*3.5. Complex Regulation of Circulating Protein Hydroperoxides Generation by Peripheral Blood Biomarkers*

Then we investigated the relation of HP generation in asthma to laboratory variables measured in the systemic circulation, including blood cell counts, inflammatory biomarkers, and atherosclerosis risk factors, such as glucose level and lipid profile.

As expected, asthma patients were characterized by increased blood eosinophilia and serum IgE compared to the controls (Table 4). Interestingly, the serum CRP concentration was also marginally elevated in the patients (*p* = 0.04). On the other hand, glucose and total cholesterol levels and blood cytokines were comparable in both study groups, except for

IL-10, which was higher in asthmatics. Similarly, the concentration of H3cit was increased in patients, suggesting neutrophil activation and the formation of extracellular traps [28].

**Figure 5.** Positive correlations between dynamics in hydroperoxide generation in the real-time coumarin boronic acid (CBA) assay and bronchoalveolar lavage fluid biomarkers (interleukin [IL]-6, IL-12, and BAL neutrophilia); for abbreviations, see the legend in Figure 2 and Table 2.


**Table 4.** Laboratory parameters in asthmatic patients and control subjects.

Variables are presented as median and interquartile range, or mean and standard deviation, as appropriate. References: § all serum measurements of interleukin (IL)-4 and IL-5 were below the assay threshold (data not shown); \*\* *p* < 0.001; \* *p* < 0.05 and *p* ≥ 0.001.

Interestingly, systemic HP generation in asthma showed a clear negative association with the red blood cell (RBC) count (Figure 6a) and hemoglobin level. This relationship applied to all CBA assay parameters and was not seen in the control grroup.

**Figure 6.** Relationships between cumulative hydroperoxide generation in the real-time coumarin boronic acid (CBA) assay and red blood cell count (**a**) peripheral blood monocyte count (**b**), circulating C-reactive protein (**c**), and fibrinogen (**d**); for abbreviations, see the legend in Figure 2 and Table 2.

Furthermore, we demonstrated a weak positive association between HP formation dynamics and blood monocyte count (Figure 6b) and some nonspecific markers of inflammation, such as serum CRP (Figure 6c) or plasma fibrinogen (Figure 6d). In contrast, it was not directly related to renowned T2 immune response measures in blood, such as eosinophilia.

In Table 5, we demonstrate the independent determinants among the measured blood biomarkers of increased susceptibility to HP formation (R factor) in a multiple regression model. Interestingly, various variables predicted an increase in the dynamics of systemic HP generation, including blood monocyte count, H3cit, and IL-17A, as well as total cholesterol levels. At the same time, the RBC count and, to a much lesser extent, the blood eosinophil count inversely impacted the R factor.

Eventually, we performed a combined analysis by using a multivariable stepwise regression model, considering both the airway and systemic measures investigated in the study that could impact HP generation. As presented in Table 6, susceptibility to increased HP formation was determined best by lower spirometry values (e.g., FEV1), elevated IL-6 in BAL, and higher circulating H3cit. As expected, the RBC count had a substantial negative contribution to that analysis.

**Table 5.** Multiple linear regression model for a relative increase of fluorescent product growth velocity (R factor) in the real-time CBA assay in asthma patients. Presented variables were documented as independent determinants; they explain 33% of R factor variability.


For data interpretation, see footnote to Table 3.

**Table 6.** Multiple linear regression model for a relative increase of fluorescent product growth velocity (R factor) in the real-time CBA assay in asthma patients, considering all study variables.


For data interpretation, see footnote to Table 3. For the abbreviations, see footnote to Table 2.

### **4. Discussion**

The present study demonstrates an increase in the generation of amino acid HP in the peripheral blood of asthmatic patients, reflecting an enhanced systemic oxidative stress response in that disease. In asthma, increased ROS formation by inflammatory cells has already been described in both the airways and systemic circulation [5,13,18]. Here, we show that it can be assessed reliably in the peripheral blood by using a recently developed technically undemanding real-time CBA assay [21,29,30]. Furthermore, we have investigated whether elevated oxidative stress in asthma, related to increased HP generation, is determined by clinical manifestations of the disease, inflammatory patterns, and various measures of airway remodeling.

In our study, increased systemic oxidative stress was associated with different variables related to asthma, including bronchial obstruction, airway geometry, and unspecific inflammatory biomarkers analyzed in the lungs and blood. Nevertheless, it was not associated with asthma severity score and symptom control, suggesting that higher blood oxidative stress in asthma is a feature of that disease per se. That is an unexpected finding since asthma is an inflammatory disease of the airways. However, many reports, including our previous research, have indicated that asthma is associated with higher inflammatory biomarkers in circulation [31]. In addition, this low-grade systemic response was related to the prothrombotic state [32] that was documented previously in that disease, as well as in the epidemiological studies [15–17]. Thus, local airway inflammation is likely associated with a variable degree of systemic response in asthma, activating blood leukocytes further and leading to higher HP production.

