1. INTRODUCTION

1.1 Diabetes:

Diabetes is a disease in which the body is unable to regulate blood sugar on its own. And

it does not produce or properly use insulin, which is a hormone that is needed to convert

sugar, starches and other food into energy needed for daily life. Although both genetics

and environmental factors such as obesity and lack of exercise appear to play a role, the

actual cause of diabetes still remains unknown. There are two major types of diabetes,

called type 1 and type 2.

Type 1 diabetes: A chronic disease in which high levels of sugar (glucose) are found in

blood. Type 1 diabetes can occur at any age but it is most often observed in children,

adolescents and young adults. The insulin hormone which is responsible for producing

specialized cells called beta cells produce little or no insulin and this results in the

formation of type1 diabetes. Without enough insulin, glucose builds up in the

bloodstream instead of entering into the cells. The body is now unable to use this glucose

as a source of energy, and this leads to the formation of symptoms of type1 diabetes. The

exact cause for type1 diabetes is not known but it is considered to be an autoimmune

disorder. In type 1 diabetes, the pancreas undergoes an autoimmune attack by the body

itself, and is rendered incapable of making insulin.

Figure 1: Schematic representation of onset of type 1 diabetes

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Type 2 diabetes: A chronic disease in which high levels of sugar (glucose) is found in

blood. It is considered to be the most common form of diabetes. During the condition of

type 2 diabetes the components like fat, liver and muscle cells do not respond properly to

the insulin. This phenomenon is called “insulin resistance”, due to which the blood sugar

cannot enter into the cells. When sugar cannot enter the cells they start building up in the

blood at high amounts. This phenomenon of building up of sugar in high amounts in the

blood stream is called “hyperglycemia”.

Type 2 diabetes usually occurs slowly over time. Most people with the disease are

overweight when they are diagnosed. Type 2 diabetes is most seen in elderly people.

Family history and genes play a large role in type 2 diabetes. Low activity level, poor

diet, and excess body weight around the waist increase your risk.

Figure 2: Schematic representation of onset of type 1 diabetes

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1.2 Causes of Diabetes:Insufficient production of insulin (either absolutely or relative to the body's needs),

production of defective insulin (which is uncommon), or the inability of cells to use

insulin properly and efficiently leads to hyperglycemia and diabetes. This latter condition

affects mostly the cells of muscle and fat tissues, and results in a condition known as

"insulin resistance." This is the primary problem in type 2 diabetes. The absolute lack of

insulin, usually secondary to a destructive process affecting the insulin producing beta

cells in the pancreas, is the main disorder in type 1 diabetes. For essentially, if someone is

resistant to insulin, the body can, to some degree, increase production of insulin and

overcome the level of resistance. After time, if production decreases and insulin cannot

be released as vigorously, hyperglycemia develops.

Insulin is a hormone that is produced by specialized cells (beta cells) of the pancreas.

(The pancreas is a deep-seated organ in the abdomen located behind the stomach). In

addition to helping glucose enter the cells, insulin is also important in tightly regulating

the level of glucose in the blood. After a meal, the blood glucose level rises. In response

to the increased glucose level, the pancreas normally releases more insulin into the

bloodstream to help glucose enter the cells and lower blood glucose levels after a meal.

When the blood glucose levels are lowered, the insulin release from the pancreas is

turned down. It is important to note that even in the fasting state there is a low steady

release of insulin than fluctuates a bit and helps to maintain a steady blood sugar level

during fasting. In normal individuals, such a regulatory system helps to keep blood

glucose levels in a tightly controlled range.

1.3 Risk Factors for Type 2 Diabetes:Type 2 diabetes occurs when the body can't use the insulin that's produced, a condition

called insulin resistance. Though it typically starts in adulthood, type 2 diabetes can begin

anytime in life. Because of the current epidemic of obesity among U.S. children, type 2

diabetes is increasingly found in teenagers. Here are the risk factors for developing type 2

diabetes.

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Obesity or being overweight. Diabetes has long been linked to obesity and being

overweight. Research at the Harvard School of Public Health showed that the single best

predictor of type 2 diabetes is being obese or overweight.

Impaired glucose tolerance or impaired fasting glucose. Prediabetes is a milder form

of diabetes that's sometimes called impaired glucose tolerance. It can be diagnosed with a

simple blood test. Prediabetes is a major risk factor for developing type 2 diabetes.

Insulin resistance. Type 2 diabetes often starts with cells that are resistant to insulin.

That means they are unable to take in insulin as it moves glucose from the blood into

cells. With insulin resistance, the pancreas has to work overly hard to produce enough

insulin so cells can get the energy they need. This involves a complex process that

eventually leads to type 2 diabetes.

Ethnic background. Diabetes occurs more often in Hispanic/Latino Americans, African-

Americans, Native Americans, Asian-Americans, Pacific Islanders, and Alaska natives.

High blood pressure. Hypertension, or high blood pressure, is a major risk factor for

diabetes. High blood pressure is generally defined as 140/90 mm Hg or higher. Low

levels of HDL "good" cholesterol and high triglyceride levels also put you at risk.

History of gestational diabetes. If you developed diabetes while you were pregnant,

you've had what is called gestational diabetes. Having had gestational diabetes puts you

at higher risk of developing type 2 diabetes later in life.

Sedentary lifestyle. Being inactive -- exercising fewer than three times a week -- makes

you more likely to develop diabetes.

Family history. Having a family history of diabetes -- a parent or sibling who's been

diagnosed with this condition -increases your risk of developing type 2 diabetes.

Polycystic ovary syndrome. Women with polycystic ovary syndrome (PCOS) are at

higher risk of type 2 diabetes.

Oxidative Stress In Type 2 Diabetes Hyperglycemia underlies the development of

diabetic complications possibly due to an increase in oxidative stress. Oxidative stress is

defined as the imbalance of oxidants and antioxidants in the favor of oxidants. This

imbalance reflects either a loss of the protective antioxidant network or the increased

production of free radicals. Oxidative stress has been strongly associated with tissue

damage in diabetic individuals. Mechanisms by which hyperglycemia can induce

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oxidative stress include enhanced glycoxidation, increased carbohydrate flux through the

polyol pathway, formation of AGEs, increased glucose flux through the hexosamine

pathway, activation of DAG-activated protein kinase C and inflammation. The unifying

event in these mechanisms is the production of free radicals, more specifically ROS and

reactive nitrogen species (Sarah Akbar et al., 2011).

