Research and Postgraduate Affairs الجامعة اإلسالمية-غ زة شؤون البحث العممي والد ارسات العميا كمية العموم ماجستير العموم الحياتية تحاليل طبية Serum Corin and Furin Levels among Hypertensive Male Adult Patients in Middle Governorate-Gaza strip مستوى إنزيمات الكورين والفيورين لدى الذكور البالغين من مرضى ارتفاع ضغط الدم في المحافظة الوسطى- قطاع غزة By Hana'a Salman Mizyed Supervised by: Dr. Baker Zabout Prof. Dr. Nutrition and Biochemistry Faculty of Science The Islamic University of Gaza Oct, 2017
اقرار
Abstract Background: Hypertension is a major global public health issue that causes cardiovascular disease (CVD), stork and other diseases that lead to death. Natriuretic peptides (NPs) are significant for balance of salt-water and blood pressure. Corin and furin are major cardiac enzymes that control of blood pressure (BP) by activating NPs, so regulating many cardiac function and hypertension. Objective: To assess serum corin and furin levels and some biochemical parameters among newly diagnosed primary hypertensive patients in Gaza Strip. Materials and Methods: Data were obtained from biochemical analysis and face to face questionnaire interview. Forty five blood samples from hypertensive patients and 45 from healthy persons were collected. ELISA techniques were used to investigate serum corin and furin levels. Enzymatic and colorimetric techniques were used to investigate urea, creatinine, total protein, albumin, potassium and lipid profile. All groups were matched for each other's in age, sex and residence. Results: The average age of the healthy individuals were (43.5±8.4) years whilst that of hypertensive patients were (44.2±7.7) years, (P >0.05). The Physical activity and diet were associated with hypertension (P=0.023, 0.008) respectively. About half of patients were hypertensive since 5 years. The main self-reported complications were retinopathy, cardiovascular diseases and recurrent infection. Body mass index, weight, diastolic & systolic BP were positively related with hypertension (P >0.05). Serum urea, creatinine, potassium (K) and albumin were higher in hypertensive (40.1±9.5 vs 31.6±6.0, 1.0±0.2 vs 0.90±0.10 mg/dl, 4.6±0.5 vs 4.3±0.4 mmol/l and 4.3±0.4 vs 4.0±0.3 g/dl, % differences=23.7, 10.5, 6.7 and 7.2, respectively and P<0.05). Triglycerides, cholesterol, low & high-density lipoprotein cholesterol were significantly elevated in hypertensive (195.6±58.3, 196.8±46.4, 196.8±46.4 and 47.5±4.0 mg/dl) than controls (166.2±63.0, 170.4±29.3, 170.4±29.3 and 45.8±3.5 mg/dl) with % differences of 14.4, 16.3, 3.6 & 18.8 % and P = 0.002, P = 0.024, P = 0.038 & P = 0.009, respectively). Corin in serum was significantly increased in controls (61.1±81.2 ng/ml, t=-4.096 and P = 0.000) compared to cases. In contrast, serum furin was scientifically increased in cases (717.3±759.4 pg/ml, t=2.143 and P = 0.035). There was statistically significant association between serum corin and smoking (t=2.360, P = 0.020), and between serum furin and education among the study population (t=3.456, P = 0.020). For the study population, there were negative correlation between corin and furin in controls (P = 0.010, r = -0.381) & cases (P < 0.001, r = -0.676). In contrast, serum corin and furin levels not correlated with SBP (mmhg), DBP (mmhg), age, weight, height, BMI, duration of hypertension, age at diagnosis of hypertension and most biochemical studied parameters among the study population. Conclusion: Serum corin and furin can be used as new biomarkers in addition to common ones of hypertension disease among hypertensive patients. Keywords: Hypertension, Corin, Furin, Biochemical parameters, Gaza Strip. II
ممخص خمفية: ارتفاع ضغط الدم هو قضية صحية عامة عالمية و كبرى يمكن أن تؤدي إلى أم ارض القمب والسكتة الدماغية وغيرها من األم ارض التي تؤدي إلى الوفاة وفي هذا السياق يعتبر استخدام بروتين ناتريوريتيك الببتايد مهما لمحفاظ عمى توازن الماء والممح وكذلك ضغط الدم الطبيعي. يعتبر الكورين والفيورين من أنزيمات القمب البروتينية التي تنظم ضغط الدم عن طريق تفعيل الناتريوريتيك ببتايد وبالتالي تنظيم ضغط الدم و وظيفة القمب. الهدف: تقييم مستوى انزيمات الكورين و الفيورين وبعض المعايير البيوكيميائية لدى الذكور البالغين من مرضى ارتفاع ضغط الدم الذين تم تشخيصهم حديثا في قطاع غزة. المواد والطرق البحثية: : البيانات المستخدمة في البحث تم الحصول عميها بمقابمة المرضى وجها لوجه وسحب عينة دم لعمل التحاليل البيوكيميائية والتي تشمل 45 مريضا من مرضى ارتفاع ضغط الدم و 45 من األصحاء. تمت د ارسة مستوى انزيمات الكورين و الفيورين وبعض العوامل البيوكيميائية في مجموعة مرضى ارتفاع ضغط الدم و المجموعة الضابطة. وتمت مطابقة نتائج كل مجموعة مع ما يماثمها في العمر والجنس ومكان اإلقامة. النتائج: متوسط عمر العينة الضابطة )األصحاء( هو 4..4.5±5 سنة ومتوسط العمر لمرضى ارتفاع ضغط الدم هو 7.7±55.4 سنة. وأوضحت النتائج أن مرض ارتفاع ضغط الدم يرتبط بالنشاط البدني والنظام الغذائي و كان حوالي نصف المرضى يعانون من ارتفاع ضغط الدم منذ 4 سنوات كما أن المضاعفات الرئيسية التي سجمت عمى لسان المرضى أوضحت وجود اعتالل الشبكية أم ارض القمب واألوعية الدموية وااللتهابات المتكررة. وكان كل من مؤشر كتمة الجسم )BMI( والوزن وضغط الدم االنقباضي )SBP( وضغط الدم االنبساطي )DBP( مرتبطين بشكل ايجابي مع ارتفاع ضغط الدم كما ان ارتفاع معدل وظائف الكمى )اليوريا والكرياتينين( والبوتاسيوم واأللبومين لدى مرضى ارتفاع ضغط الدم عنها عند أف ارد العينة الضابطة ومستوى كل من الكوليسترول الدهون الثالثية الكوليسترول عالي الكثافة )HDL-C( والكوليسترول منخفض الكثافة )LDL-C( أعمى في مرضى ارتفاع ضغط الدم عن العينة الضابطة. وقد أظهرت النتائج زيادة مستوى الكورين في العينة الضابطة مقارنة بمرضى ارتفاع ضغط الدم بفرق ذو داللة إحصائية بالمقابل زيادة مستوى الفيورين في مرضى ارتفاع ضغط الدم مقارنة بالعينة الضابطة بفرق ذو داللة احصائية. ونالحظ وجود عالقة ذات داللة احصائية بين الكورين والتدخين وبين الفيورين والمستوى التعميمي في مجتمع الد ارسة. كانت هناك عالقة ارتباط سمبية ذات داللة احصائية بين مصل الكورين ومصل الفيورين في مجتمع الد ارسة. وعمى النقيض من ذلك لم تتأثر مستويات الكورين و الفيورين بضغط الدم االنقباضي )SBP( وضغط الدم االنبساطي )DBP( والعمر والوزن والطول ومؤشر كتمة الجسم ومدة اإلصابة بضغط الدم وتاريخ التشخيص ومعظم المتغي ارت البيوكيميائية التي تم فحصها في مجتمع الد ارسة. االستنتاجات: يمكن استخدام أمصال الكورين والفيورين كعالمة بيولوجية جديدة باإلضافة إلى العالمات المتداولة لتشخيص و التننبأ مرض ارتفاع ضغط الدم لدى مرضى ارتفاع ضغط الدم. الكممات المفتاحية: ارتفاع ضغط الدم الكورين الفيورين المعايير البيوكيميائية قطاع غزة. III
IV Hana'a Mizyed
Acknowledgment No words can express my sincere gratitude to: My supervisor Dr. Baker M. Zabut, Professor of nutrition and biochemistry, Faculty of Science, Biochemistry Department, The Islamic University of Gaza for his inspiration, guidance, & true commitment to my research. I would like to thank him for his continual encouragement. All the staff members and colleagues of Medical Technology Master Program. All the laboratory staff members at laboratory General Administration of Primary Health Care in Ministry of Health for their kind help. All the staff of Directorate General of Human Resources Development. The practical part of this work was carried out at Deir EL Balah Medical Lab - Gaza. I would like to thank Mr. Naji AL Holi, Head Manager of Deir EL Balah Medical lab, thank him so much for allowing me to perform the Corin and Furin determination in his own laboratory, as well as other biochemical tests. V
Table of Contents Abstract... II Dedication. IV Acknowledgment... V Table of Contents... VI List of Tables... IX List of Figures... X List of Annex... XI List of abbreviations... XII Chapter 1 Introduction... 2 1.1 Overview... 2 1.2 General objective... 4 1.3 Specific objectives... 4 1.4 Significance... 4 Chapter 2 Literature Review... 7 2.1 Hypertension... 7 2.1.1 Definition of hypertension... 7 2.1.2 Types and causes of hypertension... 7 2.1.2.1 Essential hypertension..7 2.1.2.2 Secondary hypertension...8 2.1.3 Classification of high blood pressure for adults... 9 2.1.4 Risk factors of hypertension... 10 2.1.5 Prevalence and mortality rate of hypertension in Palestine... 10 2.1.6 Pathophysiology of hypertension... 11 2.1.7 Clinical manifestation... 11 2.2 Natriuretic peptide system... 12 2.2.1 Mechanisms of synthesis and release of natriuretic peptides... 12 2.2.1.1 Atrial natriuretic peptide 12 2.2.1.2 B-type natriuretic peptide... 13 2.2.1.3 C-type natriuretic peptide... 15 2.2.2 Natriuretic peptide receptors (NPRs)... 19 2.3 Corin... 20 2.3.1 Biology, structure and functional role of corin... 20 2.4 Furin... 23 5.2 Relation of corin and furin with hypertension... 23 Chapter 3 Subjects and Methods... 27 3.1 Study design... 27 3.2 Target population... 27 VI
3.3 Sample size... 27 3.4 Inclusion criteria... 27 5.2 Exclusion criteria... 27 3.6 Ethical consideration... 28 3.7 Data collection... 28 3.7.1 Questionnaire interview... 28 3.7.2 Body mass index... 28 3.8 Specimen collection and biochemical analysis... 28 3.8.1 Blood samples... 28 3.9 Data analysis... 44 Chapter 4 Results... 46 4.1 Baseline characteristics of study population... 46 4.2 Prevalence complications among hypertensive patients... 47 4.3 Anthropometric measures and blood pressure among study population... 48 4.4 Serum analysis... 49 4.4.1 Blood nitrogen and potassium among study population... 49 4.4.5 Lipid profile among study population... 50 4.4.3 Serum Corin and furin among study population... 51 4.5 Distribution of serum corin level according to baseline characteristics of study population... 53 4.6 Distribution of serum furin level according to baseline characteristics of study population... 55 4.7 Correlation between serum corin, furin levels and studied parameters among study population... 57 Chapter 5 Discussion... 62 5.1 Baseline characteristics of the study population... 62 5.2 Hypertension duration and complications... 63 5.3 Hypertension and body mass index (BMI)... 63 2.4 Blood nitrogen and potassium among study population... 63 5.5 Serum lipid profile among study population... 64 5.6 Serum corin and furin among study population... 64 2.5 Correlation between serum corin, furin levels and studied parameters among the study population.... 67 Chapter 6 Conclusions and Recommendations... 69 6.1 Conclusions... 69 6.2 Recommendations... 70 References... 71 Annexes... 81 VII
