Plasma levels of oxysterols 7-ketocholesterol and cholestane-3β, 5α, 6β-triol in patients with allergic asthma

Abstract 
 Objective:
 The prevalence of allergic asthma is increasing on a global scale, reflecting changes in air pollution, climatic changes, and other environmental stimulants. In allergic conditions, oxidative stress occurs as a result of immune system activation. Oxidation of cholesterol leads to the formation of oxysterols. The main purpose of the study was to compare plasma levels of two oxysterols, namely 7-ketocholesterol (7-KC) and cholestane-3β, 5α, 6β-triol (C-triol), and a lipid peroxidation product, malondialdehyde (MDA) in allergic asthma patients with those of healthy controls, in order to provide information about the involvement of lipid peroxidation in allergic asthma. 
 Methods:
 Oxysterols were quantified by LC-MS/MS in plasma samples of 120 asthma patients (90 females + 30 males) and 120 healthy controls (matched by age and sex). Plasma MDA level was analyzed by a spectrophotometric method. 
 Results:
 Plasma 7-KC (39.45 ± 20.37 ng/mL) and C-triol (25.61 ± 10.13 ng/mL) levels in patients were significantly higher than in healthy subjects (17.84 ± 4.26 ng/mL and 10.00 ± 3.90 ng/mL, respectively) (P < 0.001). Plasma MDA levels were also higher in asthmatic patients (4.98 ± 1.77 nmol/mL) than in healthy controls (1.14 ± 0.31 nmol/mL) (P < 0.001). All data support that lipid peroxidation products are involved in allergic asthma. 
 Conclusion:
 Oxysterols were quantified for the first time in allergic asthma. Since the high plasma 7-KC and C-triol levels of allergic asthma patients correlate with high IgE levels, detection of these oxysterols by LC-MS/MS may be helpful in the clinical monitoring of allergic asthma. Current data may also lead to new approaches for the prevention, diagnosis, and treatment of the disease. Supplemental data for this article is available online at at.


Introduction
Allergic asthma is the most common of many asthma phenotypes, and is characterized by the involvement of various allergic diseases such as allergic rhinitis, eczema and food or drug allergies (1). Due to air pollution, climatic changes, and other environmental stimuli, the prevalence of allergic asthma is gradually increasing worldwide, and among middle-aged people and women in particular (2). The atopic phenotype is closely associated with allergies characterized by airway hypersensitivity, predominance of eosinophil and T lymphocyte type 2 (Th2) cells in the mucosa, circulation of specific immunoglobulin E (IgE), and positive skin prick tests for common allergens (3). In allergic conditions, activation of the immune system results in oxidative stress. Many observations show that oxidative stress plays an important role in the pathogenesis of asthma. Although it is difficult to measure reactive oxygen species (ROS) directly, the exhaled gases of asthmatic patients have been investigated, and increased levels of hydrogen peroxide and nitric oxide were reported (4)(5)(6)(7). Reactive oxygen species can be produced by several inflammatory cells involved in airway inflammation, and their production may be increased in asthma. Inflammatory cells involved in asthmatic airways have an extraordinary ability to produce ROS. Activated eosinophils, neutrophils, monocytes, and macrophages can produce superoxides via the membrane-associated NADPH-dependent complex (8,9). There is also robust evidence that an imbalance between oxidants and antioxidants in favor of oxidants is predictive of asthma severity and control (10,11). Lipid peroxidation is a form of oxidative stress which involves the oxidation of membrane lipids by enzymatic and/or non-enzymatic reactions (12,13). Lipid peroxidation is defined as an event that causes oxidation of polyunsaturated fatty acids (PUFA), usually with free radical-induced chain reactions, thereby altering the structure of membrane lipids and disrupting cell structure and function. Lipid peroxidation reduces membrane fluency and affects the biophysical properties of membranes (14). In addition, oxidation of cholesterol leads to the formation of a large number of oxidation products known as oxysterols. Oxysterols resulting from spontaneous or enzymatic oxidation of cholesterol are noninvasive potential biomarkers of oxidative stress in vivo (15).
