Erythrocytes are the major intravascular storage sites of nitrite in human blood

Abstract

Plasma levels of nitrite ions have been used as an index of nitric oxide synthase (NOS) activity in vivo. Recent data suggest that nitrite is a potential intravascular repository for nitric oxide (NO), bioactivated by a nitrite reductase activity of deoxyhemoglobin. The precise levels and compartmentalization of nitrite within blood and erythrocytes have not been determined. Nitrite levels in whole blood and erythrocytes were determined using reductive chemiluminescence in conjunction with a ferricyanide-based hemoglobin oxidation assay to prevent nitrite destruction. This method yields sensitive and linear measurements of whole blood nitrite over 24 hours at room temperature. Nitrite levels measured in plasma, erythrocytes, and whole blood from 15 healthy volunteers were 121 plus or minus 9, 288 plus or minus 47, and 176 plus or minus 17 nM, indicating a surprisingly high concentration of nitrite within erythrocytes. The majority of nitrite in erythrocytes is located in the cytosol unbound to proteins. In humans, we found a significant artery-to-vein gradient of nitrite in whole blood and erythrocytes. Shear stress and acetylcholine-mediated stimulation of endothelial NOS significantly increased venous nitrite levels. These studies suggest a dynamic intravascular NO metabolism in which endothelial NOS-derived NO is stabilized as nitrite, transported by erythrocytes, and consumed during arterial-to-venous transit.

Figure 1.

Validation of assay based on ferricyanide to stabilize nitrite in whole blood. (A) Original registration of reductive tri-iodide-based chemiluminescence after injection of 200 μL plasma, treated erythrocytes, and whole blood with or without addition of acid sulfanilamide. Loss of signal after sulfanilamide indicates specificity of the assay for nitrite. (B) Recovery and linearity of nitrite spiked in whole blood to which nitrite preservation solution was added. (C) Freeze-thaw and sample processing does not influence nitrite concentration in whole blood. Open circles represent measurements after freezing, storage at -80°C for 4 days, thawing, and deproteination. With an injection volume of 200 μL, 1 pmol nitrite could be recovered at a signal-to-noise ratio of 3:1. (D) Stability of nitrite in whole blood. Spiking of nitrite to a final concentration of 1 μM and measurement of nitrite concentration of whole blood up to 24 hours after nitrite addition. Samples were either treated with nitrite preservation solution (○) or saline (□) after addition of nitrite. The inset illustrates the disappearance of nitrite in whole blood with or without nitrite preservation solution. Samples were stored at room temperature during incubation.

Figure 2.

Nitrite concentration in whole human blood and its components. (A) Distribution of nitrite in the various compartments of whole blood (WB). Measured whole blood concentration treated with nitrite preservation solution equals the calculated whole blood nitrite concentration from plasma and RBC nitrite after the allowance for hematocrit. (B) Location of nitrite within the erythrocyte. Error bars show standard error of the mean.

Figure 3.

Changes in nitrite concentration during blood flow through the human forearm circulation. (A) Infusion of acetylcholine (ACh) (7.5 μg/min) intra-arterially in the human forearm and ischemia-induced flow-mediated dilation causes a 4- to 10-fold increase in forearm blood flow. (B) The stimulation reverses the normal small uptake of nitrite to instead cause a release of nitrite into the circulation, a result consistent with NO-to-nitrite conversion during forearm circulatory passage. Horizontal bars represent mean values. (C) Comparison of nitrite formation under basal conditions (saline infusion) and NOS stimulation via ACh or shear stress. (D) Time profile of nitrite responses in whole venous blood during continuing ACh infusion (representative results). Error bars show standard error of the mean.

Figure 4.

A model of intravascular metabolism of NO. NO produced by eNOS may diffuse into the vascular lumen as well as the underlying smooth muscle. The majority of this NO enters the erythrocyte and reacts with oxyhemoglobin (Oxy Hb) to form nitrate (); a minor portion may escape the hemoglobin scavenger and react with plasma constituents to form nitros(yl)ated species (RXNO, nitrosothiols: RSNO), nitrated lipids (NO₂ lipids), and nitrite (). Each of these species is capable of transducing NO bioactivity far from its location of formation. Nitrite may diffuse into the erythrocytes where it appears in a higher concentration than in plasma. In the erythrocyte nitrite reacts with deoxyhemoglobin (Deoxy Hb) to form nitric oxide and methemoglobin (Met Hb) and other NO adducts. NO can then either diffuse out of the erythrocyte directly or via an intermediate NO metabolite. The question mark circled in white refers to the possibility of an intermediate during nitrite bioactivation. NO Hb indicates iron nitrosylhemoglobin; SNO Hb, nitrosohemoglobin; L-Arg, L-arginine; L-Cit, L-citrulline; N_xO_y: higher N oxides.