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.

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Figure 1: Schematic representation of onset of type 1 diabetes

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. 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. Pre-diabetes 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.

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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

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).

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

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

molecules listed in Table 1 can be summarized as follows:

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:

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(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

(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.

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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, 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 as shown in figure 4 and 5.

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It is a ubiquitously occurring enzyme that catalyses the decomposition of H2O2 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.

 

Catalase

2H2O2  →  2H2O + O2

CAT Gene located 11p13

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

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

Figure 5: Catalase Gene Mapping

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. 

Structure of catalase enzyme: 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

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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

1.7 Catalase and Diabetes:

The metabolic effects of oxidants, which are believed to contribute too 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

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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).

1.8 AIM AND OBJECTIVES

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.

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2. REVIEW OF LITERATURE

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. It is accepted that oxidative

stress results from an imbalance between the generations of oxygen derived radicals and

the organism’s antioxidant potential.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,

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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).

Some studies show that reactive oxygen radicals (OH, O2. and H2O2) increase tissue

damage in viral hepatitis patients. Reactive oxygen species, including hydroxyl radicals

(OH), superoxide anions (O2.-) and hydrogen peroxide (H2O2), lead to the specific

oxidation of some enzymes, protein oxidation and degradation. Cells are also equipped

with enzymatic antioxidant mechanisms that play an important role in the elimination of

ROS (sies H, 1993).

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

development of complications of both types of DM. 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

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

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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

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

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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 (PJ hisalkar et al., 2012).

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(La’szlo et al.,

2001).

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 (Maivel et al., 2012).

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3. MATERIALS AND METHOD

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 a box containing ice.

3.1 BIOCHEMICAL ANALYSIS OF CATALASE:

Separation of serum from whole blood sample

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For isolation of serum, 2-3 ml of blood was collected by vene puncture 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.

protein was estimated by 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 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 di erent proteins. In this method, Bovine Serum Albumin (BSA)ff

was used as a standard protein.

Reagent Required:

BSA stock solution (1 mg/ml)

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

NaOH+2.86g Na2Co3 in 100 ml water).

Lowry B: 1% CuSo4 in 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.

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To estimate the amount of protein in an unknown sample, a standard graph using a

known protein sample should be obtained.

Procedure for the preparation of standard graph:

Different dilutions of BSA solutions were taken in test tubes from the stock BSA solution

and F.C reagent was added in all the tubes. The final volume in each of the test tubes

should be 2 ml.

Table 2: Setup of different dilution for 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 1 ml was taken in test tube. To the reagent10 μl of serum

sample was added which was present in the test tube.

The test tubes were kept for incubation for about 30 minutes at room

temperature.

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

incubation at room temperature for another 30 minutes.

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

using spectrophometer at 595 nm. wavelength was used.

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The above steps were repeated for all samples.

3.1.2 Estimation of catalase:

Catalase enzyme converts H2O2, H2O and 1/2 O2. Catalase activity was measured by the

(Aebi H, 1983). This method was based on the hydrolyzation of H2O2 and decreasing

absorbance at 240 nm. The conversion of H2O2 into H2O and 1/2 O2 in a minute under

standard condition was considered to be the enzyme reaction velocity.

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.

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

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

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

Catalase stock solution: 840 μl of H2O2 solution was added in 299.16 ml phosphate

buffer and a stock of 300 ml was prepared.

Procedure:

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

Serum sample of 10 μl was added to cuvette.

Adjusted the wavelength to 240 nm 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:

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

concentration

3.2 MOLECULAR ANALYSIS OF CATALASE

3.2.1 Isolation of genomic DNA from human blood sample:

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

1. Autoclaved micropipettes

2. Autoclaved micro tips

3. Autoclaved distilled water

4. Eppendorff stand

Preparation of Reagents:

The reagents were prepared as described below:

Table 3: RBC Lysis Buffer/ TKM 1

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Chemicals (100 ml) (50 ml)

Tris-HCL (10 mM) 0.121 0.061

EDTA ( 2 mM) 0.0744 0.0372

KCl (10 mM) 0.0745 0.03725

MgCl2 (10 mM) 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 (100 ml) (50 ml)

Tris-HCL (10 mM) 0.121 0.061

EDTA ( 2 mM) 0.0744 0.0372

KCl (10 mM) 0.0745 0.03725

MgCl2 (10 mM) 0.2033 0.10165

NaCl (0.4 M) 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 gm of NaCl was dissolved in 25 ml of distilled water.

