Storage of erythrocytes in blood banks is associated with biochemical and morphological changes to RBCs (red blood cells). It has been suggested that these changes have potential negative clinical effects characterized by inflammation and microcirculatory dysfunction which add to other transfusion-related toxicities. However, the mechanisms linking RBC storage and toxicity remain unclear. In the present study we tested the hypothesis that storage of leucodepleted RBCs results in cells that inhibit NO (nitric oxide) signalling more so than younger cells. Using competition kinetic analyses and protocols that minimized contributions from haemolysis or microparticles, our data indicate that the consumption rates of NO increased ~40-fold and NO-dependent vasodilation was inhibited 2–4-fold comparing 42-day-old with 0-day-old RBCs. These results are probably due to the formation of smaller RBCs with increased surface area: volume as a consequence of membrane loss during storage. The potential for older RBCs to affect NO formation via deoxygenated RBC-mediated nitrite reduction was also tested. RBC storage did not affect deoxygenated RBC-dependent stimulation of nitrite-induced vasodilation. However, stored RBCs did increase the rates of nitrite oxidation to nitrate in vitro. Significant loss of whole-blood nitrite was also observed in stable trauma patients after transfusion with 1 RBC unit, with the decrease in nitrite occurring after transfusion with RBCs stored for >25 days, but not with younger RBCs. Collectively, these data suggest that increased rates of reactions between intact RBCs and NO and nitrite may contribute to mechanisms that lead to storage-lesion-related transfusion risk.

INTRODUCTION

Transfusion with RBCs (red blood cells) is the front-line therapy for massive blood loss, with ~5 million patients being transfused each year in the U.S. This figure is increasing at a rate of 6% per year. Current blood banking standards allow pRBCs (packed RBCs) to be stored for up to 42 days. However, an association between the storage age of RBCs (>14 days) and increased incidence of adverse clinical outcomes has been demonstrated in diverse patient populations including trauma patients [1,2]. In the latter, transfusion-related adverse effects are further compounded with increasing the number of RBC units transfused [3]. The potential harm from pRBCs as a function of storage time is referred to as the ‘storage lesion’. Its general concept is that the presence of an underlying inflammatory stimulus, e.g. from infection (sepsis), surgery or trauma, older RBCs, relative to younger RBCs, will provide a second hit that results in dysfunction in tissue perfusion/oxygenation and exacerbation of the inflammatory injury.

Morphological and chemical changes that occur during RBC storage are well documented and include decreases in 2,3-diphosphoglycerate and pH, as well as an increase in potassium, lactate and haemolysis and the appearance of echinocytic and smaller more dense RBCs [4]. As a consequence, stored RBCs have increased fragility and decreased deformability. How these changes are associated with toxicity remains unclear. Several hypotheses have been forwarded, including increased oxidative stress and formation of reactive lipid species, release of chemokines by contaminating leucocytes and loss of RBC chemokine scavenging potential, increased haemolysis and iron release, and formation of microparticles and other soluble mediators that agonize the host immune/inflammatory response [48].

Previous studies have demonstrated the association of in vivo toxicity with transfusion of older pRBCs despite leucoreduction [3], suggesting that mechanisms intrinsic to the RBC may also play a role in storage-lesion-dependent pathology. In this context, an emerging and unifying hypothesis to explain compromised tissue perfusion and exacerbation in inflammatory responses is that aged pRBCs cause a loss in NO (nitric oxide) signalling. NO plays important roles in vascular homoeostasis with a decrease in its bioavailability leading to hypertension, coagulation and inflammation. Specific proposed mechanisms for decreased NO signalling with transfusion of older pRBCs include a storage-dependent loss of RBC-dependent stimulation of NO signalling [via loss of S-nitrosoHb (haemoglobin) or ATP release] [911], and/or increased rates of NO scavenging by Hb in RBC-derived microparticles or after haemolysis [12,13]. This is underscored by the similarity between the RBC storage-lesion toxicities with the pathogenic effects of acellular Hb-based oxygen carriers.

The NO scavenging reactions of Hb discussed above refer to oxy-ferrous haem-dependent oxidation of NO to nitrate and deoxyferrous haem binding of NO to form nitrosylHb (Hb-Fe2+-NO) (eqns 1 and 2 respectively):

 
formula
(1)
 
formula
(2)

With cell-free Hb, both reactions occur with rate constants between 3 and 8×107 M−1·s−1 [12,1417] With erythrocytic, and hence encapsulated, Hb, the rate constant for NO scavenging is decreased by ~500–1000-fold, a property proposed to be key in allowing endothelial-derived NO to regulate signalling processes in the vasculature [14,1721]. The exact mechanism for the decreased NO scavenging rate of RBC Hb compared with cell-free Hb remains debated and involves diffusion barriers created by an unstirred layer immediately adjacent to the RBC and/or membrane-based structures, which slow down NO reactions. Interestingly, these diffusion barriers are regulated by RBC size, shape and surface area [17], biophysical properties that are known to change during RBC storage. However, the effects of these changes on reactions with NO have not been considered previously and are tested in the present study.

In addition, we also tested if reactions between RBCs and nitrite were altered during storage. Nitrite reactions with Hb are regulated by the Hb fractional saturation such that under oxygenated conditions, nitrite is oxidized to nitrate, but when deoxygenated, nitrite reduction to NO can occur to mediate hypoxic NO signalling [2226]. The balance between nitrite oxidation compared with nitrite reduction is important in regulating NO signalling [27,28] and could play a role in affecting inflammatory tissue injury, since lower nitrite levels predispose to, whereas nitrite supplementation protects against, ischaemic and inflammation-dependent tissue injury [29]. Since stored RBCs have decreased p50 (oxygen partial pressure producing 50% satn.) values and altered membrane properties which could affect nitrite transport, we reasoned that altered nitrite metabolism and NO formation by stored RBCs could also play a role in transfusion-related toxicities.

