Erythrocyte deformability, plasma viscosity and oxidative status in patients with severe obstructive sleep apnea syndrome

Sleep Medicine 7 (2006) 255–261 www.elsevier.com/locate/sleep

Original article

Erythrocyte deformability, plasma viscosity and oxidative status in patients with severe obstructive sleep apnea syndrome Neslihan Dikmenog˘lu a,*, Bu¨lent C¸iftc¸i b, Esin ˙Ileri a, Selma Fırat Gu¨ven b, Nurten Seringec¸ a, Yasemin Aksoy c, Dilek Ercil d b

a Department of Physiology, Faculty of Medicine, Hacettepe University, 06100 Ankara, Turkey Atatu¨rk Chest Diseases and Chest Surgery Research and Education Hospital, Sleep Disorders Center, Ankara, Turkey c Department of Biochemistry, Faculty of Medicine, Hacettepe University, Ankara, Turkey d Department of Pharmacognosy, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey

Received 8 June 2005; received in revised form 28 November 2005; accepted 1 December 2005

Abstract Background and purpose: In patients with severe obstructive sleep apnea syndrome (OSAS), diurnal changes of plasma viscosity and erythrocyte deformability were measured to elucidate the possible mechanism of cardiovascular diseases in OSAS patients. Patients and methods: Plasma viscosity and erythrocyte deformability was determined in 11 OSAS patients and 11 healthy subjects matched by sex and age. Plasma viscosity was measured by a cone-plate viscometer, and erythrocyte deformability was determined by filtration technique. Whole blood counts were performed and oxidative status of the patients’ plasma and erythrocytes were evaluated. Results: OSAS patients had higher plasma viscosity than controls, both in the morning (1.74G0.3 vs. 1.36G0.2 mPa s, P!0.002) and evening (1.55G0.2 vs. 1.27G0.1 mPa s, P!0.002), and morning plasma viscosity was significantly higher than the evening level (P!0.05). Morning plasma viscosity of patients was inversely correlated with their mean nocturnal SaO2. Morning plasma malonyldialdehyde level was significantly higher in the patients than in the controls (69.7G30.5 vs. 45.5G11.0 nmol/l, P!0.005). Erythrocyte deformability of the patients was slightly lower. Conclusions: We have observed that plasma viscosity is high both in the morning and in the evening in severe OSAS patients. This elevation may predispose OSAS patients to myocardial infarction and stroke by increasing blood viscosity. Low nocturnal mean SaO2 may be responsible for the high plasma viscosity in these patients. q 2006 Elsevier B.V. All rights reserved. Keywords: Hemorheology; Plasma viscosity; Erythrocyte deformability; Oxidative stress; Sleep apnea; Hematocrit; 2,3 DPG

1. Introduction Obstructive sleep apnea syndrome (OSAS) is characterized by repeating partial or complete obstructions of airways during sleep and intermittent cessation (apnea) or slowing down (hypopnea) of airflow despite ongoing respiratory efforts. Insufficient alveolar ventilation results in decreased O2 saturation and CO2 retention [1]. OSAS, one of the most

* Corresponding author. Tel.: C90 312 305 2185/1567; fax: C90 312 3052185. E-mail addresses: [email protected] (N. Dikmenog˘lu), [email protected] (N. Dikmenog˘lu).

1389-9457/$ - see front matter q 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.sleep.2005.12.005

commonly encountered respiratory disorders in humans, affects 2–4% of adults [2]. OSAS is associated with high risk of hypertension, coronary artery disease, and cerebrovascular diseases [3–10]. In addition, these vascular diseases commonly occur during morning hours [11,12], and OSAS has been blamed for this increased morning incidence [13]. OSAS has been suggested to negatively affect the functional recovery after stroke [14]. The reason underlying this association has not been clearly understood. Increased sympathetic activity, hypoxemia, hemoconcentration, changes in blood pressure, and altered cerebral blood flow are among the mechanisms that have been blamed. Hemorheologic properties of blood should also be considered when one examines circulatory problems.