No previous study has directly targeted oxysterols in allergic asthma and no randomized controlled trial has been performed to assess oxysterol levels by LC-MS/MS in patients with allergic asthma. The goal of this study is to determine the levels of two common oxysterols, 7-ketocholesterol (7-KC) and cholestan-3β,5α,6β-triol (C-triol), as well as malondialdehyde (MDA) as a by-product of lipid peroxidation, in plasma samples of allergic asthma patients and healthy individuals. In so doing, we provide information about the involvement of lipid peroxidation in allergic asthma, resulting in insights with the potential to aid in its diagnosis and treatment.

Subjects and sampling
This study was approved by the Clinical Research Ethics Committee of Istanbul University Faculty of Medicine (Date and Number: 22 December 2017, GO 2017/1421), and conducted with 120 patients (90 females + 30 males) diagnosed with allergic asthma (experimental group) and 120 healthy volunteers (control group). All procedures involving human subjects were performed in compliance with the Declaration of Helsinki of 1975, as received in 1983. The subjects were selected from among adult allergic asthma patients monitored by the Division of Immunology and Allergic Diseases of Internal Medicine Department at Istanbul Faculty of Medicine. Asthma severity diagnosed according to recent Global Initiative for Asthma (GINA) guideline (https://ginasthma.org/ w p -c o n t e n t / u p l o a d s / 2 0 2 1 / 0 5 / GINA-Main-Report-2021-V2-WMS.pdf). The level of asthma control was evaluated with the Asthma Control Test (ACT) (16). Detailed demographic and clinical features were obtained for all individuals, including: age, weight (or body mass index, BMI), tobacco and alcohol use, presence of comorbidities, an additional drug usage, presence of non-steroidal anti-inflammatory drug (NSAID)-exacerbated airway disease, and atopy. Complete blood cell count and serum total IgE (ImmunoCAP System, Phadia AB, Uppsala, Sweden) levels were recorded. Skin prick tests were performed with 12 common allergens (Allergopharma, Reinbek, Germany) to detect allergies. Atopy was defined as the presence of at least one positive result from skin prick tests with common inhalant allergens, including house dust mite (HDM), cat, dog, a mixture of molds and pollens. According to these results, patients who were allergic to house dust mite, pollens or both were included to the study. Patients with a chronic lung disease, chronic liver failure, chronic kidney disease, chronic rheumatological disease, chronic heart failure, malignancy, and those using systemic steroids and any immunosuppressive medications or those using tobacco and alcohol were excluded from the study. Healthy subjects were defined without respiratory or allergic diseases, diabetes mellitus, Alzheimer's disease, known chronic inflammatory conditions, metabolic, hepatic, cardiovascular disorders, or smoking and alcohol use. In addition, none of the patients or healthy subjects had taken any antioxidant supplements within four weeks before blood sampling.
Blood samples were taken from the experimental group and adult volunteers into two separate tubes, one containing 10 mL of ethylenediaminetetraacetic acid dipotassium (EDTA-K2) for plasma collection, and one gold-top vacuum gel-barrier tube to separate the serum. Samples were immediately centrifuged following collection, and separated parts were stored at −80 °C until use. Plasma samples were used for oxysterol and malondialdehyde analyses and serum for other biochemical parameters. All samples were transported on dry ice when needed.

Determination of malondialdehyde level in plasma
The thiobarbituric acid (TBA) method was used to assess malondialdehyde (MDA) levels in plasma (17,18). The method is based on the principle of spectrophotometric determination of the product resulting from the destruction of peroxidized lipids in the plasma, which react with TBA (TBARs). In this method, the plasma was mixed with an equal volume of assay mixture containing 0.375% thiobarbituric acid (TBA), 15% trichloroacetic acid (TCA), and 0.25 N HCl, and the mixture was then boiled in a water bath for 20 min. The tubes were centrifuged at 4.500 rpm for 15 min at RT. The absorbance of the pink supernatant was recorded at 532 nm against buffer blank on the UV-visible spectrophotometer. The lipid peroxide contents in plasma were determined by a standard curve prepared with varying concentrations of 1,1,3,3-tetraethoxypropane (TEP) between 0.312 and 10 nmol/mL.

