Introduction

Obstructive sleep apnea–hypopnea syndrome (OSAHS) is characterized by repetitive apnea and hypopnea attacks followed by arousals and affects patients’ physical, emotional, and intellectual capacities and functional quality of life. OSAHS has been linked to increased prevalence of cardiovascular and cerebrovascular diseases, stroke, metabolic syndrome, cognitive impairment, and systemic or pulmonary hypertension [1, 2]. Therefore, recent research has focused on exact mechanisms for the elucidation of the relationships between these diseases and OSAHS.

Changes in ventilatory function and concomitant sleep disruption may result in a cascade of events including oxidative stress, inflammation, vascular endothelial dysfunction, and metabolic dysregulation. Repeated apnea-related hypoxic events in OSAHS are similar to hypoxia/reperfusion injury, which initiates oxidative stress. Supporting evidence was obtained from previous studies showing increased free radical production and increased lipid peroxidation accompanying with lower antioxidant levels in OSAHS patients [3–5]. Reactive oxygen and nitrogen species are the most important free radicals causing oxidative/nitrosative stress and tissue injury related to the several diseases, which are also seen in OSAHS patients [6–8]. Nitric oxide (NO) is synthesized in mammalian cells from l-arginine through enzymatic reactions catalyzed by a family of NO synthases (NOS). Besides acting as a key vasodilator in the vascular system, it is also regulates a great variety of physiological functions including the memory process and sleep–wake cycle and inflammatory process [6, 9]. Lower NO levels were shown in OSAHS patients compared with healthy controls in previous studies [10–12].

Asymmetric dimethylarginine (ADMA), which is an endogenous NOS inhibitor, is generated by catabolism of proteins containing methylated arginine residues and metabolized by the enzyme dimethylarginine dimethyl aminohydrolase (DDAH), the activity of which regulates ADMA concentration and indirectly modulates NOS activity. During the methylation of proteins, S-adenosylmethionine is utilized as a methyl donor, and S-adenosylhomocysteine is generated as a byproduct [13]. Homocysteine is accepted as an independent cardiovascular risk factor, and its altered levels lead to endothelial dysfunction, alterations in the antithrombotic function of the endothelium [14], increase in oxidative stress [15], and decrease in NO availability [16]. Furthermore, recent studies with animal and human cell cultures indicated that ADMA is metabolically linked to homocysteine and elevated ADMA levels and may be a unifying mechanism for endothelial dysfunction during hyperhomocysteinemia [17, 18].

In the view of these research results, we focused on the changes in the oxidative status, NO, ADMA, and homocysteine levels in OSAHS patients and aimed to clarify the effects of the repetitive hypoxia resulting from OSAHS on these parameters.

Materials and methods

Study subjects

OSAHS patients and control groups involved in this study were selected from 93 consecutive subjects attended to Sleep Disorders Center in Department of Chest Diseases, Dışkapı Education and Research Hospital, for suspected sleep apnea. Before admission, all subjects were interviewed for the presence of sleep-related symptoms, snoring, witnessed apnea, and excessive daytime sleepiness. Epworth sleepiness test was used to determine the level of daytime sleepiness. After examination by an otorhinolaringologist, patients having anatomical nasal problems such as septal deviation were excluded from the study. Presence and severity of OSAHS were determined by standard overnight polysomnography (16 channels, Embla, Flaga). All data obtained from the electroencephalogram (electrodes at positions C3-A2, C4-A1, O1-A2, O2-A1), electrooculogram, and electromyogram (submental EMG and EMG tibialis), oronasal air flow measurements (cannula), breathing movements of the chest and abdomen, snoring detected by using a tracheal microphone, body position, and pulse oxymetry at the finger tip of the patients were recorded and evaluated by the Somnologica 3.2 version software program. Analysis of sleep stages were performed according to the Criteria of Rechtschaffen and Kales [19].

Apnea was defined as an absence of air flow for at least 10 s, and hypopnea was defined as a 50% reduction in airflow accompanied by a reduction in oxygen saturation by 4% from baseline or arousal. Desaturation was defined as a reduction in oxygen saturation by 4% from the baseline. Mean oxygen saturation (mean SaO2) was defined as average of oxygen saturation during the night. Lowest oxygen saturation (lowest SaO2) was defined as the lowest value of oxygen saturation during the night. The apnea–hypopnea index (AHI) was defined as the average number of apneic and hypopneic events per sleep hour. An AHI of more than 5/h was considered as diagnostic of OSAHS. Subject who had an AHI equal to or less than 5/h was placed in the control group. After excluding subjects having other diseases or taking medication, 34 OSAHS and 15 healthy control subjects were designated as the groups in this study. Altitude is 680 in Ankara, Turkey. All participants’ rights were protected, and informed consent was obtained according to the Helsinki Declaration. The study protocol was approved by the local Ethic Committee.

