1   Name of the candidate and                    Dr. SHILPASREE.A.S
    address (in block letters)
                                                 POST GRADUATE STUDENT,
                                                 DEPARTMENT OF
                                                 J.J.M. MEDICAL COLLEGE,
2   Name of the institution                      J.J.M. MEDICAL COLLEGE,
3   Course of the study and subject              M.D. BIOCHEMISTRY
4   Date of admission to the course              30-05-2009
5   Title of the topic                           “STUDY OF SERUM
                                                 APOLIPOPROTEINS AI, B AND
                                                 LIPID PROFILE IN STROKE”

    6.1 Need for the study
       Stroke is defined as rapidly developing clinical signs of focal or global disturbances
    of cerebral function lasting for more than 24 hours leading to death with no apparent
    cause other than vascular origin.1
       Stroke incidence ranges from 0.2 to 2.5/1000 population per year. There is no reliable
    information from India, but analysis of data suggests that 2% of all hospital cases, 4.5%
    of medical and 20% of neurological cases are from stroke. Stroke is the leading cause of
    death. It accounts for 10-12% of total deaths.1
       Abnormalities in plasma lipoproteins and derangements in lipid metabolism rank
among the most firmly established and best understood risk factors for atherosclerosis.
Abnormal lipid parameters like total cholesterol, LDL, HDL and triglycerides are
probable risk factors for ischemic stroke, largely by their link to atherosclerosis.2
Atherogenic index defined as log (TG/HDL-c in mmol/l) is an indicator to predict the risk
for the development of athersclerosis.
   Apolipoproteins are protein components of lipoproteins. Apolipoprotein AI and B
are the protein components of HDL and LDL cholesterol respectively. Apolipoprotein AI
and HDL are antiatherogenic whereas apolipoprotein B and LDL are atherogenic.
   Dyslipidemia, low apolipoprotein AI and high apolipoprotein B are widely accepted as
risk factors for Coronary heart disease, in contrast the relation is not well established for
   So the present study is undertaken to evaluate the relationship between apolipoprotein
AI, B and lipid profile with stroke.

6.2 Review of literature:
   Various studies have demonstrated positive and significant association between total
cholesterol, LDL cholesterol and risk of stroke and inverse relationship between HDL
cholesterol and fatal stroke.3,4,5
    Studies have also shown that linear increase in the levels of non fasting triglycerides
leads to stepwise increase in the risk of stroke.6
    Analysis of lipid and lipoprotein profile in young patients with stroke revealed that
total cholesterol, LDL cholesterol, Triglycerides, Apo AI, Apo B and Apo B/ Apo AI
ratio were significantly altered as compared to normal healthy controls.7
   Among the patients with preexisting cardiovascular disease high levels of Apo B and
low Apo A1 levels are associated with increased risk of ischemic stroke. Apolipoproteins
remained independent risk factors upon adjustment for traditional risk factors. Apo
B/Apo AI ratio as a useful indicator of ischemic stroke risk in patients with preexisting
atherothrombotic disease.8, 12
   Prospective cohort study from Taiwan showed that Apo AI but not Apo B levels serve
as an effect modifier of hypertension for the risk of stroke.9
   In contrast few studies have shown no difference between total cholesterol,
triglycerides, Apolipoprotein AI and B in patients compared to controls but higher LDL
cholesterol and lower HDL cholesterol was observed in stroke patients.11
     6.3 Objectives of the study:
        a) To study the levels of apolipoprotein AI and B in stroke patients and healthy
        b) To study the ratio of Apo B/Apo AI in stroke patients and healthy subjects.
        c) To study the lipid profile (Total cholesterol, LDL, HDL and triglycerides) and
            atherogenic index – log (TG/HDL-c in mmol/l) in stroke patients and healthy
     7.1 Source of data:
        A crosssectional 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 JJM medical college Davangere).
     Inclusion criteria:
        Cases - 50 clinically proven cases of stroke in the age group of 30 - 70 years.
        Cases diagnosed by computed tomography.
        Controls - 50 age and sex matched healthy individuals without any major illness and
        not on any medications.
     Exclusion criteria:
        Stroke patients with
           Hepatic and renal disease
           Sepsis
           Malignancy
           Patients on hypolipidemic drugs
     7.2 Methods of collection of data
        About 6 ml of venous blood will be drawn under aseptic precaution in a sterile bulb
     from selected subjects after overnight fasting. Serum separated by centrifugation will be
     used for analysis. All biochemical parameters will be estimated by semiautoanalyser.
     a) Estimation of total cholesterol by cholesterol oxidase/phenol
     aminoantipyrine method.10
        Principle: cholesterol esterase hydrolyses cholesterol esters to free cholesterol and
     fatty acids. Then cholesterol is oxidised by cholesterol oxidase forming
cholesterol4ene-3one and hydrogen peroxide. In presence of enzyme peroxidase,
hydrogen peroxide causes oxidative coupling of phenol and antipyrine to form red
coloured quinoneimine dye which is measured at 520nm.
b) Estimation of HDL cholesterol by cholesterol oxidase/phenol
aminoantipyrine method.10
   Principle: Chylomicrons, VLDL and LDL are precipitated by adding phosphotungstic
acid and magnesium ions to the sample. Centrifugation leaves only HDL in the
supernatant. Cholesterol content in it is determined enzymatically.
 c) Estimation of LDL cholesterol by Friedewald formula.10

