APPENDIX-I

6. BRIEF RESUME OF THE INTENDED WORK

APPENDIX-IA

6.1 NEED FOR THE STUDY

Diabetes, a lifelong progressive disease, is the result of body’s inability to produce insulin or use insulin to its full potential and is characterized by high circulating glucose. The sustained hyperglycemia attacks both micro vessels and macro vessels in the body1

Diabetes results in increased oxidative stress and this elevated oxidative stress plays an important role in the pathogenesis of diabetic complications such as neuropathy, nephropathy, myocardial injury and retinopathy. Oxidative stress is an imbalance between excess formation and or impaired removal of reactive oxygen species by antioxidant defense system of cells like vitamin A, vitamin E, vitamin C, superoxide dismutase, glutathione peroxidase and catalase1. This endogenous oxidative stress causes damage to proteins, lipids and DNA, which is thought to be an important etiological factor in the pathophysiology of complications of diabetes mellitus 2

Diabetic retinopathy is the leading cause of new cases of legal blindness among Americans between the ages of 20 and 74 years.3 To establish the effect of reactive oxygen species on retina, in diabetic retinopathy patients, measurement of oxidative stress and antioxidant levels is undertaken.

APPENDIX-1B

REVIEW OF LITERATURE

Diabetes mellitus comprises a group of common metabolic disorders that share the phenotype of hyperglycemia. It is due to insulin deficiency or resistance to action of insulin4

CLASSIFICATION OF DIABETES MELLITUS4

1.  Type 1 Diabetes mellitus [Insulin dependent diabetes mellitus] Type 1 diabetes mellitus is due to absolute deficiency of insulin caused by an auto immune attack on the β cells of the pancreas and its progressive destruction. It is sub classified as

a) Immune mediated

b) Idiopathic

2. Type 2 Diabetes Mellitus [Non-Insulin dependent diabetes mellitus]

Type 2 diabetes mellitus is due to insulin resistance and dysfunctional β cells. It is further classified as

a) Obese

b) Non-obese

c) Maturity onset diabetes of young

3. Diabetes Prone status

a) Gestational diabetes mellitus

b) Impaired glucose tolerance [IGT]

c) Impaired fasting hyperglycemia (IGF)

4. Secondary to other known causes

a) Endocrinopathies: - Cushing’s disease, thyrotoxicosis, and acromegally.

b) Drug induced: - pentamidine, nicotinic acid, glucocorticoids, β-adrenergic agonists, β-blockers, clozapine.

c) Diseases of exocrine pancreas: - pancreatitis, pancreatectomy, neoplasia, cystic fibrosis, and hemochromatosis.

d) Infections: - Congenital rubella, Cytomegalovirus, Coxsackie’s.

e) Uncommon forms of immune mediated diabetes:- Stiff man syndrome, anti insulin receptor antibodies

f) Genetic syndromes associated with diabetes mellitus: – Down’s syndrome, Klinefelter’s syndrome, Turner’s syndrome, Wolfram syndrome.

METABOLIC DERANGEMENTS IN DIABETES MELLITUS

CARBOHYDRATE METABOLISM

Insulin is a hypoglycemic hormone, which lowers blood glucose level by promoting utilization and storage of glucose. It stimulates glycolysis and inhibits gluconeogenesis and glycogenolysis .In diabetes mellitus, the release of insulin and the cellular response to insulin are decreased so this leads to inhibition of glycolysis and stimulation of gluconeogenesis and glycogenolysis resulting in hyperglycemia4.

LIPID METABOLISM

Insulin inhibits lipolysis, which is evidenced by fall in circulating plasma free fatty acid levels. It enhances lipogenesis and the synthesis of triacyl glycerol by providing more acetyl CoA and NADPH. The principle action of insulin in adipose tissue is to inhibit the activity of hormone sensitive lipase, reducing the release not only of free fatty acid but also of glycerol5.

