ABSTRACT. Keywords: Antioxidants, obstructive sleep apnea syndrome, oxidative stress, vascular endothelial dysfunction

Transcription

1 Vascular Endothelial Dysfunction Caused by Oxidative Stress is a Predictor Factor for Complications of Obstructive Sleep Apnea Syndrome K Kilic 1, B Aktan 2, MS Sakat 2, A Kızıltunç 3, O Yoruk 2 ABSTRACT Objective: Obstructive sleep apnea syndrome (OSAS) is characterized by repetitive episodes of upper airway obstruction that occur during sleep which leads to cardiovascular, neurovascular and metabolic diseases. The mechanisms of complications in these patients are unclear. In this report, we aimed to determine the cause of oxidative imbalance in patients with OSAS and to evaluate the vascular endothelial dysfunction as a suspect for complications of the disease. Materials and Methods: Fifty patients diagnosed as moderate or severe OSAS (AHI > 15) by polysomnography and 50 healthy volunteers are included in the study. By biochemical analysis of blood samples, serum malonyldialdehyde (MDA) levels measured to determine the oxidative stress, serum antioxidant enzymes glutathione peroxidase (GSH-Px), paraoxonase (PON) and arylesterase (ARE) activities measured to determine the antioxidant status and serum nitric oxide (NO) levels measured to show the vascular endothelial dysfunction. For statistically analysis, SPSS 15.0 is used with Mann-Whitney test to compare the results of patients and control group and Pearson Correlation Test to calculate the correlations. Results: Serum MDA levels were significantly higher in patients with OSAS than control subjects (p < 0.05). The GSH-Px, PON, NO levels were significantly lower in OSAS group than control subjects (p < 0.05). The serum ARE activity was lower in OSAS group but this decrease was not statistically significant. There was a negative correlation between serum MDA levels and GSH-Px activity and a positive correlation between serum PON activities and ARE activities. Conclusion: Obstructive sleep apnea syndrome is strongly associated with oxidative stress and abnormal lipid peroxidation. The oxidative stress depends on not only the overproducing of reactive oxygen species but also the deficiency of antioxidant defense systems. Both oxidative imbalance and vascular endothelial dysfunction lead to early development of atherosclerosis which may cause the health threatening complications of OSAS. Keywords: Antioxidants, obstructive sleep apnea syndrome, oxidative stress, vascular endothelial dysfunction 1 Department of Ear, Nose and Throat, Palandoken State Hospital, Erzurum, Turkey, 2 Department of Ear, Nose and Throat and 3 Department of Biochemistry, Faculty of Medicine, Ataturk University, Erzurum, Turkey. Correspondence: Dr K Kilic, Department of Ear, Nose and Throat, Palandoken State Hospital, Erzurum, Turkey. Telephone: korhankilic@gmail.com West Indian Med J 2016; DOI: /wimj

2 OSAS and Oxidative Stress INTRODUCTION Obstructive sleep apnea syndrome (OSAS) is characterized by repetitive episodes of upper airway obstruction that occur during sleep, usually associated with a reduction in blood oxygen saturation (1). These obstruction episodes cause apnea which is defined as cessation of airflow for at least 10 seconds, or hypopnea which is defined as substantial reduction in airflow (50%) followed by arousal. Epidemiologic studies estimate that the condition affects 2% of women and 4% of men in middle-aged adults (2). Obstructive sleep apnea syndrome is significantly associated with frequent arousals during the night that ultimately lead to sleep fragmentation and daytime sleepiness. Besides reducing the quality of life, this situation also causes significant personal and social problems through affecting work performance and driving. Polysomnography is the gold standard for the diagnosis of the syndrome and the severity of syndrome is measured as apnea-hypopnea index (AHI). Apnea-hypopnea index is defined as the number of apneas and hypopneas per hour of sleep. Apnea-hypopnea index > 5 is associated with OSAS. Apnea-hypopnea index of five to 15 indicates mild, of 15 to 30 indicates moderate and of greater than 30 indicates severe OSAS (3). In OSAS, there is a respiratory effort against total or partial obstructed airway and this effort ends by arousal. Repetitive episodes of apnea/hypopnea and hypoxia/reoxygenation cause increased sympathetic nerve activity, oxidative stress, swings in intrathorasic pressure, increased surges in arterial blood pressure, hypoxia and hypercapnia (4). All these situations may cause cardiovascular complications varying from hypertension to sudden death and serious neurovascular complications such as cerebrovascular stroke. The mechanisms of cardiovascular and cerebrovascular complications in these patients are unclear. One of the underlying mechanisms that may be involved in the development of cardiovascular and 2

