RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE



RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA

BANGALORE

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

|1. |Name of the Candidate |Dr. NALINAKSHI. S |

| |and Address |POST GRADUATE STUDENT, |

| |(in block letters) |DEPARTMENT OF BIOCHEMISTRY, |

| | |J J M MEDICAL COLLEGE, |

| | |DAVANGERE-577 004. |

|2. |Name of the Institution |J J M MEDICAL COLLEGE, |

| | |DAVANGERE – 577 004. |

|3. |Course of study and subject |M.D. – BIOCHEMISTRY |

|4. |Date of Admission to course |26-07-2010 |

|5. |Title of the Topic |“EVALUATION OF SERUM SUPEROXIDE DISMUTASE, PARAOXANASE, |

| | |MALONDIALDEHYDE AND HIGH SENSITIVITY C-REACTIVE PROTEIN IN ISCHEMIC |

| | |STROKE PATIENTS WITH AND WITHOUT TYPE II DIABETES MELLITUS’’. |

|6. |Brief Resume of the intended work : |

| | |

| |6.1 Need for the study: |

| |Stroke is defined as rapidly developed clinical signs of focal disturbance of cerebral function; lasting more than 24hrs |

| |or leading to death, with no apparent cause other than vascular origin1. |

| |Stroke is the third leading cause of death in developed countries. It ranked as the sixth leading cause of Disability |

| |Adjusted Life Years (DALY; one DALY being equal to one year of health lost due to disease) in 1990 and is projected to |

| |rank fourth by the year 20202. |

| |WHO collaborative study in 12 countries showed stroke incidence rates ranged from 0.2 to 2.5/1000 population per year. The|

| |prevalence rate of stroke in India is about 1.54/1000 and death rate about 0.6/1000. It accounts for 10-12% of total |

| |deaths1. The best strategy for reducing its burden is prevention by targeting population at risk for primary prevention |

| |and risk factor management to prevent its recurrence2. |

| |Strokes are currently classified as either ischemic or hemorrhagic. Ischemic stroke occurs when the blood supply to a part|

| |of the brain is suddenly interrupted by occlusion3. |

| |Diabetes is a strong risk factor for ischemic stroke, because of accompanying risk factors, such as obesity and high blood|

| |pressure. Also insulin resistance is implicated as an independent risk factor in the development of atherosclerosis and |

| |stroke3. |

| |Oxidative stress is probably one of the mechanisms involved in neuronal damage induced by ischemia-reperfusion. During |

| |ischemia, high amounts of free radical formation together with reduced antioxidant defense causes oxidative stress that |

| |may play a role in the pathogenesis of stroke-associated neuronal injury4. Brain is very rich in polyunsaturated fatty |

| |acids which are highly susceptible to free radical attack3. |

| |Free radicals that are formed induce lipid peroxidation of polyunsaturated fatty acids and damage biological membranes |

| |producing malondialdehyde (MDA) as one of the end product of lipid peroxidation. The pathophysiological processes involve |

| |pathological permeability of the blood-brain barrier, energy failure, loss of cell ion homeostasis, acidosis, increased |

| |intracellular calcium, apoptosis and neuronal death5. |

| |Antioxidants are exogenous or endogenous compounds acting in several ways, scavenging reactive oxygen species or their |

| |precursors, inhibiting reactive oxygen species (ROS) formation and binding metal ions needed for catalysis of ROS |

| |generation3. |

| |Enzymatic antioxidants like glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase and |

| |non-enzymatic antioxidants like ascorbic acid, tocopherols, β-carotene, bilirubin, uric acid scavenge the free radicals |

| |and protect the cells. Superoxide dismutase (SOD), present in cytosol functions mainly as a first order antioxidant enzyme|

| |by neutralizing the effect of superoxide anion which is an important precursor for oxidative stress. It dismutates |

| |superoxide anion to H2O26,9. |

| |Human serum paraoxanase or PON (aryldialkyl phosphatase) is a protein synthesized in the liver and has antiatherogenic |

| |activity due to its protective function against oxidative modification of low density lipoprotein (LDL)7. |

| |High sensitivity C-Reactive Protein (hs-CRP) is an acute-phase reactant and emerging as a new marker for coronary artery |

| |disease and associated cerebrovascular disorders. Inflammation theory of atherosclerosis suggests – CRP is not only a |

| |marker of inflammation but also proinflammatory, proatherosclerotic and prothrombotic in nature. It enhances production of|

| |MCP-1 (Monocyte chemoattractant protein) recruiting more monocytes, promoting uptake of LDL by macrophages and activation |

