There is a dire necessity to improve blood storage and prolong shelf-life of blood. Very few studies have focused on oxidative stress (OS) in blood and its influence on plasma with storage. This study attempts to (i) elucidate the continuous changes occurring in plasma during storage through oxidant levels and antioxidant status and (ii) evaluate the influence of vitamin C (VC) as an additive during blood storage. Blood was drawn from male
Blood transfusion is an irreplaceable, lifesaving, and overall safe treatment. Continued developments in storage techniques have resulted in improved storage and blood quality. Whole blood is stored in CPDA (citrate phosphate dextrose and adenine) or ACD (acid, citrate, and dextrose) solution up to a period of 35 days at 4°C [
One of the reasons for the formation of the storage lesion is oxidative stress (OS). This was evident in our earlier study on erythrocytes of stored blood [
There are many efficient antioxidants which can reduce the OS induced by storage [
Therefore, we aimed to study two aspects (i) the continuous changes occurring during storage and (ii) the influence of vitamin C as an additive in stored blood. The changes occurring in plasma isolated from stored blood were analyzed at regular intervals during a period of 25 days.
In this regard the following objectives were put forth: to analyze the antioxidant status of plasma through antioxidant enzymes: superoxide dismutase (SOD) and catalase (CAT), to evaluate the oxidant levels through lipid peroxidation (thiobarbituric acid reactive substances (TBARS)) and protein oxidation (protein carbonyls (PrC), advanced oxidation protein products (AOPP), and protein sulfhydryls (P-SH)), to determine the effects of ascorbic acid as an additive in storage solution.
Male
Epinephrine, thiobarbituric acid, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA). All other chemicals used were of reagent grade and organic solvents were of spectral grade.
Blood was drawn from male
Plasma was isolated in Eppendorf tubes by centrifuging in a fixed angle rotor for 20 min at 2000 ×g. The plasma was removed and suspended in an equal volume of isotonic phosphate buffer, pH 7.4 [
SOD was measured by the method of Misra and Fridovich [
CAT was determined by the method of Aebi [
TBARS was determined by the method of Bar-Or et al. [
PrC was measured as an index of protein oxidation as described by Uchida and Stadtman [
Spectrophotometric determination of AOPP levels was assayed as an index of dityrosine containing cross-linked protein products by Witko’s method [
The concentration of P-SH was measured as described by Habeeb [
Protein was determined in the plasma by the method of Lowry et al. [
Results are represented as mean ± SE. Values between the groups were analyzed by two-way ANOVA and were considered significant at
SOD variation was insignificant during the storage period though increments of 100%, 300%, and 200% were observed in controls on days 10, 15, and 20, respectively against day 0.
Significant differences were observed in vitamin C groups. On day 15, SOD decreased by 75% in VC (30), whereas it increased by 200% on day 25 with respect to control. In addition, increments of 100% and 300% were also observed in VC (60) against VC (10) and VC (30), respectively.
VC (10) and VC (30) showed variations in SOD activity but increased towards the end of storage. SOD in VC (60) showed an increase with storage (Figure
Superoxide dismutase activity in plasma isolated from stored blood. Values are mean ± SE of five animals/group. VC (10): vitamin C (10 mM); VC (30): vitamin C (30 mM); and VC (60): vitamin C (60 mM). Two-way ANOVA was performed between the groups and subgroups. Changes between the groups are insignificant. Changes within the groups are represented in lower case. Those not sharing the same letters are significantly different.
Catalase varied significantly with the storage. The activity increased in controls by 13-, 41-, 42-, and 18-fold on days 5, 15, 20, and 25, respectively, when compared to day 0. Similarly, increments of 6-fold were seen on days 5, 15, 20, and 25 in VC (10) and 12-fold (day 15) and 23-fold (days 20 and 25) in VC (30). CAT increased by 20-fold on days 20 and 25 in VC (60) when compared to day 0.
Variations in CAT between different concentrations were insignificant.
