reaction. Consequently, secondary products such as MDA and TBARS were maintained in young Controls. Similarly, the antioxidant defenses, including SOD, CAT, GPX, uric acid, GSH, the PMRS, superoxides, and nitrites, were maintained throughout storage in young Controls, suggesting that redox homeostasis was maintained in young cells. However, there is a gradual decrease in TAC on days 14 and 21 in young Controls, as erythrocytes experience a reduction in antioxidant capacity during storage.^51,52

Erythrocytes are susceptible to OS with cell age and storage.^15,27,50,51 This was evidenced by elevations in CD on day 14 and MDA levels on days 14 and 21, respectively, in old Controls with storage. Superoxide levels were higher in old erythrocytes than in young ones. This could be attributed to the accumulation of reactive species during cell aging, which were generated by the auto-oxidation of hemoglobin and possibly by NADPH oxidase.^11,53,54 In addition, reactive species such as nitrites and superoxides increased at the end of storage, leading to a decline in TAC. The antioxidant defenses were activated due to the accumulation of reactive species. Consequently, CAT activity elevated on days 14 and 21 in response to higher levels of H₂O₂ in old Controls.

P-SH was maintained in a reduced state, whereas AOPP levels increased throughout storage in old Controls compared with young Controls. Furthermore, MDA and CD increased on days 14 and 21 in old Controls compared with young Controls, reflecting the increased susceptibility of old cells to OS. This was also evidenced by higher levels of superoxide and nitrites on day 21 in old cells. Thereby, the antioxidant defenses were activated to counteract the reactive species and to maintain redox homeostasis. Therefore, SOD activity increased on days 1 and 21 to scavenge higher levels of superoxide radicals. GPX activity elevated on days 1 and 14 because it primarily scavenges lower levels of H₂O₂, whereas CAT activity increased on day 21 in response to higher levels of H₂O₂ at the end of storage. Similarly, endogenous antioxidants, such as uric acid and GSH, increased in old cells compared to their young cells throughout storage. Antioxidant defenses were upregulated in response to increased levels of ROS.^55,56

MDA levels increased in old Controls and VC+VE with storage; however, they were lower in old VC+VE compared with old Controls because of the synergistic action of vitamins C and E against lipid peroxidation.⁵⁷ Vitamin E, as a membrane-interacting antioxidant, decreases MDA levels and protects erythrocytes from lipid peroxidation during storage.^58,59 CAT activity elevated in old cells at the end of storage in response to higher levels of H₂O₂. Vitamin C and Vitamin E directly scavenge H₂O₂,^60–62 thereby CAT activity was lower in VC+VE compared with Controls. This was also evidenced by elevated GPX activity. Nitrites declined in old VC+VE on day 14, indicating the reaction of vitamin C⁶³ and vitamin E⁶⁴ against nitrite generation, resulting in a decrease in endogenous antioxidant levels, such as uric acid. However, the nitrites increased on day 21 in both young and old VC+VE because of the consumption of nitrites by erythrocytes under hypoxic conditions, especially after storage for 14 days or longer.⁶⁵

AOPP levels increased throughout storage in old VC+VE compared with young VC+VE because of the susceptibility of sensitive protein groups to oxidation. Thereby, the antioxidant defenses, such as GSH levels elevated throughout storage and SOD activity, and uric acid levels increased on days 1 and 21. The reactive species such as superoxides and nitrites were maintained until day 14 and increased only at the end of storage in old cells compared to their young cells, indicating that additive antioxidants could scavenge oxidants^61,63,64,66 and protected old cells up to 14 days of storage.

P-SH levels were higher in old VC+VE on day 1, indicating that additive antioxidants were effective in protecting P-SH groups against oxidation even in old cells on day 1 of storage. Vitamin C protects intracellular thiol groups,⁶⁷ thereby GSH levels were maintained in the reduced state. Higher levels of GSH protect the erythrocyte proteins from oxidation,⁶⁸ and preserved P-SH groups,²⁴ which were associated with these results.

PMRS maintains cellular redox balance by regulating the NAD(P)+/NAD(P)H ratio.⁵⁰ Vitamin C and vitamin E are involved in the cellular redox system by inhibiting lipid peroxidation and maintaining GSH in a reduced state, utilizing reducing equivalents from NADPH.^69,70 Therefore, PMRS elevated in old VC+VE on day 1 and remained higher throughout storage, indicating the effect of antioxidant additives in maintaining redox equilibrium.

Conclusion

Erythrocytes are more prone to OS during their aging and storage. This was evidenced by the elevation in oxidation products, reactive species, activation of antioxidant defenses, reduced antioxidant capacity, lower levels of SA content, and GOT activity in old cells compared to young cells during storage.