3 Kilic et al neurovascular complications in sleep apnea syndrome is the formation of hypoxia-related free radicals and increased oxidative stress due to the intermittent hypoxia (5). Changes in oxidant/antioxidant status causing vascular endothelial dysfunction followed by atherosclerosis seem to be an important reason for complications of disease. In this study, we aimed to present the vascular endothelial dysfunction in OSAS patients as a result of oxidative stress. To determine the oxidative stress, serum level of malonyldialdehyde (MDA) which is a product of lipid peroxidation, is calculated. To evaluate the changes in antioxidant defense system, activities of glutathione peroxidase (GSH-Px) which is an antioxidant enzyme that protects membrane lipids from oxidation, paraoxonase (PON) and arylesterase (ARE) which are antioxidant enzymes that protect low density lipoprotein (LDL) from peroxidation to prevent atherosclerosis, are calculated. To evaluate the vascular endothelial dysfunction which is the first step of atherosclerosis and is thought to be the predictor of complications of OSAS, serum level of nitric oxide (NO) is calculated. MATERIAL-METHOD Fifty patients (30 male, 20 female; age 50.4 ± 4.3 years) who were diagnosed as moderate or severe OSAS (AHI > 15) by an over-night polysomnography, performed at Ataturk University, Faculty of Medicine, Department of Chest Disease and 50 healthy volunteers (24 male, 26 female; age 55.4 ± 6.3 years) are included to the study. Patients who were under 18 or over 65 years of age, had a previous surgical operation for snoring, had chronic obstructive respiratory disease were excluded. 10 ml peripheral venous blood samples were obtained between 08:00 10:00 am after polysomnography. Blood was centrifuged at 3

4 OSAS and Oxidative Stress 2000 revolutions per minute (rpm) for 5 minutes and serum was immediately separated in aliquots and stored at -80 C until analysis. Biochemical studies All biochemical studies were performed at Ataturk University, Faculty of Medicine, Department of Biochemistry. The descriptions of biochemical analysis are as follows: MDA Assay: Serum MDA assay was determined by the thiobarbituric acid (TBA) method described by Ohkawa and Yagi (6). The method was based on spectrophotometrically measurement of the pink colored product of reaction of MDA and TBA at 532 nm. The results are given as nmol/ml. PON Assay: The measurement of serum PON activity was performed according to the method described by Eckerson et al (7). The method was based on the spectrophotometrically measurement of 4-nitrophenol, the product of enzymatic hydrolization of paraoxon by paraoxonase, at 37 C and 512 nm. One unit of paraoxonase activity is equal to one nmol p-nitrophenol hydrolyzed per minute per ml of serum. ARE Assay: Serum ARE activity was measured spectrophotomecrically in a reaction that phenyl acetate was used as substrate. One unit of arylesterase activity is equal to one micromole phenlyacetate hydrolyzed per minute per ml of serum. GSH-Px Assay: Glutathione peroxidase (GSH-Px) activity was measured according to the modification of the method of Paglia and Valentine (8). Glutathione peroxidase catalyses the oxidation of glutathione in the reaction of reduction of hydrogen peroxide to water. The oxidized glutathione is reducted to glutathione by glutathione reductase enzyme in the 4

