Study of Some Biochemical Changes in Serum & Saliva PDF

Republic of Iraq
Ministry of Higher Education

& Scientific Research
University of Baghdad
College of Science
Department of Chemistry
Study of Some Biochemical

Changes in Serum & Saliva of
Patients with Oral Epithelial
Tumors
A Dissertation
Submitted to the college of Science University of Baghdad, in Partial
Fulfillment of the Requirements for Degree of Doctor of Philosophy in
Clinical Biochemistry
By
Rukzan Mahmood Daoud
B.Sc., M.Sc
Supervised by
Prof. Hathama Razooki Hasan
Ph. D.
2008 1429
Abstract
The present comprehensive study was designed to investigate the
changes in some biochemical parameters, in sera and saliva samples of
patient with oral tumor (benign & malignant), and to establish the
possibility of using saliva as a supplementary to serum for diagnosis and
epidemiological testing of oral cancer.
Thirty three patients with oral tumors (benign & malignant) aged
from 15 75 years attending the specialized surgery hospital in Baghdad
medical city, were included in the present study. Also thirty two healthy
individuals of matched age and gender were utilized as control.
The main measurements included :-
1- Determination of sera and saliva contents of total proteins and
then these proteins were analyzed using conventional
electrophoresis technique. Albumin and ceruloplasmin were also
determined in sera and saliva samples; meanwhile determination
of IgA, IgG and IgM was carried out on sera samples only.
2- Determination of the concentration of urea, uric acid, calcium,
and phosphorus in sera and saliva samples of the studied groups.
3- Determination the activity and specific activity of Alkaline
phosphatase, Lactate dehydrogenase, Amylase, Alkaline and
Acid RNase, Ceruloplasmin oxidase, Total superoxide dismutase
and Total peroxidase. As well as the concentration of related
trace elements such as Fe, Cu, Zn, and Mn was also measured.
4- Follow up the variation of these enzymes by using
polyacrylamide gel electrophoresis technique.
I
The observed results could be summarized as follows;-
For malignant tumor group, the following results were obtained:-
In Serum:-
-- Significant increase in comparison with the control at level (P<0.001):
in globulin concentration and alk. RNase avtivity. at Level (P<0.01):
in total protein, ceruloplasmin concentration, IgA concentration and
peroxidase activity.at level (P<0.05) in IgG concentration, uric acid,
alk. RNase specific activity ceruloplasmin oxidase activity, Zn and
Mn concentration.
-- Non significant increase in comparison with the control at Level
(P>0.05) in:- IgM concentration, urea, alkaline phosphatase activity &
specific activity, amylase activity&specific activity, acid RNase
activity&specific activity, superoxide dismutase activity
ceruloplasmin oxidase specific activity, LDH specific activity zinc
and copper concentration.
-- Significant decrease in comparison with the control at level (P<0.001)
in: albumin level, and at level (P<0.05) in [Fe]/peroxidase activity
and iron concentration. Wherease a non significant decrease was
shown in superoxide dismutase specific activity.
In Saliva:-
-- Significant increase in comparison with the control at level (P<0.001)
in peroxidase activity, at level (P<0.01) in total protein, urea, and
copper concentration, at level (P<0.05) in ceruloplasmin
concentration, peroxidase specific activity.
-- Non significant increase in comparison with the control at(P>0.05)
was shown in:- globulin concentration, calcium concentration, alkaline
phosphatase activity and specific activity, lactate dehydrogenase
specific activity, alkaline RNase activity, acid RNase activity and
II
specific activity, superoxide dismutase activity, ceruloplasmin oxidase
specific activity, and zinc concentration.
-- Significant decrease at level (P<0.001)in:- alpha amylase activity,
manganese concentration, at level (P<0.05) in uric acid concentration,
superoxide dismutase specific activity.
For benign tumor group, the following results were obtained:-
In Serum:
--Significant increase in comparison with the control at (P<0.001) in IgA
concentration, alk RNase activity, peroxidase activity, at level (P<0.05)
in: - globulin, alkaline phosphatase activity, amylase activity, lactate
dehydrogenase activity&specific activity, alkaline RNase specific
activity, and peroxidase activity.
--Non significant increase in comparison with the control at (P>0.05) was
shown in: - total protein, ceruloplasmin concentration, IgM, IgG, urea
and uric acid, calcium & phosphorus, acid RNase activity &specific
activity, CP oxidase activity &specific activity, superoxide dismutase
activity, peroxidase specific activity and copper, zinc and manganese
concentration.
--Significant decrease at level (P<0.01) in iron concentration.
In Saliva:
--Significant increase in comparison with the control at level (P<0.001) in
phosphorus, iron and manganese concentration, at level (P<0.01) in
globulin, at level (P<0.05) in albumin, ceruloplasmin concentration,
LDH activity, SOD specific activity.
-- Non significant increase in comparison with the control at level (P>0.05)
in total protein, urea, calcium, ceruloplasmin oxidase specific activity,
peroxidase activity &specific activity, alkaline phosphatase specific
activity, superoxide dismutase specific activity.
III
--Significant decrease at level (P<0.01) in uric acid, at (P<0.05) in amylase
activity &specific activity.
pI values for peroxidase enzyme were: 5.76, 6.95, 8.1 in serum and 7.4,
8.4 in saliva.
pI value for superoxide dismutase enzyme was 3.8-6.2 in serum and
saliva.
The results were revealed positive correlation between some parameters
in sera and saliva samples of the three studied groups.
IV
List of Tables
Table No. Title Page
1-1 Major function of saliva 7
2-1 The host information of the studied groups 41
2-2 Numbers and percentage of different age groups of 41
oral tumor patients
2-3 Standard conversion of end point ring diameters to 50
concentration IgM
concentration IgG
concentration IgA
3-1 Mean value of total protein in sera &saliva samples 101
of control and patient groups
3-2 Mean value of albumin in sera &saliva samples of 102
control and patient groups
3-3 Mean value of globulin in sera &saliva samples of 103
3-4 Mean value of ceruloplasmin conc. in sera &saliva 104
samples of control and patient groups
3-5 Mean value of immunoglobulins conc. in sera 105
3-6 Mean value of urea conc. in sera &saliva samples 118
3-7 Mean value of uric acid conc. in sera &saliva 119
3-8 Mean value of calcium and phosphorus in sera 120
3-9 Mean value of calcium and phosphorus in saliva 121
3-10 Mean value of ALP activity and specific activity in 126
sera of control and patient groups
3-11 Mean value of ALP activity and specific activity in 127
saliva of control and patient groups
3-12 Mean value of LDH activity and specific activity 128
in sera of control and patient groups
3-13 Mean value of LDH activity and specific activity 128
3-14 Mean value of amylase activity and specific 136
activity in sera of control and patient groups
IX
Table No. Title Page
3-15 Mean value of salivary amylase activity and 136
specific activity of control and patient groups
3-16 Mean value of alkaline RNase activity and specific 141
3-17 Mean value of alkaline RNase activity and specific 141
activity in saliva of control and patient groups
3-18 Mean value of acid RNase activity and specific 142
3-19 Mean value of acid RNase activity and specific 143
3-20 Mean value of sera CP activity and specific 154
activity in control and patient groups
3-21 Mean value of salivary CP activity and specific 154
3-22 Mean value of Cu conc./CP oxidase activity in sera 158
and saliva samples of control and patients
3-23 Mean value of sera total SOD activity and specific 163
3-24 Mean value of salivary total SOD activity and 163
specific activity in control and patient groups
3-25 Comparison between different ratios in sera of 166
3-26 Comparison between different ratios in saliva of 167
3-27 Mean value of sera total POD activity and specific 178
3-28 Mean value of salivary total POD activity and 178
specific activity in control and patient groups
3-29 Mean value of Fe conc./POD activity in sera of 180
3-30 Mean value of Fe conc./POD activity in saliva of 180
3-31 The overall results of the current study 191

3-32 Correlations between sera and saliva parameters 193
3-33 among the studied groups
X
CHAPTER ONE Introduction
1.1 The Mouth

The mouth, otherwise known as oral cavity, is a part of the digestive
tract (1). It is composed of cheeks, lips and palate which is the roof of the
mouth, the front portion is bony (hard palate) while the back portion is
muscular (soft palate) (2).
Figure (1-1): Structure of the mouth (5)
The mouth encloses the teeth and tongue, and opens outside
interiorly through the lips and posterioly through fauces. The oral cavity
is lined with moist stratified sequamous epithelium, which provides
protection against abrasion (1).
1
Digestive juice present in the mouth is known as saliva, which is

secreted by the salivary glands (1)
1.1.1 Salivary Glands
Salivary glands are composed of specialized epithelial cells, and
their structure can be divided into two specific regions: the acinar and the
ductal regions. The acinar region is where fluid is generated and most of
the protein synthesis and secretion takes place (4).
In human, the saliva is secreted by three pairs of major (large)
glands, and by some minor (small) glands situated in the oral and
pharyngeal membrane(3). The major glands are: parotid glands,
submaxillary glands, and sublingual glands(3).
a. Parotid Glands
Parotid glands are the largest of the saliva glands; they are situated at
the side of the face, just below and in front of the ear. Each gland, weighs
about 20 to 30 gm in adult & about 25% of the daily saliva secretion is
contributed by them.
Stensens duct
Parotid gland
Figure (1.2): Major salivary gland (3)

Sublingual
gland Whartons
duct
Submaxillary
gland
Figure (1-2): Major salivary glands (3)
2
b. Submaxillary Glands
Submaxillary glands are otherwise known as submandibular glands.
These are located in submaxillary triangle medial to mandible. Each
gland weighs 8 to 10 gm & about 70% of the daily secretion of saliva is
contributed by the submaxillary glands.
c. Sublingual Glands
These are the smallest of the three major salivary glands, and are
situated in mucosa at the floor of the mouth. Each gland weighs about 2
to 3 gm. It contributes only 5% of the saliva daily secretion.
On the other hand, the minor salivary glands include: Lingual mucus
glands, lingual serous glands, buccal glands, labial glands, and palatal
glands.
The salivary glands are classified into three types based on the type
of secretion (1).
I. Serous glands: This type of glands is made up of serous cells
Predominately. These glands secrete thin and watery saliva.
Parotid glands and lingual serous glands are serous glands.
II. Mucus glands: This type of glands is made up of mucus cells
mainly. They secret thick and viscous saliva with more mucin.
Lingual mucus glands, buccal glands and palatal glands belong to
this type.
III. Mixed glands: These are made up of both serous and mucus cells.
Submandibular, sublingual and labial glands are of this type.
3
1.1.2- Saliva
The saliva is a glandular secretion; it is not one of the popular bodily
fluids. It lacks the drama of blood, the sincerity of sweat and the
(6)
emotional appeal of tears . Saliva plays a critical role in the
maintenance of oral and dental health. Whole saliva represents a mixture
of oral fluids and includes secretions from both major and minor salivary
glands (90% of saliva is produced by the major salivary gland(3),
approximately 10% produced by minor glands glustered in the oral
mucosa) in addition to several constituents of non salivary origin , such
as gingival crevicular fluid ( GCF ), serum and blood derivatives from
oral wounds, bacteria and bacteria secretions, viruses and fungi,
desquamated epithelial cells, other cellular components and food
debris(7,8,9 ). Saliva can be collected with or without stimulation (10)
.
Stimulated saliva is collected by masticatory action (i.e., from a subject
chewing on paraffin wax) or by gustatory stimulation (i.e., application of
citric acid on the subjects tongue (11)
Unstimulated whole saliva is the mixture of the secretions which
enter the mouth in the absence of exogenous gustatory, masticatory, or
mechanical stimulation. The best two ways to collect whole saliva are the
draining method, in which saliva is allowed to drip off the lower lip, and
the spitting method, in which the subject expectorates saliva into a test
tube (10).
1.1.2.1 Saliva flow rate

Unstimulated saliva flow rates in healthy individuals are in the
(12)
range of 0.3-0.5 ml/ min . There is significant difference between
genders in unstimulated flow rates (13) (men have higher flow rates than
women). The factors affecting unstimulated saliva flow rates are degree
4
of hydration, body position, exposure to light, olfactory stimulation, and

seasonal and diurnal factors. Less important factors are age, body weight,
psychic effects, variety of disease state and several pharmacological
(12) (71)
agents . During sleep, flow from major glands virtually ceases .
Diminished salivary output can have deleterious effects on oral and
systemic health (14) . The reduction of the secretion of saliva is called hypo
salivation, it is of two types:
1. The temporary hypo salivation like in fear, fever dehydration.
2. The permanent hypo salivation, like in obstruction of salivary
ducts, paralysis of facial nerve, and in congenital absence of
hypoplasia of salivary glands. Xerostomia (dry mouth)is the
subjective feeling of oral dryness.
The excessive secretion of saliva is known as hyper salivation or
(sialorrhea), and it occurs in: Pregnancy, decay of tooth, neoplasm of
mouth or tongue due to continuous irritation, disease of stomach and
intestine, nausea and vomiting and some psychological conditions(4).
1.1.2.2- Buffering capacity of saliva

Salivary buffering capacity is important in maintaining a pH level
in saliva within the pH range of (6.5 - 7.2). The buffer capacity of
unstimulated and stimulated whole saliva involves three major buffer
(15)
systems . The most important buffering system in saliva is the
carbonic acid / bicarbonate system. The dynamics of this system is
complicated by the fact that it involves the gas carbon dioxide dissolved
in the saliva (16)
CO2 + H2O H2CO3 HCO3+ H+

The saliva bicarbonate increases the pH and buffer capacity of saliva,
especially during stimulation (15).
5
The second buffering system is the phosphate system, which contributes

to some extent to the buffer capacity at low flow rate. The mechanism for
the buffering action of inorganic phosphate ion HPO4-2 to bind a
hydrogen ion and form an H2PO4-1 ion.
The third buffering system is the protein system. It has been reported that
at the low range of pH, the buffering capacity of saliva is due to the
macromolecules (proteins) containing H- binding sites (16).
1.1.2.3- Composition of Saliva

Composition of saliva depends on various factors such as, the flow
rate, type of gland and diet. Saliva is 99.5% water and 0.5% solute(17)
Among the solute are: Inorganic ions such as, Na+, K+, Cl-, HCO3-, Ca2+,
Mg2+, HPO4-2, I-, SCN- and F-(18). Proteins such as enzymes, antibacterial
substances, albumin, blood clotting factors, B2-microglobulin and
immunoglobulins, amino acids, prolin-rich proteins. And also the saliva
contain small organic molecules such as cyclic AMP, and cyclic AMP-
binding proteins, urea, uric acid various lipids and corticosteroids (19).
There are several ways by which serum constituents that are not
part of the normal salivary constituents can reach saliva. Within the
salivary glands; transfer mechanisms include intracellular and
extracellular routes. The most common intracellular route is passive
diffusion, although active transport has also been reported.
Ultrafiltration, which occurs through the tight junctions between the cells,
is the most common extracellular route (20). Serum constituents are also
found in whole saliva as a result of GCF outflow. Depending on the
degree of inflammation in the gingival, GCF is either a serum transudate
or, more commonly, inflammatory exudates that contain serum
constituents (21).
6
1.1.2.4- Major Functions of Saliva
The major functions of saliva are summarized in Table (1-1)
Table (1-1): Major functions of saliva (22)
Mucins, proline-rich proteins and H2O components

of saliva provide physical protection against
Lubrication and fluid coating
mechanical, thermal, chemical irritation
Aids in speech and swallowing
The H2O component facilitates clearance of food and

Cleaning
swallowing and bathing the teeth.
Super saturation of calcium and phosphate due to the

presence of the salivary protein( statherin) facilitates
Remineralization remineralization of the teeth. Anionic proline-rich
proteins bind calcium and phosphate and bring them
to the apatite surface.
Bicarbonate and, to a lesser extent, phosphate and

macromolecules help neutralize plaque pH after
Buffering
meals, thereby reducing time available for
demineralization.
Specific (sIgA) and non specific (lactoferrin,

Antimicrobial action lysozyme and sailoperoxidase), help to control
antimicrobial mechanisms
Digestive enzymes found in saliva include amylase,

Digestion and taste protease, lipase and nuclease. Water of saliva
functions as a solvent to facilitate taste.
7
1.2-Oral Tumors
The mouth and throat, like all organs of the body are made up of
many kinds of cells. Cells normally divided in an orderly way to produce
more cells, only when the body needs them, this process helps keep the
body healthy. Cells that divided, when new cells are not needed, form too
much tissue, the mass of extra tissue is called tumor (23). This tumor can
be benign or malignant.
Benign tumors are not cancer, they can be removed and mainly do
not grow back and most important, the cells in benign tumors do not
invade other tissues and do not spread to other parts of the body (19).
Malignant tumors are cancer, they can invade and damage nearby
tissues and organs (24). Cancer cells can break away from a malignant
tumor and enter the blood stream or lymphatic system, by this way the
cancer spreads and forms secondary tumors in other parts of the body.
The spread of the cancer is called metastasis (25).
Tumor of the oral cavity refers to any swelling in the oral cavity and
it does not necessarily imply neoplastic proliferation (19), some common
precancerous lesions and a variety of benign and malignant tumors occur
in the oral soft tissues (23).
Benign tumors represent the following cases: fibroma,
haemangioma, lymphangioma, lipoma, papilloma.
Malignant tumors represent the following cases (19): Squamons cell
carcinoma, adenocarcinoma, basal cell carcinoma mucoepidermoid
carcinoma, keratoacanthoma and malignant melanoma.
1.2.1- Oral Cancer

Oral cancer is the 6th most common human cancer in the world, with
a high morbidity rate, and a 5-year mortality rate of about 50 %( 26). It has
8
been reported in recent years to have an increasing incidence in younger

people (27, 28).
Oral cancer is described as an ulcerated lump or sore that may or
may not be associated with pain, and often involving the regional lymph
(29)
nodes . The most common intraoral malignancy is Squamous Cell
Carcinoma (OSCC), which is arising from mucosal surface epithelium(30),
most of the remainder are adenocarcinomas of minor salivary glands,
only few are undifferentiated or metastasis(29).
Despite advances in surgery, radiation and chemotherapy, more than
50% of oral cancer patients die of their malignancy, due to delay in
diagnosis which allow tumor to invade deep to local structure and spread
to regional lymph nodes in the neck resulting in this high mortality(31).
1.2.1.1-Epidemiology
Epidemiological studies showed that the incidence of oral cancer
varies considerably between different parts of the world, with the highest
level in India, and the lowest in Western Europe and North America (32).
In south eastern part of Asia, oral cancer is significantly high, this is
attributed directly to the widespread habit of chewing betel quid (or paan)
(33)
and related areca nut use . In Iraq oral carcinoma accounts for
approximately 2.7% of all cancers (34).
1.2.1.2- Etiology
A. Predisposing Factors:
1. Age: Cancer can develop in any age, though it is most common in
those over 55 years of age (35).
9
2. Heredity: Heredity plays an important role in carcinogenesis;

certain precancerous are inherited (35).
3. Environmental Factors
a. Tobacco use in all its various forms (smoking, chewing and
Snuff dipping . . . etc.) account for 80-90% of oral cancer
causes (36, 37, 38).
b. Alcohol also increases the risk of oral cancer, even for people
who do not use tobacco (39).
c. Diet and malnutrition, food constituents, metabolic
consequences of eating, and the temporal changes in the
patterns of feeding habits (40). Nutritional deficiencies, in the
form of vitamin depleting especially vitamins (A, C, E), low b-
carotene intake (41), low intake of iron, selenium, and other trace
elements have been linked to increased risk of oral cancer (42,43)
4. Acquired Precancerous Disorders

a. Certain clinical conditions are associated with increased risk of
oral cancer (section 1.2.1.4).
b. Immune status.
Oral cancer patients may have immunologic deficiencies such as
overall lumphopenia and decreased T-cell numbers (44).
B. Carcinogenic Agents
Carcinogens that cause cancer can be divided into three main
groups:
Physical: Radiation energy, ionizing radiation and ultraviolet of
sunlight causes an increase risk of skin cancer (45). Solar irradiation
is a major risk factor for cancer of the lip (46). The vast majority of
10
lip cancers occur on the lower lip of many patients have outdoor
occupations where sun exposure is increased (46).
Chemicals: Variety of chemical compounds (35) can cause cancer,
some of these can act directly and others can act as
procarcinogens, e.g. polyaromatic hydrocarbon, azo dyes, various
drugs.
Biological: Oncogenic viruses, many of viruses causes tumors like
human papilloma virus(47) (HPV).
1.2.1.3- Sites of Oral Cancer(29)

The lower lip is the most frequent site of oral cancer overall. While
the tongue is the most frequently affected site within the mouth. Within
the oral cavity, the majority of cancers are concentrated in the lower part
of the mouth, particularly the lateral borders of the tongue, the adjacent
floor of the mouth and lingual aspect of the alveolar margin forming U-
shaped area extending back towards the oropharynx.
1.2.1.4- Early Detection of Oral Cancer and Warning Signs

Two lesions that could be precursor to oral cancer are leukoplakia
and erythroplakia(30):
-Leukoplakia: is a clinical term, defined as a white patch or plaque which
cannot be removed by rubbing or scraping and cannot be characterized
clinically or pathologically as any other disease (31) .
-Erythroplakia: the term is given for a bright-red velvety plaque, which
cannot be attributed to any other diagnosable disease (31).
Other possible signs and symptoms of oral cancer could be:
11
an ulcer that does not heal, a lump of thickening of the oral soft tissue,
white of red patch on the gums, tongue or lining of the mouth, difficulty
chewing or swallowing and difficulty moving the jaw or tongue.
1.2.1.5- Diagnosis of Oral Cancer (25)

A clinical diagnosis of oral cancer should always be confirmed by
one of the following procedures:
1. Surgical biopsy.
2. Fine needle aspiration biopsy (FNA).
3. Radiography.
4. Computerized tomography (CT scan).
1.2.1.6- Clinical Staging (48)

The stage of the disease depends on several factors, including the
size of the primary lesion, local extension, lymph node involvement and
evidence of distant metastasis. Tumor size, the organ or tissue affected,
and the extents of spread are considered to be the best indicators of the
patient prognosis.
The TNM system for describing the anatomic extent of disease is based
on the assessment of three components:
T= the extent of the primary tumor
N= the absence or presence and extent of regional lymph node metastasis
M= the absence or presence of distant metastasis
According to TNM, the disease stages ranged from 0 (which has the best
prognosis) to IV (the worst prognosis).
12
1.3- Free Radicals Injury

Radicals are compounds that contain a single electron, usually in an
outside orbital, the presence of the unpaired electrons alter the chemical
reactivity of an atom or molecule, usually make it more reactive than its
corresponding non-radical. The free radical is referred as oxidizing
agents(49). They have been implicated in over 100 human diseases
ranging from rheumatoid arthritis and hemorrhagic shock to AIDS (50).
The most important radicals in biological systems are radical derivatives
of oxygen like, super oxide anion ( O.2- ), hydroxyl radical (OH), nitric
oxide (NO.), the lipid-derived peroxy radical (ROO) and alkoxy radical
(RO). These radicals are called Reactive Oxygen Species (ROS) (51),
they involved also non-radical oxygen derivatives involved in oxygen
radical production. These are oxygen containing molecules that have a
higher reactivity than the ground state oxygen. They are capable of free
radical formation in the extra oral and the intra oral environments. The
ROS that formed by reduction of O2 are: O.2- , non-radical H2O2 and OH
and their formation can be explained by the following equation:-
e- e- + 2H+ e- + H+ e- + H+
O2 O.2- H2O2 . OH H2O
H2O
Figure (1-3): Reduction of oxygen by four-one electron steps (52)
ROS are oxygen-containing compounds that are highly reactive free

radicals, or compounds readily converted to these oxygen free radicals in
the cells. Pro-oxidant is a synonym for ROS, indicating a toxic substrate
that can cause oxidative damage to biological targets.
The hydroxyl radical (OH) is probably the most potent of the ROS,
because its life time, is extremely short (0.3 ns), it can thus be expected to
13
react at, or close, to its site of formation, and is actually the one able to
attack virtually all targets (53). Also (OH) can initiates chain reactions that
form lipid peroxides and organic radicals, and also it can be added
directly to compounds (53).
The superoxide anion ( O.2- ) is also highly reactive, but has limited
lipid solubility and can not diffuse far (54).
Hydrogen peroxide, although not actually a radical, is a weak
oxidizing agent that is classified as an (ROS). Because hydrogen
peroxide is lipid soluble, it can diffuse through membranes and generate
hydroxyl radical (OH) at localized Fe+2 or Cu+ -containing sites.
Transition metal such as iron or copper catalyzes formation of (OH) from
H2O2 through the Fenton reaction (55), or from ( O.2- ) and H2O2 through the
Haber-Weiss reaction catalyzed by iron or cupper:-
The Fenton reaction _

H2O2 OH + OH
2+ 3+
Fe Fe
The Haber-Weiss reaction
metal
O.2- + H2O2 O2 + OH. + OH-
metal= Fe+2 or Cu+
Figure (1-4): Generation of hydroxyl radical by the nonenzymatic

Fenton and Haber-Weiss reactions.
ROS are generated in vivo by multiple mechanisms, including the

respiratory redox chain in mitochondria, the respiratory burst of
phagocytes and the activity of various oxidases(56).
14
The formation of (ROS) under pathological conditions could be

produced during several mechanisms (57).
1. Ionizing radiation.
2. Inflammatory cells.
3. Activation of oxidases and oxygenases.
4. Decompartmentation of transition metal ions.
5. Loss of antioxidant capacity.
The pathology associated with ROS is derived from their ability to
modify cellular and extracellular macromolecules, such as DNA, proteins
lipids and carbohydrates. ROS induce DNA strand breaks as well as
oxidation of purine and pyrimidine bases, and increase the occurrence of
mutations (58). Lipid peroxidation is a radical-mediated chain reaction, the
peroxidative membrane lipids disrupts the bilayer arrangement and
increase membrane permeability, and modifies membrane-bound
proteins(59).
1.3.1- Antioxidants and Elimination of Free Radicals

ROS are constantly generated and eliminated in the biological
system, and play important roles in a variety of normal biochemical
(60)
functions and abnormal pathological processes . Growing evidence
suggests that cancer cells exhibit increased intrinsic ROS stress due in
part to oncogenic stimulation, increased metabolic activity, and
mitochondrial malfunction (60).
To protect the cells and organs of the body against reactive species,
human body has evolved a multiple defense system called (Antioxidants)
that are present not only in intracellular fluids but also in extra cellular
(61)
fluids . So free radicals created in human bodies are balanced by
cellular antioxidant defense system (62).
15
From a biochemical point of view, an antioxidant may be defined as

a substance that when present at low concentration (compared with those
of an oxidizable substrate) significantly prevents or delay a pro-oxidant
initiated oxidation of the substrate (63). Among the different classifications
of antioxidant defenses proposed, it appears that a functional
classification of antioxidants based on the way they act, could be more
useful. According to this suggestion, antioxidant defense systems in vivo
are mainly of the following kinds (59):
a. Preventative antioxidants: which prevent the formation of new
ROS, e.g. ceruloplasmin, albumin, myoglobin, ferritin, transferrin.
b. Scavenging antioxidants: which remove ROS once formed thus
preventing radical chain reaction. This includes:
Enzymatic Antioxidants: ( superoxid dismutase, catalase ,
glutathione peroxidase and glutathione reductase , ect.), and
Non- enzymatic entities e.g., glutathione, vit.E (- Tocopherol ),
vit.C ( Ascorbate ), b-carotene, uric acid and bilirubin(66).
c. Repair enzymes that repair the damage and reconstitute
membranes.
1.3.2- Oxidative Stress

There is a balance between oxidants and antioxidant, but when this
balance is in the favor of the former, the cell or organ is consider being in
a state of oxidative stress, in which the amount of free radicals exceeds
the bodys capacity to neutralize them (65). Oxidative stress results when
the production of ROS overrides the antioxidant capability of the target
cell (66).
In most cases, free radicals are secondary to the disease process, but
(67)
in some instances free radicals are causal . Some examples of
pathological conditions include:- heart disease, cancer, atherosclerosis,
16
rheumatoid arthritis, periodontal disease (68), cataract, diabetes mellitus,

inflammatory bowel disease, retinal ischemia, AIDS, and
neurodegenerative disease such as stroke, Parkinson's disease and
Alzheimer's disease (50).
1.3.3- Oxidative stress in the cancer process

An elevated oxidative status has been found in many types of
cancer cells, and the production of chemical and enzymological
antioxidant can inhibit tumor cell proliferation, pointing to a critical role
of ROS in mediating loss of growth control (69).
Oxidative stress has also been implicated in the cancer process(70), either
by increase in the formation of free radicals or a decrease in the
antioxidant defense mechanisms. Carcinogenesis is generally divided into
three stages:-
1. Initiation.
2. Promotion.
3. Progression.
Oxidative stress is interacts with all three stages of the cancer process (71):
Initiation stage: oxidative DNA damage may produce gene
mutations and structural alterations of the DNA, resulting in the
heritable mutation.
Promotion stage: ROS and oxidative stress can contribute to
abnormal gene expression, blockage of cell-to-cell communication and
modification of second messenger systems, resulting in an increase in
cell proliferation or a decrease in apoptosis in the initiated cell
population. This result in the clonal expansion of the initiated cells to
preneoplastic focal regions.
17
Progression stage: ROS impart further DNA alterations to the

initiated cell population. These changes may result changing in enzyme
activity and make the lesions resistant to normal growth control.
1.3.4- Pro- oxidant and Antioxidant features of saliva

