Introduction

Obstructive sleep apnea-hypopnea syndrome (OSAHS), a chronic disorder characterized by sleep-related upper-airway obstruction, is strongly and independently associated with an increased risk for hypertension [1], myocardial ischemia [2–4], and stroke [5, 6]. Patients with OSAHS experience repetitive episodes of hypoxia/reoxygenation during transient cessation of breathing that promote systemic oxidative stress and inflammation, which are fundamental mechanisms underlying tissue and cell damage [7, 8].

Circulating serum cell-free DNA is present in small amounts in the serum of healthy individuals. Recently, increased concentrations of cell-free DNA have been found in various clinical conditions, including autoimmune diseases, cancer, stroke, and myocardial infarction [9–13]. Following effective treatment, circulating cell-free DNA concentrations in patients with cancer, acute pancreatitis, and other diseases could decrease, suggesting that quantification of cell-free DNA could be a novel approach to monitoring disease severity and the response to treatment [14–16]. However, the kinetics of circulating cell-free DNA concentrations either during or after therapy remains unknown, and cell death by either apoptosis or necrosis is thought to be the main source of the circulating DNA.

In this study we tested the hypothesis that elevated serum cell-free DNA concentrations are probably due to oxidative stress and inflammation in OSAHS patients, and that treatment with nasal continuous positive airway pressure (nCPAP) could reverse these alterations.

Methods

Study Population

Patients

Patients who came to the Sleep Disorders Center at Ruijin Hospital for evaluation of sleep-disordered breathing from September 2007 to January 2009 were prospectively screened for the study. Patients with newly diagnosed OSAHS, defined as an apnea-hypopnea index (AHI) of five or more obstructive events per hour of sleep, and free of conditions known to affect the serum cell-free DNA concentrations were eligible for the study. Patients with hypertension, coronary artery disease, autoimmune diseases, a history of stroke, diabetes mellitus, chronic obstructive or restrictive pulmonary disease, chronic renal disease, or tobacco use within the past 10 years were ineligible for the study. Data on age, smoking status, alcohol drinking, and medical history of diseases were obtained from questionnaire-based interviews, which were conducted by trained personnel. Diagnosis of hypertension or diabetes mellitus was determined on the basis of medical history and a comprehensive health examination, including measurement of blood pressure and assay of blood glucose.

Control Subjects

Our control subjects were nonsmoking healthy subjects who were not taking any medications or nutritional supplements. Control subjects were matched to patients for gender, age, and BMI. All control subjects underwent polysomnography (PSG) to exclude the presence of sleep-disordered breathing. The Shanghai Jiaotong University Committee on Human Research approved the study. All study participants gave written informed consent.

Study Protocol

All study participants underwent attended nocturnal PSG in the sleep disorders center. Each blood sample of peripheral venous blood was collected after overnight polysomnography from 6 a.m. to 7 a.m. Immediately after blood draw, serum was carefully removed by centrifugation at 3,000 rpm for 10 min and stored at −80°C until analysis.

Polysomnography and nCPAP Therapy

Nocturnal polysomnography was performed as previously described [17]. AHI was defined as the number of obstructive apnea plus hypopnea episodes per hour of sleep. Adherence to nCPAP was defined as nCPAP use for 4 or more hours daily. Adherence was assessed by use of an nCPAP device with compliance software.

Preparation and Measurement of Serum DNA

Serum DNA was extracted from 200 μl serum using a QIAmp Blood Kit (Qiagen, Valencia, CA, USA) according to the protocol recommended by the manufacturer. Serum DNA concentrations were measured using a real-time quantitative polymerase chain reaction (PCR) assay (Applied Biosystems, Foster, CA, USA) for the β-globin gene, which is present in all nucleated cells of the body [18]. The β-globin PCR system included the amplification primers β-globin-354F (5′-GTG CAC CTG ACT CCT GAG GAG A-3′) and β-globin-455R (5′-CCT TGA TAC CAA CCT GCC CAG-3′) [18] and iQ™ SYBR® Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA). All samples were analyzed in duplicate. Thermocycling was performed at 95°C for 30 s, followed by 45 cycles at 95°C for 15 s and 60°C for 10 s to measure the fluorescence signal. For a standard curve, serum DNA was used at concentrations of 104–108 copies/μl. Of the standard solutions, 2 μl was used in PCR as described above. DNA concentrations in the serum samples were calculated as ng DNA/ml serum.

