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Atherosclerosis
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Objective: Non-dipper hypertensives had about three times the risk of atherosclerotic events than hypertensives whose blood pressure was >10% lower at night compared to daytime (dippers). Platelet activation and inammatory response may derive from most atherosclerotic events. Mean platelet volume (MPV) is a determinant of platelet activation and high sensitive C-reactive protein (hs-CRP) is the best candidate assay to identify and monitor the inammatory response. We aimed to determine whether MPV and hs-CRP levels are elevated in non-dipper patients compared to dippers and healthy controls. In addition, we tried to nd out if MPV and CRP are related to each other or not in non-dipper hypertensives. Method: The total 126 patients study group included 86 patients with hypertension and 40 healthy subjects (16 male, mean age; 51 4) as control. Ambulatory blood pressure monitoring was performed for all patients. Hypertensive patients were divided into two groups; 46 dipper patients (18 male, mean age; 50 9) and 40 non-dipper patients (17 male, mean age; 53 11). Clinical baseline characteristics were similar between groups. We measured mean platelet volume in a blood sample collected in EDTA tubes and high-sensitive CRP was measured by using BN2 model nephlometer. Results: Non-dipper patients demonstrated higher levels of MPV compared to dippers and normotensives (9.72 0.52 vs 9.38 0.33 and 8.92 0.42 , p < 0.05, respectively). High-sensitive CRP levels were also signicantly higher in non-dippers compared to dippers and normotensives (4.9 1.7 mg/l vs 3.8 1.5 mg/l and 2.7 0.8 mg/l, p < 0.05, respectively). There was signicant positive correlation between MPV and CRP levels (p = 0.002, r = 0.482) in non-dipper hypertensives. Conclusion: Our results suggest that patients with non-dipping tend to have increased platelet activation and inammatory response. Increased platelet activation and inammatory response could contribute to increase the atherosclerotic risk in non-dipper patients compared to dippers. 2009 Elsevier Ireland Ltd. All rights reserved.
Article history: Received 14 May 2009 Received in revised form 3 September 2009 Accepted 4 September 2009 Available online 12 September 2009 Keywords: Mean platelet volume C-reactive protein Non-dipper hypertension Platelet activation
1. Introduction Ambulatory blood pressure monitoring (ABPM) correlates more closely with cardiovascular events than clinical cuff measurements [1]. The signicant circadian variation in blood pressure can be identied with ABPM. This circadian variation presents a morning increase, small postprandial decline, more than 10% decrease in systolic and diastolic blood pressure during sleep compared to daytime. However, some hypertensive subjects who have not had this nocturnal reduction have been termed non-dippers [2]. Many studies have shown that non-dippers carry a high risk of cardiovascular disease such as atherosclerotic events (myocardial or cerebral infarction) and also high incidence of target organ damage compared to dippers [37].
Corresponding author. Tel.: +90 505 3784696; fax: +90 352 4373408. E-mail address: drmgkaya@yahoo.com (M.G. Kaya). 0021-9150/$ see front matter 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2009.09.010
As known, increased platelet activation plays an important role in the development of atherosclerosis [8]. Increased platelet activity is associated with increased platelet volume. Large platelets that contain more dense granules are metabolically and enzymatically more active than small platelets and higher thrombotic potential [9]. Mean platelet volume (MPV), a determinant of platelet activation, has been recognized as an independent risk factor of hypertension, myocardial infarction, and stroke [1014]. It is also with poor clinical result among survivors from cardiovascular disease [15,16]. The high sensitive C-reactive protein (hs-CRP) is currently the best applicant assay to identify and monitor the inammatory response. High sensitive C-reactive protein is also increased with hypertension, obesity, smoking, diabetes and metabolic syndrome. It is one of the potent predictor of rst cardiovascular events and also it predicts recurrent vascular events [17,18]. In our study, we aimed to determine whether MPV and CRP levels are elevated in non-dipper patients compared to dippers and
