The value of serum asymmetric dimethylarginine levels for the determination of masked hypertension in patients with diabetes mellitus

Atherosclerosis 228 (2013) 432e437

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The value of serum asymmetric dimethylarginine levels for the determination of masked hypertension in patients with diabetes mellitus Alpaslan Taner a, *, Ali Unlu b, Mehmet Kayrak c, Mehmet Tekinalp d, Selim S. Ayhan e, Alpay Arıbas¸ c, Said Sami Erdem f a

Department of Biochemistry, Dr Faruk Sükan Maternity and Children’s Hospital, Konya, Turkey Department of Biochemistry, Selcuk University, Selcuklu School of Medicine Hospital, Selcuklu, Konya, Turkey Department of Cardiology, Necmettin Erbakan University, Meram School of Medicine Hospital, Meram, Konya, Turkey d Department of Cardiology, Beyhekim Government Hospital, Selcuklu, Konya, Turkey e Abant Izzet Baysal University, School of Medicine, Department of Cardiology, Bolu, Turkey f Department of Biochemistry, Konya Training and Research Hospital, Konya, Turkey b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 June 2012 Received in revised form 5 January 2013 Accepted 18 February 2013 Available online 13 March 2013

Background: An increased prevalence of masked hypertension (MHT) has been demonstrated among patients with diabetes mellitus (DM). MHT appears to cause cardiovascular (CV) complications similar to clinically overt hypertension. Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide inhibitor and higher plasma levels of ADMA are related to increased CV risk in both the general population and among patients with DM. The aim of this study was to evaluate the relationship between MHT and ADMA in diabetic patients. Methods: This study included DM patients (n ¼ 131) with normal office blood pressure (<140/90 mmHg). None of the participants were using antihypertensive medications. All participants utilized an ambulatory blood pressure monitor (ABPM) for 24 h. Serum ADMA and arginine levels were measured using the fluorescence detector high performance liquid chromatography method. Results: The prevalence of MHT was 24.4% among the study subjects. ADMA levels were increased in the MHT group when compared with normotensive diabetics (6.2  2.2 vs 4.2  1.7 mmol/L p ¼ 0.001, respectively). Furthermore, arginine/ADMA ratio was lower in the MHT group than among the normotensive group (29.9  12.1 vs 46.0  19.0 p ¼ 0.001). In the multivariate logistic regression model, ADMA, BMI and HDL levels were found to be independent predictors of MHT Odds ratio: 1.63 (1.28e2.06), 1.19 (1.05e1.35), and 0.95 (0.90e0.99), respectively. The cut-off value of the ADMA was 4.34 mmol/L with a sensitivity, specificity, positive predictive value, and negative predictive value of 84.4%, 59.6%, of 40.3%, and 92.2%, respectively (AUC ¼ 0.78). Conclusions: Serum ADMA may play a role in both the pathophysiology and screening of MHT in DM subjects. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: ADMA Masked hypertension Blood pressure Diabetes mellitus Type 2

1. Introduction Nitric oxide (NO) is an important critical vasoactive mediator synthesized by the vascular endothelium previously referred to as endothelium-derived relaxing factor [1]. NO is synthesized from Larginine by NO synthase. Endothelium derived NO is a powerful endogenous vasodilator and also has an important role in the maintenance of vascular homeostasis [2]. Endothelial dysfunction

* Corresponding author. Tel.: þ90 5052603129, þ90 3322244805 (office). E-mail address: [email protected] (A. Taner). 0021-9150/$ e see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2013.02.024

with a reduced bioavailability of endothelium-derived NO plays an important role in the pathogenesis of diabetic vascular disease, which is a major cause of morbidity and mortality in type 2 diabetes [3]. ADMA, a methylated L-arginine analog, is a major endogenous competitive inhibitor of NO [4]. ADMA levels are increased in several clinical conditions such as diabetes mellitus (DM) (two to three fold), hypertension (two fold), chronic renal failure, insulin resistance syndrome, dyslipidemia, stroke, congestive heart failure, and acute coronary events [5,6]. In addition, increasing ADMA levels were associated with target organ damage including retinopathy, nephropathy, cardiac hypertrophy and cardiovascular events in patients with DM [7].