An atom contains a nucleus, and electrons move around the nucleus, usually in pairs

(Woolf et al., 1998).A free radical is any atom or molecule that contains a single unpaired

electron in an outer orbital, and capable of independent existence (Gutteridge et al.,

1997). The unpaired electron alters the chemical reactivity of an atom or molecule

making it extremely reactive and unstable and enters into reactions with organic or

inorganic components in the cell (proteins, lipids, carbohydrates)particularly with key

molecules in membranes and nucleic acids (Beckman et al., 1994).

1.4 Reactive Oxygen Species (ROS):ROS include a number of chemically reactive molecules derived from oxygen. Some of

those molecules are extremely reactive, such as the hydroxyl radical, while some are less

reactive (superoxide and hydrogen peroxide). Intracellular free radicals, i.e., free, low

molecular weight molecules with an unpaired electron, are often ROS and vice versa and

the two terms are therefore commonly used as equivalents. Free radicals and ROS can

readily react with most Biomolecules, starting a chain reaction of free radical formation.

In order to stop this chain reaction, a newly formed radical must either react with another

free radical, eliminating the unpaired electrons, or react with a free radical scavenger (a

chain-breaking or primary antioxidant). In Table 1, the most common intracellular forms

of ROS are listed together with their main cellular sources of production and the relevant

enzymatic antioxidant systems scavenging these ROS molecules. The step-wise reduction

of molecular oxygen via 1-electron transfers, producing and also connecting the ROS

molecules listed in Table 1 can be summarized as follows:

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Table 1: Reactive oxygen species sources and their products

ROS

moleculeMain sources

Enzymatic defense

systemsProduct(s)

Superoxide (O2•¯)

Leakage of electrons from the

electron transport chain

Activated phagocytesXanthine oxidase

Flavoenzymes

Superoxide dismutase

(SOD)

Superoxide reductase

(in some bacteria)

H2O2 + O2

H2O2

Hydrogen

peroxide

(H2O2)

From O2•¯ via superoxide dismutase (SOD)NADPH-oxidase

(neutrophils)

Glucose oxidaseXanthine oxidase

Glutathione peroxidase

Catalase

Peroxiredoxins (Prx)

H2O + GSSG

H2O + O2

H2O

Hydroxyl

radical

(•OH)

From O2•¯and H2O2 via transition metals (Fe or Cu)

Nitric

oxide (NO)Nitric oxide

synthases

Glutathione/TrxRGSNO

1.5 Antioxidants and Antioxidant-Related Enzymes:

Defense mechanisms against free radical-induced oxidative damage include the

following:

(I) Catalytic removal of free radicals and reactive species by factors such as

Catalase (CAT), superoxide dismutase (SOD), peroxidase, and thiol-specific

antioxidants;

(II) Binding of proteins (e.g., transferring, metallothionein, haptoglobins,

caeroplasmin) to pro-oxidant metal ions, such as iron and copper;

(III) Protection against macromolecular damage by proteins such as stress or heat

shock proteins; and

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(IV) reduction of free radicals by electron donors, such as GSH, vitamin E

(tocopherol), vitamin C (ascorbic acid), bilirubin, and uric acid.

Finger 3: Schematic representation of the major players of the cellular antioxidant network.

Animal catalase is heme-containing enzymes that convert hydrogen peroxide (H2O2) to

water and O2, and they are largely localized in sub cellular organelles such as

peroxisomes. Mitochondria and the endoplasmic reticulum contain little CAT. Thus,

intracellular H2O2 cannot be eliminated unless it diffuses to the peroxisomes.

Glutathione peroxidases (GSH-Px) remove H2O2 by coupling its reduction with the

oxidation of GSH. GSH-Px can also reduce other peroxides, such as fatty acid

hydroperoxides. These enzymes are present in the cytoplasm at millimolar concentrations

and also present in the mitochondrial matrix. Most animal tissues contain both CAT and

GSH-Px activity. SODs are metal-containing proteins that catalyze the removal of

superoxide, generating water peroxide as a final product of the dismutation. Three

isoforms have been identified, and they all are present in all eukaryotic cells. The copper-

zinc SOD isoform is present in the cytoplasm, nucleus, and plasma. On the other hand,

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the manganese SOD isoform is primarily located in mitochondria. Dietary micronutrients

also contribute to the antioxidant defense system. These include carotene, vitamin C, and

vitamin E (the vitamin E family comprises both tocopherols and tocotrienols, with _-

tocopherol being the predominant and most active form). Water-soluble molecules, such

as vitamin C, are potent radical scavenging agents in the aqueous phase of the cytoplasm,

whereas lipid soluble forms, such as vitamin E and carotene, act as antioxidants within

lipid environments. Selenium, copper, zinc, and manganese are also important elements,

since they act as cofactors for antioxidant enzymes. Selenium is considered particularly

important in protecting the lipid environment against oxidative injury, as it serves as a

cofactor for GSH-Px. The most abundant cellular antioxidant is the tripeptide, GSH(L-

glutamyl -L-cysteinyl glycine). GSH is synthesized in two steps. First, glutamylcysteine

synthetase (GCS) forms a peptide bond between glutamic acid and cysteine, and then

GSH synthetase adds glycine. GSH prevents the oxidation of protein thiol groups, either

directly by reacting with reactive species or indirectly through glutathione transferases.

1.6 Catalase

Catalase was first noticed in 1811 when Louis Jacques Thénard, who discovered H2O2

(hydrogen peroxide), suggested its breakdown is caused by an unknown substance. In

1900, Oscar Loew was the first to give it the name Catalase, and found it in many plants

and animals. Catalase gene located on the short (p) arm of chromosome 11 at position 13.

More precisely, the CAT gene is located from base pair 34,460,471 to base pair

34,493,606 on chromosome 11.

It is a ubiquitously occurring enzyme that catalyses the decomposition of H 2O2 to

water and oxygen. The enzyme has one of the highest turnover rates, converting millions

of H2O2 molecules per single Catalase molecule each second. The enzyme is a tetramer

with polypeptide chains that are more than 500 amino acids long. Catalase is usually

determined in the serum.

 

Catalase8

2H2O2  →  2H2O + O2

CAT Gene located 11p13

Figure 4: Schematic Representation of Catalase gene located on chromosome 11: base

pairs 34,460,471 to 34,493,606.

