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

1

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

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.

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.

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
5

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

Enzymatic defense

Main sources

molecule

Superoxid
e (O2)

Hydrogen
peroxide

systems

Leakage of electrons from the

Superoxide dismutase

electron transport chain

(SOD)

Activated phagocytes
Xanthine oxidase
Flavoenzymes
From O2 via superoxide
dismutase (SOD)
NADPH-oxidase

Superoxide reductase
(in some bacteria)

(neutrophils)

(H2O2)

Glucose oxidase
Xanthine oxidase

Product(s)
H2O2 + O2
H2O2

Glutathione peroxidase

H2O + GSSG

Catalase

H2O + O2

Peroxiredoxins (Prx)

H2O

Hydroxyl
From O2and H2O2 via
transition metals (Fe or Cu)

radical
(OH)
Nitric
oxide (NO)

synthases

Nitric oxide

Glutathione/TrxR

GSNO

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;
6

(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.
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
7

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 (Lglutamyl -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 Thnard, 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.
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.

Catalase

2H2O2

CAT Gene located 11p13

2H2O + O2

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.
10

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
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 st

ructure

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. H 2O2 has been
reported to damage pancreatic -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 H 2O2,
and consequently, Catalase protects pancreatic -cells from damage by H 2O2 (Tiedge et
al., 1998). Low Catalase activities, which have been reported in patients with
schizophrenia and atherosclerosis (Gth et al., 1996), are consistent with the hypothesis
that long-term oxidative stress may contribute to the development of a variety of lateonset disorders, such as type 2 diabetes (Gth et al., 2000).
11

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.

12

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 organisms 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,
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, O 2. 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
13

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
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 H 2O2 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).
14

The former theory hyperglycemia, an outcome of the disease, as a secondary force that
further damages -cells. The latter theory suggests that the often-associated defect of
hyperlipidemia is a primary cause of -cell dysfunction. That patients with type 2
diabetes continually undergo oxidative stress, that elevated glucose concentrations
increase levels of reactive oxygen species in -cells, that islets have intrinsically low
antioxidant enzyme defenses, that antioxidant drugs and over expression of antioxidant
enzymes protect -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
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).

15

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 H 2O2 and
consequently, catalase protects pancreatic cells from damage by H 2O2. 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(Laszlo 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).

16

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
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
17

present and will thus vary for dierent proteins. In this method, Bovine Serum Albumin (BSA)
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.
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(l)

200

400

600

800

1000

Water(l)

1.8

1.6

1.4

1.2

0.8

FC reagent(l)

200

200

200

200

200

200

18

Procedure for the estimation of unknown sample using the standard

graph:

The serum was separated from the normal and diabetes patients serum sample.
Lowry stock reagent of 1 ml was taken in test tube. To the reagent10 l 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 l 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.

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 H 2O2 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).

19

Catalase stock solution: 840 l of H2O2 solution was added in 299.16 ml phosphate
buffer and a stock of 300 ml was prepared.

Procedure:

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

Serum sample of 10 l 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:

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:
20

1.
1.
2.
3.
4.

Autoclaved eppendorff
Autoclaved micropipettes
Autoclaved micro tips
Autoclaved distilled water
Eppendorff stand

Preparation of Reagents:
The reagents were prepared as described below:
Table 3: RBC Lysis Buffer/ TKM 1
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.
21

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

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 l of blood sample was added in it.
To the blood sample 800 l 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 l of TKM1 was added and steps 2 and 3 are repeated until a white

pellet is obtained.
To the pale pellet, 300 l of TKM2 and 80 l of 10% SDS was added and

incubated for 30 minutes.

After incubation 80 l 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 l of cold absolute Ethanol.

Centrifuged at 10,000 rpm for 5 minutes.

Supernatant was discarded and 300 l 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 l of TE buffer was added for hydration of DNA and
preserve at freezing temperature.

22

We detected the DNA in the isolated samples by using 1% of agarose gel by

electrophoresis.

Agarose gel electrophoresis 3.2.2

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 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.
2.
3.
4.
5.

Horizontal electrophoresis unit

Gel plate
Combs
Adhesive tapes
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.
23

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 l 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.
24

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 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 (g/ml). Thus, an optical density (OD) of 1
corresponds to 50 g/ml for double-stranded DNA, 38 g/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
25

Procedure:

To quantify the DNA, 99 l 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.
26

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

27

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 25l of PCR reaction:

28

Reagents

Volume

Water

18.3 l

Buffer

2.5 l

dNTPs

1 l

Forward Primers

1 l (20 pmol/l)

Reverse Primers

1 l (20 pmol/l)

Taq Polymerase

0.2 l

DNA sample

1 l

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:

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.
29

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

4.1 Biochemical Analysis:
4.1.1 Protein standard graph

30

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 g/l.
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(l)

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:

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

31

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 concentration
mg/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

Diabetic
Sample

Glucose level
mg/dl

Protein concentration
mg/ml
32

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.

33

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

34

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 (A 260/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

35

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 (g/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:
36

Table 8: showing purity and concentration of DNA of 8 normal individuals by

quantifying with UV spectrophotometer
S. No
1
2
3
4
5
6
7
8

A260
0.114
0.131
0.132
0.122
0.119
0.133
0.140
0.121

A280
0.064
0.070
0.069
0.062
0.066
0.074
0.068
0.068

Purity
1.8
1.8
1.9
1.9
1.8
1.79
2.0
1.77

Conc g/ml
17100
19650
19800
18300
17850
19950
21000
18150

Conc ng/ml
17.10
19.65
19.80
18.30
17.85
19.95
21.00
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. g/ml

Conc. ng/ml

0.367

0.182

2.0

55050

55.05

0.304

0.155

1.96

45600

45.60

0.281

0.145

1.85

42150

42.15

0.290

0.146

1.94

43500

43.50

0.261

0.141

2.08

39150

39.15

0.344

0.171

1.97

51600

51.60

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:
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
37

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).

38

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.

39

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
40

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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45