A. Taner et al. / Atherosclerosis 228 (2013) 432e437

Masked hypertension (MHT) is clinically defined as office blood pressure (BP) levels lower than 140/90 mmHg and daytime BP  135/85 using ambulatory BP monitoring (ABPM) [8]. Patients with MHT have a similar risk of developing CV disease to patients with sustained hypertension [9]. While the prevalence of MHT is accepted as 8e10% among the general population, incidence is increased about two to six times among the diabetic population [10,11]. In the current literature, MHT is associated with increased macrovascular and microvascular complications in patients with DM [10]. On the other hand, ADMA levels are related to both complications of DM and vascular tonus. Diagnosis of masked hypertension was performed by 24 h ABPM application in suspicious patients. However, ABPM is not a widely available or inexpensive method and is uncomfortable for some patients. About 20e30% of patients with normotensive DM exhibit MHT [10e12]. Since it has been clearly demonstrated that the presence of MHT increases CV risk, diagnosis of masked HT is critically important in diabetic patients. DM prevalence among adults is estimated to be about 6e7% worldwide; therefore an ABPM screening test for MHT diagnosis seems unpractical among this extremely large population. There is a need for a practical screening test for determination of MHT risk in these individuals. The purpose of this study is to compare serum ADMA levels and L-arginine/ADMA ratios of DM patients with MHT to normotensive diabetics and to investigate the predictive value of serum ADMA levels for identification of MHT in diabetic subjects. 2. Methods 2.1. Study population The present study screened a total 250 normotensive diabetics. After exclusion criteria, 131 normotensive patients with DM Type 2 remained. Most patients were referred to the study from the internal medicine outpatient clinic. ABPM was obtained in all patients whose office BP was below 140/90 mmHg. The study was approved by the local Ethics Committee of Selcuk University (date: 26.12.2008, approval number: 2008/358). Informed consent was obtained from each subject. Criteria for exclusion were as follows: 1- use of medications for hypertension and hyperlipidemia, 2- known cardiovascular diseases, such as coronary heart disease, severe valvular heart disease, or congestive heart failure, 3- electrocardiographic abnormalities, an indicator of myocardial infarction, 4- office BP measurement of >140/90 mmHg, 5- atrial fibrillation. In addition, a treadmill exercise test was performed in all patients. Inability to complete exercise test or >1 mm ST segment depression in two consecutive leads in treadmill ECG were accepted as an excluding criteria. 2.2. Office BP measurement Resting systolic BP and diastolic BP were measured from the brachial artery using a mercury sphygmomanometer (ERKA D83646 Bad Tölz, Kallmeyer Medizintechnik GmbH & Co KG, Germany) in the physician’s office. After at least 5 min of resting by sitting, BP was measured 2 times with an interval of 1 min. When readings differed by 5 mmHg, extra readings were obtained. 2.3. Ambulatory blood pressure measurement A twenty four-hour ABPM was carried out with a non-invasive automated device (Tracker NIBP2, Del Mar Reynolds Ltd, Hertford, England, UK) and the cuff of the device was fitted on the nondominant arm. Subjects were instructed to maintain the same daily routine and sleep patterns and to stop muscular activity

