Clin Oral Invest DOI 10.1007/s00784-014-1234-1
ORIGINAL ARTICLE
Effect of oral appliance on endothelial function in sleep apnea Ching-Chi Lin & Huey-Yuan Wang & Chung-Hsin Chiu & Shwu-Fang Liaw
Received: 2 August 2013 / Accepted: 17 March 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Objectives This study evaluated the effects of mandibular advancement device (MAD) on serum levels of nitric oxide derivatives and endothelial function by endotheliumdependent flow-mediated dilation (FMD) in obstructive sleep apnea syndrome (OSAS). Methods Thirty patients with moderately severe-to-severe OSAS who desired MAD and 15 healthy controls were prospectively enrolled. FMD was measured by high-resolution Bmode ultrasonography, while serum NOx level from peripheral blood samples was measured by ELISA. All subjects participated in the sleep studies, which were repeated 2 months after MAD in OSAS patients. Results Serum NOx level and FMD were lower in patients with OSAS than in controls prior to MAD. Serum NOx levels in 19 of 30 patients with OSAS, the designated MAD responders, increased from 11.8±5.8 μM pre-MAD to 22.7± 4.9 μM post-MAD. The FMD increased from 5.9±4.6 preMAD to 10.5±4.8 post-MAD. For the 11 unresponsive patients, serum NOx and FMD remained impaired after MAD. Conclusions Successful treatment of OSAS with MAD can restore serum levels of NOx and FMD. C.65 years were excluded. Fifteen healthy individuals without OSAS were also recruited as the control group. Subjects with OSAS all had a body mass index (BMI) 25 were recruited. The institutional review board approved the study, and all participants provided written informed
consent. All had healthy teeth with no abnormal dentition, temporomandibular joint disease, or an exaggerated gag reflex. All were assessed by history review, physical examination, blood tests (including pre- and post-prandial blood glucose, cholesterol, and triglycerides), chest X-ray, and electrocardiogram. All subjects underwent simple spirometry, measurement of endothelial function by endothelium-dependent flowmediated dilation (FMD), and venipuncture for measurement of NOx levels in the peripheral blood at baseline as well as an overnight sleep study. Alcohol, smoking, or sedatives were avoided for at least 1 week prior to these studies. Substances that alter metabolism (e.g., caffeine, tea, or nicotine) were also avoided for at least 2 days. Blood pressure was measured after at least 15 min of rest at 7 a.m., 8 p.m., and 10 p.m. in the sleep laboratory. At 7 a.m., after the overnight sleep study, peripheral blood samples were drawn into a clotting tube and centrifuged at 2,000 rpm for 10 min. The supernatant serum was refrigerated at −80 °C until assays for NOx. In the OSAS group, these studies were repeated 2 months after MAD treatment. Sleep studies Overnight sleep studies were performed with complete polysomnography. Sleep data and respiratory events were manually scored according to established standard criteria. Briefly, the AHI was defined as the mean number of episodes of apnea/hypopnea per hour of sleep. Desaturation event frequency was defined as the mean number of oxygen desaturation episodes per hour of sleep. Sleep apnea syndrome was diagnosed if the AHI was ≥5. Moderately severe or severe sleep apnea was defined as an AHI of ≥20. Arousal index was defined as the mean number of arousals per hour of sleep, while sleep efficiency was the percentage of total sleep time divided by total time in bed [17]. Determination of NOx serum levels The stored serum samples were deproteinated by ultrafiltration through a 10-kDa molecular weight cut-off Amicon Ultra Centrifugal Filter Devices (Millipore, Billerica, MA, USA) at 13,000×g. Deproteinated serum of 80 μL was added to 96-well plates and incubated with 10 μL nitrate reductase enzyme and 10 μL NADPH for 3 h at room temperature to reduce nitrate (NO3) to nitrite (NO2), using a nitrate/nitrite colorimetric assay kit (Cayman Chemical Co., Ann Arbor, MI, USA). Nitrite was then converted to a deep purple azo compound by reaction with 50 μL sulfanilamide (Griess reagent 1) and 50 μL N-(1-naphthyl) ethylenediamine (Griess reagent 2). After a 10-min incubation at room temperature, the absorbance of each well was determined on a microplate reader (SpectraMAX 190; Molecular Devices, Sunnyvale, CA, USA) at 540 nm, and the
