0460 : Nitric oxide and hemoglobin form a paramagnetic compound quantifiable by Electron Paramagnetic Resonance (EPR) spectroscopy in venous erythrocytes that reflects vascular NO bioavailability in vivo

Abstract

Reduced bioavailability of nitric oxide is a hallmark of endothelial dysfunction in metabolic and cardiovascular diseases, but its quantification in circulating blood remains a challenge.NO can form iron-nitrosyl complexes with hemoglobin(5-coordinate-α-HbNO) in erythrocytes(RBCs).We hypothesized that this complex reflects bioavailability of vascular NO and endothelial function in vivo .We developed a modified subtraction method using EPR to quantify it in RBCs from mouse, rat and human venous blood. NO could be supplied from vascular or erythrocytic NOS.We detected eNOS proteins in rodent and human RBC.To test eNOS functionality, we measured nitrite/ nitrate production and HbNO formation in human RBCs and from eNOS (+/+) and eNOS (-/-) mice in vitro and its sensitivity to NOS or arginase inhibitors. Nitrite and HbNO signals increased after arginase inhibition and were abrogated upon NOS inhibition in human and eNOS (+/+) but insensitive to these modulators in eNOS (-/-) RBCs. We found that upon exposure to exogenous NO-donor, the formation of HbNO was higher in hypoxic conditions(1%O2:0.018+/–0.002 compared to room air 0.0036+/–0.0004 μmol HbNO/μmolNO-donor).Accordingly, the stability of pre-formed HbNO was higher under hypoxia(17% degradation after 30 min vs 49% at room air), and preserved at 21% of O 2 by incubation with catalase(CAT:2μM HbNO vs 0.5μM HbNO in untreated controls).Conversely, CAT inhibition increased ROS formation in RBCs, measured by FACS analysis of DCFDA fluorescence. This suggested that HbNO formation is sensitive to oxidative degradation, possibly by H 2 O 2 . We compared circulating HbNO levels in venous RBCs from normal volunteers or patients with cardiovascular diseases and found decreased HbNO in patients(0.141+/–11μM vs 0.22+/–12μM in volunteers; N=38 and48).We conclude that HbNO reflects exposure of RBCs to NO in vivo and is sensitive to oxidative degradation by H 2 O 2 .HbNO could be developed as a biomarker of NO bioavailability and/or oxidative stress ex vivo . The author hereby declares no conflict of interest