Young cells exhibited minimal oxidative changes and consistent responses regardless of the presence or absence of antioxidants throughout storage. However, vitamin C and vitamin E could maintain antioxidant capacity (TAC) and scavenge nitrites in young cells up to 14 days of storage.

Vitamin C and vitamin E were effective in reducing MDA and nitrite levels up to 14 days of storage, and maintained sulfhydryls and PMRS in old cells. The onset of OS was evident from day 14 in old Controls, whereas it was evident from day 21 in old VC+VE. Therefore, employing antioxidant additives, vitamin C and vitamin E could delay the onset of oxidative changes in old erythrocytes during storage. These findings highlight the necessity for specific approaches in blood banking, particularly directed toward young and old erythrocytes.

Authors’ Contributions

M.P.: Investigation, formal analysis, data curation, writing—original draft, visualization. V.R.: Conceptualization, methodology, validation, supervision, Writing—review & editing.

Footnotes

Acknowledgments

The authors acknowledge Dr. Leela Iyengar, Dr. Soumya Ravikumar, Dr. Carl Hsieh, Dr. Manasa K, Ms. Magdaline Christina R, Anusha B A, and JAIN (Deemed-to-be University) for their support.

Author Disclosure Statement

There are no conflicts of interest to declare.

Funding Information

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author, upon reasonable request.

Ethical Approval

The study was approved by the ethics committee (IAEC/NCP/117/2022) and animal care and maintenance were in accordance with the ethical committee regulations.

References

Lledó-García

, Kalicki

, Uehlinger

, et al. Modeling of red blood cell life-spans in hematologically normal populations. J Pharmacokinet Pharmacodyn, 2012; 39(5):453–462.

Jamshidi

, Xu

, von Löhneysen

, et al. Metabolome changes during in vivo red cell aging reveal disruption of key metabolic pathways. Iscience, 2020; 23(10):101630.

D’Alessandro

, Liumbruno

, Grazzini

, et al. Red blood cell storage: The story so far. Blood Transfus, 2010; 8(2):82–88.

Yoshida

, Prudent

, D'alessandro

. Red blood cell storage lesion: Causes and potential clinical consequences. Blood Transfus, 2019; 17(1):27–52.

Magnani

, De Flora

., (eds). Red Blood Cell Aging. Plenum Press: New York and London; 1990.

Lutz

. Erythroid cells. In: Blood Cell Biochemistry ( Harris

. eds.) Plenum Press, New York and London: 1990; pp. 81–120.

Lutz

, Stringaro-Wipf

. Senescent red cell-bound IgG is attached to band 3 protein. Biomed Biochim Acta, 1983; 42(11-12):S117–121.

Kannan

, Yuan

, Low

. Isolation and partial characterization of antibody-and globin-enriched complexes from membranes of dense human erythrocytes. Biochem J, 1991; 278 (Pt 1)(Pt 1):57–62.

Bosman

, Werre

, Willekens

, et al. Erythrocyte ageing in vivo and in vitro: Structural aspects and implications for transfusion. Transfus Med, 2008; 18(6):335–347.

10.

Bizjak

, Brinkmann

, Bloch

, et al. Increase in red blood cell-nitric oxide synthase dependent nitric oxide production during red blood cell aging in health and disease: A study on age dependent changes of rheologic and enzymatic properties in red blood cells. PLoS One, 2015; 10(4):e0125206.

11.

Grau

, Kuck

, Dietz

, et al. Sub-fractions of red blood cells respond differently to shear exposure following superoxide treatment. Biology (Basel), 2021; 10(1):47.

12.

Grau

, Zollmann

, Bros

, et al. Autologous blood doping induced changes in red blood cell rheologic parameters, RBC age distribution, and performance. Biology (Basel), 2022; 11(5):647.

13.

Ermolinskiy

, Lugovtsov

, Yaya

, et al. Effect of red blood cell aging in vivo on their aggregation properties in vitro: Measurements with laser tweezers. Appl Sci, 2020; 10(21):7581.

14.

Bartosz

, Grzelinska

, Bartkowiak

. Aging of the erythrocyte. XIX. Decrease in surface charge density of bovine erythrocytes. Mech Ageing Dev, 1984; 24(1):1–7.

15.

Biasini

, Botrè

, de la Torre

, et al. Age-markers on the red blood cell surface and erythrocyte microparticles may constitute a multi-parametric strategy for detection of autologous blood transfusion. Sports Med Open, 2023; 9(1):113.

16.

Hentschel

, Wu

, Tobin

, et al. Erythrocyte cation transport activities as a function of cell age. Clin Chim Acta, 1986; 157(1):33–43.

17.

Shukla

, Hanahan

. Membrane alterations in cellular aging: Susceptibility of phospholipids in density (age)-separated human erythrocytes to phospholipase A2. Arch Biochem Biophys, 1982; 214(1):335–341.

18.

