Serum homocysteine levels and cardiovascular morbidity in obstructive sleep apnea syndrome

Respiratory Medicine (26) 1, 536 541 Serum homocysteine levels and cardiovascular morbidity in obstructive sleep apnea syndrome Oguz Kokturk, Tansu Ulukavak Ciftci, Elif Mollarecep, Bulent Ciftci Faculty of Medicine, Department of Pulmonary Disease, Gazi University, Besevler, Ankara, Turkey Received 7 March 25; accepted 3 May 25 KEYWORDS Sleep apnea; Homocysteine; Cardiovascular morbidity Summary Background: Obstructive sleep apnea syndrome (OSAS) is associated with cardiovascular morbidity and mortality. Elevated levels of serum homocysteine are also associated with cardiovascular morbidity and mortality. We aimed to investigate serum homocysteine levels and conventional cardiovascular risk factors (cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglycerides) in OSAS patients with and without cardiovascular diseases (CVD). Methods and Results: Levels of homocysteine, cholesterol, LDL, HDL and triglycerides were measured in 114 obese, male participants after overnight fasting. The presence of OSAS was determined by standard overnight polysomnography. The cases included OSAS patients (apnea hypopnea index: AHIX5) with CVD (OSAS+CVD group) (n:25) and without CVD (OSAS CVD group) (n:47). Control group was patients without OSAS (AHIo5) with CVD (CVD group) (n:42). The serum homocysteine levels were significant. & 25 Elsevier Ltd. All rights reserved. Introduction Obstructive sleep apnea syndrome (OSAS), characterized by repeated episodes of upper airways obstruction during sleep leading to significant hypoxemia, is a very prevalent disorder particularly among middle-aged, obese men, although its existence in women is increasingly recognized. 1 Epidemiological Corresponding author. Tel. +9 312 226119; fax: +9 312 212919. E-mail address: tansu.ciftci@gazi.edu.tr (T.U. Ciftci). studies estimate that 2 5% of the population meets the minimal diagnostic criteria (snoring, witnessed apnea and excessive daytime sleepiness), and two community-based studies have found that about 2% of women and 4% of men are affected by OSAS. 2 The syndrome is associated with cardiovascular morbidity, such as systemic hypertension, coronary heart disease, atherosclerosis, and stroke. Studies of random samples obtained from the general population suggest that the existence of OSAS constitute a significant risk for cardiovascular diseases (CVD) independently of other known risk factors. 3 6 954-6111/$ - see front matter & 25 Elsevier Ltd. All rights reserved. doi:1.116/j.rmed.25.5.25

Serum homocysteine levels and cardiovascular morbidity in OSAS 537 Neurohumoral and hemodynamic responses to untreated sleep apnea are likely mechanisms that produce functional and structural changes within the cardiovascular system. Obesity, higher blood pressure, and advancing age, which are common characteristics of patients with OSAS, contribute to the risk for CVD. Recent studies indicate that OSAS is associated with or aggravates other risk factors for CVD 7 and one of these risk factors is hyperhomocysteinemia. Homocysteine is an intermediate amino acid in methionine cysteine metabolism, as it represents a branching point at which it can be remethylated to methionine or converted to cysteine. 8 It was described by McCully, 9 in infants with inborn errors of metabolism, as an atherogenic compound that accelerates atherosclerosis. Many clinical and epidemiological studies confirmed this observation that mild elevation in total plasma homocysteine confers an increased risk for peripheral arterial occlusive disease, coronary artery disease, and cerebrovascular disease similar to other conventional risk factors such as hyperlipidemia or smoking. 1,11 Pertaining to the associations between OSAS and cardiovascular morbidity and between homocysteinemia and cardiovascular morbidity, we investigated the serum levels of homocysteine and conventional cardiovascular risk factors such as total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), very low-density of lipoprotein (VLDL) and triglycerides in patients diagnosed to have OSAS with and without CVD. We compared them to control subjects with CVD without OSAS. Materials and methods Patients Age and Body Mass Index (BMI) matched male subjects were included in order to eliminate the factors affecting CVD such as age, sex and obesity in the present study. All patients were obese with BMI427 kg/m 