Blood sampling

Peripheral venous blood samples were obtained in the morning after the diagnostic study night. Serum and heparinized plasma samples were immediately stored at −70°C until analysis.

Measurement of NO levels

Serum NO levels were measured as sum of nitrate and nitrite levels. After serum samples were deproteinized with ethanol, 100 μL of the supernatant were loaded in the polystyrene microtiter plate, then 100 μL saturated VCl3 solution in 1 M HCl was added to convert nitrate to nitrite. After the addition of 100 μL Griess reagent, plates were incubated at 37°C for 45 min, and absorbance of the samples were measured by using a plate reader at 540 nm [20].

Measurement of ADMA levels

Plasma ADMA levels were determined by using high-performance liquid chromatography (HPLC) with fluorescence detection. After plasma proteins were separated with 5-sulfosalicylic acid, 10 μL of the supernatant was mixed with 100 μL o-phthaldialdehyde reagent and applied to the HPLC system. Fluorescence intensities were measured with excitation at 338 nm and emission at 425 nm [21].

Measurement of oxidative status

To determine the oxidative status of plasma samples, N,N-dimethyl-p-phenylene-diamine (DMPD) was used as an indicator for oxidative status. This indicator is based on the capability of DMPD to give a stable colored solution when it is transformed into its radical cation (DMPD+) originating from the oxidation of the DMPD itself by alkoxy and peroxy radicals derived from the iron-induced decomposition of hydroperoxide. Ten microliters of the plasma sample and 20 μL 1 mM DMPD solution were added to 2 mL acetate buffer (0.1 M, pH 4.8), and the formation of the colored DMPD+ radical cation was monitored by measuring the absorbance at 505 nm. H2O2 was used as a standard for oxidative status. The oxidative status of plasma was expressed as hydrogen peroxide equivalents. Intra- and interassay CV for oxidative status were 1.8 and 4.5%, respectively. [22].

Measurement of homocysteine levels

Total homocysteine levels were measured by the flourometric HPLC methods with some modification. The method is based on the derivatization of thiols with 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid after reduction with tris(2-carboxyethyl)phosphine hydrochloride. Fluorescence intensities were measured with excitation at 385 nm and emission at 515 nm [23, 24].

Statistical methods

Statistical analyses were performed with SPSS Statistical Package Program (SPSS 8.0 version, USA). Values were expressed mean ± SD. Mann–Whithney U test, Kruskal–Wallis variance analysis, and Spearman Rank correlation analysis were applied to data. Statistical significance was set at p < 0.05.

Results

Baseline demographics characteristics and sleep study results of OSAHS patients and control group are given in Table 1. As shown, OSAHS patients and healthy controls had similar values of biochemical parameters.

Table 1 Baseline characteristics and sleep study results in the OSAHS patients and controls
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The serum levels of NO in OSAHS patients were significantly lower than that in control subjects (p < 0.05). Plasma oxidative status and serum ADMA levels did not show any significant difference between these groups. Serum homocysteine levels were higher in OSAHS patients than in control subjects, but the difference between the two groups bordered on statistical significance (Table 2). In OSAHS patients, NO levels showed positive correlations with mean SaO2 (r = 0.513, p < 0.002) and lowest SaO2 (r = 0.363, p < 0.03). NO levels were also negatively correlated with oxidative status in this group (r = -0.415, p < 0.02). No statistically significant correlation was observed between homocysteine, NO, and ADMA levels.

Table 2 Mean values of oxidative status, nitric oxide, ADMA, and homocysteine in OSAHS patients and controls
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After OSAHS patients were divided into moderate (AHI 5–29) and severe (AHI ≥ 30) OSAHS groups; increased oxidative status and homocysteine levels and decreased NO levels were observed in the severe OSAHS group; however, only decreased homocysteine levels reached statistical significance (p < 0.05; Table 3). There was also significant negative correlation between NO levels and oxidative status in the severe OSAHS group (r = −0.641, p < 0.007).

Table 3 Mean values of oxidative status, nitric oxide, ADMA, and homocysteine levels in OSAHS patient groups according to AHI index
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There was no significant difference between OSAHS patients and controls in terms of Epworth sleepiness Scale Score. Only the severe OSAHS group had a significantly higher score than control group (p < 0.04). There was no significant correlation between sleepiness score and measured parameters. Furthermore, there was no significant correlation between individual characteristics (age, BMI, SBP, and DBP) and measured parameters.

