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. (The pancreas is a deep-seated organ in the abdomen
located behind the stomach). In addition to helping glucose enter the
cells, insulin is also important in tightly regulating the level of glucose
in the blood. After a meal, the blood glucose level rises. In response to
the increased glucose level, the pancreas normally releases more
insulin into the bloodstream to help glucose enter the cells and lower
blood glucose levels after a meal. When the blood glucose levels are
lowered, the insulin release from the pancreas is turned down. It is
important to note that even in the fasting state there is a low steady
release of insulin than fluctuates a bit and helps to maintain a steady
blood sugar level during fasting. In normal individuals, such a
regulatory system helps to keep blood glucose levels in a tightly
controlled range.

1.3 Risk Factors for Type 2 Diabetes:

Type 2 diabetes occurs when the body can't use the insulin that's
produced, a condition called insulin resistance. Though it typically
starts in adulthood, type 2 diabetes can begin anytime in life. Because
of the current epidemic of obesity among U.S. children, type 2 diabetes
is increasingly found in teenagers. Here are the risk factors for
developing type 2 diabetes.

Obesity or being overweight. Diabetes has long been linked

to obesity and being overweight. Research at the Harvard School of

Public Health showed that the single best predictor of type 2 diabetes
is being obese or overweight.

Impaired

glucose

tolerance

or

impaired

fasting

glucose. Prediabetes is a milder form of diabetes that's sometimes

called impaired glucose tolerance. It can be diagnosed with a simple
blood test. Prediabetes is a major risk factor for developing type 2
diabetes.

Insulin resistance. Type 2 diabetes often starts with cells that

are resistant to insulin. That means they are unable to take in insulin
as it moves glucose from the blood into cells. With insulin resistance,
the pancreas has to work overly hard to produce enough insulin so
cells can get the energy they need. This involves a complex process
that eventually leads to type 2 diabetes.

Ethnic

background. Diabetes

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).
An atom contains a nucleus, and electrons move around the nucleus, usually in pairs
(Woolf et al., 1998).A free radical is any atom or molecule that contains a single unpaired
electron in an outer orbital, and capable of independent existence (Gutteridge et al.,
1997). The unpaired electron alters the chemical reactivity of an atom or molecule
making it extremely reactive and unstable and enters into reactions with organic or
inorganic components in the cell (proteins, lipids, carbohydrates)particularly with key
molecules in membranes and nucleic acids (Beckman et al., 1994).

1.4 Reactive Oxygen Species (ROS):

6

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 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
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.
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.

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

defense against oxidative stress. Catalase is a heme enzyme that is
present in the peroxisome of nearly all aerobic cells. Catalase converts
the reactive oxygen species hydrogen peroxide to water and oxygen
and thereby mitigates the toxic effects of hydrogen peroxide. Oxidative
stress is hypothesized to play a role in the development of many
chronic or late-onset diseases such as diabetes. Polymorphisms in this
gene have been associated with decreases in Catalase activity but, to
date, acatalasemia is the only disease known to be caused by this
gene.

1.7 Structure of Catalase Enzyme:

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
(Loew et al., 1900). In 1937 Catalase from beef liver was crystallised by
James B. Sumner and Alexander Dounce (Sumner & Dounceand, 1937)
the molecular weight was worked out in 1938 (Sumner & Dounceand,
1938). In 1969, the amino acid sequence of bovine Catalase was
worked out (Schroeder et al., 1969) then in 1981, the threedimensional structure of the protein was revealed.

12

Figure 5: Schematic Representation of 3D Catalase Structure

1.8 Catalase and Diabetes:

The metabolic effects of oxidants, which are believed to contribute to
many diseases, may influence the development of some forms of
diabetes. As we discuss earlier the oxidant hydrogen peroxide (H 2O2) 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).

13

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).

1.9 AIM AND OBJECTIVES

1.

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

Diabetes.
14

2.

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.

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
15

with chronic hyperglycemia and disturbances of carbohydrate, lipid,

and

protein

metabolism.

