1 Hoxworth Blood Center, University of Cincinnati, Cincinnati, Ohio 45267-0055; 2 Allergy and Immunology Division, Buffalo General Hospital, Buffalo, New York 14203; 3 Environmental Health Department, University of Cincinnati, Cincinnati, Ohio 45267; and 4 Department of Cell Biology, The Learner Research Foundation, Cleveland Clinic Foundation, Cleveland, Ohio 44195-0002
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ABSTRACT |
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Oxidant stress, in vivo or in vitro, is
known to induce oxidative changes in human red blood cells (RBCs). Our
objective was to examine the effect of augmenting RBC glutathione
(GSH) synthesis on 1) degenerative protein loss and
2) RBC chemokine- and free radical-scavenging functions in
the oxidatively stressed human RBCs by using banked RBCs as a model.
Packed RBCs were stored up to 84 days at 1-6°C in Adsol or in
the experimental additive solution (Adsol fortified with glutamine,
glycine, and N-acetyl-L-cysteine). Supplementing
the conventional additive with GSH precursor amino acids improved RBC
GSH synthesis and maintenance. The rise in RBC -glutamylcysteine
ligase activity was directly proportional to the GSH content and
inversely proportional to extracellular homocysteine concentration,
methemoglobin formation, and losses of the RBC proteins band 3, band
4.1, band 4.2, glyceraldehyde-3-phosphate dehydrogenase, and Duffy
antigen (P < 0.01). Reduced loss of Duffy antigen
correlated well with a decrease in chemokine RANTES (regulated upon
activation, normal T-cell expressed, and secreted) concentration. We
conclude that the concomitant loss of GSH and proteins in oxidatively stressed RBCs can compromise RBC scavenging function. Upregulating GSH
synthesis can protect RBC scavenging (free radical and chemokine) function. These results have implications not only in a transfusion setting but also in conditions like diabetes and sickle cell anemia, in
which RBCs are subjected to chronic/acute oxidant stresses.
red blood cell glutathione; Duffy antigen; chemokines; homocysteine
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INTRODUCTION |
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SURGICAL PROCEDURES AND/OR METABOLIC STRESSES, situations that normally warrant blood transfusions, result in increased free radical generation and increased chemokine production. Several recent studies have shown that red blood cells (RBCs) are important as biological carriers of glutathione (GSH) because of their circulating character and are considered to be an important mobile detoxifying system in the circulation. In fact, Brown et al. (6) have shown that the addition of RBCs during reperfusion decreases reperfusion injury and myocardial H2O2 levels in ischemic rat hearts. RBC Duffy antigen is presumed to be the sink for the circulating chemokines (7). Chemokines, secreted in response to various stress stimuli, are implicated in driving leukocyte emigration in different inflammatory reactions. In view of these important detoxifying functions of RBCs, understanding the effect of oxidant damage to the red cells and loss of specific proteins like Duffy antigen is very important and has implications not only in transfusion settings but also in conditions like diabetes or sickle cell anemia, in which RBCs are subjected to chronic/acute oxidant stresses.
Studies in our laboratory are geared toward identifying oxidative
changes that may compromise physiological function(s) of the transfused
RBCs. We have previously documented that the human RBCs banked in the
conventional medium Adsol (Baxter Healthcare, Deerfield, IL) for
transfusion purposes undergo degenerative changes including lipid
peroxidation, loss of acetylcholinesterase (AChE) (12, 13,
17), altered RBC membrane phospholipid composition (14, 15), decreased fluidity (12), altered
aminophospholipid translocase activity and aminophospholipid asymmetry
(14, 15), aggregation of band 3 and carbonylation of band
4.1 proteins (17), and loss of blood group antigens
including Duffy antigen receptor for chemokines (DARC)
(26). These oxidative changes are accompanied by
the loss of GSH and the GSH-dependent enzyme glutathione peroxidase (GSH-PX) (15, 17). Increases in chemokines and the
cytotoxic amino acid homocysteine (Hcy) in banked RBCs have been
reported (17, 34, 36). These changes are similar to those
occurring in vivo during oxidative stress. We also have observed that
in vitro augmentation of the RBC endogenous antioxidant reserve, especially GSH, provides protection against cell damage induced by
oxidative stress (16). Considering the cause-effect
relationship between various metabolic pathways, it may be inferred
from these observations that a higher demand for GSH, and/or limited
RBC GSH synthesis, increases degenerative oxidative modifications of
proteins/lipids. Human erythrocytes contain high concentrations of GSH
and maintain it by de novo synthesis (3). GSH-synthesis in
RBCs is limited by the availability of the substrate amino acids,
especially cysteine (3), and by feedback inhibition of the
ATP-dependent, rate-limiting enzyme -glutamylcysteine ligase
(
-GCL) (10). Because RBC GSH synthesis can be
manipulated easily by supplementing the storage (incubation) medium
with GSH precursor amino acids, we have used banked RBCs as a model to study the effect of sustained GSH synthesis on oxidative changes that
may diminish erythrocyte oxygen transport as well as free radical-
and/or chemokine-scavenging function. The results indicate that
maintenance of GSH in the banked RBCs reduced oxidative aberrations, including oxidation and/or loss of membrane, cytoskeletal, and cytoplasmic proteins, and curtailed increases in concentrations of
homocysteine (Hcy) and chemokines. We believe that this study provides an innovative approach for understanding the
interrelationships among cellular GSH status, degenerative protein
damage, and chemokine-initiated proinflammatory events.