VIII
List of Tables Table ( 2.1): Classification of high blood pressure levels... 9 Table ( 2.2):Biological effect of the natriuretic peptides... 17 Table ( 4.1):Baseline characteristics of study population... 47 Table ( 4.2):Prevalence complications among hypertensive patients... 48 Table ( 4.3):Anthropometric measures and blood pressure among study population... 49 Table ( 4.4):Blood nitrogen and potassium among study population... 50 Table ( 4.5):Lipid profile among study population... 51 Table ( 4.6):Serum Corin and furin among study population... 52 Table ( 4.7):Distribution of serum corin level according to baseline characteristics of study population... 54 Table ( 4.8):Distribution of serum furin level according to baseline characteristics of study population... 56 Table ( 4.9):Correlation between serum corin level and studied parameters among study population... 59 Table ( 4.10):Correlation between serum furin level and studied parameters among study population... 60 IX
List of Figures Figure ( 2.1): Schematic representation of the synthesis and half-life time of the molecular forms of the atrial natriuretic peptide... 13 Figure ( 2.2):The molecular and synthesis forms of the BNP.... 14 Figure ( 2.3): The process cleavage of pro-cnp to CNP.... 16 Figure ( 2.4): The mechanism and action of NPs in heart protection.... 18 Figure ( 2.5): The interaction between the three NPs and the three NPRs... 19 Figure ( 2.6): The processing of the two pro-cardiac NPs by corin to active NPs.... 21 Figure ( 2.7): Corin enzyme structure.... 22 Figure ( 4.1):Distribution the mean of serum corin level (ng/ml) among controls and cases... 52 Figure ( 4.2):Distribution the mean of serum furin level (pg/ml) among controls and cases.... 53 Figure ( 4.3):Correlation between corin and furin level in controls.... 58 Figure ( 4.4):Correlation between corin and furin level in cases.... 58 X
List of Annex Annex (1): Helsinki committee approval letter... 82 Annex (2) : Ministry of health permission letter... 83 Annex (3): Questionnaire... 84 XI
ACTH ADH ANOVA ANP BMI BNP CHD CHE CHF CHO CK CNP CVD DBP DM EDTA ELISA EMR GK GPO HDL-C HF HK HRP HTN K LDL-C NPRs NT-pro BNP OD PMOH SBP List of abbreviations Adrenocorticotropic hormone Antidiuretic hormone One way analysis of variance Atrial natriuretic peptides Body mass index Brain natriuretic peptide Chronic heart disease Cholesterol esterase Chronic heart failure Cholesterol oxidase Creatinine kinase C-type natriuretic peptide Cardiovascular disease Diastolic blood pressure Diabetes mellitus Ethylenediaminetetraacetatic acid Enzyme-linked immunosorbent assay Eastern Mediterranean region Glycerol kinase Glycerol phosphate oxidase High density lipoprotein cholesterol Heart failure Hexokinase Horseradish peroxidase Hypertension Potassium Low density lipoprotein cholesterol Natriuretic peptide receptor-s N-terminal pro BNP Optical density Palestine Ministry of Health Systolic blood pressure XII
SD SNPs TMB UNRWA VLDL WHO Stander division Single nucleotide polymorphisms 3,3',5,5'-tetramethylbenzidine The United Nation Relief and Works Agency sector Very low-density lipoproteins World Health Organization XIII
1 Chapter 1 Introduction 1.1 Overview The incidence and prevalence of hypertension gradually elevated in the world and become a significant health problem (Gebreselassie and Padyab, 2015). Elevated blood pressure (hypertension), is a state in that the blood vessels have high pressure. Blood is carried from the heart to other parts of the body in the vessels. Every time the heart beats, it pushes blood into the vessels. Blood pressure (BP) is formed by the power of blood pushing against the walls of blood vessels (arteries) as it is pushed by the power cardiac. The higher the pressure the harder the heart has to pump (World Health Organization (WHO), 2015). BP is regarded as the normal level when less than 120 / 80 mmhg. BP between 120/80 & 139 / 89 mmhg is considered as prehypertension (Akpa et al., 2008). The screening of hypertension is prepared for the mean of diastolic blood pressure (DBP) 90 mmhg or when systolic blood pressure (SBP) 140 mmhg. Isolated SBP is defined as 140 and DBP 90 mmhg (Chobanian et al., 2003). Hypertension a chronic state that causes cardiac disease, stroke, and other diseases that can result in premature death. Decreasing the prevalence of patients with hypertension is one of the objectives of WHO (Lee, et al., 2016). Hypertension can be classified into two groups (1) primary (essential) & (2) secondary. Over ninety-five percent of all hypertensive patients have essential hypertension, with or without identifiable cause, whilst there are identifiable risk factors. In contrast, 5% of hypertensive patients are secondary hypertension, and this type of hypertension is caused by complications of other diseases such as kidney disease and heart diseases (Gupta-Malhotra et al., 2014). Risk factors of hypertension either non-modifiable or modifiable (Mayega et al., 2012). The non-modifiable risk factors cannot be changed; hence they are not in our control, including genetic variation, family history, age & gender. On the other hand, modifiable risk factors includes lifestyle patterns which can be controlled to prevent the 2
development of the disease such as; lack of exercise or inactivity, high-fat diet, increase body weight, tobacco & alcohol consumption (Ibekwe, 2015). Brain & Atrial natriuretic peptides (BNP and ANP) are cardiac peptides produced by heart cells in the ventricle and atrium, respectively, and play primary roles in body fluid homeostasis (Cabiati et al., 2013). In response to pressure or volume overload, BNP & ANP are a secreted from the cardiac cells. The main effects of NPs include removal of water and sodium by kidney and lowering systemic vascular resistance, so decreasing blood pressure and volume (Zhou and Wu, 2014). The genetic human for BNP encodes a 134-amino acid (a.a) pre-pro-bnp precursor, which after removal of the amino-terminal 26-a.a signal peptide gives rise to a 108- a.a pro-bnp peptide (pro-bnp 1-108 ). During secretion into circulation, further processing of pro-bnp 1-108 by a proprotein convertase results in the physiologically active 32-a.a (BNP 1-32 ) with a carboxyl-terminal molecule from 77 to 108 a.a, and NT-pro-BNP 1-76 as an amino-terminal fragment, derived from amino acids 1 to 76 a.a (Dries, 2007). Proprotein convertases are a family of proteases that cleave target proproteins, generating mature, biologically active polypeptides. Two convertases enzymes, corin & furin are considered the most likely pro-bnp processing enzymes (Ichiki et al., 2013). Corin enzyme is known as a type 2 transmembrane serine protease which has been identified as the physiological pro-anp and pro-bnp convertase (Wu et al., 2002). The enzyme is highly expressed in the cardiac cells (Ichiki et al., 2011), primarily in cardiomyocytes, where it uniquely cleaves the inactive natriuretic peptide precursor molecules into biologically active peptide hormones (Wu et al., 2009). Furin is a ubiquitously expressed type I transmembrane serine protease involved in the proteolytic processing of a wide range of precursor proteins, including growth factors and their receptors, adhesion molecules, and various metalloproteinase (Creemers et al., 2007). Corin and Furin, proteases that activate ANPs, are heart hormones that played roles in salt-water balance and hypertension (Nigrovic et al., 2008 & Wang et al., 2012). In genetic studies, the minor corin allele was related to an elevated risk for increasing blood pressure and induceing heart hypertrophic sense to hypertension (Rame et al., 3
2007; Nigrovic et al., 2008 and Zhou et al., 2009). Also, furin allele was related to human hypertension (Li et al., 2010). The present study is therefore, intended to assess of serum corin and furin levels among hypertensive patients in Gaza Strip and whether these levels vary with stage of hypertension. Although there are numerous and plausible studies that have been presented by many types of research (Fang et al., 2013 and Seronde et al., 2013) about hypertension and its correlation to corin and furin, clearing relation has not fully understood yet. 1.2 General objective The general objective of the current study is to assess of serum corin and furin levels among newly diagnosed primary hypertensive patients in Gaza Strip. 1.3 Specific objectives 1. To recognize the risk factors of newly diagnosed hypertensive patients. 2. To find out other biochemical aspects including kidney functions (uric acid, urea & creatinine) & lipid profile (triglycerides, cholesterol, HDL-C, LDL-C), total protein, albumin and potassium in hypertensive patients and controls. 3. To assess corin and furin levels in hypertensive patients compared to the controls and its relation to BMI and other biochemical parameters. 4. To determine the correlation between corin and furin with the studied parameters. 1.4 Significance 1. Hypertension is a major global public health issue. It is a chronic condition that the blood pressure in the arteries is increased, requiring the heart to work harder than a normal condition to push blood through the blood vessels. 2. NPs have a major role in normal blood pressure balance and salt-water controls. Corin & furin are cardiac enzymes that control blood pressure by activating NPs, so regulating blood pressure and heart function. 4