Clin Oral Invest Table 1 Characteristics and pulmonary function test results before and after mandibular advancement device (MAD) of the OSAS patients and controls Controls (n=15)
MADS (n=19) Before
Age, years Sex (male/female) Systolic pressure, mmHg Diastolic pressure, mmHg BMI, kg/m2 FVC, % predicted FEV1/FVC Cholesterol, mg/dL Triglyceride, mg/dL
48±7 12/3 122.7±6.8 71.9±6.2 27.7±1.4 91.5±4.9 79.7±3.9 191.4±10.9 207.2±13.7
50±8 15/4 124.1±7.1 72.5±6.2 28.2±1.5 90.9±5.3 78.8±4.2 198.6±10.3 206.5±12.8
MADF (n=11) After
Before
After
123.7±7 72.3±5.9 27.9±1.6 90.6±5.1 78.6±4.4 196.4±10.4 209.4±13.2
51±7 9/2 125.1±6.8 73.1±6.1 28.5±1.5 90.3±5.4 78.3±4.6 201.3±11.3 211.2±14.1
124.6±6.7 72.8±6.3 28.2±1.4 90.1±4.8 78.1±4.2 202.4±10.7 217.8±13.7
Student’s t test was used MAD mandibular advancement device, MADS successful MAD, MADF failure MAD, BMI body mass index, FEV1 forced expiratory volume in 1 s, FVC forced vital capacity
nitrite/nitrate concentrations were deduced from a standard curve. The lower limit of detection was 2.5 μM [18]. Endothelium-dependent flow-mediated dilation The FMD was measured by high-resolution B-mode ultrasonography, with a 7.0-MHz linear array transducer and Acuson 128XP/10 system (Mountain View, CA, USA). Longitudinal scans of the brachial artery at the dominant arm were obtained at rest. Afterward, forearm blood ischemia was achieved for 5 min by inflating a cuff to suprasystolic pressures. The cuff was deflated, causing a dilation of the brachial artery during reactive hyperemia, and arterial diameter was continuously monitored over a period of 3 min. The increase in vessel
diameter observed 60 s after cuff release was determined and expressed in relative percent values compared to baseline conditions. The FMD was defined as the percentage change in diameter between the initial scan and after cuff deflation [19]. Oral appliance Patients were referred from the Department of Chest Medicine for the fabrication of mandibular advancement devices after polysomnography examination and OSAS diagnosis. The temporomandibular joints (TMJ) and masticatory muscles were examined to evaluate the bilateral temporomandibular joint and muscular condition. The overjet between the labial surface of the maxillary and mandibular incisors was
Table 2 Results of sleep study before and after MAD Controls (n=15)
MADS (n=19)
MADF (n=11)
Before
After
Before
After
AHI ODI Mean SaO2, % Baseline SaO2, % Minimum SaO2, % Sleep architecture
3.2±0.9 1.7±0.7 97.8±0.4 98.9±0.4 93.6±1.1
31.5±11.4* 25.96±11.3* 92.9±0.5* 97.8±0.9 75.4±7.6*
8.6±5.7 6.9±5.7 96.4±0.4 98.4±0.8 84.7±6.4
31.8±11.6 26.7±10.7 92.6±0.5 97.7±1 75.4±5.6
24.7±5.9 23.1±8.7 92.7±0.5 98.3±0.9 76.8±4.4
Stage 1, % Stage 2, % Stage 3+4, % REM, % AI
8.4±3.7 53.8±4.7 16.7±3.5 21.1±3.6 3.2±0.6
21.2±8.0* 54.7±8.7 9.4±5.3* 14.7±5.7* 28.7±8.3*
9.5±5.3 53.3±7.7 16.8±6.1 20.4±5.5 8.6±3.4
20.4±6.0 55.2±7.8 10.1±5.9 14.3±5.8 30.7±7.8
21.7±5.2 54.3±5.4 9.1±4.8 14.9±5.6 21.9±7.6
Student’s t test was used MAD mandibular advancement device, AHI apnea-hypopnea index, ODI oxygen desaturation index, SaO2 arterial oxyhemoglobin saturation *p5). Treatment failure (MADF) was defined as 5.
improvement in all variables except for baseline SaO2 and percentage of stage 2 sleep. In contrast, the MADF group showed no significant improvement in the sleep study results after MAD.
Serum levels of NOx and FMD before and after MAD Prior to MAD, serum NOx level and FMD were lower in patients with OSAS than in controls. The serum levels of NOx in 19 of 30 patients with OSAS, designated MAD responders, increased from 11.8±5.8 μM pre-MAD to 22.7± 4.9 μM post-MAD. The FMD increased from 5.9±4.6 preMAD to 10.5±4.8 post-MAD. For the 11 unresponsive patients, serum NOx and FMD remained impaired after MAD (Fig. 1a, b).