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(especially athletic activity) and keep their arms entirely still during BP measurements. The BP monitor was programmed to measure BP at intervals of 20 min during the day and 30 min at night. Each BP reading was recorded on a computer and rejected if the SBP was less than 80 mmHg or more than 250 mmHg or if the DBP was less than 40 mmHg or more than 140 mmHg. Recordings for each subject were accepted when more than 80% of the raw data were valid. Average values were calculated for 2 periods during the day: a 6 h period between 1 A.M. and 6 A.M. (nighttime), and a 12 h period between 9 A.M. and 9 P.M. (daytime). A daytime BP  135/ 85 mmHg was considered as hypertensive [13]. 2.4. Blood sample analysis Blood samples were collected following 12 h overnight fasting and were assayed for glucose, lipids, HbA1c, creatinine, and ADMA. Blood sample analysis was performed daily, except in the case of ADMA and L-arginine measurements. For the ADMA analysis, blood samples (5 cc) were drawn from the peripheral venous blood and collected into the tubes. The tubes were centrifuged immediately at 3,000  g for 10 min. The separated serum samples were stored at 80  C. Measurement of ADMA was accomplished by high performance lipid chromatography (HPLC), with modification of the method described by Chen et al. [14]. Briefly, 20 mg 5-sulfosalisilic acid was added into 1 ml serum, and the mixture was left in an ice bath for 10 min. The precipitated protein was removed by centrifugation at 2.000  g for 10 min 1.3 ml of the supernatant, which was filtered through a 0.22-mm pore size filter, was mixed with 8.7 ml of derivatization reagent (prepared by dissolving 10 mg ophthaldialdehyde in 0.5 ml methanol, 2 ml of 0.4 M borate buffer (pH 10.0)), and adding 30 ml of 2-mercaptoethanol and after 3 min injected into the chromatographic system by injection program. Separation of ADMA was achieved with a 150 mm  4.6 mm  5 mm ODS Hypersil column (Thermo Electron Corporation, UK) using 50 mm sodium acetate (pH 6.8), methanol, and tetrahydrofurane as the mobile phase (A, 82:0:1; B, 0:77:1) at a flow rate of 0.85 ml/min. Serum levels of ADMA were measured by HPLC (HP Agilent 1200, Agilent Technologies, Palo Alto, CA, USA) with fluorescence detection. The areas of peaks detected by fluorescent detector (excitation, 338 nm; emission, 425 nm) were used for quantification. 2.5. Statistical analysis Data were analyzed using SPSS software version 13.0 (SPSS, Chicago, IL, USA) and presented as mean  standard deviation. The distribution of the variables was analyzed with the Kolmogorove Smirnow test. Correlation analysis was carried out with Pearson’s correlation test for normally distributed variables. Independent Student’s t-tests were used for comparing differences of normally distributed variables between the two groups. Adjusted (for hip circumference and waist circumference) means of ADMA were compared using a general linear model procedure. The relationship between the categorical variables was determined by the c2-test. A stepwise logistic regression analysis was performed to detect of the predictors of MHT by using a backward elimination method. The following covariates were entered in the regression model: age, waist circumference/hip circumference ratio, BMI, total cholesterol, high-density lipoprotein (HDL) cholesterol, fasting blood glucose, HbA1c, duration of DM (year), office SBP and DBP, and ADMA level. Power analysis was performed using the Minitab 16 packet program. Sample volume was calculated as 28 for each group to determine a difference in ADMA of about 2 mmol/L with 80% power. We predict the prevalence of MHT to be about 20e25% based on previous literature in DM populations. Therefore, we planned to

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enroll about 130 normotensive diabetics to reach to the target number of MHT patients. A MedCalc 9.2.0.1 packet program was used to calculate the receiver operating curve (ROC) and to analyze specificity, sensitivity, negative and positive predictive values of ADMA for the MHT. 3. Results The study examined ABPM in 131 patients with DM who had a normal office BP at rest (<140/90 mmHg). ABPM of 32 patients with MHT are available in Table 1. The rest of the study sample was defined as true normotensive (n ¼ 99). Thus, the prevalence of MHT was found to be 24.4% in the present study (n ¼ 131). Demographics and laboratory findings of patients with MHT were compared with the normotensive group (Table 1). BMI was higher in the MHT group than among the normotensive group (p ¼ 0.02). Other demographic features such as age, gender, smoking status, and waist-hip ratio circumference were comparable between the two groups. The serum ADMA levels were increased in the MHT group compared to the controlled normotensive group (6.2  2.2 vs 4.2  1.7 mmol/L respectively, P ¼ 0.001). Also, corrected P values for ADMA according to the hip circumference and BMI were significantly different in the MHT group than in the controlled normotensive group (P ¼ 0.04 and P ¼ 0.02 respectively). The serum L-arginine levels were similar in the 2 Table 1 A comparison of demographics and laboratory findings of the groups. Parameters

