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Clinical and Experimental Hypertension
ISSN: 1064-1963 (Print) 1525-6006 (Online) Journal homepage: https://www.tandfonline.com/loi/iceh20
Total methylated arginine load as a risk parameter
in subjects with masked hypertension
Muserref Hosaf, Sedat Abusoglu, Ahmet Avci, Kenan Demir, Ali Unlu, Duygu
Eryavuz & Gulsum Abusoglu
To cite this article: Muserref Hosaf, Sedat Abusoglu, Ahmet Avci, Kenan Demir, Ali Unlu, Duygu Eryavuz & Gulsum Abusoglu (2020) Total methylated arginine load as a risk parameter in subjects with masked hypertension, Clinical and Experimental Hypertension, 42:2, 126-130, DOI: 10.1080/10641963.2019.1583246
To link to this article: https://doi.org/10.1080/10641963.2019.1583246
Published online: 22 Feb 2019.
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Total methylated arginine load as a risk parameter in subjects with masked
hypertension
Muserref Hosafa, Sedat Abusoglua, Ahmet Avcib, Kenan Demirb, Ali Unlua, Duygu Eryavuza, and Gulsum Abusogluc
aDepartment of Biochemistry, Selcuk University Faculty of Medicine, Konya, TurkeyGSM;bDepartment of Cardiology, Selcuk University Faculty of
Medicine, Konya, TurkeyGSM;cDepartment of Medical Laboratory Techniques, Selcuk University Vocational School of Health Services Medical
Services and Techniques Institution, Konya, TurkeyGSM
ABSTRACT
Asymmetric dimethylarginine, symmetric dimethylarginine, and L-monomethylarginine are originated from the subsequent proteolytic catalysis of methylated arginine residues on different proteins and inhibit the endogenous nitric oxide generation. The changes in total methylarginine load (Asymmetric dimethylarginine plus symmetric dimethylarginine plus L-monomethylarginine) may contribute to hypertension. The aim of this study was to determine serum methylarginine concentrations in patients with masked hypertension and determine the association between these biomarkers and blood pressure measurements. Control group, masked hypertension and hypertension groups consisted of 40 subjects (11 males, 28 females, mean age 48.6 ± 13.1), 28 subjects (14 males, 14 females, mean age 50.9 ± 11.0) and 36 subjects (15 males, 21 females, mean age 54.4 ± 12.3 years), respectively (P= 0.149). Serum total methylarginine load was significantly higher in hypertension group (0.63 ± 0.23) compared to masked hypertension (0.49 ± 0.16) and control groups (0.38 ± 0.13) (P= 0.008 and P< 0.001). While there was no statistically significant difference between healthy control groups [0.147 (0.03–0.29)] and masked hypertension patients [0.144 (0.05–0.42)] for serum symmetric dimethylarginine levels (P= 0.970), it was markedly elevated in hypertension group [0.25 (0.07–0.54)] compared to masked hypertension group [0.14 (0.05–0.42)] (P= 0.001). Serum total methylarginine load was positively correlated with night-time SBP (r = 0.214,P= 0.029). Serum methylarginine levels might be a useful marker for determining the courses of clinical hypertension.
ARTICLE HISTORY Received 31 January 2019 Revised 9 February 2019 Accepted 12 February 2019 KEYWORDS Symmetric dimethylarginine; masked hypertension; total methylarginine load; tandem mass spectrometry; blood pressure
Introduction
The endothelial cells release various vasoactive intermediates including nitric oxide (NO); therefore, these cells have a major role in the adjustment of the vascular tonus (1). L-arginine is the main precursor for in the NO synthesis however this synthesis is inhibited by methylated arginine derivates such as asymmetric dimethylarginine (ADMA), NG-monomethyl-L-arginine (L-NMMA), symmetric dimethylarginine (SDMA), competitively (2). Total methylarginine load might be considered as the total concentrations of all methylated forms of arginine as asymmetric dimethylarginine plus symmetric dimethylarginine plus L-monomethylarginine. ADMA and L-NMMA are produced during proteolytic degradation of methylated arginine residues on several protein molecules. This methylation reaction is cata-lyzed by enzyme groups known as protein-arginine methyltrans-ferases. Further proteolysis of proteins including methylated arginines result with the formation of free methylarginines, and these molecules inhibit NOS; thus, NO synthesis is impeded. Free methylarginines may lead to different effects on nitric oxide synthase (NOS) activity (3). Increased concentrations of ADMA inhibits NO synthesis, impairment of main endothelial function and it contributes to pathogenesis of diseases such as atherosclero-sis (4), hypertension, chronic kidney disease, insulin resistance,
dyslipidemia, acute coronary syndrome, stroke, congestive heart failure (5). A growing body of findings suggests that NO have a significant role in blood pressure homeostasis. After synthesis by endothelial cells, NO increases 3,5-cyclic-guanosine monopho-sphate (cGMP) generation then it activates cGMP-dependent protein kinase (PKG), thus provide vasodilatation in the vascular smooth muscle cells (VSMCs). Additionally, impairment of nitric oxide-mediated vasodilation in other arteries such as brachial, coronary, and renal was also identified in patients with high blood pressure (6). There is no literature data about the connec-tion between methylated arginine levels in a specific masked hypertension patient group. Our aim was to reveal serum methy-larginine levels as “total methylarginine load” in patients with masked hypertension and determine the relationship between these biomarkers and blood pressure (BP) results.
