title: Nitrosylated hemoglobin levels in human venous erythrocytes correlate with vascular endothelial function measured by digital reactive hyperemia.
abstract: 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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institut de Recherche Experimentale et Clinique (IREC), Pole of Pharmacology and Therapeutics (FATH), Université Catholique de Louvain (UCL), Brussels, Belgium.
authors.forename: Irina I
authors.initials: II
authors.lastname: Lobysheva
authors.forename: Pauline
authors.initials: P
authors.lastname: Biller
authors.forename: Bernard
authors.initials: B
authors.lastname: Gallez
authors.forename: Christophe
authors.initials: C
authors.lastname: Beauloye
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Balligand
mesh_terms: D000328:Adult; D016022:Case-Control Studies; D004578:Electron Spin Resonance Spectroscopy; D004730:Endothelium, Vascular / Q000473:pathology*; D004912:Erythrocytes / Q000378:metabolism*; D005260:Female; D005385:Fingers / Q000098:blood supply*; D005609:Free Radicals / Q000737:chemistry; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D006940:Hyperemia / Q000097:blood* / Q000378:metabolism / Q000473:pathology* / Q000503:physiopathology; D016014:Linear Models; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism*; D013382:Subtraction Technique; D014664:Vasodilation; D014680:Veins / Q000473:pathology
chemical_list: D005609:Free Radicals; D006454:Hemoglobins; D009569:Nitric Oxide