5 Kilic et al presence of NADPH. The method based on the spectrophotometrically measurement of the decrease in absorbance of NADPH at 340 nm. GSH-Px activity was given as U/L. NO Assay: Since most of the NO in human body is oxidized to nitrite (NO2-) and nitrate (NO3-), the concentrations of these anions have been used as a quantitative measure of NO production. After the conversion of NO3- to NO2-, the spectrophotometric measurement of NO2- is accomplished by using the Griess Reaction. This assay determines nitric oxide based on the enzymatic conversion of nitrate to nitrite by nitrate reductase. The reaction is followed by a colorimetric detection of nitrite as an azo dye product of the Griess reaction. The Griess reaction is based on two-step diazotization reaction in which acidified NO2- produces a nitrosating agent which reacts with sulfanilic acid to produce the diazonium ion. This ion is then coupled to N-(1-naphthyl) ethylenediamine to form the chromophoricazo-derivative which absorbs light at 540 nm (9). Statistical analysis For statistically analysis, SPSS 15.0 is used. The results are given as mean ± standard derivation and median ± standard error. Mann-Whitney test is used to compare the results of patients and controls and Pearson test is used for correlations, p-value less than 0.05 is accepted as statistically significant. RESULTS The biochemical results are given in Table 1. As seen from the table, serum MDA levels were significantly higher in OSAS patients than control subjects (p < 0.05). The activity of GSH- Px and PON and the level of NO were significantly lower in OSAS group than control 5

6 OSAS and Oxidative Stress subjects (p < 0.05). The activity of ARE was lower in OSAS group but this decrease was not statistically significant. According to Pearson Correlation test, there was a negative correlation between serum MDA levels and GSH-Px activity and a positive correlation between activities of PON and ARE. Table 1: Biochemical results; mean and median values and p-level of patient and control groups. Patients Control Mean ± SD Median ± SE Mean ± SD Median ± SE p MDA 16.6 ± ± ± ± * NO 83.2 ± ± ± ± * PON 87.4 ± ± ± ± * GPx 9.3 ± ± ± ± * ARE 12.1 ± ± ± ± MDA; malonyldialdehyde, NO; nitric oxide, PON; paraoxonase, ARE; arylesterase activities GPx; glutathione peroxidase, *: Statistically significant. DISCUSSION A free radical is defined as any species that contain one or more unpaired electrons in the outer orbit (5). Reactive oxygen species (ROS) is a new term used to include not only oxygen radicals but also some derivatives of O2 that do not contain unpaired electrons. Reactive oxygen species are products of normal oxygen metabolism that are generated during normal cellular respiration and are useful for metabolic processes. But the over-production of these radicals may be harmful for human body. Free radicals are highly chemically reactive molecules that react with nucleic acids, lipids and proteins, thereby hindering cellular metabolism resulting in cell injury. 6

7 Kilic et al The human body has several mechanisms to counteract the damage of ROS. One important mechanism includes antioxidant enzymes, such as GSH-Px, superoxide dismutase (SOD) and catalase (CAT) which decrease the concentration of ROS. The balance between production of free radicals and the antioxidant defense system is called Oxidative Balance and is important for health. When ROS generation exceeds the capacity of cellular antioxidant mechanisms to eliminate them, oxidative stress ensues (5). Oxidative stress is an imbalance between production of reactive oxygen species and the antioxidant defense system. Oxidative stress is associated with a large number of diseases. Cardiovascular diseases like myocardial infarct, neurologic diseases, asthma, diabetes mellitus, and rheumatoid arthritis are some examples of diseases associated with oxidative stress. In these diseases, oxidative stress plays a role in the pathogenesis of the disease or in the development of complications. In the present study, we aimed to investigate the effect of oxidative stress in OSAS. Obstructive sleep apnea syndrome is characterized by repetitive episodes of upper airway obstruction, increased respiratory effort against this obstructed airway and frequent awakening periods. Hypoxic asphyxia and respiratory effort against obstructed airway causes swings in intrapleural pressure, increase in intrathorasic negative pressure, hypoksemia, hyperkapnia and acidosis. Also, this apnea and arousals cause activation of autonomic nerve system. Obstructive sleep apnea syndrome leads to cardiovascular, neurovascular and metabolic diseases and decreases the quality of life. Although, the most important complications of the syndrome are about cardiovascular and neurovascular system, all systems can be affected. Saylam et al showed that, 40% of 328 patients with OSAS have an accompanying disease (10). Eight-teen per cent of these patients have hypertension, 7% have coronary artery diseases, angina or myocardial infarcts, 2.4% have diabetes mellitus, 4.5% 7