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Hemorheology is concerned with the flow properties of cellular and plasmatic components of blood. The resistance of blood to flow is known as blood viscosity. High blood viscosity slows down the blood flow and results in stasis and occlusion. Blood viscosity is determined by hematocrit, plasma viscosity, erythrocyte aggregation and cellular deformability. Since erythrocytes make up approximately 99% of the blood cells, erythrocyte deformability is an important parameter [15]. Normal erythrocytes are significantly deformable. This property enables them to adapt well to flow conditions throughout the circulation. Due to the deformability of the erythrocytes, blood viscosity is lower than the viscosity of a fluid containing non-deformable particles of the same size. When erythrocytes become less deformable the viscosity of blood increases. The deformability of an erythrocyte is determined by three factors: viscoelasticity of its membrane which is controlled by a network of cytoskeletal proteins, biconcave-disk cell shape with a large ratio of surface area/cell volume, and intracellular viscosity which is mainly determined by hemoglobin concentration [16]. The viscosity of plasma depends on its macromolecular content, the plasma proteins in particular. Among the plasma proteins, fibrinogen is the one that most affects plasma viscosity. Although fibrinogen constitutes a very small portion of the plasma proteins, its thin and long shape make it an important determinant. Simple measurement of the fibrinogen level thus may give an idea about plasma viscosity. However, protein–protein interactions also have important effects on plasma viscosity, especially during pathological conditions. Determination of plasma viscosity by a viscometer not only reflects the effect of fibrinogen but that of protein–protein interactions as well [17]. Changes in blood viscosity and its components have been reported to be related to myocardial infarction and stroke [18,19]. Whether such changes occur more commonly in the morning hours and whether OSAS is related to the condition is a matter of debate. This study aimed to reveal possible diurnal changes in plasma viscosity and erythrocyte deformability in severe OSAS patients.

2. Methods 2.1. Patients and controls Among the patients who had applied to our center (Atatu¨rk Chest Disases and Chest Surgery Research and Education Hospital, Sleep Disorders Center, Ankara, Turkey) between October 2003 and May 2004, 11 who had severe OSAS (AHIO30) and agreed to participate were included in this study. Since the male/female ratio, age distribution and BMI comply with that of the general OSAS population [1], this group may represent the severe OSAS patients. Four of the patients had diabetes mellitus, and five

had hypertension and were using various drugs. Only two of the patients were smokers. The control group consisted of the hospital personnel who volunteered to participate. They were 11 non-smoking healthy subjects, age- and sex-matched with the patient group. All the subjects were questioned about the three major symptoms of OSAS (snoring, witnessed apnea, excessive daytime sleepiness) and none of them had any of these symptoms. They were also given the Epworth Sleepiness Scale (ESS). Those with an ESS score greater than 10 were excluded; scores lower than or equal to 10 are considered to be normal, and scores higher than 10 are considered to be pathological [20]. The study was approved by the local ethics committee (Hacettepe University Faculty of Medicine, Local Ethics Committee for Clinical and Drug Research). All patients and controls signed a written informed consent form. 2.2. Polysomnography Overnight polysomnography (PSG) was performed on all patients by a computerized system (Rembrandt, Holland) and included the following variables: electrooculogram (EOG) (two channels), electroencephalogram (EEG) (four channels), electromyogram (EMG) of submental muscles (two channels) and anterior tibialis muscle of both legs (two channels); electrocardiogram (EKG) and airflow (with an oro-nasal thermistor). Chest and abdominal efforts (two channels) were recorded using inductive plethysmography, arterial oxyhemoglobin saturation (SaO2: one channel) by pulse oximetry with a finger probe. The recordings were conducted at a paper speed of 10 mm/s, and sleep stages were scored according to the standard criteria of Rechtschaffen and Kales [21]. Arousals were scored according to accepted definitions [22]. Apneas were defined as complete cessation of airflow R10 s. Hypopneas were defined as reduction of O50% in one of the three respiratory signals, airflow signal or either thoracic or abdominal signals of respiratory inductance plethysmography, with an associated fall of R3% in oxygen saturation or an arousal [23]. The apnea–hypopnea index (AHI) was defined as the number of apneas and hypopneas per hour of sleep. Patients with AHIR5 were considered OSAS. An AHI of R5 to !15 indicated mild OSAS, R15 to !30 indicated moderate OSAS, and O30 indicated severe OSAS. Patients with sleep disorders other than OSAS, such as upper airway resistance syndrome, periodic legs movement syndrome or narcolepsy, were excluded. 2.3. Hemorheological and hematologic measurements Venous blood samples were drawn in the morning (8.30–9.00) and afternoon (16.30–17.30) into heparinized injectors and were immediately taken to a room with a temperature of 4 8C, where samples were prepared for measurements. Measurements were completed within 2 h.