EXPERIMENTAL

Materials

All of the materials were purchased from Sigma–Aldrich except for MNO [MAHMA NONOate/methyl-(6-{methyl-[nitroso(oxido)amino]amino}hexyl)azanium] and L-NMMA (NG-monomethyl-L-arginine), which were obtained from Axxora Platform. Male Sprague–Dawley rats (200–250 g) were purchased from Harlan. All of the animal studies were performed following Institutional Animal Care and Use approved procedures.

RBC collection and storage

Blood was collected from healthy donors (following Institutional Review Board approved protocols), leucodepleted by filtration (Sepacell RZ-200A) and stored according to blood bank protocols (at 4°C) in Adsol. Leucodepletion decreased the white blood cells from 12000±2000 cells/ml of blood to undetectable levels determined by light microscopy. At 0, 1, 7, 14, 28 and 42 days post storage, aliquots (2 ml) of RBCs were collected and washed three times with PBS containing 0.1% BSA by centrifugation (1500 g for 10 min per wash and the supernatant discarded). Alternatively, leucodepleted pRBCs were collected from segments from pRBCs stored in the University of Alabama at Birmingham blood bank. RBCs were washed as described above prior to use.

Light microscopy of RBC

RBCs (5 μl of packed cells) were smeared on to a glass slide, fixed using methanol and then stained with Wright's stain. Slides were imaged at ×40 magnification using a Leica DM600 microscope. Images were adjusted to control for contrast.

Ex vivo aorta vasodilation

Thoracic aortas were isolated from male Sprague–Dawley rats (200–250g) and divided into approximately eight 5-mm wide sections. Rings were suspended between two hooks connected to a force transducer and placed within a vessel bath chamber containing KH (Krebs–Henseleit) buffer as described previously [27]. After two rounds of KCl-induced contractions followed by washing and 30 min equilibration, vessels were equilibrated with 21% or 1% oxygen in KH buffer at 37°C in the presence of 5% CO2. Vessels were pre-treated with indomethacin (5 μM) and L-NMMA (100 μM) and pre-contracted with phenylephrine (200 nM at 21% oxygen tension and 400 nM at 1% oxygen tension) before the addition of RBCs [0.3% Hct (haematocrit) final concentration]. Once the vessels had reached a stable tone, vasorelaxation was elicited by the addition of either sodium nitrite (3 and 10 μM) or the NO donor MHO (10 nM and 30 nM). A limited dose-dependent protocol was used to limit the time of experiment to <10 min post RBC addition; preliminary studies determined that longer durations resulted in significant RBC haemolysis which would preclude assessment of RBC-dependent effects on NO- or nitrite-dependent vasodilation. Vasorelaxation was determined in the absence and presence of RBCs, and the percentage inhibition of nitrite- or NO-dependent vasodilation by the RBCs was calculated using stable tensions at the end of vasodilation. Also, at the end of each experiment, the concentration of cell-free and RBC haem in the vessel bioassay chamber was measured.

Haem concentration measurement

Haem was measured by either the Drabkins assay or after deconvolution of visible spectra for oxyHb, deoxyHb and metHb (methaemoglobin) as described previously [27].

Fractional saturation calculation

RBCs were lysed in deionized water at a ratio of 1:5 and then diluted to 20 μM haem in PBS. This resultant suspension maintained the ratio of Hb to allosteric effectors while allowing measurement of the visible spectra (450–700 nm) without scattering associated with intact RBCs. The haemolysate (3 ml) was deoxygenated in a tonometer and equilibrated at 34°C. Wavelength scans were taken after the sequential addition of 1 ml of air until complete oxygenation of the Hb was achieved. OxyHb and deoxyHb concentrations were measured by spectral deconvolution as described previously [27] and deoxyHb compared with the pO2 was plotted to calculate the p50 value.

The p50 values for haemolysates collected from freshly isolated (day 0) RBCs were 28.9±1 mmHg (mean±S.E.M., n=4), a value which falls within the range of p50 values reported for intact freshly isolated RBCs at pH 7.4 at 37°C (27–30 mmHg). Further validation of the approach used is provided by the magnitude of storage-age-dependent decrease in the p50 value being similar to recent studies in which the oxygen-binding affinity of stored RBCs was determined by a Hemox analyser [30].

Kinetic analysis of NO scavenging by RBCs

The rate of NO dioxygenation induced by RBCs was determined using competition kinetics as described previously [20] with slight modifications. Three experimental conditions were used, PBS+0.5% BSA containing either oxyHb, oxyHb with SpNO (spermine NONOate; an NO donor) or a suspension of RBCs plus oxyHb and SpNO. The final concentrations were 7 μM for oxyHb and 7% Hct for RBCs (RBCs were added after washing three times at 1500 g for 10 min) to remove any haemolysis-derived products that may have accumulated during storage. The experiments were started by addition of SpNO (10 μM; preliminary studies established these conditions to result in linear rates of oxyHb oxidation over 60 min, results not shown). Samples were placed in six-well tissue culture plates at room temperature (25°C) and rocked gently on a rocking platform. Prior to the addition of SpNO, samples were taken to assess the free Hb concentrations. After the addition of SpNO, 0.5 ml samples were taken every 10 min for 60 min, immediately centrifuged (20 s at 2000 g) to separate the RBCs, and the supernatant collected and the concentration of oxyHb and metHb determined by visible spectroscopy. The relative rate of cell-free oxyHb oxidation to metHb by SpNO, in the presence or absence of RBCs, allows determination of kinetics of RBC-dependent NO dioxygenation reactions. In separate experiments evaluating the effects of BHT (butylated hydroxytoluene; 100 μM), SOD (superoxide dismutase) or SOD+catalase (both 100 units/ml). reagents were added and incubated for 5 min prior to the addition of SpNO.