3. In the Gaza strip, there is only one study that assessed screening use of BNP, corin and furin as biomarkers for CVD complications in type 2 diabetes mellitus (Fathy et al., 2015). This will be the first study to assess of serum corin and furin levels among hypertensive patients in Gaza Strip. 4. Early diagnostic have enormous importance in achieving the goal of lowering the prevalence of the hypertension disease. One approach to such prevention is through the struggle against the development of the risk factors. 5. Understanding corin and furin roles among hypertensive disorders may be helpful as a marker in hypertensive patients. 5
2 Chapter 2 Literature Review 2.1 Hypertension 2.1.1 Definition of hypertension Hypertension is frequently defined by the occurrence of a chronic increase of arterial pressure higher than a certain threshold scale. Two values that are used to measure BP are SBP and DBP respectively. When we measure BP, the SBP is the primary reading and the DBP is the second reading. Both SBP & DBP have their own impact. Age groups are related to hypertension incidence and their consequences depend on whether SBP or DBP or both are increased. Pulse pressure is the last scale that is the main predictor of severity of the BP (Smith et al., 2006 and Kikuya et al., 2007). Prehypertension defined as BP criteria of 120/80 mm Hg to 139/89 mm Hg as known by the Seventh Report of the Joint National Committee on Evaluation, Detection, Prevention, and Treatment of High Blood Pressure (Chobanian et al., 2003). The persistent increase of BP is called hypertension. Hypertension is an asymptomatic defect Known by increased SBP 140 mmhg and DBP at a level of 90 mmhg (Leung et al., 2016). 2.1.2 Types and causes of hypertension Hypertension can be divided into two groups either essential (primary) or secondary. 2.1.2.1 Essential hypertension Primary hypertension is known as idiopathic or essential hypertension. The cause of Primary hypertension is still unknown. That why it is called primary or idiopathic hypertension. The cause may be due to complex processes in the main body systems and organs i.e. heart, blood vessels, hormones, nerves and kidneys (Elliott et al., 2015). approximately 95 % of people with high blood pressure have no identifiable causes of primary hypertension, but there are many risk factors that can enhance increase blood pressure, such as the volume of blood pumped by the heart, amount, and the arteries 7
condition, salt & water content of the body, the hormone levels, and kidneys function in the body. Other risk factors such as; smoking, alcohol use, stress, a diet high in salt, anthropometric measurements (BMI, congenital, gender, and age) (Prathyusha et al., 2016). In recent researches, considerable evidence has suggested that changes in vascular endothelial function may induce the elevation in vascular tone. For example, the less nitric oxide in hypertensive patients was produced from the vascular endothelium with less sensitivity to the action of vascular smooth muscle that causes powerful vasodilation. There is also a raise in endothelin production that lead to vasoconstriction tone. Hyperglycemia in type 2 diabetes (NIDD) which is caused by hypoinsulinemia compelling evidence that causes endothelial dysfunction by inducing decreased nitric oxide bioavailability and oxygen free radical-mediated damage (Paravicini et al., 2008). 2.1.2.2 Secondary hypertension Prevalence of secondary hypertension is approximately five percent of all cases of hypertension and it has an identified source, with the remaining being essential hypertension. There are many risk factors that can induce secondary hypertension. Regardless of the mechanism, the arterial pressure becomes raised either due to an increase in cardiac pump, an elevated in systemic vascular resistance, or both. When the cardiac pump is raised, it is generally due to either increased blood volume or increased neurohumoral activation of the cardiac cells (Chahal and Somers, 2015). Clearly, diabetic nephropathy, polycystic kidney disease, glomerular disease, renovascular hypertension are a major risk factors of secondary hypertension. Also, endocrine disorders such as pheochromocytoma, acromegaly, Cushing syndrome, aldosteronism, and thyroid problems are considered as risk factors for secondary hypertension. In addition, some medications and supplements, coarctation of the aorta, sleep apnea, obesity, and pregnancy are considered as minor risk factors of secondary hypertension (Winer, 2012). 8
2.1.3 Classification of high blood pressure for adults The blood pressure classification used in the 2013 ESH/ESC Guidelines comprises categories of optimal (SBP < 120 mmhg and DBP < 80 mmhg), normal (SBP 120 to129 mmhg and/or DPB 80 to 84 mmhg), and high normal (SBP 130 to 139 mmhg and/or DBP 85 to 89 mmhg) blood pressure, followed by 3 levels of hypertension, and a separate category for isolated systolic hypertension (Mancia a et al., 2013). The 3 grades of hypertension (Table 2.1) correspond to (Chobanian et al., 2003): 1. Mild hypertension: SBP 140 to 159 and DBP 90 to 99 mmhg. 2. Moderate hypertension: SBP 160 to179 and DBP 100 to109 mmhg. 3. Severe hypertension: SBP 180 and DBP 110 mmhg. Isolated systolic hypertension (SBP 140 mmhg), is graded as I, II, or III, according to the SBP scale, provided that the DBP is < 90 mmhg. Table ( 2.1): Classification of high blood pressure (Chobanian et al., 2003) 9
2.1.4 Risk factors of hypertension Although the accurate cause of increase in hypertension is unknown, many risk factors have been identified. There are multifactorial causes of the non-modifiable condition, such as Anthropometric measurements (family history, ethnicity, age & gender). In contrast, modifiable risk factors including stress, increasing BMI, sodium decrease (Abed & Abu-Haddaf, 2013 and Prathyusha et al., 2016). Hypertension is the result of a raise in sympathetic nervous system activity associated with loss function of the autonomic nervous system. This cause an increased renal absorption of sodium chloride (NaCl), & water related to a gene defect in the process by which the kidney holds sodium (Esler, 2000). The renin-angiotensin which improves the activity of aldosterone system also lead to an increase of extracellular fluid and increased the resistance of systemic vascular. Functional & structural modifications in the blood vessels and heart lead to blood pressure elevation with the age (Manrique et al., 2009). 2.1.5 Prevalence and mortality rate of hypertension in Palestine CVD, are considered as one of the main causes of noncommunicable diseases (NCDs). Hypertension is an essential risk factor for CVD, renal disease, and other complications. It is a primary contributor to premature deaths. Globally, half of stroke and ischemic heart disease (IHD) suddenly deaths are caused by high SBP (WHO, 2015). There are one billion hypertensive cases, and 4,000,000 people die by a complication of hypertension (Khatib and El-Guindy, 2005). Additionally, in the Eastern Mediterranean Region (EMR), one-third of CVD deaths and 27% of the adult in the died by hypertension currently (WHO, 2015). In Palestine, hypertension related disease becomes one of the main health troubles. It is the eighth support cause of death among Palestinians in 2014 (Palestine ministry of health (PMOH), 2014). Adult and geriatrics are most vulnerable to risks of hypertension and need more care to prevent the development of hypertension. Socioeconomic changes in our regions induce major changes in lifestyle towards 10