Saliva could constitute a first line of defense against free radical-
mediated oxidative stress (72), since the process of mastication promotes a
variety of such reactions, including lipid peroxidation. Moreover, during
gingival inflammation, gingival crevicular fluid (GCF) flow increases,
adding to saliva products from the inflammatory response, this in turn
could have some role in controlling and / or modulating oxidative
damages in the oral cavity (72).
(73)
Saliva has pro-oxidant, and antioxidant properties . Physiologically
free radical/reactive oxygen species in the mouth are derived mainly from
(68)
polymorphonuclear neutrophils (PMN) . During evolution, various
defense mechanisms developed in the saliva aimed at combating
penetrating bacteria, viruses or fungi, and protecting against chemical or
medical attack. Moreover even after swelling saliva has a mucosal
(74)
protective capacity within the gastrointestinal tract . Immunologic
defense mechanism of saliva based on secretory immunoglobulin A
(sIgA), and the proteinenzymetic defense system. That, in turn is based
on the enzyme lysozyme and other components, such as histatin,
lactoferrin, prolin-rich protein, mucin, ect. Also the soft tissue integrity
defense system (74). Recently, the importance of an additional salivary
defense system has become clear; this is based on the antioxidant defense
system, which it is appears to lose efficiency with advanced age (75). The
salivary antioxidant system includes various molecules and enzymes, the
most important of which are the uric acid molecule and the peroxidase
18
enzyme, both of them water-soluble (76). Uric acid, the most important
antioxidant molecule in the saliva, contributes approximately 70% of the
total salivary antioxidant capacity, with the antioxidant role of the
ascorbic acid molecule being secondary (77). Traces of other antioxidants
(transferrin, lactoferrin and ceruloplasmin) capable of binding metal ions
found in both saliva and GCF. In fact, in healthy humans, these
compounds enable iron and copper to be safely bound: the former is
transported by the proteins transferrin and lactoferrin, and the latter is
inactivated mainly by binding to ceruloplasmin (72).
1.4- Tumor markers in cancer detection

Tumors may secret a wide range of substances into blood or other
body fluids, including hormones, enzymes, tumor antigens, nucleic acids,
polyamines and specific cell membrane and lipid (78). Therefore, effective
treatment planning would be enhanced by identification of prognostic
indicators that reflect the biologic behavior of a particular tumor in
relation to its host. In this respect, a variety of immunological,
biochemical and histological characteristic of head and neck cancer has
been done in an attempt to identify those features associated with
aggressive biological behavior which is suggested as tumor markers (79).
The evaluation of serum tumor markers in cancer patients may be a
valuable tool for the diagnosis, prognosis and treatment monitoring of the
(80)
disease . Ectopic hormone secretion; Squamous Cell Carcinoma
antigen (CA19-9 and CA-50); Carcino-Embryonic Antigen (CEA);
Tumor Necrosis Factor alpha (TNF-); Interleukins receptors; Trace
Elements; Enzymes and Immunoglobulins, are examples of serum tumor
markers(81,82) . On other hand, salivary analysis may aid in the early
detection of certain malignant tumors. Since most molecules found in
19
blood and urine has also been found in saliva but at lower
concentration(83). p53 is a tumor suppressor protein which is produced in
cells exposed to various types of DNA-damaging stress. Inactivation of
this suppressor through mutations and gene deletion is considered a
frequent occurrence in the development of human cancer (84). As a result,
accumulation of inactive p53 protein is observed, which in turn may lead
to the production of antibodies directed against this protein (85). These
antibodies can be detected in sera of patients with different types of
malignancies. p53 antibody can also be detected in the saliva of patients
diagnosed with oral Squamous Cell Carcinoma, and can thus assist in the
early detection of, and screening for, this tumor (86). Breast cancer antigen
(CA 15-3) and C-erbB-2 (erb) were detected as tumor marker in saliva of
breast cancer women, whereas, low levels of CA 15-3 were detected in
the saliva and serum of healthy individuals, erb was not detected in
healthy subjects and thus appears to hold greater promise for the early
screening and detection of breast cancer (87). Elevated salivary levels of
CA125 were detected in patient with epithelial ovarian cancer. A positive
(88)
correlation was found between salivary and serum levels of CA 125 .
Carcino-Embryonic Antigen (CEA) has also been detected as a tumor
marker in saliva (89).
1.5- The Diagnostic Uses of Saliva

Salivary diagnosis is an increasingly important field in dentistry,
immunology, clinical pathology, forensic medicine, psychology and
sports medicine(90). A growing number of drugs, hormones, and
antibodies can be reliably monitored in saliva, which is an easily
obtainable, painless, and non-invasive diagnostic medium (91).
20
It is becoming increasingly apparent to investigators and clinicians

in variety of disciplines that saliva has many diagnostic uses, in large
scale screening and epidemiologic studies. All steroids (e.g: Cortisol,
Testosterone and Progesterone ) of diagnostic significance in routine
clinical endocrinology can now be readily measured in saliva (92); multiple
specimens of saliva for steroid hormone analysis can be easily collected
by the patient, at home, to monitor fertility cycles, menopausal
fluctuations, stress and other diurnal variations (93). Also, some hormones
other than steroids have been found to be reflective of their plasma levels
and could be considered for salivary monitoring. Salivary antibody levels
can be determined to screen for infectious diseases (94). In several clinical
situations salivary analysis has provided valuable information, these
situations include digitals toxicity, affective disorders, stomatitis in
chemotherapy, specific secretory IgA deficiency, smoking, ovulation
time, relation of dietary factors to cancer and chronic pain syndromes(95)
Whole saliva however, is most frequently used for diagnosis of
systemic diseases, since it is readily collected and contains serum
constituents. These constituents are derived from the local vasculature of
the salivary glands and reach the oral cavity via the flow of gingival fluid.
An analysis of saliva may be useful for the diagnosis of hereditary
disorders(96), autoimmune diseases(97), malignant and infectious
diseases(98), viral disease (viral hepatitis)(99), and endocrine disorders(100),
as well as in the assessment of therapeutic levels of drugs and the
monitoring of illicit drug use(101).
21
1.6- The studying parameters

1.6.1- Proteins
Proteins are high molecular weight poly peptides, play a central
role in cell functions and structure, and they classified according to their
biologic functions (102). Alteration of plasma protein concentration is used
commonly in clinical practice as a nonspecific indicator of underlying
disease or monitor disease activity, since plasma proteins, including most
enzymes and tumor markers and they arise as a result of cell death or
tissue damage (103). Total protein in human plasma comprises the major
part of the solids of the plasma. The proteins in the plasma are actually a
complex mixture that includes not only simple protein, but also
conjugated proteins such as glycoproteins, lipoproteins and thousands of
antibodies (104).
Saliva also contains proteins, in concentration of approximately 3% of
plasma total protein level (17). The knowledge of body fluid proteins and
their alternations in health and disease has grown rapidly, some of the
alternations have a genetic origin, and many more reflect physiological or
pathological processes (105).
1.6.1.1- Albumin
Albumin is the most abundant serum protein, accounting for more
than 50% of all plasma proteins. Its molecular mass is approximately 66
KDa and the normal serum reference limits are 3.5-5.0 g/dl (106). Albumin
is synthesized exclusively in the liver at a rate of 100-200 mg/kg/day.
Factors that regulate albumin synthesis are nutrition, hormonal balance,
and osmotic pressure. The half life of albumin is approximately 15-20
days. About 4% is degraded per day, but synthesis can be increased by as
22
much as 100% by conditions that decrease serum albumin or lower

intravascular osmotic pressure (106).
Albumin is a significant component of most extravascular body
fluids, including CSF, interstitial fluids, and urine and during pregnancy
in amniotic fluid (107). The main biological functions of albumin include
regulation of plasma oncotic pressure, and nonspecific transport protein,
as it binds many non- polar compounds such as bilirubin, long chain fatty
acids and a number of drugs and it serves as a source of endogenous
(107)
amino acids . Albumin is an important component of plasma
antioxidant activity (61) primarily by binding free fatty acids, free ions,
hypochlorous acid (HOCI)(108). Because of its copper- binding ability,
albumin is a powerful inhibitor of copper dependent radical reactions (108).
In the oral cavity, albumin is regarded as a serum ultrafiltrate to the
mouth and it may also diffuse into the mucosal secretions (109). Salivary
albumin has been shown to increase in medically compromised patients
(110)
whose general condition gets worse . Immunosuppression,
radiotherapy, and diabetes are examples of states where high
(111)
concentrations of salivary albumin have been detected . Salivary
albumin levels have been used as a marker for the degree of mucosistis,
and inflammations in salivary glands(112). Butler et al. in their study (113)
found that albumin levels in whole saliva fluctuated in most of the elderly
patients. Albumin was regarded as one of the important salivary
antioxidant molecules (77).
1.6.1.2- Immunoglobulins
Immunoglobulins often called Antibodies are proteins synthesized in
plasma cells, as a response to the presence of foreign particles or
microorganisms. There are five major classes of immunoglobulins in the
human: IgA, IgG, IgM, IgD and IgE (114).
23
IgA: IgA forms 13% of the total body antibodies. It appears

selectively in the seromucous secretions such as saliva, tears, nasal
fluids, colostrums and secretions of the lung, genito- urinary and gastro-
intestinal tracts(103), IgA in fluids other than serum has an additional
secretary peptide, which enable it to appear in secretions(103). IgA
antibodies function by inhibiting the adherence of coated micro-
organisms to the surface of mucosal cells thereby preventing entry into
the body tissues. Increased serum IgA is found in liver disease, infections
and autoimmune disease.
Salivary IgA is an important first_ line of mucosal defense
principally by simple binding to soluble and particulate antigens (115). It
has been found that chewing stimulates epithelial cell transcytosis of IgA
and increases secretion of secretory IgA into saliva (116,117).
IgG: IgG forms 80% of the total body antibodies. It is the major
immunoglobulin to be synthesized after antigen stimulation. Through its
ability to cross the placenta it provides a major line of defense against
(115)
infection for the first few weeks of a baby's life . IgG diffuses more
readily than the other immunoglobulins into the extravascular body
spaces where as the predominant species it carries the major burden of
neutralizing bacterial toxins and of binding to micro-organisms to
enhance their phacocytosis(115). IgG increased in liver disease, infections
and collagen disease.
Salivary IgG reaches the oral cavity by leakage through various
epithelia and is mainly added to whole saliva via crevicular fluid. It is
mainly derived from serum. Salivary IgG can be expected to have the
same functional properties as circulating IgG(111).
24
IgM: IgM forms about 6%of the antibodies. It is referred to as the

macroglobulin antibodies because of their high molecular weight
(900,000 Da). IgM molecules are polymers of five four-peptide subunits
(pentamer) (115). IgM is the first antibody that appears in response to
(115)
antigenic stimulation . Increased IgM concentration is found in
toxoplasmosis, cytomegalovirus, rubella, herpes and various bacterial and
fungal diseases. Secretory IgM may function like sIgA in the first line of
mucosal defense (118).
IgD: is the immunoglobulin with the fourth highest concentration in

normal serum. Its concentration is increased in infections, liver disease,
and connective tissue disorders (103).
Traces of IgD probably reach whole saliva by passive diffusion like IgG .
IgD cannot be detected regularly in parotid fluid from normal adults but it
(119)
appears in whole saliva when present in serum .The functional
significance of salivary IgD is not known.
IgE: is the idiotypic immunoglobulin associated with allergic and

anaphylactic reactions. Its concentration is increased in allergies,
including asthma and hay fever (103).
Salivary IgE most likely reaches external secretions by passive
diffusion (120).
1.6.2- Enzymes
1.6.2.1- Superoxide Dismutase
Superoxide dismutase (SOD: E.C 1.15.1.1) is a family of
metaloisoenzymes that contain copper, zinc, manganese or iron, catalyses
25
the conversion of superoxide anion O.2- to hydrogen peroxide H2O2 and

oxygen O2 (121).
SOD
2 O.2- + 2H+ H2O2 + O2
Therefore, SOD is one of the antioxidant scavenging enzymes,

which is defense against (ROS). The conversion of O.2- to H2O2 and O2
(dismutation) by SOD is often called the primary defense against
oxidative stress (122).
SOD exists as three isoenzymes forms, copper-zinc superoxide
dismutase (CuZn-SOD) in cytosol, manganese superoxide dismutase
(Mn_SOD) in mitochondria and extracellular superoxide dismutase (EC-
SOD) (123).
(Cu-Zn SOD): This form of SOD is mainly located in the cytosol,
but it is also found in the nucleus of all cell types (124), the holoenzyme has
a molecule weight of 32 KDa, it consist of two identical subunits, each of
which contains one atom of copper and one atom of zinc, the former
serves as the redox center and the latter as a structural element, subunits
are bridged by cystine(125).
The enzyme is very sensitive to cyanide, which helps to distinguish
it from (Mn-SOD) which is relatively resistant (126).
(Mn-SOD): This form of SOD is located in mitochondrial matrix, it
is a homotetramer with a molecular weight of 22 KDa for each subunit,
having one manganese atom, and it constitutes approximately 10-15% of
total cellular SOD activity (127).
(Mn-SOD) dismute the superoxide anion that is produced in the
respiratory chain, so this enzyme seems to be necessary for electron
transport chain associated the oxidative metabolism to protect the normal
26
cells from reactive oxygen species produced during normal

metabolism(128). Therefore, because of its subcellular localization,
Mn-SOD is considered to be a first line of defense against oxidative
stress. Catalytic mechanism of this enzyme can be schematically
presented as follows (51):
k1 k2
Mn + O.2-
3+
[Mn3+- O.2- ] Mn2+ + O2 (1)
k-1
K3 k4
Mn + O.2-
2+
[Mn - O.2- ] Mn3+ + H2O
2+
(2)
k-3
It is accepted that superoxide dismutase carries out catalysis through
a redox process in which the metal cycles between its oxidized and
reduced states.
(EC-SOD): is a secretary homotetrameric CuZn-SOD containing
glycoprotein, with a molecular weight of 135 KDa, it composed of four
identical subunits and it contains one copper and one zinc atom per
subunit and both are required for enzymatic activity(129).
The enzyme has hydrophobic properties which may indicate an
affinity for cell membrane (130), also have a high affinity for heparin and
heparin sulfate (131) which is found on cell surfaces and particularly a
vessel endothelium. So it seems that (EC-SOD) is intended for
extracellular function and is partitioned between cell surface and the
(129)
extracellular fluids . This form of SOD was reported to have the
majority of the SOD activity of plasma, lymph and synovial fluid (132).
EC-SOD also occurs in tissue and in higher concentration than in plasma.
Superoxid Dismutase has been postulated to play a role in the
pathogenesis of a number of clinical disorders such as
ischemia/reperfusion injury, atherosclerosis, neurodegenerative disease,
allergy, hypertention and cancer (133).
27
1.6.2.2- Peroxidases
Peroxidase ( donor-H2O2 oxidoreductase, EC. 1.11.1.7) is a
hemoprotein catalyzing the oxidation by hydrogen peroxide of a number
of substrates, such as phenols, aromatic amines, hydroquinones and
hydroquinoid amines especially benzidine derivatives(134).
peroxidas
Substrate + H2O2 Oxidized substrate + 2H2O
Peroxidase, having a wide range of biological functions and present

in most cell organelles (Leucocytes, milk, liver, spleen, salivary glands,
stomach wall, intestinal mucosa etc), and take part in either
antibacterial actions or cellular defense against oxidative damage by
reactive oxygen species(135).
Myloperoxidase (MYO) appear to be uniquely localized in
mammalian neutrophils and monocytes(136). A major function of
neutrophils is to kill ingested bacteria (137); a function partially fulfilled by
myeloperoxidase. It is homogenous and composed of two subunits, and
have M.wt of 118-144 KDa. Myeloperoxidase is a chlorine-containing
enzyme in the azurophil granules of neutrophils and blood monocytes that
catalyzes the oxidation of chloride and reduction of H2O2 to form
hypochlorous acid (HOCl), a reactive oxygen species that can induce
peptide bond scission and the formation of low molecular weight
chloramines with bactericidal potential (138).
Lactoperoxidase (LPO) is mammalian secretary peroxidase consists
of a single polypeptide chain, have M.wt of 77-79 KDa(139).
Human salivary peroxidase "sailoperoxidase SPO" is secreted from
the major salivary glands, mainly the parotid gland (140), contributes 80%
of OPO activity. SPO resembles the lactoperoxidase because of its
28
immunological and biochemical similarity to bovine milk

lactoperoxidase(141).
Two peroxidases enzymes are found in saliva (often called oral
peroxidase OPO): salivary peroxidase from salivary glands, and
myeloperoxidase produced by leukocytes in inflammatory regions of the
oral cavity (142).
The human salivary peroxidase system ( oral peroxidase OPO)
catalyze a reaction involved in the inhibition of bacterial growth and
metabolism, and the prevention of hydrogen peroxide accumulation, thus
protecting proteins from the action of oxygen and reactive oxygen
species(143). More precisely, salivary peroxidase catalyses the oxidation of
thiocyanate ion to generate oxidation products that inhibit the growth and
metabolism of many micro organisms (144).
1.6.2.3- Ceruloplasmin oxidase

Ceruloplasmin (CP EC 1.16.3.1) is a copper-containing, a2-
glycoprotein, which has enzymatic activities (i.e. copper oxidase,
histaminase, and ferrous-oxidase). According to the literature, 90% or
more of total serum copper is associated with ceruloplasmin(103) and the
remaining 5-10% of copper is believed to be fairly loosely attached to
albumin and histidine and only a trace of copper is present as free
Cu+2(145). This high content of copper ions gives ceruloplasmin a beautiful
blue color (146).
Normally, Ceruloplasmin synthesized in the liver as a single
polypeptide chain (132 KDa) consisting of a 19 amino acid leader peptide
plus 1046 of amino acids residues, containing about six to seven copper
atoms (half as cuprous Cu+ and half as cupric Cu2+) ions are attached to
an apo-ceruloplasmin(103). CP is a multifunctional protein; it can help
transferrin to bind iron (147) which only binds the ferric form of the metal,
29
and also in copper transport role to the tissues which have separate
membrane receptors for CP and albumin-bound copper. Also
ceruloplasmin, the multifunctional copper containing enzyme, can
oxidizes iron from Fe2+ to Fe3+ throughout the ferroxidase activity, as
well as having oxidase activity toward numerous aromatic amines and
phenols(148), this oxidase activity increases during inflammation,
infection, and injury which suggest that CP possibly acts as antioxidants
and as an acute phase protein(149). The antioxidant protection of
ceruloplasmin drives mainly from its ability to oxidize highly toxic
ferrous iron to the relatively non toxic ferric form, and that helps in
preventing oxidative damage of proteins, lipids, and DNA (150).
Low levels of CP are found in Wilson's disease (107) while the elevated
levels were reported in several diseases, such as liver disease and
cancers(107) .
1.6.2.4- a-Amylase
Amylase (AMY EC 3.2.1.1) 1, 4-a-D glucanohydrolase. A group of
hydrolyases that split complex carbohydrates constituted of a-D-glucose
unit linked through carbon atom 1 and 4 located on adjacent glucose
residues. Both straight chain (linear) polyglucans such as amylose and
branched polyglucans such as amylopectine and glycogen are hydrolyzed,
although at different rates. The enzyme split the chains at alternate
a-1,4-hemiacetal (-C-O-C-) links forming maltose, glucose and a residue
of limit dextrins(151).
a-Amylase is a small heterogeneous enzyme, with a molecular
weight of 50-55 KDa, and it is exists in different isoenzyme forms,
salivary type (S-type) and pancreatic type (P-type).
30
Several different cells and tissues synthesize the salivary type

amylase, e.g. (salivary, lacrimal, sweat, lactating mammary) glands;
genital tissues, lung; bronchogenic and ovarian tumors; leukocytes and
(152)
thrombocytes cells , but the pancreatic type (P-type) amylase is
essentially tissue-specific, and semen is being the only other source (153).
-Amylase is an important enzyme in the physiologic digestion of
starches, and it requires calcium and chloride ions for its activity.
Salivary amylase activity is of short duration because, on swallowing is
inactivated by the acidity of the gastric contents (154). Pancreatic amylase
then performs the major digestive action on starch once the
polysaccharides reach the intestine. Amylase is small enough to pass
through the glomerull of the kidney, so it is the only plasma enzyme
normally found in urine (155).
It has been suggested that amylase accounts for 40-50% of the total
salivary gland-produced protein, most of the enzyme being synthesized in
the parotid gland (156). Human parotid saliva and submandibular saliva
contain about 45 mg and 30 mg of amylase, respectively, per 100 mg of
protein. It is generally considered to be a reliable marker of serous cell
function(157). Amylase also interacts specifically with certain oral bacteria
and may play a role in modulating the adhesion of those species to
teeth(158). It has been found that salivary amylase inhibits the growth of
Lgionella pneumophila and Neisseria gonorrhoeae(159). The amylase
concentration in radiation-induced hyposalivation has been found to be
reduced (157).
31
1.6.2.5- Alkaline Phosphatase

Alkaline phosphatase (ALP E.C 3.1.3.1) belongs to a group of
enzymes that catalyze the hydrolysis of various phosphomonoesters at an
alkaline medium.
ALP is a nonspecific enzyme, capable of reacting with many
different substrates. ALP function is to liberate inorganic phosphate from
an organic phosphate ester with the concomitant production of an alcohol.
The optimum pH for the reaction is 9.0-10.0, (ALP) requires Mg2+ as
an activator (160).
O O
_ ALP _
R O O P O + H2O ROH + HO P O
_ pH 9-10 _
O O
(ALP) is present in, practically, all tissues of the body, especially at

or in the cell membrane, and it occurs at particularly high levels in
intestinal epithelium, kidney tubules, bone, liver and placenta (161). The
raise of the enzyme level tends to be marked in extrahepatic obstruction
(stone or cancer tissue), and bone diseases associated with increased
(103)
osteoblastic activity like in Paget's disease .
Placental isoenzyme may appear in the sera of some patients with
malignant diseases. These carcino-placental or (Regan isoenzymes)
appear to result from the repression of the placental phosphatase gene(162).
Elevated levels of ALP are seen in primary and secondary liver
cancer; therefore, its levels may be helpful in evaluating metastatic cancer
with bone or liver involvement (163). ALP is also elevated in a variety of
malignancies, like lung, gastrointestinal and ovarian cancers and in
Hodgkin's disease (164).
32
1.6.2.6- Lactate Dehydrogenase

Lactate dehydrogenase (LDH, EC 1.1.1.27) is a tetrameric
hydrogen transfer enzyme, which plays a role during the anaerobic
glycolytic pathway. LDH catalyses the oxidation of L-Lactate to Pyruvate
in the presence of Nicotinamide Adenin Dinucleotide (NAD+) as

hydrogen acceptor(103). The reaction is reversible, and the equilibrium
strongly favors the reverse reaction, namely the reduction of pyruvate to
lactate.
L-Lactate + NAD+ pyruvate +NADH + H+
The enzyme has molecular weight of 134 KDa, and composed of four
subunits occur in two isoforms, designated H (for Heart) and M
(for Muscle)(165). It comprises five isoenzymes, which are numbered in
the order of the rapidity of their migration toward the anode of an
electrophoretic field. LD-1(H4); LD-2(H3M); LD-3(H2M2); LD4
(HM3); LD-5(M4). LD-1 moves most rapidly, whereas LD-5 is the
slowest. Marked elevation of the serum LDH activity may be observed in
the megaloplastic anemia. This elevation return to normal after
appropriate treatment. Moderate elevation occurs in myocardic infraction
and in cardiac failure with hepatic congestion, severe shock, and in
anorexia (165).
The elevation of LDH in malignancies is rather nonspecific, it has
been demonstrated in a variety of cancers including, liver, non-Hodgkin's
Lymphoma, acute leukemia, ovarian cancer, breast, colon, and lung
cancers(166,167,168). Salivary lactate dehydrogenase derived mainly from
the oral and oro pharyngeal mucosa (169).
33
1.6.2.7- Ribonucleases
Ribonucleases are best known for their ability to cleave
ribonucleic acid (RNA), which is the chemical material in the cell that
codes for different proteins (170). Different types of RNase have been
reported to be present in human, the most extensively studied one is the
bovine pancreatic ribonuclease (or RNase A; EC 3.1.27.5) because of its
ease of purification and small size (14 KDa). RNase A is the best known
member of a superfamily of secretory enzymes that operate at the
crossroads of transcription and translation by catalyzing RNA
degradation(171,172) . RNase A is secreted in large quantities by the bovine
pancreas, presumably to digest the large amount of RNA produced by
microbial residents of the rumen(173). RNase A catalyzes the cleavage of
the P-O bond of RNA on the 3 side of pyrimidine nucleosides (174). The
structure of RNase A is stabilized by four disulfide bonds that involve all
(175)
eight of its cysteine residues . Human ribonuclease is widely
(176)
distributed in various organs , such as pancreas and body fluids,
(177)
including serum, urine, saliva, and cerebrospinal fluid . RNase has
been considered as a possible tumor marker for some tumors e.g.
pancreatic tumor (178), and an elevation level of this enzyme reported in
sera of patient with benign and malignant uterine tumors (179).
1.6.3- Urea and Uric Acid

Non protein nitrogen compounds (NPN) are waste products
formed in the body as a result of the degredative metabolism of nucleic
acids, amino acids and proteins (180).
Urea is the (NPN) compound present in highest concentration in
blood; it is synthesized in the liver from CO2 and ammonia that arises
from the deamination of amino acids in the reaction of urea cycle. It is the
34
major excretory product of protein metabolism and the kidney is the only
significant route of excretion of urea. The produced urea is distributed
throughout total body water, and increase when more amino acids are
(103)
metabolized . This can occur with a high-protein diet, tissue
breakdown or decreased protein intake.
(181)
Urea is one the organic compositions of the saliva . Urea is
metabolized by the oral bacteria to ammonia and CO2 resulting in an
increase in the pH of the environment (182).
Uric acid has traditionally been considered merely an end product of
purine metabolism in human. It is insoluble and secreted in the urine as
sodium urate crystals. Other mammals have the enzyme uricase, which
coverts urate to more soluble allantoin as the end product. Uric acid is
formed primarily in the liver and then secreted by the kidney into the
urine (183). Plasma levels of uric acid are quite variable and are higher in
males than in females (184).
Uric acid acts as sacrificial antioxidant; it protects erythrocytes
against damage by singlet oxygen or t-butyl hydroxide (185). The powerful
antioxidant properties of uric acid have been long recognized, and as a
result of its comparatively high concentration, it is one of the most
abundant scavengers of free radicals in human (186).
Uric acid is the major component of the salivary antioxidant system
constituting the 70% of the total antioxidant capacity (140).
1.6.4- Calcium and Phosphorus

Almost 99 percent of the body's calcium is in the form of
hydroxyapatite (calcium phosphate with some hydroxyl groups), both
bone and teeth contain the mineral hydroxyapatite(103).
About half of the serum calcium is in ionized form and the rest is
bound to proteins (30% to albumin and 20% to -globulin). Only the
35
ionized form of calcium is biologically active, which can also complex

with anions such as citrate, phosphate and bicarbonate.
Calcium is essential to the integrity and function of cell membranes,
neuromuscular excitability, transmission of nerve impulses, multiple
enzymatic reactions, blood coagulation, and muscle contraction.
The primary factor in the regulation of extracellular calcium is
parathyroid hormone (PTH); it acts on the skeleton, small intestine and
kidney and interrelates with vitamin D and calcitonin to maintain the
extrtacellular calcium concentration within narrow limit (187). Maintenance
of calcium homeostasis depends on the balance between calcium intake
and calcium loss (188). This balance is achieved largely by the control of
calcium absorption, rather than by the regulation of calcium excretion(188).
About 80-85% of bodys phosphate are found in the bone, the
remaining 10-15% play an important biological roles in association with
other macromolecules. It is a component of structural molecules such as
nucleic acid, proteins (phosphoproteins) and lipids (phospholipids). It is
also a component of very important intracellular buffering system,
component of coenzymes (NAD+, NADH, FAD+, and FMN), and energy
transfer system (ATP, GTP, etc). Under physiological conditions, 85% of
filtered phosphates are reabsorbed by renal tubules. Calcium and
phosphorus metabolism is regulated reciprocally (114).
In saliva, phosphate system is one of the major buffering systems
which contribute to some extent to the buffer capacity at low flow rate(15).
1.6.5- Trace Elements