Measurement of Serum Interleukin-6 (IL-6) and Malonaldehyde (MDA)

IL-6 was measured by enzyme linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. MDA was measured by the thiobarbituric acid method using the MDA Test Kit (Nanjing Jiancheng, China).

Statistical Analysis

All parameters except serum DNA concentrations were expressed as mean ± standard deviation (SD). DNA concentration was not under normal distribution and was expressed as median (interval of quartile). Analysis of variance was used for the mean of samples among groups. A least significant difference (LSD) test was used for paired comparison between two groups if comparison among multiple groups with analysis of variance showed statistical significance. Univariate analysis was depicted by Pearson’s coefficient. A multilinear regression analysis was performed with AHI, oxygen desaturation index (ODI), minimal oxygen saturation (miniSaO2), MDA, and IL-6 as the independent variables and serum DNA as the dependent variable. Comparison of parameters before and after nCPAP treatment was undertaken with a paired t test. p < 0.05 was considered statistically significant.

Results

Six hundred fifty-one patients were newly diagnosed with OSAHS during the study period. Five hundred twenty-four patients were not eligible for the study because of the presence of one or more exclusion criteria. The patients in the OSAHS group were further divided into mild, moderate, and severe OSAHS subgroups based on their AHI during sleep. OSAHS and its division by degree were evaluated using international diagnostic criteria [19]. Among all OSAHS patients, ten with moderate and severe OSAHS underwent nCPAP treatment for 6 months. Fifty-two control subjects were studied.

Comparison of Baseline Characteristics Among Groups

The clinical characteristics of the study participants are summarized in Table 1. Patients and control subjects were similar in age, gender, BMI, neck circumference (NC), and systemic blood pressure (all p > 0.05). OSAHS patients had significantly lower miniSaO2 and higher ODI during sleep than control subjects. Moreover, ODI and miniSaO2 were all statistically different among the OSAHS subgroups.

Table 1 Baseline characteristics of control group and OSAHS subgroups
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Comparison of Serum DNA Among Groups

Serum DNA concentrations in the moderate and severe OSAHS subgroups were significantly higher than those in the mild OSAHS subgroup and the control group (all p < 0.05).Similarly, serum DNA concentrations in the severe OSAHS subgroup were significantly higher than those in the moderate OSAHS subgroup. No statistical difference in serum DNA was detected between the mild OSAHS subgroup and the control group (p > 0.05) (Table 1).

Comparison of Serum IL-6 and MDA Among Groups

Serum IL-6 and MDA concentrations in moderate and severe OSAHS subgroups were significantly higher than those in the mild OSAHS subgroup and the control group (all p < 0.05). Similarly, these concentrations in the severe OSAHS subgroup were significantly higher than those in the moderate OSAHS subgroup. No statistical differences in serum IL-6 and MDA were detected between the mild OSAHS subgroup and the control group (p > 0.05) (Table 1).

Relationships Between Serum DNA Concentrations and Polysomnographic Variables, Age, BMI, NC, IL-6, and MDA in all OSAHS Patients

In univariate analysis, serum DNA concentrations were positively correlated with AHI, ODI, IL-6, and MDA (r = 0.465, 0.372, 0.553, 0.601, all p < 0.001) but were negatively correlated with miniSaO2 (r = −0.345, p < 0.001). In stepwise multiple regression analysis that included AHI, ODI, miniSaO2, IL-6, and MDA, only MDA and miniSaO2 were significant independent predictors for the serum DNA concentrations (standardized regression coefficients were 0.548 and −0.151, respectively, p < 0.05). The normality test of the regressions was passed (Table 2, Figs. 1 and 2).

Table 2 Correlation between serum DNA and baseline characteristics
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Fig. 1
figure 1

Negative correlation between serum DNA level and miniSaO2 in all OSAHS patients (n = 127, r = −0.345, p < 0.001). MiniSaO 2 minimal oxygen saturation. Data were analyzed using Pearson’s correlation coefficients

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Fig. 2
figure 2

Positive correlation between serum DNA level and MDA in all OSAHS patients (n = 127, r = 0.601, p < 0.001). MDA malonaldehyde. Data were analyzed using Pearson’s correlation coefficients

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Comparison Before and on CPAP Treatment

There was a significant decrease in serum DNA, IL-6, and MDA concentrations after CPAP treatment compared with before CPAP treatment (all p < 0.001) (Table 3).