Materials and methods Study design
The present study was performed within three groups such as masked hypertension, hypertension, control and consisted of 40 subjects (11 males, 28 females, mean age 48.6 ± 13.1), 28 subjects (14 males, 14 females, mean age 50.9 ± 11.0) and 36
CONTACTSedat Abusoglu sedatabusoglu@yahoo.com Biochemistry Department, Selcuk University Faculty of MedicineSelcuk University Faculty of Medicine Alaaddin Keykubat Campus 42075 Selcuklu, Konya,TurkeyGSM
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https://doi.org/10.1080/10641963.2019.1583246
subjects (15 males, 21 females, mean age 54.4 ± 12.3 years), respectively (P= 0.149). Most of the patients involved to study were recruited from cardiology outpatient clinic. Patients with taking medications for hyperlipidemia, with a diagnosis of coronary artery disease, severe valvular heart disease, or con-gestive heart failure, electrocardiographic abnormalities, a sign of myocardial infarction or atrial fibrillation were excluded. Additionally, all of the patients were subjected to exercise stress test. Lack of exercise test accomplishment or >1 mmST segment depression in two subsequent observed in treadmill ECG were assumed as an excluding criteria. The study was performed after than local ethics committee approval was taken. A written informed consent was given to all individuals. A 24-h ambulatory blood pressure measure-ment (ABPM) was performed with a non-invasive automated apparatus OMRON RX3 (Omron Healthcare Co., Ltd., Kyoto, Japan) and the cuff of the apparatus was amputated on the nondominant arm. Participants were ordered to continue the daily routine activities and sleep periods and to stop muscular activities (particularly athletic activities) and hold their arms completely still during BP monitoring. The BP examine was designed to monitor BP at intervals of 20 min during the day and 30 min at night. Each BP measurement value was listed on a computer, and 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 was excluded from this study. Mean values were calculated for 2 time intervals during the day: a 6 h period between 1 AM and 6 AM (night-time), and a 12 h period between 9 AM and 9 PM (day-time). A daytime BP as 135/85 mmHg was accepted as hypertensive (7).
Biochemical measurements
Venous blood samples which taken from the antecubital region were drawn into serum separator tubes after overnight fasting. Serum samples were separated with using centrifugal force (2000 × g, 10 min, and 4°C) then divided into eppen-dorfs and stored at−80°C until biochemical analysis.
Serum methylated arginines, arginine, and citrulline levels were measured using the Shimadzu LC-20AD chromatography system coupled with Applied Biosystems MDS SCIEX (ABSCIEX, Canada) API 3200 Triple-Quadrupole Mass Spectrometry in electrospray ionization (ESI) positive mode by Phenomenex Luna C18 column (Catalog Number: 00G-4252-E0) with a modified method (8). Briefly, 100 µl (μL) of internal Standard [(deuterated7-ADMA) (Cambridge Isotope Laboratories Catalog No: DLM-7476-5) which was dissolved in methanol (Sigma Aldrich Cat. No.: 34860-30L-R] were added to 200μL of each serum sample then this mixture was centrifuged at 13.000 rpm during 10 min to isolate the precipitated proteins. The supernatant was taken into glass tube and evaporated under a nitrogen gas flow at 65°C. 200μL of a freshly prepared butanol (Sigma Aldrich Cat No: B7906-500ML) solution including 5% (v/v) acetyl chloride (Sigma Aldrich Cat No:320129-1KG) for the derivatization process was added into the dried sample extract and this mixture incubated at 65°C during 25 min. These samples were quickly dried under nitrogen gas at 65°C.