sedentary lifestyle and increase the incidence and morbidity of this disease (Abed and Abu-Haddaf, 2013). The PMOH report recorded approximately 49,094 hypertensive patients and 29,003 diabetic patients in Gaza strip, with a prevalence rate of 27.4 % and 15.4 %, respectively, of the adult population, have chronic diseases. In addition, the prevalence of hypertensive diseases was more in female (60%) than males (40%) (PMOH, 2014). The United Nation Relief and Works Agency sector (UNRWA) treated approximately 34,000 hypertensives and 23,000 diabetic patients in GS, with a prevalence rate of 17% and 12%, respectively, of the adult population (WHO, 2009). UNRWA continued to be the main comprehensive primary health care (PHC) provider for Palestinian Refugees in the Near East. UNRWA operates 137 PHCs scattered in Palestine (UNRWA, 2011). 2.1.6 Pathophysiology of hypertension The pathophysiology of hypertension still much unclear. A little number of patients (2% to 5%) have an essential adrenal or kidney disease as the cause for their increased blood pressure. However, no clear cause of the condition is labeled essential hypertension. A lot of physiological mechanisms are contributed in the control of normal blood pressure, and their confounding may play vital roles in the prognosis of essential hypertension. It is probable that many of factors involved in the blood pressure elevated in hypertensive patients, and their important roles may differ between subjects. Among the risk factors that have been new studied are a lot of salt intake, high body weight, the renin-angiotensin system, and insulin conflict, and the nervous system. In last few years, other factors have been assisted, including gene defect, endothelial dysfunction, intrauterine nutrition and low birth weight, and neurovascular anomalies (Wirix et al., 2015). 2.1.7 Clinical manifestation The silent killer, another name of hypertension because no symptom appears among people who have this disease. In hypertension, the primary stages of development, no signs or symptoms are noted by clients. So, damages blood vessels due to prolonged increased blood pressure in the target organs such as the eyes, brain, heart, and 11
kidneys. Hypertension is an essential risk factor for cardiac disease, stroke, retinopathy, Chronic heart failure (CHF), and renal disease. Clinical manifestations will become apparent, and clients will eventually complain about persistent headaches, fatigue, dizziness, palpitations, flushing, blurred or double vision, or epistaxis (Sawicka et al., 2011). 2.2 Natriuretic peptide system The heart is a mechanical pump and endocrine system that has special effects on blood circulation. NPs cause both diuresis & natriuresis, and they play primary roles for responding to a blood volume overload which cause either pressure or stretches on the heart (Arjamaa, 2014). NPs play an important role in controlling oxygen transport both systemically and locally, by causing volume reduction (plasma shift, diuresis, and natriuresis) that enhance increased oxygen carrying capacity (per unit volume of blood) and hemoconcentration (Arjamaa and Nikinmaa, 2013). The mammalian NPs composed of some substances: ANP; BNP and C-type natriuretic peptide (CNP) (Van Den Berge et al., 1998). Like a lot of peptides, NPs are produced as prepropeptides. After the signal peptide is removed, other cleavage enzymes are required to convert the inactive peptide to an active peptide (Wu et al., 2009). 2.2.1 Mechanisms of synthesis and release of natriuretic peptides 2.2.1.1 Atrial natriuretic peptide ANP is an amino-acid secreted by the atria in the cardiac cells. It is known as C- terminal granules of the pro-anp (Heikki, 2003). On produce, split proanp 1 126 by protease (corin) into an N-TANP 1 76 and the active ANP 1-32 (Wu et al., 2002). In the diagnostic test, NT-ANP can be used to inferences the amount of ANP from the cardiac cells. NT-ANP has a considerably longer half-life (11-15 times) in plasma compared with ANP which have a lower halflife of 3 6 min and thus has up to 12 40 times the plasma of ANP. ANP is also less stable in laboratory environment than NT- ANP. ANP is fast removed from the blood circulation by binding to receptors & hydrolysis by special enzymes. The ANP are variable in plasma, and its trustworthy detection requires a laborious extraction process. NT-ANP is considered as a 12
biomarker of lengthened cardiac overload than ANP because the NT-ANP is less changeable and has a higher half-life within blood circulation (Figure 2.1) (Heikki, 2003). Figure ( 2.1): Schematic representation of the synthesis and half-life time of the molecular forms of the atrial natriuretic peptide (Heikki, 2003). 2.2.1.2 B-type natriuretic peptide (BNP) BNP was initially revealed in the brain, it was thought to be a neurotransmitter, so it s original name, BNPs. Consequently, it was shown to be > 12-fold in the cardiac cells than in the brain, and so the term is B-type NPs (Christoffersen et al., 2002). In the previous century, small amount storage of BNP in the cardiac ventricle, that consideration is the major source. Pro-BNP is degradated in the heart to given the last peptide as BNP (32 a.a) with 77 108 of its 108-a.a pro-bnp, and an NT pro-bnp (NT-pro-BNP 1 76 a.a) (Figure 2.2). BNP is secreted by nonstop production in reply to the level of ventricular stretch & however, uncontrolled in failing ventricular. The mrna among pro-bnp is unstable, so BNP levels according to ventricular wall tension is active regulation. thus, it acts as a screening test for ventricular dilatation (Witthaut, 2004). 13
Figure ( 2.2): The molecular and synthesis forms of the BNP (Pandit et al., 2011). 14
Both NT-proBNP & BNP are formed in the same percentage (a 1:1), their concentrations are unlike because of NT-proBNP & BNP not identical half-life in vivo. mostly BNP is removed from the blood by the NPs receptor and cut by enzymes, while NT-proBNP is removed by the kidney. For that reason, NT-proBNP in plasma inversely related with the elevated by age & GFR. BNP half-life is 20-24 min, whereas the NT-pro-BNP halflife is more than 115 to 125 min with a normal GFR. In vitro, NT-proBNP is more stable than BNP if blood collected in Ethylenediaminetetraacetatic acid (EDTA) as anticoagulant tubes (Gobinet-Georges et al., 2000). ANP is less stable than BNP in the blood (McNairy et al., 2002) & BNP has a longer halflife (20-25 min), which have lesser affinity than ANP for clearance receptors and neutral endopeptidases (Lang et al., 1992). Studies illustrated NT-BNP and BNP is the same stability in stored plasma and both more reliable than ANP. BNP and NT-BNP indicate that the laboratory handling, and storage without special procedures (Thygesen et al., 2012). 2.2.1.3 C - type natriuretic peptide (CNP) CNPs is expressed highly in the heart and cytokine-exposed endothelial cells but highly in the brain. Pro-CNP in circulation included 103 amino acid, and furin as the intracellular enzymes have been demonstrated to induce inactivate procnp to the active form in vitro (Figure 2.3) (Wu et al., 2003). 15
Figure ( 2.3): The process cleavage of pro-cnp to CNP (Potter et al., 2006). 16
The biological effects of the NPs are summarized in Table (2.2) and Figure (2.4) (Nishikimi et al. 2006). Table ( 2.2): Biological effect of the natriuretic peptides (Nishikimi et al. 2006) TNF: tumor necrosis factor; IL: interleukin; ACTH: adrenocorticotropic hormone; ADH: antidiuretic hormone (Nishikimi et al., 2006). 17
Figure ( 2.4): The mechanism and action of NPs in heart protection (Nishikimi et al., 2006). 18
2.2.2 Natriuretic peptide receptors (NPRs) Three separated transmembranes NP receptors (NPRs) have been Known as NPR-A & B. It is enzyme from family transmembrane guanylyl cyclase that catalyze the production of cyclic guanosine monophosphate (cgmp) (Figure 2.5). NPR-C lacking the guanylyl cyclase domain so not included intrinsic enzymatic activity and management the plasma levels of NPs for receptor degradation and internalization. The special effects of NPs are attributed to intracellular cgmp changing by the couple to NP receptors lack cgmp domain and NPRC are a clearing by bind to NPs receptor then internalize and degradation (Potter et al., 2006). Figure ( 2.5): The interaction between the three NPs and the three NPRs (Pandey, 2011). 19
2.3 Corin 2.3.1 Biology, structure and functional role of corin Corin, a serine protease, is normally regarded as a key activator of NPs, via cleavage from pro-peptide to the active form of both ANP and BNP (Semenov et al., 2010). Corin is produced as a zymogen, which in turn important for activation by cleavage of a conserved site. The corin activator has not been identified. Corin is highly expressed in cardiomyocytes, and its promoter shares a lot of the identical transcription binding sites as BNP & ANP precursors. Thus, it could be predicted that corin and natriuretic peptides would be upregulated in response to similar stimuli (Dong et al., 2012). Corin is Known as type II transmembrane serine protease which consists of 1042 amino acids that process NPs in the heart. It consists of a transmembrane domain, an N-terminal tail, and a surface cell region with a CT domain. The transmembrane domain binds corin to the surface of cells (Qi et al., 2011). Corin has been confirmed to be the proanp/probnp convertase that exceptionally processes the NPs inactive molecules into active molecules (Yan et al., 2000). The enzyme is produced mostly in atrial and ventricular cardiac cells, where it converts inactive pro-anp & pro-bnp to active forms. These NP mediate their important actions after anchors to the BNP and generating the second messenger cgmp to promote natriuresis, diuresis, and vasodilatation (Figure 2.6) (Wu et al., 2009). 20
Figure ( 2.6): The processing of the two pro-cardiac NPs by corin to active NPs (Burnett and Olson, 2007). Clearly, last genetic research shown a defect in corin step would be expected to weaken the in-circulation activity of the NPs (Figure 2.7) (Moritoki et al., 1992). Two mutations (T555I and Q568P) are present in the corin gene, which lead to complete linkage disequilibrium in the human and co-localized in a minor allele. The two mutations of corin I555/P568 has been related to impaired NPs processing and enhance risk for the progress of heart failure (HF). These researchers suggest that the single nucleotide polymorphism (SNPs) may effect on corin processing, so weaken the antihypertrophic and antihypertensive actions of the NPs (Wang et al., 2008). A possible mechanism for this pharmacogenomic reaction is also affected on the processing of BNP in carriers of the corin SNPs compared to non-carriers (Rame et al., 2009). 21