  [LDL cholesterol] = [Total cholesterol] - [HDL cholesterol] – [Triglyceride]

d) Estimation of triglycerides by glycerol phosphate oxidase-phenol
aminoantipyrine method.10
   Principle: Triglycerides are hydrolysed by lipoprotein lipase to glycerol and fatty
acids. Glycerol is then phosphorylated to glycerol-3-phosphate followed by oxidation to
hydrogen peroxide and dihydroxyacetone phosphate by glycerol phosphate oxidase.
Hydrogen peroxide in presence of peroxidase causes oxidative coupling of 4 chlorphenol
and 4 amonophenazone to form red coloured quinoneimine dye which is measured at
e) Estimation of apolipoprotein B and AI by immunoturbidimetric
   Principle: Immunoturbidimetric method is based on the reaction of a sample
containing human Apolipoprotein A1 or B and specific antiserum to form an insoluble
complex which can be measured turbidimetrically at 340 nm.
Statistical analysis:
   Results will be subjected for stastical analysis. Students‘t’ test will be used to compare
different biochemical parameters between cases and controls.
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.
   Estimation of Apoli[poprotein AI, B, total cholesterol, LDL cholesterol, HDL
cholesterol and triglycerides in stroke patients and normal healthy controls.
7.4 Has ethical clearance been obtained from your institution in case of
       1. Park: Park’s textbook of preventive and social medicine. 19th edition. Jabalpur:
          Barasidas Bhanot; 2008. P 314
       2. Peterlibby. The pathogenesis prevention and treatment of atherosclerosis. In:
          Fauci, Braunwald, K Asper, Hauser, Longo, Jameson, Loscalzo editors.
          Harrison’s principal of internal medicine. Vol 2, 17th edition. USA: Mc Graw Hill
          Company; 2008. P 2513
       3. Tsuyoshi Imamura, Yasufumi Doi, Hisatomy Arima, Koji Yonemoto, Jun Hata,
          Michiaki kubo, et al. LDL cholesterol and the development of stroke subtypes and
          coronary heart disease in a general Japanese population: The Hisayama study.
          Stroke 2009; 40: 382-388
       4. Wanna methee, Goya S, Shaper, Gerald A, Ebrahim S. HDL- cholesterol, Total
          cholesterol and risk of stroke in middle aged British men. Stroke 2000; 31:1882-
       5. Tanne, David, Yaari, Shlomit, Goldbourt, Uri. High density lipoprotein
          cholesterol and risk of ischemic stroke mortality: A 21 year follow up of 8586
          men from isreli ischemic heart disease study. Stroke 1997; 28: 83-87.
       6. Jacob J Freiberg, Anne Tybjaerg-Hansen, Jan Skov Jensen, Borge G
          Nordestgaard. Non-fasting triglycerides and risk of ischemic stroke in general
          population. JAMA 2008; 300(18):2142-2152.
       7. Adriano paula sabino, Marinez De Oliveira Sousa, Luciana Mo Moreira Lima,
          Daniel Dias Ribeiro, Luci Maria Santano Dusse, Marie Das Gracas Carvalho et al.
          Apo B/ApoAI ratio in young patients with ischemic cerebral stroke or peripheral
          arterial disease. Transitional research 2008; 152: 113-118.
       8. Nira Koren morag, Uri Goldbourt, Eran Graff, David Tanne. Apolipoproteins B
          and AI and the risk of ischemic cerebrovascular events in patients with preexisting
          atherthrombotic disease, Journal of neurological sciences 2008; 270: 82-87.
       9. Kuo-liong chien, Fung-Chang-Sang, Hsiu-Ching Hsu, Ta-chen Su, Ruey-S Lin,
          Yuan-The Lee. Apolipoprotein AI and B and stroke events in a community based
          cohort in thaiwan. Report of Chin-Shan community cardiovascular study. Stroke
          2002; 33: 39-44.
       10. Nader Rifai, Russel Warnick G. Lipids, lipoproteins, apolipoproteins and other
   cardiovascular risk factors. In: Brutis CA, Ashwood ER, Bruns DE. editors.
   Teitz textbook of clinical chemistry and molecular diagnostics. 4th edition. New
   Delhi: Elesvier; 2006. P 942-960
11. Pedro- Botet J, Senti M, Nogues X, Rubies-Prat J, Roquer L D’Olhaberriaque J et
   al. Lipoproteins and Apolipoprotein profile in men with ischemic stroke. Role of
   lipoprotein (a) triglyceride rich lipoproteins and apolipoprotein E polymorphism.
   Stroke 1992; 23: 1556-1562.
12. Bhatia M, Howard S C, Clark T G, Neale R, Qizilbash N, Murphy M F G et al.
   Apolipiproteins as predictors of ischemic stroke in patients with a previous
   transient ischemic attack. Cerebrovascular dis 2006; 21: 323-328.
9.    Signature of the Candidate

10.   Remarks of the Guide           Study will help in identifying the
                                     individuals at risk and to prevent the
                                     stroke attack by normalizing the lipid
11.   Name and Designation of
      (in block letters)

      11.1 Guide                     DR. SAVITRI. R. SAHUKAR, MD
                                     DEPT. OF BIOCHEMISTRY
                                     JJM MEDICAL COLLEGE,

      11.2 Signature

      11.3 Co-Guide

      11.4 Signature

      11.5 Head of the Department    DR. JAYAPRAKASH MURTHY. D.S.
                                                               Bsc, MBBS, MD
                                     PROFESSSOR AND HEAD
                                     DEPT. OF BIOCHEMISTRY
                                     JJM MEDICAL COLLEGE,

      11.6 Signature

12    12.1 Remarks of the Chairman
      & the principal

      12.2 Signature

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