Due to loss of inhibitory effect on lipolysis in diabetes, there will be an increase in the free fatty acid levels in the plasma and also increased β-oxidation of fatty acids to meet the energy requirements. This will lead to high levels of acetyl CoA that cannot be completely oxidized in TCA cycle and is diverted to ketogenesis causing ketonemia and ketosis4.

PROTEIN METABOLISM

Insulin is an anabolic hormone. In diabetes mellitus there will be absence of anabolic effect of insulin. So there will be increased catabolism of proteins and amino acids for providing substrates for gluconeogenesis4.

COMPLICATIONS OF DIABETES MELLITUS2.

Chronic hyperglycemia is an important etiological factor leading to complications of diabetes mellitus. These are

Acute Complications

·  Hypoglycemia

·  Diabetic ketoacidosis

·  Hyperglycemic hyperosmolar state

Chronic Complications

a)  Micro vascular complications

Eye disease - Cataract

- Retinopathy

- Macular edema

Neuropathy - sensory and motor

- Autonomic

Nephropathy - End stage renal disease

b) Macro vascular complications

- Coronary artery disease

- Peripheral vascular disease

C) Others

- Gastrointestinal [gastro paresis, diarrhea]

- Genitourinary [uropathy / dysfunction]

- Dermatological

- Infectious

- Glaucoma

DIABETIC RETINOPATHY

Diabetic retinopathy is a well-characterized sight threatening chronic micro vascular complication that eventually afflicts all patients with diabetes mellitus3.

It is defined as the progressive dysfunction of retinal vasculature caused by chronic hyperglycemia6.

CLASSIFICATION OF DIABETIC RETINOPATHY

The American Academy of Ophthalmology established international classification of diabetic retinopathy. This international classification describes five clinical levels of diabetic retinopathy3

1)  No apparent retinopathy (No abnormalities)

2)  Mild nonproliferative diabetic retinopathy (Micro aneurysms only)

3)  Moderate nonproliferative Diabetic retinopathy (More than micro aneurysms only but less than severe nonproliferative diabetic retinopathy)

4)  Severe nonproliferative diabetic retinopathy (any of the following >20 inter retinal hemorrhages in each of 4 quadrants, prominent interretinal micro vascular abnormalities in one or more quadrants)

5)  Proliferative diabetic retinopathy (one or more of retinal neo vascularization, vitreous hemorrhages or preretinal hemorrhages

OXIDATIVE STRESS IN DIABETIC RETINOPATHY

Free radicals are defined as an atom or molecules that contain one or more unpaired electrons, making them unstable and highly reactive. The most important ROS are the superoxide anion radical (O2·¯), hydrogen peroxide (H2O2), alkoxyl (RO·), peroxyl (ROO·), hydroxyl radical (OH·) and hypochlorous acid (HOCl). ROS is continuously generated in physiological conditions and effectively eliminated by several antioxidant systems7.

The retina has high content of polyunsaturated fatty acids and has highest oxygen uptake and glucose oxidation relative to any other tissue. This phenomenon renders retina more susceptible to oxidative stress1.

Hyperglycemia increases ROS production3. In chronic hyperglycemia production of reducing equivalents is more which saturates the electron transport chain leading to the production of more superoxide radical beyond its threshold3.

As a result, in diabetes mellitus there will be increased ROS production. These reactive oxygen species can activate a number of other superoxide production pathways such as3,

- Formation of advanced Glycation end products

- Sorbitol pathways

- Activation of protein kinase C

- Hexosamine pathway

The endogenous oxidative stress causes damage to proteins, lipids and DNA. This is likely to occur only after the production of reactive oxygen species has exceeded the body or cell’s capacity to protect itself and effectively repair oxidative damage. Normally the body has an abundant supply of antioxidants such as superoxide dismutase, glutathione peroxidase, catalase, vitamin A, vitamin E and vitamin C etc., which are naturally occurring substances that delay or inhibit oxidation and neutralize oxygen free radicals. In diabetes oxidative stress seems to be caused by both increased production of ROS and sharp reduction in antioxidant defenses and altered cellular redox status8.