Figure ( 2.7): Corin enzyme structure (Moritoki et al., 1992). corin secretion from renal cells and specially secretion from the proximal tubule and collecting duct cells, also renal corin expression was notably lowering in rat had renal disease. However, maybe contribute to sodium retention in that rat (Creemers et al., 2007). In additional, corin have a function in the kidney in controlling sodium excretion by the urinary system (Fang et al., 2013). Clearly, corin knockout mice have impaired sodium removed from blood circulation and hypertension induced (Wang et al., 2012). 22
2.4 Furin Furin is a ubiquitously expressed proprotein convertase (Molloy et al., 1994). A diversity of proproteins have been illustrated to be the substrates of furin (Wu et al., 2003). Both proteolytic enzymes (furin and corin) are required in the probnp pathway, giving final forms of BNP. Furin cleavage of probnp resulted in active BNP (32 amino acid), whereas corin cleavage involved in the production of other active BNP 28 amino acid) (Semenov et al., 2010). Raise in blood circulating furin activity as proteolytic enzymes, lead to an optimized production of BNP & NT-proBNP. Additionally, Blood circulating furin activity shown as a promising companion biomarker for NT-proBNP to more reliable measure acuity of HF (Vodovar et al., 2014). 2.5 Relation of corin and furin with hypertension The NPs system plays a good role in controlling blood pressure and salt water balance. Corin is a protease that activates ANP, a cardiac hormone central in the control of saltwater balance and blood pressure (Wang et al., 2012). To exam the associations between deficiency of corin and hypertension, (Chan et al., 2005) Hypertension in mice lacking the corin were investigated, they illustrated that corin lacking (Cor -/- ) mice enhance normally for survive to postnatal life & embryogenesis. They concluded corin is the convertase for pro-anp and appear that decrease of corin may lead to hypertension & then heart disease. Dries et al., (2005) exam two SNPs mutations that common in blacks corin gene minor allele and it defined by and related to hypertension. Corin I555 (P568) allele (SNPs) was still independently related with high risk for prevalent hypertension (OR, 1.63; P = 0.013). Also, the corin allele was linked with hypertension in people not using antihypertensive medication (P = 0.029; 134±20.7 versus 129±17.4 mm Hg, respectively) and with adjusted (P = 0.029; 132±1.6 vs 129±0.6 mm Hg, respectively) analyses and the independent association of the minor corin allele with increased risk for hypertension was confirmed in the Multi-Ethnic Study of Atherosclerosis (OR, 1.50; P = 0.014). However, the association of the minor corin allele with hypertension was confirmed in the adjusted analysis in the Chicago Genetics of Hypertension Study 23
(P = 0.03; 126±2.0 versus 121±0.7 mm Hg, respectively). In conclusion, they illustrated that the corin gene variation is common in blacks and increase significantly blood pressure with increased risk for incidence hypertension. In research in last year s, corin levels were found to significantly decrease in patients with hypertension compared to there without hypertension. The reduction in plasma corin levels associated with the severity of hypertension (Jiang et al., 2011; Dong et al., 2010 & Zhou et al., 2014). Also for hypertension, corin null mice also had heart hypertrophy. This heart phenotypes were established in a naturally occurring SNPs mouse, Kit Wsj, in which the corin gene was damaged by genetic engineering. It is unclear if this phenotype is essential for hypertension in these mice or due to change in corin enzymes structure & morphology (Nigrovic et al., 2008). Previous researches illustrated the minor corin SNPs was associated with hypertension and induced heart hypertrophic response to elevated blood pressure (Rame et al., 2007 and Nigrovic et al., 2008). Patients with these SNPs variations had a higher left ventricular mass than that in normal with wild-type alleles but similar SBP. In all recent study, recombinant corin variants for most common corin SNPs (T555I/Q568P) were found to have a decreasing activity in processing NPs (Wang et al., 2008). The furin SNPs variation may have important play in the renin-angiotensin system and in controlling the electrolyte balance. Because of its effect and decay of regulation of blood pressure. Li et al., (2010) investigated whether there is a relationship between a genetic defect in the furin alleles and primary hypertension in a Xinjiang Kazakh people. They have studied furin variation in ninety-four hypertensive patients to identify SNPs linked with hypertensive patients. They found 9 novels and 7 known SNPs were identified in the furin alleles. The results shown that 1970C > G was related with a hypertension for additive, dominant & allele model (P = 0.091; P = 0.031 & P = 0.030, respectively), and after adjustment with multivariate regression analysis, OR=1.5, P = 0.008 & 1.5, P = 0.010 among additive & dominant models, respectively. However, the relationship between 1970C > G and hypertension was replicated in Uygur people for additive, dominant & allele (P = 0.042; P = 0.102; model, P = 0.027) after adjustment in additive and dominant models, (OR = 1.3; P = 0.01 and 1.307; P = 0.038), respectively. G allele carriers exhibited considerable lower 24
urinary sodium excretion rate than Kazakh Chinese population that were non-carriers the (153±76 um/min vs 173± 90 um/min, P = 0.007). the researcher recommended the gene of furin may be an applicant gene occupied in hypertension patients, and that the G allele of 1970C > G may be a simple risk factor for hypertension patients in Uygur & Xinjiang Kazakh populations. 25
3 Chapter 3 Subjects and Methods 3.1 Study design The current study is an observational case-control investigation. The advantage it studies is frequently used to exam risk factors that may induce to a medical condition by comparing groups who have that disease (patients group) with others who healthy individual (controls group). this type of studies more common because relatively inexpensive to implement, fast, widely application, require comparatively fewer cases, and agree to risk factors to be assessed for one outcome (Song and Chung, 2010). 3.2 Target population The study population was comprised of hypertensive patients aged 30-65 years attending Hypertension clinics in Gaza Strip. Control group was apparently healthy persons. 3.3 Sample size The sample size calculations by EPI-INFO new version 3.5 was used with 95% confidence interval, 20% type 2 error (100-power) and 50% prevalence and an odds ratio less than 2. The sample size 45 cases and 45 controls. Cases and controls were matched for age, gender and residence. 3.4 Inclusion criteria All of the newly diagnosed hypertensive patients aged 30-65, regardless of the type of later treatment. 3.5 Exclusion criteria The following people were excluded from the current study to eliminate potential confounding factors: Cases and controls who are aged < 30 years and > 65 years old. Renal failure patients. Subjects with a history of cancer. Patients who take hormone replacement therapy or corticosteroid therapy. Patient with liver cirrhosis or hematologic disorder. 27
3.6 Ethical consideration Approvals for performing the present study were given by Helsinki committee and ministry of health in the Gaza Strip. 3.7 Data collection 3.7.1 Questionnaire interview Face to face meeting was used to fill in questionnaire. The questionnaire included questions on personal data (Age, education, employment, family income/month and family history of Hypertension), lifestyle (Smoking, diet, physical activity), and clinical data (and duration & age at diagnosis of hypertension, DM, neuropathy, CVD). However, During the study, the interviewer answered all requirements and questions to the participants and most questions were the no / yes question which offers dichotomous choices and multiple choice (Backestrom and Hursh-Cesar, 2012). 3.7.2 Body mass index BMI was calculated from the general equation that (body weight in Kg)/height in m 2. The participants were asked to remove heavy clothes & shoes before measurement scales of weight and height. Normal weight for the participant with 18.0 to 24.9, overweight from 25.0 to 29.9 & obese 30.0 kg/m 2. (WHO, 2012). 3.8 Specimen collection and biochemical analysis 3.8.1 Blood samples Blood samples were collected from 45 hypertensive patients and 45 healthy individuals. Fasting blood sample was drawn by the researcher herself into plan tubes from each participant. The sample was allowed to clot. Then obtained serum sample by centrifugation at 3000 rpm/ 5 min for biochemical tests. serum creatinine, urea, triglyceride, uric acid, cholesterol, total protein, albumin, potassium, corin, furin HDL-C and LDL-C, was analyzed. 28