DM with normotensive (n ¼ 99) (Mean  SD)

Age (years) 49.5  8.8 Females n (%) 61 (62) Smoker (%) 8 (8.1) 29.3  3.5 BMI (kg/m2) Waist-hip ratio 0.94  0.07 Glucose (mg/dl) 154.7  55.9 BUN, mg/dL 18.6  7.4 Creatinine (mg/dl) 0.74  0.14 TC (mg/dl) 197.8  40.0 LDL-C (mg/dl) 119.9  32.7 HDL-C (mg/dl) 44.4  13.8 Trigliceride (mg/dl) 179.6  82.2 4.2  1.7 ADMA (mmol/L) L-Arginine (mmol/L) 169.9  55.3 L-Arginine/ADMA ratio 46.0  19.0 HbA1c 7.4  1.3 Office BP measurements SBP 120.0  9.9 DBP 78.5  7.8 Ambulatory BP measurements Daytime SBP 121.7  7.0 Daytime DBP 73.0  5.3 Nighttime SBP 114.7  10.4 Nighttime DBP 64.2  7.3 24 h SBP 119.3  7.5 24 h DBP 69.8  5.6 Early morning SBP 120.5  12.3 Early morning DBP 73.5  10.2

DM with MHT (n ¼ 32) (Mean  SD)

p value

50.5  7.5 24 (75) 2 (6.3) 32.7  8.0 0.93  0.05 162.1  55.6 18.4  6.9 0.70  0.11 192.3  31.6 121.1  25.8 39.1  7.3 160.3  61.8 6.2  2.2 166.8  38.6 29.9  12.1 7.5  1.5

0.55 0.17 0.74 0.02 0.33 0.51 0.88 0.20 0.42 0.83 0.001 0.13 0.001 0.72 0.001 0.86

120.6  11.1 77.7  6.5

0.78 0.54

138.3  5.4 83.1  4.0 124.7  10.9 72.4  5.3 135.3  6.6 79.7  3.6 130.1  12.0 80.5  14.0

0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.01

Table 2 Linear relationship between blood pressure measurements and ADMA values.

Daytime SBP Daytime DBP Nighttime SBP Nighttime DBP 24-h SBP 24-h DBP Early morning SBP Early morning DBP HR (mean of the 24 h) Office SBP Office DBP

Arginine/ADMA ratio r (P)

0.38 0.34 0.21 0.26 0.35 0.35 0.25 0.23 0.10 0.02 0.11

L0.42 L0.35 0.16 L0.22 L0.35 L0.34 L0.19 L0.17 0.11 0.02 0.04

(0.001) (0.001) (0.02) (0.003) (0.001) (0.001) (0.01) (0.01) (0.25) (0.80) (0.20)

(0.001) (0.001) (0.07) (0.01) (0.001) (0.001) (0.03) (0.04) (0.23) (0.83) (0.66)

SBP: systolic blood pressure, DBP: diastolic blood pressure, HR: heart rate, r: Pearson correlation coefficient, P: level of statistical significance. Bold values signify BMI was higher in the MHT group than among the normotensive group (p ¼ 0.02). The serum ADMA levels were increased in the MHT group compared to the controlled normotensive group (6.2  2.2 vs 4.2  1.7 mmol/L respectively, PP ¼ 0.001). ADMA levels were positively correlated with most of the ABPM measurements. L-Arginine/ ADMA ratio and HDL cholesterol levels were significantly decreased in the MHT group vs normotensive group. In the multivariate logistic regression model, ADMA, BMI and HDL levels were found to be independent predictors of MHT.