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MATERIALS AND METHODS |
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Samples. Blood donors acceptable to criteria of the American Association of Blood Banks and the Food and Drug Administration were used. The protocol was approved by the University's Institutional Review Board. Platelet-reduced packed RBCs (PRBCs) were prepared by using standard blood bank procedures (12, 14, 15). After 100 ml of Adsol were added per the manufacturer's guidelines, the units were leukoreduced by filtering (Sepacell filter, code 4C2300; Baxter Healthcare). Each filtered unit was mixed and divided into two aliquots by weight in 300-ml polyvinyl chloride transfer bags (code 4R2001; Baxter Healthcare). After filtration, a sample was taken for leukocyte and platelet counts, performed with a flow cytometer (EPIC XL-MCL; Coulter, Miami, FL) and CD45 and CD41 antibodies, respectively. No leukocytes or platelets were detectable with the use of this technique. However, the leukocyte count obtained by using manual counting with a Nageotte chamber was 1-3 white blood cells/µl blood.
Of the two aliquots, one was a control stored in Adsol only. The other aliquot was stored in an experimental additive solution (EAS) consisting of Adsol fortified with 2.5 mM glutamine, 2.5 mM glycine, and 2.5 mM N-acetyl-L-cysteine (NAC). The 10× stock solutions of the amino acids glutamine, glycine, and NAC were prepared in Adsol. These solutions were filter sterilized (2 µm Acrodisc; Gelman, Ann Arbor, MI) and aseptically introduced into the storage container to obtain a 2.5 mM final concentration of amino acids in the blood-additive mixture. To obtain comparable hematocrits, we added an equivalent small volume of Adsol to the controls. Samples for study were removed aseptically at 0, 42, and 84 days. Sterility was confirmed by inoculating 1.0 ml of the final samples into tubes of thioglycolate and tryptic soy broth and then monitoring for bacterial and fungal growth for 7 days.Preparation of RBC ghosts. The RBCs were lysed in 30 volumes of 5 mM phosphate buffer, pH 8.0. The ghosts were isolated by centrifugation at 38, 000 g for 20 min and washed five times in the same buffer to obtain hemoglobin (Hb)-free white ghosts (12, 16, 17).
Membrane proteins. Total proteins were determined by using the bicinchoninic acid method (Micro BCA Kit; Pierce, Rockford, IL).
Antioxidant status and GSH synthesis in banked RBCs. To evaluate the antioxidant status of the fresh and stored RBCs, we assayed GSH concentration and the activities of GSH-PX and catalase by using procedures described previously (16, 17).