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Venous blood samples were centrifuged in order to obtain plasma for viscosity measurements. Buffy coat was first removed manually; the remaining erythrocytes were suspended in isotonic phosphate buffer (pH 7.4) and were filtered twice through Imugard III-RC filter to remove the remaining leukocytes. Finally, 10% erythrocyte suspensions were prepared in isotonic phosphate buffer. Erythrocyte deformability was measured by a device (developed at the Department of Physiology with Hacettepe University Grant—project no. 96.01.101/002) that works on the basis of the filtration technique. It uses a fiber–optic system to measure the time required for 1 ml of erythrocyte suspension to pass through a polycarbonate filter with pores of 5 mm diameter (Whatman–Nuclepore), under gravitational force, at 37 8C. The pores of the filter provide a good approximation to the capillary system. A shorter filtration time represents easy passage through the pores of the filter due to better erythrocyte deformability. Time required for the passage of erythrocyte suspension was referred to as t1, and for that of the buffer solution as t2. Deformability indices (DI) were calculated by the formula DIZt1/t2. An increase in DI indicates a decrease in erythrocyte deformability. Less deformable erythrocytes pass through the pores with difficulty, which increases the filtration time and increases the DI [24]. Plasma viscosity was measured by a cone-plate viscometer (Wells-Brookfield LVT), at 37 8C and at six different shear rates ranging between 11.25 and 450 sK1. For each sample, the average of the six measurements was taken. Complete blood count was made for each sample. Hemoglobin (Hb), hematocrit (Htc), leukocyte, erythrocyte counts, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW) were determined by an electronic blood counter (Coulter). Blood 2,3-DPG level was determined spectrophotometrically via enzymatic reactions (Roche-Kit, Roche Diagnostics Cat. no. 148 334). Reduced glutathione (GSH) was measured through total sulfhydryl (–SH) groups using Elman Reagent (DTNB). Hemolysate (1:10, v/v) was deproteinized with 5% trichloroacetic acid. The resulting precipitate was removed by centrifugation, and supernatant was used for GSH determination at 412 nm. GSH levels were calculated as micromole per gram of hemoglobin [25]. Plasma and erythrocyte malondialdehyde (MDA) levels were determined by high-performance liquid chromatography (HPLC), after the thiobarbituric acid reaction [26]. HPLC investigations were performed on a HP Agilent 1100 Series HPLC instrument. A 15 cm length!4.6 mm internal diameters (ID) reversed-phase column (HiChrom, Nucleosil 120-5C18) was used under ambient temperature conditions. A guard column (HiChrom, NC120-5C18-10C) was used to safeguard the analytic column. The mobile phase was isotonic phosphate buffer/methanol (65:35, v/v), flow rate 1 ml/min, was prepared freshly and filtered through a

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0.45 mm filter (agilent nylon filter membranes 47 mm 0.45 mm) and degassed for 15 min in an ultrasonic bath before using. Furthermore, 20 ml samples were first filtered through 0.45 mm filter and then were applied to the HPLC column. Quantification was made by measuring the absorbance at 532 nm as established from the threedimensional chromatogram. Peak areas were integrated automatically by computer using the HPChemStation software program. 2.4. Statistics Comparison of morning and evening values within each group was made using Wilcoxon signed ranks test. Comparisons between groups were made using Mann– Whitney U-test. Correlation between different parameters was determined by Spearman correlation test. All statistical analyses were done using SPSS 11.01 for windows. Results are expressed as meanGSD. P values smaller than 0.05 were accepted to be statistically significant. When the morning plasma viscosity values of control and patient groups are considered, the power of 95% sample size of 11 will be enough to detect a difference of K0.4. When the evening plasma viscosity values of control and patient groups are considered, the power of 95% sample size of 11 will be enough to detect a difference of K0.3.

3. Results General characteristics of patients and controls are given in Tables 1 and 2. All the patients had severe OSAS, and their mean AHI value was found to be 55.2G24.5 hK1. Their mean nocturnal SaO2 was 91.0G2.2% and daytime SaO2 was 94.8G2.0%. ESS was 3.9G2.1 in controls (!12 in all) and 10.8G5.8 (!10 in 45.5%, 10–12 in 9%, O12 in 45.5%) in patients. The groups were age- and sex-matched. The mean body mass index (BMI) value of the patient group (31.1G3.7) was significantly (P!0.05) higher than the control group (26.6G3.1). Hematologic profiles of the groups are given in Table 3. There was no significant difference in the morning and evening values of leukocyte and erythrocyte counts, Hb, Htc, MCV, MCH, MCHC of both groups. Leukocyte counts of the patients were slightly higher than the controls, although this difference did not reach statistical significance. Morning RDW value of the patient group was significantly higher compared with the control group (P!0.05). When the plasma viscosity of patients and controls were compared, both morning and evening values of the patients were found to be significantly higher (P!0.002). Within the patient group, plasma viscosity in the morning was significantly higher than the evening (P!0.05). There was no significant difference between the morning and evening

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258 Table 1 General characteristics of the patients Patient no.