BLOOD SUGAR LEVEL

Blood sugar level measurement is most commonly used for detecting Diabetes mellitus. Fasting blood sugar concentration ≥ 126mg/dl on more than one occasion are diagnostic of Diabetes mellitus

Post prandial blood sugar level of 200mg/dl or higher at 2 hrs on two occasions is suggestive of diabetes mellitus9.

GLYCATED HEMOGLOBIN

It is the standard measure of long-term control of blood glucose level. Glycation is a non-enzymatic process where the glucose after entering RBC forms a Schiff base with N terminal amino group of protein by an aldamine linkage which changes to a ketamine linkage by an irreversible amadori rearrangement. This glycated hemoglobin remains inside erythrocytes throughout its life span4.

HbA1c reveals the mean glucose level in last 8-10 weeks. Normal Glycated hemoglobin (HbA1c) level is 4-7%. Reduction in 1% of HbA1c will decrease long-term complications to an extent of 30%4.

The use of the HbA1c level can play a major role in detecting diabetes in hospitalized patients with random hyperglycemia, as it does not require fasting, necessitates fewer blood draws, unaffected by recent food intake or recent changes in blood sugar levels, reflects glucose metabolism over several weeks and can be done at admission rather than the minimum of 3 days required for traditional methods like oral glucose tolerance tests (OGTT).

SERUM MALONDIALDEHYDE [MDA] LEVELS

Malandialdehyde is a highly toxic product formed by lipid peroxidation by free radicals. The concentration of MDA is high in Diabetes mellitus correlating with poor glycemic control as shown by many studies7. The membrane lipids are more liable to attack by free radicals and produce damage to the integrity of the membrane.

SERUM VITAMIN A

It is a Fat-soluble vitamin, stored in liver. Normal serum level is 25-50 μg/dl. It plays an important role in vision, reproduction and maintenance of epithelial tissues4.

Active form is present only in animal tissue, provitamin, beta carotene is present in plant tissues. Three different forms of vitamin A are retinol, retinal and retinoic acid. There is a correlation between the occurrence of epithelial cancers and vitamin A deficiency. The anticancer activity has been attributed to the natural antioxidant property of carotenoids 9.


SERUM VITAMIN C [ASCORBIC ACID]

It is a water-soluble vitamin. Source of vitamin C are Lime, Orange, Gooseberry.

Normal serum level is 0.4 to 1.5mg/dl and daily requirement is 75mg/day4.

Ascorbic acid is one of the most effective water-soluble antioxidant in biological fluids and can scavenge physiologically important reactive oxygen species. Both ascorbate and ascorbyl radical have low redox potential and react with most other biologically relevant radicals. Thus it prevents oxidative damage to biological macromolecules including DNA, lipids and protein4.

SERUM VITAMIN E (TOCOPHEROL)

It is a fat soluble vitamin the source of vitamin E are vegetable oils like wheat germ oil, sunflower oil and cotton seed oil. Recommended daily allowance is 8-10mg/day. Normal serum vitamin E level is 0.5-0.8mg/day. Vitamin E is a membrane antioxidant. The free radicals would attack biomembranes. Vitamin E protects red cells by preventing the peroxidation of lipids in membranes. It keeps the structural and functional integrity of all cell membranes. Gradual deterioration of aging process is due to the cumulative effects of free radicals. Vitamin E also boosts immune system4.

APPENDIX-1C

6.3. OBEJECTIVES OF THE STUDY

-  To know the oxidative stress in diabetic retinopathy patients by measuring serum MDA levels.

-  To know the antioxidants status in diabetic retinopathy patients by measuring serum vitamin A, vitamin E and vitamin C

-  To know the correlation between glycated hemoglobin and oxidative status in cases of diabetic retinopathy.

APPENDIX-1D

INCLUSION AND EXCLUSION CRITERIA.

INCLUSION CRITERIA

50 clinically diagnosed cases of Diabetic Retinopathy who will attend out patient department and also who are admitted in wards will be taken for case study and 50 numbers of age and sex matched healthy persons will be taken as controls.