Oxidized glutathione (GSSG) was measured by the cycling assay of Tietze after 100 µl of PRBCs had been treated with 100 µl of 10 mM N-ethylmaleimide (NEM) to prevent oxidation during sample preparation (35). Briefly, 200 µl of NEM-treated RBCs were precipitated with 200 µl of 10% trichloroacetic acid (TCA), and the supernatants containing GSSG were treated five times with 0.5 ml of diethyl ether to remove TCA. After the last extraction, residual ether was removed under a stream of nitrogen gas. To 500 µl of 100 mM PBS containing 5 mM disodium EDTA (pH 7.5), we added 100 µl of GSSG sample, 20 µl of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB; 5 mM in 0.5% NaHCO3), and 20 µl of reduced nicotinamide adenine dinucleotide phosphate (NADPH; 10 mM in 0.5% NaHCO3). The reaction was initiated by adding 20 µl of GSH-reductase (20 units in 0.4 ml of 100 mM PBS containing 5 mM EDTA, pH 7.5). The linear increase in optical density was recorded at 412 nm every 10 s for 3 min. The reference cuvette contained equal concentrations of DTNB, NADPH, and the enzyme but no GSSG sample. For the standard curve, varying concentrations of GSSG from 0 to 150 ng were used. The activity ofMiscellaneous assays. AChE activity was determined as a marker of an indirect measure of membrane integrity (12, 13, 15-17). Extracellular Hcy was analyzed by using a previously described HPLC method (17, 22). Methemoglobin was measured by using the method described by Hegesh et al. (21).
Western blot analysis of RBC proteins: bands 3, 4.1, and 4.2, glyceraldehyde-3-phosphate dehydrogenase, and DARC. Domain-specific monoclonal anti-band 3-IVF12 and anti-Duffy 6 antigen-i3A antibodies were generous gifts from Dr. M. L. Jennings (Univ. of Arkansas, Little Rock, AR) and Dr. Yves Colin (Institut National de la Santé et de la Recherche Médicale U76, Institut National de Transfusion, Paris, France), respectively. Polyclonal anti-band 4.1 and anti-band 4.2 antibodies were provided by Drs. C. M. Hamilton and C. M. Cohen (St. Elizabeth Medical Center, Brighton, MA). Monoclonal antibody MAB374 anti-mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from Chemicon International (Temecula, CA).
Because DARC sites per cell are limited (~5,000/cell), the ghosts were extracted with 4-5 volumes of 1% Triton X-100 in PBS, pH 7.5, for 1 h at room temperature and then precipitated with 4-5 volumes of cold acetone in 50 mM Tris · HCl, pH 8.3, atChemokine RANTES and interleukin-8 estimation. At different storage intervals, supernatant samples were analyzed for C-C chemokine, RANTES (regulated upon activation, normal T-cell expressed, and secreted), and C-X-C chemokine interleukin-8 (IL-8) by using an ELISA procedure via a commercially available kit (R&D Systems, Minneapolis, MN). The samples read well within the range of the curve: IL-8 ranged from 0 to 25 pg/ml, and RANTES ranged from 0 to 2,000 pg/ml. The inter- and intra-assay precision was determined from several replicates, and the coefficient of variation was <10%. Linearity of the assay was tested, and linear regression analysis of sample vs. expected concentrations yielded a correlation coefficient of 0.999.
Statistical analysis. Data showing the effect of storage interval and storage media were analyzed and compared (Statistix Analytical Software, St. Paul, MN). The Student's t-test for paired samples was used for paired means. Nonparametric ANOVA was used wherever applicable.
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RESULTS |
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When human blood is stored under standard blood-banking conditions, RBCs undergo changes that lead, in vivo, to a loss of viability and the capacity to offload oxygen normally.
Multiple studies have attempted to correlate ATP levels, RBC shape, and deformability with posttransfusion viability (5, 11). In contrast, Wood and Beutler (38) failed to observe significant improvement in posttransfusion survival of banked RBCs despite maintaining ~40% higher ATP levels. Results of this and other studies suggest that additional mechanisms leading to the loss of posttransfusion viability must occur in concert with ATP changes (23). The identity of these mechanisms is unclear. Free radical reactions are postulated to be major contributors to the degenerative processes responsible for decreasing the defense systems, causing damage to cell membrane and function and eventually leading to a cellular breakdown (16, 17, 37). The three main conditions that favor free radical formation are a rich supply of oxygen, the presence of a transition metal catalyst, and a high degree of unsaturation in the lipid substrate. Human erythrocytes meet all three requirements and are very susceptible to oxidative/peroxidative damage. Because mature erythrocytes lack protein synthesis machinery and cannot replace damaged components, much of their metabolic activity is geared toward reductive processes that combat the threat of oxidation. If these reductive processes are deficient or overwhelmed, oxidative damages to cellular constituents occur, leading to hemolysis (19). Accordingly, as a first step, we examined the effect of banking RBCs in the new fortified additive on some of the traditional parameters of RBC health, namely, RBC ATP content, methemoglobin formation, degree of hemolysis, and AChE activity.