Trace elements have been extensively studied in recent years to
assess whether they have any modifying effects in the etiology of cancer.
It has become well established that many trace elements play
essential role in a number of biological processes (189). They are usually
36
associated with an enzyme (metalloenzyme) or protein (metalloprotein)

as an essential components or cofactor (190).
The action of a very small amount of trace elements is necessary for
optimal performance of the whole organism. The basis for the
amplification of trace element action is related to their interaction with
enzymes and hormones, which regulate the metabolism of much larger
amounts of biochemical substrates (191).
1.6.5.1 Iron
Iron is one of the most essential metal ions in human (192), most of
the iron in the body is in hemoglobin, a moderate amount of iron is in
myoglobin, a small amount, but extremely important Poole is in tissue
where iron is bound to several enzymes that require iron for full
activity(193). These include peroxidase(194) , catalase and cytochromase(193).
Iron is also stored as ferritin and hemosiderin, primarily in the bone
marrow, spleen and liver. This critical pool of iron may be the first to
become diminished in iron deficiency states (195). A small amount of iron
is found in plasma associated with transferrin and albumin (196).
Iron is a transition element capable of reduction-oxidation (redox)
activity, i.e. play a role as a pro-oxidant, since it can enhance production
of free radicals through Fenton & Haber-Weiss reactions (Figure 1-4) and
subsequent oxidative stress and cell damage (197). Therefore, iron had
(198)
been found to be involved in the carcinogenic process . Recently,
haemotological abnormalities in oral leukoplakia were reported (199), and
occurrence of iron deficiency is known to present in oral cancer (200).
37
1.6.5.2- Zinc
Next to iron, zinc is the most abundant trace element. The
biological functions of zinc can be divided into three categories: catalytic,
structural and regulatory. Zinc is a component of > 200 different
enzymes(201), such as alkaline phosphatase, lactate dehydrogenase
carbonate anhydrase, nucleoside phosphorlylase, cytosol and
EC-superoxide dismutase, RNA and DNA polymerases, and DNA
transcription factors (202). Zinc enzymes are essential for growth, wound
healing, integrity of connective tissue, reproductive function of the
(203)
immune system and protection from free radical damage .
Zinc deficiency is associated with poor growth, loss of appetite, skin
lesions, delayed wound healing, and impaired immune response and
malignancies (107). Zinc has been shown to reduce copper absorption (204).
Varghese et al. found a significant reduction in the serum copper and zinc
levels in oral submucous fibrosis and depressed in oral cancer (205).
1.6.5.3- Copper
Copper is the third most abundant trace element in the human
body, following zinc and iron (206). According to the literature, about 5%
of human plasma copper is bound to albumin or to amino acids such as
histidine, and the rest is bound to ceruloplasmin(207). Ceruloplasmin may
be able to supply copper within cells for incorporation into other copper
proteins like superoxide dismutase , cytochrom C oxidase, Lysyl oxidase,
Tyrosinase, Dopamine hydroxylase and Clotting factor V(208,209). This
copper donor role of ceruloplasmin is often referred to as a copper
transport function. Metallothioneins is the copper storage protein (210).
Copper levels are normally constant, increase in patients with acute
myocardial infraction, leukemia, solid tumors, infections and with
38
cirrhosis of the liver(211,212), also elevated levels were found in carcinoma

of breast, colon, ovary, gynecological and larynx(208,213).
High levels of copper in areca nut (a major etiological factor in oral
cancer) plays an initiating role in stimulation of fibrogenesis by up
regulation of lysyl oxidase and thereby causing inhibition of degradation
of collagen(214).
The copper/zinc ratio appears to be more decisively important than
either of the individual metals alone. Zinc deficiency often results in
elevated blood levels of copper, due to the dynamic competition of these
metals in the body (214).
1.6.5.4- Manganese
Manganese is an essential nutrient involved in the formation of bone
and amino acid, cholesterol and carbohydrate metabolism (215).
Manganese high concentration is neurotoxic, accumulating
particularly in the globus pallidus, while the clinical symptoms are typical
(215)
of Parkinsons disease . Both Mn2+and Mn3+ ions are present in
biologic systems largely as protein-bound ions. Manganese ion is an
activator of several enzymes, including arginase, pyruvate carboxylase,
(216)
and superoxide dismutase . Manganese is transported in plasma by
albumin, a2-macroglobuline, and transferrin and excreted in bile and
pancreatic secretions (216).
A possible relationship between low intake of manganese and
susceptibility to cancer has been proposed and relate it partly to the
decreased activity of manganese-SOD in tumor cells (217).
39
Aim of the study
The overall aim of the current study was to investigate various

aspects of sera and saliva diagnosis in healthy and oral tumor patients. An
attempt was made to assess these parameters as predictors for disease
occurrence and progression. Also the aim of the current study was to
shed further light on the role of salivary analysis in oral cancer diagnosis.
This diagnostic capacity is based on the ever continuous and intimate
contact between saliva and the mucosa (where this cancer evolves).
More specifically, the purposes were:
1. To investigate sera and saliva proteins alterations in oral tumor
(benign and malignant) patients. And follow such changes by
polyacrylamide gel electrophoresis ( PAGE ). The proteins under study
were (TP, Alb., Glob., and CP concentration ) in sera and saliva
samples, and ( IgA, IgM, IgG )in sera samples only.
2. To investigate some constituents in sera and saliva samples, like urea,
uric acid, calcium &phosphorus, and evaluate their levels in benign
and malignant oral tumors, and their relation to some enzymes.
3. To investigate the effect of oral tumor on the enzymatic activity profile
of some diagnostic enzymes and some hydrolytic enzymes like
alkaline phosphatase, lactate dehydrogenase, amylase, and
ribonucleases (alkaline and acid). Conventional PAGE was carrying
out to evaluate the changes in these enzymatic profiles.
4. To investigate the effect of the oral tumor on the oxidant/antioxidant
status, through measuring the activity of some antioxidant enzymes
like ceruloplasmin oxidase, total superoxide dismutase, and total
peroxidase and their related trace elements. Also distinguish the
variation in their isoenzymes on polyacrylamide gel electrophoresis.
40
Chapter Two Material &Methods
2.1 Subjects, Samples, Chemicals, & Instruments

2.1.1 Subjects
Sixty five individuals were included in the present study; thirty three
of them were clinically and histologically diagnosed as patients with oral
epithelial tumors (benign or malignant). The samples were collected
during the period April-2005 till January-2006 from the hospital of
Specialized Surgery in Baghdad Medical City. Patients were evaluated by
full medical history to exclude any existing of systemic disease or taking
any drug that may affect the parameters to be examined. They were
compared to a control group of thirty two healthy individuals who
matched in age and gender.
The information of all patients and control subjects is summarized
in Table (2-1), while the percentage of different age groups of oral
epithelial tumor patients is summarized in Table (2-2).
Table (2-1): The host information of the studied groups
Age (year)
Group No. gender Samples used
MeanSD
64
17 males
Control 35.6611.46 32 32 blood, 32
15 females
saliva
28
6 males
Benign 35.7815.63 14 14 blood, 14
8 females
saliva
38
10 males
46.5715.21 19 19 blood, 19
Malignant 9 females
saliva
41
Table (2-2): Numbers and percentage of different age groups for oral
tumor patients
Age group 15-40 > 40
(years)
group No. % No. %
Benign 10 71.42 4 28.57
Malignant 7 36.84 12 63.15
2.1.2 Samples
2.1.2.1 Blood Samples
Six milliliters of venous blood were taken without using tornique
from each individual, collected in plane polyethylene tube, allowed to
stand at room temperature for thirty minutes, then the sample was
centrifuged at (2000xg ) for 10 minutes, the obtained serum transferred
immediately to another test tube. These samples were estimated directly
for enzymes activities or frozen at 20 C for subsequent analysis.
Hemolyzed samples were discarded.
2.1.2.2 Saliva Samples

unstimulated (resting) whole saliva was collected after the diagnosis,
under resting conditions between 8.0-10.0 A.M. Patients were asked to
rinse their mouth with water and to generate saliva in their mouth and to
spit into a wide test tube(10). The collection period was twenty minutes.
Following the collection, the saliva was centrifuged at (2000 xg) for 10
minutes. The resulting supernatant was stored at 20 C in polyethylene
tubes until assayed.
42
2.1.3- Chemicals
Common laboratory chemicals and reagents (analar grade) were
used; they were obtained from the following companies:
-BDH Company
Ammonium sulphate, ammonium persulphate, acetic acid, copper
sulphate-pentahydrate, dipotassium hydrogen orthophosphate-trihydrate,
ethanol, ethylene diamine tetra acetic acid disodium salt dehydrate,
glacial acetic acid, hydrochloric acid, orthophosphoric acid, folin
ciocalutes phenol reagent, N,N,N`,N`-tetramethyl-ethylene diamine,
phenol, potassium dihydrogen orthophosphate, potassium cyanide
periodic acid, sodium hydroxide, sodium-potassium tartarate, sodium
metabisulphate, sodium carbonate, sodium cyanide, sodium acetate,
Triton X-100, glycine, dipotassium hydrogen orthophosphate, glycerol,
sulphanilic acid, silver nitrate, formaldehyde, hydrogen peroxide,
acrylamide, acetone.
-Bio Merieux
Albumin kit, Urea kit, Uric acid kit, Ca kit, and P kit
-Biomaghreb
Immunoglobulin (IgA, IgM, IgG ) kit.
-Chem supply company
Trichloroacetic acid
-Fluka Company
Nitroblue tetrazolium, 4-aminoantipyrine, tolidine blue, 1,4-
phenylenediamine-dihydrochloride, 3,3`,5,5`-tetramethyl benzidin,
sodium phenyl phosphate, sodium chloride, beta-alanin
-Hopkin & Williams company

Bromophenol blue, basicfuchsine,potassium ferrocyanide ,methylene
green.
43
-LKB-Switzerland
Ampholine pH (3.5-10)
-Merck Company
Coomassie brilliant blue G-250 and R-250, L-Methionine.
-Sigma Company
Bovine serum albumin, , sephadex G-25.
-Randox
LDH kit
-Riedel-de Haen Company

Riboflavin-5-phosphate, darco charcoal
2.1.4- Instruments
The following list includes the instruments, which were used
throughout this work:
pH-meter (pH 521 WTW). Germany
Electronic analytical balance (Sartorius BL 210S).
Bench centrifuge (MSE). England
Vortex (Hook and Tucker). Germany
Water bath (Memmert). England
Spectrophotometer (LKB Ultrospeck 4053).
Orbital shaker (lab- lin orbit environ shaker).
Electrophoresis instrument (power supply 2197, multiphor 2117
and multitemp 2209) from LKB.
Magnetic stirrer (Baind and Tatlock). Germany
Atomic absorption Spectrophotometer GBC 933 plus.
High vacuum pump.
44
2.2- Methods
2.2.1- Determination of Total Protein Concentration
The total protein concentration of all samples (serum and saliva) was
(218)
determined using Hartree method (modified Lowry method), and
bovine serum albumin (BSA) as standard. Protein concentrations of
serum and saliva were expressed in g/dl.
Reagents
1. Solution A
A weight of 2 gm potassium-sodium tartarate and 100 gm sodium
carbonate were dissolved in 500 ml (1N) NaOH and diluted with distilled
water to 1 liter.
2. Solution B
A weight of 2 gm potassium-sodium tartarate and 1 gm copper
sulphate pentahydrate were dissolved in 90 ml distilled water, and then 10
ml of (1N) sodium hydroxide was added.
3. Solution C
One ml of folin-cioealtue reagent was diluted with 15 ml of distilled
water. This solution was prepared daily.
4. Standard protein
The standard protein (BSA) was prepared as follows:
a. A stock solution of 1 mg/1 ml was prepared by dissolving 100 mg
of BSA in 100 ml distilled water.
b. From the stock solution, the following concentrations were
prepared by serial dilution with distilled water (20, 40, 60, 80, 100,
120, 140 mg/ml)
Procedure
A- Standard curve preparation
1-Different volumes (0, 10, 20, 40, 60, 80, 100, 120, 140) l of
45
standard BSA (1mg/ml) were pipetted into a set of test tubes, the
volumes were made up to 1 ml with distilled water to give a final
Concentration of (0, 10, 20, 40, 60, 80, 100, 120, 140) g/ml of
protein.
2- A volume of 0.9 ml of solution A was added to 1 ml of standard
protein, and then the tubes were incubated in a water bath at 50 C
for 10 minutes, and then cooled to room temperature. A volume of
0.1 ml of solution B was added, and the tubes were left at room
temperature for 10 minutes.
3- A volume of 3 ml of solution C was added in rapidly and shaken
vigorously (prefer vortex), then the tubes were left in water bath at
50 C for 10 minutes, then cooled to room temperature, and the
absorbance was read at wave length = 650 nm using 1 cm cuvette.
4- The standard curve of protein was obtained by plotting the
absorbance of the protein standard solutions against their
concentrations using the zero concentration of protein as a blank.
The equation of the straight line obtained from this standard curve
was used to determine the unknown protein concentration .
Absorbance (=650 nm)
Conc. g/ml
Figure (2-1 ): Standard curve for protein determination
46
B- Serum & Saliva Total Protein Determination

Total serum protein was measured in 1 ml diluted samples
(1:1000 for serum & 1:40 for saliva) with distilled water. These samples
were treated using the same steps in section A (steps 2-4). The protein
concentration was calculated from the equation that derived from the
standard curve.
2.2.2- Determination of Albumin

Serum and saliva albumin was determined by dye-bindin
method(219) using kit manufactured by bioMerieux.
Principle
The measurement of albumin is based on its quantitative binding
at pH 4.2 with bromocresol green (BCG) to form a blue-green complex
which its absorbance was measured at wave length = 628 nm.
Albumin + BCG (Albumin-BCG) complex
Reagents
1- Reagent 1 (standard) consisted of:
Bovine albumin (50 g/l)
Sodium merthiolate (0.1 g/l)
2- Reagent 2 (color reagent) consisted of:
Bromocresol green (0.23 mmol/l)
Succinate buffer (pH4.2) (75 mmol/l)
3- Brij 35 (2.1 g/l)
4- Sod. Merthiolate (0.1 g/l)
47
Procedure
1- To three test tubes the following were pipetted:
Blank Standard Sample
Standard - 10 ml -
Sample - - 10 ml
Color reagent 2.51 ml 2.5 ml 2.5 ml
2- Solutions in the blank, standard and sample tubes were mixed, and
incubated in a water bath at (20-25) C for 5 minutes.
3- The absorbance of the sample and the standard was measured at
= 628 nm against the blank.
Calculations
The albumin concentration of each sample was calculated, using the
following equation:
A
Albumin concentration (g/dl) = concentration of standard
S
A = absorbance of the sample
S = absorbance of the standard
2.2.3- Determination of Globulin

The concentration of globulin in serum & saliva samples of control
and oral tumor patient groups was calculated, using the following
equation:
Cglob (gldl.) = CTP Calb
Cglob. = concentration of globulin
CTP = concentration of Total Protein
Calb. = concentration of albumin
48
2.2.4- Determination of Immunoglobulins

The serum immunoglobulines were determined using Radial
Immuno Diffusion plates (RID) of Mancini(220), for quantitative
determination of these proteins in serum and other biological fluids.
Principle
This technique involves immuno precipitation in agarose, between
an antigen and its homologous antibody. It was performed by
incorporating one of the two immune reactant (usually antibody)
uniformly throughout a layer of agarose gel, and then the other reactant
(usually antigen) was introduced into wells punched in the gel. Antigen
diffused radially out of the well into the surrounding gel-antibody
mixture, and the visible ring of precipitation was formed where the
antigen and antibody had reacted.
A quantitative relationship does exist between ring diameter and
antigen concentration, while the precipitate is expanding, the ratio
between ring diameter and antigen concentration logarithm is
approximately linear.
Procedure
1. The plate was opened and let to stay for five minutes at room
temperature to allow any possible condensation to evaporate.
2. The well was filled with (5 ml) of serum, and then the plate was closed
tightly.
3. The plate was allowed to stay flat at room temperature, until the
precipitation ring reach the maximal possible size, which often
required 48-72 hours of diffusion. The end point of diffusion was
indicated by a sharp precipitation ring at the end of incubation time.
The diameter of the ring was measured by a lens, and then the results
49
were calculated using the reference Tables (2-3), (2-4), (2-5) included
in the leaflet of the kit:-
Table (2-3): Standard conversion of endpoint ring diameters to
Concentration IgM
D(mm) mg/dl D(mm) mg/dl
3.6 12.3 6.4 143.1

3.7 15.7 6.5 149.1
3.8 19.2 6.6 155.2
3.9 22.8 6.7 161.4
4.0 26.5 6.8 167.7
4.1 30.3 6.9 174.1
4.2 34.2 7.0 180.6
4.3 38.1 7.1 187.2
4.4 42.2 7.2 193.9
4.5 46.4 7.3 200.7
4.6 50.6 7.4 207.5
4.7 55.0 7.5 214.5
4.8 59.4 7.6 221.6
4.9 63.9 7.7 228.7
5.0 68.6 7.8 235.9
5.1 73.3 7.9 243.3
5.2 78.1 8.0 250.7
5.3 83.0 8.1 258.2
5.4 88.0 8.2 265.8
5.5 93.1 8.3 273.5
5.6 98.3 8.4 281.3
5.7 103.5 8.5 289.2
5.8 108.9 8.6 297.2
5.9 114.4 8.7 305.3
6.0 119.9 8.8 313.5
6.1 125.6 8.9 321.7
6.2 131.3 9.0 330.1
6.3 137.1 9.1 338.5
50

Concentration IgG
3.6 125.2 6.4 1360.6

3.7 157.4 6.5 1417.5
3.8 190.5 6.6 1475.3
3.9 224.5 6.7 1533.9
4.0 259.4 6.8 1593.5
4.1 295.1 6.9 1653.9
4.2 331.7 7.0 1715.3
4.3 369.2 7.1 1777.5
4.4 407.6 7.2 1840.6
4.5 446.9 7.3 1904.5
4.6 487.0 7.4 1969.4
4.7 528.0 7.5 2035.1
4.8 570.0 7.6 2101.8
4.9 612.8 7.7 2169.3
5.0 656.4 7.8 2237.6
5.1 701.0 7.9 2306.9
5.2 746.4 8.0 2377.1
5.3 792.8 8.1 2448.1
5.4 840.0 8.2 2520.0
5.5 888.0 8.3 2592.8
5.6 937.0 8.4 2666.5
5.7 986.9 8.5 2741.0
5.8 1037.6 8.6 2816.5
5.9 1089.2 8.7 2892.8
6.0 1141.7 8.8 2970.0
6.1 1195.1 8.9 3048.1
6.2 1249.4 9.0 3127.1
6.3 1304.5 9.1 3206.9
51

Concentration IgA
3.6 21.7 6.4 264.1

3.7 28.0 6.5 275.3
3.8 34.5 6.6 286.6
3.9 41.2 6.7 298.1
4.0 48.0 6.8 309.8
4.1 55.0 6.9 321.7
4.2 62.2 7.0 333.7
4.3 69.6 7.1 346.0
4.4 77.1 7.2 358.3
4.5 84.8 7.3 370.9
4.6 92.7 7.4 383.6
4.7 100.7 7.5 396.5
4.8 109.0 7.6 409.6
4.9 117.4 7.7 422.8
5.0 125.9 7.8 436.3
5.1 134.7 7.9 449.9
5.2 143.6 8.0 463.6
5.3 152.7 8.1 477.5
5.4 161.9 8.2 491.7
5.5 171.4 8.3 506.0
5.6 181.0 8.4 520.4
5.7 190.8 8.5 535.0
5.8 200.7 8.6 549.8
5.9 210.9 8.7 564.8
6.0 221.2 8.8 580.0
6.1 231.6 8.9 595.3
6.2 242.3 9.0 610.8
6.3 253.1 9.1 626.5
52
2.2.5- Determination of Urea

The concentration of urea in serum, saliva samples was determined
by an enzymatic method (221,222) using kit manufactured by bioMeriux.
Principle
The enzymatic determination of urea was carried out according to
the following reaction:
urease
Urea + H2O 2NH3 + CO2
Where ammonium ions in an alkaline medium react with the salicylate
and hydrochlorite to form a green colored indophenol (2, 2-dicarboxyl-
indophenol which absorbance was measured at wave length =580 nm.
Reagents
1- Reagent 1 consisted of:
Standard urea (0.5 g/dl)
2- Reagent 2 consisted of:
Enzyme urease ( 5000 u/l)
Phosphate buffer pH 8.0 (40 mmol/l)
Sod. Salicylat (52 mmol/l)
Sod. Nitroprusside (2.83 mmol/l)
Alkaline EDTA (1 mmol/l)
4- Reagent 4 (alkaline reagent) consisted of:
Sodium carbonate (83 mmol/l)
Sodium hypochlorite (3.75mm)
53
Procedure
1- To these test tubes, the following were pipetted :
Standard - 10 ml -
Sample - - 10 ml
Working solution
(enzyme + color reagent)
1 ml 1 ml 1 ml
2- All tubes were shaked carefully, and incubated for at least 3

min at 37 C .
3- A volume of 200 ml of alkaline reagent was added to all tubes.
4- The tubes were shaked and incubated for at least 5 min at
37C.
5- The absorbance of the sample and standard was measured against
blank at 580 nm.
Calculations
A
Urea concentration (mg/dl) = concentration of standard
S
2.2.6- Determination of Uric Acid

Serum and saliva Uric acid concentration was measured (223) using
colorimetric bioMeriux Kit
54
Principle
Uric acid is oxidized by uricase to allantoine and hydrogen peroxide.
In the presence of peroxidase, a mixture of 3, 5-dichloro-2-hydroxy
benzene and 4-aminoantipyrine was oxidized by hydrogen peroxide to
form quinoneimine dye.
Uric acid + 2H2O + O2 allantoin + 2H2O2 + CO2
peroxidase
H2O2 + (3,5-dichloro-2-hydroxy benzene) + (4-aminoantipyrine)
quinoneimin + HCl + 2H2O
The intensity of the colored compound (quinoneimine) is

proportional to the concentration of uric acid in the sample.
Reagents
1- Reagent 1: (standard) was consisted of
Uric acid (80 mg/l)
2- Reagent 2: (chromogen buffer) consisted of:
Tris buffer pH 8.0 (50mmol/l)
3, 5-dichloro-2-hydroxy benzene sulfunic acid (2 mmol/l )
3- Reagent 3: Enzymes (lyophilized)
Uricase (80 u/l)
Peroxidase ( 200 u/l)
Ascorbate oxidase ( 1000 u/l)
4- Aminoantipyrine (0.25 mmol/l)
5- Potassium ferricyanide (0.03 mmol/l)
Procedure
1-The contents of reagent 3 were reconstituted with 25 ml of reagent 2
prior to use.
2-To the test tubes the following were pipetted:
55
Distilled water 20 ml - -
Standard - 20 ml -
Sample - - 20 ml
Reconstituted reagent 3 1 ml 1 ml 1 ml
3- The tubes were mixed and incubated at 37 C for 5 minutes.

4- The absorbance of the sample and standard was measured against
blank at = 520 nm.
Calculation
A
Concentration of uric acid (mg/dl) = concentration of standard
S

2.2.7- Determination of calcium

Colorimetric determination of total calcium concentration in
serum& saliva samples without deproteinization(224), was carried out by
using bioMeriux kit.
Principle
The calcium ions react with methyl thymol blue indicator (MTB) in
an alkaline medium.
Ca2+ + MTB Ca-MTB complex
56
The color intensity of the Ca-MTB complex was measured at = 612 nm,
which is proportional to the quantity of calcium present in the sample. To
eliminate the interference of magnesium & protein, 8-hydroxy quinoline
and polyvinyl pyrrolidone (PVP) were used respectively.
Reagents
1- Reagent 1
Calcium standard (Ca+2) 25mmol/L (10mg/dl).
2- Reagent 2 (color reagent) which contained:
Methyl thymol blue (0.092 mmol/l)
P-hydroxy quinoline (11 mmol/l)
PVP (3 g/l)
3- Reagent 3 (alkaline reagent) which contained:
Reagent pH > 11, and monoethanolamine(MEA) ( 200ml/L )
Procedure
1- The following solution were pipetted into three test tubes:
Standard - 10 ml -
Sample - - 10 ml
Color reagent 0.5 ml 0.5 ml 0.5 ml
Alkaline reagent 0.5 ml 0.5 ml 0.5 ml
2- All the tubes were mixed well, and after one minute the absorbance
was read at = 612 nm against blank.
57
Calculations
Sample Ca+2 concentration (mg/dl) =
Absorbance of sample
Conc. Of the standard
Absorbance of s tan dard
2.2.8- Determination of Inorganic Phosphate
The inorganic phosphate concentration was determined(225, 226)
using colorimetric bioMeriux kit.
Principle
The inorganic phosphate was determined without deproteinization,
using a single reagent, which formed a phospho-molybdate complex in
the presence of a reducing reagent (ferrous sulphate).
Reagents
1- Reagent 1 (standard) consisted of:
Phosphorous (1.61mmol/L) (5 mg/dL)
Sodium azide (1 g/l)
2- Reagent 2 (reducing agent) consisted of:
Sulfuric acid (1.06 N)
Ferrous ammonium sulphate (100 g/l)
Ferrous nitrate (2 g/l)
Sulfuric acid (1.06 N)
Ammonium hepta-molybdate (4.5 g/l)
4- Working solution, consisted of:
One volume of reducing agent was mixed with one volume of
color reagent.
58
Procedure
1- The following solutions were pipetted into three test tubes:
Sample - - 100 ml
Standard - 100 ml -
Distilled water 100 ml - -
Working solution 2.5 ml 2.5 ml 2.5 ml
2- The tubes were mixed well, and left to stand for 10 minutes.
3- The absorbance of sample and standard was measured against
blank at 690 nm.
Calculations
Absorbance of sample
Inorganic phosphate conc. (mg/dl) = conc. of the standard
Absorbance of s tan dard
2.2.9- Determination of the Trace Elements

The most popular technique for the determination of the trace
elements in biological specimens is the Atomic Absorption
Spectrophotometry (AAS). The AAS technique detects only one element
at a time. Elemental determination is quick and accurate once the sample
is in the proper form, besides offering good specificity and sensitivity (227).
Iron (Fe), copper (Cu), Zinc (Zn), manganese (Mn) have been
reported to relate to some enzymes activities which included in the
present study and also they play a role in oxidant /antioxidant status,
59
therefore their concentration were determined using flame atomic

absorption spectrophotometer type GBC 933 plus. Working standard of
the element to be analyzed were prepared from zero concentration and
upwards of at least five concentrations, then they were analyzed
sequentially from the most dilute to the most concentrated, and the
resulting values were used to establish the calibration curve.
Procedure
A volume of serum and saliva were diluted (1:10) with deionized
water, and directly aspirated into the flame. The concentration of the
metal was determined from the appropriate calibration standard curve that
was prepared. The results of the trace elements were expressed in mol/l.
The determination of (Fe), (Cu), (Zn), and (Mn) using Atomic
Absorption Spectrophotometry ( AAS) was carried out at wave lengths
248.3 nm, 324.7 nm, 213.8 nm 279.5 nm respectively.
2.2.10- Determination of a- Amylase Activity (228)
Principle
Both straight chain (linear) polyglucans such as amylose and
branched polyglucans such as amylopectin and glycogen are hydrolyzed
by a-1,4 amylase. In the case of amylose, the enzyme splits the chains at
alternate a-1,4-hemiacetal (-C-O-C-) links, forming maltose and some
residual glucose; maltose, glucose, and a residue of limit dextrins are
formed if branched-chain polyglucans are used as substrate
The samples were incubated with buffered starch substrate at controlled
temp. (37C) for 15 minutes, then subsequently react with iodine which
produced a blue color with starch. The decrease in the color compared
60
with that obtained in the absence of amylase, provides a measure of

amylase activity.
Reagents
1. Buffered starch substrate pH 7.0:

A weight of 13.3 gm of dry anhydrous disodium hydrogen
phosphate, and 4.3 gm of benzoic acid were dissolved in 250 ml of
distilled water and then was bring to boil.
A weight of 0.2 gm of soluble starch was dissolved in 10 ml cold
water, and then was added to the boiling mixture, and the boiling was
continued for 1 min. The mixture was left at room temperature to cool
down after which it was diluted to 500 ml with distilled water and kept in
bottles at 4 C.
2. Stock iodine solution (0.1 N):

A weight of 3.5 gm of pure sublimed iodine was dissolved in a
solution of 24 gm of potassium iodide in about 100 ml of distilled water
and was made up to 1 liter with distilled water. This solution should be
stored in a brown bottle.
3. Working iodine solution (0.01 N):

A weight of 50 gm of potassium fluoride was dissolved in a little
distilled water, then a volume of 100 ml of stock iodide solution was
added, the volume was made up to 1 liter with distilled water. This
solution should be stored at 4 C in a brown bottle.
61
Procedure
1. A volume of 1.0 ml of buffered starch substrate was placed in a test
tube (test) and (control). Then the tubes were placed in a water bath
at 37 C for 3-5 minutes.
2. To the test tube, 0.1 ml of diluted sample (1:10 with normal saline)
was added, mixed gently, and incubated for exactly 15 min at 37 C.
A volume of 0.1 ml of distilled water was added to control tube.
3. The tubes were removed from the water bath. Then a volume of 0.4
ml iodine working solution was added, and mixed well, followed by
addition of 8.5 ml of distilled water. Mixed well for several times by
inversion.
4. The absorbance of the test and control was measured at wave length
of 660 nm.
Calculations
The amylase unit is defined as the amount of enzyme that digest
(5mg) of starch under the condition of the test (37 C and for 15 min).
C-T
(Amylase activity unit/100ml) = conc. of st. dilution factor
C
C = absorbance control
T = absorbance test
Amylase activity U/L = 1.85 Amylase activity in unit/100ml

1.85 is the conversion factor to SI unit
The specific activity of the enzyme was expressed in U per mg of protein.
62
2.2.11- Determination of Alkaline Phosphatase activity
Principle
The alkaline phosphatase (ALP) activity was determined by
colorimetric method (229), according to the following reaction:
O O
_ ALP _
O P O + H OH o OH + HO P O
_ 37 C , pH = 10 _
O O
The phenol librated was measured in the presence of 4-amino-

antipyrin and potassium ferricyanide according to the following equation:
Ph Ph
Me N Me N
N K3Fe(CN)6 N
OH + O O
NaHCO3
Me Me
NH2 N C C O
Red quinon color

complex
Reagents
1. Sodium carbonate bicarbonate buffer 0.1 M, pH10
A weight of 6.3 gm of anhydrous sodium carbonate, and 3.36 gm of
sodium bicarbonate were dissolved in distilled water, and made up to
1L. this solution was kept at 4 C.
2. Substrate solution (0.01 M)

A weight of 2.18 gm of disodium phenyl phosphate was dissolved in
litter of distilled water. The solution was brought quickly to boil, to kill
any organism, then it was cooled and preserved by little chloroform
(4 ml/L). This solution was kept at 4 C.
63
3. Sodium bicarbonate (0.5 N):

A weight of (4.2 gm) Sodium bicarbonate was dissolved and made up
to 100 ml with distilled water.
4. Sodium hydroxide (0.5 N):
A weight of (2g) Sodium hydroxide was dissolved in distilled water,
and made up to 100 ml with distilled water.
5. 4-Aminoantipyrine (0.03 N)
A weight of 0.6 gm of 4-aminoantipyrine was dissolved per 100 ml
distilled water, and stored in brown bottle.
6. Potassium ferricyanide (2.4%)

A weight of 2.4 gm of potassium ferricyanide was dissolved in
100ml distilled water, and stored in brown bottle.
7. Stock phenol standard (1 mg/1 ml)

A weight of 1 gm of pure crystalline phenol was dissolved per 1 liter
of (0.1N) HCl.
8. Working phenol standard (0.01 mg/ ml)

