Académique Documents
Professionnel Documents
Culture Documents
Graduate College
Medical and Health Studies Board
By:
Yousif Osman Yousif
BDS , MSc (Uof K)
A Thesis Submitted for the Degree of PhD in Oral and Maxillofacial Surgery
Supervisor:
Prof. A. M. SULIMAN
BDS, MSc, FRCDS, Ph.D
2014
Dedication
and
Yousif
ACKOWLEDGEMENTS
I raise my heart in gratitude to Allah Almighty for all the blessings Allah has
showered on me throughout this study.
I sincerely acknowledge with deep sense of gratitude and humble respect, the
valuable guidance and suggestions from my respected teacher and supervisor
Prof. A.M.Suliman. I am deeply indebted to him for his help, guidance, endless
support and constant encouragement throughout the period of this study.
My sincere thanks and appreciation are due to the Ministry of High Education &
Scientific Research for funding this research work. Without their help, this work
would not have come to existence.
I would like to extend my sincere thanks to the staff of Jaber Abu El Izz Diabetic
Centre & El Ribat University Hospital for their endless co- operation.
I express my sincere gratitude to Dr. Yasir Osman, Dr.Yasir Siddig and
Dr.Inaam for their help in the collection of the data.
Special thanks & gratitude to Dr.Mohamed Ali & miss Wafa for their continuous
support and encouragement.
My sincere gratitude to Miss Aisha Mohammed and Dr.Marium EL Hadi for
their great effort in the statistical analysis and for finalizing the computer work.
I heart- fully thank my senior colleagues at the department of Oral &
Maxillofacial Surgery for ever being helpful and supportive.
Last but not the least; I thank all the participants who formed an important part of
this study, without whose co-operation this study would not have been possible.
II
LIST OF ABBREVIATION
AGEs
BEC
BMI
C
CFU
D.M
EDTA
G
GLUT
HbA1c
IDDM
Ig
IL
NIDDM
P
r
RAGE
RT-PCR
SS
SD
SPSS
T
WHO
III
LIST OF CONTENTS
Item
Page no
CHAPTER ONE
1.1 Introduction & Literature Review
1 33
34 36
CHAPTER TWO
2.1 Methodology of the Biochemical Analysis of Saliva
37 42
43 46
CHAPTER THREE
3.1 Results of the Biochemical Analysis of Saliva
47 52
53 54
CHAPTER FOUR
4.1. Discussion-The Biochemical Analysis of Saliva
55 65
66 72
4.3. Conclusions:
73
4.4 Recommendations:
74
Annexures
References
75 98
99 101
IV
Title
Page No
1.1
Classification of D.M
1.2
1.3
31
3. 1
3.2
3.3
3. 4
3. 5
3. 6
3. 7
49
49
49
50
50
50
51
3. 8
51
3.9
51
3.10
52
3. 11
52
3. 12
Fig. 3. 1
53
54
ABSTRACT
Background:
Diabetes mellitus is a group of metabolic disorders that share the common
underlying feature of hyperglycemia. Hyperglycemia in diabetes results from
defects in insulin secretion, insulin action, or most commonly from both of them.
In Sudan the prevalence of the disease in 1996 was 3.4% but it has increased
dramatically to 14.5 % in 2010.
Saliva is a complex fluid consisting mainly of water, essential electrolytes,
glycoproteins, antimicrobial enzymes and numerous other important constituents.
Diabetes has been consistently documented to be associated with altered salivary
composition and function. This disrupts the homeostasis of the oral cavity, making
it susceptible to various oral infections, with candidosis reported to be the
commonest one.
The present study consisted of two parts: the first one was designed to investigate
the saliva composition; mainly glucose, amylase, total protein and urea; among
type- 2 diabetic patients in comparison with non-diabetics. The second part aimed
to estimate the salivary candidal counts among both groups.
Methodology:
The study was conducted at Jabir Abu EL Iz Diabetic Centre-Khartoum during the
period May to August 2010. A total of 120 age and sex matched participants were
divided into 3 groups: uncontrolled diabetics, controlled diabetics and healthy nondiabetics. Salivary glucose was measured using glucose oxidase end-point method,
while amylase was measured by direct substrate kinetic enzymatic method.