TE Buffer

Chemical Amount

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

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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.

3.2.2 Agarose gel electrophoresis

Electrophoresis is a technique used to separate and sometimes purify macromolecules

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

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Fragments of linear DNA migrate through agarose gel 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.

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: 19.36 gm of Tris was dissolved in 50 ml of distilled water.

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

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

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

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

make 1:10 dilution.

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

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

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

readymade.

24

Figure 7: Agarose Gel Electrophoresis Figure 8: Gel Documentation System

Procedure:

Preparation of 1% agarose gels:

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

conical flask.

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

dissolved.

Then 7l of Ethidium bromide was added to the gel 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 4 l 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 45 minutes, After 45 min DNA was visualized

under gel documentation system.

3.2.3 Spectrophotometer:

25

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 9: Spectrophotometer

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 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). Thus, an optical density (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.

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

Procedure:

26

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

spectrophotometer at 260 nm as well as 280 nm.

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

Then 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.

Cycling conditions

There are different steps in PCR reaction program, which are automatically controlled by

the automated thermal cycles and the steps are as follows:

1) Initial denaturation: The template present in the mixture gets initially denatured

to remove the secondary structures present in it.

2) Denaturation: This is the important step in which it is repeated and start point for

every cycle. In this step the strands formed in the previous cycle get denatured to

form single strands.

27

3) Annealing: As the temperature is low compared to denaturation step the primers,

which are specific to the DNA in the mixture get anneal to the single strands to

the complementary sequence. This forms the attachment of polymerase to stand.

The temperature of this step is depends on the Tm (melting temperature) of

primers.

4) Extension: In this step the polymerase get bind to the DNA and extends

(polymerizes) the DNA strands complementary to the template strands. After this

step the cycle again starts from the initiation step to get exponential fold of

strands.

5) Final extension: In this final step, complete extension of the complementary

strands occurs to form expected band size.

The following conditions were maintained to perform PCR:

Stage 1: 94oC – 5 min

Stage 2: 35 cycles

Step 1: 95oC – 1 min

Step 2: 60oC – 1 min

Step 3: 72oC – 1 min

Stage 3:

Step 1: 72oC – 5 min

Step 2: 15oC 

28

Figure 10: Polymerase Chain Reaction

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 11.

Figure 11: The exponential amplification of the gene in PCR.

Procedure for making 25µl of PCR reaction:

29

Reagents Volume

Water 18.3 µl

Buffer 2.5 µl

dNTPs 1 µl

Forward Primers 1 µl (20 pmol/µl)

Reverse Primers 1 µl (20 pmol/µl)

Taq Polymerase 0.2 µl

DNA sample 1 µl

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. The amplified samples were stored at freezing

temperature for further analysis.

Agarose gel electrophoresis:

Procedure:

Preparation of 1.5% agarose gels:

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

conical flask.

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

Then 7 l 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:

30

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

the electrophoresis chamber containing 1x TAE.

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

to the wells.

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

Samples were run at 75 volts for 45 minutes.

After 45 minutes DNA was visualized under gel documentation system.

4. RESULTS AND DISCUSSION

31

4.1 Biochemical Analysis:

4.1.1 Protein standard graph

As demonstrated in figure 12, protein concentration shows a direct correlation with

absorbance. The absorbance was determined for BSA conc. ranging from 0 to 1000

μg/μl. The standard graph was constructed to convert the absorbance readings for the

experimental samples into a protein amount or concentration. Over this range the

absorbance increased in a linear fashion.

Table 5: Setup of different dilution of 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 12: Standard graph

4.1.2 Estimation of total serum protein analysis:

32

Randomly 16 serum samples of diabetic and normal individuals protein content is

estimated by using spectrophotometer with an absorbance of 595 nm. The results of the

present study showed that the levels of total serum protein were significantly higher when

compared with the normal individual as shown in figure 13.

Figure 13: Histogram showing the levels of total protein serum in normal and diabetic

individuals.