A volume of 1 ml of stock standard was diluted to 100ml with
distilled water. The working standard should be preserved with 2 or 3
drops of chloroform.
64
Procedure
The following tubes were sat up:
Test Control Standard Blank
Buffer pH 10 1 ml 1 ml 1 ml 1 ml
Substrate 1 ml 1 ml 1 ml 1 ml
The tubes were placed at 37 C for 3 min.
Sample 0.1 ml - - -
Standard - - 0.1 ml -
Distilled water - - - 0.1 ml
Then the solution were incubated at 37 C for 15 min.
0.5 N NaOH 0.8 ml 0.8 ml 0.8 ml 0.8 ml
0.5 N NaHCO3 1.2 ml 1.2 ml 1.2 ml 1.2 ml
The tubes were mixed well , followed by the addition of
4-aminoantipyrine 1.0 ml 1.0 ml 1.0 ml 1.0 ml
Potassium cyanide 1.0 ml 1.0 ml 1.0 ml 1.0 ml
All tubes were mixed well and let stand for 10 minutes in the dark, and
the absorbance was measured at =510 nm.
Calculation
The activity of alkaline phosphatase was expressed in U/L according
to the following equation:
A/min Vt 106 (mol/mol)

U/L=
Vs l
65
Where:-
U/L = mol / minute /liter of the sample
A/min = Absorbance change (Test-Control /Standard Blank)
Vt = Total reaction volume (6.1 ml)
Vs = sample volume (0.1 ml)
l = Cuvette path length (1 cm)
= Extiniction coefficient of phenol (50.000 L/mol/cm)
2.4.12- Determination of Lactate Dehydrogenase Activity

Lactate dehydrogenase (LDH) was assayed (230) using Randox kit.
Principle
The determination is based on the reduction of pyruvate to lactate in
the presence of NADH by the action of LDH.
Pyruvate + NADH + H+ L-lactate + NAD+
The pyruvate that remains unchanged reacts with 2, 4-dinitro phenyl

hydrazine to give the corresponding phenyl hydrazone, which is
determined colorimetrically in an alkaline medium.
Reagents
1. Phosphate buffer PH 7.45 (100 mmol/l)

Sodium pyruvate ( 0.8 mmol/l)
2. NADH (1 mg/ml)
3. Color reagent consists of:
2, 4-dinitrophenyl hydrazine (1 mmol/l)
Hydrochloride acid (1 mmol/l)
4. Sodium hydroxide (0.4 N)
66
Procedure
1. One vial of NADH was reconstituted with 10 ml of buffer.
2. Into test tube, 0.9 ml of the (NADH + buffer) was added and
placed in water bath at 37 C for 2-3 min.
3. A volume of sample (0.1 ml) was added to the test tube and was
mixed well, then incubated at 37 C for exactly 30 min.
4. One ml of color reagent was added with mixing and was allowed to
stand at room temperature for 20 minutes.
5. Then ten ml of 0.4 N sodium hydroxide was added, with mixing,
and was allowed to stand for 10 min.
6. The absorbance of samples was measured at 510 nm against D.W.
The enzyme activity was determined by reference to the calibration
curve.
Calibration curve
Six test tubes were prepared, and the following solutions were added:
Test tube No. 1 2 3 4 5 6
(NADH + buffer)
ml
0.9 0.7 0.5 0.3 0.2 0.1
D.W
ml
0.1 0.3 0.5 0.7 0.8 0.9
Color reagent
ml
1.0 1.0 1.0 1.0 1.0 1.0
Solutions were mixed and allowed to stand for 20 min.
NaOH (0.4 N)
ml
10 10 10 10 10 10
After the (NaOH) addition, the solutions were mixed and allowed to
stand at room temperature for 10 minutes. The absorbance was measured
against distilled water at wave length =510 nm. The corresponding
67
values in U/L were obtained from the supplier standard curve and found
to be as shown in the following table:
Test tube 1 2 3 4 5 6
U/L 0 111.4 223.1 334.9 390.6 446.3
2.2.13- Determination of Total (SOD) activity

The activity of SOD was determined by the riboflavin/NitroBlue
(231)
Tetrazolium method , which is based on the formation of blue
formazan dye (lmax = 560 nm), by the reaction with O.2- .
Principle
In the presence of light, and suitable hydrogen donor, such as methionine
or TEMED, riboflavin is photoreduced, and by subsequent spontaneous
reoxidation generates O2.-, NitroBlue Tetrazolium (NBT) is used as
indicating scavenger, and reduced by O2.-, the stepwise addition of
electrons forming the blue formazan. NBT reduction causes in
absorbance at = 560 nm, is inhibited by the presence of SOD.
Reagents
1. Working phosphate buffer (50 mM) pH 7.8 containing EDTA

(0.1 mM) and Triton X-100 (0.025%)
This solution was prepared as follows:
Solution A: Dipotassium hydrogen orthophosphate (50 mM).
A weight of 8.709 gm of K2HPO4 was dissolved in about 250 ml
deionized water, then the volume was made up to 1 liter.
Solution B: Potassium dihydrogen orthophosphate (50 mM).
68
A weight of 6.805 gm of KH2PO4 was dissolved in about 250 ml

deionized water, and the volume was made up to 1 liter.
a- A volume of (800ml) of solution A and (200ml) of solution B was
mixed, and the pH adjusted to 7.8 .
b- The working phosphate buffer solution was prepared by mixing
0.0373 gm of EDTA-2Na and 0.25 ml of Triton X-100 in
phosphate buffer (50 mM) pH 7.8, and then the volume was made
up to 1 liter with the same way.
2. L-Methionine solution (0.2 M)
A weight of 0.3 gm of L-methionine was dissolved in small amount
of deionized water, then the volume was made up to 10 ml.
3. NBT-2HCl solution (1.73 mM)
A weight of 0.0141 gm of NBT-2HCl was dissolved in small amount
of deionized water, then the volume was made up to (10 ml), then
the solution was stored cold in a brown bottle.
4. Triton X-100 1% (w/v) in deionized water.
5. Riboflavin solution (117 mM)
A weight of 0.0011 gm of riboflavin was dissolved in small amount
of deionized water, then the volume was made up to 25 ml with
deionized water, it must be kept in a brown bottle.
6. Sodium cyanide solution (2 mM)
A weight of 0.001 gm of sodium cyanide was dissolved in small
amount of deionized water; the volume was made up to 10 ml with
deionized water.
7. Working mixture reaction:
This solution was prepared by mixing 17.0 ml of reagent(1), 1.5 ml
of reagent (2), 1.0 ml of reagent (3), and 0.75 ml of reagent (4).
69
Procedure
1. Two glass test tubes were prepared, as follows:
Solutions Test Blank
Working mixture solution 1 ml 1 ml
Sodium cyanide 0.015 ml 0.015 ml
Deionized water 0.3 ml 0.3 ml
Phosphate buffer pH 7.8 0.1204 ml 0.1204 ml
Sample (serum, saliva) 50 ml -
Deionized water - 50 ml
The tubes were mixed after each addition
Riboflavin 0.0146 ml 0.0146 ml
Then the solution was mixed immediately, and the absorbance of

test and blank were measured at (l = 560 nm).
2. All tubes were illuminated for 7 minutes at 25 C in an aluminum
foil lined box containing two 20-W fluorescent lamps.
3. At the end of illumination time, the tubes were removed, and the
absorbance was measured immediately at l = 560 nm.
4. The difference in the absorbance between test and control was due
to the enzymatic activity
5. In order to determine the SOD unit, different volumes of serum
samples (20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240ml)
were used and the enzymatic activity for SOD was expressed as a
percentage of inhibition of NBT reduction. One unit of SOD is
defined as that amount of the serum which causes a 50% decrease
of the SOD-inhibitable NBT reaction.
70
The inhibition (%) caused by SOD was plotted as a function of serum and
saliva volume Figures (2-2), (2-3)
60
50
Inhibition (%)
40
30
20
10
0
0 50 100 150 200 250
Volume of serum (ul)
Figure (2-2): The inhibition of reaction by SOD for sera samples
60
50
40
inhibition%
30
20
10
0
0 50 100 150 200 250
volume of saliva (ul)
Figure (2-3): The inhibition of reaction by SOD for saliva samples
Calculations
Maximum inhibition was calculated from the inhibition curve, and
the SOD activity was calculated as follows:
71
(A 2 B - A 1 B) - (A 2 S - A 1S)
SOD activity (inhibition %) = 100
(A 2 B - A 1 B)
Where:
A1S = absorbance of sample tube before illumination
A2S = absorbance of sample tube after illumination
A1B = absorbance of blank tube before illumination
A2B = absorbance of blank tube after illumination
One unit of SOD is defined as that amount of sample which causes a
50% decrease of the SOD-inhibition for NBT reduction in this assay.
Therefore, the SOD activity in the sample can be expressed in riboflavin /
NBT assay unit (U) as following:
sample inhibition % 2 1000
SOD activity (u/ml) =
Max. inhibition % Vs(ml)
Where: U= riboflavin/NBT assay unit,Vs =sample volume (serum, saliva)

unit of SOD (u / ml)
SOD specific activity =
protein conc. (mg / ml)
2.2.14- Determination of Total Peroxidase activity
Peroxidase activity was determined colorimetrically. Wide variety of

hydrogen donors have been utilized in peroxidase assay systems .In this
study an improved assay was adopted using 4- aminoantipyrine as
hydrogen donor (232). The activity is determined by measuring the increase
in absorbance at l = 510 nm resulting from the decomposition of
hydrogen peroxide per time of incubation (A/min).
Reagents
1. Phosphate buffer (0.2 M) pH 7.0
a- A weight of 2.72 gm of KH2PO4 was dissolved in 100 ml deionized
water.
72
b- A weight of 3.48 gm of K2HPO4 was dissolved in 100ml of

deionized water. 60 ml of solution b is adjusted to pH 7.0 by adding
appropriate amount of solution a.
2. Hydrogen peroxide (0.0017 M)

This solution was prepared by diluting 1 ml of 30% hydrogen
peroxide to 100 ml with deionized water; further dilution was carried
out where 1 ml of this was diluted to 50 ml with (0.2M) potassium
phosphate buffer (PH 7.0). This solution was prepared fresh daily.
3. 4-Aminoantipyrine (0.0025 M) with phenol (0.17 M)

This solution was prepared by dissolving 0.810 gm phenol in 40 ml
deionized water, and then 0.025 gm of 4-aminoantipyrine was added,
and diluted to a final volume of 50 ml with deionized water. This
solution should be kept in a brown bottle.
Procedure
1- The following solutions were pipetted into test tube:
Solution Test tube
Phenol / 4-aminoantipyrine solution 1.4 ml
0.0017M hydrogen peroxide 1.5 ml
2- The test tube was incubated at 25C for 3-4 min. to achieve
temperature equilibration.
3-The reaction was initiated by the addition of (0.1ml) of the sample
(serum, saliva), with mixing. The increase in the absorbance at wave
length = 510 nm, was recorded for 5 minutes, to obtain A/min.
73
Calculations
(DA/min) was calculated, since DA is the difference in absorbance
between zero time and 5 minutes. One unit represent the decomposition
of one mmole of hydrogen peroxide per min. at 25 C and pH = 7 under
the specified conditions.
DA / min Vt
peroxidase activity U/L= 106
e Vs
Where:-
Vt = total volume
Vs = sample volume
DA/min = (Abs.at 5 min Abs. at the zero time)/incubation time (5min.)
2.2.15- Determination of Ceruloplasmin Oxidas activity

The activity of ceruloplasmin oxidase was determined in serum and
saliva using the modified Rice method(233) whereas ceruloplasmin
catalyzed the oxidation of p-phenylenediamine (substrate) to give blue-
violet color that measured at l = 525 nm.
Reagents
1. Substrate (p-phenylene diamine.2HCl)
The commercial PPD.2HCl salt was purified by dissolving 3 gm of
the salt in a minimum volume of hot water (60 C), darco charcoal was
added and left for 5 minutes, and then the mixture was filtered while hot.
The purified salt was precipitated from the filtrate by the addition of
acetone until the turbidity was appeared.
The mixture was refrigerated for several hours, filtered off the
crystals, then it was dried in the dark in a vacuum desicator over
anhydrous calcium.
74
2. Acetate buffer (0.4 M) pH 5.2 containing EDTA (0.4 mM)

This buffer was prepared by mixing 800 ml of solution (A), and 200 ml
of solution (B). The pH was adjusted to pH 5.2 & a weight of 0.015 gm
of EDTA was included in this buffer.
Solution (A): Sodium acetate (0.4 M)
A weight of 32.812 gm of sodium acetate was dissolved in small
amounts of deionized water, then the volume was made up to 1 liter.
Solution (B): glacial acetic acid (0.4 M)
A volume of 23.04 ml of glacial acetic acid was diluted to 1 liter
with deionized water.
3. Buffered substrate solution (5.5 mM)

This solution was freshly prepared by dissolving (0.1 gm) of
PPD.2HCl in 100 ml of acetate buffer.
4. Stock inhibition solution: sodium azide (0.1 M) and sodium

chloride (0.5 M)
A weight of (0.65 gm) sodium azide and (3 gm) sodium chloride
were dissolved in a small amount of deionized water, the volume was
made up to 100 ml, and this solution should be kept at 4 C.
5. Working inhibition solution

This solution was prepared by diluting (3 ml) of stock inhibition
solution with deionized water, to 100 ml to give a final concentration
of (3 mM) of sodium azide and (18.4 mM) of sodium chloride.
Procedure
1- In two glass test tubes, the following solutions were pipetted:
75
Solution Test Control
Buffered substrate 1 ml 1 ml
The tubes were incubated at 37C for 5 min.
Sample 0.1 ml ---
The tubes were incubated at 37C for 15 min.
Working inhibition 3 ml
Solution(cold)
3 ml
---- 0.1ml
Sample
Note: the solutions was mixed vigorously after each addition

2-The absorbance of the test and control were measured at wave
Length =540 nm .
Calculations
(Test absorbance Control absorbance) Final multiplication factor
10000
Final multiplication factor =
e incubation time
= molar absorbtivity of the base which equal to 1.91 L/mol.Cm
incubation time = 15 min.

Ceruloplasmin oxidase concentration was determined by measuring the
absorbance of the test and control tubes at wavelength (l = 605 nm)
(Atest Acontrol) Holmberge Laurell factor(234)
Where:-
A = absorbance
Holmberge Laurell factor = 87.5
76
2.2.16- Determination of RNases activity
2.2.16.1- Determination of Alkaline RNase activity

Alkaline RNase was estimated according to Bardon et.al.
method (235)with the following modification:
1- A volume of 1 ml of Davis buffer was used.
2- A volume of total reaction mixture was 1.1 ml instead of 0.2 ml
which was reported in the original method.
Reagents
- Davis buffer (236)
The following weights were dissolved in appropriate amounts of
distilled water:
Citric acid (21.01gm); Potassium dihydrogen orthophosphate (13.61);
Sodium tetraborate (19.07gm); Tris (hydroxymethyl aminomethane)
(12.11gm); and Potassium chloride (7.46gm) .The PH of the solution
was adjusted to (8) using sodium hydroxide (0.4N), and then the
volume was completed to 1 liter with water .
- Buffered substrate solution
A weight of (0.0275gm) yeast RNA was dissolved in (10ml) of
Davis buffer, then the volume was made up to (25ml) with the same
buffer.
-HCl (1M) in ethanol (70%)
A volume of (8.62ml) of concentrated HCl was added to (70ml) of
ethanol, and the volume was made up to (100ml) with distilled water.
Procedure
1- Reaction mixture of (1.1ml) volume was consisted of:
a-for saliva
A volume of (1ml) buffer substrate solution pH=8 and 0.1ml of saliva
77
b- for serum
A volume of (1.05ml) buffer substrate solution pH=8 and 0.05 ml
of serum.
2- The mixture was incubated for (15min.) at 37C .After the
incubation period, the reaction mixture was cooled to (0C).
3- An equal volume (1.1ml) of HCl (1M) in (70%) ethanol was
mixed with the reaction mixture, and was left for (30min).
4- The samples were centrifuged at (2000xg) for (10 min).
5- The supernatant was diluted with distilled water (1:5), then the
Absorbance was measured at (=260nm).
Note: the control was treated as the test except the sample was
added to control after the addition of HCl (1M) in (70%) ethanol.
Calculation
RNase activity was calculated by the following equation:
RNase activity (U/L) = (A/t Vt/Vs dilution factor 1000)/ Vs
Where :
A= Samples absorbance Controls absorbance
(at = 260nm )
Vt= the total volume
Vs= the volume of the sample (serum, saliva)
t = the incubation time (min)
2.2.16.2- Determination of Acid RNase activity

The method described in (2.4.16.1) was used to determine the acid
RNase activity in serum & saliva, except that the pH of Davis buffer was
adjusted to (5) and the substrate was also dissolved in this buffer. The
78
RNase activity in the acidic pH was calculated by using the same

equation that was used to calculate alkaline RNase activity.
2.2.17- Saliva samples concentrated procedure:

The crude saliva samples were concentrated by using Sephadex
G-25 method (237) in order to use it in the electrophoresis technique.
Dry Sephadex G-25 medium (0.042gm) was weighed in a 1.5-ml
Eppendorf tube which was previously pierced the base of the tube
carefully by a fine needle, the resulting hole is small enough to ensure
that Sephadex powder is unable to leak from the bottom of the tube. The
eppendorf tube was mounted on a vial, and both the eppendorf tube and
the vial was put into plastic centrifuge tube. Sample was added to the
sephadex powder, and left at (4C) for 10min allowing the sephadex to
swell. Then the assembly was centrifuged for 2 min at (1000g). The
concentrated saliva was collected in the lower vial.
2.2.18- Polyacrylamide Gel Electrophoresis (PAGE)

2.2.18.1- Conventional polyacrylamide gel electrophoresis
In this study, Conventional polyacrylamide gel electrophoresis
(7.5%) was carried out according to LKB 2117 note, for separation of:
proteins, glycoproteins, superoxide dismutase, lactate dehydrogenase,
ceruloplasmin oxidase, peroxidase, amylase, and alkaline RNase.
Reagents
1. Tris-glycine buffer stock solution (0.15 M) pH 8.9
A weight of 22.89 gm of glycine was dissolved in a liter of
distilled water. The pH of the solution was adjusted to pH 8.9 with tris
79
(hydroxyl-amino methane), the volume was made up to 2 liters with

distilled water.
2. Electrode buffer
One part of the stock solution was diluted with an equal amount of
distilled water.
3. Polyacrylamide solution 7.5%

A Weight of 22.2 gm of acrylamide and 0.6 gm of N,N`-methylene
bis acrylamide, were dissolved in 60 ml distilled water and then the
volume was made up to 100 ml with distilled water.
4. Ammonium persulphate 1.5% (w/v)

This solution was prepared on day of use.
5. Bromophenol blue 0.25% (w/v)
6. N,N,N`,N`-tetramethyl ethylene diamine (TEMED) (2.6%)
7. Samples used throughout this experiment, include crude pooled

serum and saliva samples from control, benign and malignant groups.
Procedure
1. Polyacrylamide gel 7.5% total monomer (T) and 2.6% cross-linked

(C), was prepared by mixing the following solution respectively:
7.5 ml of distilled water
33 ml of solution 1
22.2 ml of solution 3
The mixture was de-gassed in a 250 ml vacuum flask for 15 minutes,
then 3.2 ml of solution 4 and 0.1 ml of solution 6 were added to the
mixture solution
2. The gel was immediately poured into the mould, where the
polymerization reaction was completed within 40 minutes. after
80
polymerization , the mould was put in the refrigerator for 15

minutes.
3. The buffer tanks of the multiphor were filled with the electrode
buffer, 1L for each tank. The gel glass plate was placed on the
cooling plate and connected with the buffer by means of 8-10
layers of electrode wicks. They were soaked in the buffer, then
placed on the edge of the gel, covering 10-12mm of it.
4. According to (application note 306) LKB, pre electrophoresis was
carried out at 50 mA and 15 v/cm for 30 min.
5. A volume of (10 ml) of the samples were applied into the wells in
the gel, and concentrated for 5-10 min. with a current of 20 mA,
electrophoresis was continued using 40 mA, until the
Bromophenol Blue dye reached the gel margin.
2.2.18.2- Cathodic polyacrylamide gel electrophoresis

The method of Reisfeld et. al. (238) was used to separate and detect
Peroxidase isoenzymes in samples (serum and saliva) of the three studied
groups.
Reagents
1- Electrode buffer (pH4.5):
A weight of (6.24gm) of -alanin was dissolved in deionized
water, then a volume of (1.6ml) of glacial acetic acid was added.
The volume was madeup to 2 liters with deionized water.
2- Polyacrylamide solution 7.5%

A weight of 7.5gm acrylamide and 0.2gm of methylene
bisacrylamide were dissolved in distilled water, and diluted to
100ml.
81
3- Running buffer solution

A volume of 4.8 ml of 1N KOH and 0.4ml of tetra
methylethylene diamine (TEMED) were mixed with 1.7 ml of
glacial acetic acid, and diluted to 10 ml with distilled water.
4- Ammonium persulphate 0.028% (w/v) in distilled water .

This solution was prepared freshly in distilled water.
Procedure
1- To polymerize acrylamide 7.5% (pH 4.3) to form the gel:
8.75ml distilled water
8.75ml solution 3
17.5ml solution 2
35ml solution 4
This solution was gently mixed and loaded into the gel plate .
2- After the polymerization was completed , pre electrophoresis was
carried out at 20mA for 30 minutes .
3- The samples were applied to the gel (10 l) , electrophoresis was
continued using 30mA , until the dye (methylene green) reached
nearly the gel margin.
2.2.18.3- Discontinuous Polyacrylamide Gel Electrophoresis

The methode of Laemmeli(239), using PAGE 12.5% was used to
separate alkaline RNase isoenzymes in sera and saliva samples of control
and patient groups
82
Reagents
1- Electrode buffer pH (8.2) (Tris 0.025M- glycine 0.192 SDS 0.1%)
This buffer was prepared by weighing 28.826 gm of glycine,
6.056gm of Tris and 2gm of SDS, which were dissolved in an
adequate amount of distilled water, then the volume, was made up to
two liters with distilled water.
2- Acrylamide stock solution (30% acrylamide, 0.8%bisacrylamide):

A weight of 30 gm of acrylamide and 0.8 gm of N,N-methylene
bisacrylamide were dissolved in an adequate amount of distilled
water, and then the volume was made up to 100ml with distilled
water.
3- Ammonium persulphate solution 0.5% (w/v ) (freshly prepared ):

A weight of 2.5gm of ammonium persulphate was dissolved in an
adequate amount of distilled water , and then the volume was made up
to 5ml .
4- Bromophenol blue solution 0.25% (w/v).
5- N,N,N,N,tetramethylendiamine (TEMED)
6- Separating buffer (Tris 1.5 M) pH 8.8 :
A weight of 18.25gm of Tris was dissolved in an adequate amount
of distilled water, the pH was adjusted to 8.8 with hydrochloric acid
solution (0.2N), the volume was made up to 100ml with distilled water.
7- Stacking buffer (Tris 0.5 M) pH 6.8:

A weight of 6gm of Tris was dissolved in distilled water , then the
pH was adjusted to 6.8 with hydrochloric acid solution (0.2N) , and
then the volume was made up to 100ml with distilled water .
83
Procedure
Polyacrylamide gel (separating gel) 12.5% total monomer and 2.6
cross- linker was prepared by mixing the following solutions (50 ml total
volume):
15.85 ml distilled water
0.5 gm RNA
12.5 ml separating buffer
20.8 ml acrylamide solution
0.5 ml 10% SDS
0.25 ml ammonium persulphate solution
0.1 ml TEMED
The solution was gently mixed and loaded into the gel plates up to
the mark delimiting the separating gel. A little mixture of distilled water
and n- butanol was carefully layered on top of the gel solution to prevent
a curved meniscus from forming as the gel polymerized . The gel was
allowed to polymerize for about 3 hrs. To prepare the stacking gel (4%)
the following solutions were mixed (30 ml total volume):
18 ml distilled water
0.3ml 10% SDS
7.5ml stacking buffer
4ml acrylamide solution
0.15ml ammonium persulphate solution
0.05 ml TEMED
After removing the layer of saturated butanol and washing the upper
surface of the gel for several times with distilled water, the stacking gel
was mixed and poured on the top of the separating gel, and then allowed
to polymerize for 30-45min.The optimum conditions mentioned in the
application note 306 of LKB-electrophoresis instruction were exactly
followed . After the pre-electrophoresis with a current of 50mA, for
84
30min, the samples were loaded into the well in the stacking gel, after
that the current was changed to 20mA for 30min, and finally changed to
30mA. The electrophoresis continued until the bromophenol blue stain
reached nearly the gel margin. The gel was divided in order to be stained
for protein and alkaline RNase activity.
2.2.19- Gel Staining Methods

2.2.19.1- Protein stain
The polyacrylamide gel was stained for protein, using either
Coomassie Brillant Blue G-25 or silver stain and as follows:
2.2.19.1 A- staining with Coomassie Brillant Blue G-250

(CBB)(240)
Reagents
1. Fixing solution: TCA 12% (w/v)
2. Staining solution:
A weight of 0.1 gm of CBB G-250, 1.2 ml phosphoric acid and
10 gm of ammonium sulphate were dissolved in distilled water up to
100ml, a volume of 80 ml of this solution was adjusted to 100 ml
with methanol.
3. Washing solution: methanol 25% (v/v) in distilled water
4. Destaining solution
Volumes of 300 ml ethanol and 100 ml glacial acetic acid were
thoroughly mixed, then the volume was made up to liter distilled
water.
5. Storage solution: Ammonium sulphate 20-25% (w/v)
85
Procedure
1. Following electrophoresis, the gel was placed in the fixing solution
for an hour for fixation and removal of substances potentially
interfere with staining.
2. After fixation, the gel was immersed in the staining solution
overnight with gentle rocking until the required sensitivity; staining
dishes should be sealed carefully to avoid loss of methanol.
3. After staining step, the gel was washed with the washing solution,
then the gel was destained using destaining solution for several
times until the background was cleared.
4. Finally, the gel was placed in the storage solution for 60 min., then
it was kept in a plastic sheet at 4C.
2.2.19.1 B- Staining with silver stain (241)

Reagents
1. Fixing solution:
This solution consist of (200 ml) of 50 %( v/v) methanol,
12 %( v/v) glacial acetic acid and 100 ml of 35% formaldehyde.
2. Washing solution: 35% ethanol.

3. Sensitizing solution:
This solution consist of 0.02 %( w/v) sodium thiosulphate
This solution consist of (200 ml) of 0.2 %( w/v) silver nitrate
and 152 ml of 35% formaldehyde.
86
5. Developing solution:
This solution consist of (400 ml) of 6 %( w/v) sodium
Carbonate, (8 ml) of 0.02% sodium thiosulphate, and (200 ml) of
35% formaldehyde.
6. Stop solution:
This solution consist of (200 ml) of 50% (v/v) methanol and
12%(v/v) glacial acetic acid.
Procedure
1. Following electrophoresis, the gel was placed into the fixing

solution for 2 hours or overnight.
2. After fixation, the gel was washed 3 times with washing solution
for 20 minutes for each.
3. For sensitizing, the gel was soaked in sensitizing solution for 2
minutes.
4. After sensitizing, the gel was washed 3 times with D.W for 5
minutes for each.
5. After that the gel was soaked in the staining solution for 20
minutes.
6. After staining, the gel was washed 2 times with D.W for 1 min. for
each.
7. The Color was developed by developing solution until the band
being clear.
8. For stopping color development, the gel was soaked in stop
solution for 5 minutes. Then the gel was left at 4 C in 1% glacial
acetic acid.
87
2.2.19.1 C- Glycoprotein Staining (242)

Reagents
1. The fixation solution: 12.5% (w/v) TCA.
2. The fixation solution: 1%, periodic acid and 7%. TCA
3. Sodium metabisulfate 0.5% in 0.1N HCl
Sodium metabisulfate (0.5gm) was dissolved in a little amount of
distilled water, and then 0.86ml of concentrated HCl was added
after that the volume was made up to 100ml with distilled water.
4. Staining solution (Schiffs reagent)

A weight of 1 gm basic fuchsine was dissolved in 200 ml distilled
water with heating, after cooling, a volume of 20 ml of (1N) HCl,
and 2 gm of sodium metabisulphate was added with stirring, then
this solution was left at 4 C for six hours or overnight, and then 2
gm of activated charcoal was added, followed by filtration through
Whatman paper No.1. This solution should be kept in dark bottle.
Procedure
1. The gel was immersed in the fixing solution (solution 1) for 1 hour,
followed by overnight immersion in the other fixing solution
(solution 2).
2. Excess periodic acid was removed by washing the gel with several
changes with 250 ml of (solution 3), for 60 minutes or until the gel
became colorless, and followed by washing with distilled water.
3. Staining with Schiffs reagent was performed by submersing the
gel in a small amount of this reagent, and allowed the pink color to
develop in the dark at 4 C.
88
2.2.19.2- SOD Activity Staining

The electrophoretic gel was stained for SOD activity using the
modified riboflavin /NBT method (243).
Reagents
1. Phosphate buffer solution (100 mM) pH 7.0 containing riboflavin
(28mM) and TEMED (28 mM)
Phosphate buffer solution (100ml) was prepared as following:
A volume of 90ml of solution (a) was mixed with solution (b) until the
pH of the mixture achieved 7.0 .
Solution (a):
Weights of 1.369 gm of KH2PO4 and 0.0011 gm of riboflavin were
dissolved in 90 ml of deionized water, then 0.42 ml of TEMED was
added and the volume was made up to 100 ml with deionized water.
Solution (b):
Weights of 1.7418 gm of K2HPO4 and 0.0011 gm of riboflavin were
dissolved in 90 ml deionized water, then 0.42 ml TEMED was added;
the volume was made up to 100 ml with deionized water.
2. NBT solution (1.225 mM)

This solution was prepared by dissolving 100 gm of NBT in 100 ml
deionized water.
Procedure
After electrophoresis, the gel was first soaked in (NBT solution)
for 15 minutes with shaking. Briefly washed with deionized water, and
then soaked in a 100 ml (phosphate buffer solution pH 7.0) for another
89
15 minutes with shaking. Briefly washed with deionized water and it

was illuminated on a light box containing four 18-w fluorescent lamps
for 15 minutes to initiate the photochemical reaction. After the bands
were appeared, the gel was kept in a plastic sheet away from light.
Note: All procedures were carried out at room temperature.
2.2.19.3- Ceruloplasmin Oxidase Activity Staining(244)
Reagents
1. Acetate buffer (0.1 M) pH 5.7
This solution was obtained by mixing 130 ml solution (A) with a
volume of solution (B) to adjust pH 5.7.
Solution (a): sodium acetate solution (0.1 M)
A weight of 1.6404 gm of sodium acetate was dissolved in deionized
water, then the volume was made up to 200ml with deionized water.
Solution (b): glacial acetic acid (0.1 M)
A volume of 1.146 ml of glacial acetic acid was dilute to 200 ml with
deionized water.
2. Buffered substrate solution (13.8 mM)

A weight of 0.25 gm of PPD.2HCl was dissolved in 100 ml acetate
buffer (0.1 M) pH 5.7. This solution was prepared freshly prior to use.
Procedure
After electrophoresis, the gel was washed with acetate buffer (0.1 M,
pH 5.7) at room temperature. After that the gel was soaked in buffered
substrate solution for an hour at 37 C with gentle shaking, and in dark
place. The appearance of purple bands were indicated the location of Cp
oxidase activity.
90
2.2.19.4- LDH Activity Staining (245)
Reagents
1. Tris buffer (0.056 M) pH 7.4
A weight of 0.078 gm of tris (hydroxyl-amino methane) was
dissolved in distilled water, the pH was adjusted by using HCl
(0.1N) to pH 7.4, then the volume was made up to 10 ml.
2. Sodium lactate solution (2 M)
3. Phenazine methosulphate (1 mg/1 ml).
4. NBT solution (1 mg/1 ml)
Note: Solution 3, 4 was kept at 4 C.
5. Fixing solution
This solution was prepared as follows:
Ethanol 50% + distilled water 40% + glacial acetic acid 10%.
Reaction mixture solution

The reaction mixture solution was prepared as follows:
Volumes of: (1 ml) sodium lactate solution, (3 ml) NBT
solution,(0.3ml) ml phenazin methosulphate solution and (1 ml) tris
buffer (pH 7.4) solution, were mixed well, and a weight of 10 gm
of NAD+ was added to the mixture solution. This solution was
prepared prior to use and was kept away from light.
Procedure
After electrophoresis, the gel was covered with the reaction mixture
solution, and left at 37 C for 30 minutes, until the violet bands were
appeared, which indicated the LDH activity. Then the gel was placed
in fixing solution for 10 minutes, then it was kept in plastic sheet.
91
2.2.19.5- Amylase Activity Staining (246)

Reagents
1. Phosphate buffer (0.02 M) pH 6.9, containing 0.0067 M NaCl.