In total study group, homocysteine positively correlated with AHI (r = 0.797, p < 0.002) and with lowest SaO2 (r = 0.673, p < 0.02). NO levels negatively correlated with desaturation number (r = −0.415, p < 0.003) and positively correlated with mean SaO2 (r = 0.556, p < 0.0001) and lowest SaO2 (r = 0.431, p < 0.002).

Discussion

Although previous epidemiological studies have shown an association between OSAHS and coronary heart disease, heart failure, and pulmonary hypertension [25–27], exact causes of this association is still unknown. Hypercoagulability [28], polymorphonuclear neutrophil activation [29], increased inflammatory markers [30], and impaired endothelium dependent vasodilatation [31], were reported in OSAHS patients. Because the repetitive episode of nocturnal apnea leading to intermittent hypoxia and recurrent reoxygenation secondary to reperfusion are similar to ischemia/reperfusion injury, OSAHS is also considered to have links to oxidative stress and endothelial dysfunction.

Previous studies showed increased oxidative stress in OSAHS patients and in some of these studies, decreases in oxidative stress markers were observed after treatment [3–5, 32, 33]. Consistent with these studies, we observed increased oxidative status in patients, especially with severe OSAHS. Probable free radical sources in OSAHS have been suggested as hypoxia/reoxygenation insult, mitochondria, inflammatory leukocytes, and oxidation of small molecules such as homocysteine and additional sources [7]. Increased oxidative stress is one of the factors leading to endothelial dysfunction and also decreasing NO levels, which are characteristics of atherosclerotic process. In the present study, decreased NO levels and negative correlation between NO and oxidative status, especially in severe OSAHS patients, indicate that oxidative stress depending on the severity of hypoxia might have an important role in the cardiovascular abnormalities seen in OSAHS patients. In addition, because oxygen is a cosubstrate of NOS, OSAHS-related nocturnal desaturation might result in depressed synthesis of NO. Results of our study show significant positive correlations between NO and with mean SaO2 and negative correlation with lowest SaO2 in OSAHS patients. These results are also supportive of adverse cardiovascular effects.

Several research have indicated relationship between high ADMA, homocysteine and endothelial dysfunction [34, 35, 36]. In addition to as oxidative stress, elevated ADMA and homocysteine levels are important factors leading to impairment of NO availability and diminished endothelium-dependent vasodilatation, and their increased levels have been reported in cardiovascular disease [37, 38]. There is only one study reporting ADMA levels in obstructive sleep apnea patients [11]. In that study, Ohike et al. showed that plasma ADMA levels of OSAHS patients were decreased inversely to the improvement of flow-mediated vasodilatation after CPAP therapy, but the difference in ADMA levels before and after treatments did not reach statistical significance. In addition, increased homocysteine levels have been reported in OSAHS patients [39] and declined with CPAP therapy [40]. Kokturk et al. [41] showed that OSAHS patients with and without cardiovascular disease had significantly higher homocysteine levels, and its levels were independently associated with severity of OSAHS. In agreement with these previous studies, we observed elevated homocysteine levels even higher than the normal fasting range (5–15 μmol/l) especially in severe OSAHS patients. However, other studies reported similar mean homocysteine values in OSAHS and non-OSAHS groups [42, 43].

There are metabolic relationships between ADMA and homocysteine. ADMA levels and ADMA/l-arginine ratios were increased under hypoxic conditions [44]. Shear stress to the vessel occurs in response to repetitive apnea, and shear stress enhances the gene expression of arginine methyltransferase and ADMA release. Homocysteine produced during the synthesis of ADMA can alter ADMA metabolism by inhibiting DDAH. In addition, homocysteine was shown to decrease endothelial NOS and reduce DDAH expression in microvascular endothelial cells [45]. Despite these relationships, we did not observe any correlation between homocysteine and ADMA levels, and in contrast to our expectation, we did not find any difference between OSAHS patients and controls in terms of ADMA levels.

OSAHS patients have a high incidence of cardiovascular disease and mortality rate increases with severity of OSAHS. Although we think that a larger study group are necessary to clarify the exact interactions among NO, ADMA, homocysteine, and oxidative status, hyperhomocysteinemia and diminished NO production may be a causal factors in endothelial dysfunction seeing in OSAHS and may explain the association between OSAHS and cardiovascular diseases. These modifiable factors should be monitored in patients suspected of having OSAHS.