Bartosz

. Erythroid Cells. In: Blood Cell Biochemistry ( Harris

. eds.) Plenum Press: New York; 1990; pp. 45-79.

19.

Jain

. Evidence for membrane lipid peroxidation during the in vivo aging of human erythrocytes. Biochim Biophys Acta, 1988; 937(2):205–210.

20.

Huang

, Wu

, Mehrishi

, et al. Human red blood cell aging: Correlative changes in surface charge and cell properties. J Cell Mol Med, 2011; 15(12):2634–2642.

21.

Fischer

, Walter

. Aspartate amino transferase (GOT) from young and old human erythrocytes. I Lab Clin Med, 1971; 78:736–745.

22.

Seaman

, Knox

, Nordt

, et al. Red cell aging. I. Surface charge density and sialic acid content of density-fractionated human erythrocytes. Blood, 1977; 50(6):1001–1011.

23.

Jakubowska-Solarska

, Solski

. Sialic acids of young and old red blood cells in healthy subjects. Med Sci Monit, 2000; 6:871–874.

24.

Çimen

MYB

. Free radical metabolism in human erythrocytes. Clin Chim Acta, 2008; 390(1-2):1–11.

25.

Antonelou

, Tzounakas

, Velentzas

, et al. Effects of pre-storage leukoreduction on stored red blood cells signaling: A time-course evaluation from shape to proteome. J Proteomics, 2012; 76 Spec No:220–238.

26.

Hsieh

, Prabhu

, Rajashekaraiah

. Age-related modulations in erythrocytes under blood bank conditions. Transfus Med Hemother, 2019; 46(4):257–266.

27.

Mykhailova

, Olafson

, Turner

, et al. Donor‐dependent aging of young and old red blood cell subpopulations: Metabolic and functional heterogeneity. Transfusion, 2020; 60(11):2633–2646.

28.

Arduini

, Minetti

, Ciana

, et al. Cellular properties of human erythrocytes preserved in saline–adenine–glucose–mannitol in the presence of L‐carnitine. Am J Hematol, 2007; 82(1):31–40.

29.

Pallavi

, Rajashekaraiah

. Synergistic activity of vitamin-C and vitamin-E to ameliorate the efficacy of stored erythrocytes. Transfus Clin Biol, 2023; 30(1):87–95.

30.

Dodge

, Mitchell

, Hanahan

. The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch Biochem Biophys, 1963; 100:119–130.

31.

Rennie

, Thompson

, Parker

, et al. Human erythrocyte fractionation in “Percoll” density gradients. Clin Chim Acta, 1979; 98(1-2):119–125.

32.

Veale

, Healey

, Sran

, et al. AS‐7 improved in vitro quality of red blood cells prepared from whole blood held overnight at room temperature. Transfusion, 2015; 55(1):108–114.

33.

Misra

, Fridovich

. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem, 1972; 247(10):3170–3175.

34.

Aebi

. Catalase in vitro. Methods Enzymol, 1984; 105:121–126.

35.

Flohe

, Gunzler

. Assays of glutathione peroxidase. Methods Enzymol, 1984; 105:114–121.

36.

Witko-Sarsat

, Friedlander

, Capeillère-Blandin

, et al. Advanced oxidation protein products as a novel marker of oxidative stress in uremia. Kidney Int, 1996; 49(5):1304–1313.

37.

Habeeb

AFSA

. Reaction of protein sulfhydryl groups with Ellman’s reagent. Methods Enzymol, 1972; 25:457–464.

38.

Ohkawa

, Ohishi

, Yagi

. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem, 1979; 95(2):351–358.

39.

Olas

, Wachowicz

. Resveratrol and vitamin C as antioxidants in blood platelets. Thromb Res, 2002; 106(2):143–148.

40.

Bar-Or

, Rael

, Lau

, et al. An analog of the human albumin N-terminus (Asp-Ala-His-Lys) prevents formation of copper-induced reactive oxygen species. Biochem Biophys Res Commun, 2001; 284(3):856–862.

41.

Yegin

, Yur

, Çetın

, et al. Effect of lycopene on serum nitrite-nitrate levels in diabetic rats. Indian J Pharm Sci, 2015; 77(3):357–360.

42.

Campos

, Guzmán

, López-Fernández

, et al. Evaluation of the copper (II) reduction assay using bathocuproinedisulfonic acid disodium salt for the total antioxidant capacity assessment: The CUPRAC-BCS assay. Anal Biochem, 2009; 392(1):37–44.

43.

Avron

, Shavit

. A sensitive and simple method for determination of ferrocyanide. Anal Biochem, 1963; 6:549–554.

44.

Beutler

, Duran

, Kelley

. Modified procedure for the estimation of reduced glutathione. J Lab Clin Med, 1963; 61:882–888.

45.