2. Starch 1% (w/v) in phosphate buffer.
3. KI-iodine solution, this solution prepare as follows:-
Five gm KI and 1.0 ml of stock solution (iodine 2 g/100 ml and
potassium iodide 6 g/100 ml), and then adjusted to a final volume of
125 ml.
Procedure
After electrophoresis, the part of the gel which contained saliva
samples was incubated in starch solution for 15 min at room temperature,
followed by immediate staining with KI-iodine solution, the bands which
represented the amylase activity was rapidly appeared. The other part of
gel which contained serum sample was incubated in starch solution for 2
hours at 37 C. The gel was brief water-rinsed and stained with KI-iodine
solution, until resolution of the amylase banding was evident.
Photographs were taken immediately after bands appearance.
The incubation times for gels was varied inversely with the amylase
activity of the sample.
2.2.19.6- Peroxidase Activity Staining
In the current study, two different stains (substrates) were used to

detect peroxidase activity on poly acrylamide gel.
92
A- Tetramethylbenzidine ( TMBZ ) Stain

The gel was stained for peroxidase activity using TMBZ according
to Thomas et.al.(247)
Reagents
1. TMBZ solution (6.3 mM) was freshly prepared in methanol.
Aweight of 0.1514gm of TMBZ was dissolved in 100ml methanol.
2. Staining mixture: this solution prepared immediately before use: 3

parts of the TMBZ solution were mixed with 7 parts of sodium
acetate buffer 0.25 M (pH 5.0) (as described in sec. 2-2-15).
3. H2O2 solution with final concentration of 30 mM.
Procedure
After electrophoresis, the gels were immersed in staining mixture for
1-2 hours, at room temperature in the dark with occasional mixing
(every 10-15 min). H2O2 solution was added to a final concentration
of 30 mM.
The staining was visible within 3 minutes and increased in intensity
over the next 30 minutes. Photographs should be taken after bands
appearance.
B- o-dianisidine stain
The gel was stained for peroxidase activity by using o-dianisidine
stain(248)
Reagents
1- -dianisidine 1mM
2- H2O2 10 mM
93
Procedure
After electrophoresis, the gel was incubation at room temperature in
a solution of 1mM of o-dianisidine for 5 minutes, then followed by
incubation for 5 minutes in 10 mM H2O2 .
2.2.19.7- Alkaline RNase activity staining (249)
Reagents
1. The incubation buffer, Tris-HCl buffer (0.1M;pH 8.5)
This buffer was prepared as described in (2.2.18.3)
2. Isopropanol 10% (v/v) and Tris buffer 10mM at pH 8

Volume of 10ml isopropanol was made up to 100ml with 10mM Tris
buffer at pH 8.
3. Staining solution, toluidine blue (0.2 %) (w/v)

A weight of (0.2gm) of toluidine blue was dissolved in (50ml) of Tris-
HCl buffer (0.01M; pH8.5), then the volume was made up to (100ml)
with the same buffer.
Procedure
The SDS was removed from the gel with 10mM Tris-HCl, pH8, and
10% isopropanol. The gel was then incubated in the activity buffer (0.1M
Tris-HCl, pH8) for an hour to allow enzymatic digestion of the embedded
substrate. The gel was stained with 0.2% (w/v) toluidine blue in (10mM
Tris-HCl, pH8) for 10 minutes, after that the solution was removed, and
the gel was destained with water until the positive bands appeared. The
gel was kept in plastic sheet at 4C.
94
2.2.20- Isoelectrofocusing point (pI) In polyacrylamide gel

Electrofocusing is a well established technique for separating
biological macromolecules in a pH gradient which forms as a result of
the migration of carrier ampholytes to determine the isoelectric
points(250).
Reagents
1. Anode solution 1M H3PO4:
2. Cathode solution 1M NaOH:
3. Gelling solutions
a. Acrylamide-bisacrylamide solution: 22 gm of acrylamide and 0.6 g
of N,N`-methylene bisacrylamide, were dissolved in 100 ml distilled
water
b. Glycerol 25% (v/v)
c. Ampholine 40% (pH 3.5-10)
d. Ammonium persulphate solution 1.5% (w/v)
e. N,N,N`,N`-tetramethylene diamine (TEMED)
f. Riboflavin-5-phosphate 0.1% (w/v) solution
Procedure
5% polyacrylamide gel containing Ampholine PH range of (3.5-10)
was prepared by mixing (31.57 ml) distilled water, (14.8 ml) of (3a)
solution, (12.7 ml) of (3b) solution, and ( 3.3 ml ) of (3c) solution in 250
ml vacuum flask, the mixture was degassed for 15 minutes, then (3.2 ml)
of (3d), (0.1 ml) of (3e) and (0.33 ml) of (3f) solutions were added. The
mixture was sttirled gently then poured into the mould, which was then
exposed directly to a fluorescent lamp, about 3-4 cm far from the gel so
95
that the photopolymerization could be intiated. The gel could be kept in a

refrigerator up to 24 hours before use.
Gel was placed on the cooling plate of LKB 2103 multiple systems
and the temperature maintained at 10 C. Electrode strips soaked with
electrode solutions, and then they were applied close to the long edges of
the gel (cathode strip to the left, anode strip to the right). Pre
electrofocusing was performed with electrical conditions of power = 30
watt, voltage = 1500 volt, current = 50 mA. When a constant watt was
achieved, samples 20 ml (serum, saliva) were applied on a (5 10 mm)
sample application filter pieces placed on gel, electrophoresis was
continued for 2 hours.
Note: The sample application pieces should be removed after
approximately half the focusing time.
After the time was finished, the electrode strip was removed, and the
electrofocused gel was divided into 4 parts, to be detected for protein and
enzymatic activity of SOD and peroxidase.
A narrow strip of gel was cut into (1 1 cm) slices from cathode to
anode, and solubilized in 1 ml boiled cooled distilled water and left
overnight at 4 C. Then the pH was measured and a pH gradient curve
was made by plotting pH values versus distance from cathode. Then the
gel was divided and stained.
A-Protein stain (251)

Reagents
1. Fixing solution:
Weights of 17.3 gm sulphosalicylic acid and 57.5 gm of
trichloroacetic acid (TCA) were dissolved in 500 ml D.W.
96
A weight of 0.460 gm coomassie brilliant blue R-250 was
dissolved in 400 ml of destaining solution.
3. Destaining solution:
500 ml ethanol and 160 ml acetic acid were mixed, and volume
was made up to 2 L with D.W.
4. Preserving solution:
50 ml glycerol was added to 500 ml destaining solution.
Procedure
Proteins were fixed by immersing the gel in fixing solution for one
hour. The solution precipitates the proteins and allows the
Ampholine to diffuse out. Before staining, the gel was washed with
destaining solution for 5 minutes, and then stained with staining
solution for 10 minutes at 60C. Then the gel was destained with
destaining solution several times, until the background became clear.
The gel was preserved for 1 hour in preserving solution, then
wrapped with cellophane sheet and stored at 4 C.
B- Enzymatic activity
SOD activity staining (231)
The gel was stained for SOD activity as described in 2.14.3.1
Peroxidase Activity Staining (247)
The gel was stained for peroxidase activity as described in 2.14.3.4
97
2.2.21-Statistical Analysis
The findings were expressed as the mean standard deviation. The
data were analyzed with student's independent t test. All statistical
analyses were performed with the program Statistical Package for
the Social Science (SPSS for windows, version 10.0).
A P value of <0.05 was accepted as statistically significant.
98
Chapter Three Results& Discussion
Proteins play a central role in cell functions and cell structure;

(102)
they are classified according to their biological functions . The
knowledge of body fluid proteins and their alternations in health and
disease has grown rapidly, some of the alternations have a genetic origin,
and many more reflect physiological or pathological processes (252).
Measurement of total protein (TP) concentration provides general
information, reflecting disease states in many organs systems(103), so
alteration in serum total protein concentration is used commonly in
clinical practice as a nonspecific indicator for underlining disease or
monitor disease activity(253). Increased concentration in TP can be due to
either increase in an individual protein, or group of proteins, or increased
in all proteins (17). So analysis of certain proteins and enzymes of blood are
widely used for diagnostic purpose (254)
Saliva contains proteins, in concentration of approximately 3% of
plasma protein level (17). Human saliva proteins can be informative for
disease detection and surveillance of oral health (105).
This part of the study, deals with the determination of total protein,
albumin, globulins, and ceruloplasmin in sera and saliva samples, and
immunoglobulins in sera samples of control and patients with different
oral tumors (benign and malignant). Meanwhile, polyacrylamide gel
electrophoresis (PAGE) was used to differentiate between protein patterns
in both serum and saliva samples among the studied groups. The gel was
also stained for glycoprotein to evaluate as well the changes in
glycoprotein contents.

Results
3.1.1- Total Protein

Total protein concentration was measured, according to Hartree
method (247), in sera and saliva samples, and as described in (2.2.1).
The results presented in Table (3 1) show the mean value of TP
concentration in serum and saliva samples, and revealed high significant
increase (P 0.01) in sera and saliva samples of malignant group, but the
increase was non- significant (p > 0.05) in sera and saliva samples of
benign group, in comparison to that of the control group.
Table (3-1) Mean value of total protein in sera & saliva samples of
( MeanSD)
Samples Age(year) g/dl
Group
Numbers (MeanSD)
Serum Saliva
Control 32 35.6611.64 7.72 1.45 0.221 0.062
Benign 14 35.7815.63 8.8 2.015 0.326 0.26
Malignant 19 46.5715.21 9.742.309 ** 0.5040.387 **

** Significant difference in comparison to control at (P 0.01)
3.1.2- Albumin
The albumin concentration in sera and saliva samples of control
and patient groups was measured as described in (2.2.2). The results are
presented in table (3 2), refer to the sera and salivary albumin
concentration, show the presence of highly significant decrease
(p =0.001), and significant decrease (P<0.05) in sera albumin
concentration in the case of malignant and benign groups respectively in
comparison to that of the control group.

In contrast, the results of salivary albumin concentration which

presented also in table (3 2), show significant increase (p < 0.05) in both
benign and malignant groups in comparison to that of the control group.
Table (3-2) Mean value of sera & salivary albumin concentration in

( MeanSD)
Group
Numbers (MeanSD)
Serum Saliva
Control 32 35.6611.64 4.49 0.72 0.027 0.019
Benign 14 35.7815.63 3.68 0.92* 0.069 0.047*
Malignant 19 46.5715.21 3.420.87** 0.0780.076*

** Highly significant difference in comparison to control at (P 0.001)
* Significant difference in comparison to control at (p < 0.05
3.1.3 Globulin
Globulin concentration in sera and saliva samples of the studied
groups in this study was calculated from the following equation:
Globulin conc. (g/dl) = Total protein conc. Albumin conc.
The results in Table (3 3) indicates that there is a high significant
increase (P = 0.001) in sera globulin concentration of malignant group,
and a significant increase (P<0.05) in the case of benign group in
comparison to that of control group.
Meanwhile, the results indicate that salivary globulin was
significantly increased (P<0.01) in malignant group, and non significantly
increased (P>0.05) in benign group, in comparison to that of control
group.

Table (3-3) Mean value of sera & saliva globulin concentration in

( MeanSD)
Group
Numbers (MeanSD)
Serum Saliva
Control 32 35.6611.64 3.23 0.74 0.194 0.04
Benign 14 35.7815.63 5.12 1.12* 0.257 0.21**
Malignant 19 46.5715.21 6.321.44*** 0.426 0.311

***Highly Significant difference in comparison to control at (P 0.001)
** Significant difference in comparison to control at (P<0.01)
* Significant difference in comparison to control at (P<0.05)
3.1.4- Ceruloplasmin( CP)
Ceruloplasmin is a multifunctional protein, which is able to

transport copper, as well as perform several enzymatic activities (288). It is
classified as one of the positive acute phase reactant proteins (APR).
Ceruloplasmin concentration in sera & saliva samples of control and oral
tumor patient groups was measured as described in (2.2.15).
The mean value of CP concentration in sera and saliva samples are
presented in Table (3 4) and shows a significant increase (P < 0.01), in
sera samples of malignant group, and non significant increase (P>0.05) in
benign group. While this concentration shows a significant increase
(P<0.05) in both sera and saliva samples of malignant group, in
comparison to that of the control.

Table (3-4): Mean value of CP concentration in sera & saliva samples

( MeanSD)
Samples Age(year) mg/dl
Group
Numbers (MeanSD)
Serum Saliva
Control 32 35.6611.64 8.65 2.45 0.300.28
Benign 14 35.7815.63 12.50 9.70 0.320.21*
Malignant 19 46.5715.21 13.725.73** 0.600.54*

** Significant difference in comparison to control at (P < 0.01)
* Significant difference in comparison to control at (P< 0.05)
3.1.5- Immunoglobulines
The evaluation of sera (IgG, IgM, IgA,) concentration in different

oral tumor patients and normal controls were measured as described in
(2.2.4). The results in Table (3 6) show a highly significant increase
(P<0.001) and a significant increase (P<0.01) in IgA concentration in
benign and malignant group respectively. The concentration of IgG show
a significant increase (P<0.05) in malignant group, but IgM concentration
show a non- significant increase (P> 0.05) in both benign and malignant
groups, in comparison to that of the control.

Table (3-5) Mean value of sera immunoglobulin concentration in

Control and patient groups
Mean SD mg/dl
Sample Age(year)
Group IgA IgG IgM
number MeanSD
Control 32 35.6611.64 149.0221.44 1219.93128.2 174.9572.57
Benign 14 35.7815.63 288.7175.75*** 1300.29406.07 184.0691.87
Malignant 19 46.5715.21 256.46126.49** 1387.7378.71* 203.1897.65

***Highly significant difference in comparison to control at (P < 0.001)
**Significant difference in comparison to control at (P<0.01)
*Significant difference in comparison to control at (P< 0.05)
3.1.6- Total Protein Electrophoresis
In order to detect the differences in total protein and glycoprotein

present in the studied groups. Conventional polyacrylamide gel
electrophoresis (PAGE) was carried out, as described in (2.4.1) on sera
and saliva samples of control and oral tumor patient groups, by which the
separation of different proteins is based on the differences of both
molecular size and the charge of these proteins.
To detect the location of the protein bands and to identify the differences
in protein profile. CBB-G250 stain and silver stain were used as described
in (2.5.1.1) and (2.5.1.2) respectively. While, Schiff's reagent was used as
described in (2.5.1.3) to detect the glycoprotein profile.
The electrozymogram in Figures (3 -1), (3 - 2) and (3 ) show the
protein separation profiles using CBB-G250 and silver stain. While Figure

(3 4) shows the glycoprotein profile that were separated from sera and
saliva samples of the studied groups.
11 10
10
9 9
8 8
7
7
6 6
5
4
3 4
3
2 2
1 1
The
Origin
point
Figure (3-1 ): Conventional poly acrylamide gel electrophoresis 7.5%,

using tris-glycin pH 8.9 as electrode buffer.
Electorophoresis was carried out for 3 hours at 4C
using a constant current of 40 mA and a voltage of
15 V/Cm the gel was stained for protein. The samples
used were as follows:
1 crude saliva (individual malignant)
2 pooled crude saliva (malignant)
3 pooled crude saliva (benign)
4 pooled crude saliva (control)
5 crude serum (individual malignant)
6 pooled crude serum (malignant)
7 pooled crude serum (benign)
8 pooled crude serum (control)

Figure (3-2 ) Conventional poly acrylamide gel electrophoresis 7.5%,

using Tris- glycin buffer, pH 8.9 as electrode buffer.
Electrophoresis was carried out for three hours at 4 C
using a constant current of 40 mA & voltage of 15v/cm.
The gel was stained for protein. The samples used were:
1 crude sera (control)
2, 3, 4 crude sera (benign)
5, 6, 7 crude sera (malignant)

The presence of distinct variations in the proteins and

glycoprotein separation profile was observed among the studied groups,
where it is obvious from the comparison between protein profile of the
three studied groups, Figure (3 1) that the proteins in sera of benign and
malignant groups (Lane5, 6) was separated to 10 bands and only 7 bands
were detected in sera of the control group (Lane 8). These differences
were confirmed when the gel was stained with silver stain (Figure 3 3).
The differences were also clear among the studied groups, when the gel
was stained for glycoprotein; Figure (3 4).
Salivary total protein profile, Figure ( 3 1) show distinct differences
between the three studied groups, where in malignant group (Lane 1)
eleven protein bands were appeared, in comparison to only six bands
detected in the normal group (Lane 4).
The sera glycoprotein profile, Figure ( 3 4) also shows obvious
differences between the studied groups, and the new bands that appeared
in benign and malignant groups, most of them were concentrated in the
middle part of globulin region.

The origin
point
Figure (3-3 ) Conventional poly acrylamide gel electrophoresis (PAGE)

7.5%, using Tris- glycin buffer, pH 8.9 as electrode
buffer solution. Electrophoresis was carried out for 3
hours at 4 C by using a constant current of 40 mA &
voltage of 15v/cm. The gel was stained for protein with
silver. The samples applied were:
1 crude serum (control)
2 crude serum (benign)
3 crude serum (malignant)
4 crude serum (malignant)
5 crude serum (control)

Figure (3-4 ) Conventional poly acrylamide gel electrophoresis 7.5%,

using Tris- glycin pH 8.9 as an electrode buffer.
The gel was stained for glycoprotein using Schiff
reagent .
The samples used were :
1. crude pooled serum (control)
2, 3, 4 crude pooled serum (benign)
5, 6, 7 crude pooled serum (malignant)

Discussion
Comprehensive analysis and identification of the proteins in sera and
saliva samples is a necessary first step toward the discovery of protein
change for human disease detection (105).
In the current study, the total protein in sera samples of patient group
(Table 3-1) was found markedly increased, when compared with that of
the control group. This result is accordant with several studies referred to
the presence of significant increase in TP concentration in patients with
different types of cancer (252, 255).
Such increase in TP concentration can be explained as follows: - the
whole body of cancer patient is engaged in protein synthesis of variable
forms, like globulins, immunoglobulin, enzymes and other proteinous
material (256). Among these proteins a group of several proteins called
positive acute phase reactants (APR)(257), like 1-antitrypsin, 1- acid
glycoprotein, C- reactive protein and ceruroplasmin, were reported to
increase significantly during acute inflammation, surgery, infection and
tumors(184). This was confirmed by the elevation of ceruloplasmin
concentration measured in this study (Table 3- 4) in the case of oral tumor
patients. Moreover, there are group of proteins in the body were reported
to decrease in concentration, and known as negative acute phase proteins
like albumin and prealbumin(184).
The synthesis of positive (APR) proteins exceeded the synthesis of
negative APR (albumin, prealbumin), such imbalance lead to a marked
increase in serum total protein (258).
Salivary total protein levels was also markedly increased (Table 3 1)
and this is in agreement with AL-rawi & Ibrahim studies on oral
sequamous cell carcinoma (OSCC) patients(259, 260)
.This increase may
explained on the basis that saliva in general, contain arrays of proteins that
have distinct biological function, most of them have antibacterial,

antimicrobial(261), and antibodies properties(262, 263)

defend the oral
environment against any noxious agents, and such proteins were reported
to increase in case of inflammation and tumors(264). Also it has been
reported that many serum-derived proteins transferred to the saliva during
inflammation (265).
The variations in the protein profile (Figure 3 1) is obvious from the
comparison among the sera and saliva samples of the different oral tumor
groups and control group, that the protein in saliva of benign and
malignant groups (Lane 2, 3) and sera of both groups (Lane 6, 7) were
separated into more protein bands than those were detected in pooled
saliva and sera of control group (Lane 4) and (Lane 8) respectively. The
alteration in the numbers and position of protein bands detected in sera
and saliva samples of the different studied groups may reflect altered gene
expression in cancer patient which lead to produce altered proteins (266). It
is clear from the electrozymogram that the protein bands detected in saliva
samples were more than those detected in the serum samples, some of the
bands migrate to the same distance in both serum and saliva samples of
the studied groups. This agrees with what the literature mention about that
some proteins are transported from serum to saliva (serum derived
proteins) whereas the others are derived from salivary glands. And the
significant increase observed in this study agree with the suggestion of
leakage of serum components into the mouth (saliva) (111).
Through out this study, a reduction in albumin level was observed
in sera of oral tumor patients (Table 3 2). Generally cancer patients have
lower serum albumin concentration than the healthy individuals (267). Many
references reported that a reduction in albumin concentration occurred in
inflammatory processes, chronic inflammatory disease, and in neoplastic
disease(161) such as lung cancer(268), breast cancer(269, 270)
, malignant

lymphoma(271), leukemia(272), melanoma(273), prostatic cancer(274) and brain

cancer(275).
The reduction in sera albumin concentration that observed in the current
study may be explained as follows:-
Liver is the site for the synthesis of most proteins including both
negative APR (pre albumin and albumin) and positive APR (276). It is well
known that the concentration of positive APR in serum increased in
malignancy, which mean that liver will be busy with synthesis of this type
of APR leaving behind the synthesis of other proteins like albumin (258),
also the synthesis of inflammatory cytokines, such as tumor necrosis
factor- (TNF-) and interlukines and C-reactive protein seems to cause a
reduction in albumin concentration (277).
Moreover, nutritional deficiency and weight loss in association with
psychological distress and lower quality of life may cause reduction in
sera albumin concentration (278, 279).
The other cause of the observed reduced albumin level in sera of
patient group, may be due to the role of albumin as one of the extracellular
antioxidants(280, 281) where albumin constitute up to 49% of total plasma
antioxidant status(282). Meanwhile albumin acts as sacrificial antioxidant
by inhibiting the generation of free radicals through an immediate attacks
of albumin molecule itself, so the radical reaction continue on albumin
surface and cause damage to albumin molecule(283, 284), such damage is
probably biologically insignificant, due to that the albumin is present in
plasma in high concentration(285).
In contrast to sera albumin, throughout the present study, salivary
albumin concentration showed a significant increase (Table3 2) in patient
groups in comparison to that of the control. This increase is in agreement
with other studies on (OSCC) patients where an increase in salivary
albumin concentration was also recorded (286, 287).

In the oral cavity, albumin in saliva was reported to be blood

derived protein (288), and it may diffuse into the mucosal secretions (289), so
the marked increase level of albumin may be related to the increase of
tissue damage and loss of epithelial barrier function and increased
vascular permeability leading to leakage or escape of many plasma
proteins including albumin to extracellular spaces (interstiasial) and
through cervicular fluid to saliva causing increase in salivary albumin (290).
Salivary albumin has been shown to increase in medically
(110)
compromised patients whose general condition gets worse .
Immunosuppression, radiotherapy, and diabetes are examples of states
where high concentrations of salivary albumin have been detected(111,291).
It may be hypothesized that salivary albumin can be used to assess the
integrity of mucosal function in the mouth(292), where salivary albumin
levels have been used as a marker for the degree of mucositis(112) and
inflammation in salivary glands(293).
Albumin in the whole saliva may be a marker and predictor of
stomatitis(290), since an increase in albumin concentration in whole saliva
was always detected prior to the clinical appearance of stomatitis.
In the current study, globulin concentration showed a significant
increase in patient groups (Table 3 3) and this may attribute to that most
of the positive (APR ) are globulins (i.e. glycoprotein), so the increased
level of globulins is due to the increased synthesis of positive APR in
malignancies(257).This change in globulins concentration was confirmed
by the significant increase in glycoprotein bands observed upon
electrophoresis of benign and malignant groups when compared with that
of the control group, Figure (3 4 ). The additional bands that appear in
glycoprotein profile, particularly in the globulins region result from the
synthesis of the positive APR during inflammation.

The increase in CP concentration in different tumoural processes is a

well known factor (294) .Ceruloplasmin is considered as one of the positive
acute-phase reactants (APR) which their concentration increases upon
(257)
different diseases including tumors . The measured increase in
ceruloplasmin concentration in the present study, in sera samples of
patient groups (Table 3 4) is in agreement with many studies on different
(291)
types of cancer, such as oral sequamous cell carcinoma , brain
cancer(275), breast cancer (295), pancreatic cancer (296), lung cancer (297), and
colon cancer(298). The increased CP concentration in sera samples result
from its increased synthesis by liver, as one of the acute phase proteins
which their concentration increases upon different diseases including
tumors (299).
Ceruloplasmin is an enzyme which has a role as an oxidant or
(300)
antioxidant . The unbalanced production of reactive oxygen
intermediates have been postulated to play a role in the pathogenesis of
cancer(301), so the high level of CP in neoplasia may be due to the host
response against such high levels of reactive oxygen intermediates which
recorded to be present in patients having different tumors(302).
Salivary CP concentration also showd increased levels. This could
be explained on the basis that many molecules including CP are capable of
penetrating the gingival tissue through the intercellular spaces of the
(303)
junctional epithelium to the saliva . Also its claimed that CP
synthesized by the tumor cell itself (304).
In this study immunoglobulins ( IgA, IgG and IgM) concentrations
were measured in sera samples of healthy individuals (control) and oral
tumor patient groups, and show a significant increase in IgA and IgG
(Table 3-6 ). This result is in agreement with AL- Dleemy study
(305)
concerning immunoglobulins in breast cancer patients . Circulating
immunoglobulins have detected in 75% of patients with head and neck

carcinoma (306). The major immunoglobulin produced by plasma cell is

IgG, makes up to 70-75% of the total immunoglobulins, and diffuses more
(115)
readily than other immunoglobulins into the extravascular spaces .
Unlike IgA which constitute only 10-15% of serum immunoglobulins, its
unique ability to be selectively transported across mucosal barrier into the
lumen of mucosa lined organs, make it important to early warning defense
system (115).
The reproducible elevation of sera IgA and IgG of oral tumor
patients observed in this study, may be also a result of the natural antibody
response to the presence of antigens of oral cancer, or as a defense
reaction against increasing tumor load, or may be due to the secretion of
immunoglobulin by the tumor itself (307, 308 ).
Kashmoola(262) on his study on salivary immunoglobulins (IgA, IgG
and IgM ) reported an elevation in saliva IgG and IgM and a reduction in
secretory salivary IgA . He suggested that such increased levels of IgG
and IgM was attributed to their leakage from interstitial fluids through the
damaged oral mucosa.
From the results of this part of the work, a conclusion may be
achieved, that oral tumor (benign & malignant) affects the sera and
salivary proteins composition and concentration.

Many of biochemical constituents are among the composition of

serum and saliva, such as urea & uric acid. Urea and uric acid are a non-
protein nitrogen compounds (NPN), where urea is the major excretory
product of protein metabolism(309), while uric acid which is regarded as the
most abundant scavenger of free radical in human(310, 311)
is the final
breakdown product of purine metabolism(309).
Saliva, the most accessible and non-invasive biofluid of our body,
harbors a wide spectrum of biological analytes informative for clinical
diagnostic applications (286). Saliva may constitute a first line of defense
against free-radical mediated oxidative stress. Many studies confirm that
saliva is rich in antioxidant, mainly uric acid, which is the major aqueous
antioxidant component of whole saliva (77), with lesser contributions from
albumin and ascorbic acid.
Salivary urea is elevated as a result of some disease (312). It was
reported that in saliva, urea is metabolized by oral bacteria to ammonia
and CO2 resulting in an increase in the pH of the environment (313, 314) .It
has been suggested that in certain metabolic conditions, mainly patients
with renal dysfunction, urea is accumulated, and it could be easily
monitored in saliva (315).
Both bone and teeth contain the mineral hydroxyapatite (calcium
with hydroxyl groups) associated with a cartilage (protein matrix).
Calcium and phosphorus are the major mineral components of the body.
They occur as free ions or in combination with organic and inorganic
compounds. Calcium, potassium, chloride, and phosphorus are the major
salivary complex-forming ions (315).
The goal of this part of the study is to look for the changes in urea, uric
acid, calcium and phosphorus concentration, of patients with different
types of oral tumors (benign & malignant) in comparison to healthy
individuals (control), and their affect on salivary pH.