groups (166.8  38.6 vs 169.9  55.3 mmol/L respectively, P ¼ 0.72). ratio and HDL cholesterol levels were significantly decreased in the MHT group vs normotensive group. Other laboratory findings such as creatinine, HbA1c, and total cholesterol were comparable between the two groups. The main characteristics and laboratory findings of subjects are summarized in Table 1. The relationship between ABPM and ADMA was examined. At all ABPM periods, both SBP and DBP were significantly higher among patients with MHT than in normotensive patients with DM. ADMA levels were positively correlated with most of the ABPM measurements (Table 2). The best correlation between ADMA and ABPM was seen in the daytime period and 24 h period records. The correlation of the nighttime and early morning ABPM with ADMA levels was weak and/or not present. In addition, ADMA levels were not correlated with office BP measurements. L-Arginine/ADMA ratio was negatively correlated with ABPM as a concordant with L-Arginine/ADMA

Table 3 The independent predictors of MHT in the stepwise logistic regression model.

The first step

MHT, masked hypertension; BMI, body mass index; BUN, blood urea nitrogen; TC, total cholesterol; LDL, low-density lipoprotein cholesterol; HDL, high-density lipoprotein cholesterol; ADMA, asymmetric dimethylarginine; hsCRP, high sensitive Creactive protein; SBP, systolic blood pressure; DBP, diastolic blood pressure; BP, blood pressure. Bold values signify BMI was higher in the MHT group than among the normotensive group (p ¼ 0.02). The serum ADMA levels were increased in the MHT group compared to the controlled normotensive group (6.2  2.2 vs 4.2  1.7 mmol/L respectively, PP ¼ 0.001). ADMA levels were positively correlated with most of the ABPM measurements. L-Arginine/ADMA ratio and HDL cholesterol levels were significantly decreased in the MHT group vs normotensive group. In the multivariate logistic regression model, ADMA, BMI and HDL levels were found to be independent predictors of MHT.

ADMA r (P)

The last step

Age Duration of DM BMI HbA1c Total Cholesterol HDL SBP at the office DBP at the office ADMA Waist-hip ratio BMI ADMA HDL

Beta

Odds (95,0% C.I. for odds)

P

0.020 0.037 0.184 0.107 0.002 0.068 0.009 0.018 0.495 0.025 0.172 0.486 0.054

1.021 0.96 1.20 1.11 1.00 0.93 1.01 0.98 1.64 1.05 1.19 1.63 0.95

0.51 0.58 0.006 0.57 0.74 0.03 0.76 0.68 0.001 0.16 0.01 0.001 0.04

(0.96e1.09) (0.84e1.10) (1.06e1.37) (0.77e1.60) (0.99e1.02) (0.88e0.99) (0.95e1.07) (0.90e1.07) (1.27e2.11) (0.98e1.11) (1.05e1.35) (1.28e2.06) (0.90e0.99)

Nagelkerke R Square: 0.36, Model statistics P ¼ 0.001, DM: diabetes mellitus. BMI: body mass index, SBP: systolic BP obtaining with sphygmomanometer by physician, DBP: diastolic BP obtaining with sphygmomanometer by physician, ADMA, HDL. Bold values signify BMI was higher in the MHT group than among the normotensive group (p ¼ 0.02). The serum ADMA levels were increased in the MHT group compared to the controlled normotensive group (6.2  2.2 vs 4.2  1.7 mmol/L respectively, PP ¼ 0.001). ADMA levels were positively correlated with most of the ABPM measurements. L-Arginine/ADMA ratio and HDL cholesterol levels were significantly decreased in the MHT group vs normotensive group. In the multivariate logistic regression model, ADMA, BMI and HDL levels were found to be independent predictors of MHT.

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Fig. 1. Receiver operating curve of ADMA levels to predict of MHT.