VI
Pyrogallol red dye and diacetyl monoxime, were the methods used to measure total
protein and urea levels respectively.
Salivary Candida albicans counts were estimated using real- time PCR.
Results:
Salivary glucose and urea levels were found to be significantly elevated in both
uncontrolled and controlled diabetics, as compared to non-diabetics. (P < 0.05).
There was no significant difference when diabetics and non-diabetics were
compared for salivary amylase and total protein. (P > 0.05).
The study showed an increased Candida albicans counts among diabetics, in
comparison to non-diabetics, with a positive correlation between salivary candidal
counts and salivary glucose levels. (P < 0.0.1, r= 0.5).
Conclusion:
Diabetics saliva showed higher levels of glucose and urea in addition to increased
candidal counts. The latter, might be a factor behind the frequent candidosis seen
among those patients. Saliva may be used as a non-invasive diagnostic, as well as a
monitoring tool to assess the glycemic status of diabetic patients. Further studies
on larger populations and in different geographic areas, are required to establish
such a role.
VII
:
.
1996 3.4 14.5
.2010
.
.
: :
.
.
:
.2010 120
. :
.
.
.
.
:
.
.
.
:
.
.
.
VIII
CHAPTER ONE
Introduction and Literature Review
1. Diabetes Mellitus
1.1.1 Definition of Diabetes Mellitus
Diabetes mellitus is defined by the American Diabetes Associations Expert
Committee as a group of metabolic disorders characterized by hyperglycemia
resulting from defects in insulin secretion or insulin action or both. The chronic
hyperglycemia is associated with long-term damage, dysfunction and failure of
various organs, especially the eyes, kidney, nerves, heart and blood vessels. Thus,
Diabetes covers a wide range of heterogeneous diseases. (1, 2)
healthcare expenditures to treat and prevent diabetes and its complications were at
least 376 billion US Dollars (USD) in 2010. By the year 2030, this number is
projected to exceed some 490 billion USD (6).
The reported prevalence of type 2 diabetes varies from zero in Togo (7), 9% in
Egypt (8) to 50% in pima Indians (USA) (9). Particular ethnic groups (e.g. South
Asian, Native American and Mexican- Americans) are highly susceptible to type 2 Diabetes, and this may be revealed when such groups migrate into relatively
affluent settings. Regional and ethnic differences in the prevalence of type-2
diabetes probably related to both the lifestyle and the underlying genetic
susceptibility (10).
Prevalence rates are relatively high (9-8%) in Tunisia as well as Egypt (8).
However, recently most Arabic countries have experienced an increase in the
disease prevalence, with the evolving trend particularly alarming in Jordan and
Egypt. (8, 11)
In the Kingdom of Saudi Arabia the prevalence of 4.95% and 4.30% was reported
during 1985 to 1987(12). In Yemen, the crude prevalence of known diabetes was
6.57% (13).
mellitus
is
classified
by
four
distinct
categories
based
on
generated. The terms type-1 and type-2 diabetes mellitus were retained, with
Arabic number being used. (18).
Type- 2 diabetes mellitus is the most prevalent form of diabetes, which results from
insulin resistance, with or without a secretory defect. It primarily occurs with
increasing age and is associated with genetic and environmental risk factors. Type-2
diabetes is commonly preceded by a long period of abnormal glycaemic control and
is part of the metabolic syndrome associated with hypertension, dyslipidaemia and
hyperglycaemia. The condition has a stronger genetic aetiology than Type-1 DM
although environmental factors such as diet, exercise, obesity and smoking have an
impact on the development of type 2 diabetes (19).
Clinical:
Clinical:
Casual is defined as any time of the day without regard to time since last
meal.
Fasting is defined as no caloric intake for at least 8hours.
Criteria 2 and 3 should be confirmed by repeat testing on a separate day.