The relative rate constants for RBCs (kRBC) compared with cell-free oxyHb (kHb) were calculated as described previously [20] using eqn (3):

 
formula
(3)

where [metHb]c is the concentration of cell-free metHb in the preparation containing cell-free oxyHb and SpNO (no RBCs), Hct is the RBC Hct, [oxyHb]RBC is the concentration of total Hb in RBCs, kRBC and kHb are the rate constants for the RBC- and Hb-dependent NO dioxygenation reactions respectively, and [totalHb]ex and [oxyHb]ex are the total cell-free and oxyHb concentrations respectively in preparations containing RBCs. At each time point sampled, the ([metHb]c-[(1-Hct)]ex)/[oxyHb]RBC term (y-axis) was plotted against the ln([totalHb]ex/[oxyHb]ex) term (x-axis) and kRBC/kHb was determined by the gradient. Initial studies indicated that significant haemolysis occurred during the experiment when assessing RBCs were stored for more than 14 days. The protocol was therefore modified to collect samples every 3 min for 12 min, a time period over which the above described plot remained linear and cell-free Hb concentration changed by <6%.

Microparticle measurement

Stored RBCs were left unwashed (60% Hct) or washed (three times) and brought to 60% Hct in PBS+0.1% BSA. RBCs were then incubated with anti-glycophorin A-FITC conjugated antibody (0.17 μg/ml) for 30 min in the dark at room temperature. Samples were then analysed by flow cytometry using a Becton Dickinson FACSCalibur and events acquired using CellQuest software. Approximately 100000 events were collected per measurement. All of the analyses were done with FlowJo software.

Nitrite consumption by RBCs

Nitrite consumption by RBCs (washed to remove microparticles and cell-free haem) was determined as described previously [31] at different oxygen tensions in a controlled-atmosphere chamber (Plas Labs) using atmospheric gas combined with nitrogen gas to produce 21% or 2% oxygen tensions. RBC suspensions at 5% Hct in Tris buffer containing 0.1% BSA (pH 7.4) were equilibrated for 30 min in six-well tissue culture plates with gentle rocking. To initiate experiments, nitrite (100 μM) was added (nitrite stock solutions were prepared in deoxygenated PBS) and aliquots (500 μl) removed at 0, 5 and 15 min. Aliquots were taken out of the chamber and immediately centrifuged at 2000 g for 30 s. The extracellular fraction (supernatant) was collected and vortex-mixed with equal volumes of methanol then frozen in liquid nitrogen. In all of the experiments, parallel incubations of nitrite alone in Tris-BSA buffer were included. Nitrite consumption was determined as the difference between its concentration in the samples with and without RBCs.

Trauma patient studies

Patients admitted into the trauma intensive care unit at the University of Alabama at Birmingham with orders to receive 1 pRBC unit transfusion were enrolled into the study. The study protocol, patient enrolment, exclusion criteria and demographics were as described recently [32]. Approval for this study was granted by the University of Alabama at Birmingham Institutional Review Board. Blood samples (400 μl) were collected immediately before RBC transfusion and at 1 h after the completion of transfusion and immediately processed for whole-blood nitrite measurements by methanol extraction as described previously [33]. Methanolic extracts were stored (−80°C) prior to nitrite measurement.

Nitrite and nitrate measurements

Methanolic extracts were thawed on ice and in the dark and then centrifuged (15,000 g for 5 min at 4°C). The supernatant volume was measured and nitrite and nitrate concentrations determined using triodide-based chemiluminesence as described previously on a Sievers NO analyser and with comparison with respective standard curves [33].

Statistical analysis

Storage time-dependent changes were analysed by one-way repeated measures ANOVA with Tukey's post-hoc test or by two-way ANOVA when also assessing effects of pO2. Changes in circulating nitrite levels in trauma patients before and after RBC transfusion were analysed by unpaired Student's t test. P values less than 0.05 were considered significant. All of the analyses used GraphPad Prism Software.

RESULTS

Validation of storage-dependent changes in RBCs

RBC segments of pRBCs of different storage ages collected from the University of Alabama at Birmingham blood bank or RBCs isolated from healthy volunteers, leucodepleted and stored according to standard blood banking conditions at our institution were used. Initial studies were performed to validate that these RBC preparations exhibited biochemical and morphological changes that typify the storage lesion. Supplementary Figure S1 (at http://www.BiochemJ.org/bj/446/bj4460499add.htm) shows that the RBC shape changed from a normal discoidal form (day 0) to echinocytes characterized by spiculated membranes (days 28 and 42). Furthermore, the RBC p50 value decreased and haemolysis increased with storage time (Figure 1), consistent with previous studies [13,30].

RBC storage increases oxygen affinity

Figure 1
RBC storage increases oxygen affinity

RBCs were collected from healthy volunteers, leucodepleted, stored for the indicated times and their p50 value (A) and haemolysis (B) measured. (A) Results are means±S.E.M. (n=3–4); *P<0.05 compared with day 0, #P<0.05 compared with days 0, 1 and 7 by one-way ANOVA with Tukey's post-hoc test. (B) Results are means±S.E.M. (n=3); *P<0.01 compared with day 1 and 14 by one-way ANOVA with Tukey's post-hoc test.

Figure 1
RBC storage increases oxygen affinity

RBCs were collected from healthy volunteers, leucodepleted, stored for the indicated times and their p50 value (A) and haemolysis (B) measured. (A) Results are means±S.E.M. (n=3–4); *P<0.05 compared with day 0, #P<0.05 compared with days 0, 1 and 7 by one-way ANOVA with Tukey's post-hoc test. (B) Results are means±S.E.M. (n=3); *P<0.01 compared with day 1 and 14 by one-way ANOVA with Tukey's post-hoc test.