This gene encodes Catalase, a key antioxidant enzyme in the bodies’ defense against

oxidative stress. Catalase is a heme enzyme that is present in the peroxisome of nearly all

aerobic cells. Catalase converts the reactive oxygen species hydrogen peroxide to water

and oxygen and thereby mitigates the toxic effects of hydrogen peroxide. Oxidative stress

is hypothesized to play a role in the development of many chronic or late-onset diseases

such as diabetes. Polymorphisms in this gene have been associated with decreases in

Catalase activity but, to date, acatalasemia is the only disease known to be caused by this

gene. 

1.7 Structure of Catalase Enzyme: 

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Catalase was first noticed in 1811 when Louis Jacques Thénard, who discovered H2O2

(hydrogen peroxide), suggested its breakdown is caused by an unknown substance. In

1900, Oscar Loew was the first to give it the name Catalase, and found it in many plants

and animals (Loew et al., 1900). In 1937 Catalase from beef liver was crystallised by

James B. Sumner and Alexander Dounce (Sumner & Dounceand, 1937) the molecular

weight was worked out in 1938 (Sumner & Dounceand, 1938). In 1969, the amino acid

sequence of bovine Catalase was worked out (Schroeder et al., 1969) then in 1981, the

three-dimensional structure of the protein was revealed.

Figure 5: Schematic Representation of 3D Catalase Structure

 

1.8 Catalase and Diabetes:

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The metabolic effects of oxidants, which are believed to contribute to many diseases,

may influence the development of some forms of diabetes. As we discuss earlier the

oxidant hydrogen peroxide (H2O2) is a by-product of normal cellular respiration and is

also formed from superoxide anion by the action of superoxide dismutase. H2O2 has been

reported to damage pancreatic β-cells (Murata et al., 1998) and inhibit insulin signaling

(Hausen et al., 1999).

The enzyme Catalase has a predominant role in controlling the concentration of H2O2,

and consequently, Catalase protects pancreatic β-cells from damage by H2O2 (Tiedge et

al., 1998). Low Catalase activities, which have been reported in patients with

schizophrenia and atherosclerosis (Góth et al., 1996), are consistent with the hypothesis

that long-term oxidative stress may contribute to the development of a variety of late-

onset disorders, such as type 2 diabetes (Góth et al., 2000).

1.9 AIM AND OBJECTIVES

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1. To estimate the effect of antioxidant (Catalase) activity in Type2 Diabetes.

2. To Identify and Isolate human genomic DNA and amplify the antioxidant

(catalase) gene using specific primers for Catalase gene with polymerase

chain reaction.

3. To use antioxidant (catalase) as a potential biomarker for type 2 diabetes.

2. REVIEW OF LITERATURE

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Recently it has been studied that it is characterized by absolute or relative deficiencies in

insulin secretion and insulin action associated with chronic hyperglycemia and

disturbances of carbohydrate, lipid, and protein metabolism. As a consequence of the

metabolic derangements in diabetes, various complications develop including both macro

and micro-vascular dysfunctions. It is accepted that oxidative stress results from an

imbalance between the generations of oxygen derived radicals and the organism’s

antioxidant potential. Various studies have shown that diabetes mellitus is associated with

increased formation of free radicals and decrease in antioxidant potential. Due to these

events, the balance normally present in cells between radical formation and protection

against them is disturbed. This leads to oxidative damage of cell components such as

proteins, lipids, and nucleic acids. In both insulin dependent (type 1) and non-insulin-

dependent diabetes (type 2) there is increased oxidative stress (Roja Rahimi et al., 2005).

Recent studies report that oxidative stress plays a major role in the pathogenesis and

development of complications of both types of DM. However, the exact mechanism by

which oxidative stress could contribute to and accelerate the development of

complications in diabetic mellitus is only partly known and remains to be clarified. On

one hand, hyperglycemia induces free radicals; on the other hand, it impairs the

endogenous antioxidant defense system in patients with diabetes. Endogenous antioxidant

defense mechanisms include both enzymatic and non-enzymatic pathways. Their

functions in human cells are to counterbalance toxic reactive oxygen species (ROS).

Common antioxidants include the vitamins A, C, and E, glutathione (GSH), and the

enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx),

and glutathione reductase (GRx). The importance of endogenous antioxidant defense

systems, their relationship to several path physiological processes and their possible

therapeutic implications in vivo (Fatimah et al., 2011).

This study was undertaken to investigate the association between gene polymorphisms of

selected antioxidant enzymes and vascular complications of DM. Significant differences

in allele and genotype distribution among T1DM, T2DM and control persons were found

in SOD1 and SOD2 genes but not in CAT gene (p < 0,01). Demonstrate that oxidative

stress in DM can be accelerated not only due to increased production of ROS caused by

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hyperglycaemia but also by reduced ability of antioxidant defense system caused at least

partly by SNPs of some scavenger enzymes (Milan et al., 2008).

An imbalance in antioxidant enzymes has been related to specific pathologies such as

diabetic complications. Catalase catalyzes the reduction of hydroperoxides, thereby

protecting mammalian cells against oxidative damage. In addition, catalase is active in

neutralizing reactive oxygen species and so removes cellular superoxide and peroxides

before they react with metal catalysts to form more reactive species. The status of

Catalase activity in erythrocytes of streptozotocin (STZ)-induced diabetic rats. Catalase

activity was measured by using spectrophtometric techniques. Catalase activity increased

in diabetic rats compared to control group [25.7 ± 2.8 vs. 16.3 ± 2.1 mmol H2O2 per min/

mg of protein, mean ± SD, p < 0.05]. Catalase activity increased significantly in the

erythrocytes of STZ-induced diabetic rats (Durdi et al., 2007).

The former theory hyperglycemia, an outcome of the disease, as a secondary force that

further damages β-cells. The latter theory suggests that the often-associated defect of

hyperlipidemia is a primary cause of β-cell dysfunction. That patients with type 2

diabetes continually undergo oxidative stress, that elevated glucose concentrations

increase levels of reactive oxygen species in β-cells, that islets have intrinsically low

antioxidant enzyme defenses, that antioxidant drugs and over expression of antioxidant

enzymes protect β-cells from glucose toxicity, and that lipotoxicity, to the extent it can be

attributable to hyperlipidemia, occurs only in the context of preexisting hyperglycemia,

whereas glucose toxicity can occur in the absence of hyperlipidemia (R.Paul et al., 2004).

