Nitrosylated hemoglobin levels in human venous erythrocytes correlate with vascular endothelial function measured by digital reactive hyperemia

PLoS One. 2013 Oct 10;8(10):e76457. doi: 10.1371/journal.pone.0076457. eCollection 2013.


Impaired nitric oxide (NO)-dependent endothelial function is associated with the development of cardiovascular diseases. We hypothesized that erythrocyte levels of nitrosylated hemoglobin (HbNO-heme) may reflect vascular endothelial function in vivo. We developed a modified subtraction method using Electron Paramagnetic Resonance (EPR) spectroscopy to identify the 5-coordinate α-HbNO (HbNO) concentration in human erythrocytes and examined its correlation with endothelial function assessed by peripheral arterial tonometry (PAT). Changes in digital pulse amplitude were measured by PAT during reactive hyperemia following brachial arterial occlusion in a group of healthy volunteers (50 subjects). Erythrocyte HbNO levels were measured at baseline and at the peak of hyperemia. We digitally subtracted an individual model EPR signal of erythrocyte free radicals from the whole EPR spectrum to unmask and quantitate the HbNO EPR signals.

Results: Mean erythrocyte HbNO concentration at baseline was 219+/-12 nmol/L (n = 50). HbNO levels and reactive hyperemia (RH) indexes were higher in female (free of contraceptive pills) than male subjects. We observed a dynamic increase of HbNO levels in erythrocytes isolated at 1-2 min of post-occlusion hyperemia (120+/-8% of basal levels); post-occlusion HbNO levels were correlated with basal levels. Both basal and post-occlusion HbNO levels were significantly correlated with reactive hyperemia (RH) indexes (r = 0.58; P<0.0001 for basal HbNO).

Conclusion: The study demonstrates quantitative measurements of 5-coordinate α-HbNO in human venous erythrocytes, its dynamic physiologic regulation and correlation with endothelial function measured by tonometry during hyperemia. This opens the way to further understanding of in vivo determinants of NO bioavailability in human circulation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Case-Control Studies
  • Electron Spin Resonance Spectroscopy
  • Endothelium, Vascular / pathology*
  • Erythrocytes / metabolism*
  • Female
  • Fingers / blood supply*
  • Free Radicals / chemistry
  • Hemoglobins / metabolism*
  • Humans
  • Hyperemia / blood*
  • Hyperemia / metabolism
  • Hyperemia / pathology*
  • Hyperemia / physiopathology
  • Linear Models
  • Male
  • Nitric Oxide / metabolism*
  • Subtraction Technique
  • Vasodilation
  • Veins / pathology


  • Free Radicals
  • Hemoglobins
  • Nitric Oxide

Grants and funding

This work was supported by grant ARC11-16/035 of the Federation Wallonie Bruxelles (to JLB), a PPP "NOHEMIE" from the Region Wallonne (to JLB), the Fonds Special de Recherche-UCL and Fondation Roger Havaux. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.