As

consequence

of

the

metabolic

derangements in diabetes, various complications develop including

both macro and micro-vascular dysfunctions. It is accepted that
oxidative stress results from an imbalance between the generations of
oxygen derived radicals and the organisms antioxidant potential.
Various studies have shown that diabetes mellitus is associated with
increased formation of free radicals and decrease in antioxidant
potential. Due to these events, the balance normally present in cells
between radical formation and protection against them is disturbed.
This leads to oxidative damage of cell components such as proteins,
lipids, and nucleic acids. In both insulin dependent (type 1) and noninsulin-dependent diabetes (type 2) there is increased oxidative stress
(Roja Rahimi et al., 2005).
Recent studies report that oxidative stress plays a major role in the
pathogenesis and development of complications of both types of DM.
However, the exact mechanism by which oxidative stress could
contribute to and accelerate the development of complications in
diabetic mellitus is only partly known and remains to be clarified. On
one hand, hyperglycemia induces free radicals; on the other hand, it
impairs the endogenous antioxidant defense system in patients with
diabetes. Endogenous antioxidant defense mechanisms include both
enzymatic and non-enzymatic pathways. Their functions in human cells
are to counterbalance toxic reactive oxygen species (ROS). Common
antioxidants include the vitamins A, C, and E, glutathione (GSH), and
the enzymes superoxide dismutase (SOD), catalase (CAT), glutathione
peroxidase (GPx), and glutathione reductase (GRx). The importance of
endogenous antioxidant defense systems, their relationship to several
path

physiological

processes

and

their

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

16

possible

therapeutic

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
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
17

-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).
18

Oxidative stress (OS) results when production of reactive oxidative

species (ROS) exceeds the capacity of cellular antioxidant defenses to
remove these toxic species. (Jorge et al., 2008).
Decreased activity of these antioxidant enzymes may increase the
susceptibility of diabetic patients to oxidative injury. An appropriate
support of antioxidant supplies may help in preventing clinical
complications of diabetes. Estimations of these antioxidant enzymes
might be used as marker in the management of glycemic control and
the development of diabetic complications.

The enzyme catalase has a predominant role in controlling the

concentration of H2O2 and consequently, catalase protects pancreatic
cells from damage by H2O2. Low catalase activities, which have been
reported in patients with schizophrenia and atherosclerosis, are
consistent with the hypothesis that long-term oxidative stress may
contribute to the development of a variety of late-onset disorders, such
as type 2 diabetes().
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 ().

19

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 cool box
containing ice.

3.1 BIOCHEMICAL ANALYSIS OF CATALASE:

20

Separation of Serum from whole blood sample

Serum is made up of non-clotting proteins, glucose, nutrients,
electrolytes, hormones, antigens, antibodies and other particles.
Separation of serum is more tedious and time-consuming than plasma
extraction. For isolation of serum, first a blood sample is allowed to
clot, after which the coagulated blood is centrifuged. The liquid
supernatant formed at the top portion is serum. The procedure for
extraction of plasma is very simple; blood sample is spun by using a
centrifuge apparatus. The heavier blood cells settle at the bottom, and
blood plasma is collected from the upper layer. 2-3 ml of blood was
collected by venepuncture in EDTA vacuumed tubes. The tubes were
centrifuged for 10 minutes at 4000 rpm. Supernatant serum was
pipette out with a micropipette, transferred to an Eppendorf tube, and
stored in deep freeze. Biochemical estimation was performed from
these sera in the following methods.

3.1.1 Estimation of Protein in total serum sample:

Serum is isolated from blood samples of both normal and diabetic patients
and performed protein analysis to evaluate the usefulness of serum total
protein.
We have estimated the protein using the Lowry method. The Lowry
assay-Protein by Folin Reaction (Lowry et al., 1951) has been the most
widely used method to estimate the amount of protein in biological
samples. The phenolic group of tyrosine and tryptophan residues
(amino acid) in a protein will produce a blue purple color complex ,
with maximum absorption in the region of 595 nm wavelength, with
Folin-

Ciocalteau

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

21

different proteins. Estimation techniques use Bovin Serum Albumin

(BSA) universally as a standard protein.

Reagent Required:
1.