Overproduction or insufficient removal of these free radicals results in vascular

dysfunction, damage to cellular proteins, membrane lipids and nucleic acids. Despite

overwhelming evidence on the damaging consequences of oxidative stress and its role in

experimental diabetes, large scale clinical trials with classic antioxidants failed to

demonstrate any benefit for diabetic patients. As our understanding of the mechanisms of

free radical generation evolves, it is becoming clear that rather than merely scavenging

reactive radicals, a more comprehensive approach aimed at preventing the generation of

these reactive species as well as scavenging may prove more beneficial. Therefore, new

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strategies with classic as well as new antioxidants should be implemented in the

treatment of diabetes (Jeanette et al., 2005).

It has been suggested that enhanced production of free radicals and oxidative stress is

central event to the development of diabetic complications. This suggestion has been

supported by demonstration of increased levels of indicators of oxidative stress in

diabetic individuals suffering from complications. Therefore, it seems reasonable that

antioxidants can play an important role in the improvement of diabetes. There are many

reports on effects of antioxidants in the management of diabetes. The relationships

between diabetes and oxidative stress and use of antioxidants in the management of

diabetes and its complications have been well reviewed. Oxidative stress is involved in

the pathogenesis of diabetes and its complications. Use of antioxidants reduces oxidative

stress and alleviates diabetic complications (Roja et al., 2005).

Oxidative stress (OS) results when production of reactive oxidative species (ROS)

exceeds the capacity of cellular antioxidant defenses to remove these toxic species. (Jorge

et al., 2008).

Decreased activity of these antioxidant enzymes may increase the susceptibility of

diabetic patients to oxidative injury. An appropriate support of antioxidant supplies may

help in preventing clinical complications of diabetes. Estimations of these antioxidant

enzymes might be used as marker in the management of glycemic control and the

development of diabetic complications.

The enzyme catalase has a predominant role in controlling the concentration of H2O2 and

consequently, catalase protects pancreatic cells from damage by H2O2. Low catalase

activities, which have been reported in patients with schizophrenia and atherosclerosis,

are consistent with the hypothesis that long-term oxidative stress may contribute to the

development of a variety of late-onset disorders, such as type 2 diabetes().

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CAT and SOD activities, glycated hemoglobin, and insulin and lipid profiles were

assessed. CAT and SOD activities were significantly decreased in T2DM compared with

the control subjects. T allele of CAT and C allele of SOD1 were significant risk factors

for T2DM. No effects of CAT or SOD1 gene polymorphisms on glycated haemoglobin or

on HOMA-IR were found. The enzymes activities, only +35 A/C of SOD1 were related

to SOD activity. Genetic variants C1167T of CAT gene and +35 A/C of SOD1 gene has

no role in insulin resistance in T2DM ().

3. MATERIALS AND METHOD

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Blood Sample Collection

Blood samples were collected after obtaining the patients consent and ethical

clearance from the Ethical Committee by Jawaharlal Nehru Institute of Advanced Studies

(JNIAS). Blood samples were collected in vials containing anticoagulant EDTA

vacuumed tubes and kept at -20oC within 30 minutes after removal from patients from

Mahaveer hospital, Hyderabad and brought to JNIAS in cool box containing ice.

3.1 BIOCHEMICAL ANALYSIS OF CATALASE:

Separation of Serum from whole blood sample

Serum is made up of non-clotting proteins, glucose, nutrients, electrolytes,

hormones, antigens, antibodies and other particles. Separation of serum is more tedious

and time-consuming than plasma extraction. For isolation of serum, first a blood sample

is allowed to clot, after which the coagulated blood is centrifuged. The liquid supernatant

formed at the top portion is serum. The procedure for extraction of plasma is very simple;

blood sample is spun by using a centrifuge apparatus. The heavier blood cells settle at the

bottom, and blood plasma is collected from the upper layer. 2-3 ml of blood was

collected by venepuncture in EDTA vacuumed tubes. The tubes were centrifuged for 10

minutes at 4000 rpm. Supernatant serum was pipette out with a micropipette, transferred

to an Eppendorf tube, and stored in deep freeze. Biochemical estimation was performed

from these sera in the following methods.

3.1.1 Estimation of Protein in total serum sample:

Serum is isolated from blood samples of both normal and diabetic patients and performed protein

analysis to evaluate the usefulness of serum total protein.

We have estimated the protein using the Lowry method. The “Lowry assay-Protein by

Folin Reaction” (Lowry et al., 1951) has been the most widely used method to estimate

the amount of protein in biological samples. The phenolic group of tyrosine and

tryptophan residues (amino acid) in a protein will produce a blue purple color complex ,

with maximum absorption in the region of 595 nm wavelength, with Folin- Ciocalteau

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reagent which consists of sodium tungstate molybdate and phosphate. Thus the intensity

of color depends on the amount of these aromatic amino acids present and will thus vary

for different proteins. Estimation techniques use Bovin Serum Albumin (BSA)

universally as a standard protein.

Reagent Required:

1.      BSA stock solution (1mg/ml)

2.      Sodium Carbonate anhydrous

3.      Sodium hydroxide

4.      Copper sulphate

5.      Sodium potassium tartarate

6.      F.C reagent (Phenol reagent)

Reagents Prepared:

Lowry A: 2% Sodium Carbonate anhydrous in 0.1M Sodium hydroxide. (0.56g

NaOH+2.86g Na2Co3 in 100 ml water).

Lowry B: 1% CuSo4 in distilled water ( 0.28g of CusO4 in 20ml distilled water).

Lowry C: Sodium potassium tartarate( 0.56g in 20ml of distilled water).

Lowry stock reagent: 49ml of Lowry A + 0.5ml of Lowry B+ 0.5ml of Lowry C

F.C reagent: Phenol reagent (2N) was diluted in water in 1:1 ratio.

To estimate the amount of protein in an unknown sample, we should first prepare a

standard graph using a known protein sample. We have used Bovine serum albumin

(BSA) as known sample to obtain the standard graph.

Procedure for the Preparation of Standard Graph:

We have prepared different dilutions of BSA solutions by mixing stock BSA

solution (1 mg/ ml) water and F.C reagent in the test tube. The final volume in each of the

test tubes is 2 ml.

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Table 2: preparation of standard graph 

BSA(μl) 0 200 400 600 800 1000

Water(μl) 1.8 1.6 1.4 1.2 1 0.8

FC reagent(μl) 200 200 200 200 200 200

Procedure for the Estimation of unknown sample using the Standard

Graph:

The serum was separated from the normal and diabetes patient’s serum sample.

Lowry stock reagent of 1ml was taken in test tube. To the reagent10μl of serum

sample was added which was present in the test tube.