2. Subjects were recruited from those who were referred for suspected sleep apnea to our Sleep Disorders Center, Gazi University, Faculty of Medicine. The study was approved by the institutional Ethics Committee, and study was performed in accordance with the guidelines of declaration of Helsinki. The control subjects and OSAS patients were examined with polysomnography (PSG) and classified according to data of the apnea hypopnea index (AHI). BMI was calculated as weight in kilograms divided by the square of the height in meters measured by a scale. Patients with hypertension (HT) and/or ischemic heart disease (IHD) were enrolled as patients with CVD. None of the patients has used folat or other food supplements like it, but data about lipid lowering medications of patients were not available. Before enrollment, all subjects gave written informed consent. Sleep study Overnight PSG was performed in all patients by a computerized system (Somnostar alpha; Sensormedics, USA) and included the following variables: electrooculogram (two channels), electroencephalogram (four channels), electromyogram of submental muscles (two channels), electromyogram of the anterior tibialis muscle of both legs (two channels), and electrocardiogram and airflow (with an oro-nasal thermistor). Chest and abdominal efforts (two channels) were recorded using inductive plethysmography, arterial oxyhemoglobin saturation (SaO 2 : 1 channel) by pulse oximetry with a finger probe. The recordings were conducted at a paper speed of 1 mm/s, and sleep stage were scored according to the standard criteria of Rechtschaffen and Kales. 12 Arousals were scored according to accepted definitions. 13 Apneas were defined as complete cessation of airflow X1 s. Hypopneas were defined as reduction of 45% in one of three respiratory signals, airflow signal or either respiratory or abdominal signals of respiratory inductance plethysmography, or with a fall of X3% in oxygen saturation or an arousal. The AHI was defined as the number of apneas and hypopneas per hour of sleep. Patients with AHIo5 were included in control group. Patients with AHIX5 were considered as OSAS. Patients were divided into three groups: 1. OSAS+CVD group: patients with OSAS and CVD (HT and/or IHD) 2. OSAS CVD group: patients with OSAS without CVD 3. CVD group: patients without OSAS with CVD (HT and/or IHD). Circulating parameters assay All subjects had fasting blood samples taken between 7: AM and 8: AM. Blood samples were immediately sent to the hospital laboratory. Cholesterol and triglyceride concentrations in serum and lipoprotein fractions were determined enzymatically using standard laboratory procedures. Serum homocysteine levels were measured using Chromsystems HPLC with Agilent 11 series

538 fluorescence detector. Normal range is considered as 5 14 mmol/l for homocysteine levels. Statistical analysis Means and standard errors of measurement (SEM) were determined for continuous variables and percentage for categorical variables. The significance of differences between three groups was analyzed with the Kruskal Wallis variance analysis. Differences between two groups were analyzed with the Mann Whitney U test. We applied a Bonferroni correction for multiple comparisons. Categorical data were analyzed by x 2 with Fisher Exact Probability test. The correlation was analyzed with Spearman correlation coefficient. To assess the relative strength of association of OSAS as well as possible confounding factors with homocysteine, we employed a multiple regression analysis to the patients with OSAS as a single group. In this analysis, we used serum levels of homocysteine as dependent variable and evaluated the order of inclusion in the model of the following independent variables: AHI, BMI, total cholesterol, HDL, LDL, and VLDL. All statistical analyses were carried out using statistical software (SPSS, version 11. for Windows; SPSS Inc; Chicago, IL). Differences were considered significant at Po:5. Results Newly diagnosed obese (BMI427 kg/m 2 ) men with OSAS and age- and BMI matched male control subjects with HT and/or IHD were enrolled in present study. O. Kokturk et al. After 157 obese, male subjects underwent PSG, 72 were considered to have OSAS, which included 25 patients with CVD and 47 patients without CVD. Eighty-five subjects were considered not to have OSAS and of these, 42 patients, who were with CVD, were enrolled as control subjects. The 114 patients