Heme-nitrosylated hemoglobin and oxidative stress in women consuming combined contraceptives. Clinical application of the EPR spectroscopy

Free Radic Biol Med. 2017 Jul:108:524-532. doi: 10.1016/j.freeradbiomed.2017.03.039. Epub 2017 Apr 7.


An increased risk of venous thromboembolism was identified in young women consuming combined contraceptive pills (CP) suggesting a disturbance of vascular homeostasis but the impact of CP on endothelial function and redox status of the vasculature was not thoroughly analyzed. We measured the bioavailability of nitric oxide (NO), a main mediator of vascular homeostasis in a cohort of young female subjects (n=114) and compared the results in users or not of CPs containing ethinyl estradiol and synthetic progestogens. Vascular NO availability was measured by quantification of the heme-nitrosylated hemoglobin (5-coordinate-α-HbNO) concentrations in venous erythrocytes using Electron Paramagnetic Resonance spectroscopy (EPR). Vascular oxidative status was assessed by measurement of peroxides in plasma, and of the thiol redox state in erythrocytes. In addition, endothelial function was assessed by digital reactive hyperemia pulse tonometry using EndoPAT. We observed that the HbNO level was significantly lower in erythrocytes of subjects consuming CPs versus controls (162±8 and 217±12 nmol/L). This correlated with significantly increased levels of plasma peroxides (1.8±0.1mmol/L versus 0.8±0.1mmol/L in controls) and decreased concentrations of erythrocyte reduced thiols (by 12%). Interestingly, the level of oxidized ceruloplasmin-Cu(II) was also significantly higher in the group consuming CPs. The EndoPAT index showed a trend towards impairment in CP users, and was significantly lower in subjects that consumed CPs containing drospirenone, and had lowest erythrocyte HbNO levels.

Conclusion: This cross-sectional cohort study demonstrates that a decrease of HbNO measured by quantitative EPR in human venous erythrocytes is correlated with the development of endothelial dysfunction under CPs consumption, in parallel with increased vascular oxidative stress.

Keywords: Contraceptives; Electron Paramagnetic Resonance; Endothelial function; Heme-nitrosylated hemoglobin; Reactive hyperemia response; Vascular oxidative status.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Cells, Cultured
  • Cohort Studies
  • Contraceptive Agents / adverse effects*
  • Contraceptive Agents / therapeutic use
  • Cross-Sectional Studies
  • Drug-Related Side Effects and Adverse Reactions / metabolism*
  • Electron Spin Resonance Spectroscopy
  • Endothelium, Vascular / pathology*
  • Erythrocytes / metabolism*
  • Ethinyl Estradiol / adverse effects*
  • Ethinyl Estradiol / therapeutic use
  • Female
  • Glycated Hemoglobin / metabolism*
  • Humans
  • Nitric Oxide / metabolism*
  • Oxidation-Reduction
  • Oxidative Stress
  • Peroxides / blood
  • Progesterone Congeners / adverse effects*
  • Progesterone Congeners / therapeutic use
  • Venous Thromboembolism / etiology
  • Venous Thromboembolism / metabolism*
  • Young Adult


  • Contraceptive Agents
  • Glycated Hemoglobin A
  • Peroxides
  • Progesterone Congeners
  • hemoglobin A, glycosylated-nitric oxide complex
  • Nitric Oxide
  • Ethinyl Estradiol