**1. Introduction**

Asthma is a chronic disease of the airways that is characterized by variable bronchial obstruction and hyperresponsiveness, often accompanied by structural remodeling [1]. Disease pathogenesis involves various cell types and mediators that participate in airway inflammation, trigger asthma symptoms, and contribute to disease progression. Airway inflammation can be exacerbated by viral infections and exposure to inhaled allergens or airway pollutants. Epidemiological studies demonstrate a clear relationship between air quality and control of asthma symptoms. Exposure to tobacco smoke, ozone, and environmental pollution, such as diesel exhaust, generates reactive oxygen species (ROS) and other oxidative stressors, initiating and augmenting inflammation and sensitizing the airways to other triggers of symptoms [1,2]. Additionally, the inflammatory cells present in the asthmatic airways are considered the primary local source of ROS [3]. Furthermore, ROS itself may play a role in asthma pathogenesis, as they promote type-2 (T2) responses in the lungs and activate nuclear factor (NF)-κβ, a potent pro-inflammatory gene inducer [4].

ROS are produced continuously in a small amount by all cells. Still, if delivered in higher amounts, for example, during inflammation, they alter the pro/antioxidant balance, causing oxidative stress and tissue damage [5]. One signature of increased oxidative stress is the hydroperoxides of amino acid residues (HP), unstable derivatives formed during exposure of proteins to ROS [6]. Previous experimental studies confirmed that oxidative damage to proteins, lipids, or nucleic acids might lead to pathological changes in airway epithelial cells, resulting in increased permeability, mucus secretion, and enhanced airway hyperresponsiveness [5,7–9].

In asthmatic airways, many cell types enhance ROS production, including epithelial and endothelial cells and infiltrating leukocytes; therefore, ROS are necessary components of the innate immune system [5]. However, the lungs and blood provide an efficient defense system against oxidative stress, mediated by two essential elements. The first contains nonenzymatic dietary antioxidants, including tocopherols, carotenes, and lycopene. The second refers to the endogenous system of antioxidant enzymes, such as superoxide dismutase, catalase, and lipoprotein-associated phospholipase A2 (Lp-PLA2), which combat biochemically oxidative stress [10]. Despite these mechanisms, asthma patients show increased lung oxidative stress, as evidenced by elevated nitric oxide and carbon monoxide concentrations in the exhaled air [3]. Therefore, it has been suggested that asthma is characterized by a decreased ability to respond to oxidative stress [5,10]. For example, patients with severe asthma show decreased plasma activity of Lp-PLA2 [11]. On the contrary, others point to the upregulation of antioxidative mechanisms, albeit with still an overwhelming prooxidative capacity [3,12,13]. Nevertheless, the role of oxidative stress in asthma pathology and airway remodeling has not been comprehensively studied, including how it impacts endothelial injury and early atherosclerosis [14] and increases the risk of prothrombotic and cardiovascular events, as previously reported in that disease [15–17].

Numerous studies on asthma indicate an increased prooxidative potential of peripheral blood leukocytes, mainly neutrophils, and upregulation of oxidative biomarkers in airways, e.g., nitric oxide, or in circulation, e.g., malondialdehyde and uric acid [3,5,13,18]. However, scarcer data analyzed oxidative stress globally in circulating blood, probably due to the lack of reliable research methods that could be successfully applied to serum or plasma samples. Current assays are based mainly on the oxidation of ferrous ions, monitored with orange xylenol or the iodometric test [19,20], and are demanding from a technical point of view. Additionally, they cannot be used in real-time measurements. However, recently Michalski et al. [21] developed and validated a novel real-time fluorescent assay that fits this purpose. In this assay, the profluorescent coumarin boronic acid (CBA) probe reacts with amino acid and protein hydroperoxides to form the corresponding fluorescent product, 7-hydroxycoumarin, which is easily detectable by a fluorescent reader.

Considering the available data on the possible link between airway inflammation, local oxidative stress, premature atherosclerosis, and increased risk of cardiovascular events in asthma, we sought to evaluate the CBA assay in the circulating blood of those subjects. We also examined its relation to asthma severity; lung function and morphometry; blood and bronchoalveolar lavage fluid (BAL) inflammatory biomarkers; and histological measures of airway remodeling, including reticular basement membrane (RBM) thickness and collagen I deposits.

To date, such studies have not yet been performed.
