S-nitrosohemoglobin deficiency: a mechanism for loss of physiological activity in banked blood

Abstract

RBCs distribute oxygen to tissues, but, paradoxically, blood transfusion does not always improve oxygen delivery and is associated with ischemic events. We hypothesized that storage of blood would result in loss of NO bioactivity, impairing RBC vasodilation and thus compromising blood flow, and that repleting NO bioactivity would restore RBC function. We report that S-nitrosohemoglobin (SNO-Hb) concentrations declined rapidly after storage of fresh venous blood and that hypoxic vasodilation by banked RBCs correlated strongly with the amounts of SNO-Hb (r(2) = 0.90; P < 0.0005). Renitrosylation of banked blood during storage increased the SNO-Hb content and restored its vasodilatory activity. In addition, canine coronary blood flow was greater during infusion of renitrosylated RBCs than during infusion of S-nitrosothiol-depleted RBCs, and this difference in coronary flow was accentuated by hypoxemia (P < 0.001). Our findings indicate that NO bioactivity is depleted in banked blood, impairing the vasodilatory response to hypoxia, and they suggest that SNO-Hb repletion may improve transfusion efficacy.

Fig. 1.

SNO-Hb in stored human whole blood. Shown is the change in levels over time in human RBCs (n = 4) stored in citrate phosphate dextrose/additive solution 1. Packed cells were kept in vacutainers at 4°C and serially sampled by using an aseptic technique at the times indicated. , P < 0.05 (significant difference from fresh blood). At day 21, the level of SNO-Hb was below the sensitivity of the assay (ND, not detected).

Fig. 2.

SNO-Hb and bioactivity of banked RBCs. (A) Cohort A: SNO-Hb concentrations (n = 4–10), expressed per Hb tetramer, and corresponding vasodilatory activity of RBCs [expressed as changes in tension in rabbit aortic rings (mean ± SD; n = 11–22; arc-sine transformed)] for fresh (Day 0) and stored blood. Bars marked with are time points where SNO-Hb levels were significantly reduced compared with fresh blood (P < 0.05). Similarly, vasorelaxations marked with were significantly less (P < 0.01) than those produced by fresh RBCs. Each time point is an independent sampling comprising different individuals. (B) Cohort B: Change in SNO-Hb concentration over time from serially sampled blood bags obtained at day 2 (n = 6); SNO-Hb is significantly reduced at all time points compared with fresh venous blood. Blood in cohorts A and B was obtained from the same commercial source.

Fig. 3.

NO content of human RBCs during storage and after NO repletion. Shown is the effect of renitrosylation on levels of SNO-Hb, Hb[FeNO], and total NO per Hb tetramer (HbNO). At all time points (n = 5–16), renitrosylation, demarcated by the filled bars, significantly increased the levels of both SNO-Hb and Hb[FeNO] vs. untreated cohorts (open bars). , time points where SNO-Hb and HbNO levels in untreated blood were significantly reduced compared with fresh blood (P < 0.05); †, the time point (day 43) where SNO-Hb and total NO levels in treated blood remained significantly reduced compared with fresh blood (P < 0.05).

Fig. 4.

SNO-Hb content and vasodilatory activity of renitrosylated RBCs. Shown is hypoxic vasodilatory activity of RBCs in bioassays. (A) Representative tracings showing the degree of vasorelaxation as percent change in tension in rabbit aortic rings produced by fresh, stored (expired and day 1), or renitrosylated (day 1) RBCs. (B) Relaxation data (mean ± SD; n = 11–25) and corresponding SNO-Hb concentrations for renitrosylated fresh and stored RBCs. At all storage time points SNO-reconstituted RBCs exhibited relaxations equivalent to fresh blood.

Fig. 5.

Hypoxic vasodilation by stored and renitrosylated RBCs in vivo. Shown are changes in canine coronary artery blood flow (mean ± SD; n = 7) produced by infusion of SNO-depleted or renitrosylated RBCs. Increases in flow elicited by renitrosylated RBCs were significantly greater than those produced by SNO-depleted RBCs, and the degree of change was greater under hypoxic (5% FiO₂) than normoxic (21% FiO₂) conditions.