Effect of storage media on RBC ATP, methemoglobin formation,
hemolysis, and AChE activity.
At 42 days, the ATP levels were better maintained and the degree of
hemolysis was modestly lower (1.71 ± 0.3 in EAS vs. 2.11 ± 0.65 in Adsol) in the RBCs banked in the amino acid-fortified EAS than
in RBCs banked in Adsol. A similar but more accentuated trend was
observed at 84 days (Table 1). Hb
oxidation and AChE activity were determined as an initial screen of
oxidative damage.
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Antioxidant status and GSH synthesis in banked RBCs.
GSH-PX and catalase are two of the three main antioxidant enzymes in
RBCs. GSH-PX activity is regulated by the availability of reduced GSH.
After 42 days of storage, GSH levels remained unchanged in the RBCs
banked in EAS (Table 3), while those
banked in Adsol showed an ~20% decline in GSH content.
Interestingly, even after 84 days of storage, only an 8-10% GSH
loss in EAS vs. a 33% GSH loss in Adsol suggested increased GSH
synthesis in these cells. Indeed, the activity of -GCL, the
rate-limiting enzyme of GSH synthesis, was upregulated by the presence
of the precursor amino acids in the medium (Table 3). At 42 days of
storage, the enzymatic activity in the RBCs stored in Adsol was not
significantly different from that of fresh RBCs but was significantly
lower (P < 0.02) than that of RBCs stored in EAS
(Table 3). However, after 84 days of storage, RBC
-GCL activity was
elevated in Adsol compared with that in EAS (Table 3). The ratio of GSH
to GSSG is considered a marker of antioxidative defense in RBCs
(2). This ratio was significantly lower (P < 0.001) in the RBCs banked in Adsol than in RBCs stored in EAS after
42 as well as 84 days of storage (Table 3). A time-dependent increase
in extracellular Hcy was observed in both media, although its
concentration was significantly lower (P < 0.01) in
EAS than in Adsol (Table 4).
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Western blot analysis of RBC proteins: bands 3, 4.1, and 4.2, GAPDH, and DARC. In erythrocytes, proteins constitute an important target for oxidative modifications (4, 9, 12, 16, 17). It is now well established that membrane-bound proteinases, the secondary antioxidant defense mechanism, protect erythrocytes by preferentially degrading the oxidatively damaged proteins (4, 9). We therefore assessed the oxidative degradation/loss of proteins in banked RBCs as an indicator of oxidative stress. We examined the effect of storage on proteins critical for RBC structure and function including band 3, band 4.1, band 4.2, Duffy blood group antigen, and the glycolytic enzyme GAPDH. The time-dependent loss of all the proteins was more severe in RBCs banked in Adsol than in those stored in EAS. There was less loss of membrane proteins from RBCs stored in EAS compared with those banked in Adsol, indicating better preservation. Percent changes in EAS vs. Adsol are as follows: band 3, 20 ± 10%; band 4.1, 15 ± 2.8%; band 4.2, 13.8 ± 5.5%; GAPDH, 40 ± 5.7; and DARC, 23 ± 7.5% (mean ± SE, n = 4). Actin (membrane protein band 5) remained unaltered and served as an internal control (identity confirmed by immunoblotting with monospecific antibody). Better maintenance of GSH by the RBCs banked in the EAS probably countered the oxidative stress. Differential sensitivity of the proteins to oxidative stress may be due to the abundance and location of their modifiable sulfhydryl groups and other amino acid side chains. Because the striking change was seen in the conservation of DARC, we assessed whether this decreased loss of Duffy antigen in EAS would translate into improved RBC chemokine-scavenging function.