ADMA levels. The linear association between BP measurements and ADMA levels are available in Table 2. The serum ADMA levels and the Arginine/ADMA ratio were not related with other demographics and laboratory finding such as age, BMI, waist/hip ratio, duration of DM, HbA1c, lipid profile, and creatinine (p > 0.10). In the multivariate logistic regression model, ADMA, BMI and HDL levels were found to be independent predictors of MHT (Table 3). ADMA level was the most powerful predictor of MHT among the variables evaluated (Odds: 1.63 (1.28e 2.06, 95% CI)). A cut off value was examined to determine the positive predictive value. The ROC curve is shown in Fig. 1. When ADMA levels of 4.5 mmol/L were accepted as a cut off for MHT, the following results were obtained: sensitivity ¼ 78.1%, specificity ¼ 60.6%, positive predictive value ¼ 39.2%, negative predictive value ¼ 90.3%. When ADMA levels 5.0 mmol/L were accepted as a cut off, the following results were obtained: sensitivity ¼ 68.8%, specificity ¼ 72.7%, positive predictive value ¼ 44.9%, negative predictive value ¼ 87.8%. However, the optimal cut off value was determined to be > 4.34 mmol/L of ADMA using the MedCal 9.2.01 program (Fig. 1). Area under the curve (AUC) was found to be 0.78 (SE ¼ 0.043 p ¼ 0.001) for this cut off. The following results were obtained for the cut off of >4.34 mmol/L ADMA: sensitivity ¼ 84.4%, specificity ¼ 59.6%, positive predictive value ¼ 40.3%, and negative predictive value ¼ 92.2%. 4. Discussion The main finding of this study is that the serum ADMA levels were significantly increased in MHT patients with DM compared to normotensive diabetic subjects. ADMA levels, BMI, and low HDL levels were independent predictors of MHT in patients with DM. ADMA levels positively correlated with ABPM measurements, especially daytime measurements, but were not related to office BP measurements. ADMA levels may play a role in prediction, with a good sensitivity and negative predictive value but poor specificity and positive predictive value. To the best of our knowledge, this is the first study to examine the relationship between ADMA and MHT in the current literature. Ng et al. studied in a total of 133 DM patients, 18% of whom had MHT. It was reported that MHT is associated with a higher prevalence of albuminuria, left ventricular diastolic dysfunction, and hypertrophy compared with normotensive DM [11]. Also, the prevalence of target organ damage was comparable in the MHT group with sustained hypertension in this study. Eguchi et al. reported MHT prevalence of 46.7% among 81 clinically normotensive Japanese diabetic persons. In this study, MHT was related to

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significantly increased incidence of silent brain infarcts and albuminuria [12]. In another cross-sectional study enrolling a total 135 normotensive patients with type 2 diabetes, the prevalence of MHT was reported as 30% [10]. Based on the current literature, MHT prevalence was higher in type 2 DM subjects and is correlated with target organ damage relative to normotensive diabetics. Therefore, these subjects may have an increased risk of cardiovascular events. However, the value of cardiovascular risk markers to determine MHT has not been well studied. ADMA has been shown to inhibit endothelial NOS in vitro, in animals, and in the human forearm arterial bed [15]. Surdacki et al. also suggested that increased serum ADMA levels may lead to increased vascular resistance and elevated BP via a decrease in NO bioavailability [16]. In a well designed study, intravenously administered suppressor doses of ADMA resulted in increased arterial stiffness and decreased cerebral blood flow in young healthy men [17]. In another elegant human trial, Achan et al. demonstrated that ADMA infusion increases systemic vascular resistance by 24%, and mean arterial BP by 6% in healthy subjects and that a bolus dose of ADMA decreases cardiac output by 15% [18]. In a second part of that study, it was shown that a handgrip maneuver increased cardiac output in control subjects by 96%, but in subjects given ADMA, cardiac output increased by only 35%. ADMA concentration 30 min after the infusion was 2.6 mol/L, however, the baseline value was not provided. In addition, several studies have shown that serum ADMA levels are increased in essential hypertension about two fold and are related to impaired endothelial function [5,16]. Perticone and colleagues found that ADMA in essential hypertensive subjects was strongly and inversely associated with the peak increase in forearm blood flow [5]. These studies demonstrated that ADMA plays an important role in the regulation of vascular tone. It is known that ADMA levels are increased among type 2 DM patients [19]. On the contrary, in a small study, Paiva et al. reported decreased ADMA level in diabetics due to renal hyperfiltration [20]. It seems that the prognostic value of ADMA is more important in patients with DM [21]. Krzyzanowska et al. reported that ADMA predicted cardiovascular events and enhanced the predictive role of CRP in patients with type 2 diabetes. In addition, ADMA was related to increased hs-CRP levels in the MHT group [22]. While the regulatory role in vascular tone and the prognostic value of ADMA are known, levels ADMA in patients with MHT have not been studied yet. The role of ADMA in the pathophysiology of MHT remains unclear. Two well designed studies indicate that FMD is impaired in MHT both diabetic and non-diabetic patients. Takeno et al. revealed that FMD is impaired (5.65  2.00% for NT, 4.26  1.88% for MHT, 3.90  1.71% for HT, P < 0.001) and brachial-ankle pulse wave velocity (baPWV) is increased (1514.2  212.7 cm/s for NT, 1749.9  339.7 cm/s for MHT, and 1768.6  302.8 cm/s for HT, P < 0.001) in type 2 diabetic patients with MHT [23]. Among nondiabetic African-Americans, Veerabhadrappa et al. revealed that those with MHT have significantly lower FMD% than the trueprehypertension group (6.5  4.0% vs 8.4  3.8%; P ¼ 0.03), which may indicate impaired endothelial-dependent dilatation [24]. However, while these studies showed impaired FMD among the MHT group, serum ADMA levels were not examined. On the other hand, several studies have shown that elevated ADMA levels are associated with endothelial dysfunction in patients with DM [25]. Based on these studies, we speculated that ADMA may play a pivotal role in the development of MHT via endothelial dysfunction. The most important limitation of the present study is that endothelial functions were not measured due a limited budget. Previous work has demonstrated that ADMA causes endothelial dysfunction via suppression of endothelial progenitor cells (EPCs) [26]. EPCs are circulating immature cells that contribute to vascular homeostasis