On the other hand, lower FEV1/VC values, reflecting more advanced airway obstruction and thicker bronchial walls in RB1, were independent determinants of higher susceptibility to HP generation. This observation suggests that objective indicators of more severe disease forms may be related, indeed, to the higher circulating oxidative stress capacity. At the same time, various laboratory variables in peripheral blood and

BAL predicted higher HP formation. Among them, the most important were those related to innate immunity and unspecific inflammation, such as BAL neutrophilia and blood monocyte count, as well as circulating CRP, fibrinogen, IL-17A, and H3cit. The latter is a novel biomarker of neutrophil activation and extracellular trap formation [28]. The relation of circulating oxidative stress to CRP in asthmatics is not surprising. A similar association was previously shown in other conditions, e.g., in healthy heavy workers [33]. Conversely, in controls, we did not record such a relationship. However, the six control subjects who had HP generation very high, above the fourth-quartile cutoff point in the asthmatics (Figure 2b), were characterized by the highest CRP values (all within the normal range), as compared to the remaining control subjects (2.65 [1.4–4.6] vs. 1 [1–1.7] mL/L, *p* = 0.006). Interestingly, those six controls have not reported any chronic inflammatory or acute disease or significant clinical symptoms and did not differ in the prevalence of internal comorbidities or other laboratory variables, except for CRP. Therefore, they were not excluded from the control group.

Interestingly, in our study, a higher oxidative stress response in asthma was determined by an increased total cholesterol level in serum. Since hypercholesterolemia and oxidative stress are well-known factors leading to endothelial dysfunction and atherosclerosis [9,14], we speculate that elevated HP generation in asthma might unfavorably affect the cardiovascular system. However, advanced observational and experimental studies are needed to verify this hypothesis.

On the other hand, the negative association of HP generation with blood eosinophilia in a multiple regression model suggests that oxidative stress in asthma is not related to the T2 response [1,12]. Thus, our report is consistent with several epidemiological studies that indicate that an increased risk of cardiovascular diseases in asthma occurs primarily in the late-onset asthma phenotype [34,35] or even in women with adult asthma [17], more frequently representing the non-T2 phenotype [36,37].

The lack of a relationship between HP formation and the histological characteristics of airway remodeling is another surprising finding that deserves a comment. Protein and lipid oxidation were previously linked to pro-inflammatory airway epithelial and endothelial cell modification [5]. Therefore, increased oxidative stress could lead to airway structural changes in the airways, such as the thickening of the RBM. However, the characteristics and role of structural alterations of the bronchial wall in asthma remain unknown. Previously, we have shown that RBM thickening did not depend on the asthma duration or lung function [12]. The current study likewise documents that it is also not linked to asthma severity. Notably, our reports align with former data, such as those published by Payne et al. [38]. They demonstrated that RBM thickening was present even in young children with asthma to a similar extent as seen in milder adult asthmatics and independently of asthma duration, severity, and lung function [38]. Therefore, it seems that changes in RBM occur early during the disease, e.g., as a response to ongoing airway inflammation, and do not progress further, due to the implemented anti-inflammatory treatment. Therefore, airway inflammation may promote ROS overproduction during a stable course of asthma but not further RBM thickening.

One of the strongest associations in our study is a clear inverse relationship between the potential for oxidative stress and RBC count. It indicates a substantial contribution of the antioxidant system of RBCs in balancing enhanced ROS generation. In the systemic circulation, the RBCs are continuously exposed to exogenous and endogenous sources of ROS, e.g., released from activated locally inflammatory cells. However, they possess an extensive antioxidant system, involving nonenzymatic antioxidants, for example, glutathione and enzymes, such as superoxide dismutase, catalase, glutathione peroxidase, and peroxiredoxin-2 [39]. Our data suggest that the antioxidative potential of RBC could diminish the lung-originated oxidative stress in asthma by counteracting ROS, at least to some extent. Therefore, it might be particularly insufficient in asthma patients with anemia.

Whether enhanced oxidative stress in asthma requires any therapeutic modifications is still unknown. However, some interventions have been suggested, including dietary changes, antioxidant vitamins, other antioxidant drugs and supplements, and even radon exposure, with varying results [40]. For example, nutritional studies suggest that asthmatic children with low dietary intake of vitamins C and E and other antioxidants have worse asthma symptoms; however, therapy with these vitamins was ineffective [40]. Another large epidemiological study prospectively documented the lower prevalence of asthma in those with higher α-tocopherol and Lp-PLA2 activity in peripheral blood at baseline [10]. At the same time, two longitudinal studies of dietary intake demonstrated inconsistent results with asthma risk. The Nurses' Health Study showed a higher asthma rate in those with a lower dietary intake of vitamin E [41], while in the E3N study, no such association was documented [42].

Finally, GINA [1] does not recommend any antioxidant supplements to decrease asthma-induced oxidative stress. Therefore, proper disease control, dietary intake of natural antioxidants in fruits and vegetables, and minimizing exposure to environmental pollution and tobacco smoke remain paramount.