Gender

Age (years)

BMI (kg/m2)

Epworth sleepiness scale

AHI

Mean SaO2 night (%)

Mean SaO2 day (%)

1 2 3 4 5 6 7 8 9 10 11

M F M M M M M F M F M

46 61 43 42 50 56 42 39 57 59 57

30.1 38.7 31.7 29.7 33.8 24.9 28.4 33.6 30.5 27.3 33.0

5 1 11 16 14 8 6 17 13 8 20

56.5 51.3 41.8 33.4 92.4 34.0 56.0 56.9 34.7 41.4 109.0

90 92 94 92 86 92 90 92 93 91 89

96 97 96 97 94 97 93 93 95 94 91

values in controls. Erythrocyte deformability indices, 2–3 DPG, GSH, and erythrocyte MDA levels were similar. Patient plasma MDA levels in the morning were significantly higher when compared with evening levels (P!0.005). The results of the two hemorheologic parameters and the factors that may affect them are given in Table 4. The morning plasma viscosity values of the patients revealed a negative correlation with the mean nocturnal SaO2 (P!0.05; rZK0.648) (Fig. 1) and minimum SaO2 (P!0.05; rZK0.731), and a positive correlation with the time spent below 80 and 90% SaO2 (P!0.05; rZ0.651 and P!0.05; rZ0.655, respectively).

4. Discussion In this study, plasma viscosity, erythrocyte deformability and a group of factors, which may alter these two hemorheologic parameters were examined in severe OSAS patients. Plasma viscosity in these patients was higher both in the morning and in the evening. It improved during the day but did not reach the level observed in the control group. Morning plasma viscosity in the patient group is inversely correlated with the mean nocturnal SaO2. Morning plasma MDA level was significantly higher in the Table 2 General characteristics of the controls Control

Gender

Age (years)

BMI (kg/m2)

Epworth sleepiness scale

1 2 3 4 5 6 7 8 9 10 11

M M F M F M F M M M M

49 39 39 40 54 60 58 47 36 49 38

32.0 27.8 23.7 24.9 26.6 30.0 28.0 23.8 29.0 24.1 21.8

4 8 6 0 4 3 2 3 5 3 5

patient group. Erythrocyte deformability and other hematologic parameters revealed no significant difference. The high plasma viscosity observed in our patients may be due to a rise in fibrinogen concentration. Nobili et al. and Chin et al. have reported that plasma fibrinogen concentration in OSAS patients in the morning was significantly higher than that in the afternoon [27,28]. This could be a transient elevation due to high sodium and water output during the night, or it may be due to a continuous increase in fibrinogen formation. This transient condition may explain the high morning levels and the partial improvement of plasma viscosity during the day but not the elevated viscosity we have observed in the evening. Another mechanism is needed to explain the continuous rise in plasma viscosity. In accordance with this view, Chin et al. have reported that nocturnal urine volume did not correlate with the difference in the plasma fibrinogen levels between morning and afternoon, and other factors should be considered [28]. A continuous rise in fibrinogen concentration may be due to increased production of fibrinogen during the inflammatory process that is present in these patients [29]. Reinhart et al. have reported that plasma viscosity and fibrinogen levels were higher in OSAS patients being treated with nasal continuous positive airway pressure (nCPAP), both in the evening and in the morning [30]. No change was observed when nCPAP was discontinued for 1 day. Since, nCPAP only partially abrogates local and systemic inflammation in these patients [29], results of their study support our view. The second possible explanation for high plasma viscosity may be a change in protein–protein interactions in the plasma. In the present study, high morning plasma MDA level in severe OSAS patients supports this possibility. MDA, which is an end product of lipid peroxidation, is known to cross-bind or induce secondary oxidative damage in the plasma proteins [31]. To our knowledge our results are the first to report high plasma viscosity measured directly in untreated and severe OSAS cases. We also report a negative correlation between nocturnal mean SaO2 and plasma viscosity. The intermittent hypoxia in OSAS is believed to initiate oxidative stress,

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Table 3 Hematologic profile Control

Leukocyte (103/ml) Erythrocyte (106/ml) Hb (g/dl) Hct (%) MCV (fl) MCH (pg) MCHC (g/dl) RDW (%)

Patient

Morning

Evening

Morning

Evening

6.1G1.7 4.9G0.5 14.3G0.9 42.1G3.0 85.6G3.0 29.2G1.5 34.1G0.8 13.0G0.4

6.4G1.2 4.5G0.5 13.8G1.0 38.7G4.2 85.5G3.3 30.6G2.6 35.9G3.1 13.0G0.3

7.8G2.6 4.8G0.5 13.6G1.6 40.6G4.0 85.3G8.3 28.9G2.7 33.4G1.2 13.7G1.0*

7.8G1.4 4.7G0.6 13.4G1.8 40.0G4.7 84.9G8.0 28.4G3.2 33.4G1.0 13.6G1.0

Evening

Morning

*P!0.05.