| |and thus plaque formation leading to atherosclerosis and subsequently stroke2. |

| |Inflammatory course of atherosclerosis is more severe in diabetics than non-diabetics. Serum CRP levels increase with the |

| |stage of beta cell dysfunction and insulin resistance. CRP induces upregulation of NADPH oxidase and uncoupling of |

| |endothelial nitric oxide synthase (eNOS) in blood vessels of diabetics leading to endothelial dysfunction and excessive |

| |superoxide anion (O2.-) production causing decreased NO (nitric oxide) bioavailability which has an important role in |

| |homeostatic vasodilatation and regulation of blood flow8. |

| |High CRP levels have poor prognostic significance in ischemic stroke patients. Statins or fibrate therapy may be used |

| |which are helpful in controlling, both dyslipidemia and elevated CRP levels. Monitoring the levels of CRP may help in the |

| |prevention and treatment of stroke2. |

| |Human studies on stroke and oxidative stress are limited and controversial. Hence the present study is undertaken to |

| |evaluate by measuring the serum levels of superoxide dismutase, paraoxanase as antioxidant enzymes, hs-CRP as inflammatory|

| |marker and malondialdehyde as lipid peroxidation marker and correlate between serum levels of SOD, PON with serum levels |

| |of hs-CRP and MDA in ischemic stroke patients with and without type II diabetes mellitus. |

| |6.2 Review of literature : |

| |Changes in enzymatic antioxidative defence systems after acute stroke are controversial5. Cherubini et al9 and Demirkaya |

| |et al10 have demonstrated a significantly reduced SOD activity in AIS (acute ischemic stroke) while others have |

| |demonstrated an increased SOD activity11 still others have demonstrated an unchanged SOD activity in AIS4. |

| |Significant reduction in paraoxanase activity was found in ischemic stroke patients with age and oxidized LDL |

| |significantly increased7. |

| |Studies have shown that ischemic stroke is associated with increased oxidative stress reflected by the lipid peroxidation |

| |product malondialdehyde which is seen to be increased and the reduced antioxidant status in ischemic stroke patients with |

| |and without diabetes3. |

| |Demirkaya et al showed that patients with acute ischemic stroke had significantly higher levels of MDA in their red blood |

| |cells compared with controls which also correlated with infarct size and initial stroke severity3, 10. |

| |A meta – analysis of studies with long term follow up showed that the risk for stroke in healthy individuals with the |

| |highest quartile of CRP concentrations increased nearly 1.7 fold compared with those with the lowest quartile2, 12. |

| |The Framingham study, a cohort follow up study stated that independent of other cardiovascular risk factors, elevated |

| |plasma CRP levels significantly predict the risk of future ischemic stroke and TIA (Transient Ischemic Attack) in the |

| |elderly13. |

| |The Hisayama study, a cohort follow up study in middle aged and elderly Japanese men & women found that elevated serum |

| |hs-CRP levels are an independent risk factor for future ischemic stroke in men and that the coexistence of another risk |

| |factor with high hs-CRP levels increased the risk further14. |

| |Admission CRP after acute ischemic stroke is associated with stroke severity and long-term mortality. High CRP levels may |

| |be considered as a “red flag” marker of high mortality15. |

| |6.3 Objectives of the study : |

| |To evaluate the serum levels of superoxide dismutase and paraoxanase as antioxidant enzymes. |

| |To evaluate the serum levels of hs-CRP as inflammatory marker. |

| |To study the serum levels of malondialdehyde as marker of lipid peroxidation. |

| |To evaluate the correlation of the serum levels of SOD and PON with hs-CRP and MDA in ischemic stroke patients with and |

| |without type II diabetes mellitus and in healthy individuals (as control). |

|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 are attached to the teaching institute J.J.M. Medical College, |

| |Davangere). |

| |The study group consists of acute ischemic stroke patients with and without type II diabetes mellitus. The control group |

| |consists of age and sex matched healthy individuals. Both cases and controls will be interviewed to obtain relevant data |

| |after taking informed consent. Based on inclusion and exclusion criteria minimum of 60 cases will be selected. Among 60 |

| |cases approximately 30 cases will be with type II diabetes mellitus and the rest will be without type II diabetes |

| |mellitus. About 30 age and sex matched healthy individuals will be taken as controls. |

| |Inclusion criteria : |

| |Newly diagnosed acute ischemic stroke patients, diagnosed by Computed Tomography scan, clinical signs and symptoms and |

| |biochemical parameters in the age group of 30-80 years admitted within first 48 hours of stroke onset. |