BSA stock solution (1mg/ml)

2.

Sodium Carbonate anhydrous

3.

Sodium hydroxide

4.

Copper sulphate

5.

Sodium potassium tartarate

6.

F.C reagent (Phenol reagent)

Reagents Prepared:
Lowry A: 2% Sodium Carbonate anhydrous in 0.1M Sodium hydroxide.
(0.56g NaOH+2.86g Na2Co3 in 100 ml water).
Lowry B: 1% CuSo4 in distilled water ( 0.28g of CusO 4 in 20ml distilled
water).
Lowry C: Sodium potassium tartarate( 0.56g in 20ml of distilled
water).
Lowry stock reagent: 49ml of Lowry A + 0.5ml of Lowry B+ 0.5ml of
Lowry C
F.C reagent: Phenol reagent (2N) was diluted in water in 1:1 ratio.
To estimate the amount of protein in an unknown sample, we should
first prepare a standard graph using a known protein sample. We have
used Bovine serum albumin (BSA) as known sample to obtain the
standard graph.

Procedure for the Preparation of Standard Graph:

22

We have prepared different dilutions of BSA solutions by

mixing stock BSA solution (1 mg/ ml) water and F.C reagent in the test
tube. The final volume in each of the test tubes is 2 ml.
Table 2: preparation of 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.
Lowry stock reagent of 1ml was taken in test tube. To the
reagent10l of serum sample was added which was present in

the test tube.

The test tubes were kept for incubation for about 30mins at

room temperature.
After incubation 200l of FC reagent was added. The test tubes
were kept for incubation at room temperature for another

30mins.
After incubation, 2ml of the mixture was taken in a cuvette to
read the OD value using spectrophometer at 595nm. wavelength

was used.
The above steps were repeated for all samples.

3.1.2 Estimation of Catalase:

Chemicals Required:
1 Potassium di-hydrogen orthophosphate
23

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 9ml water). This diluted solution is again diluted
3 times (1ml of diluted H2O2 in 2ml of water) bringing it to 1% solution
(30mM).
Catalase stock solution: 840 micro liter of H2O2 solution was added
in 299.160ml phosphate buffer and a stock of 300ml was prepared.

Procedure:

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

were added.
Serum sample of 10l was added to cuvette.
Adjusted the wavelength to 240nm and noted down the OD
values

using

the

time

scan

measurement

in

the

spectrophotometer.
Using the obtained OD values of protein and Catalase.
We found out the activity of the catalase enzyme by substituting
in the formula given below:

Catalase activity = Final OD/extinction coefficient*volume

of sample*protein

24

concentration

25

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
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 :
26

The reagents were prepared as described below:

Table 3: RBC Lysis Buffer/ TKM 1

Chemicals

(100ml)

(50ml)

Tris-HCL (10mM)

0.121

0.061

EDTA ( 2mM)

0.0744

0.0372

KCl

0.0745

0.03725

0.2033

0.10165

(10mM)

MgCl2 (10mM)

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

is adjusted to 7.6. Then EDTA is dissolved followed by other chemicals.

Table 4:

Cell Lysis Buffer/ TKM2

Chemicals

(100ml)

(50ml)

Tris-HCL (10mM)

0.121

0.061

EDTA ( 2mM)

0.0744

0.0372

27

KCl

(10mM)

0.0745

0.03725

MgCl2 (10mM)

0.2033

0.10165

NaCl (0.4M)

2.3376

1.1688

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

is adjusted at 7.6. Then EDTA is dissolved followed by other chemicals
and the volume is made up to 100 ml with distilled water.
10% SDS (10 ml): 1gm of SDS was dissolved in 10 ml of autoclaved
distilled water.
0.6M NaCl: 0.8765 g of NaCl was dissolved in 25 ml of distilled water.
TE Buffer
Chemical

Amount

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.

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.