Data analysis Student’s t test and analysis of variance (ANOVA) were used for statistical analysis where appropriate. If ANOVA showed statistically significant differences, Scheffe’s test was performed. All values were expressed as the mean±standard deviation. Significance was set at p
ORIGINAL ARTICLE
Effect of oral appliance on endothelial function in sleep apnea Ching-Chi Lin & Huey-Yuan Wang & Chung-Hsin Chiu & Shwu-Fang Liaw
Received: 2 August 2013 / Accepted: 17 March 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Objectives This study evaluated the effects of mandibular advancement device (MAD) on serum levels of nitric oxide derivatives and endothelial function by endotheliumdependent flow-mediated dilation (FMD) in obstructive sleep apnea syndrome (OSAS). Methods Thirty patients with moderately severe-to-severe OSAS who desired MAD and 15 healthy controls were prospectively enrolled. FMD was measured by high-resolution Bmode ultrasonography, while serum NOx level from peripheral blood samples was measured by ELISA. All subjects participated in the sleep studies, which were repeated 2 months after MAD in OSAS patients. Results Serum NOx level and FMD were lower in patients with OSAS than in controls prior to MAD. Serum NOx levels in 19 of 30 patients with OSAS, the designated MAD responders, increased from 11.8±5.8 μM pre-MAD to 22.7± 4.9 μM post-MAD. The FMD increased from 5.9±4.6 preMAD to 10.5±4.8 post-MAD. For the 11 unresponsive patients, serum NOx and FMD remained impaired after MAD. Conclusions Successful treatment of OSAS with MAD can restore serum levels of NOx and FMD. C.65 years were excluded. Fifteen healthy individuals without OSAS were also recruited as the control group. Subjects with OSAS all had a body mass index (BMI) 25 were recruited. The institutional review board approved the study, and all participants provided written informed
consent. All had healthy teeth with no abnormal dentition, temporomandibular joint disease, or an exaggerated gag reflex. All were assessed by history review, physical examination, blood tests (including pre- and post-prandial blood glucose, cholesterol, and triglycerides), chest X-ray, and electrocardiogram. All subjects underwent simple spirometry, measurement of endothelial function by endothelium-dependent flowmediated dilation (FMD), and venipuncture for measurement of NOx levels in the peripheral blood at baseline as well as an overnight sleep study. Alcohol, smoking, or sedatives were avoided for at least 1 week prior to these studies. Substances that alter metabolism (e.g., caffeine, tea, or nicotine) were also avoided for at least 2 days. Blood pressure was measured after at least 15 min of rest at 7 a.m., 8 p.m., and 10 p.m. in the sleep laboratory. At 7 a.m., after the overnight sleep study, peripheral blood samples were drawn into a clotting tube and centrifuged at 2,000 rpm for 10 min. The supernatant serum was refrigerated at −80 °C until assays for NOx. In the OSAS group, these studies were repeated 2 months after MAD treatment. Sleep studies Overnight sleep studies were performed with complete polysomnography. Sleep data and respiratory events were manually scored according to established standard criteria. Briefly, the AHI was defined as the mean number of episodes of apnea/hypopnea per hour of sleep. Desaturation event frequency was defined as the mean number of oxygen desaturation episodes per hour of sleep. Sleep apnea syndrome was diagnosed if the AHI was ≥5. Moderately severe or severe sleep apnea was defined as an AHI of ≥20. Arousal index was defined as the mean number of arousals per hour of sleep, while sleep efficiency was the percentage of total sleep time divided by total time in bed [17]. Determination of NOx serum levels The stored serum samples were deproteinated by ultrafiltration through a 10-kDa molecular weight cut-off Amicon Ultra Centrifugal Filter Devices (Millipore, Billerica, MA, USA) at 13,000×g. Deproteinated serum of 80 μL was added to 96-well plates and incubated with 10 μL nitrate reductase enzyme and 10 μL NADPH for 3 h at room temperature to reduce nitrate (NO3) to nitrite (NO2), using a nitrate/nitrite colorimetric assay kit (Cayman Chemical Co., Ann Arbor, MI, USA). Nitrite was then converted to a deep purple azo compound by reaction with 50 μL sulfanilamide (Griess reagent 1) and 50 μL N-(1-naphthyl) ethylenediamine (Griess reagent 2). After a 10-min incubation at room temperature, the absorbance of each well was determined on a microplate reader (SpectraMAX 190; Molecular Devices, Sunnyvale, CA, USA) at 540 nm, and the