The specimens were dissolved in 100μL of water–metha-nol (90:10, v/v) mixture including 0.1% (v/v) formic acid
(Sigma Aldrich Cat No: 5438040100) and mixed then 40μL was injected into the chromatography system. 50 μM stan-dards of ADMA, SDMA, L-NMMA, Arginine and citrulline were infused to device for optimal multiple reaction mon-itoring parameters were to determined thus preliminary frag-mentation studies were performed. For this application method, the intra-day coefficient of variation (CV) and the inter-day CV were 8.6% and 10.1%, respectively. The limit of detection and the limit of quantification were below or equal than the lowest calibration point: LOD was 0.01 µM for all compounds in serum. The LOQ was 0.05 µM for ADMA, SDMA, L-NMMA, citrulline, and arginine. The observed bias for all added concentrations was <±17% and recoveries were between 80% and 92% (80% for L-NMMA).
Statistical analysis
Statistical evaluation was carried out using the SPSS v16 Statistical Package for Social Sciences (SPSS Inc, USA). Statistical difference between in the groups were evaluated with a Mann–Whitney U test when the variance was heterogeneous. Spearman's correlation analysis was performed.P< 0.05 were accepted as significant.
Results
Smoking incidence were 17.5%, 28.5%, and 19.4% in control, masked hypertension, and hypertension groups, respectively, with an insignificant difference (P= 0.472). Demographic values and blood pressure measurements, serum methylated arginine levels of three groups are presented inTables 1and2, respectively.
Serum total methylarginine concentrations were signifi-cantly higher in hypertension group (0.63 ± 0.23) compared to masked hypertension (0.49 ± 0.16) and control groups (0.38 ± 0.13) (P= 0.008 and P< 0.001). Also, the levels of serum methylated arginine concentrations were statistically lower in control group according to masked hypertension group (P= 0.006) (Figure 1).
While there was a significant positive correlation between serum L-NMMA and serum arginine (r = 0.593,P< 0.001), this was slightly statistically significant for serum L-NMMA and day-time SBP (r = 0.193,P= 0.049); total SBP (r = 0.195, P= 0.047). Serum arginine levels were found to have powerful positive corre-lation with Arginine/ADMA ratio (r = 0.657,P< 0.001), day-time SBP (r = 0.263,P= 0.007), night-time SBP (r = 0.263, P= 0.007) and total SBP (r = 0.267,P= 0.006). Also, there was an inverse weak correlation for BMI (r =−0.202, P= 0.039). Serum total methylar-ginine load was positively correlated with night-time SBP (r = 0.214,P= 0.029) (Figure 2).
Discussion
Our study exhibited an important increase in serum total methy-lated arginines in patients with hypertension according to healthy subjects. This study is also important in terms of to be the first study evaluating the levels of serum methylated argi-nines in patients with masked hypertension.
Table 1.Blood pressure measurements and demographic data of all groups.
Control (n = 40) Masked hypertension (n = 28) Hypertension (n = 36) p Total systolic pressure (mmHg) 117 (104–134) 137 (130–154) 134 (98–162) a:0.001
b:0.001 c:0.319 Total diastolic pressure (mmHg) 74 (59–88) 83 (69–106) 82 (49–105) a:0.001 b:0.001 c:0.386 Day-time systolic pressure (mmHg) 118 (105–136) 138 (128–164) 135 (98–166) a:0.001 b:0.001 c:0.313 Day-time diastolic pressure (mmHg) 75 (60–93) 83 (69–111) 84 (51–105) a:0.001 b:0.001 c:0.409 Night-time systolic pressure (mmHg) 113 (96–133) 134 (122–154) 131 (98–168) a:0.001 b:0.001 c:0.486 Night-time diastolic pressure (mmHg) 70 (51–89) 81 (65–99) 78 (47–107) a:0.001 b:0.001 c:0.247 Age (years) 48.6 ± 13.1 50.9 ± 11.7 54.4 ± 12.3 0.690 Weight (kg) 84.3 ± 17.1 86.6 ± 16.8 84.5 ± 16.4 0.166 Height (cm) 162 ± 14.4 164 ± 11.9 162 ± 9.87 0.163 BMI (kg/m2) 32.2 ± 6.7 31.8 ± 4.5 31.8 ± 5.2 0.478
Table 2.Serum methylarginine levels of all groups.
Control (n = 40) Masked hypertension (n = 28) Hypertension (n = 36) p ADMA (µmol/L) 0.215 (0.06–0.29) 0.326 (0.13–0.64) 0.301 (0.13–0.57) a:0.008
b:0.001 c:0.383 L-NMMA (µmol/L) 0.0196 (0.005–0.118) 0.0269 (0.002–0.071) 0.034 (0.008–0.083) a:0.032 b:0.001 c:0.236 SDMA (µmol/L) 0.147 (0.03–0.29) 0.144 (0.05–0.42) 0.251 (0.07–0.54) a:0.970 b:0.025 c:0.001 Arginine (µmol/L) 78.1 (11.9–181) 112 (35.8–235) 103(39.9–218) a:0.002 b:0.002 c:0.588 Citrulline (µmol/L) 24.9 (3.87–66.6) 22.4 (2.6–58.3) 20.4 (7.4–48) a:0.397 b:0.025 c:0.234 Arginine/ADMA ratio 368.5 (199.2–1601) 388.1 (102.4–1175) 342.8 (111.9–1043) a:0.881 b:0.399 c:0.333 Data were presented as median (minimum-maximum) and mean ± standard deviation. a = control group vs masked hypertension group; b = control group vs
hypertension group; c = masked hypertension group vs hypertension group. BMI = body mass index.