Table 4 Hemorheologic parameters and factors which may affect them Control Morning Plasma viscosity (mPa s) Deformability index 2–3 DPG (mmol/l erythrocytes) GSH (mmol/g Hb) Erythrocyte MDA (nmol/g Hb) Plasma MDA (nmol/l)

Patient

1.36G0.2 1.61G0.3 5.33G1.0

1.27G0.1 1.59G0.3 5.94G1.2

10.36G2.8 410G117

10.40G3.0 472G137

45.5G11.0

43.6G13.0

Evening ,

1.74G0.3* ** 1.69G0.6 5.21G1.1

1.55G0.2* 1.67G0.5 5.58G0.7

10.58G5.2 364G124

12.35G4.1 401G159

69.7G30.5**

45.2G8.9

*P!0.002, **P!0.05.

found no clinical polycythemia, but have found minor elevations in hematocrit value, and they have concluded that these small elevations were unlikely to be useful as markers of hypoxic stress associated with sleep apnea. The higher leukocyte count in our patients is in concordance with the finding of Hoffstein et al. [43]. Since RDW is a measurement of the variability of red cell size, higher values indicate greater variation in size. There may be two explanations for the high RDW in OSAS patients. High RDW may indicate anisocytosis that may be 2.5

Plasma Viscosity (mPa.s)

similar to hypoxia-reperfusion injury [32]. The high plasma MDA levels of the OSAS patients may reflect such an effect. The DI in the patient group was slightly higher, suggesting lower erythrocyte deformability. In addition, 2, 3 DPG and MDA which have been reported to adversely affect the deformability of erythrocytes [33–36], revealed no significant difference between severe OSAS patients and controls. Although there are reports about a morning increase in blood 2,3-DPG levels in OSAS patients [37,38], we have not observed an appreciable change. Our findings on erythrocyte MDA and GSH levels support the findings of Ozturk et al. [39], who have compared the morning levels of OSAS patients with controls and have found no difference. MCV and MCHC are two important determinants of erythrocyte deformability. Neither the MCHC, which determines the internal viscosity of the erythrocytes nor the MCV, which determines the surface area/volume relationship have revealed any significant difference. There was no significant difference between the morning and evening Htc levels in both patients and controls. Chin et al. have reported higher morning Htc levels in OSAS patients, which were restored to normal after nCPAP treatment [28]. Other studies that have examined Htc levels in OSAS patients have not measured the evening values but have reported decrease in morning Htc levels after nCPAP treatment [40–42]. More importantly, Hoffstein et al. have

2 1.5 1 0.5 0 84

86

88

90

92

94

96

Mean nocturnal O2 Saturation (%) Fig. 1. Correlation between mean nocturnal O2 saturation and plasma viscosity.

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due to reticulocytosis. The other explanation is hemorheological. RDW is determined by the measurement of the MCV of a group of erythrocytes. MCV is determined by electronic cell counter (Coulter) by the measurement of impedence while the red cells pass through an orifice. This method is very similar to the filtration method by which erythrocyte deformability is determined. Therefore, the MCV and RDW measurements are influenced by erythrocyte deformability [44]. The high RDW values we have observed in our OSAS patients may reflect the slight increase in the erythrocyte deformability indices. Although PSG was not performed in the control group, the subjects were evaluated with the ESS and questioned about the three major symptoms of OSAS. The results of these interpretations minimized the possibility of a breathing disorder during sleep. Another weakness of this study is that diabetic and hypertensive patients in the OSAS group are not matched with the control group. This is a pilot study, and a better conclusion may be reached with further studies with a greater patient population with no diabetics and hypertensives. These studies should as well include fibrinogen and lipid profile determinations in order to explain the causes of the high plasma viscosity levels. In conclusion, we have observed that plasma viscosity is high both in the morning and in the evening in severe OSAS patients. Being especially high in the morning, this condition may predispose patients to myocardial infarction and stroke by increasing the blood viscosity. Low nocturnal mean SaO2 may be responsible for high plasma viscosity in these patients.

Acknowledgements Hacettepe University Grant, Project no. 03.02.101.008.

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