3.8.1.1 Chemicals and reagents Chemicals reagents used in the present study are shown in the following Table: Reagent Supplier Urea Diasys Diagnostic Systems, Germany Creatinine Diasys Diagnostic Systems, Germany Uric acid BioSystems, Spain Cholesterol Diasys Diagnostic Systems, Germany Triglycerides Diasys Diagnostic Systems, Germany HDL-C Diasys Diagnostic Systems, Germany Total protein Quimica Clinica Aplicada S.A, Spain Albumin Diasys Diagnostic Systems, Germany Potassium Lab-Care Diagnostic, India Corin Cusabio Biotech Company, China Furin Cusabio Biotech Company, China 3.8.1.2 Biochemical analysis 1- Determination of serum urea Principle 29
Reagents Concentrations are those in the final test mixture. Assay procedure The mixing 4 parts of Reagent 1 with 1 part of Reagent 2 for prepared working reagent (WR). Wavelength = 340 nm Temperature = 37 ºC 10 µl of sample/control were added to 1 ml of WR and then mix with incubated for I min then absorbance (A1) was recorded and after 2 nd min (A2) was measured. Calculation Reference value (serum urea) 30
2- Determination of serum creatinine Principle Orange-red complex colored were forms for creatinine by an alkaline picrate solution. The variation in absorbance at fixed times during change is relative to the concentration of creatinine in the sample. Reagents Concentrations are those in the final test mixture. Assay procedure Mixing 4 parts of Reagent 1 with 1 part of Reagent 2 to the prepared working solution. then added 50 µl of sample/control were added and mixed. The incubated the mixture for 60 sec then absorbance (A1) was measured (Wavelength 340 nm) and after 2 nd min (A2) was measured Calculation Note: Creatinine in urine was analyzed by the same procedure for serum creatinine after diluting urine 1+ 19 of distilled water and the result was multiplied by 50. 31
3- Determination of serum cholesterol Principle Cholesterol was determined of after enzymatic reaction. The indicator is quinoneimine that is form by phenol by hydrogen peroxide and 4-aminoantipyrine under the catalytic reactions. Reagents Concentrations are those in the final test mixture. Assay procedure 10 µl of control/sample was added to 1 ml of reagent, incubated at 37 ºC for 5 min and then measured within 10 min (Wavelength 500 nm) Calculation 32
Reference value (serum cholesterol) Levels in term of risk for coronary heart disease 4- Determination of serum triglycerides Principle Determination of triglycerides after enzymatic splitting with lipoprotein lipase Indicator is quinoneimine which is generated from 4-aminoantipyrine and 4-chlorophenol by hydrogen peroxide under the catalytic action of peroxidase Reagents Concentrations are those in the final test mixture. 33
Assay procedure 10 µl of control/sample was added to 1 ml of reagent, incubated at 37 ºC for 5 min and then measured within 10 min (Wavelength 500 nm) Calculation Reference value (Triglycerides) 5- Determination of serum High density lipoprotein cholesterol Principle Phosphotungstic acid and magnesium ions were precipitated LDL, very low-density lipoproteins (VLDL), and Chylomicrons when added to the sample. only the HDL were left by Centrifugation, their cholesterol content is determined enzymatically using cholesterol reagent. Reagents Assay procedure 1- Precipitation 100 µl of sample/control were added to 250 µl of the reagent, allowed to stand for 10 min and then centrifuged for 5 min. 2- Cholesterol determination Assay procedure 10 µl of control/sample was added to 1 ml of reagent, incubated at 37 ºC for 5 min and then measured within 10 min (Wavelength 500 nm) 34
Calculation Reference value (HDL-C) 6- Determination of serum low density lipoproteins Calculated LDL-C by using the empirical equation (Friedewald et al., 1972). The Equation 7- Uric acid determination Principle Uric acid in the sample originates, by means of the coupled reactions described below, a colored complex that can be measured by spectrophotometry. 35
Reagents Assay Procedure 10 µl of control/sample was added to 1 ml of reagent, incubated at 37 ºC for 5 min and then measured within 20 min (Wavelength 500 nm) Calculation Reference value (serum uric acid) 36
8- Determination of serum albumin Principle Color modifies of the indicator from Yellow-green to green-blue will be formed in the presence of bromocresol green at slightly acid Ph, serum albumin produces. Reagents Assay Procedure 10 µl of control/sample was added to 1 ml of reagent, incubated at 37 ºC for 5 min and then measured within 10 min (Wavelength 540 nm) Calculation Reference value (serum albumin) 37
9- Determination of serum total protein Principle In alkaline solution, the proteins form with copper(ii) ions a colored complex, highly stable, which is spectrophotometrically measurable and proportional to the concentration of protein in the sample. Reagents Reagent NaOH Potassium iodide Copper (II) sulfate Sodium- Potassium Tartrate Standard Concentration 0.47 mol/l 23.3mmol/l 6.5 mmol/l 22.1 mmol/l 5 g/dl Assay Procedure 20 µl of control/sample was added to 1 ml of reagent, incubated at 37 ºC for 5 min and then measured within 10 min (Wavelength 540 nm) Calculation Total protein [g/dl] = A sample X concentration of standard A standard Reference value (total protein) 38
10- Determination of serum potassium Principle Potassium ions in a protein-free alkaline medium react with sodium tetraphenyl boron to produce a finely dispersed turbid suspension of potassium tetraphenyl boron. The turbidity produced is proportional to the potassium concentration and read photometrically. Reagents Reagent Concentration Potassium Reagent Standard 5.0mmol/l Assay Procedure 20 µl of control/sample was added to 1 ml of reagent, incubated at 25 ºC for 20 min and then measured within 10 min (Wavelength 630 nm) Calculation Potassium [mmol/l] = A sample X concentration of standard A standard Reference value (serum potassium) Adult 3.6 5.5mmol/l 39
11- Determination of corin Principle The quantitative sandwich enzyme immunoassay technique was used in corin assay. a microplate pre-coated with specific for corin antibody were used. samples are pipetted into the wells and any corin present is bound by the immobilized antibody. After removing any unbound substances, a specific antibody for corin is added to the wells, followed by a wash and a substrate solution is added to the wells. The amount of corin proportion to color development in wells. Reagents and materials provided 40
Reagent preparation All reagents were brought to room temperature (18-25 C) before use for 30min. 1. Biotin-antibody (1x) was centrifuged the vial before opening. Biotin-antibody was diluted 100-fold. 10 μl of Biotin-antibody was added to 990 μl of Biotin-antibody Diluent. 2. HRP-avidin (1x) was centrifuged the vial before opening. HRP-avidin was diluted a 100- fold. HRP-avidin 990 was added to μl of HRP-avidin Diluent. 3. Wash Buffer(1x)- 20 ml of Wash Buffer Concentrate (25 x) was added to deionized to prepare 500 ml of Wash Buffer (1 x). 4. Standard The standard vial was centrifuged at 6000-10000rpm for the 30s. The Standard was reconstituted with 1.0 ml of Sample Diluent to produce a stock solution of 40 ng/ml. 250 μl of Sample diluent was pipetted into each tube (S0-S6). The stock solution to was used to produce a 2-fold dilution series (below). each tube was mixed thoroughly before the next transfer. The undiluted Standard was served as the high standard (40 ng/ml). Sample Diluent was served as the zero standards (0 ng/ml) 41
Assay procedure 1. 100 μl of standard/sample was added per well. The adhesive strips were covered and incubate to 2 hours at 37 C. 2. 100 μl of Biotin-antibody(1x) was added to each well. Cover with a new adhesive strip. 3. 100μl of HRP-avidin (1x) was added to each well. The microtiter plate was covered with a new adhesive strip and it was incubated for 1 hour at 37 C. 4. The aspiration/wash process was repeated five times as in step 5. 5. 90 μl of TMB substrate was added to each well and Incubated for 15minutes. 6. 50 μl of stop solution was added to each well and mixed gently tap the plate to ensure thorough mixing. 7. reader set to 450 nm. 12- Determination of furin Principle The quantitative sandwich enzyme immunoassay technique was used in furin assay. a microplate pre-coated with specific for furin antibody was used. samples are pipetted into the wells and any furin present is bound by the immobilized antibody. After removing any unbound substances, a specific antibody for furin is added to the wells, followed by a wash and a substrate solution is added to the wells. The amount of furin proportion to color development in wells. 42
Reagents and materials provided Reagent preparation All reagents were incubated at room temperature. 2. The standard vial was centrifuged at 6000rpm for the 30s and then dilution in 1 ml diluents as corin: 43