The disease usually has a gradual and insidious onset. The diagnosis is made
incidentally (during insurance screening or during hospital visits for other medical
problems) in almost one-third of cases, and almost one-half do not complain of
obvious diabetic symptoms. Common presentations are with genital candidosis
(particularly in women), or urinary tract or skin infections (23). A significant
number of type-2 diabetic patients already have one or more chronic complications
at time of diagnosis notably diabetic retinopathy and macro vascular disease (24,
25). It has been observed that type -2 diabetes generally starts 4-7 years before the
diagnosis is made (26).
10
11
12
islets release less insulin in response to glucose and this is accompanied by altered
expression of glucose transporters (in particular GLUT2) and that of glucokinase (37).
It was found that arginine and glipenclamide induced an early insulin secretion
phase from the diabetic islets. Both arginine (by increasing intracellular positive
charge) and sulfonylurea (by inhibiting K+ loss from -cell) cause depolarization of
-cell membrane. This event is followed by Ca2+ entry, increased cytosolic Ca+
concentration, translocation and exocytosis of insulin granules. In summary it is
believed that the defects of glucose-stimulated insulin release in type 2 diabetic cells lie between glucose transport and depolarization of cellular membrane (36).
acinar cells first secrete isotonic primary saliva and then the striated duct cells
actively extract ions to render the saliva progressively more hypotonic as it passes
down the ducts towards the mouth (40).
Inorganic Composition of Saliva:
The most abundant component in saliva is water (approximately 99%), followed
by ions Na+,Cl Ca2+,K+,HCO3 ,H2PO4 , F, I,Mg2+ and thiocyanate.
The hypotonicity facilitates taste sensitivity and hydrates various organic
compounds that form a protective coating on the oral mucosa.
The bicarbonate serves as a buffering agent and calcium and phosphate neutralize
acids that would otherwise compromise tooth mineral integrity (41, 42).
The Organic Composition of Saliva:
Saliva includes a large number of organic compounds such as: urea, ammonia,
uric acid, glucose, cholesterol fatty acids, mono, di, and triglycerides, phosphor
and neutral lipids, glycolipids, amino acids, steroid hormones and proteins that aid
in the protection of the oral cavity tissues, including mucins, amylases, agglutinins,
glycoproteins, lysozymes, peroxidases, lactoferrin and secretory IgA. Non-immune
factors include lactoferrin, lysozyme, myeloperoxidase, histatins, cystatins, mucin
G1 and G2, and defensins (43-49). In addition, these macromolecules form a
viscoelastic mucosal coat and tooth enamel pellicle and aggregate and cleanse
14
bacteria and debris from the oral cavity (50). Saliva also contains a variety of
antimicrobial constituents and growth factors (51, 52).
Salivary antibody levels can be determined to screen for infectious diseases. AntiHIV antibody immunocapture assays have also been developed and tested for
saliva, which could be useful in high-risk groups under field conditions in
developing countries (61). Salivary assays have been used for monitoring of
hepatitis A, B and C, measles, Epstein-Barr virus, rubella, parvovirus B 19, human
herpes virus 6, Helicobacter pylori and rotavirus infection (55, 62). In addition to
measuring antibody, it is possible to identify a number of viral antigens in saliva,
for example mumps and cytomegalo virus. Saliva has also proven to be a
convenient source of host and microbial DNAs (57).
There has been growing interest in the use of saliva in pharmacokinetic studies of
drugs and in therapeutic drug monitoring in a variety of clinical situations. It has
been suggested that drug levels in saliva reflect the free, non-protein-bound portion
in plasma and hence may have a greater therapeutic implication than the total
blood levels (55).
Lipid solubility is a determining factor in saliva excretion of drugs, and the degree
of acidity and basicity of a drug will determine its salivary/plasma ratio. The
salivary flow rate, pH, sampling conditions, contamination and many other
pathophysiological factors may influence the concentrations of drugs in saliva (63).
Drugs currently monitored in saliva include anticonvulsants, theophylline,
16
This phenomenon can be used as a diagnostic test by measuring the flow from
labial glands of the lower lip (55). The sodium (Na) and potassium (K)
concentrations of saliva are markedly affected by corticosteroids, especially
aldosterone. The Na/K ratio of stimulated whole saliva can be used in diagnosing
and monitoring Cushings syndrome and Addisons disease. Investigators have also
demonstrated the diagnostic value of Na/K ratio in primary aldosteronism (69).