Results
3.2.1 - Urea
Urea concentration was measured in sera and saliva samples of
control and oral tumor patient groups using BioMeriux kit, and as
described in (2.2.5).
The results presented in Table (3 6) show the mean values of urea
concentration in sera and saliva samples. The results reflected a non
significant increase (P > 0.05) in sera samples of both benign and
malignant groups in comparison to that of the control.
Meanwhile the results of salivary urea concentration of control and
patient groups, Table (3 6) show a significant increase (P< 0.01) in case
of the malignant group, whereas the increase was non significant
(P> 0.05) in salivary urea concentration of benign group in comparison
to that of the control.
Table (3-6) Mean value of urea concentration in sera & saliva samples
( MeanSD)
Group
Numbers (MeanSD)
Serum Saliva
Control 32 35.6611.64 25.21 4.94 32.839.3
Benign 14 35.7815.63 29.15 9.15 44.2522.87
Malignant 19 46.5715.21 39.7511.4 69.1253.42*


3.2.2 - uric acid

Uric acid concentration in sera and saliva samples of control and
oral tumor patient groups was measured using BioMerux kit and as
described in (2.2.6). The results in table (3 7) reflect a significant
increase (P < 0.01 )in sera samples of malignant group, and a non-
significant increase ( P > 0.05) in benign group in comparison to that of
the control group.
The results of salivary uric acid show a significant decrease (P< 0.01)
and (P< 0.05) in benign and malignant groups respectively, in comparison
to that of control
Table (3- 7) Mean value of uric acid concentration in sera & saliva
Samples of Control and patient groups
( MeanSD)
Group
Numbers (MeanSD)
Serum Saliva
Control 32 35.6611.64 4.761.87 2.311.31
Benign 14 35.7815.63 5.131.14 1.310.506**
Malignant 19 46.5715.21 6.142.68* 1.670.75*

**Significant difference in comparison to control at (P < 0.01 )
*Significant difference in comparison to control at ((P< 0.05)
3.2.3 - Calcium & Phosphorus
Calcium and phosphorus are the major mineral components of the

body, they occur as free ions or in combination with organic and inorganic
compounds. Calcium which is very important cofactor for some enzymes

activity, like amylase, therefore calcium and phosphorus concentration

were measured in sera and saliva samples of control and oral tumor patient
groups, using colorimetric method, and as described in (2.2.7) and (2.2.8)
respectively.
The results in table (3 8) show the mean values of sera total calcium
and phosphorus concentrations, and revealed a non significant differences
(P>0.05) in both benign and malignant groups in comparison to that of the
control group.
Table (3- 8) Mean value of total calcium and phosphorus
concentrations in Sera of control and patient groups
MeanSD (mg/dl)
Sample Age (year)

Group Calcium Phosphorus
number MeanSD
Control 32 35.6611.64 9.161.44 3.640.8
Benign 14 35.7815.63 9.831.69 4.42.25
Malignant 19 46.5715.21 9.671.53 3.722.11
Salivary calcium and phosphorus concentration results are presented in

table (3 9). The results show non significant increase (P>0.05) in salivary
calcium in both benign and malignant groups in comparison to that of the
control.
In contrast, to sera phosphorus level, the salivary phosphorus results
reflect a highly significant increase (P<0.001) in both benign and
malignant groups, in comparison to that of the control.

Table (3- 9) Mean value of salivary calcium and phosphorus

Concentrations in control and patient groups
MeanSD (mg/dl)
Sample Age (year)

Group Calcium Phosphorus
number MeanSD
Control 32 35.6611.64 1. 911.0 7.762.8
Benign 14 35.7815.63 2.541.94 12.365.9*
Malignant 19 46.5715.21 2.942.85 17.168.9*

*Highly significant difference in comparison to control at (P<0.001)
3.2.4- Salivary pH
The pH of saliva samples of the control and oral tumor patient groups
was measured, based on the color change of the indicator paper; the
buffering capacity is assessed in comparison with a color chart. The
results represent in Figure (3 5) show a significant increase (P<0.05) in
comparison to that of the control group.
Control
8.2
Benign
8
Malignant
7.8
7.6
7.4
pH
7.2
7
6.8
6.6
6.4
Saliva
Figure (3-5): Salivary pH for the three studied groups

Discussion
In the current study, there was a slight increase (non significant) in
serum urea level in malignant group in comparison to that of the control.
Generally the elevation of urea concentration is accompanied by renal
disease, but there are many reasons to elevate urea concentration, such as
the amount of protein metabolism, high-protein diet, increased protein
catabolism such as that occur in fever, major illness, dehydration and
stress (103).
In contrast, salivary urea mesurment reflected an elevation levels in
oral tumor patients in comparison to that of the control. As known salivary
urea is metabolized by the oral bacteria to ammonia and CO2 resulting in
an increase in the pH of the environment (313, 314), and this may be the cause
for the increased salivary pH of the patient groups when it was compared
with that of the control (Figure 3 5). AL Nowaiser et al. (315) has been
suggested that salivary urea is accumulated and it could be easily
monitored in saliva in certain metabolic conditions, mainly patients with
renal dysfunction,
The elevated level of salivary urea may be attributed to the flow rate
of the saliva, whereas the relative concentration of the organic salivary
constituents are known to be dependant inversely on salivary flow rate(316).
This is confirmed by the observation that almost all the patients included
with the current study, had low salivary flow rate. This slow rate of saliva
leads to the accumulation of urea in the mouth leading to its hydrolysis
into CO2 and ammonia by the bacteria in the mouth and so affected the pH
of the environment. This lead to the observed increase in the salivary pH
in the present study.
In the current study, an increase in sera uric acid concentration was
observed in patients with oral tumor. This is in agreement with study on
patients with lung cancer where an increase in plasma uric acid

concentration (317) was found. Also with a study on patients with pancreatic
cancer which reported to have a high uric acid concentration (318). Uric acid
levels in breast cancer patients were found to be significantly higher than
those of controls (319). Also there is a report referred to presence of an
increase in serum uric acid in patients with leukemia (320).
As known uric acid is produced in the body as an end product of
purine metabolism. The increased level of sera uric acid in this study
could be explained as follows: -
The metabolic disturbance of the cancerous cells will lead to an
increase in the metabolism of purine, causing an increase in sera uric acid
level (321). Also the lysis of tumor cells leads to the increase in the release
of intracellular contents like (uric acid, potassium, phosphorus) into the
extra cellular components, and hence caused on elevation of uric acid
concentration (322).
Salivary uric acid throughout this study was found to decrease
significantly in oral tumor patients. And this result disagrees with Ibrahim
study (260) on OSCC patients, where he found an increase in salivary uric
acid. The reduction in salivary uric acid level may be explained on the
basis of that, uric acid is the major antioxidant in saliva(140), which is
capable especially of reacting with hydroxyl radical and hypochlorous
acid, as well as preventing H2O2 formation(323), and sinc uric acid is one of
the sacrificial antioxidants(324) thus the radical reaction continue on uric
acid surface, so the increased of its utilization to scavenge free radicals
cause a reduction in its concentration in the saliva.
In the present study, non significant increase of calcium level was
observed. The elevation of calcium level has been reported to occur in up
to 20 to 30 % of patients with cancer at some time during the course of
their disease (325, 326, 327). Humoral hypercalcemia of malignancy (HHM)
caused by systemic secretion of parathyroid hormone related protein

(PTH-rP) by malignant tumor (328, 329). (PTH-rP) increased resorption and

enhance renal retention of calcium (330). Head and neck tumor is one of the
tumors that most commonly cause HHM (331) (as many as 4% of patients
with cancer in the head and neck may have elevated serum calcium
level(332). It has been reported that hypercalciemia is associated with
OSCC patients (333). In the current study 27.7% of the malignant oral tumor
patients were found to have an elevated serum calcium level.
Salivary calcium reveal a non significant increase, the increased salivary
calcium level may be due to decrease in salivary flow rate, since the
concentration of calcium of unstimulated saliva is inversely related to
saliva flow rate (334).
Phosphorus level in sera samples of oral tumor patients show non
significant difference in oral patients group. Meanwhile, salivary
phosphorus shows a highly significant increase. Many causes may
contribute to such elevation, among them the lysis of tumor cells lead to
increase the release of intracellular components like phosphorus; this
could increase the level of phosphorus in the nearby environment
(saliva)(322). Also the increase may be attributing to the increased ALP
activity in saliva (Table3-11) which leads to increase release of inorganic
phosphate in the environment (saliva).
And as known salivary phosphorus has the main buffering effect on
salivary pH(15), so such increase in Pi concentration is another reason of
the observed elevated pH (Figure 3 5 ), beside the effect of elevated
salivary urea that observed among the three studied groups (Table 3 6).

A variety of enzymes and proteins have been studied in patients with

different oral tumors in the hope of finding useful diagnostic markers in
biological fluids of oral tumors. Enzymes had been used as tumor markers,
since they present in higher concentration inside the cell than outside and
their released into the circulation as a result of tumor necrosis causes an
increase in their serum levels which can be used as a sign for the presence
of malignancy (335).
Alkaline phosphatase ALP and lactate dehydrogenase LDH have
been found elevated in a variety of malignances (336). The level of ALP was
suggested to be elevated in primary and secondary liver cancer. Therefore,
its level may be helpful in evaluating metastatic cancer with bone or liver
involvement (337).
The elevation of LDH activity had been often used in clinical
diagnosis, although this elevation in malignancies is rather non specific. It
seems more helpful to measure the salivary LDH beside serum LDH for
clinical diagnosis (338) of oral cancer, since saliva is the fluid that obtained
from glands near or adjacent to oral tumors,
Previously, the activity of these two enzymes were measured in
patient with OSCC(259) (type of malignant tumor) where an elevation of
their activities were observed. Accordingly, in the current work ALP &
LDH activities were followed in patient with different kinds of oral tumors
(benign &malignant) to follow the changes in their activities upon the
progress of the oral tumor disease.
Results
3.3.1- Alkaline Phosphatase (ALP)
Alkaline phosphatase activity in sera and saliva samples of normal

individuals (control) and oral tumor patient (benign and malignant) was
measured according to Kind & King method and as described in (2.2.11).

The results in Table (3 10 ) show the mean value of activity (U/L)

and its specific activity (U/mg) in sera, and reveal a significant increase
(P<0.05) in benign group, whereas the increase was non significant
(P>0.05) in the malignant group, in comparison to that of the control
group. The specific activity was non significant increased (P>0.05) in both
benign and malignant groups, in comparison to the control group
Table (3 -10 ) Mean value of ALP activity and specific activity in sera
Of control and patient groups
MeanSD
Specific
Sample Age (year) Activity
GROUP Activity10
Number MeanSD U/L
U/mg
Control 32 35.6611.64 61.7549.09 0.7000.256
Benign 14 35.7815.63 84.5334.82* 1.020.43
Malignant 19 46.5715.21 77.8345.99 0.880.79

*Significant difference in comparison to control at (P<0.05)
The results in Table (3 11) show the mean value of ALP activity (U/L)
and specific activity (U/mg) in saliva samples of the studied groups. The
presence of non significant increase (P>0.05) in the activity of both benign
and malignant groups is obvious, in comparison to that of the control.
Meanwhile, the results show a non significant decrease (P>0.05) in
specific activity among the studied groups.

Table (3 11) Mean value of ALP activity and specific activity in saliva
MeanSD
Specific
GROUP Activity10
Number MeanSD U/L
U/mg
Control 32 35.6611.64 3.382.31 1.921.56
Benign 14 35.7815.63 4.204.11* 1.621.92*
Malignant 19 46.5715.21 4.665.94* 1.551.81*
*Non significant difference in comparison to control at (P>0.05)
3.3.2- Lactate Dehydrogenase (LDH)
Lactate dehydrogenase (LDH) activity was measured in sera and

saliva samples of the control and oral tumor patient groups, as described in
(2.2.12).
The results of sera LDH activity (U/L) and specific activity (U/mg)
are presented in Table (3 12) & show a highly significant increase
(P=0.001) in malignant group, and a non significant increase (P>0.05) in
benign group in comparison to that of the control. While the specific
activity show a non significant increase (P>0.05) in both benign and
malignant groups in comparison to that of the control.
Table (3 13) shows the results of LDH activity and specific activity
in saliva samples of control and oral tumor patients. The results reflect a
significant increase (P<0.05) in the activity of benign and malignant
groups in comparison to that of the control. While a non significant

differences (P>0.05) was observed in their enzyme specific activity in

both benign and malignant groups in comparison to that of the control.
Table (3 - 12 ) Mean value of LDH activity and specific activity in sera

MeanSD
Specific
GROUP Activity10
Number MeanSD U/L
U/mg
Control 32 35.6611.64 182.0827.40 2.410.56
Benign 14 35.7815.63 211.6950.53 2.530.85
Malignant 19 46.5715.21 278.3285.48* 3.041.33

*Highly significant difference in comparison to control at (p0.001)
Table (3 - 13) Mean value of LDH activity and specific activity in

Saliva of control and patient groups
MeanSD
Specific
GROUP Activity
Number MeanSD U/L
U/mg10
Control 32 35.6611.64 90.3939.76 54.7626.78
Benign 14 35.7815.63 143.14101.67* 61.4349.05
Malignant 19 46.5715.21 166.04137.42* 48.6142.15


3.3.3 - Lactate dehydrogenase Isoenzyme
Throughout this part of the work, and to illustrate the differences in the
sera and saliva LDH isoenzymes profile in the different studied groups, a
conventional polyacrylamide gel electrophoresis (7.5%) was used, as
described in (2..18.1), and stained for LDH activity as described in
(2.2.19.4). Figure (3 6) show the sera LDH isoenzymes of the three
studied groups (control, benign, malignant). It is obvious from the
zymogram that there are many differences in the distribution and level of
the activity between the isoenzymes of the studied groups. The
electrophoretic migration of these isoenzymes was quite similar. The
Figure shows an increase in LDH1 & LDH2 . The appearance of such
differences may be due to the alteration in the LDH biosynthesis, total
activity in addition to the alteration in LDH isoenzymes distribution in
human cancerious tissues.

(+)
LDH1
LDH2
LDH3
(-) LDH4
LDH5
1 2 3
Figure (3- 6 ) Conventional poly acrylamide gel electrophoresis (PAGE)

buffer. Electrophoresis was carried out for 3 hours at
4 C by using a constant current of 40 mA & voltage of
15v/cm. The gel was stained for LDH activity. The
samples applied were as follows:
1 crude pooled serum (malignant)
2 crude pooled serum (benign)
3 crude pooled serum (control)

Discussion
It seems that increased ALP activity is a constant feature in neoplastic
transformation, and this was noted in all cases of breast carcinoma(258).
Increased sera ALP is seen in state of increased osteoblastic activity
(Osteomalacia, primary and metastatic neoplasms)(258). In this study, the
activity of ALP in sera samples shows an increase. These results are in
agreement with previous reports in different types of cancer, where ALP
activity is markedly increased in ovarian (339), lung (340), bone(341), gastro
intestinal cancer(342), Seminoma and Hodgkin's disease(343).
The observed increase in this study could be explained by the fact
that the changes in the membrane permeability of the tumor cells lead to
the release of the enzyme into the circulation (332).
Salivary ALP, throughout this study, shows non significant
increase. This result disagree with Al-Rawi(259) study on OSCC patients
where he found a decrease salivary ALP activity with no explanation for
such reduction. The observed increase in salivary ALP here could be
explained on the basis that the induction of salivary ALP activity has been
demonstrated in periosteal fibroblasts in response to demineralised bone
matrix(344).
In the current study, LDH activity showed an increase in sera of the
oral tumor patients. This result is in agreement with many studies (259, 287)
OSCC patients, also the result agrees with a study on patients with
malignant tumors of oral mucosa (345). The elevation in LDH activity was
also demonstrated in serum of patients with different malignant neoplasm,
such as, Hodgkin's lymphoma, acute lymphoblastic leukemia, colorectal
carcinoma, lung cancer, breast cancer, and in various other
cancers(307,346,347).
The importance of estimating the LDH level as a prognostic factor
and direct indicator of tumor burden has emerged in several clinical

studies, and the association between the elevation of LDH and the
presence of malignancy was significant, because the abnormal elevation
will disappear shortly after surgical removal of tumor and after
chemotherapy or radiotherapy began (348).
Almost every type of cancer as well as many diseases can cause LDH
levels to be elevated. Therefore, It was suggested that LDH cannot be used
to diagnose a particular type of cancer, but can be used to monitor
treatment of some cancers like sarcoma and some types of Leukemia(332) .
Cancer cells will relay on anaerobic oxidation as a source of energy
by forming lactate. It was originally thought there is an hypoxia areas only
in the center of tumors and remained relatively static and eventually
became necrotic, while latter it was known that hypoxic areas actually
come and go in a tumor as perfusion varies and as new blood vessels form,
fade away, and then reform(349). Lactate acidosis has been shown to
activate the biosynthesis of some acid hydrolyases(350), to cause release of
lysozomal enzymes(350), to disturb calcium metabolisim(351), and to change
the permeability of cell membranes(352). Further, it has been shown that
decreased intracellular pH and a decreased ATP level will cause an
increase release of proteins from tissues into different body fluids(352, 353).
which are in concordance with the results obtained in the present study.
Lactate is a by-product of anaerobic metabolism, as oxygen delivery
decrease below a critical level, blood lactate concentration rise rapidly and
indicate tissue hypoxia , and the accumulation of excess lactate in blood is
an early sensitive and quantitative indicator of oxygen deprivation leading
to cell death(103). Rapidly proliferating populations of euplastic cells
depend largely upon glycolytic mechanisms for derivation of energy, with
a large requirement for LDH to regenerate NAD+ from NADH in order to

support continued glycolysis(354).

In the current study, salivary LDH show an increase in oral tumor

patients in comparison to that of the control. This result is in agreement
with many studies oral cancer patients (259, 287). Salivary LDH is known to
be mainly derived from exofoliative oral epithelial cells (169) and as such,
LDH may also be used as a general salivary marker for the diseased
mucosa. It has been suggested that the tumor cells are the source of the
elevated LDH level (355).
Number of changes in the biochemical characteristic of malignant cell
surface have been observed, these include the appearance of new surface
antigen proteoglycans, glycolipids, and mucins and altered cell-cell and
cell-extracellular matrix communication (356). Such changes may lead to
transport different enzymes from the blood to the saliva via salivary
glands. An increased LDH activity in sera and saliva of benign and
malignant oral tumor patients may be a sign of tissue damage and cell
death, their LDH is released and finds its way into the circulation (103).
Figure (3- 6) show the electrophoretic patterns of LDH isoenzymes.
The same isoenzyme bands were observed to be present in the sera of all
studied groups with alteration of their intensity especially LDH1 & LDH2
when compared to the control group. The appearance of such differences
(357)
may be due to the alteration in the LDH biosynthesis . The
simultaneous high activity of the same basic bands might serve as a useful
parameter of disease activity (354).
For the known relationship between alkaline phosphatase and
inorganic phosphate and the relation between the latter with calcium, it
was necessary to estimate calcium & phosphorus concentration for the
studied groups and check the relation between these parameters.
From the results in (Table 3-11), the observed elevated salivary ALP
activity seems to be consequence with the increased concentration of
salivary inorganic phosphate Table (3 - 9).

Human alpha amylase enzyme is the most important digestive

enzymes. It consists of two families of isoenzymes, pancreatic amylase
(P-type) and salivary amylase (S-type) (103). The human alpha amylase(s)
enzyme is calcium metalloenzymes, completely unable to function in the
absence of calcium (258).
Human salivary alpha amylase is the only enzyme in saliva capable
of degrading oligosaccharides, most of the enzyme being synthesized in
(156)
the parotid gland . Human parotid saliva and submandibular saliva
contain about 45 and 30 mg of amylase respectively per 100mg of
protein(358). However, it has been claimed that amylase makes up about 1/3
of the total protein content in parotid saliva, and the content in whole
saliva would be lower(154). To our knowledge there is no literature deal
with the measurment of amylase activity in sera and saliva of oral tumor
(287)
patient (benign & malignant), except a recent one , which measure
amylase activity in saliva of OSCC patients.
Ribonuclease is a type of enzyme which catalyzes the breakdown of
bonds in ribonucleic acid (RNA) which is the chemical material in a cell
that codes for different proteins (170). It is known that RNA is more labile
than DNA and is presumed to be highly susceptible to degradation by
RNase(359).
Throughout the past few years, several studies on RNase were
carried out in our laboratory in patient with different types of cancer, such
as ovarian cancer (360), uterus cancer (361), and breast cancer (362). The results
of these studies showed a presence of significant increase in RNases
activity, as well as, variation in their forms. No available literature has
been found which concerning this enzyme in saliva and sera of patients
with oral tumor (benign and malignant).

This part of the study was undertaken to determine the activity and
specific activity of both sera and salivary amylase and RNases, with
studying their isoenzymes using PAGE to evaluate the changes of these
isoenzymes affecting by the presence of benign and malignant oral tumor.
Results
3.4.1 - - Amylase activity & specific activity
Amylase activity was measured in sera and saliva samples of
normal individuals and oral tumor patient groups, as described in (2.2.10).
The amylase activity (U/L), and specific activity (U/mg) of sera
samples were presented in Table (3- 14) and reveal a non significant
increase (P>0.05) in both benign and malignant groups in comparison to
that of the control. The specific activity results show a non significant
decrease in both benign and malignant groups in comparison to that of the
control.
The results of salivary amylase activity and specific activity were
presented in Table (3- 15), and show a significant decrease (P<0.05) in
amylase activity of the malignant group. The specific activity of the same
group show a highly significant decrease (P<0.001). Meanwhile the
activity& specific activity results of benign group show non significant
decrease (P>0.05) in comparison to that of the control.

Table (3- 14) Mean value of amylase activity and specific activity
In sera of control and patient groups
MeanSD
Sample Age (year) Activity Specific Activity

GROUP
Number MeanSD U/L U/mg
Control 32 35.6611.64 331.6970.44 4.441.32
Benign 14 35.7815.63 336.37104.89 3.851.31
Malignant 19 46.5715.21 352.91142.46 3.621.41
Table (3-15) Mean value of salivary amylase activity and specific

Activity of control and patient groups
MeanSD
Sample Age (year) Activity103 Specific Activity

GROUP
Number MeanSD U/L U/mg
Control 32 35.6611.64 1.460.04 0.7380.216
Benign 14 35.7815.63 1.340.36* 0.6460.300*
Malignant 19 46.5715.21 1.330.24* 0.3780.284**

**Highly significant difference in comparison to control at (P<0.001)
* Significant difference in comparison to control at (P>0.05)

3.4.2- - Amylase Isoenzyme
In this part of the work, a conventional PAGE (7.5%) was used as

described in (2.2.18.1). The gel was stained for amylase activity as
described in (2.2.19.5) to illustrate the differences in profile of amylase
isoenzymes in the sera and saliva samples of the different studied groups.
Figure (3 - 7) and Figure (3 - 8), show the electrophoresis pattern of
amylase in sera & saliva samples respectively of control and oral tumor
patient groups (benign & malignant).
In refer to Figure (3 - 7) it is clear that the serum amylase activity
located in two different parts of the gel, one with slow mobility (S-type)
and the other with fast mobility (P-type), and each of them in turn consist
of more than one band. Upon comparison of the amylase separation
pattern it is obvious that there are interesting differences between the three
studied groups. The isozymes in benign and malignant groups (Lane 2, 3)
respectively began to increase in both the slowest and the fastest fraction
(according to their mobility to the anode) i.e. the pancreatic type and the
salivary type were increased (number of bands and their intensity) in the
sera of benign and malignant oral tumor patients in comparison to the
control (Lane 1).
It is obvious from the salivary amylase zymogram Figurer (3 - 8) that
there are several interesting differences between the three studied groups.
The electrophoretic pattern for salivary amylase shows an alternating light
bands and heavy bands with decreasing intensity toward the anode. Four-
five bands were visualized in control group (Lane 1), these bands start in
reduction (faint) in benign group (Lane 2), meanwhile, only three four
bands were detected in the malignant group (Lane 3) and they seem to be
heavy bands, i.e. the light bands disappeared in the malignant group.

A 10
9
8 P- type
7
(+)
6
5
4 S- type
3
2
1
(-)
A- Positive B- Negative
Figure (3-7 ): Conventional poly acrylamide gel electrophoresis (PAGE)
buffer. Electrophoresis was carried out for at
C using a constant current of 40 mA & voltage of
15v/cm. The gel was stained for amylase activity. The
samples were applied as follows:
1 crude pooled serum (control)
2 crude pooled serum (benign)
3 crude pooled serum (malignant)

A B
(+)
5
4
3
2
1
(-)
Figure (3-8): Conventional poly acrylamide gel electrophoresis (PAGE)

buffer. Electrophoresis was carried out for at
C using a constant current of 40 mA & voltage of
15v/cm. The gel was stained for amylase activity. The
samples were applied as follows:
1 crude pooled saliva (control)
2 crude pooled saliva (benign)
3 crude pooled saliva (malignant)

3.4.3- Ribonuclease ( RNase)

3.4.3.1- Alkaline RNase activity& specific activity
The activity of alkaline RNase in sera and saliva samples of normal
individuals and oral tumor patients (benign and malignant) was measured
as described in (2.2.16.1). The results of sera alkaline RNase activity
(U/L) and specific activity (U/mg) are presented in Table (3 - 16). These
results indicated the presence of a highly significant increase (P<0.001) in
the activity of both benign and malignant groups in comparison to that of
the control. The specific activity results show also a significant increase
(P<0.05) in both benign and malignant groups.
Table (3 - 17) presented the results of alkaline RNase activity and
specific activity in saliva samples of control and oral tumor patients
groups. The results reflect a non significant difference (P>0.05) in the
activity of both benign and malignant groups, while the specific activity
show a significant decrease (P=0.01) in malignant group and a non
significant decrease (P>0.05) in benign group, in comparison to that of the
control group.