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and compensatory angiogenesis [26]. Many cardiovascular risk factors, including hypertension, have been associated with decreased and/or dysfunction of circulating EPCs [26]. NO partially mediates the regulation of EPC function and effects the differentiation process of EPCs [26]. Thum et al. demonstrated that ADMA was an endogenous inhibitor of mobilization, differentiation, and function of EPCs. They also showed that rosuvastatin abolished the detrimental effects of ADMA on EPCs [27]. Based on prior studies, we hypothesize that endothelial dysfunction due to increased ADMA levels may result in MHT in diabetic subjects. Interestingly, HbA1c, a marker of glycemic regulation, was not related to MHT in the present study. Hyperglycemia affects both endothelial function and ADMA levels [28]. Recently, Paiva et al. showed that ADMA levels were inversely related to HbA1c levels [20]. In the present study we did not find a relationship between HbA1c and ADMA. Based on the potentially controversial results, we think that HbA1c may be a weak marker of long-term glycemic control considering the chronic nature of DM. Office BP measurements failed to predict cardiovascular events in hypertensive patients and recent guidelines have emphasized value of ABPM, advising more frequent use of ABPM than is currently used daily practice among the general population [8]. Concordantly, in the present study, serum ADMA levels were not correlated with office BP measurements. On the contrary, serum ADMA levels had a moderately linear relationship with ABPM, especially using daytime and 24 h measurements. In literature, it has been shown that ABPM records are superior to office BP measurements for predicting target organ damage in DM [10]. On the other hand, patients with MHT had a cardiovascular risk similar to those with sustained hypertension and MHT risk was increased 2to 6-fold in DM subjects [29]. At this point, the essential question is in which patients with DM should ABPM be applied? Due to the time consuming nature of the technique and the negative effect on sleep quality during ABPM, physicians prefer not to apply ABPM without necessary indications. The results of the present study may be useful in answering this question. Patients with ADMA levels below 4.34 are probably truly normotensive. However, patients with increased ADMA levels may be at risk for the development of MHT and increased cardiovascular risk. In this area, clinically practical biochemical markers are required to predict MHT, especially in patients with DM. The present study may be accepted as a preliminary investigation in this area. The gold standard method for measuring ADMA, HPLC, has widespread availability but limited use due to the time consuming nature of the assay and the need for laboratory training. In the present study, the total cost was $ 2.1 per patient for the measurement of ADMA levels by HPLC. Alternately, commercial ELISA kits are commonly available for the measurement of ADMA level and may be more readily applicable than the HPLC method. A total cost of one ELISA kit is $ 495. The measurement of ADMA from about 84 to 86 patients can be completed using one ELISA kit. Therefore, the total cost of measuring ADMA levels by ELISA is approximately $ 6 (495/85) per patient measurement. Currently, there are no specific therapies to reduce serum ADMA levels. However, there are promising results from small, short-term follow-up clinical studies. For example, McLaughlin et al. demonstrated that plasma ADMA levels are elevated in obese insulinresistant women, and that ADMA levels fall with weight loss [30]. In addition, there are publications demonstrating that ADMA levels decrease with exercise in type 1-DM and metabolic syndrome [31]. These data are supported by the present study. Increased BMI and lower HDL levels were found in the MHT group. It is known that ADMA levels are increased in obesity and increased BMI is correlated with MHT. However there is no data on the relationship between low HDL levels and MHT and there is no exact explanation for decreased HDL-cholesterol levels in the MHT group. Since BMI