Assay procedure 1. 100 μl of standard/sample was added per well. The adhesive strips were covered and incubate to 2 hours at 37 C. 2. 100 μl of Biotin-antibody(1x) was added to each well. Cover with a new adhesive strip. 3. 100μl of HRP-avidin (1x) was added to each well. The microtiter plate was covered with a new adhesive strip and it was incubated for 1 hour at 37 C. 4. The aspiration/wash process was repeated five times as in step 5. 5. 90 μl of TMB substrate was added to each well and Incubated for 15minutes. 6. 50 μl of stop solution was added to each well and mixed gently tap the plate to ensure thorough mixing. 7. reader set to 450 nm. 3.9 Data analysis Data was collected, summarized, tabulated and analyzed using Statistical Package for Social Sciences (SPSS) software version 23.0. Data regarding patients was collected throughout face to face questionnaire which includes questions that will help in the study. The % difference was calculated: The following statistical tests were t-test, Pearson's correlation test, Chi-Square Test & ANOVA Probability values (P) were significance at P 0.05. The maximum & minimum values were used. Graphs were plotted using excels. 44
45
4 Chapter 4 Results The current study is a case-control which included 90 adult males (45 hypertensive patients and 45 controls). The average age of the controls was 43.5±8.4 years whereas that of hypertensive patients was 44.2±7.7 years (P>0.05). 4.1 Baseline characteristics of study population Table (4.1) illustrates Baseline characteristics of the study population. Analysis of the educational status of the controls and cases showed that 16 (35.6%) and 18 (40.0%) have diploma or university degree, 19 (42.2%) and 20 (44.4%) have finished secondary school, 1 (2.2%) and 3 (6.7%) have finished preparatory school, 9 (20.0%) and 4 (8.9%) have passed primary school, respectively. The educational level among populations was found to be not associated with hypertension ( 2 =3.066, P = 0.382). In addition, the number of controls and cases who have employment were 33 (73.3%) and 26 (57.8%), respectively, ( 2 =2.411, P = 0.120). The results also showed that 12 (26.7%) of controls and 19 (42.2%) of cases have monthly income less than 1000 NIS. 1000 2000 NIS comprised 28 (62.2%) of controls and 19 (42.2%) of cases. Controls and cases who have more than 2000 NIS monthly were 5 (11.1%) and 7 (15.6%), respectively, ( 2 =3.637, P = 0.162). Regarding to physical activity, the number of cases and controls on lazy physical activity were 4 (8.9%) and 12 (26.7%), respectively, whereas those who have intermediate physical activity were 10 (22.2%) 14 (31.1%), and the active physical activity were 31 (68.9%) and 19 (42.2%), respectively, ( 2 =7.547, P = 0.023). In addition, the number of controls and cases on diet were 4 (8.9%) and 14 (31.1%), respectively there was a significant difference between controls and cases ( 2 =6.944, P = 0.008). Also, Table 4.1 illustrates the smoking status where 19 (42.2%) controls and 19 (42.2%) cases were smokers, there was no significant difference between controls and cases ( 2 =0.000, P = 1.000). Regarding family history of hypertension, 37 (82.2%) controls and 36 (80.0%) cases reported that they have a family history of hypertension, there was no significant difference between controls and cases ( 2 =0.073, P = 0.788). 46
Table ( 4.1): Baseline characteristics of study population General characteristics Controls (n=45) n (%) Cases(n=45) n (%) 2 P-value Education Diploma or University Secondary school Preparatory school Primary school 16 (35.6) 19 (42.2) 1 (2.2) 9 (20.0) 18 (40.0) 20 (44.4) 3 (6.7) 4 (8.9) 3.066 0.382 Employment Yes No 33 (73.3) 12 (26.7) 26 (57.8) 19 (42.2) 2.411 0.120 Family income per month (NIS) Less than 1000 1000 to 2000 More than2000 12 (26.7) 28 (62.2) 5 (11.1) 19 (42.2) 19 (42.2) 7 (15.6) 3.637 0.162 Physical activity Lazy Intermediate Active 4 (8.9) 10 (22.2) 31 (68.9) 12 (26.7) 14 (31.1) 19 (42.2) 7.547 0.023* Particular diet Ýes No 4 (8.9) 41 (91.1) 14 (31.1) 31 (68.9) 6.944 0.008* Smoking Yes No 19 (42.2) 26 (57.8) 19 (42.2) 26 (57.8) 0.000 1.000 Family history of hypertension Yes No 37 (82.2) 8 (17.8) *P-value significant at P 0.05; NIS: new Israeli shekel. 36 (80) 9 (20) 0.073 0.788 4.2 Prevalence complications among hypertensive patients Table (4.2) summarizes the Prevalence complications among hypertensive patients compared to controls. The percentages of retinopathy, CVD and recurrent infections were higher in cases compared to controls (46.7, 15.6 and 8.9% vs. 37.8, 17.8 and 2.2%, respectively) with no statistically significant differences ( 2 = 0.729, P = 0.393; 2 =1.800, P = 0.180 and 2 =1.906 P = 0.180, respectively). On the other hand, the percentages of diabetes mellitus and neuropathy were higher in controls compared to cases (17.8 and 26.7% vs. 15.6 and 22.2%, respectively) with no statistically significant differences ( 2 = 0.080, P = 0.777 and 2 = 0.241, P = 0.624, respectively). 47
Table ( 4.2): Prevalence complications among hypertensive patients Complication Controls (n=45) n (%) Cases (n=45) n (%) 2 P-value Retinopathy Ýes No 17 (37.8) 28 (62.2) 21 (46.7) 24 (53.3) 0.729 0.393 Cardiovascular diseases Yes No 3 (6.7) 42 (93.3) 7 (15.6) 38 (84.4) 1.800 0.180 Recurrent infection Ýes No 1 (2.2) 44 (97.8) 4 (8.9) 41 (91.1) 1.906 0.180 Diabetes mellitus Yes No 8 (17.8) 37 (82.2) 7 (15.6) 38 (84.4) 0.080 0.777 Neuropathy Ýes No 12 (26.7) 33 (73.3) 10 (22.2) 35 (77.8) 0.241 0.624 *P-value significant at P 0.05. 4.3 Anthropometric measures and blood pressure among study population Table (4.3) summarizes the Baseline characteristics of the study population according to age, height, weight, BMI and systolic and diastolic blood pressure. The means of age were (43.5±8.4) years of controls and (44.2±7.7) years of cases, there were no significant differences between controls and cases with age (t=0.419, P = 0.676). Weight, height and BMI of controls are (84.5±12.2 kg), (177.5±7.3 cm) and (26.8±3.6 kg/m 2 ), respectively while those of cases are (93.8±17.9 kg), (175.2±8.4 cm) and (30.6±5.8 kg/m 2,), respectively. There were significant differences between controls and cases regarding weight and BMI (t=2.880, P = 0.005) and (t=3.673, P = 0.000). while there were no significant differences between controls and cases regarding height (t=-1.352, P = 0.180). The mean levels of systolic / diastolic blood pressure were (120.3±7.9) / (77.1±6.3), (138.7±10.2) / (89.7±9.4) mmhg for controls and cases respectively. There were significant differences between controls and cases with both systolic and diastolic blood pressure among study population (t=9.598, P = 0.000), (t=7.475, P = 0.000), respectively. 48
Also, table 4.3 shows the mean values of hypertension duration and age at diagnosis of hypertension for cases (1.9±1.0), (42.2±8.0) years, respectively. Table ( 4.3): Anthropometric measures and blood pressure among study population Controls (n=45) Cases (n=45) % General characteristics t P-value Mean±SD Mean±SD difference Age (years) (min-max) 43.5±8.4 (31-65) 44.2±7.7 (30-63) 1.6 0.419 0.676 Weight (kg) (min-max) 84.5±12.2 (58-112) 93.8±17.9 (53-150) 10.4 2.880 0.005* Height (cm) (min-max) 177.5±7.3 (163-194) 175.2±8.4 (155-190) -1.3-1.352 0.180 BMI (kg/m 2 ) (min-max) 26.8±3.6 (20.7-35.8) 30.6±5.8 (19-51.9) 13.2 3.673 0.000* SBP (mmhg) (min-max) 120.3±7.9 (100-135) 138.7±10.2 (120-160) 14.2 9.598 0.000* DBP (mmhg) (min-max) 77.1±6.3 (60-85) 89.7±9.4 (64-110) 15.1 7.475 0.000* Duration of HTN (years) (min-max) - 1.9±1.0 (1-5) Age at diagnosis HTN (years) (min-max) - 42.2±8.0 (27-61) *P-value significant at P 0.05; BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure; HTN: hypertension. 4.4 Serum analysis 4.4.1 Blood nitrogen and potassium among study population Table (4.4) points out that the mean serum urea concentrations were significantly increased in cases compared to controls (40.1±9.5 vs 31.6±6.0 mg/dl, % difference=23.7, t=5.014, P = 0.000). similar trend was found for creatinine (1.0±0.2 vs 0.90±0.10 mg/dl, % difference=10.5, t=4.275, P = 0.000). This change was also significant (t=4.275, P = 0.000). In contrast, the mean serum uric acid and total protein concentrations were increased in controls compared to cases (6.24±1.5 vs 6.19±1.4 mg/dl, % difference=0.8, t=-0.165, P = 0.870) and (7.2±0.4 vs 7.1±0.3g/dl, % difference=-1.4, t=-0.176, P = 0.861), respectively. There was no significant difference in the mean levels of uric acid 49
and total protein among the study population. On the other hand, the mean serum K and albumin concentrations were significantly increased in cases compared to controls (4.6±0.5 vs 4.3±0.4 mmol/l, % difference=6.7, t=3.224, P = 0.002), (4.3±0.4 vs 4.0±0.3 g/dl, % difference=7.2, t=3.851, P = 0.000), respectively. Table ( 4.4): Blood nitrogen and potassium among study population Parameter Controls (n=45) Mean±SD Cases (n=45) Mean±SD % difference T P-value Urea (mg/dl) 31.6±6 40.1±9.5 23.7 5.014 0.000* (min-max) (20-44) (27-66) Creatinine (mg/dl) 0.9±0.1 1±0.2 10.5 4.275 0.000* (min-max) (0.7-1.2) (0.7-1.4) Uricacid (mg/dl) 6.24±1.5 6.19±1.4 0.8-0.165 0.870 (min-max) (3.5-11.3) (4.3-9.2) K (mmol/l) 4.3±0.4 4.6±0.5 6.7 3.224 0.002* (min-max) (3.5-5.1) (3.7-5.6) Total protein (g/dl) (min-max) 7.2±0.4 (6-7.8) 7.1±0.3 (6.5-8) -1.4-0.176 0.861 Albumin (g/dl) 4.0±0.3 4.3±0.4 7.2 3.851 0.000* (min-max) (3.5-4.8) (3.4-5.1) *P-value significant at P 0.05; K: potassium. 4.4.2 Lipid profile among study population Serum lipid profile including cholesterol, triglycerides, HDL-C and LDL-C of cases and controls is illustrated in Table (4.5). The average levels of cholesterol, triglycerides, HDL-C and LDL-C were found to be higher in cases (196.8±46.4, 195.6±58.3, 47.5±4.0 and 196.8±46.4 mg/dl, respectively) compared to controls (170.4±29.3, 166.2±63.0, 45.8±3.5 and 170.4±29.3 mg/dl, respectively) with % differences of 14.4, 16.3, 3.6 and 18.8 %, respectively). This elevation was statically significant (t=3.236, P = 0.002; 50