8 OSAS and Oxidative Stress have asthma or chronic obstructive lung disease, 2% have physichiatric problems and 1% has epilepsy. The underlying mechanism of these complications is not fully understood. Recent studies showed that OSAS is associated with oxidative stress. Some studies focussed on the overproduction of reactive oxygen species in patient with OSAS. Lavie et al studied MDA in OSAS patients and found MDA was significantly higher in patient with OSAS than control subjects. Lavie also mentioned that c-pap treatment may normalize serum MDA levels (11). Similarly, Barcelo et al found abnormal lipid peroxidation in patients with OSAS (12). Yamauchi et al studied urinary 8-OHdG extraction as a marker of oxidative stress and found urinary 8-OHdG extraction in OSAS patients higher than control subjects. Also, they found positive correlation between urinary 8-OHdG extractions and severity of OSAS (13). Christou et al used D-ROM as a marker of oxidative stress and found D-ROM levels higher in patients with OSAS (14). In other studies about OSAS and oxidative stress, Dyugoskava et al showed overproduction of reactive oxygen species from leukocytes and Schulz et al showed overproduction of superoxide from PNL in patients with OSAS (15, 16). In the present study, we used MDA as a marker of oxidative stress and we found MDA significantly higher in patients with OSAS than control subjects. This result showed that, OSAS is associated with high levels of lipid peroxidation which indicates oxidative stress. The antioxidant defense system counteracts the harmful effects of reactive oxidative species. In oxidative stress situation, there may also be a deficiency in antioxidant systems. Some other studies focussed on the deficiency in antioxidant systems in patient with OSAS. Barcelo et al studied the total antioxidant capacity of patients with OSAS and found that total antioxidant capacity was lower in patients than control subjects (17). Christou et al studied 17 patients with OSAS and showed that total antioxidant capacity is significantly 8

9 Kilic et al lower in severe OSAS (18). In the present study, we investigate the activity of glutathione peroxidase in patients with OSAS to determine the changes in the antioxidant system. Glutathione peroxidase is an antioxidant enzyme that both eliminates hydrogen peroxide and protects membrane lipids from peroxidation. In our results, the activity of glutathione peroxidase in patients with OSAS was significantly lower than control subjects. These results showed the deficiency in antioxidant status of patients with OSAS. Both the overproduction of reactive oxygen species and deficiency in antioxidant system increase the effect of oxidative stress. We also found a negative correlation between serum MDA levels and serum GSH-Px activity. While GSH-Px levels are decreased, the membrane lipids become unprotected and the increase of lipid peroxidation causes increase in MDA levels. According to the present study and the studies mentioned above, it is clear that OSAS is associated with oxidative stress. But, how does oxidative stress cause complications of OSAS? Researchers focussed on vascular endothelial dysfunction and atherosclerosis about this subject. Vascular endothelial dysfunction, the first pathologic sign of atherosclerosis, is defined as deficiency or loss of endothelial dependent vasodilatation. There are many studies about OSAS and vascular endothelial dysfunction. Ip et al evaluated the flow mediated dilation (FMD) of brachial artery in 28 men with OSAS and 12 men without OSAS and Grebe et al studied the FMD of brachial artery of 10 patients with OSAS and 10 healthy subjects (19, 20). They both found that subjects with OSAS have lower FMD compared to subjects without OSAS. These findings demonstrated that men with severe or moderate OSAS have endothelial dysfunction and treatment of OSAS could reverse the dysfunction. Grebe et al also found that endothelial dysfunction in patients with OSAS is linked to oxidative stress (20). Similarly; Carlson et al studied forearm blood flow and vascular resistance of 16 patients with OSAS after intraarterial infusion of asetilcoline and showed that the endothelial dependent vasodilatation of patients with OSAS was reduced 9

