Oxidative stress perturbs vascular homeostasis leading to endothelial dysfunction and cardiovascular diseases. Vascular reactive oxygen species (ROS) reduce nitric oxide (NO) bioactivity, a hallmark of cardiovascular and metabolic diseases. We measured steady-state vascular NO levels through the quantification of heme nitrosylated hemoglobin (5-coordinate-α-HbNO) in venous erythrocytes of healthy human subjects using electron paramagnetic resonance (EPR) spectroscopy. To examine how ROS may influence HbNO complex formation and stability, we identified the pro- and anti-oxidant enzymatic sources in human erythrocytes and their relative impact on intracellular redox state and steady-state HbNO levels. We demonstrated that pro-oxidant enzymes such as NADPH oxidases are expressed and produce a significant amount of ROS at the membrane of healthy erythrocytes. In addition, the steady-state levels of HbNO were preserved when NOX (e.g. NOX1 and NOX2) activity was inhibited. We next evaluated the impact of selective antioxidant enzymatic systems on HbNO stability. Peroxiredoxin 2 and catalase, in particular, played an important role in endogenous and exogenous H₂O₂ degradation, respectively. Accordingly, inhibitors of peroxiredoxin 2 and catalase significantly decreased erythrocyte HbNO concentration. Conversely, steady-state levels of HbNO were preserved upon supplying erythrocytes with exogenous catalase. These findings support HbNO measurements as indicators of vascular oxidant stress and of NO bioavailability and potentially, as useful biomarkers of early endothelial dysfunction.

氧化应激干扰血管稳态，导致内皮功能障碍和心血管疾病。血管活性氧（ROS）会降低一氧化氮（NO）的生物活性，这是心血管疾病和代谢疾病的标志。我们通过使用电子顺磁共振（EPR）光谱对健康人静脉血红细胞中的血红素亚硝化血红蛋白（5-坐标-α-HbNO）进行定量测量来测量稳态血管NO水平。为了检查ROS如何影响HbNO复合物的形成和稳定性，我们鉴定了人红细胞中的抗氧化剂和抗氧化剂酶源及其对细胞内氧化还原状态和稳态HbNO水平的相对影响。我们证明了诸如NADPH氧化酶之类的前氧化酶在健康的红细胞膜上表达并产生大量的ROS。另外，当抑制NOX（例如NOX1和NOX2）活性时，HbNO的稳态水平得以保留。接下来，我们评估了选择性抗氧化剂酶体系对HbNO稳定性的影响。过氧化物酶2和过氧化氢酶尤其在内源性和外源性H中起重要作用₂ O ₂分别降解。因此，过氧化物酶2和过氧化氢酶的抑制剂显着降低了红细胞HbNO浓度。相反，在向红细胞供应外源过氧化氢酶后，HbNO的稳态水平得以保持。这些发现支持HbNO测量作为血管氧化应激和NO生物利用度的指标，并有可能作为早期内皮功能障碍的有用生物标志物。