28

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 by 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 gels with a mobility that is inversely
proportional to the log10 of their molecular weight. Bromophenol blue is used as
loading dye to track the movement of the sample. It is mixed well with
the sample. In addition, it increases the density of the mixture, so that,
they reside down at the bottom of the well and are diffused in the gel.
By using gels with different concentrations of agarose, DNA fragments of different sizes
can be resolved. Higher concentrations of agarose facilitate separation of small DNA
fragments, while low agarose concentrations allow resolution of larger DNAs (Maniatis T
.et al., 1989)

Materials Required:
1
2
3
4

Horizontal electrophoresis unit

Gel plate
Combs
Adhesive tapes
29

5 10T micropipette and autoclaved tips

Reagent Preparation:
10 x TAE Buffer (100) ml:

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

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

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

acetic acid was

added. Then the volume was

made up to 100ml with distilled water.

1X TAE Buffer: 30ml of 10X TAE Buffer was dissolved in 270 ml of
distilled water to make 1:10 dilution.
1% Agarose: 0.25g of Agarose was dissolved in 25ml of 1X TAE
Buffer.
1% Ethidium

bromide solution: 0.1g

of

ethidium bromide was

dissolved in 10ml distilled water. Gel loading solution and dye used was
6X concentrated and was obtained readymade.

Figure 6: Agarose Gel Electrophoresis

Documentation System
30

Figure 7: Gel

Procedure:
Preparation of 1% agarose gels:

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

conical flask.

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

After boiling 7l of Ethidium bromide was added to the solution and was allowed
to cool, Poured into the gel-casting tray.

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

Sample Loading and Electrophoresis:

After solidification of the gel, the comb was removed and the gel was placed in
the electrophoresis chamber containing 1x TAE.

Now 4l 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 30 minutes, After 30 min DNA was visualized
under gel documentation system.

3.2.3 Spectrophotometer:
In chemistry, spectrophotometer is the quantitative measurement of
the reflection or transmission properties of a material as a function of
wavelength. It is more specific than the general term electromagnetic
spectroscopy in that spectrophotometer deals with visible light, 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.
31

Figure 8: Spectrophotometer

Because DNA and RNA absorb ultraviolet light, with a absorption peak
at 260nm wavelength, spectrophotometers are commonly used to
determine

the

concentration

of

DNA

in

solution.

Inside

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)-1 cm-1; for single-stranded DNA and RNA it is 38
(g/ml)-1 cm-1. Thus, an optical density (or 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.

32

Figure 9: A spectrophotometer measures how much light of a certain

wavelength is absorbed by a liquid

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

Procedure:

To quantify the DNA, 99 l of TE buffer was taken in a cuvette

and calibrated the spectrophotometer at 260nm as well as

280nm. 1 l of each
DNA sample of 1l each to 99 l TE (Tris-EDTA buffer) and mixed

well.
Now 2.9 ml of water was added to the cuvette.
TE buffer and water used as a blank in the other cuvette of the

spectrophotometer.
The OD260 and OD280 values on spectrophotometer were noted.

3.2.4 Polymerase Chain Reaction:

33

Polymerase chain reaction in vitro was designed first by Karry Mullis in

1983. It follows the process of DNA replication using temperature
variations with a help of a thermo cycler. This process include five
major steps , at specific accurate temperature for each step for exact
specificity of the amplification or duplication of the specific DNA
sequence or gene out of the whole genomic DNA sequence. This is
possible by using specific complementary forward and reverse primers
that specified the region of duplication. The enzyme used for the
amplification is generally consists of 3 end to 5 end extension and 5
end to 3 end exonuclease activity. The enzyme used is called Taq
polymerase, which is extracted from thermo stable bacteria Thermus
aquaticus, generally found in hot springs.
The purpose of a PCR (Polymerase Chain Reaction) is to make a huge
number of copies of a gene. This is necessary to have enough starting
template for sequencing.

The cycling reactions:

There are three major steps in a PCR, which are repeated for 30
or 40 cycles. This is done on an automated cycler, which can heat and
cool the tubes with the reaction mixture in a very short time as shown
in figure 11.

1 Denaturation at 94C :
During the denaturation, the double strand melts open to single
stranded DNA, all enzymatic reactions stop (for example: the extension
from a previous cycle).

