Need for the Study s1

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA BANGALORE. ANNEXURE II PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. NAME OF THE CANDIDATE Dr. SAHANA G R, AND ADDRESS POST-GRADUATE STUDENT, (in block letters) DEPARTMENT OF BIOCHEMISTRY, J.J.M. MEDICAL COLLEGE, DAVANGERE- 577004, KARNATAKA. 2. NAME OF THE INSTITUTION J.J.M. MEDICAL COLLEGE, DAVANGERE- 577004. 3. COURSE OF STUDY AND M.D. BIOCHEMISTRY SUBJECT 4. DATE OF ADMISSION TO 30-08-2013 COURSE “SERUM LIPID PROFLIE, CARBONIC 5. TITLE OF THE TOPIC ANHYDRASE AND ZINC LEVELS IN PATIENTS WITH ESSENTIAL HYPERTENSION.” 6. BRIEF RESUME OF THE INTENDED WORK : 6.1 Need for the study: Essential hypertension is defined as chronic elevation in blood pressure (BP) ≥140/90 mm Hg with no definable cause.1, 2. In India, the prevalence of hypertension is 59.9 and 69.9 per 1000 males and females respectively in urban population and 35.5 and 35.9 per 1000 males and females respectively in rural population1 Various epidemiological studies have highlighted the increasing incidence of hypertension, which is the commonest, asymptomatic, readily detectable and one of the major risk factor in the causation of CVD, Stroke and other vascular complications. The incidence of cardiovascular disease is raising and they are predicted to be the biggest cause of death by 2020 in India. Hypertension accelerates the atherosclerotic process along with other factors leading to CHD. It is a serious public health problem in the industrialized world as well as in the developing countries and one of the leading causes of morbidity and mortality. Even though effective treatment measures have been extended against hypertension, it still remains inadequately managed. Essential hypertension is the most prevalent hypertension type, accounting to 90– 95% of the patients suffering from hypertension. Even though no direct cause has been identified factors such as sedentary lifestyle, stress, obesity, hypokalemia, salt sensitivity, alcohol intake, and vitamin D deficiency are reported to increase the risk of developing essential hypertension Hypertension is also associated with elevated reactive oxygen species and the impairment of endogenous antioxidant mechanisms. Increased levels of serum cholesterol, TG and VLDL have been observed in patients with hypertension. It has been shown that oxidized lipoprotein inactivates NO and aggravates hypertension.3 The association of carbonic anhydrase with hypertension has been established and its activities are obvious with altered metabolism, especially in hypertension, diabetes mellitus and hyperlipidaemia.4 Carbonic anhydrase is Zn metalloenzyme, which catalyzes the reversible hydration and dehydration of CO2, a product of cellular aerobic energy production. carbonic anhydrase H2O + CO2 H2CO3 Carbonic anhydrase inhibitors such as acetazolamide are used as a class of weak diuretic agents .These agents are used along with other thiazide diuretics as a therapy in hypertension. These therapeutic measures which are used in hypertension, can dose- dependently elevate serum total cholesterol levels, modestly increase low-density lipoprotein cholesterol (LDL-C) levels and raise triacylglycerol (TG) levels, while minimally changing high-density lipoprotein cholesterol (HDL-C) concentrations. All diuretics, including loop diuretics, cause these lipid changes. The mechanisms of diuretic- induced dyslipidemia remain uncertain, but have been related to worsened insulin sensitivity and/or the reflex activation of the Renin-Angiotensin-Aldosterone system (RAAS) and the sympathetic nervous system in response to volume depletion. Considering the view points and the relationship between hypertension, lipid profile, carbonic anhydrase modulation in diuretic therapy and scanty literature reports in this region, the current study will be focused to determine the association between lipid profile and carbonic anhydrase activity in essential hypertension patients. The essential trace element zinc [Zn] is an important component of bio membranes and an essential co factor in a variety of enzymes. Zn has an antioxidant like properties thus it can stabilize macromolecules against radical production5.We also know that Zn is a component of the carbonic anhydrase molecule and plays an important role in maintaining blood pressure.6

6.2 Review of literature : Hypercholesterolemia, hypertension and obesity are considered as standard coronary risk factors.7 There are numerous studies highlighting significant alteration in serum cholesterol, triglyceride, HDL-C and LDL-C in hypertensive patients.8 It has been established that there will be changes in carbonic anhydrase activities with altered metabolism in essential hypertension.5 Altered Zn levels in serum results in abnormal physiological disorder and in addition to other factors may have a role in hypertensive disease.9 Various experiments have been done to evaluate the relationship between serum carbonic anhydrase activity and serum Zn levels in many pathological conditions.10

6.3 Objectives of the study : To estimate serum lipid profile, carbonic anhydrase activity and Zn levels in essential hypertension. To determine the correlation between lipid profile, carbonic anhydrase activity and Zinc in essential hypertension. To study relationship of carbonic anhydrase activity with the degree of severity of hypertension.