Determination of oxysterols
Oxysterol analysis was performed by LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry) based on the previously reported method (19) carried out in a triple quadrapole system (Shimadzu 8040). Plasma 7-KC and C-triol were derived from N, N-dimethylglycine esters to increase ionization and cleavage of 7-KC and C-triol for mass determination of oxysterol species in human plasma. As internal standards, 3β, 5α, 6β-trihydroxycholastane D7 (Toronto Research Chemicals, Toronto, Canada) and 3β-hydroxy-5-cholesten-7-one D7 (Avanti Polar Lipids, Inc., Alabaster, AL, USA) were used for 7-KC and C-triol measurements. Eight-point calibrators (3.12-400 ng/mL) were prepared for measurement. Plasma quality control samples were prepared by raising the known amounts of 7-KC and C-triol standards to obtain endogenously 40/40 and 150/150 ng/ mL levels, respectively. Chromatographic separation was performed on a Symmetry C18 column (100 mm × 2.1 mm, 5 mm) using a linear water and acetonitrile gradient (pH 3; 1 mM ammonium formate) (Thermo Fisher Scientific, Waltham, MA, USA). A mass spectrometer was connected in series with a 2010 A single quadrupole detector containing interchangeable ESI. The system was controlled by a computer. The nitrogen supply for the MS detector was provided via aeration from a liquid nitrogen tank. 7-KC and C-triol levels were determined in 50 mL of plasma. Sample preparation consisted of three phases: protein precipitation, separation, and drying; the second phase was the derivatization phase; and the third step was included in the sampling by LC.

Statistical analysis
The conformity of the variables to normal distribution was examined using visual (histogram and detrended) plots and Kolmogorov Smirnov's normality test. Descriptive statistics of categorical variables are shown with frequencies while numerical variables are represented using mean-standard deviation or median (interquartile range). Pearson's or Fisher's Exact χ2 tests were used to analyze the differences in categorical variables between groups. The independent samples t-test or the Mann-Whitney U test was used for numerical comparison of the two groups, depending on the distribution structure. In order to measure the success of 7KC, C-triol and MDA in distinguishing between asthma and controls, receiver operator characteristic (ROC) analysis was performed, and AUC (area under curve), sensitivity and specificity scores were calculated. The ROC is a visual method for representing the tradeoff between the true-positive rate and false-positive rate of a test. In this way, it was aimed to find the best cutoff score for these three basic parameters in the study. While determining the best cutoff point, the point with the highest sum of sensitivity and specificity, therefore the point with the lowest misclassification rate was chosen as the optimal value. Relationships between oxysterols and other parameters were examined with either Pearson's correlation coefficient or Spearman's correlation coefficient, depending on the normality. Linear regression analysis was performed based on the backward variable selection method to determine the factors affecting 7-KC and C-triol levels. The IBM SPSS statistical package program version 22 (Armonk, NY: IBM Corp) was used for all statistical analyses. GraphPad Prism software (San Diego, CA, USA) was used for graphical analysis. Values of p < 0.05 were considered statistically significant.
The results of comparisons between plasma 7-KC, C-triol and MDA levels and major biochemical and clinical parameters of the control and patient groups, such as total cholesterol, total IgE, HDL, triglyceride, Tprot, Fe, and oxysterols, are summarized in Table 2.
Significant differences were found between the groups in terms of 7-KC, C-triol, MDA, Total IgE, high, low, and very low-density lipoproteins (HDL, LDL, and VLDL, respectively), triglyceride, ALT, Tprot, Tbil, Ggt, and Fe levels ( Table 2). However, plasma sterols of the patients were not associated with asthma severity such as mild, moderate or severe asthma or presence of NSAID hypersensitivity (p > 0.05) (Suppl. Figs. 1 and 2). In addition, when the relationship between inflammatory markers such as WBC, RBC, eosinophil count and CRP and plasma sterols of the patients was examined, a weak correlation was found only between MDA and RBC (r = 0.279, p < 0.01). Besides, there were no statistically significant differences between the presence of comorbid diseases including arterial hypertension, coronary artery disease, diabetes, hypothyroidism or hypercholesterolemia, and the use of additional drugs such as ACE-inhibitors, anti-platelet drugs, ARBs (angiotensin II receptor blockers), beta-blockers, statins, metformin or DPP-4 inhibitors and plasma sterols (p > 0.05).