34

2 Annealing at 54C :
The primers are jiggling around, caused by the Brownian motion. Ionic
bonds are constantly formed and broken between the single stranded
primer and the single stranded template. The more stable bonds last a
little bit longer (primers that fit exactly) and on that little piece of
double stranded DNA (template and primer), the polymerase can
attach and starts copying the template. Once there are a few bases
built in, the ionic bond is so strong between the template and the
primer, that it does not break anymore.

3 Extension at 72C :
This is the ideal working temperature for the polymerase. The primers,
where there are a few bases built in, already have a stronger ionic
attraction to the template than the forces breaking these attractions.
Primers that are on positions with no exact match, get loose again
(because of the higher temperature) and don't give an extension of the
fragment. The bases (complementary to the template) are coupled to
the primer on the 3' side (the polymerase adds dNTP's from 5' to 3',
reading

the

template

from

3'

complementary to the template).

35

to

5'

side,

bases

are

added

Figure 10: Polymerase Chain Reaction

Figure 11: The different steps in PCR

36

Because

both

strands

are

copied

during

PCR,

there

is

an exponential increase of the number of copies of the gene.

Suppose there is only one copy of the wanted gene before the cycling
starts, after one cycle, there will be 2 copies, after two cycles, there
will be 4 copies, three cycles will result in 8 copies and so on as shown
in figure 12.

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

Procedure for making 25l of PCR reaction:

Reagents

Volume

Water

18.3l

Buffer

2.5l

dNTPs

1l

Forward Primers

1l (20 pmol/l)

Reverse Primers

1l (20 pmol/l)

Taq Polymerase

0.2l

DNA sample

1l

37

Above content was mixed well gently by tapping. The amplification was
carried out in a thermo cycler for 30-35 cycles. After amplification,
amplified samples were analyzed using agarose gel electrophoresis.
Store the amplified samples were stored at freezing temperature for
further analysis.

Agarose Gel by Electrophoresis

Procedure:
Preparation of 1.5% Agarose gels:

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

conical flask.
The flask was kept in a microwave oven and boiled until the agarose dissolved.
After boiling 7l of ethidium bromide was added to the solution and was allowed

to cool, Poured into the gel-casting tray.

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

Sample Loading and Electrophoresis:

After solidification of the gel, the comb was removed and the gel was placed in
the electrophoresis chamber containing 1x TAE.

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

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

Samples were run at 75 volts for 30 minutes.

After 30 min DNA was visualized under gel documentation system.

38

4 RESULTS AND DISCUSSION

4.1 Biochemical Analysis:
4.1.1 Protein Standard graph
As demonstrated in Figure 13, protein concentration shows a direct
correlation with absorbance. The absorbance was determined for BSA
protein concentrations ranging from 0.0 to 1000 g/l. Over this range
the absorbance increased in a linear fashion, the standard curve was
generated. By this standard curve can be used to convert the
Absorbance readings for the experimental samples into a protein
amount or concentration.

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

for Standard Graph

BSA(l)

200

400

600

800

1000

OD(595nm)

0.039

0.13

0.216

0.30

0.39

0.51

39

Figure 13: Standard graph

4.1.2 Estimation of Total protein serum

analysis
We have taken 16 random serum samples of diabetic patients and normal group and
estimated the protein content in them by using Spectrophotometer with an absorbance at 595nm.
As a positive control we used Bovine Serum Albumin (BSA) method. The results of the present
study showed that the levels of serum total protein were significantly higher compared with the
normal group.

Figure 14: Total Protein in Serum

Histogram showing the levels of serum of total protein in normal and diabetic patients as
shown in figure 14.
Table 6: Spectrophotometer value of Normal serum sample of catalase
activity
Normal
Sample
01