RBC storage increases NO scavenging and inhibits NO-dependent vasodilation

The rate constant for NO reactions with oxyHb in RBCs can be determined using a competition assay, in which the NO donor SpNO is added to a mixture of cell-free oxyHb and RBCs, and then following the time-dependent formation of cell-free metHb. At non-limiting SpNO concentrations, an increase in the rate of RBC Hb reactions with NO is reflected as a decrease in the rate of cell-free metHb formation. Figure 2(A) shows representative kinetic traces and demonstrates that RBC-dependent inhibition of cell-free metHb formation increases with the length of RBC storage. The transition from linear to curved kinetic traces for metHb formation in the presence of RBCs is similar to previous studies [20], and reflects the competition between RBC and cell-free Hb for reaction with NO. Figure 2(B) plots the calculated ratio of rate constants for the NO dioxygenation reaction between erythrocytic and cell-free Hb (kRBC/kHb) and demonstrates that RBC-dependent NO scavenging increases ~40-fold over 42 days of storage. Significance by one-way ANOVA was observed with 42-day-old RBCs relative to all other ages. A trend towards increasing NO scavenging rates was also noted by 14–28 days, which was not significant due to variance within any given RBC age, especially from day 14 and onwards (probably reflecting donor to donor differences). Exclusion of outlier data (n=1 each from the 14 and 28 day datasets) detected by Grubbs' test resulted in an observation of a significant increase in NO-scavenging kinetics between both day 14 and 28 RBCs relative to day 0 (P<0.05 by Student's t test, results not shown). Addition of SOD, SOD+catalase or BHT had no effect on NO-dioxygenation rates by 42-day-old RBCs (results not shown) suggesting that increased superoxide or lipid alkoxyl/peroxyl radicals are not responsible for accelerated NO consumption.

Effects of RBC storage time on NO-scavenging kinetics

Figure 2
Effects of RBC storage time on NO-scavenging kinetics

(A) Representative traces for SpNO-dependent formation of cell-free metHb from oxyHb (7 μM) in the presence and absence of RBC (7% Hct) of different storage ages. ●, cell-free oxyHb alone; □, day 0 RBCs; and ◇, day 7 RBCs). Inset shows data with older RBCs (Δ, day 14; ▽, day 28; and ○, day 42) where kinetics were determined over shorter times due to haemolysis (as described in the Experimental section). (B) Scatter plot of calculated ratio of rate constants for NO dioxygenation by RBCs relative to cell-free Hb. RBCs of different ages were collected from the University of Birmingham at Alabama blood bank. For day 0 samples, RBCs were collected from healthy volunteers, leucodepleted and processed as described in the Experimental section. Experiments were performed in PBS (pH 7.4) at 20°C. Results are means±S.E.M., n=5–10. *P<0.01 relative to day 42 by one-way ANOVA (P=0.0005) and Tukey's post-hoc test.

Figure 2
Effects of RBC storage time on NO-scavenging kinetics

(A) Representative traces for SpNO-dependent formation of cell-free metHb from oxyHb (7 μM) in the presence and absence of RBC (7% Hct) of different storage ages. ●, cell-free oxyHb alone; □, day 0 RBCs; and ◇, day 7 RBCs). Inset shows data with older RBCs (Δ, day 14; ▽, day 28; and ○, day 42) where kinetics were determined over shorter times due to haemolysis (as described in the Experimental section). (B) Scatter plot of calculated ratio of rate constants for NO dioxygenation by RBCs relative to cell-free Hb. RBCs of different ages were collected from the University of Birmingham at Alabama blood bank. For day 0 samples, RBCs were collected from healthy volunteers, leucodepleted and processed as described in the Experimental section. Experiments were performed in PBS (pH 7.4) at 20°C. Results are means±S.E.M., n=5–10. *P<0.01 relative to day 42 by one-way ANOVA (P=0.0005) and Tukey's post-hoc test.

NO-dependent dilation of isolated rat thoracic aortas was used to test if storage-dependent increased NO scavenging by RBCs translates to a greater degree of inhibition of NO signalling. Aortas were pre-constricted with PE (as described in the Experimental section) and L-NMMA, the latter to inhibit endogenous NO formation from endothelial NO synthase. RBCs of different ages were then added and followed by the addition of MNO (10 nM and 30 nM). Figure 3(A) shows representative vessel tension traces showing that MNO (30 nM) stimulated ~40% dilation, which was inhibited in the presence of day 0 and day 42 RBCs, the latter having a greater inhibitory effect. Figure 3(B) shows the percentage inhibition of MNO-dependent vasodilation by RBCs as a function of storage time. MNO-dependent vasodilation at both 21% and 1% O2 was inhibited by 42-day-old RBCs consistent with increased NO-scavenging kinetics. No significant effect of oxygen tension (P=0.1, by two-way ANOVA) was observed.

Effects of RBC storage time on NO-dependent vasodilation

Figure 3
Effects of RBC storage time on NO-dependent vasodilation

RBCs (0.3% Hct) of different ages were added to vessel bioassay chambers followed by addition of MNO to rat aortic segments. Experiments were performed at 21% and 1% O2. (A) Representative vessel tension compared with time traces (21% O2). RBCs were added at time 0, and MNO (30 nM) addition is indicated by the arrow. (B) Percentage inhibition of MNO-dependent vasodilation by RBCs of different storage ages at 1% O2 (open bars) and 21% O2 (closed bars). Data are normalized to the RBC haem concentration in the vessel bioassay chamber measured at the end of each experiment and are means±S.E.M. (n=5–6); *P<0.05 relative to day 0, 1 and 14, #P<0.05 relative to day 0 by one-way ANOVA with Tukey's post-hoc test. (C) Percentage inhibition of MNO-dependent vasodilation by RBCs as a function of calculated fractional saturation. ○, data collected at 1% O2; ●, data collected at 21% O2.

Figure 3
Effects of RBC storage time on NO-dependent vasodilation

RBCs (0.3% Hct) of different ages were added to vessel bioassay chambers followed by addition of MNO to rat aortic segments. Experiments were performed at 21% and 1% O2. (A) Representative vessel tension compared with time traces (21% O2). RBCs were added at time 0, and MNO (30 nM) addition is indicated by the arrow. (B) Percentage inhibition of MNO-dependent vasodilation by RBCs of different storage ages at 1% O2 (open bars) and 21% O2 (closed bars). Data are normalized to the RBC haem concentration in the vessel bioassay chamber measured at the end of each experiment and are means±S.E.M. (n=5–6); *P<0.05 relative to day 0, 1 and 14, #P<0.05 relative to day 0 by one-way ANOVA with Tukey's post-hoc test. (C) Percentage inhibition of MNO-dependent vasodilation by RBCs as a function of calculated fractional saturation. ○, data collected at 1% O2; ●, data collected at 21% O2.