Catalase activity increased in all groups with storage (Figure
Catalase activity in stored plasma. Values are mean ± SE of five animals/group. VC (10): vitamin C (10 mM), VC (30): vitamin C (30 mM), and VC (60): vitamin C (60 mM). Two-way ANOVA was performed between the groups and subgroups. A–F values between the groups are significantly different at
Significant changes were observed in TBARS during storage. In controls, TBARS decreased by 80% and 40% on days 5 and 20, respectively, whereas they increased by 160% and 60% on days 10 and 15, respectively, when compared to day 0. TBARS also reduced on days 10, 20, and 25 by 40%, 90%, and 80%, respectively, and increased by 100% on day 5 when compared to day 0 in VC (10) samples. There were increments of 300% (day 5) and 100% (day 25) in VC (30) and decrements of 42%, 85%, and 65% on days 5, 10, and 25, respectively, in VC (60) against day 0.
TBARS elevated by 3-fold on day 0 in VC (60) with VC (30). On day 5, TBARS increased by 12-fold in VC (30) whereas on day 10, it decreased by 77% in VC (10), VC (30), and VC (60) when compared to control.
TBARS decreased in VC (10) but was maintained in VC (30) and VC (60) with storage (Figure
Thiobarbituric acid reactive substances in plasma isolated from stored blood. Values are mean ± SE of five animals/group. VC (10): vitamin C (10 mM), VC (30): vitamin C (30 mM), and VC (60): vitamin C (60 mM). Two-way ANOVA was performed between the groups and subgroups. A–F values between the groups are significantly different at
Carbonyls of controls increased significantly by 1-, 2-, 12-, 8-, and 2-fold, respectively, from days 5 to 25 with respect to day 0. In VC (10), decrements of 43%, 89%, 42%, and 91% were observed on days 5, 15, 20, and 25, respectively, while an increment of 72% was observed on day 10 against day 0. A similar trend was noticed in VC (30) as PrC reduced by 85%, 77%, 48%, and 95% on days 5, 10, 15, and 25. But, in VC (60), PrC showed increments of 176%, 71% and 62%, and 80% on days 10, 15, and 20 and 25, respectively, with respect to control.
PrC increased by 23-fold on day 0 in VC (30) with respect to control, while it decreased by 1-fold in VC (60) against VC (30) on day 0.
Although there were variations in the levels of PrC in all groups, it was maintained towards the end of storage (Figure
Protein carbonyls in plasma isolated from stored blood. Values are mean ± SE of five animals/group. VC (10): vitamin C (10 mM), VC (30): vitamin C (30 mM). and VC (60): vitamin C (60 mM). Two-way ANOVA was performed between the groups and subgroups. A–F values with different superscripts between groups are significantly different at
AOPP increased by 300% on days 15 and 25 and by 400% on day 20 in controls. AOPP also increased by 100% (days 5, 10, and 15), 200% (day 20), and 300% (day 25) in VC (10). A similar trend was observed in VC (30) as AOPP increased by 100% on days 15 and 25, 70% on day 5, and 200% on day 20. AOPP elevated by 100% on day 10 and 200% on day 15 in VC (60) with respect to control.
AOPP reduced by 69% and 72% on days 15 and 25, respectively, in VC (10) against control. AOPP elevated by 100% and 200% on days 15 and 20, respectively, in VC (30) in comparison with VC (10). Increments of 69% and 74% were observed on days 20 and 25 when VC (30) was compared with VC (60). Decrements of 72% and 74% on days 20 and 25 were observed in VC (60) against controls.
AOPP increased in VC (30) and was maintained in VC (10) and VC (60) (Figure
Advanced oxidation protein products in plasma isolated from stored blood. Values are mean ± SE of five animals/group. VC (10): vitamin C (10 mM), VC (30): vitamin C (30 mM), and VC (60): vitamin C (60 mM). Two-way ANOVA was performed between the groups and subgroups. A–F values between the groups are significantly different at
Sulfhydryls varied significantly during storage. P-SH increased in controls by 3-, 2-, 8-, 10-, and 9-fold on days 5, 10, 15, 20, and 25, respectively, with day 0. P-SH also elevated by approximately 4-fold on days 5, 10, and 20, and 45-fold on day 15, respectively, in VC (30), when compared to day 0. An increment of 6-fold on day 15 and 1-fold on days 20 and 25 and a decrement of 54% were observed in VC (60) on day 10 against day 0.