The test tubes were kept for incubation for about 30mins at room temperature.

After incubation 200μl of FC reagent was added. The test tubes were kept for

incubation at room temperature for another 30mins.

After incubation, 2ml of the mixture was taken in a cuvette to read the OD value

using spectrophometer at 595nm. wavelength was used.

The above steps were repeated for all samples.

3.1.2 Estimation of Catalase:

Chemicals Required:

1. Potassium di-hydrogen orthophosphate

2. Di-potassium hydrogen phosphate

3. Hydrogen peroxide solution

Reagents Prepared:

Phosphate buffer: Potassium di-hydrogen orthophosphate was mixed with di-potassium

hydrogen phosphate with pH maintained at 7.

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Hydrogen peroxide solution: 30% H2O2 was diluted 10 times in water (1 ml of H2O2 in

9ml water). This diluted solution is again diluted 3 times (1ml of diluted H2O2 in 2ml of

water) bringing it to 1% solution (30mM).

Catalase stock solution: 840 micro liter of H2O2 solution was added in 299.160ml

phosphate buffer and a stock of 300ml was prepared.

Procedure:

To cuvette, 790μl of water was taken, to it 200 μl of reagent were added.

Serum sample of 10μl was added to cuvette.

Adjusted the wavelength to 240nm and noted down the OD values using the time

scan measurement in the spectrophotometer.

Using the obtained OD values of protein and Catalase.

We found out the activity of the catalase enzyme by substituting in the formula

given below:

Catalase activity = Final OD/extinction coefficient*volume of sample*protein

concentration

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3.2 MOLECULAR ANALYSIS OF CATALASE

3.2.1 Isolation of Genomic DNA from Human Blood Sample:

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DNA was isolated from the blood samples by a rapid non-enzymatic method by salting

out cellular proteins with saturated solution and precipitation by dehydration (Alluri et

al., 2005). Since RBC has no charge on their plasma membrane, non- ionic detergent

called, Triton X 100 removes them out. KCl and MgCl2 in TKM1 helps in lysis of the

RBC cell membrane and EDTA acts as a divalent ion chelator (it contains di-sodium

atom). Hence, it helps in de-activating the metallozymes as DNAses. Tris acts as a

buffering agent maintaining the pH at 7.6 for the proper function of the lysis buffer. In

addition, it helps in solubility of the ions so that they do not precipitate out. TKM2 or

Cell lysis buffer has a higher concentration of MgCl2, KCl and NaCl to lyse both the cell

and the nuclear membrane. KCl also acts as solubilizer of proteins. NaCl acts as extractor

of RNA and used in salting out of proteins .SDS acts as anionic detergent and both acts

on anionic lymphocytic cell membranes and help in their lysis deactivate the negatively

charged proteins.

Materials Required:

1. Autoclaved eppendorff

2. Autoclaved micropipettes

3. Autoclaved micro tips

4. Autoclaved distilled water

5. Eppendorff stand

Preparation of Reagents :The reagents were prepared as described below:

Table 3: RBC Lysis Buffer/ TKM 1

Chemicals (100ml) (50ml)

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Tris-HCL (10mM) 0.121 0.061

EDTA ( 2mM) 0.0744 0.0372

KCl (10mM) 0.0745 0.03725

MgCl2 (10mM) 0.2033 0.10165

Tris is first dissolved in few ml of autoclaved distilled water and the pH is adjusted to

7.6. Then EDTA is dissolved followed by other chemicals.

Table 4: Cell Lysis Buffer/ TKM2

Chemicals (100ml) (50ml)

Tris-HCL (10mM) 0.121 0.061

EDTA ( 2mM) 0.0744 0.0372

KCl (10mM) 0.0745 0.03725

MgCl2 (10mM) 0.2033 0.10165

NaCl (0.4M) 2.3376 1.1688

Tris is first dissolved in few ml of autoclaved distilled water and the pH is adjusted at 7.6.

Then EDTA is dissolved followed by other chemicals and the volume is made up to 100

ml with distilled water.

10% SDS (10 ml): 1gm of SDS was dissolved in 10 ml of autoclaved distilled water.

0.6M NaCl: 0.8765 g of NaCl was dissolved in 25 ml of distilled water.

TE Buffer

Chemical Amount

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Tris hydrogen chloride (HCl) (10mM) pH 8 0.030 g

Ethylene diamine tetra acetic acid (EDTA) (1mm) 0.009 g

Tris is dissolved in few ml of autoclaved distilled water, after adjusting the pH, EDTA is

dissolved, and the volume is made up to 25 ml.

70 % Ethanol – Dissolve 7 ml of absolute ethanol in 10 ml of distilled water.

Procedure: A sterilized eppendorff was taken and 300 μl of blood sample was added in it.

To the blood sample 800 μl of TKM1 and 1 drop of 100% Triton X 100 was

added, mixed well, and incubated for 5 minutes.

Centrifuged at 10,000 rpm for 5 minutes, and then supernatant was discarded. To

the pellet 800 μl of TKM1 was added and steps 2 and 3 are repeated until a white

pellet is obtained.

To the pale pellet, 300 μl of TKM2 and 80 μl of 10% SDS was added and

incubated for 30 minutes.

After incubation 80 μl of 6M NaCl was add and mixed well by tapping for 5

minutes. Centrifuged at 10,000 rpm for 5 minutes.

Supernatant was transfered carefully to 680 μl of cold absolute Ethanol.

Centrifuged at 10,000 rpm for 5 minutes.

Supernatant was discarded and 300 μl of 70% absolute Ethanol was added to the

DNA pellet. Centrifuged at 10,000 rpm for 5 minutes and the pellet was air dried.

To the dried pellet, 50 μl of TE buffer was added for hydration of DNA and

preserve at freezing temperature.

We detected the DNA in the Isolated Samples by Using 1% of Agarose Gel by

Electrophoresis.

24

3.2.2 Agarose Gel by Electrophoresis

Electrophoresis is a technique used to separate and sometimes purify macromolecules

especially proteins and nucleic acids - that differ in size, charge or conformation.

Fragments of linear DNA migrate through agarose gels with a mobility that is inversely

proportional to the log10 of their molecular weight. Bromophenol blue is used as loading

dye to track the movement of the sample. It is mixed well with the sample. In addition, it

increases the density of the mixture, so that, they reside down at the bottom of the well

and are diffused in the gel.

By using gels with different concentrations of agarose, DNA fragments of different sizes

can be resolved. Higher concentrations of agarose facilitate separation of small DNA

fragments, while low agarose concentrations allow resolution of larger DNAs (Maniatis T

et al., 1989).