Glucose
level
mg/dl
76.106

Protein
concentration
mg/ml
0.21
40

OD at 240
nm

Catalase
activity

0.3252

2181.08

02
03
04
05
06

52.21
89
80
86
85

0.19
0.16
0.09
0.15
0.15

0.1941
0.2041
0.1201
0.1577
0.1392

1438.34
1901.46
1879.49
1480.75
1307.04

07
08

89
82

0.16
0.16

0.2160
0.1235

1796.65
2099.47

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 eight different serum samples of both normal as well as
diabetic individuals with different glucose concentrations ranging from less than 90mg/dl
glucose value and more than 130mg/dl glucose value. The range less than 90mg/dl
glucose value will consider as normal individuals and more than 130mg/dl glucose value
will consider as Diabetes individuals. Catalase activity was determined as H2O2
consumption measured as the decrease in absorbance at 240 nm (Aebi et al., 1983). The
assay contained 50 mM KH2PO4/K2HPO4 (pH 7.0), 10 mM H2O2 in phosphate buffer.
Extinction coefficient of 39.4 mM-1cm-1 was used to calculate activity. Catalase activity
41

was expressed as (Unit). The Correlation of enzyme activity with Glucose concentration
was measured as shown in Table 6 & 7 as well as histogram result shown the catalase
activity with respect to glucose concentration as shown in graph 3.

figure 15: Catalase activity in normal and diabetic

4.2 Molecular analysis

4.2.1 Isolation of DNA from normal and diabetic blood samples:
After the above experiment we took eight normal blood samples in eppendorff tubes and
extracted DNA from them by following the given protocol and ran it on 1% agarose gel at

42

75v for 45 minutes. On gel documenting the gel we got nice band indicating the presence
of the DNA; as shown in the figure 16.
.

Figure 16: Isolated genomic DNA from normal blood samples

The following bands show the DNA of 8 normal samples which was performed
on 1% Agarose gel. Regarding the quality of the DNA extracted, no

differences in band patterns were observed in the agarose gel. Light

DNA bands found in normal sample 6&8 (N6&N8).
Similarly seven diabetic blood samples in eppendorf tubes were taken and DNA was
extracted from them by following the given protocol and ran it on 1% agarose gel at 75v
for 45 mintutes. On gel documenting the gel we got nice band indicating the presence of
the DNA; as shown in the figure 17.

43

Figure 17: Isolated genomic DNA diabetic blood samples

The following bands show the DNA of 7 diabetic samples which was also
performed on 1% Agarose gel . Regarding the quality of the DNA

extracted, no differences in band patterns were observed in the

agarose

gel,

but

remaing

samples

showed

some

degree

of

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

4.2.2 Quantification of DNA by Spectrophotometer:

After isolating the blood samples of both normal and diabetic we have
analyzed the DNA concentration by using the spectrophotometer. For
confirming the concentration and to check the purity of DNA, 1%
Agarose gel electrophoresis was performed and DNA analyzation was
done by UV Spectrophotometer at both 260nm and 280 nm. Finally, we
calculated the DNA concentration by using the formulae:
DNA Concentration (g/ml) = A260 x 50 x dilution factor (where A260 =
optical

density reading at 260 nm)

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

individuals are given below:

44

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
o
1
2
3
4
5
6
7

A260
0.367
0.304
0.281
0.290
0.261
0.344
0.384

A280
0.182
0.155
0.145
0.146
0.141
0.171
0.195

Purity

Conc

Conc

2.0
1.96
1.85
1.94
2.08
1.97
2.0

g/ml
55050
45600
42150
43500
39150
51600
57600

ng/ml
55.05
45.60
42.15
43.50
39.15
51.60
57.60

4.2.3 Identification of Catalase Gene by PCR

Amplification
In the present study, we have isolated DNA from blood of normal group
and diabetic patients and performed PCR reaction first a reaction mix is
prepared by adding all the contents required for amplification. It is
45

distributed in different PCR tubes and then Different DNA samples are
added accordingly in different PCR tubes, all these preparations are
done under laminar air flow chamber to avoid contamination.
Then temperature and all PCR conditions are adjusted in the PCR
machine and the PCR is kept for about 35 cycles. The cycles are done
on an automated cycler, a device which rapidly heats and cools the
test tubes containing the reaction mixture. During this process it
undergoes three major steps, they are- Denaturation (alteration of
structure), annealing (joining), and extension and they takes place at a
different temperature.
Within one cycle, a single segment of double-stranded DNA template is
amplified into two separate pieces of double-stranded DNA. These two
pieces are then available for amplification in the next cycle. As the
cycles are repeated, more and more copies are generated and the
number of copies of the template is increased exponentially.
Figure 18: Catalase Gene Mapping