were subdivided into three groups: 1. OSAS+CVD group: patients with OSAS and CVD (HT and/or IHD) (n ¼ 25) 2. OSAS CVD group: patients with OSAS without CVD (n ¼ 47) 3. CVD group: patients without OSAS with CVD (HT and/or IHD) (n ¼ 42). The demographic and clinical data of the three groups are presented in Table 1. The serum homocysteine levels were significantly elevated in OSAS+CVD group more than both the other two groups (Po:1). When differences between two groups were analyzed, we showed that serum homocysteine levels significantly increased more in the OSAS CVD group compared to the CVD group (Table 1, Fig. 1). Hyperhomocysteinemia (414 mmol/l) was found in 17 patients (68%) of OSAS+CVD group, in 22 patients (46.8%) of OSAS CVD group and in seven (16.7%) of CVD group. Therefore, hyperhomocysteinemia was found highest in OSAS+CVD groups (Po:5). No significant difference was detected among the different groups with regard to serum total cholesterol, HDL, LDL, VLDL and triglyceride levels (Table 1). When we evaluated the association between the serum homocysteine level and AHI as a severity measure of OSAS, homocysteine levels were positively correlated with the severity of OSAS both in OSAS+CVD (r ¼ :73, Po:1) and OSAS CVD Table 1 The demographic and clinical data of study groups. Mean7SD OSAS CVD (n ¼ 47) OSAS+CVD (n ¼ 25) CVD (n ¼ 42) P-value Age (years) 48.5671.2 51.7678.67 48.86711.55 4.5 BMI (kg/m 2 ) 3.5375.1 32.2774.89 27.1774.9 4.5 AHI 32.93728.7 44.92741.53 2.1171.56 o.1 Total cholesterol 218.47784.99 24.9765.18 197.37739.45 4.5 HDL 48.5717.87 46.2678.69 47.2671.4 4.5 LDL 129.51741 113.52728.3 122.42735.59 4.5 VLDL 29.48711.87 32.91718.69 29.36716.1 4.5 Triglyceride 168.227123.44 172.277121.78 154.92797.64 4.5 Homocysteine 14.774.52 y 17.276.58 y 1.3573.63 o.1 OSAS: obstructive sleep apnea syndrome; CVD: cardiovascular disease; BMI: body mass index; AHI: apnea hypopnea index; HDL: high-density lipoprotein; LDL: low-density lipoprotein; VLDL: very low-density lipoprotein; SD: standart deviation. Po:1, OSAS CVD group vs. CVD group and OSAS+CVD group vs. CVD group. y Po:1, OSAS CVD group vs. CVD group and OSAS+CVD group vs. CVD group.

Serum homocysteine levels and cardiovascular morbidity in OSAS 539 (r ¼ :53, Po:1) (Fig. 2). There was no correlation between serum lipid and homocysteine levels in all groups. Evaluation of the relative strength of association using multiple regression analysis between homocysteine and OSAS severity as well as the other possible confounding variables showed that homocysteine levels were independently associated with OSAS severity both in OSAS+CVD (F ¼ 3:1, Po:5) and OSAS CVD group (F ¼ 1:7, Po:5). homocysteine 5 4 3 2 1 N = 25 OSAS+CVD 47 OSAS-CVD groups 42 CVD Figure 1 Serum homocysteine levels in the study groups. Discussion The acute hemodynamic alterations of obstructive sleep apnea include systemic and pulmonary hypertension, increased right and left ventricular afterload, and increased cardiac output. Earlier reports attributed the coexistence of OSAS with CVD to the shared risk factors such as age, sex, and obesity. However, recent epidemiologic data confirm an independent association between OSAS and the different manifestations of CVD. Possible mechanisms may include a combination of intermittent hypoxia and hypercapnia, repeated arousals, sustained increase in sympathetic tone, reduced baroreflex sensitivity, increased platelet aggregation, elevated plasma fibrinogen levels and also hyperhomocysteinemia. 14 Pertaining to the associations between OSAS and cardiovascular morbidity and between homocysteinemia and cardiovascular morbidity, our study confirms the previous finding by Lavie et al. 15,16 that levels of homocysteine are elevated in OSAS patients. The most important difference in our study is that it consists of obese males with completely similar age and weights. Therefore, some parameters affecting cardiovascular morbidity such as sex, age and BMI has been eliminated in this study. In Lavie s study, however, age and BMI have been differed between groups. We showed that OSAS patients with and without CVD had significantly higher levels of homocysteine when compared with patients with CVD without OSAS, and serum homocysteine levels 5 3 4 2 Homocysteine 3 Homocysteine 1 2 1 1 AHI Observed Linear 2 2 4 6 AHI 8 Observed Linear 1 12 Figure 2 Simple correlation between serum homocysteine levels and AHI in OSAS+CVD and OSAS CVD groups.