As shown in Figure 1(A), the p50 value of RBCs decreases with storage time. Since the rate of NO scavenging by oxyHb is slightly (~1.5–2-fold) faster compared with deoxyHb [14,15,17,34], the increased inhibition of NO-dependent vasodilation observed with older RBCs (~2–3 fold for 0-day-old compared with 42-day-old RBCs) could reflect the presence of more oxyHb compared with deoxyHb. This is unlikely, however, since the rate of NO consumption by RBCs is zero order with respect to Hb [17]. Another possibility is that when deoxygenated, the RBC membrane permeability to NO is increased and thereby contribute, in part, to differential NO-scavenging kinetics by RBCs compared with cell-free Hb [14]. Inhibition of MNO-dependent vasodilation was replotted as a function of oxygen fractional saturation of RBCs in vessel bioassay chambers (Figure 3C). At 1% O2, fractional saturation was higher for 42-day-old compared with 0-day-old RBCs (P<0.05 by one-way ANOVA with Tukey's post-hoc test) and inhibition of MNO-dependent vasodilation paralleled storage time (Figure 3B), suggesting that higher concentrations of oxyHb in older RBCs mediate increased inhibition of MNO-dependent vasodilation. At 21% O2, however, no difference in fractional saturation was observed (P=0.21 by one-way ANOVA) between RBCs of different age. This suggests that the increased inhibition of NO scavenging and rate of NO reactions are due to storage-dependent changes in RBC morphology or biochemistry that are independent of oxygen affinity.

Stored RBCs are more sensitive to haemolysis and produce more Hb-containing microparticles, both of which exhibit increased NO-scavenging kinetics [13]. Microparticle formation and haemolysis was measured before and after washing. Microparticles increased with storage age, but washing removed them to ≤0.5% of total glycophorin-positive events (Figure 4). Moreover, washing also decreased cell-free Hb to <0.5 μM (results not shown). Finally, and since haemolysis may occur during the time over which vessel relaxation is assessed, cell-free Hb was measured in vessel bioassay chambers at the end of each experiment. No significant correlation between the concentration of cell-free haem and the extent of inhibition of MNO-dependent vasodilation was observed (results not shown). Collectively, these data suggest that using the described protocols, neither microparticles nor cell-free Hb contributed to the enhanced NO scavenging observed by stored RBCs.

Testing a role for microparticles or cell-free Hb in enhanced stored-RBC-dependent inhibition of NO signalling

Figure 4
Testing a role for microparticles or cell-free Hb in enhanced stored-RBC-dependent inhibition of NO signalling

(A and B) Representative histograms for microparticle analysis in 42-day-old RBCs by FACS before and after washing respectively. Events in the upper left-hand quadrant represent microparticles and the upper right-hand quadrant intact RBCs. (C) Changes in microparticle levels during storage before (closed bars) and after (open bars) washing. Results are means±S.E.M. (n=3); *P<0.02 relative to before washing by Student's t test.

Figure 4
Testing a role for microparticles or cell-free Hb in enhanced stored-RBC-dependent inhibition of NO signalling

(A and B) Representative histograms for microparticle analysis in 42-day-old RBCs by FACS before and after washing respectively. Events in the upper left-hand quadrant represent microparticles and the upper right-hand quadrant intact RBCs. (C) Changes in microparticle levels during storage before (closed bars) and after (open bars) washing. Results are means±S.E.M. (n=3); *P<0.02 relative to before washing by Student's t test.

Effects of RBC storage on nitrite metabolism

Nitrite was added to RBCs of different storage ages pre-equilibrated at either 21% or 2% O2. Figure 5 shows how RBC storage affects nitrite consumption and nitrate formation profiles. The key results from these experiments were: (i) consistent with our previous data [31], nitrite consumption was faster under deoxygenated compared with oxygenated conditions with freshly isolated (day 0) RBCs (Figure 5A); (ii) Figures 5A–5E show that as a function of storage time, the difference between low and high oxygen-dependent nitrite consumption kinetics increases up to 14 days, and then decreases thereafter, with no oxygen-dependent difference evident with 42-day-old RBCs; (iii) comparison of how storage age affected nitrite con-sumption at each pO2 showed that at low O2, the rates of nitrite consumption were higher with RBCs stored for 14 days or longer compared with 0-day-old RBCs (Supplementary Figure S2 at http://www.BiochemJ.org/bj/446/bj4460499add.htm). Similarly, significant increases in rates of nitrite consumption under oxygenated conditions were observed by stored RBCs compared with 0-day-old RBCs (Figure 6A and Supplementary Figure S3 at http://www.BiochemJ.org/bj/446/bj4460499add.htm); (iv) both low and high oxygen conditions resulted in similar rates of nitrate formation from nitrite with 0-day-old RBCs (Figure 5F). As a function of storage time, however, an oxygen-dependent effect became apparent with nitrate formation being greater at the lower relative to the higher oxygen tensions with RBCs stored for 14 days or longer (Figures 5F–5J). Importantly, however, nitrate formation increased as a function of storage time at both high and low oxygen conditions; Figure 6(B) shows rates at 21% O2.

RBC storage age effects on nitrite metabolism

Figure 5
RBC storage age effects on nitrite metabolism

Nitrite (100 μM) was added to RBCs (5% Hct) of different storage ages in PBS+0.1% BSA pre-equilibrated at either 21% O2 (●) or 2% O2 (□). At 5 and 15 min after nitrite addition, RBCs were pelleted and nitrite and nitrate levels measured in the extra-erythrocytic fractions. Data show nitrite consumption (AE) and nitrate formation (FJ) normalized to haem. P values indicated on each panel demonstrate effects of pO2 on nitrite consumption or nitrate formation rates determined by two-way ANOVA. Calculated oxygen fractional saturations for 2% O2 condition for storage times 0, 7, 14, 28 and 42 days were 0.27, 0.46, 0.56, 0.66 and 0.69 respectively.