7. MATERIALS AND METHODS 7.1. Source of data: A cross sectional study will be carried out for a period of one year. The patients will be selected from Chigateri General Hospital and Bapuji Hospital, Davangere (both hospitals attached to the teaching institute J.J.M Medical College, Davangere). The study group consists of patients with essential hypertension. The control groups consists of normotensive subjects both in the age group 30-50yrs. Cases and controls will be interviewed to obtain relevant data after taking informed consent. Based on inclusion and exclusion criteria minimum of 50 cases will be selected and about 50 age matched controls will be selected.

Inclusion Criteria: Cases: Patients with blood pressure of ≥140/90mm of hg as measured with standard mercury sphygmomanometer will be considered as having essential hypertension and taken as cases. Patients who are diagnosed within 6 months of onset, with or without antihypertensive therapy will also be included. Control: Healthy age matched normotensive subjects without any major illness and not under any medications.

Exclusion criteria : subjects  Who are smokers  Who are on lipid lowering drugs  Who are on anti oxidant vitamin supplements  Who are obese  Who are chronic hypertensives on anti-hypertensive medications for over six months or more

7.2 Method of collection of data : A 5ml of venous blood collected after overnight fasting from cases and healthy volunteers under aseptic precautions into a vacutainer with clot activator. Clear serum is separated by centrifugation and analyzed for the following biochemical parameters.

A)Estimation of total cholesterol by cholesterol oxidase /phenol aminoantipyrine method:11 Principle: Cholesteryl esterase hydrolyses cholesterol esters to free cholesterol and fatty acid. Then cholesterol is oxidized by cholesterol oxidase forming cholesterol-4-ene-3-one and hydrogen peroxide. In presence of enzyme peroxidase, hydrogen peroxide causes oxidative coupling of phenol and 4-aminoantipyrine to form red colored quinoneimine dye. The intensity of red colour is measured at 520nm in a spectrophotometer.

B)Estimation of HDL-cholesterol by cholesterol oxidase/phenol aminoantipyrine method:11 Principle: Chylomicrons, VLDL and LDL are precipitated by adding phosphotungstic acid and magnesium ions to the serum sample. After Centrifugation the supernatant containing HDL-cholesterol is determined enzymatically. The cholesterol ester is hydrolyzed by cholesteryl estarase to free cholesterol and fatty acid. Then cholesterol is oxidized by cholesterol oxidase forming cholesterol-4-ene-3-one and hydrogen peroxide. In presence of enzyme peroxidase, hydrogen peroxide causes oxidative coupling of phenol and 4- aminoantipyrine to form red coloured quinoneimine dye. The intensity of red colour is measured at 520nm in a spectrophotometer.

C)Estimation of triglycerides by glycerol phosphate oxidase-phenol aminoantipyrine method:11 Principle: Triglycerides are hydrolyzed by lipoprotein lipase to glycerol and fatty acid. Glycerol is then phosphorylated to glycerol-3-phosphate followed by oxidation to dihydroxyacetone phosphate and hydrogen peroxide by glycerol phosphate oxidase. Hydrogen peroxide in presence of peroxidase causes oxidative coupling of 4-chlorophenol and 4-aminoantipyrine to form red colored quinoneimine dye. The intensity of red color is measured at 520nm in spectrophotometer.

D)Estimation of LDL cholesterol by Friedwald formula:11 LDL cholesterol = Total cholesterol – (HDL cholesterol+Triglyceride/5)

E)Estimation of Carbonic anhydrase activity by Rackers method.12 The assay system consisted of 100 µl of sample (serum) containing 1.4 ml of 0.05 M Tris-SO4 buffer (pH 7.4) and 1.5 mL of3mM p-nitro phenyl acetate. The change in absorbance at 348 nm will be measured over a period of 3 min., before and after adding the sample. One unit of enzyme activity will be expressed as 1 µmol of released p-nitro phenol per minute at room temperature.