Clin Oral Invest Table 1 Characteristics and pulmonary function test results before and after mandibular advancement device (MAD) of the OSAS patients and controls Controls (n=15)
MADS (n=19) Before
Age, years Sex (male/female) Systolic pressure, mmHg Diastolic pressure, mmHg BMI, kg/m2 FVC, % predicted FEV1/FVC Cholesterol, mg/dL Triglyceride, mg/dL
48±7 12/3 122.7±6.8 71.9±6.2 27.7±1.4 91.5±4.9 79.7±3.9 191.4±10.9 207.2±13.7
50±8 15/4 124.1±7.1 72.5±6.2 28.2±1.5 90.9±5.3 78.8±4.2 198.6±10.3 206.5±12.8
MADF (n=11) After
Before
After
123.7±7 72.3±5.9 27.9±1.6 90.6±5.1 78.6±4.4 196.4±10.4 209.4±13.2
51±7 9/2 125.1±6.8 73.1±6.1 28.5±1.5 90.3±5.4 78.3±4.6 201.3±11.3 211.2±14.1
124.6±6.7 72.8±6.3 28.2±1.4 90.1±4.8 78.1±4.2 202.4±10.7 217.8±13.7
Student’s t test was used MAD mandibular advancement device, MADS successful MAD, MADF failure MAD, BMI body mass index, FEV1 forced expiratory volume in 1 s, FVC forced vital capacity
nitrite/nitrate concentrations were deduced from a standard curve. The lower limit of detection was 2.5 μM [18]. Endothelium-dependent flow-mediated dilation The FMD was measured by high-resolution B-mode ultrasonography, with a 7.0-MHz linear array transducer and Acuson 128XP/10 system (Mountain View, CA, USA). Longitudinal scans of the brachial artery at the dominant arm were obtained at rest. Afterward, forearm blood ischemia was achieved for 5 min by inflating a cuff to suprasystolic pressures. The cuff was deflated, causing a dilation of the brachial artery during reactive hyperemia, and arterial diameter was continuously monitored over a period of 3 min. The increase in vessel
diameter observed 60 s after cuff release was determined and expressed in relative percent values compared to baseline conditions. The FMD was defined as the percentage change in diameter between the initial scan and after cuff deflation [19]. Oral appliance Patients were referred from the Department of Chest Medicine for the fabrication of mandibular advancement devices after polysomnography examination and OSAS diagnosis. The temporomandibular joints (TMJ) and masticatory muscles were examined to evaluate the bilateral temporomandibular joint and muscular condition. The overjet between the labial surface of the maxillary and mandibular incisors was
Table 2 Results of sleep study before and after MAD Controls (n=15)
MADS (n=19)
MADF (n=11)
Before
After
Before
After
AHI ODI Mean SaO2, % Baseline SaO2, % Minimum SaO2, % Sleep architecture
3.2±0.9 1.7±0.7 97.8±0.4 98.9±0.4 93.6±1.1
31.5±11.4* 25.96±11.3* 92.9±0.5* 97.8±0.9 75.4±7.6*
8.6±5.7 6.9±5.7 96.4±0.4 98.4±0.8 84.7±6.4
31.8±11.6 26.7±10.7 92.6±0.5 97.7±1 75.4±5.6
24.7±5.9 23.1±8.7 92.7±0.5 98.3±0.9 76.8±4.4
Stage 1, % Stage 2, % Stage 3+4, % REM, % AI
8.4±3.7 53.8±4.7 16.7±3.5 21.1±3.6 3.2±0.6
21.2±8.0* 54.7±8.7 9.4±5.3* 14.7±5.7* 28.7±8.3*
9.5±5.3 53.3±7.7 16.8±6.1 20.4±5.5 8.6±3.4
20.4±6.0 55.2±7.8 10.1±5.9 14.3±5.8 30.7±7.8
21.7±5.2 54.3±5.4 9.1±4.8 14.9±5.6 21.9±7.6
Student’s t test was used MAD mandibular advancement device, AHI apnea-hypopnea index, ODI oxygen desaturation index, SaO2 arterial oxyhemoglobin saturation *p5). Treatment failure (MADF) was defined as 5.
improvement in all variables except for baseline SaO2 and percentage of stage 2 sleep. In contrast, the MADF group showed no significant improvement in the sleep study results after MAD.
Serum levels of NOx and FMD before and after MAD Prior to MAD, serum NOx level and FMD were lower in patients with OSAS than in controls. The serum levels of NOx in 19 of 30 patients with OSAS, designated MAD responders, increased from 11.8±5.8 μM pre-MAD to 22.7± 4.9 μM post-MAD. The FMD increased from 5.9±4.6 preMAD to 10.5±4.8 post-MAD. For the 11 unresponsive patients, serum NOx and FMD remained impaired after MAD (Fig. 1a, b).
Data analysis Student’s t test and analysis of variance (ANOVA) were used for statistical analysis where appropriate. If ANOVA showed statistically significant differences, Scheffe’s test was performed. All values were expressed as the mean±standard deviation. Significance was set at p