18
19
causing cell lysis and death. Many organisms, however, have cell capsules or other
cell wall protective material, which confer resistance against lysozyme attack (83).
Lysozyme has been proposed as a lytic factor for bacteria which immunoglobulins
have bound, mimicking in some respects the complement system in serum.
Lysozyme aggregates cell suspensions of some bacterial species (78). It is also
known that lysozyme contributes to mucosal protection and modulates Candida
populations in the oral cavity (84).
21
1.2.2.2.c Lactoferrin
Lactoferrin is present in plasma and in mucosal secretions (87). Salivary lactoferrin
has antibacterial activity. Lactoferrin binds iron, making it unavailable for
microbial use (83). Lactoferrin, in its unbound state, also has a direct bactericidal
effect on some microorganisms including Streptococcus mutans strains.
1.2.2.2 .d.Histatins
Histatins comprise a group of small histidine-rich proteins present in the saliva.
The most significant function of histatins may be their anti-fungal activity against
Candida albicans (88, 89). Oral candidiasis may also modulate the levels of
salivary histatin (90, 91). It has been suggested that histatins could be used as
components of artificial saliva for patients with salivary dysfunction (88).
Histatins have been shown to be tannin-binding proteins in human saliva (92, 93).
Histatins also bind to enamel surfaces and hydroxyapatite in a complex manner (94).
1.2.2.2.e. Agglutinins
Salivary agglutinins are glycoproteins which have the capacity to interact with
unattached bacteria, resulting in clumping of bacteria into large aggregates which
are more easily flushed away by saliva and swallowed (95). Bacterial binding to
salivary proteins may in part account for individual differences in the colonization
22
1.2.3. AMYLASES
Amylase ( amylase) is one of the most important salivary digestive enzymes. It
consists of two families of isoenzymes, of which one set is glycosylated and the
other contains no carbohydrate (71). Salivary amylase is a calcium metalloenzyme
which hydrolyses the alpha bonds of starches, such as amylose and amylopectin
(78). Maltose is the major end-product.
It has been suggested that amylase accounts for 40 to 50% of the total salivary
gland-produced protein, most of the enzyme being synthesized in the parotid gland
(82). Human parotid saliva and submandibular saliva contain about 45 mg and 30
mg of amylase, respectively, per 100 mg of protein (71). However, it has also been
claimed that amylase makes up about 1/3 of the total protein content in parotid
23
saliva, and the content in whole saliva would be lower (99). The concentration of
amylase increases with the salivary flow rate, and it is generally considered to be a
reliable marker of serous cell function (100).
In addition to its well-known function as a digestive enzyme, amylase has been
reported to act as an antimicrobial enzyme. Amylase activity exists also in tears,
nasal and bronchial secretions, milk, serum, urine and in the secretions of the
urogenital tract (83). Amylase also interacts specifically with certain oral bacteria
and may play a role in modulating the adhesion of those species to teeth (101). It
has been found that salivary amylase inhibits the growth of Legionella
pneumophila and Neisseria gonorrhoeae (83). Amylase is also present in human
acquired pellicle in vivo (102). Fasting has been found to decrease whole saliva
amylase levels and activity (71). The amylase concentrations in radiation-induced
hyposalivation have been found to be reduced (100).
24
hormonal balance, and osmotic pressure. The half life of albumin is approximately
15 - 20 days. About 4% of albumin is degraded per day, but synthesis can be
increased by as much as 100% by conditions that decrease serum albumin or lower
intravascular osmotic pressure (103). Nephrotic syndrome is the best known
example of systemic disorders with characteristic proteinuria and subsequent
hypoalbuminaemia which lead to oedema (104). In the oral cavity, albumin is
regarded as a serum ultrafiltrate to the mouth (105), 106) and it may also diffuse
into the mucosal secretions (107).