The 7-KC and C-triol levels of the patients were significantly higher than those of the control group (p < 0.001 for each) (Figure 1). Patients' MDA levels (4.98 ± 1.77 nmol/mL) were also significantly higher (1.14 ± 0.31 nmol/mL) (p < 0.001) (Figure 2A). Similarly, total IgE ( Figure 2B) and lipoproteins were higher in patients. However, ALT, Tprot and Fe levels were significantly lower in patients than in healthy subjects ( Table 2). The ROC analysis was applied to determine cutoff values, which are the critical cutoff points for the 7-KC, C-triol, and MDA scores that are thought to be susceptible to asthma. As a result of the analysis, the cutoff values providing the best sensitivity and specificity values were determined. The AUC, sensitivity and specificity for determined cutoff values were also calculated and are presented in Table 3. The parameter with the best discriminating performance was achieved by MDA score. Diagnostic success for MDA scores of 2.155 and higher was as follows; area under the curve: AUC = 0.99 (0.984-1.00), sensitivity= 0.97, and specificity= 0.99. Additionally, the ability to identify controls by 7-KC and C-triol were quite high (specificity: 1.00 for both of them). When the best cutoff point for the 7-KC was taken as 22.65 ng/mL, the success rate of the diagnosis was obtained as AUC = 0.91 (0.863-0.949), with a sensitivity= 0.83, and specificity= 1.00. Similarly, when the best cutoff point for C-triol was taken as 17.35, the success rate of asthma recognition, in which the C-triol value is 17.35 or more, was obtained as; AUC = 0.90 (0.853-0.944), sensitivity= 0.83, and specificity= 1.00.
The distributions of some parameters most strikingly related to oxysterols in asthma patients and all individuals are given in Figure 3.
A multivariable stepwise regression analysis was used to predict 7-KC and C-triol levels in asthma patients. While the biochemical parameters assessed in relation to 7-KC were HDL, ALT, AST, VLDL, Tprot, Tbil, and Ggt, the parameters included in the C-triol model were total IgE, HDL, total cholesterol, VLDL, triglyceride, AST, Tprot, Uric, Tbil, Ggt, and Fe (Table 4).
In examining the results, we found that MDA, AST, and total cholesterol affected 7-KC level. The parameters with a significant effect on C-triol were MDA, Tbil, and total IgE (p < 0.05). A unit increase in MDA caused an average increase of 3.66 units on 7-KC. A unit increase in AST resulted in a 0.59-unit increase in 7-KC. A unit increase in total cholesterol level had a reducing effect of −0.08 units in 7-KC.  For C-triol, a unit increase in MDA caused an average 2.84-unit increase in C-triol. While a unit increase in total IgE level increased the average level of C-triol 0.006 times, a unit increase in Tbil decreased the average level of C-triol 0.48 times. However, when the estimation success of the models was evaluated, we find that MDA, AST, and total cholesterol explain 28% of the variability in 7-KC, while MDA, Tbil, and total IgE together explain 47% of the variability in C-triol.

Discussion
Increased oxidative stress has been reported in various studies on asthmatic patients (20). It has been previously established that high levels of oxidative stress accompany allergic asthma as a result of increased pulmonary inflammation, ROS production, and decreased antioxidant defense (21)(22)(23). Macrophages, eosinophils and neutrophils produce various types of ROS that damage cellular molecules such as DNA, proteins, carbohydrates and lipids (10,24). One of the consequences of oxidative stress is lipid peroxidation, which involves the oxidation of fatty acids in vivo (25). Lipid peroxidation reduces membrane fluidity and affects the biophysical properties of membranes. MDA, formed during lipid peroxidation of PUFAs and arachidonic acid metabolism, is a chemically active molecule that can easily diffuse into surrounding cells and tissues, resulting in harmful effects, especially on proteins (26). It is the most commonly used biomarker of oxidative stress in many health conditions, such as cancer, psychiatric disorders, chronic obstructive pulmonary disease, asthma and cardiovascular diseases (27). Allergic asthma patients are reported to have exceptionally high rates of lipid peroxidation and protein oxidation even under controlled conditions (11).