46

+1Forward
primer

126 bp Fragment of
Catalase gene

Reverse
primer

+12
6

PCR amplification of normal samples of CAT gene:

Agarose gel electrophoresis was performed for above DNA product of
normal samples and quality of DNA was checked by performing PCR for
Catalase specific primers. The obtained product is Catalase gene which
is of 126bp. The expression of DNA in all normal samples indicates the
good quality of DNA. We have taken C- negative control, L- ladder is
about 1000 bp and N- normal individual as shomn in figure 19.

Figure 19: PCR product of normal individual

By this analysis of PCR with specific primers provided amplicons of the
expected size of 126bp as shown in a figure 19; six samples of normal
individual amplify with the specific primers and a negative control.

PCR amplification of Diabetes samples of CAT gene:

Similarly here also Agarose gel electrophoresis was performed for above DNA
product of diabetic samples and quality of DNA was checked by performing
47

PCR for Catalase specific primers. The obtained product is Catalase gene
which is of 126bp. The expression of DNA in all normal samples indicates the
good quality of DNA. Here we assumed that all the PCR material such as buffer,

dNTPs and Taq polymerase along with 1000bp DNA ladder shown positive results we
have taken C- negative control, L- ladder is about 1000 bp and DDiabetic individual as shown in figure 20(a) & (b).

Figure 20(a): PCR product of diabetic individual

48

Figure 20(b): PCR product of diabetic individual

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

expected size of 126bp as shown in a figure 20; six samples of diabetic
individual amplify with the specific primers and a negative control. No
results were found in diabetic sample1 (D1) as shown in figure 20(a).
Similarly we performed with some more diabetic individual the results
are shown in figure 20(b).

49

5 Conclusion
Diabetes is one of the pathological processes known to be related to an unbalanced
production of ROS, such as H2O2. Therefore, cells must be protected from this oxidative
injury by antioxidant enzymes. In this investigation, Catalase activity was measured in
serum samples of both normal as well as diabetic individuals with different glucose
concentrations ranging from less than 90mg/dl glucose value and more than 130mg/dl
glucose value. We found in our study increasing glucose level shows decrease Catalase
activity as compare to normal individuals.

50

The regulation of Catalase gene is complex and appears to occur through different
pathways. In this study, we have isolated the genomic DNA from normal as well as
Diabetes blood sample. PCR results of catalase gene on 1.5% agarose gel showed band at
126bp. In both samples we successfully amplified the catalase gene.
Therefore, finally we conclude that our finding of this Catalase gene may be used as
Biomarker for Type 2 Diabetes.

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Toronto Sydney Tokyo 1998.

Association of Polymorphic Markers of the Catalase and Superoxide

Dismutase Genes with Type 2 Diabetes Mellitus
To cite this article:
Maivel H. Ghattas and Dina M. Abo-Elmatty. DNA and Cell Biology. November 2012, 31(11): 15981603. doi:10.1089/dna.2012.1739.
Published in Volume: 31 Issue 11: October 25, 2012
Online Ahead of Print: September 12, 2012
Our study aims at determining whether genetic polymorphisms of catalase (CAT 1167C/T) and
superoxide dismutase (SOD +35 A/C) could be associated with type 2 diabetes mellitus (T2DM).
The study was conducted on 105 Egyptian patients with T2DM and 115 control subjects. Genotypes
were done by polymerase chain reaction-restriction fragment length polymorphism methods.
Homeostatic model assessment of insulin resistance (HOMA-IR),

CAT and SOD activities,

glycated hemoglobin, and insulin and lipid profiles were assessed. CAT
and SOD activities were significantly decreased in T2DM compared
with the control subjects. T allele of CAT and C allele of SOD1 were
significant risk factors for T2DM. No effects of CAT or SOD1 gene
polymorphisms on glycated haemoglobin or on HOMA-IR were found.
With regard to the enzymes activities, only +35 A/C of SOD1 was
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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.

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