M.G. Kaya et al. / Atherosclerosis 209 (2010) 278282 Table 1 Characteristics, blood pressure, and laboratory parameters of study groups. Non-dippers (n = 40) Men/women Age, yrs Clinic SBP, mmHg Clinic DBP, mmHg Smoker/nonsmoker Fasting glucose, mg/dl Creatinine, mg/dl Total cholesterol, mg/dl LDL cholesterol, mg/dl HDL cholesterol, mg/dl Triglyceride, mg/dl Body mass index, kg/m2 MPV, CRP, mg/lt Platelet count, 103 /l ACE inh. use/not use ARB use/not use Beta-blocker use/not use Ca channel blocker use/not use Diuretic use/not use 17/23 53 11 149.2 30.2* 95.4 12.4* 9/31 84 9 0.80 0.16 196.2 38.0* 134.4 25.5* 44.4 10.2# 154.5 62.8 27.6 4.6* 9.72 0.52* , # 4.9 1.7* , # 302.1 110.0 22/18 12/28 10/30 8/32 19/21 Dippers (n = 46) 18/28 50 9 145.2 44.6* 92.6 13.3* 18/28 81 8 0.85 0.21 205.5 42.8* 140.5 32.2* 39.4 9.2 148.3 76.5 28.9 6.1* 9.38 0.33* 3.8 1.5 295.5 98.2 24/22 10/36 14/32 12/34 23/23
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Normotensives (n = 40) 24/16 51 6 116.8 9.7 68.6 12.2 16/24 80 10 0.76 0.20 182.0 17.5 108.2 16.2 42.4 7.2 123.4 11.4 26.8 3.7 8.92 0.42 2.7 0.8 328.8 93.2 /40 /40 /40 /40 /40
SBP: systolic blood pressure, DBP: diastolic blood pressure, MPV: mean platelet volume, CRP: C-reactive protein, ACE: angiotensin converting enzyme, ARB: angiotensin receptor blocker. * p < 0.05 non-dippers and dippers vs normotensives. # p < 0.05 non-dippers vs dippers.
healthy controls. In addition, we tried to nd out if MPV and CRP levels are related to each other or not in non-dipper hypertensive patients. 2. Method 2.1. Study population The total 126 patients study group included 86 patients with hypertension and 40 healthy subjects (16 males, mean age; 51 4) as controls in this prospective study. This study complied with the Declaration of Helsinki, was approved by the Ethics Committee and the institutional review board of Erciyes University Medical School, and informed consent was obtained from each patient. Physical examination and ambulatory blood pressure monitoring was performed for all patients. Hypertensive patients were divided into two groups; 46 dipper patients (18 males, mean age; 50 9) and 40 non-dipper patients (17 males, mean age; 53 11). Exclusion criteria for entry into the study were evidence of coronary artery disease, chronic heart failure, diabetes mellitus, renal or hepatic dysfunction, hematological disease, cancer, thrombocytopenia, systemic inammatory conditions, auto-immune disease and antithrombotic agents or serotonin reuptake inhibitory drug use. 2.2. Diagnostic criteria for hypertension and non-dipper Hypertension was considered to be present if the systolic pressure was >140 mm Hg and/or diastolic pressure was >90 mmHg or if the individual was taking antihypertensive medication [19]. According to the ambulatory monitoring, patients with less than 10% decrease in either systolic or diastolic pressure was considered to be non-dipper in this study [2]. 2.3. Biochemical measurements Blood samples were drawn in the morning after 20-min rest following a fasting period of 12 h. Glucose, creatinine, and lipid prole were determined by standard methods. Tripotassium EDTA based anticoagulated blood samples were drawn in the morning after 20-min rest, stored at 4 C and assessed
by Sysmex K-1000 auto analyzer within 30 min of sampling. Highsensitive CRP was measured by using BN2 model nephlometer (Dade-Behring). The expected values for CRP in our laboratory ranged from 0 to 6 mg/l. 2.4. Statistical analysis Statistical analysis was done by using SPSS 13.0 statistical software. Adequacy of all parameters to normal distribution, was tested by using KolmogorovSmirnov Test. Parametric tests were applied to with normal distribution, non-parametric tests were used to without normal distribution. Variables that match with normal distribution were given as mean SD. ANOVA test is used for statistical comparing of data that match with normal distribution, Kruskal Wallis test is used for data that not match with normal distribution among three groups. MannWhitney U test is applied to compare data that without normal distribution between to groups. Results of data that without normal distribution are shown by median (minimummaximum) expression. Spearman correlation coefcients examined the degree of association between examined variables. Statistical signicance was dened as p < 0.05. 3. Results The patient characteristics for non-dipper, dipper and normotensives groups are presented in Table 1. Age, gender distribution, creatinine, fasting glucose levels, and platelet count were similar among the groups. There were no signicant differences in triglyceride levels in non-dippers and dippers compared to normotensives (154.5 62.8 mg/dl and 148.3 76.5 mg/dl vs 123.4 11.4 mg/dl) but total cholesterol and LDL cholesterol levels were signicantly higher in non-dippers and dippers compared to normotensives (196.2 38.0 mg/dl and 205.5 42.8 mg/dl vs 182.0 17.5 mg/dl, p < 0.05 and 134.4 25.5 mgr/dl and 140.5 32.2 mg/dl vs 108.2 16.2 mg/dl, p < 0.05, respectively). Non-dippers demonstrated a signicantly higher HDL levels than dippers surprisingly (44.4 10.2 mg/dl vs 39.4 9.2 mg/dl, p < 0.05). Dipper and non-dipper patients had similar body mass indexes but higher than normotensives (27.6 4.6 kg/m2 and 28.9 6.1 kg/m2 vs 26.8 3.7 kg/m2 , p < 0.05).