Table 3 Comparison before and after 6 months of CPAP treatment
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Discussion

The β-globin gene measured in our study is the most frequently used gene in previous cell-free DNA studies [18, 20]. Our results showed that cell-free DNA measured in serum of patients with severe and moderate OSAHS was significantly higher than in subjects with mild OSAHS and those without OSAHS. This is the first report that shows that nCPAP treatment can effectively decrease serum DNA concentrations in OSAHS patients.

The mechanisms underlying the presence of circulating DNA are generally unknown. Apoptosis, necrosis, and impaired clearance have been implicated [21–23], but information regarding their role in OSAHS is scarce. Shin et al. [24] first reported that cell-free DNA concentrations were increased in patients with severe obstructive sleep apnea (OSA). However, they did not study the association between oxidative stress, inflammation, and circulating cell-free DNA concentrations. Obstructive sleep apnea syndrome with its hypoxia/reoxygenation episodes may be viewed as similar to ischemic/reperfusion injury, causing the generation of reactive oxygen species (ROS) [25]. MDA is regarded as an appropriate biomarker of oxidative stress [26]. Intermittent hypoxia has been associated with systematic inflammation, which is likely to play a crucial role in the activation of proinflammatory factors with consequent production of proinflammatory cytokines such as IL-6 and tumor necrosis factor-α [27, 28].

In this study we found elevated serum DNA concentrations in patients with moderate and severe OSAHS. The serum DNA concentrations were positively correlated with AHI and ODI but negatively correlated with miniSaO2, suggesting that serum DNA concentrations increased when OSAHS became severe. The severity of OSAHS could thus be reflected by the concentration of serum DNA. In particular, our study demonstrated that the serum IL-6 and MDA concentrations significantly increased in moderate and severe OSAHS patients, and these concentrations were positively correlated with serum DNA concentrations. Then we performed a multiple linear regression analysis for predicting serum DNA concentrations. MDA and miniSaO2 were significant independent predictors for serum DNA concentrations. It has been suggested that higher cell-free serum DNA concentrations of OSAHS patients may be attributed to increased oxidative stress that occurs under more severe illness conditions. Free serum DNA in OSAHS patients is supposed to originate from the apoptosis or necrosis of tissue [29, 30], and the correlation of serum IL-6, MDA, and serum DNA concentrations also supports this hypothesis. Therefore, we can infer that the serum DNA concentrations of patients with mild disease were not significantly different from those of the controls because they have only limited or recovered oxidative stress. Greater tissue necrosis or apoptosis levels would cause a significant influx of free DNA into circulation, with its concentrations rising during chronic intermittent hypoxia. Although we have excluded patients with other possible sources of free DNA (e.g., infection, trauma, myocardial infarction), we did not provide direct evidence that the free serum DNA originates from tissue necrosis or apoptosis.

Because serum DNA concentrations were positively correlated with AHI and ODI and negatively correlated with miniSaO2, we evaluated whether reversion of hypoxia using nCPAP treatment could alter serum DNA concentrations. After 6 months of follow-up of ten OSAHS patients on CPAP treatment, there was a remarkable improvement not only in IL-6 and MDA levels but also in serum DNA (all p < 0.05). This observation provides further evidence that the elevated serum DNA in OSAHS patients might result from intermittent hypoxemia caused by repeated apnea and hypopnea events, since oxidative stress and inflammatory reactions became weak as abnormally high AHI and low SaO2 were corrected by CPAP treatment [31]. There were potential limitations in our study. It was not designed as a randomized controlled trial. Of all the OSAHS patients, only ten moderate and severe OSAHS patients underwent nCPAP treatment. Therefore, the present findings need to be confirmed in randomized controlled trials with larger sample sizes.

In conclusion, we have shown that OSAHS patients have significantly increased serum DNA concentrations, which were positively correlated with disease severity. Moreover, the present investigation is the first to report that elevated serum DNA concentrations might be gradually reversed by nCPAP treatment. Consequently, the serum DNA may become an important parameter for monitoring the disease severity of OSAHS and therapeutic effectiveness. The kinetics of serum cell-free DNA concentrations in OSAHS patients need to be further investigated.