Recently studies demonstrate an important increase in treat-ment cost of hypertension and it is a common health problem. During the next 20 years, the rate of the adult individuals that will be affected by hypertension is predicted to raise by 60% to a total of over 1.5 billion. In developed countries, the monitoring of sufficient blood pressure (BP) (<140/90 mm Hg) among patients receiving antihypertensive intervention ranges from nearly 30–50% (9).
Arginine residues on protein molecules are methylated by a type of enzyme class known as protein arginine methyltrans-ferases (PRMT) to synthesis protein-bound monomethylargi-nine (MMA), symmetric dimethylargimonomethylargi-nine (SDMA), or asymmetric dimethylarginine (ADMA). Increased ADMA levels have been reported in the serum of patients with idiopathic pulmonary artery hypertension (PAH), chronic thromboem-bolic pulmonary hypertension (CTEPH) or PAH related to sickle cell disease or systemic sclerosis, thus a strong correlation was found between serum methylarginine concentrations and PAH pathogenesis (10).
Chien et al. (11) presented that systolic blood pressure is related with arginine/ADMA ratio (AAR) and blood pressure variability was widespread and connected with AAR in pediatric patients with early chronic renal failure (CKF). In our hyperten-sion group without CKD there was a decrease in AAR compared
to healthy group however this was not statistically significant [342.8 (111.9–1043) vs 368.5 (199.2–1601), P= 0.333].
Poreba et al. (12) demonstrated that in the individuals suffering from essential hypertension (EH) coupled with left ventricular diastolic dysfunction (LVDD), mean ADMA levels were statistically higher and the ratio of arginine to ADMA was statistically significant low according to patients with EH without LVDD.
Decreased NO synthesis, which is called as a sign of endothelial dysfunction (13), acts as a key molecule in con-trolling blood pressure alterations. A clear identification of the biochemical pathways related to nitric oxide signaling under pathological situations is of great importance for discovering new medical approaches. Such that, Henrohn et al. (14) detected that a temporary reduction of serum ADMA and SDMA concentrations after administration of oral vardenafil and plasma arginine concentration tend to increase as well as the arginine/ADMA ratio elevated gradually.
ADMA is the major endogenous inhibitor of nitric oxide synthase (NOS) enzyme and competes with L-arginine for bind-ing to active regions on endothelial form (eNOS) of this enzyme. ADMA and SDMA impede with y+-regulated transfer of argi-nine into cells (15). Inan et al. (16) reported a positive correlation between the systolic blood pressure (SBP) and diastolic blood
Figure 2.Correlations for serum arginine and methylated arginines with blood pressures and body-mass index.
pressure (DBP) levels with ADMA in patients with masked hypertension. In consistency with their results, in this study, it was found that both masked or clinical hypertensive patients have statistically higher ADMA levels compared to healthy con-trols (Table 2). However, serum ADMA levels were similar in masked and hypertensive groups (P =0.383). As a new finding in our study, SDMA may be considered as a marker for disease progression from masked to hypertension. Due to the advantage of determining total methylarginines in only a single chromato-gram in liquid chromatography–tandem mass spectrometry, it might be better to evaluate these molecules as total arginine load for comparison between groups. According to this point of view, total methylarginine levels tend to increase from healthy to hypertensive groups (Figure 1).
Conclusion
Serum symmetric dimethylarginine levels might be useful mar-ker for determining the transition from masked to clinical hypertension. Additionally, this is the first study evaluating the other methylarginine levels in addition to asymmetric dimethy-larginine in hypertension groups. However, further studies with larger clinical groups are necessary to identify the possible rela-tion between total methylarginine load and etiopathogenesis of masked hypertension. The number of patient groups and lack of serum nitric oxide analysis were a limitation of this study. This might be explained by the difficulty of diagnosing the patients with masked hypertension.
Acknowledgments
The authors would like to thank the Scientific Investigation and Project Foundation of Selcuk University, Konya, Turkey (Project number: 4202014) for funding this project.
Funding
Our study was financially supported by Scientific Investigation and Project Foundation of Selcuk University [Project number: 4202014].
Declaration of interest statement
The authors declare that they have no conflict of interest relevant to the content of this manuscript.
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