Figure 5
RBC storage age effects on nitrite metabolism

Nitrite (100 μM) was added to RBCs (5% Hct) of different storage ages in PBS+0.1% BSA pre-equilibrated at either 21% O2 (●) or 2% O2 (□). At 5 and 15 min after nitrite addition, RBCs were pelleted and nitrite and nitrate levels measured in the extra-erythrocytic fractions. Data show nitrite consumption (AE) and nitrate formation (FJ) normalized to haem. P values indicated on each panel demonstrate effects of pO2 on nitrite consumption or nitrate formation rates determined by two-way ANOVA. Calculated oxygen fractional saturations for 2% O2 condition for storage times 0, 7, 14, 28 and 42 days were 0.27, 0.46, 0.56, 0.66 and 0.69 respectively.

RBC storage increases nitrite consumption and nitrate formation under oxygenated conditions

Figure 6
RBC storage increases nitrite consumption and nitrate formation under oxygenated conditions

Nitrite was added to RBC at 21% O2 as described in the legend to Figure 5, and nitrite consumption (A) and nitrate formation (B) were measured after 5 min. Results are means±S.E.M. (n=4–5). *P<0.03 by Student's t test.

Figure 6
RBC storage increases nitrite consumption and nitrate formation under oxygenated conditions

Nitrite was added to RBC at 21% O2 as described in the legend to Figure 5, and nitrite consumption (A) and nitrate formation (B) were measured after 5 min. Results are means±S.E.M. (n=4–5). *P<0.03 by Student's t test.

Effects of RBC storage on nitrite-dependent vasodilation

Nitrite-dependent vasodilation was assessed in the presence and absence of RBCs and at 21% and 1% O2; Figures 7(A) and 7(B) show representative tension compared with time traces. Consistent with previous studies, nitrite alone is a more potent vasodilator at low oxygen tensions [28,35,36]. RBCs inhibited nitrite-dependent vasodilation when oxygenated, but promoted vasodilation when deoxygenated. Figure 7(C) shows how storage duration affected RBC-dependent inhibition or potentiation of nitrite-dependent vessel relaxation. Low oxygen tensions resulted in a relative potentiation of nitrite-dependent vasodilation compared with high oxygen tensions consistent with a deoxygenation-dependent nitrite reductase activity of RBCs. This relative effect was not affected by RBC storage age.

Effects of RBC storage on nitrite-dependent vasodilation of rat thoracic aorta

Figure 7
Effects of RBC storage on nitrite-dependent vasodilation of rat thoracic aorta

Nitrite (3 and 10 μM) was added to aortic baths containing Krebs buffer with or without RBCs (0.3% Hct) of different storage ages and at 1% or 21% O2. (A and B) Representative vessel tension traces. Arrows indicate nitrite addition. (C) Percentage change in vasodilation elicited by RBCs relative to nitrite alone at 1% (open bars) and 21% (closed bars) O2 and storage age. Data are normalized to the concentration of RBC haem in each vessel bioassay chamber. A positive value denotes inhibition, and a negative value indicates potentiation of nitrite-dependent vasodilation. Results are means±S.E.M. (n=3–6). #P<0.005 by two-way ANOVA for the effects of oxygen.

Figure 7
Effects of RBC storage on nitrite-dependent vasodilation of rat thoracic aorta

Nitrite (3 and 10 μM) was added to aortic baths containing Krebs buffer with or without RBCs (0.3% Hct) of different storage ages and at 1% or 21% O2. (A and B) Representative vessel tension traces. Arrows indicate nitrite addition. (C) Percentage change in vasodilation elicited by RBCs relative to nitrite alone at 1% (open bars) and 21% (closed bars) O2 and storage age. Data are normalized to the concentration of RBC haem in each vessel bioassay chamber. A positive value denotes inhibition, and a negative value indicates potentiation of nitrite-dependent vasodilation. Results are means±S.E.M. (n=3–6). #P<0.005 by two-way ANOVA for the effects of oxygen.

Effects of RBC storage and transfusion on nitrite levels in trauma patients

Figure 8(A) shows that whole-blood nitrite levels decreased significantly after transfusion of stable trauma patients with 1 unit of pRBCs. Each unit was transfused over ~60–90 min and resulted in ~60% decrease in blood nitrite levels. Moreover, the decrease in nitrite levels was greater when transfusion occurred with RBCs stored for >25 days compared with <25 days (Figure 8B). Since the same volume (1 unit, ~500 ml) of RBCs were transfused, these data also suggest that decreased nitrite levels are not due to resultant dilution of blood (~10%).

RBC transfusion decreases circulating nitrite levels in stable trauma patients

Figure 8
RBC transfusion decreases circulating nitrite levels in stable trauma patients

(A) Whole-blood nitrite was measured pre- and post- transfusion with 1 unit of RBCs in stable trauma patients. Results are means±S.E.M. (n=31); *P<0.01 by paired Student's t test. (B) Data were separated by the storage age of transfused RBCs (0–25 days, n=14 or 26–42 days, n=17) and changes in whole-blood nitrite (pre-post transfusion) plotted for these groups. *P<0.02 by unpaired Student's t test.

Figure 8
RBC transfusion decreases circulating nitrite levels in stable trauma patients

(A) Whole-blood nitrite was measured pre- and post- transfusion with 1 unit of RBCs in stable trauma patients. Results are means±S.E.M. (n=31); *P<0.01 by paired Student's t test. (B) Data were separated by the storage age of transfused RBCs (0–25 days, n=14 or 26–42 days, n=17) and changes in whole-blood nitrite (pre-post transfusion) plotted for these groups. *P<0.02 by unpaired Student's t test.