t=2.295, P = 0.024; t=2.101, P = 0.038 and t=2.660, P = 0.009, respectively). This result of lipid profile may be used as a strong predictive of developing of hypertension. Table ( 4.5): Lipid profile among study population Parameter Controls(n=45) Mean±SD Cases(n=45) Mean±SD % difference T P-value Cholesterol (mg/dl) 170.4±29.3 196.8±46.4 14.4 3.236 0.002* (min-max) (114-238) (141-361) Triglycerides (mg/dl) 166.2±63 195.6±58.3 16.3 2.295 0.024* (min-max) (79-384) (124-354) HDL-C (mg/dl) 45.8±3.5 47.5±4.0 3.6 2.101 0.038* (min-max) (40-56) (41-61) LDL-C (mg/dl) 170.4±29.3 196.8±46.4 18.8 2.660 0.009* (min-max) (114-238) (141-361) *P-value significant at P 0.05; HDL-C: high density lipoprotein; LDL-C: low density lipoprotein 4.4.3 Serum Corin and furin among study population Table (4.6), Figures (4.1) and (4.2) demonstrate the mean levels of serum corin and furin in controls and cases. The mean of serum corin levels was decreased in cases (11.0±11.2 ng/ml) than controls (61.1±81.2 ng/ml) and the statistical test showed a significant difference between cases and controls (t=-4.096 and P = 0.000). In contrast, Serum furin was significantly elevated in cases than controls (717.3±759.4 vs 415.0±563.9 pg/dl; respectively t=2.143 and P = 0.035). The high level of SD values of corin and furin indicated that the variation in the results was very high (heterogenous values). 51
Table ( 4.6): Serum Corin and furin among study population Parameter Controls (n=45) Mean±SD Cases (n=45) Mean±SD % difference T P-value Corin (ng/ml) 61.1±81.2 11.0±11.2 139.0-4.096 0.000* (min-max) (8.1-364.0) (0.3-43.6) Furin (pg/ml) 415.0±563.9 717.3±759.4 53.4 2.143 0.035* (min-max) (4.6-2275) (10.6-3443.0) Figure ( 4.1): Distribution the mean of serum corin level (ng/ml) among controls and cases 52
Figure ( 4.2): Distribution the mean of serum furin level (pg/ml) among controls and cases. 4.5 Distribution of serum corin level according to baseline characteristics of study population Table (4.7) demonstrated the distribution of serum corin level according to baseline characteristics of study population. There was no statistically significant association between serum corin and education, employment, family income per month, physical activity, particular diet, family history of hypertension, retinopathy, diabetes mellitus, neuropathy, cardiovascular diseases and recurrent infection among study population (P>0.05). In contrast, there was a statistically significant association between serum corin and smoking among study population (t=2.360, P = 0.020). 53
Table ( 4.7): Distribution of serum corin level according to baseline characteristics of study population General characteristics Serum corin level (ng/ml) Mean±SD (min-max) Statistical test Value P-value Education Diploma or University Secondary school Preparatory school Primary school Employment Ýes No Family income per month (NIS) Less than 1000 1000 to 2000 More than 2000 Physical activity Lazy Intermediate Active Particular diet Ýes No Smoking Yes No Family history of hypertension Ýes No Retinopathy Ýes No Diabetes mellitus Yes No Neuropathy Yes No Cardiovascular diseases Yes No Recurrent infection Yes No 15.3±12.1(0.4-46.4) 91.8±148.4(8.1-314) 46.4±77.9(0.6-364) 25.6±29.7 (0.3-105) 39.2±67.3 (0.3-364) 30.1±54.5 (0.4-302) 21.2±20.9 (0.4-93.1) 51.3±82.5 (0.3-364) 14.9±18.0 (0.6-57) 851.3±655.0 (21.5-1875) 620.3±744.1 (12.9-2275) 449.0±642.0 (4.6-3443) 20±24.4 (0.3-93.1) 40.1±68.9 (0.4-364) 54±89.6 (0.6-364) 23.0±26.2 (0.3-122) 34.6±60.0 (0.3-364) 42.2±76.2 (0.6-314) 40.0±77.9 (0.6-364) 33.2±50.0 (0.3-314) 40.0±77.9 (0.6-364) 33.2±50.0 (0.3-314) 30.1±62.3 (0.6-302) 38.0±63.5 (0.3-364) 14.2±15.0 (0.6-43.6) 38.8±66.1 (0.3-364) 11.4±4.7 (8.1-19.6) 37.5±64.5 (0.3-364) *P-value significant at P 0.05; NIC: new Israeli shekel. F 2.270 0.086 t 0.653 0.515 F 3.050 0.052 F 1.082 0.171 t -1.212 0.229 t 2.360 0.020* t -0.445 0.657 t -0.503 0.619 t -0.865 0.398 t -0.514 0.609 t -1.169 0.246 t -0.900 0.371 54
4.6 Distribution of serum furin level according to baseline characteristics of study population Table (4.8) provides the distribution of Serum furin level according to baseline characteristics of study population. There was a statistically significant association between serum furin and education among study population (P = 0.020). In contrast, there was no statistically significant association between serum furin and employment, family income per month, physical activity, particular diet, smoking, family history of hypertension, retinopathy, diabetes mellitus, neuropathy, cardiovascular diseases and recurrent infection among study population (P>0.05). 55
Table ( 4.8): Distribution of serum furin level according to baseline characteristics of study population General characteristics Education Diploma or University Secondary school Preparatory school Primary school Employment Yes No Family income per month (NIS) <1000 1000-2000 >2000 Physical activity Lazy Intermediate Active Particular diet Yes No Smoking Yes No Family history of hypertension Yes No Retinopathy Yes No Diabetes mellitus Yes No Neuropathy Yes No cardiovascular diseases Yes No Recurrent infection Yes No *P-value significant at P 0.05 Serum furin level (pg/ml) Mean ±SD (min-max) 1035.3±761.0 (109.9-2275) 167±175.2(23.9-412) 413.7±545.5(10.6-1974) 608.8±751.0 (4.6-3443) 568.9±707.5 (4.6-3443) 561.2±642.7 (20.8-2275) 567.5±642.3 (10.6-2275) 494.5±698.3 (4.6-3443) 843.5±704.4 (27.6-2066) 9.2±10.6 (0.6-39.8) 41.1±75.4 (0.4-364) 42.3±64.9 (0.3-314) 653.5±879.1 (14.9-3443) 544.4±629.3 (4.6-2275) 508.6±604.1 (12.9-2066) 608.3±737.0 (4.6-3443) 578.8±686.4 (4.6-3443) 512.2±681.9 (10.6-1974) 558±649.9(10.6-2275) 572.2±711.2 (4.6-3443) 558±649.9 (10.6-2275) 572.2±711.2 (4.6-3443) 610.6±699.2 (13.6-2275) 551.9±681.3 (4.6-3443) 647.6±724.3 (13.6-1974) 556±680.9 (4.6-3443) 400.9±208.5 (140.5-718.6) 575.9±699.6 (4.6-3443) statistical test value P-value F 3.456 0.020* t 0.050 0.960 F 1.258 0.289 F 2.273 0.109 t 0.604 0.547 t -0.683 0.497 t 0.360 0.719 t -0.097 0.923 t 1.702 0.092 t 0.349 0.728 t 0.398 0.692 t -0.555 0.580 56
4.7 Correlation between serum corin, furin levels and studied parameters among study population Correlations between serum corin, furin level and studied parameters among study population are pointed out in Figures (4.3) and (4.4) and Tables (4.9), (4.10). The Pearson correlation test showed statistically significant negative correlation between corin level and furin level among controls (r=-0.381, P = 0.010) and cases (r=-0.676, P = 0.000). However, there was no statistically significant correlation between serum corin and furin levels with SBP, DBP, age, weight, height, BMI, duration HTN, age at diagnosis HTN and most biochemical studied parameters among study population (P>0.05). 57
Figure ( 4.3): Correlation between corin and furin level in controls. Figure ( 4.4): Correlation between corin and furin level in cases. 58
Table ( 4.9): Correlation between serum corin level and studied parameters among study population Serum corin level (ng/ml) Parameters Controls Cases Pearson Pearson P-value P-value correlation (r) correlation (r) SBP (mmhg) -0.127 0.408-0.142 0.352 DBP (mmhg) -0.237 0.117-0.079 0.605 Age (years) -0.234 0.122 0.275 0.067 Weight (kg) 0.181 0.233 0.047 0.760 Height (cm) 0.095 0.535 0.052 0.734 BMI (kg/m 2 ) 0.132 0.387 0.013 0.934 Duration HTN (years) -- -- 0.046 0.762 Age at diagnosis HTN (years) -- -- 0.262 0.082 Urea (mg/dl) -0.215 0.155-0.018 0.907 Creatinine (mg/dl) -0.183 0.228 0.071 0.644 Uric acid (mg/dl) -0.254 0.092 0.033 0.829 K (mmol/l) -0.035 0.821 0.056 0.713 Total protein (g/dl) -0.017 0.913 0.045 0.767 Albumin (g/dl) -0.031 0.841-0.089 0.563 Cholesterol (mg/dl) -0.086 0.573-0.161 0.290 Triglyceride (mg/dl) -0.101 0.507-0.073 0.635 HDL-C (mg/dl) 0.100 0.514-0.095 0.534 LDL-C (mg/dl) -0.063 0.679-0.153 0.315 Furin (pg/ml) -0.381 0.010* -0.676 0.000* *P-value significant at P 0.05; SBP: systolic blood pressure; DBP: diastolic blood pressure; HTN: hypertension; k: potassium; HDL-C: high density lipoprotein; LDL-C: low density lipoprotein. 59
Table ( 4.10): Correlation between serum furin level and studied parameters among study population Serum furin level (pg/ml) Parameters Controls Cases Pearson Pearson P-value P-value correlation (r) correlation (r) SBP (mmhg) 0.121 0.427 0.144 0.346 DBP (mmhg) 0.258 0.087-0.021 0.893 Age (years) 0.187 0.218-0.165 0.278 Weight (kg) -0.065 0.672 0.033 0.832 Height (cm) -0.017 0.913-0.034 0.826 BMI (kg/m 2 ) -0.075 0.626 0.067 0.662 Duration HTN (years) -- -- 0.084 0.585 Age at diagnosis HTN (years) -- -- -0.175 0.251 Urea (mg/dl) 0.139 0.362 0.008 0.958 Creatinine (mg/dl) 0.128 0.404-0.106 0.490 Uric acid (mg/dl) 0.061 0.689-0.056 0.713 K (mmol/l) -0.063 0.681-0.055 0.719 Total protein (g/dl) 0.111 0.470-0.160 0.294 Albumin (g/dl) -0.278 0.064-0.087 0.572 Cholesterol (mg/dl) 0.016 0.915 0.205 0.176 Triglyceride (mg/dl) 0.139 0.363-0.008 0.957 HDL-C (mg/dl) 0.042 0.784 0.160 0.295 LDL-C (mg/dl) -0.056 0.715 0.221 0.145 Corin (ng/ml) -0.381 0.010* -0.676 0.000* P-value significant at P 0.05; SBP: systolic blood pressure; DBP: diastolic blood pressure; HTN: hypertension; k: potassium; HDL-C: high density lipoprotein; LDL-C: low density lipoprotein. 60