In the study of Abboud et al. in 2021, the changes in serum and saliva oxidative markers were investigated for the first time in patients with bronchial asthma (28). According to their findings; all asthma patients also showed a significant increase in serum levels of total glutathione (tGSH) and malondialdehyde (MDA) compared to their serum concentrations in the control group. Similarly, in this study, MDA levels of patients were also found to be significantly higher than those of the control group. Also, in their work; ROC plot expresses the sensitivity versus 1-specificity, was used to test the efficiency of serum and salivary oxidative markers to discriminate asthma disease. They obtained a sensitivity of approximately 0.57 versus a specificity of 0.45 for MDA. Moreover, only 48 adults and 55 children constituted their asthma patient group. Although it does not allow some advanced multivariable analyzes, in our study that we carried out with our sample consisting of 120 control and 120 asthma patients, the best cutoff value for MDA was 2.555, and the diagnostic performances of this point were obtained as sensitivity = 0.97, specificity = 0.99, and AUC = 0.99.  Oxysterols are defined as the final products of cholesterol oxidation. Generally, they are present in very low concentrations in mammalian tissues. These metabolites play key roles in health and disease, particularly in the development and regulation of immune cell responses (29). The roles of specific oxysterols as mediators, and their use as appropriate markers for diagnosing certain diseases, such as diabetes mellitus, Alzheimer's disease, multiple sclerosis, osteoporosis, lung cancer, breast cancer, and infertility have been reported in various studies (30)(31)(32)(33). Measuring 7-KC in combination with C-triol levels may be helpful in the prediction of oxidative stress in several diseases. For example, 7-KC and C-triol have been found to increase in diabetes mellitus (34) and neurodegenerative diseases (35). However, there has been no  clinical study on oxysterols in allergic asthma to date, although it is known that they are directly related to oxidation, inflammation, and immunity (36). This study is the first report on plasma levels of two oxysterols, 7-KC and C-triol, in allergic asthma patients. ROC analysis for 7-KC and C-triol was also performed for the first time in this study. According to our results, the plasma levels of 7KC and C-triol in asthmatic patients were significantly higher than those of healthy subjects. In addition, multivariable regression analysis demonstrated that MDA affects these oxysterols. The parameters we found to have a significant effect on C-triol were MDA, Tbil, and total IgE, while the factors affecting 7-KC were MDA, AST, and total cholesterol. These findings are evidence of the close interaction between lipid metabolism and allergic asthma, mediated by oxysterols and lipid peroxidation end products, which has been previously documented (37). Low serum levels of AST and ALT are reportedly associated, to a significant degree, with asthma severity (38). Here, only ALT levels in patients were lower than in controls, presumably due to the mild severity of the experimental group's condition. Liver X receptors (LXRs) play a prominent role in lipid metabolism and play anti-inflammatory roles in acute lung injury (39). LXRs are also known to be involved in the control of IgE secretion (40). A study in mice has shown that inhibition of LXR over-activation can reduce allergic airway inflammation and airway hyper-responsiveness (41). The results of these studies indicate that the development of cell-and receptor-selective LXR agonists that more specifically modulate B-cell activation could contribute to the development of new protocols for anti-allergic therapy. Therefore, inhibition of LXRs may be a potential treatment for allergic asthma.
Oxysterols are dangerous because of their cytotoxic effects, such as the ability to induce oxidative and inflammatory activities, cell death, and a lysosomal storage disorder characterized by the excess accumulation of phospholipids in tissues (phospholipidosis) (42). Elevated levels of oxysterols may also prove to be important because of the associated production of sulfated derivatives affecting human health and disease (43). We know that lipid peroxidation plays a key role in allergic asthma, but more detailed studies are needed.
Our data on iron corroborates a previous report that found a close relationship between iron deficiency and asthma (44) since Fe levels were significantly lower in patients in our study.

Conclusions
Plasma 7-ketocholesterol and cholestane-3β, 5α, 6β-triol levels were measured for the first time in allergic asthma patients using a highly sensitive and specific LC-MS/MS method. The systemic increase in oxysterol levels appears to be a reliable biomarker of allergic asthma that may be useful in routine clinical practice, and a target for treatment. However more comprehensive studies on other types of oxysterols, their common sources, their interaction partners, and the molecules they damage, are needed in order to get more detailed information about the role of lipid peroxidation and cholesterol oxidation in allergic asthma. This study provides precise and reliable quantitative data that draws attention to lipid oxidation in the pathogenesis of asthma.