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As expected, clinical blood pressure was signicantly higher in non-dipper and dipper patients than normotensives (systolic blood pressure; 149.2 30.2 mmHg and 145.2 44.6 mmHg vs 116.8 9.7 mmHg, diastolic blood pressure; 95.4 12.4 mmHg and 92.6 13.3 mmHg vs 68.6 12.2 mmHg, p < 0.05, respectively) but was similar between dippers and non-dippers. As reported in the Table 1 and Fig. 1, non-dipper patients demonstrated higher levels of MPV compared to dippers and normotensives (9.72 0.52 mmHg and 9.38 0.33 mmHg vs 8.92 0.42 mmHg, p < 0.05, respectively). As shown in Table 1 and Fig. 2, non-dippers had signicantly higher CRP levels compared to dippers and normotensives (4.9 1.7 mg/lt vs 3.8 1.5 mg/lt and 2.7 0.8 mg/lt, p < 0.05 respectively). There was signicant positive correlation between MPV and CRP levels (p = 0.002, r = 0.482) in non-dipper hypertensives, as seen in Fig. 3.
As shown in Table 2, in non-dipper patients, 24-h systolic blood pressure (142.5 10.6 mmHg vs 134.3 8.3 mmHg, p < 0.001), 24h diastolic blood pressure (90.2 8.9 mmHg vs 82.3 8.8 mmHg, p < 0.01), and 24-h average blood pressure (107.2 9.5 mmHg vs 98.7 8.5 mmHg, p < 0.001) are signicantly higher than dippers. Although, daytime measurements were similar between dippers and non-dippers, there was a signicant difference between each group during nighttime measurements (nighttime systolic BP; 139.4 10.5 mmHg vs 125.2 10.0 mmHg, p < 0.001 and nighttime diastolic BP; 88.3 7.5 mmHg vs 74.8 8.2 mmHg, p < 0.001). There was no signicant difference for antihypertensive medications taken per dipper and non-dipper groups. In non-dippers group, 9 patients took only one drug, 31 patients took two drugs together and nobody took three drugs treatment. In dippers group, 13 patients took only one drug, 29 patients took two drugs together and 4 patients took three drugs treatment. Normotensives did not take any medicine. 4. Discussion Hypertension is a common chronic condition and important factor for heart attacks, strokes and other vascular and target organ damage [19,20]. During last 20 years clinical investigations have demonstrated an important correlation between ABPM readings and prevalence and extent of cardiovascular events. In the rst published study of outcomes in central Italy, ABPM was the best predictor of atherosclerotic events; non-dipper hypertensives had approximately three times the risk of hypertensives whose blood pressure was >10% lower at night compared to daytime (dippers) [21,22]. It is very important to nd responsible pathophysiological conditions which may be cause of this risk rise. Platelet activation and inammatory response may derive from most atherosclerotic events. Hemostatically reactive platelets mean larger platelets have more granules and adhesion receptors that have resulted in increased platelet volumes [23]. The MPV correlates closely with platelet size and activity. It has become a prognostic factor in coronary heart disease [24]. There are some studies about MPV and hypertension relationship. Nadar et al. found that MPV in patient with hypertension was signicantly higher than normotensives control subjects, and within hypertensive group those with larger platelets with greater mass than those without target organ damage [25]. Giles and Inglis et al. reported that the MPV in patients with gestational hyper-
Table 2 Comparison of ambulatory blood pressure monitoring results between dippers and non-dippers. Non-dippers (n = 40) Daytime systolic (mmHg) Daytime diastolic (mmHg) Nighttime systolic (mmHg) Nighttime diastolic (mmHg) 24-h SBP (mmHg) 24-h DBP (mmHg) 24-h Average BP (mmHg) 146.4 88.9 139.4 88.3 142.5 90.2 107.2 9.6 10.2 10.5 7.5 10.6 8.9 9.5 Dippers (n = 46) 142.8 88.3 125.2 74.8 134.3 82.3 98.7 9.2 9.6 10.0 8.2 8.3 8.8 8.5 p value 0.52 0.68 <0.001 <0.001 <0.001 <0.01 <0.001
SBP: systolic blood pressure, DBP: diastolic blood pressure, BP: blood pressure.