Erythrocyte chemokine-scavenging function. Because it has been suggested that febrile nonhemolytic transfusion reaction and/or other transfusion-related inflammatory reactions may be associated with chemokine levels in the transfused product (31), we estimated the levels of IL-8 and RANTES, representative proinflammatory and regulatory chemokines, respectively, in the RBC supernatants. The results were correlated with changes in RBC DARC content. During storage, the RANTES levels were significantly greater (P < 0.04) in Adsol than in EAS. The RANTES concentration in EAS compared with that in Adsol was 30.5 and 35.1% less after 42 and 84 days of storage, respectively. The results clearly demonstrate that the greater loss of DARC and consequent reduction in RBC chemokine-scavenging function results in higher RANTES levels in Adsol than in EAS. The possible implication of this result in a transfusion setting warrants further exploration. In our study, the IL-8 levels were very inconsistent, and therefore the results obtained were hard to interpret.
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DISCUSSION |
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The present study has demonstrated the following: human RBCs
banked in the experimental medium supplemented with glutamine, glycine,
and a cysteine analog, NAC, show 1) a significant increase in -GCL activity, GSH content, and GSH/GSSG ratio; 2) an
increase in GSH and a decrease in Hcy concentration in the supernatant; 3) decreased Hb oxidation to methemoglobin; 4)
decreased loss of membrane proteins including DARC; and 5)
decreased RANTES concentration compared with banking in conventional
medium, Adsol.
We have observed that banking RBCs in the hyperglycemic additive solution Adsol (122 mM glucose) subjects them to oxidative stress, leading to a decline of cellular GSH, GSH-PX activity, and the oxidative modification of proteins and lipids (12-15, 17, 26).
Direct evidence for the role of GSH as the major antioxidant defense in the oxidatively stressed RBC can be obtained by augmenting GSH synthesis or depleting the existing thiols, followed by assessment of the oxidative lesions. Using banked human RBCs as a model allowed us to moderately deplete/augment cellular GSH without severe manipulations. Therefore, the changes observed have significant physiological implications. Normally, RBCs for transfusion purposes are banked for up to 6 wk. Because we wanted to evaluate the effect of enhanced oxidative stress, we examined the RBCs at 42 as well as 84 days of storage. In general, the results at 6 and 12 wk were parallel, except that the differences between the EAS and Adsol were more accentuated.
Increased GSH synthesis and -GCL activity has been suggested to be
an adaptive response to oxidative stress (33). Because RBCs are enucleated,
-GCL activity is regulated by substrate availability and/or feedback regulation by GSH. At 42 days, despite lower GSH (Table 3) and comparable ATP (Table 1) content, the
-GCL
activity was significantly lower (Table 3) in RBCs banked in Adsol than
in those banked in EAS. However, by 84 days of storage, the
-GCL
activity of RBCs banked in Adsol exceeded that of RBCs stored in the
EAS. This can be explained on the basis of our previous observation
that oxidation and proteolysis of proteins after prolonged storage of
RBCs in Adsol may generate needed amino acids for
-GCL activity
(12, 13, 17). The absence of a corresponding increase in
cellular GSH content at 84 days suggests direct loss through increased
oxidation to GSSG and/or export out of cells in response to oxidative
stress, and/or indirectly through repair processes requiring GSH, such
as the reduction of oxidized membrane protein thiol groups. This is
reflected in a decreased GSH/GSSG ratio, an indicator of antioxidative
defense (2). Interestingly, an inverse correlation among
enhanced
-GCL activity, RBC GSH content, and the extracellular
concentration of the cytotoxic amino acid Hcy was observed (Table 4).
In vitro studies have shown that the plasma Hcy increases after blood
storage (1, 36). Because RBCs lack remethylation and
transsulfuration pathways, the homocysteine they produce from
methionine is exported (25). Our results support the
hypothesis that Hcy accumulation in banked blood may be the result of
incomplete conversion of methionine to cysteine and reflect the
increased oxidative stress and thus the escalated need of cysteine for
GSH synthesis. The results demonstrate that the availability of GSH
substrate amino acids regulates both GSH and Hcy synthesis.