Materials Required:

1. Horizontal electrophoresis unit

2. Gel plate

3. Combs

4. Adhesive tapes

5. 10T micropipette and autoclaved tips

Reagent Preparation:

10 x TAE Buffer (100) ml:

Solution A: 19.36g of Tris was dissolved in 50 ml of distilled water.

Solution B: 1.86g of EDTA was dissolved in 10ml of distilled water.

Solution C: 8 ml of B solution was added to solution A and 4.36ml of acetic acid was

added. Then the volume was made up to 100ml with distilled water.

1X TAE Buffer: 30ml of 10X TAE Buffer was dissolved in 270 ml of distilled water to

make 1:10 dilution.

1% Agarose: 0.25g of Agarose was dissolved in 25ml of 1X TAE Buffer.

25

1% Ethidium bromide solution: 0.1g of ethidium bromide was dissolved in 10ml

distilled water. Gel loading solution and dye used was 6X concentrated and was obtained

readymade.

Figure 6: Agarose Gel Electrophoresis Figure 7: Gel Documentation System

Procedure:

Preparation of 1% agarose gels:

Agarose powder of 0.5gm was added to 50ml of 1X TAE buffer in a 100 ml

conical flask.

The flask was kept in a microwave oven and boiled until the agarose dissolved.

After boiling 7l of Ethidium bromide was added to the solution and was allowed

to cool, Poured into the gel-casting tray.

The comb was kept in place and the gel was allowed to solidify at room

temperature.

Sample Loading and Electrophoresis:

After solidification of the gel, the comb was removed and the gel was placed in

the electrophoresis chamber containing 1x TAE.

26

Now 4l of the DNA sample was mixed with 3l of loading dye and 7 μl of the

mixture was loaded into the well.

Samples were run at 75 volts for 30 minutes, After 30 min DNA was visualized

under gel documentation system.

3.2.3 Spectrophotometer:

In chemistry, spectrophotometer is the quantitative measurement of the reflection or

transmission properties of a material as a function of wavelength. It is more specific than

the general term  electromagnetic spectroscopy in that spectrophotometer deals

with visible light, near-ultraviolet, and near-infrared, but does not cover time-resolved

spectroscopic techniques. A spectrophotometer is a photometer that can measure intensity

as a function of the light source wavelength. Important features of spectrophotometers are

spectral bandwidth and linear range of absorption or reflectance measurement.

Figure 8: Spectrophotometer

Because DNA and RNA absorb ultraviolet light, with a absorption peak at 260nm

wavelength, spectrophotometers are commonly used to determine the concentration of

DNA in a solution. Inside a spectrophotometer, a sample is exposed to ultraviolet light at

260 nm, and a photo-detector measures the light that passes through the sample. The

27

more light absorbed by the sample, the higher the nucleic acid concentration in the

sample.

Using the Beer-Lambert law it is possible to relate the amount of light absorbed to

the concentration of the absorbing molecule. At a wavelength of 260 nm, the extinction

coefficient for double-stranded DNA is 50 (μg/ml)-1 cm-1; for single-stranded DNA and

RNA it is 38 (μg/ml)-1 cm-1. Thus, an optical density (or “OD”) of 1 corresponds to 50

µg/ml for double-stranded DNA, 38 µg/ml for single-stranded DNA and RNA. This

method of calculation is valid for up to an OD of at least 2.

Figure 9: A spectrophotometer measures how much light of a certain wavelength is absorbed by a liquid

DNA concentration (g/ml) = OD260 x dilution factor x 50 g/ml

Procedure:

To quantify the DNA, 99 μl of TE buffer was taken in a cuvette and calibrated the

spectrophotometer at 260nm as well as 280nm. 1 l of each

28

DNA sample of 1l each to 99 l TE (Tris-EDTA buffer) and mixed well.

Now 2.9 ml of water was added to the cuvette.

TE buffer and water used as a blank in the other cuvette of the spectrophotometer.

The OD260 and OD280 values on spectrophotometer were noted.

3.2.4 Polymerase Chain Reaction:

Polymerase chain reaction in vitro was designed first by Karry Mullis in 1983. It follows

the process of DNA replication using temperature variations with a help of a thermo

cycler. This process include five major steps , at specific accurate temperature for each

step for exact specificity of the amplification or duplication of the specific DNA

sequence or gene out of the whole genomic DNA sequence. This is possible by using

specific complementary forward and reverse primers that specified the region of

duplication. The enzyme used for the amplification is generally consists of 3 ’ end to 5’

end extension and 5’ end to 3’ end exonuclease activity. The enzyme used is called Taq

polymerase, which is extracted from thermo stable bacteria Thermus aquaticus, generally

found in hot springs.

The purpose of a PCR (Polymerase Chain Reaction) is to make a huge number of copies

of a gene. This is necessary to have enough starting template for sequencing.

The cycling reactions:

There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This

is done on an automated cycler, which can heat and cool the tubes with the reaction

mixture in a very short time as shown in figure 11.

1. Denaturation at 94°C :

During the denaturation, the double strand melts open to single stranded DNA, all

enzymatic reactions stop (for example: the extension from a previous cycle).

29

2. Annealing at 54°C :

The primers are jiggling around, caused by the Brownian motion. Ionic bonds are

constantly formed and broken between the single stranded primer and the single stranded

template. The more stable bonds last a little bit longer (primers that fit exactly) and on

that little piece of double stranded DNA (template and primer), the polymerase can attach

and starts copying the template. Once there are a few bases built in, the ionic bond is so

strong between the template and the primer, that it does not break anymore.

3. Extension at 72°C :

This is the ideal working temperature for the polymerase. The primers, where there are a

few bases built in, already have a stronger ionic attraction to the template than the forces

breaking these attractions. Primers that are on positions with no exact match, get loose

again (because of the higher temperature) and don't give an extension of the

fragment. The bases (complementary to the template) are coupled to the primer on the 3'

side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side,

bases are added complementary to the template).

30

Figure 10: Polymerase Chain Reaction

Figure 11: The different steps in PCR

Because both strands are copied during PCR, there is an exponential increase of the

number of copies of the gene. Suppose there is only one copy of the wanted gene before

the cycling starts, after one cycle, there will be 2 copies, after two cycles, there will be 4

copies, three cycles will result in 8 copies and so on as shown in figure 12.

31

Figure 12: The exponential amplification of the gene in PCR.