F)Estimation of serum Zinc by Nitro PAPS method.13 Principle: Zinc in an alkaline medium reacts with nitro-PAPS to form a purple coloured complex. Intensity of the complex formed is directly proportional to the amount of Zinc present in the sample and will be measured at 578nm by a colorimeter.

Zinc+ Nitro –PAPS Alkaline medium Purple colored complex

Statistical analysis: Results will be subjected to appropriate statistical analysis unpaired‘t test will be used to compare between groups. Correlation analysis will be done to measure the relationship between different parameters.

7.3 Does the study require any investigation or intervention to be conducted on patients or other humans or animals? If so, please describe briefly. Yes. Estimation of lipid profile, carbonic anhydrase activity, zinc levels will be done using blood sample from patients with essential hypertension and normotensive subjects as controls.

7.4 Has ethical clearance been obtained from your institution in case of 7.3? Yes. 8. LIST OF REFERENCES : 1. Park K. Epidemiology of chronic non communicable diseases and conditions. In: Text Book of Community Medicine. 21sted, Jabalpur(India):M/s Banarasidas bhanot publishers,2011: 344-348.

2. Ahlawat SK, Singh MM et al. JNC 5 guidelines, Time trends in the prevalence of hypertension and associated risk factors in Chandigarh. Indian Med Assoc 2002; 100(9):547-52,554-5, 572.

3. Beg M, Gupta A et al. Oxidative Stress in Essential Hypertension and Role of Antioxidant.Journal, Indian academy of clinical medicine 2010; 11(4): 287-293.

4. Biswas UK AND Kumar A. Study on lipid profile, oxidation stress and carbonic anhydrase activity in patients with essential hypertension Journal of Clinical and Diagnostic Research 2010 Dec;4:3414-3420.

5. HI Afridi ,TG Khazi et al.Evaluation of cadmium, lead, nickel and zinc status in biological samples of smokers and nonsmokers hypertensive patients. Journal of Human Hypertension 2010;24: 34–43

6. Kheilin.D and Mann.T. Carbonic anhydrase: Purification and nature of the enzyme. Biochem. J 1940; 34:1163

7. Gupta R, Gupta VP et al. Prevalence of coronary heart disease and risk factors in an urban Indian population: JaipurHeartWatch-2. Indian Heart J 2002; 54(1):59- 66.

8. Sarkar D, Latif SA et al. Studies on serum lipid profile in hypertensive patient. Mymensingh Med J 2007 Jan; 16(1):70-6.

9. Hennig B, Meerarani P et al. Antioxidant-like properties of zinc in activated endothelial cells. J Am Coll Nutr 1999; 18: 152–158

10. Bert L, Vallee et al .The relationship between carbonic anhydrase activity and zinc content of erythrocytes in normal, in anemic and other pathological condition. Blood Journal, American society of hematology 1949;4:467-478

11. Rifal N, GR. Lipids, Lipoproteins, Apo lipoproteins and other cardiovascular risk factors. In: Burtis CA, Ashwood ER, Bruns DE. Teitz textbook of clinical chemistry and molecular diagnostics. 4th edition, New Delhi: Elesvier, 2006:942- 949.

12. Racker, E., in S. P. Colowick and N. 0.Kaplan (Editors), Methods in Enzymology, Vol.ZZZ, Academic Press; New York; 1963; 283. 13. Makino T .A sensitive, direct colorimetric assay of serum zinc using nitro-PAPS and using micro well plates .Clin chem acta1991; 197:209-220. SIGNATURE OF THE 9 CANDIDATE

REMARKS OF THE The present study is relevant and feasible in our set 10 GUIDE up 11 NAME & DESIGNATION. Dr. SAVTHRI R SAHUKAR MD. 11.1 GUIDE PROFESSOR, DEPARTMENT OF BIOCHEMISTRY, J.J.M. MEDICAL COLLEGE, DAVANGERE-577004.

11.2 SIGNATURE

11.3 CO-GUIDE Dr. GURUSHANTAPPA MD (if any)

PROFESSOR, DEPARTMENT OF MEDICINE, J.J.M. MEDCAL COLLEGE, DAVANGERE-577004.

11.4 SIGNATURE

11.5 HEAD OF THE Dr. MANJUNATH M TEMBAD DEPARTMENT M.D. PROFESSOR & HOD, DEPARTMENT OF BIOCHEMISTRY, J.J.M. MEDICAL COLLEGE, DAVANGERE.

11.6 SIGNATURE

12 REMARKS OF THE CHAIRMAN & PRINCIPAL

12.1 SIGNATURE