54 were independently associated with severity of OSAS. There was no difference in conventional risk factors for CVD among the three groups, but there was also a limitation in our study because we had no data about lipid lowering medications of patients. Therefore, the indifference between groups may be explained by decreasing high lipid levels of some patients with medication. We found that only 16.6% of CVD group had hyperhomocysteinemia (414 mmol/l) and the mean homocysteine levels in CVD group was 1.3573.63 mmol/l. This mean value is similar to the level of homocysteine observed in seven studies investigating large groups of cardiovascular patients (12.271.3 mmol/l) 17 and in Lavie s study investigating patients with IHD (11.9275.77 mmol/ L). 15 Our CVD group contained both HT and IHD patients. Although it is known to increase in CVD morbidity by hyperhomocysteinemia, considering the other factors also affecting CVD, it would not be surprising to find low homocysteine levels in this CVD group. In patients with OSAS and CVD, serum homocysteine levels were higher than patients with CVD without OSAS. Besides, in OSAS patients with CVD, the number of patients whose homocysteine levels were above the limits was found to be significantly higher. This suggests that, among CVD patients, patients with OSAS may have higher homocysteinemia due to OSAS. The essential issue deserving to be researched is the mechanism that is responsible for the increased serum homocysteine levels in OSAS. In patients with OSAS, cyclical alterations of arterial oxygen saturation are observed with oxygen desaturation developing in response to apnea followed by the resumption of oxygen saturation during hyperventilation. This phenomenon has been referred to as hypoxia/reoxygenation and might alter the oxidative balance through the induction of excess oxygen free radicals quite like in the sequelae of ischemia/reperfusion injury. Some authors have reported that systemic biomarkers of oxidative stress has increased in patients with sleep apnea suggesting a possible role in the pathologic consequences of OSAS. 18 Excess superoxide production due to hypoxia/reoxygenation may also inactivate nitric oxide resulting in the formation of the toxic peroxynitrite. This may further prevent the inactivation of homocysteine leading to an increase in its levels, putting OSAS patients with cardiovascular morbidity at a greater risk. 16 Endothelial dysfunction, which is the first step in the development of atherosclerosis according to the response to injury hypothesis, was initially implicated with hypercholesterolemia. However, endothelial dysfunction could result from many seemingly unrelated pathological conditions such as increased plasma homocysteine levels. Cumulative data in vivo and in vitro clearly demonstrate that homocysteine affects multiple vascular functions such as promoting a prothrombotic phenotype of the endothelium by increasing platelet aggregation and activation, and stimulating vascular smooth muscle cell proliferation. Currently, the leading mechanisms proposed for the adverse effects of homocysteine on endothelial function implicate oxidative stress and depletion in nitric oxide bioavaliability. 11,16,19 In conclusion, the impact of alleviating OSAS on cardiovascular risk factors such as hyperhomocysteinemia has not been fully elucidated. Given the compelling epidemiological and mechanistic evidence implicating increased homocysteine in CVD, homocysteine may be an important factor linking OSAS to cardiovascular morbidity. Since CPAP is a very efficient treatment method which eliminates the oxygen desaturation and other consequences resulting from obstructive respiratory events, we speculate that CPAP-related improvement in OSAS also translates into lower serum homocysteine level and, by inference, a decrease in cardiovascular risk. Further studies are needed to evaluate the effect of CPAP on the serum homocysteine levels. References O. Kokturk et al. 1. Bassiri AG, Guilleminault C. Clinical features and evaluation of obstructive sleep apnea hypopnea syndrome. In: Kryger MH, Roth T, Dement WC, editors. Sleep medicine. 3rd ed. Philadelphia: W.B. Saunders Company; 2. p. 869 78. 2. Young T, Palta M, Dempsey J, et al. The occurrence of sleepdisordered breathing among middle-aged adults. N Engl J Med 1993;328:123 5. 3. Grote L, Hedner J, Peter JH. Sleep-related breathing disorder is an independent factor for uncontrolled hypertension. J Hypertens 2;18:679 85. 4. Shahar E, Whitney CW, Redline S, et al. 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