Thomas

., (eds.). Clinical laboratory diagnostics: use and assessment of clinical laboratory results. TH-books Verlagsgesellschaft; 1998.

46.

Warren

. The thiobarbituric acid assay of sialic acids. J Biol Chem, 1959; 234(8):1971–1975.

47.

Lowry

, Rosebrough

, Farr

, et al. Protein measurements with Folin-phenol reagent. J Biol Chem, 1951; 193(1):265–275.

48.

Soumya

, Vani

. CUPRAC–BCS and antioxidant activity assays as reliable markers of antioxidant capacity in erythrocytes. Hematology, 2015; 20(3):165–174.

49.

Tuo

, Wang

, Liang

, et al. How cell number and cellular properties of blood-banked red blood cells of different cell ages decline during storage. PLoS One, 2014; 9(8):e105692.

50.

Kumar

, Rizvi

. Markers of oxidative stress in senescent erythrocytes obtained from young and old age rats. Rejuvenation Res, 2014; 17(5):446–452.

51.

Soumya

, Vani

. Vitamin C as a modulator of oxidative stress in erythrocytes of stored blood. Acta Haematol Pol, 2017; 48(4):350–356.

52.

Huyut

, Şekeroğlu

, Balahoroğlu

, et al. Characteristics of resveratrol and serotonin on antioxidant capacity and susceptibility to oxidation of red blood cells in stored human blood in a time-dependent manner. J Int Med Res, 2018; 46(1):272–283.

53.

Mohanty

, Nagababu

, Rifkind

. Red blood cell oxidative stress impairs oxygen delivery and induces red blood cell aging. Front Physiol, 2014; 5:84.

54.

George

, Pushkaran

, Konstantinidis

, et al. Erythrocyte NADPH oxidase activity modulated by Rac GTPases, PKC, and plasma cytokines contributes to oxidative stress in sickle cell disease. Blood, 2013; 121(11):2099–2107.

55.

. Modulation of skeletal muscle antioxidant defense by exercise: Role of redox signaling. Free Radic Biol Med, 2008; 44(2):142–152.

56.

Didier

, Stiene

, Fang

, et al. Antioxidant and anti-tumor effects of dietary vitamins A, C, and E. Antioxidants, 2023; 12(3):632.

57.

Claro

, Leonart

MSS

, Comar

, et al. Effect of vitamins C and E on oxidative processes in human erythrocytes. Cell Biochem Funct, 2006; 24(6):531–535.

58.

Antosik

, Czubak

, Cichon

, Nowak

, Zbikowska

. Vitamin E analogue protects red blood cells against storage-induced oxidative damage. Transfus Med Hemother, 2018; 45(5):347–354.

59.

Hajizamani

, Atarodi

, Deyhim

, et al. Antioxidative effects of α-tocopherol on stored human red blood cell units. Asian J Transfus Sci, 2023

60.

Knight

, Blaylock

, Searles

. The effect of vitamins C and E on lipid peroxidation in stored erythrocytes. Ann Clin Lab Sci, 1993; 23:51–56.

61.

Lassnigg

, Punz

, Barker

, et al. Influence of intravenous vitamin E supplementation in cardiac surgery on oxidative stress: A Double Blinded, Randomized, Controlled Study. Br J Anaesth, 2003; 90(2):148–154.

62.

Pehlivan

. Vitamin C: An antioxidant agent. Vitamin C, 2017; 2:23–35.

63.

May

, Qu

, Xia

, et al. Nitrite uptake and metabolism and oxidant stress in human erythrocytes. Am J Physiol Cell Physiol, 2000; 279(6):C1946–1954.

64.

Chow

, Hong

. Dietary vitamin E and selenium and toxicity of nitrite and nitrate. Toxicology, 2002; 180(2):195–207.

65.

Stapley

, Owusu

, Brandon

, et al. Erythrocyte storage increases rates of NO and nitrite scavenging: Implications for transfusion-related toxicity. Biochem J, 2012; 446(3):499–508.

66.

Halliwell

. Vitamin C: Antioxidant or prooxidant in vivo? Free Radic Res, 1996; 25:439–454.

67.

Carr

, Frei

. The role of natural antioxidants in preserving the biological activity of endothelium-derived nitric oxide. Free Radic Biol Med, 2000; 28(12):1806–1814.

68.

Carroll

, Raththagala

, Subasinghe

, et al. An altered oxidant defense system in red blood cells affects their ability to release nitric oxide stimulating ATP. Mol Biosyst, 2006; 2(6-7):305–311.

69.

Traber

, Atkinson

. Vitamin E, antioxidant and nothing more. Free Radic Biol Med, 2007; 43(1):4–15.

70.

Blaner

, Shmarakov

, Traber

. Vitamin A and vitamin E: Will the real antioxidant please stand up? Annu Rev Nutr, 2021; 41:105–131.