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

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.
3

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

Hispanic/Latino

Americans,

occurs

more

African-Americans,

Native

often

in

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 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

Product(s

defense systems

Leakage of electrons

Superoxide

H2O2 + O2

from the electron

dismutase (SOD)

molecul

Main sources

e
Supero
xide
(O2)

transport chain
Superoxide
Activated phagocytes

reductase (in some

7

H2O2

Xanthine oxidase
Flavoenzymes

bacteria)

From O2 via
superoxide dismutase
(SOD)

Glutathione

Hydroge

NADPH-oxidase

(neutrophils)

Hydroxy
l radical
(OH)

Glucose oxidase

Peroxiredoxins

Xanthine oxidase

(Prx)

H2O + O2
H2O

From O2and H2O2 via

transition metals (Fe or
Cu)

Nitric
oxide

GSSG

Catalase

peroxid
e (H2O2)

H2O +

peroxidase

synthases

Nitric oxide

Glutathione/TrxR

(NO)

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),

(II)

peroxidase, and thiol-specific antioxidants;

Binding of proteins (e.g., transferring,

metallothionein,

haptoglobins, caeroplasmin) to pro-oxidant metal ions, such

as iron and copper;
8

(III)

Protection against macromolecular damage by proteins such

(IV)

as stress or heat shock proteins; and

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 H 2O2

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
9

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 -Lcysteinyl

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
10

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 H2O2 to water and oxygen. The enzyme has one of
the highest turnover rates, converting millions of H 2O2 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

11

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
12

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 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 (H 2O2) is
a by-product of normal cellular respiration and is also formed from

13

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 H2O2, and consequently, Catalase protects pancreatic
-cells from damage by H2O2 (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 late-onset disorders, such as type 2 diabetes (Gth et al.,
2000).

1.8 AIM AND OBJECTIVES

14

1.

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

2.

Diabetes.
To Identify and Isolate human genomic DNA and amplify the
antioxidant (catalase) gene using specific primers for Catalase

3.

gene with polymerase chain reaction.

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

15

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
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
16

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 H2O2 per min/ mg of protein, mean SD, p < 0.05].
Catalase activity increased significantly in the erythrocytes of STZinduced diabetic rats (Durdi et al., 2007).
The former theory hyperglycemia, an outcome of the disease, as a
secondary force that further damages -cells. The latter theory
17

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
18

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(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

19

+35 A/C of SOD1 gene has no role in insulin resistance in T2DM (Maivel
et al., 2012).

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:

20

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 present and will thus
vary for different 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

hydroxide. (0.56g NaOH+2.86g Na2Co3 in 100 ml water).

Lowry B: 1% CuSo4 in distilled water
21

Sodium

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

200

200

200

200

200

200

reagent(l)

Procedure for the estimation of unknown sample

using the standard graph:

The serum was separated from the normal and diabetes patients
serum sample.
22

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 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).

23

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 MgCl 2 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
24

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

Materials Required:
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

0.0745

0.03725

0.2033

0.10165

(10 mM)

MgCl2 (10 mM)

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

25

EDTA ( 2 mM)

0.0744

0.0372

KCl

0.0745

0.03725

MgCl2 (10 mM)

0.2033

0.10165

NaCl (0.4 M)

2.3376

1.1688

(10 mM)

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

Ethylene diamine tetra acetic acid (EDTA)

0.009 g

(1mm)

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.

26

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.

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:
27

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.

Figure 7: Agarose Gel Electrophoresis

Documentation System

Procedure:
Preparation of 1% agarose gels:
28

Figure 8: Gel

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.

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, nearultraviolet,

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.

29

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

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.
30

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.

31

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.
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

32

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.

33

Procedure for making 25l of PCR reaction:

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:

34

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

35

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 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

0.13

0.216

0.30

0.39

0.51

Figure 12: Standard graph

36

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
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
Norma
l
Sampl
e

Glucose
level
mg/dl

Protein
concentration
mg/ml

OD at 240
nm

Catalas
e
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

37

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
Diabet
ic
Sampl
e

Glucose
level
mg/dl

Protein
concentration
mg/ml

OD at 240
nm

Catalas
e
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
38

Correlation of enzyme activity with Glucose concentration was measured and represented
in figure 14.

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).

39

4.2 Molecular analysis

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

40

DNA integrity of diabetic individuals

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:
41

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

A260

A280

Purity

Conc

Conc

No
1
2
3
4
5
6
7
8

0.114
0.131
0.132
0.122
0.119
0.133
0.140
0.121

0.064
0.070
0.069
0.062
0.066
0.074
0.068
0.068

1.8
1.8
1.9
1.9
1.8
1.79
2.0
1.77

g/ml
17100
19650
19800
18300
17850
19950
21000
18150

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.N

A260

A280

Purity

Conc.

Conc.
ng/ml
55.05

o
1

0.367

0.182

2.0

g/ml
55050

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

42

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 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:

43

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).

Figure 18(a): PCR product of diabetic individual

44

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.

45

5 Conclusion
Diabetes is one of the pathological processes known to be related to an unbalanced
production of ROS, such as H2O2. Therefore, cells must be protected from this oxidative
injury by antioxidant enzymes. In this investigation, catalase activity was measured in
serum samples of both normal as well as diabetic individuals with different glucose 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.
46

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51