Salivary albumin is selectively adsorbed by different materials in the oral cavity,
which may enable the attachment of specific bacteria and thus alter the
composition of dental plaque (108). Salivary albumin has been shown to increase
in medically compromised patients whose general condition gets worse (109).
Immunosuppression, radiotherapy, and diabetes are examples of states where high
concentrations of salivary albumin have been detected (110, 111).
Salivary albumin levels have been used as a marker for the degree of mucositis and
inflammations in salivary glands (112). Butler et al. (113) found that albumin
levels in whole saliva fluctuated in most of the elderly patients in their study.
Cuida et al. (114) found that albumin concentrations were higher in both parotid
and whole saliva in patients with primary Sjgrens syndrome (SS) than in the
control group. However, the output/min of albumin was lower in SS patients.
25
Lenander- Lumikari et al. (115) found that albumin concentrations were higher in
patients with celiac disease than in healthy controls. It may be hypothesized that
salivary albumin can be used to assess the integrity of mucosal function in the
mouth (116). In periodontitis patients, significantly increased levels of salivary
albumin have been reported, and a significant correlation between salivary albumin
and gingival index in diabetic patients has been found (110).
On the other hand, Sweeney and co-workers (117) did not find any difference in
serum albumin concentrations in elderly patients with mucosal pathology in the
mouth when compared with those with healthy mouths.
Yoshihara et al. (118) found that there is a relationship between root caries and
serum albumin concentrations in elderly subjects. Terrapon et al. (119) found that
the low salivary albumin of old edentulous people was similar to that in a group of
younger individuals with a healthy periodontium.
26
glucose levels have been reported (127, 128, 129, 130), but no such correlations
have been found by others (131, 132, 133, 134, 135). Tenovuo et al. (1986) (136)
analyzed glucose levels in more than one hundred simultaneously taken stimulated
whole saliva samples and blood samples of seven patients. The variations in
salivary glucose were found to be extensive, and the correlation between salivary
and blood glucose was highly individual: some subjects showed high correlations,
while some others showed no change in salivary glucose, even when their blood
glucose levels were very high.
Glucose in parotid saliva or in gingival crevicular fluid is more strongly related to
blood glucose levels than glucose in mixed saliva (137, 138). Borg Andersson et
al. (130) reported that, after a standardized carbohydrate load, glucose levels in
parotid saliva increased in diabetic patients.
In an experimental animal study by Reuterving (139), the salivary secretion rate
was found to be decreased in diabetic rats, and the flow rates correlated inversely
with blood glucose levels.
Salivary microbial counts in relation to glucose control have hardly been
investigated. Reuterving et al. (127) observed that the counts of mutans
streptococci in mixed saliva decreased significantly as the metabolic control of 11
diabetic patients improved, but the counts of lactobacilli remained stable. Twetman
28
et al. (140) reported an increase of salivary lactobacilli counts along with a rise of
salivary glucose levels. During a two-year follow-up of newly diagnosed patients
with type 1 diabetes, salivary glucose levels tended to be lower during the second
than the first year, and the counts of lactobacilli dropped significantly during the
first six months, while the counts of mutans streptococci remained stable (141).
High salivary glucose levels might, however be connected with an increase of
microbial colonization, as also indicated by Darwazeh et al. (129).
facilitate their clearance from the mouth, adsorbing to teeth surface to form an
acquired pellicle to which microorganisms can attach, serving as a primary source
of nutrients, and mediating microbial inhibition or killing (143). In addition to
saliva, the gingival crevicular fluid, a plasma derived fluid that flows through the
junctional epithelium, provides microbes in the gingival crevice with nutrients and
carries host immune components that play an important role in regulating the
microflora (142).
Oral microbes are predominantly bacteria but fungi, viruses, mycoplasmas and
even protozoa (142) and archaea (144) can also be found.
The bacteria found in the oral cavity are presented in T.1.3. Below:
30
31
flow rates and Candida counts in 112 subjects who reported xerostomia.