We further examined whether the improved RBC GSH content and antioxidative defense (GSH/GSSG) would provide better protection against oxidative stress and would maintain the structure and function of banked RBCs by assessing the oxidative modification/loss of cytosolic protein (Hb), structural, functional, and transport proteins (bands 3, 4.1, 4.2, and GAPDH), and RBC DARC. These proteins were selected for the following reasons. 1) Band 3 protein, identified as an anion transport protein, is an integral membrane protein that binds the RBC cytoskeletal proteins ankyrin and band 4.2, and it is also associated with GAPDH. 2) Band 4.1 serves a dual function in the erythrocyte membrane skeleton: it promotes a high-affinity association between spectrin and F-actin, and it links the skeleton to the membrane by virtue of its association with glycophorin and band 3. 3) Band 4.2 is bound to the inner membrane surface through interactions with band 3 protein and has been shown to be essential for normal erythrocyte function (8). 4) GAPDH is an important enzyme of the anaerobic glycolytic pathway. In RBCs, 90% of the ATP is provided by this pathway. 5) Duffy blood group antigen (DARC), also an integral membrane protein, is postulated to be a sink to bind and inactivate excess chemokines released in the circulation. Indeed, improved RBC GSH and antioxidative defense translated into less loss of peripheral, cytosolic, and transmembrane proteins in EAS than in Adsol.
In RBCs, Hb is a powerful promoter of oxidative processes and can autooxidize to methemoglobin with concomitant production of superoxide radicals. The free radicals generated by Hb can further oxidize themselves, with a resultant increase in accumulation of damaged, nonfunctional proteins (Heinz bodies) and/or attack membrane proteins and lipids. These oxidatively damaged RBCs are susceptible to recognition by macrophages (18). Formation of band 3 clusters following Hb denaturation has been reported (24). We observed that the EAS maintained GSH levels efficiently and reduced methemoglobin formation in the RBCs banked in EAS compared with that in RBCs banked in Adsol. More recently, Rettig et al. (27) reported increased membrane-bound globin and GSH loss (up to 70%) in 100- to 115-day-old dog RBCs. One of the outcomes of Hb denaturation and free radical generation is protein oxidation including clustering of integral RBC membrane protein, band 3 (24, 27, 28).
The current study demonstrates that maintaining cellular GSH protects RBC proteins and Hb from oxidative stress and establishes GSH as the major player in antioxidant defense in banked RBCs. Improved free radical scavenging and reduced protein oxidation in RBCs banked in the EAS should improve their posttransfusion survival. Indeed, Lachant et al. (23) have documented that the ability to maintain a normal reduced GSH concentration during oxidant stress is an important determinant of RBC survival in the peritransfusion period.
It is apparent from the results that GSH effectively prevents loss of DARC from oxidatively stressed RBCs. Besides their involvement in transfusion incompatibility and the hemolytic disease of the newborn, the biological importance of DARC stems from their role as receptors for chemokines including IL-8 and RANTES. These molecules, released by several cell types that contact blood, namely, T cells, monocytes, and endothelial cells, are involved in immunoregulatory and inflammatory processes (29). Recently, despite filtration and leukoreduction, age- and storage-dependent increases in chemokines in banked blood have been reported (31, 34). In our studies, an inverse correlation between the preservation of DARC and RANTES concentration in plasma was noted. RBCs banked in the amino acid-fortified Adsol showed improved GSH reserve, better maintenance of erythrocyte DARC, and reduced plasma chemokine concentration. This observation has an important biological implication. Heddle et al. (20) reported a correlation between the age of the RBCs and the risk of an acute transfusion reaction. Although this association was not as strong for RBCs as for platelets, their results suggest that some age-related factors (possibly chemokines) may play a role in RBC transfusion reactions (20). The significance of preserving RBC chemokine-scavenging function in a transfusion setting warrants further assessment.
In summary, the results clearly demonstrate the pivotal role of GSH in preventing oxidative damage and maintaining the free radical- and chemokine-scavenging function of stored human RBCs. Because RBCs act as mobile detoxifying units, and their GSH concentrations can be easily manipulated by substrate amino acids, these observations have wide ramifications in a transfusion setting and in any situation of acute/chronic oxidant stress. This study also provides a novel approach to explore the significance of RBC Duffy antigen in the regulation of chemokine levels.
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ACKNOWLEDGEMENTS |
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We thank Margaret O'Leary for contributing to the organization and preparation of this manuscript.
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FOOTNOTES |
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This research was supported in part by American Heart Association Grants SW9708S and 9951502V-UJD (Ohio Valley).
Address for reprint requests and other correspondence: U. J. Dumaswala, Research Dept., Hoxworth Blood Center, 3130 Highland Ave., Cincinnati, OH 45267-0055 (E-mail: umakant.dumaswala{at}uc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 July 2000; accepted in final form 26 October 2000.
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