DISCUSSION

Decreased NO signalling has emerged as a central mechanism underlying the RBC storage lesion [3740]. This hypothesis is further supported by data from the present study, that show RBC-dependent scavenging of NO and nitrite is enhanced during storage. We propose that this contributes to an overall deficit in NO bioavailability and predisposition to circulatory dysfunction and tissue inflammatory injury, two features emblematic of transfusion-related toxicity [4,6,41].

Decreased RBC-dependent stimulation of NO signalling and increased NO scavenging (secondary to haemolysis and increased microparticle formation) with stored cells have been reported to contribute to a deficit in NO bioactivity [911,13,42,43]. How the storage-time-dependent changes in these processes translate to the clinical manifestation of storage-lesion-related toxicity is difficult to ascertain directly. Retrospective studies indicate that adverse effects are observed with transfusion of RBCs stored for >14 days [44]. Consistent with this, ATP release and subsequent activation of eNOS (endothelial nitric oxide synthase) is diminished, with deficits observed with 7–14-day-old RBCs, and increased haemolysis and microparticle formation occurs in a similar storage-time-dependent manner, with significant increases and effects on NO scavenging evident after ~14–21 days (Figures 1 and 4, and [13]). Reported deficits in S-nitrosoHb bioactivity occur much faster and within 1 day of storage [9,10], leading to suggestions that the RBC lesion occurs even earlier than expected or that there is no direct cause and effect relationship in this case [45]. How intact stored RBC interactions with NO are affected by storage has received little attention. Data from the present study indicate that significant increases in NO-scavenging occur with RBCs stored for 42 days, but with trends being observed by 14–28 days. How intact RBCs affect NO homoeostasis is an important consideration since: (i) biophysical and biochemical properties of the RBCs are key in controlling NO diffusion [12,19,20] and slowing NO scavenging by encapsulated haem; and (ii) RBC storage leads to smaller less flexible cells with a greater surface area to volume ratio [4]. The latter is predicted to decrease NO-diffusion barriers and thus increase NO-scavenging rates (see below). To test this hypothesis, we used in vitro competition kinetic assays to determine the rate of NO dioxygenation by RBCs stored for different times. The kRBC/kHb increased ~40-fold over 42 days, which was reflected by an enhanced inhibition of NO-dependent vasodilation. Importantly, experimental conditions that allowed exclusion of contributions from storage-dependent haemolysis and microparticles were used, although we note that whereas washing decreased microparticles to control levels, complete removal was not achieved precluding a definitive exclusion of these species in contributing to increased NO-dioxygenation kinetics, or the inhibited MNO-dependent vasodilation observed. Although the rate constant of RBC-dependent scavenging of NO increased during storage, this remained significantly less than cell-free Hb (by ~20-fold, Figure 2B). In a unit of stored RBCs with 60% Hct, however, the concentration of erythrocytic Hb is ~150-fold greater compared with cell-free Hb (~12 mM RBC haem compared with ~80 μM for cell-free Hb, see Figure 1B). Since the rate of NO scavenging is the product of the rate constant and concentration, this suggests that intact stored RBCs may effectively contribute to NO scavenging. According to current blood banking guidelines, at least 75% of transfused RBCs are expected to be present in the circulation at 24 h post-transfusion. This suggests that RBCs with enhanced NO-scavenging properties may be persistent for many hours post-transfusion. The potential role for intact RBC-dependent effects on NO function is further underscored by the fact that we used leucodepleted RBCs, which may decrease (although not completely prevent) haemolysis and microparticle formation. We also note that at least in trauma patients, evidence for storage-lesion-dependent toxicity still remains despite the use of leucodepleted pRBCs [46]. It is difficult to ascertain the relative contributions of erythrocytic compared with microparticle compared with cell-free Hb towards inhibited NO signalling, since steady states of the above species are unlikely in a transfusion setting. Many factors will affect the concentrations of intact RBCs, microparticles and cell-free Hb, including RBC turnover, variable Hct, number of pRBC units transfused, and the balance between formation of microparticles and haemolysis compared with clearance of these species (e.g. cell-free Hb is cleared by haptoglobin- and CD163-dependent pathways) [8,47,48]. The calculation presented above also does not account for differences in NO scavenging that occurs due to the RBC-free zone. Irrespective of the relative contributions, we posit that intact RBCs are important partners with microparticles and cell-free Hb, which collectively inhibit endogenous NO-dependent signalling. In addition our data suggest that therapeutic strategies aimed at limited NO scavenging should also target intact RBC-dependent NO reactions.

How storage increases RBC-dependent scavenging of NO is unclear. Storage changes RBC size, shape, membrane permeability and extracellular diffusion, all of which can regulate the kinetics of NO scavenging [12,14,1720,49]. Moreover, the rate limit may be controlled by distinct factors as exemplified by recent experimental and modelling studies that indicate the unstirred layer is the primary component regulating NO scavenging by RBCs, whereas membrane permeability is the key controlling factor for NO reactions with microparticles [12]. Although it has been proposed that an additional factor is an intracellular diffusion barrier due to the very high Hb concentration [21,49], this has been challenged on experimental and theoretical grounds [14]. In addition, the fact that NO consumption by RBCs is zero order with intracellular Hb concentration (which was actually the initial observation that prompted the concept of an extracellular diffusion barrier) is also inconsistent with this conclusion [17]. During storage, the size of RBCs decreases under certain conditions [5052]. The half-life of NO (t1/2) in the presence of RBCs has been modelled previously [17] according eqn 4:

 
formula
(4)

where N=number of cells/ml, DNO=aqueous diffusion constant for NO and r=cell radius. According to this equation, as the cell radius increases, there will be a proportional decrease in the NO half-life or an increase in RBC scavenging kinetics. This is consistent with the concept of an unstirred layer, where at non-limiting Hb concentrations, the rate of NO dioxygenation is proportional to the RBC surface area [17]. During storage, RBCs change from a biconcave disc to echinocytes with spiculated membranes (see Supplementary Figure S1). Although the cells become smaller, these morphological changes are likely to increase the total surface area comparing like volumes of old RBCs to young RBCs, which would result in increased NO-consumption rates. Another consideration is that the internal cell volume of RBCs decreases during storage [4]. However, Hb concentration does not change (results not shown) indicating that during storage, Hb packaging is altered. How this may affect the rate of NO dioxygenation can be derived from eqns (5) and (6):

 
formula
(5)

or

 
formula
(6)

where the ratio of internal volume of the cell (Vi) to total volume occupied by all cells (VT) is equal to the number of cells/ml (N) multiplied by the volume of a sphere (where r is expressed in cm).