On day 15, increases of 1-, 7-, and 3-fold were observed in VC (60) when compared with control, VC (10), and VC (30), respectively. On day 25, decrements of 1-fold were observed in VC (10) and VC (30) against controls.
Sulfhydryls were maintained in all groups throughout storage (Figure
Protein sulfhydryls in plasma isolated from stored blood. Values are mean ± SE of five animals/group. VC (10): vitamin C (10 mM); VC (30): vitamin C (30 mM), and VC (60): vitamin C (60 mM). Two-way ANOVA was performed between the groups and subgroups. A–F values between the groups are significantly different at
The effects of vitamin C as an additive in blood during storage were evaluated through plasma. Although SOD levels were insignificant during storage, VC (10) and VC (30) decreased SOD levels on the days when ROS was found to be higher [
Blood plasma is considered well equipped with both chain-breaking and preventive antioxidants to cope with OS and prevent peroxidative damage to circulating lipids. The antioxidants do not exert their functions by merely scavenging radicals but also by inducing/activating enzymes counteracting OS or by modulating redox-sensitive metabolic pathways.
Superoxide dismutases are enzymes that convert superoxide radical to oxygen and hydrogen peroxide. These enzymes carry out catalysis via general mechanism that involves the sequential reduction and oxidation of the metals like copper, iron, manganese, and nickel, at the active site [
Catalase rapidly catalyzes the decomposition of hydrogen peroxide to less reactive gaseous oxygen and water molecules. CAT exhibits a high
TBARS increased in the earlier stage of storage period but later decreased in controls. The earlier increase may be correlated to the latent phase of antioxidant activation and the decrease may be justified by the amelioration of the endogenous antioxidant system in the plasma. Vitamin C (ascorbic acid) is an important antioxidant in human plasma, where it acts as a scavenger of free radicals and protects against lipid peroxidation. Ascorbate plays a pivotal role in protecting plasma lipids from peroxidative damage initiated by aqueous peroxyl radicals [
The quantification of oxidative damage to proteins has been studied almost exclusively by assessing the total carbonyl content. The oxidants responsible for carbonyl formation within the proteins
Oxidation of proteins can lead to a whole variety of amino acid modifications. Action of chloraminated oxidants, mainly hypochlorous acid and chloramines, produced by myeloperoxidase, forms dityrosine containing cross-linked protein products known as AOPP and is also considered as one of the biomarkers to estimate the degree of oxidative modifications of proteins [
Our results on carbonyls in controls proved that during storage period, there was production of ROS leading to oxidant damage of proteins. Ascorbyl-free radical reductase increases the ascorbic acid recycling in human plasma and is reported as a compensatory/protective mechanism that operates to maintain the ascorbic acid level in plasma and thereby minimize OS [
Plasma is endowed with an array of antioxidant defense mechanisms. One of the important plasma antioxidants appears to be ascorbate. Protein sulfhydryl groups have also been suggested to contribute significantly to the antioxidant capacity of plasma. In particular, oxidative modification of sulfhydryl groups in proteins can be a two-faceted process: it could lead to impairment of protein function or, depending on the redox state of cysteine residues, may activate specific pathways involved in regulating key cell functions [
Oxidation of sulfhydryls of the membrane protein to disulfides causes reversible changes. This may be due to the disulfide exchange reactions carried out by a class of thioltransferases that catalyze reactions between glutathione and thioredoxin to regenerate the protein sulfhydryls [
Plasma has an efficient antioxidant system and can minimize the levels of oxidants during storage of 25 days. Vitamin C at the concentrations of 10, 30, and 60 mM also enhanced the antioxidant defenses but could not protect susceptible protein groups. Our study gives an insight into the interactions of different oxidants and antioxidants (both endogenous and exogenous). Vitamin C alone could not sufficiently attenuate OS and hence this opens the possibilities for further studies on vitamin C in combination with other antioxidants, in storage solutions.
The authors declare that there is no conflict of interests.
The authors like to thank Professor Leela Iyengar, Ms. Manasa K, and Jain University for their support.