| |Acute ischemic stroke patients with type II diabetes mellitus. |

| |Acute ischemic stroke patients without type II diabetes mellitus. |

| |Age and sex matched healthy individuals without any major illness and not on any medications as controls. |

| |Exclusion criteria : |

| |Patients with recent clinical infection, chronic inflammatory condition, or neoplastic disease. |

| |Patients with surgery or major trauma in the previous month. |

| |Patients with any major renal or hepatic disease. |

| |Patients on antioxidant medications. |

| |Individuals who are chronic smokers or chronic alcoholics. |

| | |

| |7.2 Method of collection of data (including sampling procedure if any): |

| |About 5ml of venous blood will be drawn from subjects (from large peripheral veins) under aseptic precautions in a sterile|

| |bulb. Serum will be separated by centrifugation and used for analysis of MDA, SOD, PON and hs-CRP. |

| |Estimation of serum malondialdehyde (MDA) by Nadiger et al method16. |

| |Principle: Serum is treated with 10% trichloroacetic acid (TCA) followed by addition of 0.67% thiobarbituric acid (TBA) |

| |and to that solution 1ml of distilled water is added. The mixture is centrifuged and clear pale pink supernatent is |

| |collected. Colour intensity is measured at 530nm in a colorimeter. |

| |Estimation of serum superoxide dismutase (SOD) by Marklund and Marklund method6. |

| |Principle: Superoxide anion is involved in auto-oxidation of pyrogallol at alkaline pH (8.5). The superoxide dismutase |

| |inhibits auto-oxidation of pyrogallol, which can be determined as an increase in absorbance at 420nm. |

| |Estimation of serum paraoxanase (PON) as done by Sarkar7. |

| |Principle: PON activity is measured by using 5.5 mmol/l p-nitrophenyl acetate as a substrate. The increase in the |

| |absorbance at 412nm due to formation of p-nitrophenol is measured using spectrophotometer. |

| |Estimation of serum high sensitivity C-Reactive protein (hs-CRP) by Immunoturbidimetry17. |

| |Principle: Turbidimetry measures the reduction in the light transmission caused by particle formation and it quantifies |

| |the residual light transmitted. It is based on the principle of agglutination reaction. The test specimen is mixed with |

| |the activation buffer (R1) and the antihuman antibody solution (R2) and allowed to react. |

| |Presence of hs-CRP in the test specimen forms an insoluble complex producing a turbidity, which is measured at wavelength |

| |600nm. The resulting turbidity corresponds to the concentration of the hs-CRP in the test specimen. |

| |Statistical analysis : |

| |Results will be subjected to appropriate statistical analysis. Unpaired ‘t’ test will be used to compare between two |

| |groups. Correlation analysis will be done using Pearson’s correlation co-efficient. |

| | |

| |7.3 Does the study require any investigations or interventions to be conducted on patients or other humans or animals? If|

| |so, please describe briefly. |

| |Yes |

| |Estimation of malondialdehyde (MDA), superoxide dismutase (SOD), paraoxanase (PON), high sensitivity C-Reactive Protein |

| |(hs-CRP) using human blood sample in ischemic stroke patients with and without type II diabetes mellitus and in healthy |

| |individuals as controls. |

| | |

| |7.4 Has ethical clearance been obtained from your institution in case of 7.3? |

| |Yes |

|8. |List of References : |

| |Park K. Epidemiology of chronic non communicable diseases and conditions. In: Park’s textbook of preventive and social |

| |medicine. 20th ed. Jabalpur: Banarsidas Bhanot; 2009. p. 327,328. |

| |Dhamija RK, Arora S, Gaba P, Jais M, Kaintura A, Kumar M and Bhattacharjee J. Study of genetic, metabolic and inflammatory|

| |risk factors in patients of acute ischemic stroke. IJCB 2008;23(2):136–143. |

| |D’souza B, D’souza V, Sowmya S, Seema G, Hemalatha CN, Anu M et al. A comparative study on oxidative stress and |

| |antioxidant status in ischemic stroke patients with and without diabetes. IJCB 2008;23(3):218-222. |

| |Abdul-Fattah D, Abdel-Ghani AM, El-Azouni OMMA, Eltarhouny SA, Gharieb AF. Acute ischemic stroke: clinical, radiological |

| |and antioxidant profile study. Egypt J. Neurol. Psychiat. Neurosurg. 2006;43(1):159-166. |

| |Aygul R, Kotan D, Demirbas F, Ulvi H and Deniz O. Plasma oxidants and antioxidants in acute ischemic stroke. Journal of |