Table (3 - 16) Mean value of alkaline RNase activity and specific

Activity in sera of control and patient groups
MeanSD
Specific
Sample Age (year) Activity103
GROUP Activity
Number MeanSD U/L
U/mg
Control 32 35.6611.64 18.853.67 0.420.34
Benign 14 35.7815.63 55.9724.64** 0.620.24*
Malignant 19 46.5715.21 62.0817.65** 0.650.16*
**Highly significant difference in comparison to control at (P<0.001)

Table (3 - 17) Mean value of alkaline RNase activity and specific

MeanSD
Sample Age (year) Activity103 Specific

GROUP
Number MeanSD U/L Activity U/mg
Control 32 35.6611.64 15.603.89 7.672.23
Benign 14 35.7815.63 15.519.20 6.625.23
Malignant 19 46.5715.21 15.847.27 4.272.61*
*Significant difference in comparison to control at(p 0.01)

3.4.3.2- Acid RNase activity & specific activity

The acid RNase activity in sera and saliva samples of control and oral
tumor patient groups was measured as described in (2.2.16.2). The
results of sera acid RNase activity (U/L) and specific activity (U/mg) are
present in Table (3 - 18) and reflect a non significant increase (P> 0.05) in
activity and specific activity in both benign and malignant groups in
comparison to that of the control group. The results in Table ( 3 - 19)
show the activity and specific activity of acid RNase in saliva of the three
studied groups, and reveal a non significant difference ( P>0.05 ) in both
activity and specific activity of benign and malignant groups in
comparison to that of the control.
Table (3 -18 ) Mean value of acid RNase activity and specific activity
MeanSD

GROUP
Control 32 35.6611.64 17.424.27 0.340.22
Benign 14 35.7815.63 24.9614.89 0.300.19
Malignant 19 46.5715.21 24.6517.65 0.300.20

Table (3 - 19) Mean value of acid RNase activity and specific activity
In saliva of control and patient groups
MeanSD

GROUP
Control 32 35.6611.64 5.982.20 4.171.47
Benign 14 35.7815.63 8.306.65 4.013.20
Malignant 14 46.5715.21 6.605.59 2.643.21
3.4.3.3- Alkaline RNase isoenzymes

In order to obtain an idea about the variation in alkaline RNase
profile in sera and saliva samples of the studied groups, a discontinues
electrophoresis using Lammali system in the presence of 12.5%
acrylamide as a separating gel was used as described in (2.2.18.3), the gel
was stained for RNase activity as described in (2.2.19.7 ).
Figure (3 - 9) and Figure (3 - 10) show the electrophoresic patterns
of the protein and RNase activity respectively of sera and saliva samples
among the studied groups. It is clear from the Figure (3 - 10) that the
enzyme present in different forms in sera samples of the studied groups.
Reflect the different sources of this enzyme in the sera. These forms are
different in their relative mobilities, most of them are with relatively low
mobility on the gel. Also the electrozymogram show that there are
RNase's bands with different intensities that move faster than the other in
all sera samples. which reflect changes in their molecular weight and

charges. When this enzyme activity was localized on the gel for the saliva
samples, It is obvious from Figure (3-10) that the enzyme present in one
form in saliva samples of each group with different mobility in the gel.
Separating gel 12.5%
Stacking
gel 4%
Figure (3-9): Conventional poly acrylamide gel electrophoresis 12.5%,

using tri-glycin pH 8.2 as electrode buffer. The gel
was stained for protein. The samples used were as
follows:

(+)
Separating gel 12.5%

(-)
Stacking
gel 4%
using tri-glycin pH 8.2 as electrode buffer. The gel
was stained for alkaline RNase activity. The samples
used were as follows:
1 pooled crude serum (malignant) 4 pooled crude saliva (control)
2 pooled crude serum (benign) 5 pooled crude saliva (benign)
3 pooled crude serum (control) 6 pooled crude saliva (malignant)

Discussion
In the current study, alpha- amylase activity was measured in sera and
saliva samples of normal individuals (control) and oral tumor patient
groups. The results (Table 3 - 14) reveal a non significant increase in
amylase activity in sera samples of patients group in comparison to that of
the control.
Hyperamylasemia has been reported to occur in many malignancies
such as lung (363), pancreas (152), and to lesser extent ovary (364) and also with
multiple myeloma(365). The alpha amylase present in the sera samples
actually it is the total amylase i.e. consist of both P-type and S-type,
therefore the elevation of sera alpha amylase (total amylase) may be
attributed to the (S-type) that is present within the sera alpha amylase,
which secreted from variety of tissues. Therefore, the elevation of sera
amylase (total amylase ) may not only occur in pancreatic disorders
(referred to elevated P-type), but also in mumps, intestinal obstruction and
other intestinal disorders and also in cases of malignancies.
In addition to its well- known function as a digestive enzyme,
salivary alpha amylase has been reported to act as an antimicrobial
enzyme(366), and interacts specifically with certain oral bacteria and may
play a role in modulating the adhesion of those species to teeth(367). In the
current study, salivary amylase activity showed a significant decrease in
oral tumor patient groups (Table 3 - 15) in comparison to that of the
control group. This result is in agreement with Shpitzer et.al(287) on their
study on OSCC patients and disagree with Bssalyk(345) where he observed
a significant increase in salivary alpha amylase in patient with malignant
tumors of the oral mucosa. .
It has been suggested that amylase make up about 1/3 of the total
(154)
protein content in parotid saliva so amylase is thought to be an
indicator of acinar cell function in parotid glands (368). It has been found

earlier that amylase increases with saliva flow rate, and it is generally
(157)
considered to be a reliable marker of serous cell function . The
observed reduced amylase activity in this study may be due to the low
salivary flow rate noticed in almost all the patients under the present
study.
The altered concentration and activity of various enzymes,
electrolytes, and ions in saliva may compromise various salivary
functions, so the reduced salivary amylase activity may impair salivary
digesting ability (287) and indicate the presence of dysfunction in parotid
glands.
The separation of amylase isoenzymes by gel electrophoresis has
been shown to be reliable and reproducible (368). The differences in sera
and saliva amylase activity was confirmed by the elecrtophoretic profile
(Figures 3-7, 3-8) respectively, where it was obvious from the
electrophoresis zymogram that the sera amylase consist of ( P-type & S-
type) which they differe in their mobility toward the anode, where the
increased isozymes referred to the secretion of S-type from variety of
tissues including malignant one(369).
Salivary amylase zymogram show a decreased in bands number and
intensity in benign and malignant groups when compared with the control
group, and this may due to the dysfunction of parotid gland which is the
major source of salivary alpha amylase in the saliva. Human alpha
amylases as known are calcium metalloenzymes(247), and the results of
increased calcium concentration (Table 3 - 8 ) is with the increased sera
amylase activity.
Throughout this study, acid and alkaline RNases activities were
measured in sera and saliva samples of control and oral tumor patient
groups. The results Table (3-16) reveal that oral tumor patient groups have
highly significant increased RNase activity in sera samples. The observed

increased in sera alkaline RNase is in agreement with many other studies

(360)
in different kinds of cancer such as ovarian cancer , pancreatic
cancer(370), leukemia (371), multiple myeloma(372), and uterine cancer(361). It
have been suggested by a number of authors that the levels of RNase
activity in human body fluids, such as serum, urine, and saliva may be an
indicative of disease states (373).
Generally it is known that cancer cells are characterized by
uncontrolled increase in the number and size(374) , which mean that there
will be an increase in the synthesis of different proteins and this explain
the need for high RNase activity.
Although many human tissues express ribonucleases, the origin of the
serum RNase has not been established (375). The increase in RNase activity
may be attributed to many reasons such as the lack of a host defence
mechanisms, or it may be produced by malignant cells, a secondary
destructive process in other cell or tissue (375), & this is confirmed by the
appearance of different bande on the gel which have different mobilities in
the acrylamide gel.
RNase activity in saliva during this study, was also investigated, and
the results Table (3 17) revealed a non significant increase in RNase
activity and this result disagree with AL-Issa(362) study on breast cancer
patients where he found a highly significant increase in RNase activity.
There are some reports on human RNase which indicate the presence of an
elevation in the activity of this enzyme in saliva of patients with different
diseases such as cystic fibrosis (CF)(235), influenza(376), and ovarian and
breast tumors(360,362).
Actually little is known about human salivary RNases & its molecular
nature and properties (377) . Saliva is known to contain ribonucleases from
various sources(378,379). It is not clear how RNA and RNase can coexist in
the saliva. RNA can enter the oral cavity through various routes, including

saliva secretions from the three major salivary glands and minor glands,
like many micro and macro molecules, RNA in salivary glands secretions
could originate from acinar cells(380), or from gingival crevice fluid (GCF),
because many blood cells and their cellular components are released into
the oral cavity from GCF(381), or from desquamated oral epithelial cells,
where turnover of epithelial cells in the oral cavity could be another
source of RNA. Also micro wounds inside the oral cavity could release the
RNA directly from the blood (382).
The cause of such elevation in activity may be attributed to the
alteration in cell permeability of tumor cell membranes where a number of
changes in the biochemical characteristic of malignant cell surface have
been observed. Such changes lead to transport of enzymes from the blood
to the saliva via salivary glands, and one of these enzymes is RNases.
Furthermore, some divalent cations or other parameters that activated the
RNase may be transported from the blood to the saliva with the disease
such as cystic fibrosis (235), and this increase in their level in saliva, may
lead to the observed elevation in salivary RNase.
Salivary RNA is protected from degradation by the association with
macromolecules (382). Saliva contains mucin (proteins that forms oligomers
which are highly glycosylated)(383), and it is possible that in saliva some
RNA is protecting from degradation by their association with these
oligomers. This may be the reason for the reduction of salivary RNase
specific activity.
Another reason may be the reduction of some salivary trace
elements concentration like manganese Mn (Figure 3- 16 ) which was
suggested(360) as one of the RNase activators.
The electrophoretic profile of RNase in sera and saliva samples
Figure (3-10) reflect the increase in RNase activity in the oral tumor
patient groups, where it is obvious the presence of increased isoenzymes

in benign and malignant groups and also these isoenzymes ( forms) are
different in their mobility in the gel, that mean different in their molecular
weight.

Oral cancer is the sixth most common cancer worldwide (26). The
disease is characterized by a high rate of morbidity and mortality
(about 50%) (384). The burst of reactive oxygen species (ROS) has been
implicated in the development of oral cavity cancer (385). (ROS) such as
superoxide radicals (O2), hydroxyl radicals(OH) and hydrogen
peroxide(H2O2) play a key role in human cancer development(66, 386). The
pathology associated with ROS is derived from their ability to modify
cellular and extracellular macromolecules, such as protein, lipid, and
DNA, and to disrupt cellular function(387,388). Cells have developed
elaborate antioxidant defense system to prevent oxidative damage and to
allow survival in an aerobic environment(310). This system includes
enzymatic activities such as superoxide dismutase (SOD), catalase (CAT),
peroxidase, and ceruloplasmin oxidase as well as non-enzymatic
antioxidants including vitamins E, C, A, melatonin, uric acid, albumin
glutathione(64, 389)
. Antioxidant defense system work cooperatively to
alleviate the oxidative stress caused by enhanced free radical
production(390).
In the enzymatic salivary antioxidant system, peroxidase is by far
the most important enzyme (391). However, superoxide dismutase exists in
a few isoenzymes in saliva and has a secondary antioxidant role (392). Other
detectiable salivary enzymes, are catalase, glutathione peroxidase and
glutathione reductase and ceruloplasmin oxidase.
Sun Y. Concluded that detection of antioxidant enzymes may be a
useful future marker in the molecular diagnosis of the oral cancer and that
it may be possible to monitor oral cancer and the effectiveness of chemo
preventive and therapeutic strategies in oral cancer and tumor
recurrence(393).
Neoplastic diseases activate antioxidant systems. As a result,
concentration of redoxal enzymes and their co-factor elements appear to

change (394). Trace elements have been extensively studied in recent years
to assess whether they have any modifying effects in the etiology of
cancer. Copper, zinc, manganese, and iron are essential for numerous
enzymes and therefore it is reasonable to assume that variations in sera
and saliva level of these biochemical markers may be associated with the
pathogenesis of oral cancer.
To our knowledge, there is no report in the literature regarding to sera
and saliva oxidant /antioxidant system in patients with different oral
tumors (benign and malignant).
This part of the present work was conducted to investigate the
alternation in the activities of antioxidant enzymes in sera and saliva of
healthy individuals and oral tumor patients. To achieve this aim,
ceruloplasmin oxidase (CP), total superoxide dismutase (SOD), and
peroxidase activities were followed, and explained their relation to some
trace elements was tested. The study also included the electrophoretic
profile of such enzymes.
3.5.1- Ceruloplasmin Oxidase

Ceruloplasmin is a multifunctional protein, it is able to transport
copper as well as perform its (oxidase & ferroxidase) enzymatic
activity(184). Its synthesis and secretion is related to inflammation (149)
,
therefore the measurement of ceruloplasmin can be useful to monitoring
the progress of inflammation or response to treatment (395). Ceruloplasmin
is one of the important antioxidants, its antioxidants protection derives
mainly from its oxidase activity, since it can oxidize polyamines, which in
turn are associated with tumor progression. Ceruloplasmin are found to be
increased in breast, cervix and endometrium cancers and oral
leukoplakia(184, 396). To our knowledge there is one study (260) measured

ceruloplasmin concentration in sera and saliva of OSCC patients but not

its activity and specific activity.
In this part of the study, ceruloplasmin oxidase activity and specific
activity were measured in sera and saliva samples. And the variations in
ceruloplasmin oxidase isoenymes were observed by using conventional
PAGE.
Determination of copper concentration in sera and saliva samples and
its related to ceruloplasmin oxidase activity was also included in the
current study.
Results
3.5.1.1- Ceruloplasmin Oxidase activity & specific activity
Modification of Rice method(233) using PPD-2HCL as a substrate
was used in the current study to measure ceruloplasmin oxidase activity in
sera and saliva samples of control and oral tumor patients as described in
(2.2.15). The results in Table (3 - 20) presented the mean value of
ceruloplasmin oxidase activity U/L and the specific activity U/mg in sera
samples. The results reflect the presence of a significant increase (P<0.05)
in CP oxidase activity of malignant group, and a non significant increase
(P> 0.05) in benign group, in comparison to that of the control group.
Regarding the specific activity, non significant increase (P> 0.05) was
found in both benign and malignant groups in comparison to that of the
control.
Mean value of the activity and specific activity of ceruloplasmin
oxidase in saliva samples of control and oral tumor patient groups, are
present in Table ( 3 - 21 ), and reveal the presence of a significant
increase (P<0.05) in malignant group, with non significant increase in
benign group (P>0.05) , in comparison to that of the control group.

Meanwhile, the results of specific activity, show non significant

differences (P>0.05) in saliva samples of both benign and malignant
group, when compared with the control group.
Table (3- 20 ) Mean values of sera CP activity and specific

MeanSD
Specific
GROUP Activity 10
Number MeanSD U/L
U/mg
Control 32 35.6611.64 55.212.13 0.720.23
Benign 14 35.7815.63 65.9253.92 0.760.63
Malignant 19 46.5715.21 75.5830.87* 0.820.52
Table (3- 21 ) Mean value of salivary CP activity and specific

MeanSD

GROUP
Number MeanSD U/L 10 U/mg
Control 32 35.6611.64 2.731.35 1.230.75
Benign 14 35.7815.63 3.22.01 1.211.12
Malignant 19 46.5715.21 4.133.51* 1.110.97

3.5.1.2- ceruloplasmin oxidase electrophoretic profile

A conventional polyacrylamide gel electrophoresis (7.5%) was used
as described in (2.2.18.1), to detect the differences in ceruloplasmine
oxidase isozymes (forms) in sera and saliva samples among the studied
groups.
The gel was stained for CP oxidase activity as described in (2.2.19.3).
Figure (3 - 11) represent the electrophoretic profile of CP oxidase in sera
and saliva samples, where Lanes 1, 2, 3, 4 represent the sera samples, and
Lanes 5, 6, 7 represent the saliva samples. From the comparison between
the activity bands in the zymogram of the studied groups, it is clear that
band no.1 present in the sera of all the studied groups. Bands no. 2, 3, 4
are present in benign and malignant groups only (Lane 2, 3) but band no. 5
is present in malignant group only (Lane 3, 4).
Band no. 6 is present in the sera of all the studied groups , but it seems to
be more compact in the malignant group (Lane 3 ), than in control and
benign (Lane 1,2 ).
For saliva samples, distinct differences were observed between
zymograms of the studied groups, where band no.1' is present in the
control and benign groups only (Lane 5, 6). Band no.2' is present in the
control group (Lane 6) only. Bands 3'& 4' (Lane 7) are present in
malignant group only and are absent in control and benign groups.

7
6 6'
5 5'
4
3 4'
3'
2 1'
2'
1
Figure (3-11) Conventional poly acrylamide gel electrophoresis (PAGE)

15v/cm. The gel was stained for CP oxidase activity.
The samples applied were as follows:
1 pooled crude serum (control) 5 pooled crude saliva (control)
2 pooled crude serum (benign) 6 pooled crude saliva (benign)
3 pooled crude serum (malignant) 7 pooled crude saliva (malignant)
4 crude serum (individual 8 crude saliva (individual
malignant) malignant)

3.5.1.3 Ceruloplasmin oxidase activity & copper concentration

The results in Figure (3- 12) and Figure (3- 13) show the results of
copper level in sera and saliva of control and patient groups. The increase
in serum copper was non significant (P> 0.05) in both benign and
malignant groups in comparison to that of control. While the increase in
salivary copper was significant (P< 0.01) in malignant group, in
comparison to control group.
C= 5.880.70
B= 5.991.07
M=6.240.73
Figure (3-12): Mean values of serum Cu level (mol/l)

in control and oral tumor patient groups
7
Control(n=32)
Benign(n=14)
6
Malignant(n=19)
5
Cu Conc. (umol/l)
Control=4.510.42
1
Benign=4.620.55
0 Malignant=5.110.81
Saliva
Figure (3-13): Mean values of saliva Cu level (mol/l)

in control and oral tumor patient groups

Copper and ceruloplasmin are usually closely correlated with each other,
since over 95% of plasma copper is bound to CP.
Throughout this study the ratio Cu Conc./ CP activity was calculated
and presented in Table (3 - 22) and shows a significant decrease (P<0.05)
in both sera and saliva samples of oral tumor patient and control groups.
Table (3 -22): Mean value of Cu concentration / CP oxidase activity in

sera and saliva samples of control and patient groups
MeanSD
Sample Age (year) Sera Saliva

GROUP
Number MeanSD [Cu]/CPactivity [Cu]/CPactivity
Control 32 35.6611.64 0.1060.059 1.650.31
Benign 14 35.7815.63 0.090.019* 1.450.27*
Malignant 19 46.5715.21 0.0820.023* 1.240.23*

*Significant differences in comparison to control at (P<0.05)
Discussion
In this study, CP oxidase activity and specific activity were
increased, and this is in agreement with many studies such as on brain
(275) (397)
cancer , and gastrointestinal cancer . In cancerous processes an
enzymatic activation of ceruloplasmin was occurred (149). The increase of
ceruloplasmin activity may be associated with enhanced synthesis of
ceruloplasmin in the liver as one of the positive acute phase reactant
protein (149) which their levels increased upon malignancies.

It was reported that ceruloplasmin plays an important role in

protecting a variety of tissues from free radical injury (398), the antioxidant
protection of CP drives mainly from its ability to oxidize poly amines
which in turn associated with tumor progression (399), also it can play an
important role in preventing the formation of free radicals by controlling
the levels of highly toxic iron. Most evidence points to CP ferroxidase
activity as an antioxidant activity; conversion of Fe+2 to Fe+3 may reduce
the oxidation by inhibition of the Fenton reaction (which requires reduced
metal), by decreasing the amount of the pro-oxidant Fe+2or an Fe+2 / Fe+3
complex or by causing iron sequestration by apo-transferrin(108, 400).
Copper and ceruloplasmin are usually closely correlated with each
other; since ceruloplasmin is the major copper- binding protein (over 95%
of plasma copper is bound to CP). Copper ions are involved in the
sequence of events leading to angiogenesis, and when copper bound to
CP, this binding increase angiogenic activity and correlate with tumor
incidence burden and malignant progression (401). Ceruloplasmin can also
induced angiogenesis but only when Cu was bound to it(401).
Generally increased Cu observed with malignancies and that it is likely
associated with the increased neoplastic synthesis of CP and/or decreased
catabolism of this protein in cancer patients (402).
Copper plays an important role as a component of enzymes involved
in redox reactions, such as ceruloplasmin and superoxide dismutase(403).
The ability of Cu to cycle between a stable oxidized Cu (II) and unstable
reduced Cu (I) states is used by the cuproenzyme to directly bind and react
with molecular oxygen in a number of oxidation-reduction reactions. On
the other hand, copper has an oxidant property (404) by generating free
radicals such as (OH) and (O2) through Fenton and Haber-Weiss
reactions (Figure 1- 4).

In the present study, an increase in sera and saliva copper level was
observed in oral tumor patients group as compared to that of control. And
(259, 263)
this result in agreement with many studies on oral SCC , while
Varghese et.al. Concluded a significant reduction in serum copper in oral
(205)
cancer and leukoplakia patients . Serum copper was found to be
elevated in different kinds of cancer, like oral leukoplakia and squamous
(405) (406)
cell carcinoma lung cancer , osteosarcoma(407, 408)
, Hodgkin's
disease(409), breast cancer(410), carcinoma of larynx(411), and carcinoma of
cervix uteri(412).
Copper has been found to behave as a molecular switch for
activating cytokines, interleukin-1(IL-1), tumor necrosis factor (TNF-)
and growth factor such as basic fibroblast growth factor, all these factors
have shown to be angiogenic, and copper seems to act as co-factor
allowing for the angiogenic activator to become function(294) . Moreover
the role of copper ions in biological damage is caused by superoxide
radicals or other reducing agents as ascorbate, which reduce the copper
complexes and these complexes react with hydrogen peroxide to form
hydroxyl radicals that cause damage to protein, RNA, DNA that are not
repairable by cellular mechanisms thus intiating the malignant process(413).
The rise in sera and saliva copper level may be due to increased turnover
of ceruloplasmin in the sera and saliva of the carcinoma patients (405).
Throughout this study [Cu]/ CP activity ratio show a decrease in both sera
and saliva samples of oral tumor patients in comparison to that of the
control. The [Cu]/CP activity ratio (Table 3-22) reflect the concentration
of Cu that is either bound to albumin or free copper. This form of Cu is
consider to be one of the pro- oxidants in the body, where transition
metals such as Fe+2 or Cu+, catalyze formation of the hydroxyl radical
(OH) from hydrogen peroxide in the nonenzymatic Fenton reaction

(Figure 1 - 4). So the decreased free copper may be a defense mechanism

against tumor presence.
The increase in the ceruloplasmin oxidase activity in sera and saliva
samples, is confirmed by the poly acrylamide gel electrophoresis (PAGE)
patterns, where the gel reveal the presence of additional bands in benign
group as well as in malignant groups (Lane 3, 4 respectively), these
isoenzymes seems to be with high molecular weight (slow mobility)
Whereas, the saliva pattern show also an additional bands in the benign
and malignant groups (Lane 6, 7 respectively). But in malignant group the
isoenzymes seem to have a low molecular weight, because they move
faster than those in the benign group. So such differences are responsible
of the variation which observed in the cancer cells.
3.5.2- Total Superoxide Dismutase(SOD)

The aerobic metabolism is the most important source of Othat
contributes to the production of other ROS involved in many pathological
mechanisms. O can be a precursor of various metabolites that induce a
deleterious effect on biological structure. The cell defense system is very
sophisticated and the SOD is one of the first antioxidant lines of defense
against ROS, protecting the cell against the production of other deleterious
metabolites (414).
Superoxide dismutase SOD is a family of antioxidative enzymes that
act as a first cell defense against oxidative stress, catalyzes the dismutation
of O to H2O2(54, 67)
. Fiaschi et.al. (415) who found low level of SOD
activity in blood and tissue of oral cancer patients, explained such
reduction to the increased utilization of antioxidants to scavenge the
reactive oxygen species. Karincaoglu et.al.(416) found salivary SOD levels
are high, whereas plasma level are low in patients with recurrent aphthous

stomatitis, it has been thought that, salivary defense mechanisms via

antioxidant agents may be stimulated against to the ulcerous lesion.
In this part of the work, total SODs activity was measured in sera and
saliva samples of healthy individuals and oral tumor (benign& malignant)
patient groups. Also the concentration of related trace elements was
measured among the studied groups. Furthermore the study includes
electrophoresis technique to detect the variations in SOD isoenzymes upon
the presence of oral tumors.
Results
3.5.2.1- SOD activity and specific activity
Total SODs enzymatic activities in sera and saliva samples were
determined by the indirect spectrophotometric method, and employing the
NBT reduction assay, as described in (2.2.13).
The mean value of sera total SOD activity (u/ml) and specific
activity (u/mg) in control and oral tumor patient groups are presented in
Table (3 - 23). These results show the presence of non significant increase
(P> 0.05) in both benign and malignant groups in comparison to that of
the control group. On other hand, when the activities expressed by means
of the specific activity, the results show a non significant decrease
(P>0.05) in both benign and malignant groups in comparison to the
control group. Salivary SOD activity results were presented in
Table (3 - 24), and show non-significant differences in both benign and
malignant groups when compared with the control group. While the
salivary specific activity results reflect the presence of a significant
decrease (P< 0.05) in the malignant group, and was non significant
difference (P> 0.05) for the benign group, in comparison to that of the
control.

Table (3-23) Mean values of sera total SOD activity & specific activity
In Control and patient groups
MeanSD

GROUP
Number MeanSD U/ml U/mg
Control 32 35.6611.64 15.275.2 0.2040.08
Benign 14 35.7815.63 17.496.23 0.1980.08
Malignant 19 46.5715.21 17.1710.76 0.1880.12
Table (3-24) Mean values of salivary total SOD activity & specific
Activity in control and patient groups
MeanSD

GROUP
Number MeanSD U/ml U/mg
Control 32 35.6611.64 16.89.28 8.414.52
Benign 14 35.7815.63 12.57.36 6.184.88*
Malignant 19 46.5715.21 17.811.85 5.044.41

3.5.2.2- SOD activity & related trace elements

Zinc level was determined in sera and saliva of control and oral
tumor patient groups, and the results presented in Figure (3- 14), (3- 15).
The results in Figure (3- 13) reveal the presence of significant increase
(P<0.05) in serum zinc level in malignant group, and non significant
increase in benign group. But with saliva samples, the results show non

significant difference (P> 0.05) in zinc level in patient groups in

comparison to that of the control
Control( n=32)
6 Benign(n=14)
Malignant(n=19)
5
Zn Conc.(umol/l)
4
1
Control=3.890.75
Benign=4.230.41
Serum
Figure (3-14): Mean values of serum Zn level (mol/L) in

Control and oral tumor patient groups
Control:n=32
5
Benign:n=14
4.5 Malignant:n=19
4
Zn Conc.(umol/l)
3.5
3
2.5
2
1.5
1 Control=3.220.84
0.5 Benign=3.291.17
Saliva
Figure (3-15): Mean values of saliva Zn level (mol/L) in

Figures (3- 16) and (3- 17) show the results of manganese level in
sera and saliva of control and oral tumor patient groups. In sera samples,
the increase of manganese level was significant (P< 0.05) in malignant
group, but it was non significant (P> 0.05) with benign group in

comparison to that of the control. In saliva samples, manganese level show

a highly significant decrease (P < 0.001) in both benign and malignant
groups in comparison to that of the control.
Control(n=32)
9
Benign(n=14)
8 Malignant(n=19)
7
6
Mn Conc.(umol/l)
2 Control=6.750.89
1 Benign=6.911.70
0
Malignant=8.040.80
Serum
Figure (3-16): Mean values of serum Mn level (mol/L) in

Control(n=32)
12
Benign(n=14)
Malignant(n=19)
10
8
Mn Conc.(umol/l)
2
Control=9.261.38
Benign=5.080.61
Saliva
Figure (3-17): Mean values of serum Mn level (mol/L) in

In order to draw a conclusion concerning the significance role

played by Cu, Zn, and Mn in oral tumor patient, the ratios of element
concentration to SOD activity which represent the non SOD metal
fractions were calculated.

The ratios of [Cu] /SOD activity, [Zn] /SOD activity and [Mn] /SOD
activity were determined in sera and saliva samples of the three studied
groups. The results in Table (3 - 25) show the comparison between [Cu]
/SOD activity [Zn] /SOD activity and [Mn] /SOD activity ratios in sera of
the control and oral tumor patient groups. Non significant differences
were observed (P> 0.05) in these ratios between control and patient
groups.
Table (3-25 ) Comparison between different ratios in sera of

Control and patient groups
MeanSD (umol/l) 10
Sample Age (year) [Cu] / SOD [Zn] / SOD [Mn] / SOD

Group activity
Number MeanSD activity activity
Control 32 35.6611.64 0.560.23 0.370.14 0.650.28
Benign 14 35.7815.63 0.410.14 0.280.15 0.540.31
Malignant 19 46.5715.21 0.540.29 0.410.27 0.610.31
On other hand, the ratios of [Cu]/SOD activity, [Zn]/SOD activity,

and [Mn]/SOD activity in saliva sample of control and oral tumor patient
groups are represented in table (3 - 26). These results show a significant
decrease ( P< 0.05 ) in [Mn]/SOD activity ratio in malignant and benign
group in comparison to that of the control, but non significant differences
(P> 0.05) were observed in both [Cu]/SOD activity & [ Zn]/SOD activity
ratios in saliva of control and patient groups.

Table (3-26) Comparison between different ratios in Saliva of Control

and patient groups
MeanSD (umol/l) 10
Sample Age (year) [Cu] / SOD [Zn] / SOD [Mn] / SOD

Group
Number MeanSD activity activity activity
Control 32 35.6611.64 0.530.29 0.410.35 0.980.59
Benign 14 35.7815.63 0.520.39 0.340.24 0.610.47*
Malignant 19 46.5715.21 0.640.48 0.330.24 0.530.41*
*Significant difference in comparison to control at ( P< 0.05 )
3.5.2. 3- The electrophoresis patterns of SOD

Isoenzymes
Conventional polyacrylamide gel electrophoresis (PAGE) 7.5%

was used, as described in (2.2.18.1) to illustrate the differences in the
separation profile of SOD isoenzymes in sera and saliva of the different
studied groups. Localization of SOD activity in the gel was carried out
using a system containing NBT and riboflavin as described in (2.2.19.2)
where achromatic zones localize the SOD activity.
Figure (3 - 18) reveals the electrophoretic profile of the crude
serum and saliva of the studied groups. Upon the comparison of SOD
activity zymogram in sera samples of the control and patient groups ( lane
1, lane 2 & lane 3) it become obvious that there is a distinct variations
between the studied groups, where band no. 1 is present in the sera of all

the studied groups, whereas band no. 2 disappear in benign and malignant
groups ( lane 2 and lane 3 respectively), bands 3&4 are present in all the
studied groups, bands 5 &6 are present in control & benign groups ( lane 1
& 2 respectively ) and disappear in malignant group ( lane 3 ). Bands 8 &9
are present in the sera of all the groups. Finally band 10 is present in the
sera of the control and benign groups (lane 1& lane 2) and not in the sera
of the malignant group (lane 3). The lanes 4, 5 & 6 represent the SOD
activity zymogram in saliva samples of the control and the patient groups.
It is obvious that there is a variation between the three studied groups,
where bands no. 1', 2', 3' & 4' are present in the saliva of the all studied
groups, but bands 5', 6' are present in the saliva of benign and malignant
groups ( lane 5 & 6 respectively) only and not in the control group (lane4).