Substituting eqn (4), the half-life of NO in the presence of RBCs with different volumes is shown in eqn (7):

 
formula
(7)

Thus if we compare a suspension of RBCs of equal total Hb concentration, but different sizes similar to our experimental protocol to assess reaction kinetics, the rate of NO consumption by the smaller cells will be greater in proportion to the square of the ratio of the radii. Thus RBCs with smaller volumes provide another possible explanation for why stored RBCs reacted faster with NO. A limitation of this modelling is that it assumes spherical RBCs, whereas storage induces a spectrum of cells with different shapes and sizes. Thus it is likely that individual RBCs possess heterogenous NO-scavenging potential in stored pRBC units.

We also investigated the effects of storage time on reactions between RBCs and nitrite under oxygenated and deoxygenated conditions to test whether deoxyHb-dependent nitrite reduction and NO formation is altered. Storage age had no effect on how RBCs modulated nitrite-dependent relaxation. At high oxygen tension, RBCs of all storage ages inhibited, whereas at low oxygen, no change or a potentiation of relaxation was observed, reflecting the balance between NO scavenging by oxyhaem and nitrite reduction to NO by deoxyhaem [28]. However, as shown in Figure 3, 42-day-old RBCs inhibited NO-dependent vasodilation more than 0-day-old RBCs. Since nitrite-dependent vasodilation occurs via NO formation [28], we speculate that to observe no storage-age effect on nitrite dilation at low oxygen, increased rates of nitrite reduction must be occurring to counter increased NO scavenging. Indeed, increased rates of nitrite consumption were observed at low oxygen by RBCs stored for greater than 14 days (see Supplementary Figure S2). Another consideration in these experiments is that the initial rates of nitrite reduction follow a bell-shape dependence with respect to Hb oxygen fractional saturation, with maximal rates achieved close to the p50 value [25,53]. Owing to decreasing RBC p50 values, calculated oxygen fractional saturations for RBC in nitrite consumption studies (at 2% O2) indicated that maximal rates were observed with 14-day-old RBCs, which were also closest to the p50 value (see the legend to Figure 5). These data underscore the fact that the precise RBC oxygen fractional saturation, a product of local pO2 and RBC oxygen affinity, will be key determinants of how quickly nitrite may be reduced to NO.

RBC storage also increased rates of nitrate formation under oxygenated conditions (Figures 5F–5J), which was paralleled by increased rates of nitrite consumption (Figure 6). A higher concentration of oxyHb (due to a lower p50 value) may also underlie increased nitrate formation kinetics observed with stored RBCs at 2% oxygen (Supplementary Figure S3). OxyHb-mediated nitrite oxidation to nitrate is an autocatalytic reaction that proceeds via intermediate formation of hydrogen peroxide and nitrogen dioxide radical [26]. Therefore antioxidant enzymes or reductants that can scavenge these reactive species slow down nitrite oxidation [26]. Oxidative damage concomitant with loss of antioxidants has been reported in RBCs during storage [54]. Thus the increase in nitrite oxidation to nitrate observed with stored RBCs is probably the result of lower endogenous reductant systems. The lower blood nitrite levels in stable trauma patients transfused with older compared with younger pRBCs may reflect increased rates of nitrite consumption by deoxygenated and/or oxygenated RBCs. However, since the p50 value decreases with RBC storage (implying greater oxyHb concentrations) we speculate that RBC-dependent oxidation of nitrite predominates over reduction pathways.

In summary, we show that in addition to increased NO scavenging by haemolysed cell-free Hb and microparticles, changes to the RBC itself that occur during storage can lead to decreased NO bioavailability. We posit that the combination of stored RBC-dependent increased NO scavenging and nitrite oxidation disposes tissues to inflammatory stress during and following transfusion and underscores the potential therapeutic benefit for NO repletion strategies, with recent studies [43] showing promise in this concept.

Abbreviations

     
  • BHT

    butylated hydroxytoluene

  •  
  • Hb

    haemoglobin

  •  
  • Hct

    haematocrit

  •  
  • KH

    Krebs–Henseleit

  •  
  • L-NMMA

    NG-monomethyl-L-arginine: metHb, methaemoglobin

  •  
  • MNO

    MAHMA NONOate

  •  
  • p50

    oxygen partial pressure producing 50% satn.

  •  
  • RBC

    red blood cell

  •  
  • pRBC

    packed RBC

  •  
  • SpNO

    spermine NONOate

  •  
  • SOD

    superoxide dismutase

AUTHOR CONTRIBUTION

Ryan Stapley, Benjamin Owusu, Angela Brandon and Cilina Rodriguez were responsible for experimental design, collecting and analysing data and writing the paper; Marianne Cusick, Marisa Marques, Jeffrey Kerby and Jordan Weinberg were responsible for clinical sample collection and writing the paper; and Scott Barnum, Jack Lancaster Jr. and Rakesh Patel were responsible for experimental design, data analysis and writing the paper.

We thank Scott Tanner for assistance with flow cytometry studies.

FUNDING

This work was supported the National Institutes of Health [grant numbers HL095468 (to R.P.P., J.A.W. and S.R.B.) and CA131653 (to J.R.L.)].

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Author notes

1

Rakesh Patel is a co-inventor on a patent for use of nitrite salts for the treatment of cardiovascular conditions.

Supplementary data