Airway peroxidases catalyze nitration of the β₂-agonist salbutamol and decrease its pharmacological activity

β₂-Agonists are the most effective bronchodilators for the rapid relief of asthma symptoms, but for unclear reasons, their effectiveness may be decreased during severe exacerbations. Because peroxidase activity and nitrogen oxides are increased in the asthmatic airway, we examined whether salbutamol, a clinically important β₂-agonist, is subject to potentially inactivating nitration. When salbutamol was exposed to myeloperoxidase, eosinophil peroxidase or lactoperoxidase in the presence of hydrogen peroxide (H₂O₂) and nitrite (NO₂ ^-), both absorption spectroscopy and mass spectrometry indicated formation of a new metabolite with features expected for the nitrated drug. The new metabolites showed an absorption maximum at 410 nm and pK_a of 6.6 of the phenolic hydroxyl group. In addition to nitrosalbutamol (m/z 285.14), a salbutamol-derived nitrophenol, formed by elimination of the formaldehyde group, was detected (m/z 255.13) by mass spectrometry. It is noteworthy that the latter metabolite was detected in exhaled breath condensates of asthma patients receiving salbutamol but not in unexposed control subjects, indicating the potential for β₂-agonist nitration to occur in the inflamed airway in vivo. Salbutamol nitration was inhibited in vitro by ascorbate, thiocyanate, and the pharmacological agents methimazole and dapsone. The efficacy of inhibition depended on the nitrating system, with the lactoperoxidase/H₂O₂/NO₂^- being the most affected. Functionally, nitrated salbutamol showed decreased affinity for β₂-adrenergic receptors and impaired cAMP synthesis in airway smooth muscle cells compared with the native drug. These results suggest that under inflammatory conditions associated with asthma, phenolic β₂-agonists may be subject to peroxidase-catalyzed nitration that could potentially diminish their therapeutic efficacy.