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 52.21 0.19 0.1941 1438.34

02 76.106 0.21 0.3252 2181.08

03 80 0.09 0.1201 1879.49

04 82 0.16 0.1235 2099.47

05 89 0.16 0.2041 1901.46

06 85 0.15 0.1392 1307.04

07 86 0.15 0.1577 1480.75

08 89 0.16 0.2160 1796.65

Table 7: Spectrophotometer value of diabetic serum sample of catalase activity

33

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

03 205 0.16 0.1109 976.23

04 139 0.16 0.2160 644.36

05 347 0.27 0.0701 365.67

06 139 0.16 0.2256 1985.91

07 152 0.15 0.2034 1909.85

08 193 0.20 0.1475 1549.29

4.1.3 Relationship between catalase activity and glucose level

Catalase activity was measured in 16 different serum samples of both normal as well as

diabetic individuals with varying concentrations of glucose. The conc. of glucose in

normal individual is ranging from 52-90 mg/dl are listed in the table 6, and average value

of catalase activity of the samples is calculated and represented in figure 14. Similarly the

conc. of glucose in diabetic individuals is ranging from 130-340 mg/dl are listed in table

7 and average value of catalase activity of the samples was calculated and represented in

figure 14.

The Catalase activity was determined by H2O2 consumption and found that resultant

catalase activity of diabetic individual is less than that of normal individual. The

Correlation of enzyme activity with Glucose concentration was measured and represented

in figure 14.

34

Figure 14: Catalase activity in normal and diabetic

In this study, our results show that blood serum catalase activity in the type 2 diabetic subjects

was decreased (OD mean value - 1290) as compared with that in the nondiabetic subjects (OD

mean value - 1760). The values represented in the bracket is based on the glucose concentration

that is less than 90 represent the nondiabetic subjects and more than 90 represents diabetic

subjects Both these parameters indicated an oxidative stress. The oxidative stress is more during

uncontrolled stage of diabetes. Our findings are in accordance with the observations made earlier

(Goth et al., 2001; Mukhopadhyay et al., 2006).

4.2 Molecular analysis

35

4.2.1 Isolation of DNA from normal and diabetic blood samples:

On molecular level 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. The purity of the DNA samples was confirmed by absorbance (A260/A280) ratio,

which was 1.8-2.0. In order to further check the quality of the genomic DNA extracted by this

method, PCR amplification was performed. Agarose gel analysis revealed that 1.5% and 1000 bp

DNA fragments were amplified from the extracted DNA.

DNA integrity of normal individuals

DNA of normal individual is extracted from 3.2.1 procedure and were loaded on 1%

agarose gel at 75 volts for 45 minutes and band were observed indicating the presence of

the genomic DNA; as shown in the figure 15.

Figure 15: Isolated genomic DNA from normal blood samples

DNA integrity of diabetic individuals

36

DNA of diabetic individual is extracted from 3.2.1 procedure and were loaded on 1%

agarose gel at 75volts for 45 minutes and band were observed indicating the presence of

the genomic DNA; as shown in the figure 16.

Figure 16: Isolated genomic DNA diabetic blood samples

4.2.2 Quantification of DNA by spectrophotometer:

After isolating the blood samples of both normal and diabetic individual 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 260 nm 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 260 nm and 280 nm of normal individuals are

given below:

37

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

PCR amplification of normal samples of CAT gene:

38

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 126 bp. 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 shown in figure 17.

Figure 17: PCR product of normal individual

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

126 bp as shown in a figure 17.

PCR amplification of diabetes samples of CAT gene:

Similarly here also agarose gel electrophoresis were 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 126 bp. The

expression of DNA in all normal samples indicates the good quality of DNA.

C- negative control, L- ladder is about 1000 bp and D- Diabetic individual as shown in

figure 18(a) & (b).

39

Figure 18(a): PCR product of diabetic individual

Figure 18(b): PCR product of diabetic individual

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

of 126 bp as shown in a figure 18; by this amplification finding of this catalase gene may

be used as biomarker for type 2 diabetes.

40

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

41

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 conc.

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.

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

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

of this catalase gene may be used as biomarker for type 2 diabetes.

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