| |International Medical Research 2006;34:413-18. |

| |Nischal HK, Sharma MP, Goyal RK and Kaushik GG. Serum superoxide dismutase levels in diabetes mellitus with or without |

| |microangiopathic complications. JAPI 1998;46(10):853-55. |

| |Sarkar PD and Rautaray SS. Oxidized LDL and Paraoxanase status in ischemic stroke patients. Indian J Physiol Pharmacol |

| |2008;52(4):403-7. |

| |Khan DA and Qayyum S. Evaluation of cardiac risk by oxidative stress and inflammatory markers in diabetic patients. Pak J |

| |Med Sci 2009;25(5):776-781. |

| |Cherubini A, Polidori MC, Bregnocchi M, Pezzuto S, Cecchetti R, Ingegni T et al. Antioxidant profile and early outcome in |

| |stroke patients. Stroke 2000;31:2295-2300. |

| |Demirkaya S, Topcuoglu MA, Aydin A, Ulas UH, Isimer AI and Vural O. Malondialdehyde, glutathione peroxidase and |

| |superoxide dismutase in peripheral blood erythrocytes of patients with acute cerebral ischemia. European Journal of |

| |Neurology 2001;8(1):43-51. |

| |Kocaturk PA, Akbostanci MC, Isikay C, Ocal A, Tuncel D, Kavas GO et al. Antioxidant status in cerebrovascular accident.|

| |Biol Trace Elem Res 2001;80(2):115-24. |

| |Hsu-Ko Kuo MPH, Dr Chung-Jen Yen, Chia-Hsuin Chang, Chen-Ko Kuo, Jen-Hau Chen and Farzaneh Sorond. Relation of C-reactive |

| |protein to stroke, cognitive disorders, and depression in the general population: systematic review and meta-analysis. The|

| |Lancet Neurology 2005;4(6):371-380. |

| |Rost NS, Wolf PA, Kase CS, Kelly-Hayes M, Silbershatz H, Massaro JM et al. Plasma concentration of C- reactive |

| |protein and risk of ischemic stroke and transient ischemic attack: the Framingham Study. Stroke 2001;32:2575-2579. |

| |Wakugawa Y, Kiyohara Y, Tanizaki Y, Kubo M, Ninomiya T, Hata J et al. C-reactive protein and risk of first-ever ischemic |

| |and hemorrhagic stroke in a general Japanese population: the Hisayama Study. Stroke 2006;37:27-32. |

| |Idicula TT, Brogger J, Naess H, Waje-Andreassen U and Thomassen L. Admission C-reactive protein after acute ischemic |

| |stroke is associated with stroke severity and mortality: The ‘Bergen stroke study’. BMC Neurology 2009;9:18. |

| |Kalavacherla US, Ishaq M, Rao URK, Sachindranath A and Hepsiba T. Malondialdehyde as a sensitive marker of inflammation in|

| |patients with rheumatoid arthritis. JAPI 1994;42(10):775-776. |

| |Hind CRH and Pepys MB. The role of serum C-Reactive protein (CRP) measurement in clinical practice. Int Med |

| |1984;5:112-151. |

|9. |Signature of candidate | |

|10. |Remarks of the guide |Human studies on stroke and its consequent oxidative stress are limited. |

| | |Therefore, the present study is undertaken to evaluate antioxidant status and|

| | |hs-CRP as inflammatory marker and it may be helpful in understanding the use |

| | |of antioxidants as therapeutic agents in the treatment of ischemic stroke. |

|11. |Name & Designation of (in block letters) | |

| |11.1 Guide | |

| | |Dr. P.M. GANGADHARA SWAMYM.Sc., PhD., |

| | |PROFESSOR, |

| | |Department of BIOCHEMISTRY, |

| | |J.J.M. Medical College, |

| | |DAVANGERE - 577 004. |

| |11.2 Signature | |

| | | |

| |11.3 Co-Guide | |

| | |Dr. P.M. VINAY SWAMYM.D., |

| | |PROFESSOR, |

| | |DEPARTMENT OF MEDICINE, |

| | |J.J.M. MEDICAL COLLEGE, |

| | |DAVANGERE - 577 004. |

| |11.4 Signature | |

| | | |

| |11.5 Head of the Department | |

| | |Dr. D.S. JAYAPRAKASH MURTHYM.D., |

| | |Professor and Head, |

| | |Department of BIOCHEMISTRY, |

| | |J.J.M. Medical College, |

| |11.6 Signature |DAVANGERE - 577 004. |

| | | |

| | | |

|12. |12.1 Remarks of the | |

| |Chairman & Principal | |

| | | |

| |12.2 Signature | |

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