Procedure for making 25µl of PCR reaction:

Reagents Volume

Water 18.3µl

Buffer 2.5µl

dNTPs 1µl

Forward Primers 1µl (20 pmol/µl)

Reverse Primers 1µl (20 pmol/µl)

Taq Polymerase 0.2µl

DNA sample 1µl

Above content was mixed well gently by tapping. The amplification was carried out in a

thermo cycler for 30-35 cycles. After amplification, amplified samples were analyzed

using agarose gel electrophoresis. Store the amplified samples were stored at freezing

temperature for further analysis.

Agarose Gel by Electrophoresis

32

Procedure:

Preparation of 1.5% Agarose gels:

Agarose powder of 0.75gm was added to 50ml of 1X TAE buffer in a 100 ml

conical flask.

The flask was kept in a microwave oven and boiled until the agarose dissolved.

After boiling 7l of ethidium bromide was added to the solution and was allowed

to cool, Poured into the gel-casting tray.

The comb was kept in place and the gel was allowed to solidify at room

temperature.

Sample Loading and Electrophoresis:

After solidification of the gel, the comb was removed and the gel was placed in

the electrophoresis chamber containing 1x TAE.

Now 5l of the PCR product was mixed with 3l of loading dye and loaded in to

the wells

In one of the wells 5l of 1000bp DNA ladder was loaded.

Samples were run at 75 volts for 30 minutes.

After 30 min DNA was visualized under gel documentation system.

4. RESULTS AND DISCUSSION

4.1 Biochemical Analysis:

4.1.1 Protein Standard graph

As demonstrated in Figure 13, protein concentration shows a direct correlation with

absorbance. The absorbance was determined for BSA protein concentrations ranging

33

from 0.0 to 1000 μg/μl. Over this range the absorbance increased in a linear fashion, the

standard curve was generated. By this standard curve can be used to convert the

Absorbance readings for the experimental samples into a protein amount or

concentration.

Table 5: Setup of Different dilution of buffer, Reagents and protein for Standard

Graph

BSA(μl) 0 200 400 600 800 1000

OD(595nm) 0.039 0.131 0.216 0.305 0.397 0.517

 

Figure 13: Standard graph

4.1.2 Estimation of Total protein serum analysis

We have taken 16 random serum samples of diabetic patients and normal group and

estimated the protein content in them by using Spectrophotometer with an absorbance at 595nm.

As a positive control we used Bovine Serum Albumin (BSA) method. The results of the present

34

study showed that the levels of serum total protein were significantly higher compared with the

normal group.

Figure 14: Total Protein in Serum

Histogram showing the levels of serum of total protein in normal and diabetic patients as

shown in figure 14.

Table 6: Spectrophotometer value of Normal serum sample of catalase activity

Normal Sample

Glucose level mg/dl

Protein concentrationmg/ml

OD at 240 nm Catalase activity

01 76.106 0.21 0.3252 2181.0802 52.21 0.19 0.1941 1438.3403 89 0.16 0.2041 1901.4604 80 0.09 0.1201 1879.4905 86 0.15 0.1577 1480.7506 85 0.15 0.1392 1307.0407 89 0.16 0.2160 1796.6508 82 0.16 0.1235 2099.47

Table 7: Spectrophotometer value of Diabetic serum sample of Catalase activity

Diabetic Sample

Glucose level mg/dl

Protein concentrationmg/ml

OD at 240 nm

Catalase activity

01 149.55 0.20 0.2062 1452.11

02 337 0.17 0.2200 1445.73

35

03 205 0.16 0.1109 976.2304 139 0.16 0.2160 644.3605 347 0.27 0.0701 365.6706 139 0.16 0.2256 1985.91

07 152 0.15 0.2034 1909.8508 193 0.20 0.1475 1549.29

4.1.3 Relationship between Catalase activity and Glucose level

Catalase activity was measured in eight different serum samples of both normal as well as

diabetic individuals with different glucose concentrations ranging from less than 90mg/dl

glucose value and more than 130mg/dl glucose value. The range less than 90mg/dl

glucose value will consider as normal individuals and more than 130mg/dl glucose value

will consider as Diabetes individuals. Catalase activity was determined as H2O2

consumption measured as the decrease in absorbance at 240 nm (Aebi et al., 1983). The

assay contained 50 mM KH2PO4/K2HPO4 (pH 7.0), 10 mM H2O2 in phosphate buffer.

Extinction coefficient of 39.4 mM-1cm-1 was used to calculate activity. Catalase activity

was expressed as (Unit). The Correlation of enzyme activity with Glucose concentration

was measured as shown in Table 6 & 7 as well as histogram result shown the catalase

activity with respect to glucose concentration as shown in graph 3.

36

figure 15: Catalase activity in normal and diabetic

4.2 Molecular analysis

4.2.1 Isolation of DNA from normal and diabetic blood samples:

After the above experiment we took eight normal blood samples in eppendorff tubes and

extracted DNA from them by following the given protocol and ran it on 1% agarose gel

at 75v for 45 minutes. On gel documenting the gel we got nice band indicating the

presence of the DNA; as shown in the figure 16.

.

Figure 16: Isolated genomic DNA from normal blood samples

The following bands show the DNA of 8 normal samples which was performed on 1% Agarose

gel. Regarding the quality of the DNA extracted, no differences in band patterns were

observed in the agarose gel. Light DNA bands found in normal sample 6&8 (N6&N8).

Similarly seven diabetic blood samples in eppendorf tubes were taken and DNA was

extracted from them by following the given protocol and ran it on 1% agarose gel at 75v

37

for 45 mintutes. On gel documenting the gel we got nice band indicating the presence of

the DNA; as shown in the figure 17.

Figure 17: Isolated genomic DNA diabetic blood samples

The following bands show the DNA of 7 diabetic samples which was also performed on 1%

Agarose gel. Regarding the quality of the DNA extracted, no differences in band patterns

were observed in the agarose gel, but remaing samples showed some degree of

degradation. No result are found in diabetic sample 1 (D1).