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tension was signicantly higher than in normal pregnant women [26]. Coban et al. found that MPV was positively correlated with ambulatory diastolic blood pressure in essential hypertension and white coat hypertension groups [27]. And Tavil et al. reported that MPV in hypertensive or pre-hypertensive patients with metabolic syndrome is associated with coronary artery disease [28]. To our knowledge, this is the rst study to evaluate MPV levels in non-dipper. We found that the MPV levels were signicantly higher in the non-dipper group than in dipper and healthy group. Our results suggest that patients with non-dipping tend to have increased platelet activation. Increased platelet activation could contribute to increase the atherosclerotic risk in non-dipper patients compared to dippers. In non-dippers we also found higher C-reactive protein levels which are one of the best indicators for inammation that typify all phase of atherothrombosis like widespread coronary inammation in unstable angina [29,30]. We used hs-CRP levels to show inammatory response because recent studies have focused on the use of hs-CRP, a marker of inammation in the detection of patients at increased risk for cardiovascular disease. Several prospective studies have demonstrated that hs-CRP is an independent predictor of future risk for cardiovascular events among healthy individuals, as well as among patients with acute coronary syndromes [31]. Previous studies show that essential hypertensive non-dippers compared to dippers exhibit higher hs-CRP values [32]. Our ndings have conrmed this previous study. The present study also demonstrates, for the rst time, the signicant positively correlation between MPV and hs-CRP levels in non-dipper hypertensive patients. As we know, most of vascular complications depend on endothelial damages which initiate thrombosis. This process involves thrombus formation, which is contributed to increased platelet activation and activated inammatory cells. Several studies focused on molecular interaction between activated platelets and inammatory cells [3336]. Activated platelets play a predominant role in the initiation and progression of atherogenesis [33]. According to the many studies investigating the role of platelets in atherosclerosis, involves many of the molecules belongs to activated platelets. In the one of animal study, P-Selectin is determined as adhesion molecule of platelets leading to prolonged contact with endothelial cells that activate monocytes and aggravate atherosclerotic lesions in mice [34]. The strong proinammatory mediator IL-1 is secreted by activated platelets and has an impact on atherosclerosis that induces alterations of the adhesive and chemotactic properties of endothelial cells [35]. Primarily described proatherogenic chemokine MCP-1 are also found in activated platelets. The involvement of glycoprotein Ib (GPIb) in activated platelets may encompass binding to von Willebrand factor to support platelet adhesion or to leukocyte Mac-1 to support platelet/leukocyte aggregates. [36]. As these all studies has shown the effects of platelets in leukocytes functions, it is not astonishing to determine positive correlation between MPV, the indicator of platelet activation and CRP, the indicator of inammatory response, in our study. One of the limitations of this study may be method of MPV measurement. Tripotassium EDTA based anticoagulated blood samples were used to measure MPV levels in our study. Most laboratories use EDTA for anticoagulation of whole blood prior to automated cell counting but due to platelet swelling, mean platelet volume (MPV) values may increase with its use. Dastjerdi et al. found that MPV can be measured accurately by using both methods of anticoagulation, EDTA and citrate if analysis be performed within 1 h of sampling [37]. Macey et al. also showed the changes in MPV, which reect platelet sphering and swelling, were greatest between 30 and 60 min in blood stored at ambient temperature [38]. Whereas in our study, blood samples were drawn in the morning after 20-
min rest, stored at 4 C and measurement performed within 30 min. So, our study had adequate condence about results. Moreover, we did not offer a cut-off value for MPV; we compared groups between each other in the terms of MPV levels which were determined by using same methodology for all patients. The other limitation of this study is absence of clinical follow up for non-dipper patients with elevated MPV levels. Our ndings may inspire to further clinical studies with follow-up. In conclusion, our results suggest that patients with nondipper tend to have increased platelet activation and inammatory response. Increased platelet activation and inammatory response could contribute to increase the atherosclerotic risk in non-dipper patients compared to dippers. Conict of interest The authors have no conicts of interest to disclose. References
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