EXCLUSION CRITERIA

Patients with acute and chronic inflammatory conditions, other metabolic conditions like ketoacidosis, cerebrovascular accidents or renal diseases as well as smokers, alcoholics, patients with psychiatric disorders and primary hypertensives were excluded from the study. None of the subjects were on antioxidant supplementation.

APPENDIX-II

7. MATERIALS AND METHODS

APPENDIX-IIA

7.1 SOURCE OF DATA

All clinically diagnosed cases of diabetic retinopathy irrespective of age and sex who attends out patient department and admitted cases of Adichunchanagiri hospital and research center will be included in the study. Age and sex matched healthy individuals will be taken as control group. Study span is about 18 months.


APPENDIX-IIB

7.2 METHOD OF COLLECTION OF DATA

LABORATORY INVESTIGATION IN DIABETES MELLITUS

Blood samples were collected in fasting state and will be analyzed for fasting blood glucose, serum malondialdehyde, serum vitamin A, serum vitamin E, serum vitamin C and glycated hemoglobin.

10ml of Blood will be drawn under aseptic precautions from clinically diagnosed cases of diabetic retinopathy and divided into 3 test tubes, marked as 1, 2 and 3.

1. Test tube 1 contains 2ml of blood with anticoagulant, which is used for estimation of Blood glucose. (Glucose Oxidase Method)

2. Test tube 2 contains whole blood that is used for estimation of glycated hemoglobin (Affinity chromatography)

3. Test tube 3 contains 6 ml of blood with no anticoagulant that is allowed to clot and Serum is separated serum is used for the measurement of

a)  Serum Malondialdehyde. (Thiobarbuturic acid method)

b)  Serum Vitamin A. (Spectrophotometric Method)

c)  Serum Vitamin E. (Baker and Frank method)

d)  Serum Vitamin C. (2,6,dichlorophenol indophenol method)

4. Test tube 4 contains 2ml of blood, which will be collected with anticoagulant after 2 hrs of meals, which is used for estimation of post prandial blood sugar.

STATISTICAL ANALYSIS:

Statistical analysis will be done using student “t” test and statistical significance will be compared between the cases and the control group.


APPENDIX-IIC.

7.3 DOES THE STUDY REQUIRE ANY INVESTIGATIONS OR

INTERVENTION TO BE CONDUCTED ON PATIENTS OR

OTHER ANIMALS, IF SO PLEASE DESCRIBE BRIEFLY

YES, the patient will require the following blood investigations

1. Blood Sugar level

2. Serum Malondialdehyde

3. Serum Vitamin A.

4. Serum Vitamin E.

5. Serum Vitamin C.

6. HbA1c

APPENDIX-IID

7.4 HAS THE ETHICAL CLEARANCE BEEN OBTAINED FROM

YOUR INSTITUTION IN CASE OF 7.3?

YES, the investigations are carried out after the ethical clearance from the Institution. All investigations are carried out after taking the written consent from the Patients.


APPENDIX-III

LIST OF REFERENCES

1. Renu A. Kowrluru and Pooi-see chan; Review article “Oxidative stress and diabetic

Retinopathy.” Experimental diabetes research Hindawi publishing corporation volume

2007, article ID 43603.

2. Dennis L. Kasper., Eugene Braunwald., Anthony S. Fauci., Stephen L. Hauser., Dan L.

Lango., J. Larry Jameson., Joseph Loscalzo., Harrison’s Principles of Internal Medicine. 16th Ed. McGraw-Hill Medical publishing division; 2008.

3.Henry M.Kronenberg., Shlomo melmed., Kenneth S. Polonsky., P.Reed Larsen.,“Williams textbook of endocrinology” 11th edition Saunders Elsevier publishing Division; 2008

4. Vasudevan D.M and Srekumari S “Text Book of Bio-chemistry for Medical Students”. 5th Ed. Jaypee brother’s medical publishers (P) Ltd; 2007.