4.2.2 Quantification of DNA by Spectrophotometer:

After isolating the blood samples of both normal and diabetic we have analyzed the DNA

concentration by using the spectrophotometer. For confirming the concentration and to

check the purity of DNA, 1% Agarose gel electrophoresis was performed and DNA

analyzation was done by UV Spectrophotometer at both 260nm and 280 nm. Finally, we

calculated the DNA concentration by using the formulae:

DNA Concentration (µg/ml) = A260 x 50 x dilution factor (where A260 = optical density

reading at 260 nm)

The spectrophotometer results for both 260nm and 280nm of normal individuals are

given below:

38

Table 8: showing purity and concentration of DNA of 8 normal individuals by quantifying with UV spectrophotometer

S. No A260 A280 Purity Conc µg/ml Conc ng/ml

1 0.114 0.064 1.8 17100 17.10

2 0.131 0.070 1.8 19650 19.65

3 0.132 0.069 1.9 19800 19.80

4 0.122 0.062 1.9 18300 18.30

5 0.119 0.066 1.8 17850 17.85

6 0.133 0.074 1.79 19950 19.95

7 0.140 0.068 2.0 21000 21.00

8 0.121 0.068 1.77 18150 18.15

Similarly integrity of DNA of diabetic individuals was performed by Agarose gel

electrophoresis and quantification was done by using UV spectrophotometer at

A260/280.

Table 9: Showing DNA Purity and concentration of 7 diabetic individuals by quantifying with UV spectrophotometer

S.No A260 A280 Purity Conc µg/ml Conc ng/ml

1 0.367 0.182 2.0 55050 55.05

2 0.304 0.155 1.96 45600 45.60

3 0.281 0.145 1.85 42150 42.15

4 0.290 0.146 1.94 43500 43.50

5 0.261 0.141 2.08 39150 39.15

6 0.344 0.171 1.97 51600 51.60

7 0.384 0.195 2.0 57600 57.60

4.2.3 Identification of Catalase Gene by PCR Amplification

39

In the present study, we have isolated DNA from blood of normal group and diabetic

patients and performed PCR reaction first a reaction mix is prepared by adding all the

contents required for amplification. It is distributed in different PCR tubes and then

Different DNA samples are added accordingly in different PCR tubes, all these

preparations are done under laminar air flow chamber to avoid contamination.

Then temperature and all PCR conditions are adjusted in the PCR machine and the PCR

is kept for about 35 cycles. The cycles are done on an automated cycler, a device which

rapidly heats and cools the test tubes containing the reaction mixture. During this process

it undergoes three major steps, they are- Denaturation (alteration of structure), annealing

(joining), and extension and they takes place at a different temperature.

Within one cycle, a single segment of double-stranded DNA template is amplified into

two separate pieces of double-stranded DNA. These two pieces are then available for

amplification in the next cycle. As the cycles are repeated, more and more copies are

generated and the number of copies of the template is increased exponentially.

Figure 18: Catalase Gene Mapping

40

126 bp Fragment of Catalase geneForward primer Reverse primer

+1 +126

PCR amplification of normal samples of CAT gene:

Agarose gel electrophoresis was performed for above DNA product of normal samples

and quality of DNA was checked by performing PCR for Catalase specific primers. The

obtained product is Catalase gene which is of 126bp. The expression of DNA in all

normal samples indicates the good quality of DNA. We have taken C- negative control,

L- ladder is about 1000 bp and N- normal individual as shomn in figure 19.

Figure 19: PCR product of normal individual

41

By this analysis of PCR with specific primers provided amplicons of the expected size of

126bp as shown in a figure 19; six samples of normal individual amplify with the specific

primers and a negative control.

PCR amplification of Diabetes samples of CAT gene:

Similarly here also Agarose gel electrophoresis was performed for above DNA product of

diabetic samples and quality of DNA was checked by performing PCR for Catalase specific

primers. The obtained product is Catalase gene which is of 126bp. The expression of DNA in all

normal samples indicates the good quality of DNA. Here we assumed that all the PCR

material such as buffer, dNTPs and Taq polymerase along with 1000bp DNA ladder

shown positive results we have taken C- negative control, L- ladder is about 1000 bp and

D- Diabetic individual as shown in figure 20(a) & (b).

Figure 20(a): PCR product of diabetic individual

42

Figure 20(b): PCR product of diabetic individual

By this analysis of PCR with specific primers provided amplicons of the expected size of

126bp as shown in a figure 20; six samples of diabetic individual amplify with the

specific primers and a negative control. No results were found in diabetic sample1 (D1)

as shown in figure 20(a). Similarly we performed with some more diabetic individual the

results are shown in figure 20(b).

43

5. Conclusion

Diabetes is one of the pathological processes known to be related to an unbalanced

production of ROS, such as H2O2. Therefore, cells must be protected from this oxidative

injury by antioxidant enzymes. In this investigation, Catalase activity was measured in

serum samples of both normal as well as diabetic individuals with different glucose

concentrations ranging from less than 90mg/dl glucose value and more than 130mg/dl

glucose value. We found in our study increasing glucose level shows decrease Catalase

activity as compare to normal individuals.

44

The regulation of Catalase gene is complex and appears to occur through different

pathways. In this study, we have isolated the genomic DNA from normal as well as

Diabetes blood sample. PCR results of catalase gene on 1.5% agarose gel showed band at

126bp. In both samples we successfully amplified the catalase gene.

Therefore, finally we conclude that our finding of this Catalase gene may be used as

Biomarker for Type 2 Diabetes.

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Association of Polymorphic Markers of the Catalase and Superoxide Dismutase Genes with Type 2 Diabetes Mellitus

To cite this article:Maivel H. Ghattas and Dina M. Abo-Elmatty. DNA and Cell Biology. November 2012, 31(11): 1598-1603. doi:10.1089/dna.2012.1739.

Published in Volume: 31 Issue 11: October 25, 2012Online Ahead of Print: September 12, 2012

Our study aims at determining whether genetic polymorphisms of catalase (CAT 1167C/T) and superoxide dismutase (SOD +35 A/C) could be associated with type 2 diabetes mellitus (T2DM). The study was conducted on 105 Egyptian patients with T2DM and 115 control subjects. Genotypes were done by polymerase chain reaction-restriction fragment length polymorphism methods. Homeostatic

model assessment of insulin resistance (HOMA-IR), CAT and SOD activities, glycated hemoglobin, and insulin and lipid profiles were assessed. CAT and SOD

activities were significantly decreased in T2DM compared with the control subjects. T allele of CAT and C allele of SOD1 were significant risk factors for T2DM. No effects of CAT or SOD1 gene polymorphisms on glycated haemoglobin or on HOMA-IR were found. With regard to

the enzymes activities, only +35 A/C of SOD1 was related to SOD activity. Genetic variants C1167T of CAT gene and +35 A/C of SOD1

gene has no role in insulin resistance in T2DM.

49