Nitrosyl-hemoglobin formation in rodent and human venous erythrocytes reflects NO formation from the vasculature in vivo

<div><p>Reduced bioavailability of nitric oxide (NO) is a major feature of endothelial dysfunction characteristic of cardiovascular and metabolic diseases but the short half-life of NO precludes its easy quantification in circulating blood for early diagnosis. In erythrocytes, NO can react with hemoglobin to form an iron-nitrosyl complex (5-coordinate-α-HbNO) directly quantifiable by Electron Paramagnetic Resonance spectroscopy (EPR) in mouse, rat and human venous blood <i>ex vivo</i>. However, the sources of the nitrosylating species <i>in vivo</i> and optimal conditions of HbNO preservation for diagnostic use in human erythrocytes are unknown. Using EPR spectroscopy, we found that HbNO stability was significantly higher under hypoxia (equivalent to venous pO₂; 12.0±0.2% degradation of HbNO at 30 minutes) than at room air (47.7±0.2% degradation) in intact erythrocytes; at 20°C (15.2±0.3% degradation after 30 min versus 29.6±0.1% at 37°C) and under acidic pH (31.7±0.8% versus 62.2±0.4% degradation after 30 min at physiological pH) at 50% of haematocrit. We next examined the relative contribution of NO synthase (NOS) from the vasculature or in erythrocytes themselves as a source of nitrosylating NO. We detected a NOS activity (and eNOS expression) in human red blood cells (RBC), and in RBCs from eNOS^(+/+) (but not eNOS^(-/-)) mice, as measured by HbNO formation and nitrite/nitrate accumulation. NO formation was increased after inhibition of arginase but abrogated upon NOS inhibition in human RBC and in RBCs from eNOS^(+/+) (but not eNOS^(-/-)) mice. However, the HbNO signal from freshly drawn venous RBCs was minimally sensitive to the inhibitors <i>ex vivo</i>, while it was enhanced upon caveolin-1 deletion <i>in vivo</i>, suggesting a minor contribution of erythrocyte NOS to HbNO complex formation compared with vascular endothelial NOS or other paracrine NO sources. We conclude that HbNO formation in rodent and human venous erythrocytes is mainly influenced by vascular NO sources despite the erythrocyte NOS activity, so that its measurement by EPR could serve as a surrogate for NO-dependent endothelial function.</p></div

Effect of oxygen on HbNO (5-coordinate alpha-nitrosyl-hemoglobin) formation in human RBCs.

<p>A. Typical EPR spectra recorded in human erythrocyte samples exposed to 1% (a, c) or 21% (b) of O₂ and treated with 50 μmol/L of Spermine-NONOate for 1 hour (a, b); or with a NO-generating mixture (4 μmol/L of NO₂^- and 50 mmol/L of Na₂S₂O₄) (c). Spectra were acquired as described in Materials and Methods. Arrows point to the triplet hyperfine structures (hfs). A(I) corresponds to the amplitude of the first hf component used for HbNO quantification. B. The concentrations of HbNO complex accumulated in human reconstituted RBCs (50% of haematocrit in isotonic buffer) under 21% (square) or 1% (triangle) of O₂ after addition of graded concentrations of Spermine-NONOate. Data are shown as mean values ± SEM; ^$ P < 0.01 ^§P < 0.0001; n = 4 different RBC preparations.</p

Temperature and pH influence on HbNO stability.

<p>A. Stability of HbNO complex accumulated in human RBCs pre-incubated with Spermine-NONOate (100 μmol/L, 60 minutes) under venous O₂ level, and subsequently incubated at 20°C (circle) or 37°C (triangle) during 50 minutes under 21% of O₂. B. Effect of pH on HbNO stability in human RBCs pre-incubated with NO-donor and reconstituted at 50% haematocrit with isotonic buffer at acidic pH (6.2–4.7) or at physiological pH (7.4–7.2) at 21% of O₂ and 37°C. Data are shown as mean values ± SEM; * P < 0.05; n = 4 different RBC preparations.</p

Effect of oxygen on stability of HbNO formed <i>ex vivo</i>.

<p>A. Typical EPR spectra, and B. concentrations of HbNO complex accumulated in human RBCs pre-incubated with Spermine-NONOate (100 μmol/L, for 60 minutes, under venous O₂ level) and subsequently exposed for 60 minutes to 21% (square) or 1% (triangle) of O₂ as described in Methods. Arrows in (A) point to the triplet hyperfine structures (hfs); A(I) indicates the amplitude of the first hf component. Data in B are shown as mean values ± SEM; ^* P < 0.05; n = 4 different RBC preparations. C-D. Correlation between HbNO concentrations in RBCs and percentage of oxy/deoxy hemoglobin in venous blood during incubation at 21% (C); and 1% of O₂ (D).</p

Minimal influence of erythrocyte eNOS activity on HbNO content.