was higher in the MHT group than among the normotensive group, we speculated that patients in the MHT group may be more sedentary than the patients in normotensive group and the decrease of HDL-cholesterol levels may be attributed to a sedentary life style [32]. Interestingly, two studies have shown that fenofibrats and niasin decrease ADMA levels [33,34]. Both of these agents are used in hypertriglyceridemia therapy. However the use of these drugs effectively increased HDL levels. Hence, future examination will be required to determine the role of HDL levels in pathogenesis of the MHT. In the current literature, some studies have examined the effect of cardiovascular drugs on the ADMA level. Chen et al. demonstrated that ACE inhibition decreased ADMA levels in patients with diabetes, hypertension, or syndrome X [35]. It was also shown that angiotensin receptor blockers (ARBs) can reduce plasma ADMA levels in patients with essential hypertension [36]. Rosuvastatin, a statin class drug, has been demonstrated to cause a decrease in ADMA levels [37]. Asagami et al. demonstrated that metformin treatment lowers ADMA concentrations in patients with type-2 diabetes [38]. Yasuda et al. showed that intensive treatment of risk factors not only reduced ADMA levels, but also improved endothelial functions in patients with type 2 DM [39]. Long term follow-ups clinical studies are needed to determine the value of non-specific ADMA reducing therapies. 5. Limitations Primarily, a causative relationship between ADMA and MHT was not identified due to the cross-sectional nature of the study. It would be interesting to compare ADMA to other inflammatory markers that have been assessed in the literature for masked hypertension, such as hsCRP. One of the important limitations of this study is that FMD parameters were not measured. Measurement of endothelial dysfunction parameters would allow for the assessment of the causative effect of ADMA on development of MHT via endothelial dysfunction. However, this could not be done due to limited budget of the project. Also, the specific effects of the combination of ADMA elevation and MHT should be evaluated among patients with ADMA elevation or MHT alone or healthy subjects in large randomized controlled trials. Another limitation is that dietary habits of the patients were not examined. Diet may affect Larginine and ADMA levels. However, none of the patients was on a special diet. The difference in ADMA levels between the 2 groups cannot be fully accounted due to the difference in dietary habits among subjects in the present study. 6. Conclusion Despite of these limitations, the present study demonstrates that increased ADMA levels are independently associated with MHT in diabetic patients. The measurement of ADMA levels may be a useful tool for the screening of MHT in diabetic patients with acceptable sensitivity and good negative predictive value. Author contributions Taner A, Unlu M and Kayrak M: designed and performed research, analyzed data, wrote paper. Tekinalp M, Ayhan SS and Aribas A: collected data, performed research. Erdem SS designed and performed research. Conflict of interest The authors declare that there is no conflict of interest.

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