Erythrocyte storage increases rates of NO and nitrite scavenging: implications for transfusion-related toxicity

Abstract

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.

Figure 1. RBC storage increases oxygen affinity

RBC were collected from healthy volunteers, leukodepleted, stored for the indicated times and P₅₀ (Panel A) and hemolysis (Panel B) measured. For panel A data show mean ± SEM (n=3-4) *P < 0.05 vs day 0; ^#P < 0.05 vs days 0, 1, 7 by 1-way ANOVA with Tukey’s post test. For Panel B data show mean ± SEM (n=3) *P < 0.01 vs day 1 and 14 by 1-way ANOVA with Tukey’s post test.

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

Panel A shows representative traces for SperNO dependent formation of cell-free metHb from oxyHb (7μM) in the presence and absence of RBC (7% Hct) of different storage ages (●, cell free oxyhemoglobin alone; □, day 0 RBC, ◇, day 7 RBC). Inset shows data with older RBC (△, day 14; ▽, day 28; ○, day 42) where kinetics were determined over shorter times due to hemolysis (as described in methods). Panel B shows scatter plot of calculated ratio of rate constants for NO-dioxygenation by RBC relative to cell-free Hb. RBC of different ages were collected from UAB blood bank. For day 0 samples, RBC were collected from healthy volunteers, leukodepleted and processed as described in methods. Experiments were performed in PBS, pH 7.4 at 20°C. Data represent mean ± SEM, n = 5-10. *P < 0.01 relative to day 42 by 1-way ANOVA (P = 0.0005) and Tukey’s post test.

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

RBC (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%O₂ and 1% O₂. Panel A shows representative vessel tension vs. time traces (21%O₂). RBC were added at time 0 and MNO (30nM) addition indicated by arrow. Panel B shows percent inhibition of MNO-dependent vasodilation by RBC of different storage ages at 1% O₂ (□) and 21% O₂ (■). Data are normalized to the RBC heme concentration in the vessel bioassay chamber measured at the end of each experiment and are mean ± SEM (n=5-6). *P < 0.05 relative to day 0, 1, 14 and ^#P < 0.05 relative to day 0 by 1-way ANOVA with Tukey’s post test. Panel C plots percent inhibition of MNO-dependent vasodilation by RBC as a function of calculated fractional saturation. ○, data collected at 1%O₂; ●, data collected at 21%O₂.

Figure 4. Testing a role for microparticles or cell-free hemoglobin in enhanced stored RBC dependent inhibition of NO-signaling

Panel A and B show representative histograms for microparticle analysis in 42d RBC by FACS before and after washing respectively. Events in upper left quadrant represent microparticles and upper right quadrant intact RBC. Panel C shows changes in microparticle levels during storage before (■) and after (□) washing. Data are mean ± SEM (n=3). *P<0.02 relative to before washing by t-test.

Figure 5. RBC storage age effects on nitrite metabolism

Nitrite (100μM) was added to RBC (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, RBC were pelleted and nitrite and nitrate levels measured in the extra-erythrocytic fractions. Data show nitrite consumption (Panels A-E) and nitrate formation (Panels F-J) normalized to heme. P-values indicated on each panel demonstrate effects of pO₂ on nitrite consumption or nitrate formation rates determined by 2-way ANOVA. Calculated oxygen fractional saturations for 2% O₂ condition for storage times 0, 7, 14, 28, 42 were respectively 0.27, 0.46, 0.56, 0.66, 0.69.

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

Nitrite was added to RBC at 21% O₂ as described in Figure 5 legend and nitrite consumption (panel A) and nitrate formation (panel B) measured at 5 min. Data show mean ± SEM (n=4-5). * P < 0.03 by t-test.

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

Nitrite (3 and 10μM) was added to aortic baths containing Krebs buffer with or without RBC (0.3%Hct) of different storage ages and at 1% O₂ or 21% O₂. Panel A-B show representative vessel tension traces. Arrows indicate nitrite addition. Panel C shows percent change in vasodilation elicited by RBC relative to nitrite alone at 1% O₂ (□) and 21% O₂ (■) and storage age. Data are normalized to the concentration of RBC heme in each vessel bioassay chamber. A positive value denotes inhibition, and negative value indicates potentiation of nitrite-dependent vasodilation. Data are mean ± SEM (n = 3-6). ^#P < 0.005 by 2-way ANOVA for effects of oxygen.

Figure 8. RBC transfusion decreases circulating nitrite levels in stable Trauma patients

Panel A: Whole blood nitrite was measured pre- and post transfusion with 1 unit of RBC in stable trauma patients. Data show mean ± SEM (n =31), *P<0.01 by paired t-test. Panel B: Data were separated by the storage age of transfused RBC (0-25d, n = 14 or 26-42 d, n = 17) and changes in whole blood nitrite (pre – post transfusion) plotted for these groups. *P<0.02 by unpaired t-test.