(+) 11
10
9 6'
5'
8
7
6
5 4'
4 3'
3 2'
2
1 1'
(-)

15v/cm. The gel was stained for SOD activity. The
samples applied were as follows:
1 crude pooled serum (control) 4 crude pooled saliva (control)
2 crude pooled serum (benign) 5 crude pooled saliva (benign)
3 crude pooled serum (malignant) 6 crude pooled saliva (malignant)
7 Albumin

3.5.2.4 - Determination of SOD isoelectric point

The isoelectric point (pI) is an important physical parameter for
protein. In order to determine the pI for SOD, a pH gradient (3.5-10.0)
was produced electrophoretically in 5% polyacrylamide gel, as described
in (2 .2.20). Figure (3 - 20 ) presents the focused gel which stained for
protein and for SOD activity. The pI points were determined by measuring
the distance between anode and the visualized band, and by extrapolating
the pI from the pH gradient curve Figure (3 - 19). It is obvious that several
activity bands appear, in both serum and saliva samples, and they have a
wide range of pI expanding between 3.8 and 6.2 .
12
10
8
pH
6
4
2
0
0 2 4 6 8 10 12 14 16
Distance from cathod(cm)
Figure (3-19 ): pH gradient profile on 5% polyacrylamide gel

containing Ampholine(3.5-10)

(-)
Sample
Application
(+)
Figure (3-20): Elelctrofocusing banding pattern stained for SOD

activity. Analytical isoelectric focusing was carried out on
5% ampholine polyaerylamide gel with a PH range 3.5-10
using 1.0 M H3PO4 as anode electrode solution , and 1.0
M NaoH as cathode electrode solution.
The samples applied were:
1 pooled crude saliva (malignant) 4 pooled crude serum (malignant)
2 pooled crude saliva (benign) 5 pooled crude serum (benign)
3 pooled crude Saliva (malignant) 6 pooled crude serum (malignant)

Discussion
Antioxidant enzymes such as SOD provide the first line of cellular
defense against ROS, protecting the cell against the production of other
deleterious metabolites (414). Reactive oxygen species such as O2, OH&
H2O2 play a key role in human cancer development.
Total SOD activity, in the current study, show an increase
(non significant) in sera samples of the patient groups (Table 3- 23 ). This
is in agreement with AL-Habal(417) , who reported an increase in plasma
total SOD activity of breast cancer patients, whereas the result disagree
with AL-Ani(418) who found a decrease in serum total SOD activity in
brain cancer patients.
SOD metabolizes free radicals and dismutates superoxide anion
(O2) to (H2O2), and protect the cell against O2 mediated lipid
peroxidation(419), Since there is an enhanced of free radical activity that
which cause endothelial damage, the body raise the level of its
antioxidants in order to combat such oxidative stress or oxidative
damage(420). Several studies point out the relation between the
inflammatory process and free radical mechanisms(394). Enzymes of the
oxidative system may be induced by free radicals, which are produced as a
result of infiltration and activation of lymphocytes. Human peripheral
blood lymphocytes have been shown to be more sensitive to such oxidant
agents as H2O2, and exhibiting more damage (DNA strand breaks) than
other cell types (421). The presence of SODs enzymes in plasma is in all
probability the result of passive leakage from cells (422), where proteolysis
process may attribute to the observed increase of total SOD activity in sera
samples.
Potent free radicals attack on the oral mucosa leading to various
alternations in a wide spectrum, from infection to lethal cancer(140). In the
current work, a reduction in salivary SOD activity was observed, and this

might be due to the depletion of the antioxidant defense system. This

could occur as a consequence of overwhelming free radicals in the
(423)
circulation of oral cavity cancer patients . Infiltration of immune
system defense cell into lesion area of oral tumor has been reported, and
killer activity emerged from these cells result in an increase in the
concentration of free radicals (416). Dismutation of increased superoxide
radicals, in particular, can be achieved by high SOD activity (424).
The increased activity of SOD result in overproduction of H2O2 which
is the product of dismutation reaction, and it has been reported that H2O2
suppresses SOD activity and superoxide radicals O2 inhibit catalase
activity (one of the antioxidant enzymes that remove H2O2 )(425). This
may explain the observed reduction in salivary SOD specific activity.
Another reason could be due to the presence of phosphate with high
concentration in the saliva of oral tumor patients (Table 3- 9) since
phosphate was reported to be an inhibitor of Cu-Zn SOD (426).
The results of the present study clearly reveal a decreased in SOD
specific activity , when compared with the control group ( Table 3 - 23 )
this may be due to the reduction in produce of this enzyme, whereas, the
oxidative stress (reactive species) is reported to affect mRNA
expression(427, 428). The differences in total SOD activity in sera and saliva
were confirmed in the electrophoretic pattern (Figure 3 - 18) where there
are many differences in SOD isoenzymes electromobility was observed on
the gel. These differences means that antioxidant enzymes show different
patterns during neoplastic transformation and tumor cells exhibit abnormal
activities of antioxidant enzymes, when compared to their appropriate
normal cell counterparts.
The present study showed a significant increase in zinc level in sera
samples (Figure 3-14). This result is agree with Al-Rawi(259) study on
OSCC patients. Although such elevations of sera zinc levels have been

seen in patients with different malignancies such of stomach, bronchus,

and sarcomas(429), a decreased levels were also encountered in carcinoma
of larynx and lung(430). The elevated sera zinc levels probably aid in
scavenging toxic free radicals which are librated from tumor tissue (213).
Zinc may exert its antioxidant effect by decreasing the susceptibility of
essential sulfhydryl group of proteins to oxidation, and by competing with
pro-oxidant metals such as iron and copper for biological binding sites(107).
Zinc ensures the stability of the fullness of the d orbital and this makes
oxidation- reduction impossible in any environment containing Zn(258).
Zinc and copper are considered as part of antioxidant enzymes and a
significant portion of the body defense against free radical mediated
injury, through cascading enzymes, where superoxide free radicals are
reduced to hydrogen peroxide by SOD in the presence of copper and zinc
cofactors, hydrogen peroxide is then reduced to water by selenium-
glutathione peroxidase couple(431). Zinc level in saliva samples
(Figure3-15) show a non significant decrease in malignant group, and this
disagree with Kashmoola study(263) who found a significant increase in
zinc level in OSCC patients. The reduction in zinc level in bio-membranes
underlies some of the disorders associated with zinc deficiency and with a
loss of zinc from the membrane resulting in an increase susceptibility to
oxidative damage(432) and deficiency in the performance of the immune
system(433).
Manganese level (Figure 3-16) in sera samples show an increase in
patient groups in comparison to that of the control, and this is in
agreement with Al-Rawi study on OSCC patients (259). Azin et al., found
significant low Mn level in all cancer cases studied(430).
Manganese have activity against progression of carcinomatous
process, since Manganese is considered as anticarcinogenic element which

required for the activity of several enzymes like Mn-SOD that have a role
in the protection of mitochondria against oxidative stress(424).
Salivary manganese throughout this study show a significant
decrease (Figure 3 -17) and this, result disagree with Kashmoola(263) and
Ai-Rawi(259) studies on OSCC patients, where they found a significant
increase in salivary Mn. The decrease in salivary Mn level that observed
in this study may explained as follows: Mn is a required element for the
activity of many enzymes like SOD which during this study show an
increased activity in the saliva, and also for RNases activity which also
show an increased activity (Table 3- 16 ). And since the reactive oxygen
species (ROS) production can induce the expression of several genes of
antioxidant enzymes such as SOD, Catalase(427). So the decreased level of
salivary Mn may be attributed to the increased consumption of this metal
ion by tumor tissue as co factor of antioxidant enzymes to scavenge the
elevated ROS in the affected area in the oral cavity.
Throughout this study, the relationships between the ratios of
concentration of transition metal copper, zinc and manganese and total
SOD activity in sera and saliva samples of oral tumor patients were
calculated, in order to show the role played by these metal ions and SOD
in oral tumor.
Neoplastic disease activate antioxidant defense system, as a result,
concentration of redox enzymes and their co-factor elements appear to
change, so the ratios in (sera & saliva) of these elements level to SOD
activity ([Cu]/SOD activity, [Zn]/SOD activity, and [Mn]/SOD activity)
which represent the non- superoxide dismutase metal fractions were
calculated. Non-significant differences were observed (Table 3-25)
between the ratios in sera samples of the three studied groups. While only
the salivary [Mn]/SOD activity ratio (Table 3-26) show a highly
significant decrease in oral malignant tumor. This is in consequence with

the depletion of manganese concentration in the saliva of oral tumor

patients (Figure 3- 17). The observed decline in Mn level could be related
to increase of Mn up take by the tissues, since Mn is an activator of
several enzymes including, mitochondrial superoxide dismutase.
Throughout this study, the males among the different studied groups
(control, benign, and malignant) show an elevated total sera SOD activity
(75%) in comparison to that of females (25%) (results not shown). The
decrease in SOD activity in females in comparison with male indicates the
genetic differences between the two genders (428). This difference may be
attributed to the protective role played by estrogen hormone against ROS
(435)
effects . Thus the role of estrogen as antioxidant may act as a
substitution of SOD activity in females.
3.5.3- Total Peroxidases

Peroxidase is one of the important antioxidant enzymes. It is a
hemoprotein catalyzing the oxidation by hydrogen peroxide of number
of substrates. Peroxidases take part in either antibacterial actions or
cellular defense against oxidative damage by reactive oxygen
species(135). Peroxidase is the most important enzyme in the salivary
antioxidant system. It is one of the nonimmunoglobulin defense
factors(436). Two peroxidase enzymes are found in saliva: salivary
peroxidase secreted from the major salivary glands, mainly the partid
gland(140) contributes 80% of oral peroxidase activity, and
myeloperoxidase produced by leukocytes in inflammatory regions of the
oral cavity(142) contributes the remaining 20%of the oral peroxidase
activity. Oral peroxidase plays a dual role: a) control the level of
hydrogen peroxide secreted by bacteria and leukocytes present in the
oral cavity, and b) has a specific antibacterial activity, inhibiting the
metabolism and proliferation of various bacteria in the oral cavity.

Peroxidase is a metalloenzyme (haemoprotein), so it is worthy to

calculate the iron concentration to peroxidase activity ratio to evaluate
the non peroxidase iron.
3.5.3.1- Total peroxidase activity & specific activity

The activity of total peroxidase was measured in sera and saliva
samples of healthy individuals and oral tumor patient groups, as described
in (2.2.14). The specific activity was calculated in respect to protein
concentration in the samples of the studied groups.
Mean values of the activity and specific activity of total peroxidase in sera
samples are presented in Table (3- 27). The results reveal a highly
significant increase (P< 0.001), and significant increase (P<0.01) in
benign and malignant groups respectively in comparison to that of the
control. On other hand, non significant difference (P > 0.05) in the
specific activity was observed of benign and malignant groups, when
compared with the control group.
The results in Table (3- 28) present the mean values of the activity and
specific activity of salivary peroxidase of control and patient groups. The
results indicate presence of highly specific increase (P<0.001) in
malignant groups in comparison to the control. While the specific activity
results show a significant increase (P<0.05) in malignant group in
comparison to that of the control group. Whereas the difference was non
significant (P > 0.05) in activity and specific activity in benign group.

Table (3-27 ) Mean values of sera total POD activity and Specific
MeanSD
Specific Activity
GROUP 10
Number MeanSD U/L
U/mg
Control 32 35.6611.64 13.399.20 0.24.0.23
Benign 14 35.7815.63 28.8313.49** 0.330.18
Malignant 19 46.5715.21 25.6120.26* 0.270.24

**Highly significant difference in comparison to control at (P< 0.001)
*Significant difference in comparison to control at (P<0. 01)
Table (3- 28) Mean values of salivary total POD activity and
Specific activity in control and patient groups
MeanSD
Specific
GROUP Activity 10
Number MeanSD U/L
U/mg
Control 32 35.6611.64 24.2418.85 1.711.38
Benign 14 35.7815.63 29.8923.7 1.741.53
Malignant 19 46.5715.21 79.2673.26** 4.015.56*

**Highly significant difference in comparison to control at (P< 0.001)
*Significant difference in comparison to control at (P<0. 05)

3.5.3.2- Total peroxidase activity and Iron concentration

Iron level in sera and saliva samples of control and oral tumor
patient groups are represented in Figure ( 3 - 20 ), (3 - 21) respectively and
show in sera samples a highly significant decrease ( P=0.01 ), ( P > 0.01 )
in benign and malignant groups respectively in comparison to the control
group. Salivary iron show a highly significant increase (P = 0.001) in
benign group, also in malignant group the increase was significant
(P=0.05) in comparison to that of the control.
Control(n=32)
50
Benign(n=14)
45 Malignant(n=19)
40
35
Fe Conc.(umol/l)
30
25
20
15
10 Control=42.493.94
5 Benign=39.585.27
0
Malignant=39.594.25
Serum
Figure (3- 21): Mean values of serum Fe level (mol/L) in

Control(n=32)
60
Benign(n=14)
Malignant(n=19)
50
40
Fe Conc.(umol/l)
30
20
Control=39.142.57
10
Benign=44.944.19
0
Malignant=43.568.80
Saliva
Figure (3- 22): Mean values of saliva Fe level (mol/L) in


Table (3 - 29) show the mean value of [Fe] / peroxidase activity in sera
samples, and reflect a significant decrease, (P< 0.05), (P<0.01) in
malignant and benign groups respectively, in comparison to that of the
control. Meanwhile the results in Table (3 - 30) show the [Fe] /
peroxidase activity in saliva samples, and reveal a non significant increase
( P> 0.05 ) in both benign and malignant groups in comparison to the
control group.
Table (3- 29): Mean value of Fe concentration / POD activity in sera

Of Control and patient groups
Fe conc./POD
Sample Age (year)
Group activity ( mol/U)
number MeanSD
MeanSD
Control 32 35.6611.64 7.105.08
Benign 14 35.7815.63 1.690.91**
Malignant 19 46.5715.21 3.043.21*

** Significant difference in comparison to control at (P<0.01)
* Significant difference in comparison to control at (P<0.05)
Table (3- 30): Mean value of Fe concentration / POD activity in saliva

Of Control and patient groups
Fe conc./POD
Sample Age (year)
Group activity ( mol/U)
number MeanSD
MeanSD
Control 32 35.6611.64 2.362.3
Benign 14 35.7815.63 3.454.7
Malignant 19 46.5715.21 3.704.9

3.5.3.3- Peroxidase Electrophoretic Profile

peroxidase enzyme was reported to consist of acidic and basic
isoenzymes , so anodic and cathodic conventional PAGE (7.5%) were
used as described in ( 2.2.18.1 ), and (2.2.18.2 ) respectively, to illustrate
the differences in separation profile in the sera and saliva peroxidase
isoenzymes of the control and oral tumor patient groups.
The presence of peroxidase activity was detected by using two
different staining methods, Tetra methyl benzidine (TMBZ) stain as
described in (2.2.19.6A), and o-dinasidine stain as described in
(2.2.19.6B). The electrozymogram of the two different staining methods
indicate that both methods are suitable for the detection of the peroxidase
activity zone, since they gave clear distinct bands. Meanwhile it was
noticed that when TMBZ stain was used, the resolution of the bands were
better, but these bands disappeared quickly. On contrast, with o-dinasidine
stain, was produced less resolution, but the bands were stable by time.
Throughout the current study and from the observed results, o-dinasidine
stain seems to be better than TMBZ stain for detecting peroxidase
isoenzymes for its easy in use and stability.
Upon the comparison of the localized activity bands in the
zymograms of the studied groups, Figure (3 - 22 A), (3 - 22 B ), it is clear
that band no. 1 is present in the sera of all the studied groups (Lane 1, 2, 3)
In malignant group (Lane 3) more aggregated bands which migrate faster
in to the gel than in the control and benign groups (lane 1, 2).
In the saliva samples (Lane 4, 5, 6), the peroxidase isoenzymes seems
to migrate faster (low molecular weight) than the isoenzymes in the sera
samples, especially in the benign and malignant groups (Lane 5& Lane 6)
respectively.

(+)
(-)
(+)
(-)

using Tris- glycin buffer, pH 8.0 as electrode buffer.
Electrophoresis was carried out for three hours at 10 C.
The gel was stained for peroxidase using two types of
substrates:
A) TMBZ B) o- dianisidine
The samples used were:
1 crude pooled serum (control) 4 crude pooled saliva (control)
2 crude pooled serum (benign) 5 crude pooled saliva (benign)
3 crude pooled serum (malignant) 6 crude pooled saliva (malignant)
7 horseradish peroxidase enzyme ( standard)

A B
(-)
(+)

7.5%, using B-alanine / glacial acetic acid pH 4.5 as
electrode buffer. Electrophoresis was carried out for 3
hours at 4 C by using a constant current of 30 mA.
The gel was stained for peroxidase activity using two
types of substrates.
A) o- dianisidine B) TMBZ
The samples used were:
4 pooled crude saliva (control)
5 pooled crude saliva (benign)
6 pooled crude saliva (malignant)

3.5.3.4- Peroxidase Isoelectrofocusing Point (pI)

In order to determine the isoelectric point (pI) for peroxidase
isoenzymes, isoelectric point (pI) in sera and saliva samples of the
different studied groups, a pH gradient (3.5-10) was produced
electrophoretically in 5% polyacrylamide gel, as described in (2.2.20).
Figure (3- 23) presents the focused gel which stained for peroxidase
activity, as described in (2.2.19.6 A). The pI points were determined by
measuring the distance between the cathode and the visualized band and
by extrapolating the pI from the pH gradient curve (Figure 3 - 19).
The approximate pI of salivary peroxidase which was separated into
two bands was found to equal to (7.4 & 8.4), while sera peroxidase bands
were at pI (8.1, 6.95, and 5.67).

(-)
Sample
Application
(+)
Figure (3-25): Electrofocusing banding pattern stained for peroxidase

activity. Analytical electrofocusing was carried out on
5% ampholine polyacrylamide gel with a pH range 3.5-
10, using 1.0 M H3PO4 as anode electrode solution, and
1.0M NaOH as cathode electrode solution.
The samples applied were:
1 pooled crude saliva (malignant) 4 pooled crude serum (malignant)
2 pooled crude saliva (benign) 5 pooled crude serum (benign)
3 pooled crude saliva (control) 6 pooled crude serum (control)

Discussion
Reactive oxygen species (ROS) are constantly generated and
eliminated in the biological system, and play important role in a variety of
normal biochemical functions and abnormal pathological processes (60).
In the current study, total peroxidase activity in sera samples show
an increased level, this result seem to agree with those obtained by other
investigators on oral squamous cell carcinoma patients(389). As well as in
patients with advanced laryngeal carcinoma (437) and breast cancer (438).
The elevation of sera total peroxidase which observed in this study
might be due to the enhanced free radical activity which causes
endothelial damage. So, in order to combat this oxidative stress, the body
raises the level of its antioxidants. The synthesis of some enzymes of the
oxidative system were reported to be induced by free radicals which being
increased at oxidative stress(394). In other words, over production of
hydrogen peroxide (as a result of both dismutation of superoxide radicals
by SOD and immune cells) leading to increase peroxidase activity.
During this part of the study, salivary peroxidase show a highly
significant increase in malignant group, and this is in agreement with
many studies on OSCC patients(140,287).
Two peroxidase enzymes are found in saliva. Salivary
peroxidase(SPO) produced by salivary glands(140) and myeloperoxidase
(MPO) produced by leukocytes in inflammatory region of oral cavity(142).
In the reaction catalyzed by salivary peroxidase:
SCN + H2O2 + H+ HOSCN + H2O

thiocyanate is the electron-donating component (similar to glutathione in
other biological system), and the reaction is catalyzed by proxidase(439)).
Two potent antibacterial oxidizing products evolve out of this reaction:
hypothiocyanous acid (HOSCN) and its conjugated hypothiocyanite anion

(OSCN-). The acculamated antibacterial activity of the combination of

peroxidase, hydrogen peroxide and thiocyanate is much more potent than
that of hydrogen peroxide alone(440).
Salivary peroxidase and Myeloperoxidase, catalyze the reaction
involved in the inhibition of bacterial growth and metabolism, and the
prevention of hydrogen peroxide accumulation, thus protecting proteins
from the action of oxygen and reactive oxygen species (441). However, the
elevated level of peroxidase activity may be attributed to that : in the
inflamed tissue the elevated activity of the peroxidase contributed by
myeloperoxidase activity .The gingival cervicular fluid (GCF) is
constantly mixed with saliva and its flow rate increase with gingival
inflammation, this increased in GCF flow relates to increased
polymophonuclear nutrophil (PMN) levels which is rich with
Myeloperoxidase, and as a result, the overall peroxidase activity increased
contributing to the entrance of Myeloperoxidase to the saliva(442).
Higher Myeloperoxidase levels was suggested to be present in low
flow rate whole saliva supernants of subject with sever gingival
inflammation probably owing to the enhanced number of PMN which
enter the oral cavity(442) .it is worth to mention that almost all the patients
included in the current study had a low flow rate of saliva.
Serum iron levels are considered as biochemical indicators for
nutritional assessment. In most cases clinical anemia may be a
contributing factor. Iron deficiency is known to be occured in oral
cancer(310). In the current study, there was a highly significant decrease in
Iron level in sera samples of oral tumor patients, and this is in accordant
with many studies on oral cancer (200, 259) different malignancies such as
gastric cancer (443) and larynx cancer (444). Inadequate intake of food due to
burning sensation and vesiculation in the oral cavity might also be an

important factor. Reduction in the serum iron level may be due to

malnutrition caused by the tumor burden in cancer patients (445).
Salivary iron show an increased level, and this is disagree Al-rawi and
Kashmoola studies on OSCC patients (259, 263), where they explained the
decrease in salivary Fe level to the malnutrition of cancer patients.
Excess iron as with excess copper can cause free radicals production and
more cell damage (446).
Iron is regarded as co-factor of peroxidase enzyme, which its
concentration was observed to increase in oral cavity fluid in oral tumor
patients as described previously. In order to indicate the relationship
between iron concentration and peroxidase activity, the ratio of this
element concentration to peroxidase activity ([Fe] / peroxidase activity)
was calculated (Table 3 - 29). This ratio represent the non- peroxidase
metal fraction and show a significant decrease in sera samples of benign &
malignant patients, this finding confirm the depletion of iron level in the
sera of oral cancer patients ( Figure 3- 20 ) and as discussed in this part
of the study. On the contrary, the ratio [Fe] / peroxidase activity in saliva
show a non significant increase, which mean increased non- peroxidase
iron, such increase and in the presence of overproduction of H2O2 in lesion
area lead to more free radical production through Fenton reaction and
Haber-weiss reaction Figure (1- 4) since Fe is one of the pro-oxidant
elements, excess iron as with excess copper can cause free radical
production and cell damage (447).

Saliva
Serum derived protein a-Amylase Salivary flow

activity rate
[Albumin] -
Serum derived [Uric acid] - [Urea]
protein
Oral
[TP] - Bacteria
Oxidative Stress - Sacrificial
F.R. Scavenger
[Free radicals] - - NH3 + CO2
Defens
- system e.g. -[ O.2-] [H2O2]-
[globulins]-
- PH
- [CP] [Cu] - Peroxidase -

SOD
activity activity - Pi
[Mn] - ALP activity

- [Fe]
- CP oxidase
activity
- Permability of
the membrane
- Alk. RNase
activity - Lysis Cancer cell
- anoxia
- LDH activity
Scheme 2

Serum
- Synthesis of Synthesis of
[TP] -
positive acute negative acute-
phase protein phase protein e.g
e.g [CP] - [Albumin]
- [Immunoglobulins]
[Cu] - Sacrificial
F.R. Scavenger
- Oxidative Stress
- CP oxidase - Uric acid
activity - [Free radicals]
- [H2O2] - [ O.2- ] - Purin neucleotide
degredatin
Peroxidase - SOD - - LDH Activity

activity activity - RNase
activity
Anaerobic
- a-Amylase activity oxidation
Lysis
- [Ca+2] - [PTHrp] induce

Cancer cell
- ALP - Permability of DNA synthesis

activity the membrane Protein syn.
ATP syn.
- [Pi]
Neucleoside Neucleotide
Scheme 1

Table (3-31): The overall results of the current study
Serum Saliva
Parameter
Benign Malignant Benign Malignant
Total protein * ** * **
Albumin ** *** ** **
Globulin ** *** * **
Ceruloplasmin * ** ** **
IgA *** **
IgM * *
IgG ** **
Alp Activity ** * * *
Alp S.A ** ** * *
LDH Activity * *** ** **
LDH S.A * * * *
Amylase activity * * * **
Amylase S.A * * * ***
Alk.RNase A. *** *** * *
Alk. RNase S.A ** ** * **
Acid RNase A. * * * *
AcidRNase SA * * * *
CP Oxidase A * ** * **
CP OxidaseS.A * * * *
SODS Activity * * * *
SODS S.A * * * **
Peroxidase A. *** ** * ***
Peroxidase S.A * * * **
Urea * * * **
Uric acid * ** ** **
Calcium * * * *
Phosphorus * * *** ***
Copper * * ** **
Zinc * ** * *
191
Serum Saliva
Parameter
Benign Malignant Benign Malignant
Manganese * ** *** ***
Iron *** *** *** **
[Cu]/CPActivity ** ** ** **
[Cu]/SODActivity * * * *
[Zn]/SODActivity * * * *
[Mn]/SODActivity * * ** **
[Fe]/Peroxidase
** ** * *
Activity
*Non significant difference
** Significant difference
***Highly significant difference
A = Activity
S.A =Specific activity
192
Table (3-32): Correlation between sera and saliva parameters in
Malignant group
Saliva
Total Globul Uric
Alb CP Urea Ca PO4
protein in acid
Serum
Total r
protein 0.039
r
Albumin
0.319
r
Globulin
0.006
r
CP
0.365
r
Urea
0.186
r
Uric acid *
0.565
r
Calcium
-0.342
r
Phosphorus
0.196
*Correlation is significant at the 0.05 level (2-tailed)
Saliva Amylase Alk. Alk. Acid Acid ALP

Amylase ALP
specific RNase RNase RNase RNase specific
Activity Activity
Serum Activity Activity S.A Activity S.A Activity
Amylase r
Activity -.110
Amylase r
S.A -.338
Alk.
r
RNase
Activity -.227
Alk.
r
RNase
S.A 0.105
Acid
r
RNase
Activity 0.064
Acid
r
RNase
S.A 0.125
ALP r
Activity 0.000
r
ALP S.A
-.230
193
Saliva Peroxid-
LDH LDH CP CP SOD SOD Peroxid-
Ase
Activity S.A Activity S.A Activity S.A Ase
Serum S.A
LDH r
Activity -.001
LDH r
S.A -.296
CP
r
oxidase
Activity .365
CP
r
oxidase
S.A -.204
SOD r
*
Activity 0.545
SOD r
specific *
Activity 0.495
Peroxid
r
ase
activity 0.108
Peroxid
ase r
*
spesific 0.485
activity
* Correlation in significant at the 0.05 level (2-tailed)
194
Benign group
Saliva
Total Uric Phosph-
Albumin Globulin Urea Calcium
protein acid orus
Serum
Total r
protein 0.821**
r
Albumin -0.044
r
Globulin 0.820**
r
Urea 0.
r
Uric acid 0.
r
Calcium -0.259
r
Phosphorus -0.01
**Correlation is significant at the 0.0 10 level (2-tailed)

Amylase ALP
Activity Activity
Amylase r
Activity -.0238
Amylase r
S.A 0.319
Alk.
r
RNase
Activity 0.245
Alk.
r
RNase
S.A 0.111
Acid
r
RNase
Activity 0.197
Acid
r
RNase
S.A 0.587*
ALP r
Activity -0.195
r
ALP S.A
0.008
195
Saliva Peroxid-
Ase
Serum S.A
LDH r
Activity -0.048
LDH r
S.A 0.203
CP
r
oxidase
Activity 0.
CP
r
oxidase
S.A .
SOD r
Activity 0.
SOD
r
specific
Activity 0.498
Peroxid
r
ase
activity 0.702**
Peroxid
ase r
spesific 0.146
activity
**Correlation is significant at the 0.0 level (2-tailed)
196
Control group
Saliva
Total Uric Phosph-
Albumin Globulin Urea Calcium
protein acid orus
Serum
Total r
protein -0.048
r
Albumin 0.249
r
Globulin -0.224
r
Urea 0.268
r
Uric acid 0.495**
r
Calcium -0.147
r
Phosphorus 0.286
**Correlation is significant at the 0.0 level (2-tailed)

Amylase ALP
Activity Activity
Amylase r
Activity 0.319
Amylase r
S.A 0.000
Alk.
r
RNase
Activity -0.230
Alk.
r
RNase
S.A -0.186
Acid
r
RNase
Activity 0.129
Acid
r
RNase
S.A 0.341
ALP r
Activity -0.298
r
ALP S.A
-0.205
197
Saliva Peroxid-
Ase
Serum S.A
LDH r
Activity 0.24
LDH r
S.A 0.415*
CP
r
oxidase
Activity -0.118
CP
r
oxidase
S.A .112
SOD r
Activity 0.
SOD
r
specific
Activity -0.040
Peroxid
r
ase
activity -0.148
Peroxid
ase r
spesific -0.089
activity
198
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245

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Republic of Iraq

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Study of Some Biochemical

3-31 The overall results of the current study 191

1.1 The Mouth

Figure (1-1): Structure of the mouth (5)

Digestive juice present in the mouth is known as saliva, which is

Figure (1.2): Major salivary gland (3)

Figure (1-2): Major salivary glands (3)

1.1.2.1 Saliva flow rate

of hydration, body position, exposure to light, olfactory stimulation, and

1.1.2.2- Buffering capacity of saliva

CO2 + H2O H2CO3 HCO3+ H+

The second buffering system is the phosphate system, which contributes

1.1.2.3- Composition of Saliva

1.1.2.4- Major Functions of Saliva

The major functions of saliva are summarized in Table (1-1)

Table (1-1): Major functions of saliva (22)

Mucins, proline-rich proteins and H2O components

The H2O component facilitates clearance of food and

Super saturation of calcium and phosphate due to the

Bicarbonate and, to a lesser extent, phosphate and

Specific (sIgA) and non specific (lactoferrin,

Digestive enzymes found in saliva include amylase,

1.2.1- Oral Cancer

been reported in recent years to have an increasing incidence in younger

2. Heredity: Heredity plays an important role in carcinogenesis;

4. Acquired Precancerous Disorders

1.2.1.3- Sites of Oral Cancer(29)

1.2.1.4- Early Detection of Oral Cancer and Warning Signs

1.2.1.5- Diagnosis of Oral Cancer (25)

1.2.1.6- Clinical Staging (48)

1.3- Free Radicals Injury

Figure (1-3): Reduction of oxygen by four-one electron steps (52)

ROS are oxygen-containing compounds that are highly reactive free

The Fenton reaction _

The Haber-Weiss reaction

Figure (1-4): Generation of hydroxyl radical by the nonenzymatic

ROS are generated in vivo by multiple mechanisms, including the

The formation of (ROS) under pathological conditions could be

1.3.1- Antioxidants and Elimination of Free Radicals

From a biochemical point of view, an antioxidant may be defined as

1.3.2- Oxidative Stress

rheumatoid arthritis, periodontal disease (68), cataract, diabetes mellitus,

1.3.3- Oxidative stress in the cancer process

Progression stage: ROS impart further DNA alterations to the

1.3.4- Pro- oxidant and Antioxidant features of saliva

1.4- Tumor markers in cancer detection

1.5- The Diagnostic Uses of Saliva

It is becoming increasingly apparent to investigators and clinicians

1.6- The studying parameters

much as 100% by conditions that decrease serum albumin or lower

IgA: IgA forms 13% of the total body antibodies. It appears

IgM: IgM forms about 6%of the antibodies. It is referred to as the

IgD: is the immunoglobulin with the fourth highest concentration in

IgE: is the idiotypic immunoglobulin associated with allergic and

the conversion of superoxide anion O.2- to hydrogen peroxide H2O2 and

Therefore, SOD is one of the antioxidant scavenging enzymes,

cells from reactive oxygen species produced during normal

Peroxidase, having a wide range of biological functions and present

immunological and biochemical similarity to bovine milk

1.6.2.3- Ceruloplasmin oxidase

Several different cells and tissues synthesize the salivary type