<p>Time-dependent decay of EPR HbNO signal formed <i>in vivo</i> in mouse (A and B) and in human (D and E) erythrocytes after <i>ex vivo</i> treatment with L-NAME or vehicle (A and D) or with nor-NOHA or vehicle (B and E) under L-Arginine supplementation at 21% of O₂ as described in Materials and Methods. C. Typical EPR spectra recorded in mouse erythrocyte samples before (a) and after incubation with L-Arginine alone (b) or with nor-NOHA (c) or L-NAME (d) for 20 minutes at 21% of O₂. Sample aliquots were frozen at 0, 5, 20 and 40 minutes. The EPR spectra were acquired as described in Material and Methods. Data are shown as mean values ± SEM; n = 3 different RBC preparations. * <i>P</i> <0.05 at 20 min.</p

Detection of erythrocyte eNOS activity by EPR spectroscopy and by nitrite and nitrate colorimetric assay.

<p>A. Typical procedure used for subtraction analysis: the EPR spectrum of RBC sample before (a) and after (b) subtraction of PFR EPR spectrum (c) obtained as described in Material and Methods. Typical HbNO EPR signal from immediately frozen mouse whole blood (d); the hyperfine structure (hfs) of HbNO is shown by the arrows. (B-F). Quantification of erythrocyte HbNO EPR signals (B) and products of NO oxidation, nitrite and nitrate formed in isolated RBCs of eNOS^(+/+)and eNOS^(-/-) mice (C and D) and in human erythrocytes (E and F), after incubation <i>ex vivo</i> with norNOHA and/or L-NAME as described in Materials and Methods. Data are shown as mean values ± SD; *<i>P</i> < 0.05, ^$P<0.01, ^§ P < 0.0001; n = 3–7 different mouse RBC preparations and n = 10 different human preparations.</p

Human RBCs express eNOS proteins.

<p>A. Representative flow cytometric two parameter dot plot of isolated RBCs from a healthy subject co-stained with primary anti-Aquaporin-1 and secondary Alexa Fluor-488-conjugated anti-IgG antibodies; and primary anti-eNOS and secondary Alexa Fluor-647-conjugated anti-IgG antibodies. Panel on the left shows co-staining only with conjugated secondary antibodies, panel in the middle shows staining only with mouse isotype IgG1,k Alexa-647-conjugated (negative controls). Panel on the right shows the identification of RBCs as eNOS–positive and Aquaporin1-positive events in the upper right quadrant. The percentage of RBCs double positive is indicated in the upper right quadrant. (B-E) Representative images of eNOS (B) and AQP1 (D) detection in healthy human RBCs using immunofluorescence microscopy. As negative controls, RBCs were stained only with conjugated secondary antibodies (Alexa Fluor-488 anti-mouse IgG for eNOS (C) and Alexa Fluor-568 anti-rabbit IgG for AQP1 (D)).</p

Sensitivity of blood HbNO formation to <i>in vivo</i> administered L-NAME or a nitrovasodilator.

<p>A. Concentration of HbNO in venous mouse RBCs after seven days of treatment with L-NAME or vehicle as described in Materials and Methods. B. Typical EPR spectra of HbNO recorded from isolated RBCs after one week administration of vehicle (a) or L-NAME (b) in drinking water. C. Kinetics of relative <i>in vivo</i> changes of HbNO EPR signal in venous rat blood after <i>in vivo</i> isosorbide dinitrate (ISDN) or vehicle injection. D. Typical HbNO EPR spectra after 5 and 10 minutes i.v. administration of vehicle (a-b) or isosorbide dinitrate (ISDN) (c-d). Data are shown as mean values ± SD; ^* P < 0.05 and ^$ P < 0.01; n = 4 different RBC preparations.</p

Hb-NO levels correlate with endothelial function in normal subjects.

<p>(A) Linear regression analysis between basal HbNO level in RBCs isolated from venous blood and FRHI calculated as described in Methods (r = 0.58, P<0.0001). (B) Analysis of HbNO levels in RBCs isolated from venous blood of subjects distributed in different tertiles of FRHI as measured in the same arm; one-way analysis of variance (ANOVA) shows significant difference of mean values between groups (P = 0.0002).</p

Hb-NO levels correlate with reactive hyperemia both in males and females.

<p>Linear regression analysis between basal HbNO level (nmol/L) in RBCs isolated from venous blood of male (A) and female (B) subjects and FRHI calculated as described in Methods (r = 0.57; P<0.005; n = 23 and r = 0.55; P<0.005; n = 27 respectively).</p