10 OSAS and Oxidative Stress independently of hypertension (21). These studies showed vascular endothelial dysfunction in OSAS but they did not reveal the cause of this dysfunction. The most important mediator that acts for vasodilatation is NO which is also a member of reactive oxygen species family. Vascular endothelial cells produce both NO and superoxide. Nitric oxide reacts with superoxide at physiological ph to yield a nonradical product, peroxynitrite. Interaction of superoxide and NO might represent a physiological regulatory process affecting vascular muscle tone. Thus, it has been suggested that vascular endothelial cells might produce both superoxide and NO as "antagonistic agents", to give fine control of vascular tone (5). In oxidative stress situation, the production of superoxide increases. Increased levels of superoxide remove more NO, and this interaction results in loss of vasodilatation. Schulz et al studied the serum NO level of 21 patients with OSAS and found NO levels reduced in patients with OSAS and can be increased by treatment (22). Similar results are found from other studies (23, 24). In the present study, we studied the serum NO levels of patients with OSAS and found it significantly lower than control subjects. The cause of vascular endothelial dysfunction in OSAS is provided as the decrease of NO levels because of oxidative stress. As vascular endothelial dysfunction is the first step of atherosclerosis, another step is the oxidation of low-density lipoprotein (LDL). Low-density lipoprotein, when modified by oxidation, is a major cause of injury to endothelial cells and underlying smooth muscle cells (5). In oxidative stress not only the membrane lipids, but also the plasma lipids are susceptible to peroxidation. There are protective mechanisms that prevent LDL from oxidation such as paraoxonase-1 (PON-1). Paraoxonase-1 is a calcium-dependent esterase which is physically associated with high-density lipoproteins (HDL) and has been shown to protect both LDL and HDL against lipid peroxidation and, thereby, is postulated to at least 10

11 Kilic et al partially protect against atherosclerosis (11). Shih et al studied mice which has deficiency of PON-1 and found these mice develop significantly larger atherosclerotic lesions. These data strongly support the notion that PON-1 plays an important role in preventing atherosclerosis (25). Other studies showed the LDL of patients with OSAS is more susceptible to oxidation (12). Paraoxonase-1 has both ARE and PON activity. In the present study, we studied serum ARE and PON activity of patients with OSAS and found PON activity was significantly lower in patients with OSAS than control subjects and ARE activity was lower in OSAS group but this decrease was not statistically significant. The deficiency in these protective enzymes against atherosclerosis may explain the early development of atherosclerosis in OSAS patients. Atherosclerosis may be the suspect for complications of OSAS. Additionally, we found positive correlation between serum PON activity and serum ARE activity. This correlation suggests that, the enzyme level decrease in OSAS because of over-use. CONCLUSION Obstructive sleep apnea syndrome is strongly associated with oxidative stress and abnormal lipid peroxidation. The oxidative stress depends on not only the overproducing of reactive oxygen species but also the deficiency of antioxidant defense systems. Oxidative stress cause vascular endothelial dysfunction in these patients by decreasing serum NO levels. The activity of the protective mechanisms against atherosclerosis, such as PON-1, reduces in OSAS. Both vascular endothelial dysfunction and deficiencies of protective mechanisms lead to early development of atherosclerosis. The health threatening complications of OSAS may depend on atherosclerosis caused by vascular endothelial dysfunction as a result of oxidative stress. As the number of study and control group is small and there may be other factors that affect the status of atherosclerosis, further studies are needed to evaluate the accurate effect of atherosclerosis in OSAS. 11

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