Stimulated whole saliva was collected and streaked in Candida agar plates and
counted over 72 hours. The results showed a significant inverse relationship
between salivary flow and Candida colony forming units (CFU). Salivary
alterations in type-2 diabetes mellitus were investigated by Dodds et al. (157). The
authors assessed the salivary flow rates and yeast carriage in 233 subjects with type
2 diabetes mellitus and 240 healthy control subjects. The results showed that
diabetic patients had reduced output of both stimulated and unstimulated saliva;
and had high oral yeast counts compared with healthy subjects. The authors
concluded that diabetics may be more prone to oral dryness and infections than
non-diabetics.
33
34
35
36
CHAPTER TWO
2.1 STUDY (1): METHODOLOGY
The Biochemical Analysis of Saliva
37
Group (2):
-A controlled diabetic group (40 patients).
-23 males, 17 females with a mean age: 49.50 10.88 years.
(T. 2.1& 2.2).
Group (3):
-A healthy non-diabetic group (40 subjects).
-20 males, 20 females with a mean age: 46.12 10. 25 years.
(T.2.1 & 2.2).
The diabetic patients were divided according to the disease duration into:
-Short duration diabetics:
-included those patients who have been diabetic up to 6 years.
-long duration diabetics:
-included those patients who have been diabetic for more
Than 6 years. (T.2.3).
2.1.4 Inclusion and exclusion criteria:
- Cases of type-2 diabetes mellitus who were diagnosed clinically with features of
polyuria, polydypsia and polyphagia and elevated blood glucose levels, as per the
criteria established by the Expert Committee on Diagnosis and Classification of
Diabetes Mellitus in 1998 (1) were included in the study.
- Volunteers workers at EL Ribat University Hospital, with no features of diabetes
38
mellitus and blood glucose levels within normal limits, confirmed by checking their
fasting blood glucose level twice within a two weeks interval were included as a
control group (3).
-Patients with severe diabetic complications, with any other systemic illnesses or on
medications other than those for diabetes were excluded.
- Individuals who were:
-Alcoholics.
- Tobacco users.
-Denture wearers.
Were excluded.
2.1.5 Ethical Considerations:
- Ethical clearance & approval from the Postgraduates Board of the Faculty of
Dentistry-University of Khartoum, and the administrators of Jaber Abu EL
Izz Diabetic Centre and EL Ribat University Hospital were received.
- The study protocol was explained for the subjects involved and an informed
consent was obtained.
position in a quiet
4-PCR Real time Kit for Candida albicans of Primer Design Lt. Company (U.K).
40
41
42
Volume
10 l
1 l
1 l
4 l
Final Volume
15 l
Then a 15 l was pipetted into each well according to real-time PCR experimental
plate set up.
- RNAse/DNAse free water has been prepared in the concentration of (10ul of
sample DNA and 190ul of water),
- An moment of 5l of this preparation was pipetted in to the wells.
-The final volume in each well is 20 l.
-A series of standard curve dilution were done in the following manner:
1) 900ul of RNAse/DNAse free water pipetted into 5 tubes and labeled 2-6.
2) 100ul positive control Template pipetted into tube 2.
3) The volume was vortexed thoroughly.
4) Pipette tip was changed and 100ul pipette from tube 2 into tube 3
44
Copy Number
2 105 Per l
Tube 2
2 104 Per l
Tube 3
2 103 Per l
Tube 4
2 102 Per l
Tube 5
20 Per l
Tube 6
2 Per l
-Finally a 5 l standard template was pipetted into each well, according to the
experimental plate set up. Making the final volume in each well 20l
Amplification Protocol:
The amplification protocol proceeded according to the table below:
50 Cycles
Step
Time
Temp
Denaturation
10s
95 C
DATA COLLECTION
60s
60 C
45
46
CHAPTER THREE
3.1 : Results of The Biochemical Analysis of Saliva
3.1.1 Salivary glucose:
-Means salivary glucose level were:
-Group (1): The uncontrolled diabetics: 8.096.45 mg/dl.
-Group (2): The controlled diabetics:
7.646.44 mg/dl.
T. 2.5.
47
T. 6.
T. 6.
According to
gender (Chisquare-test, P
value=0.12-NS)
Male (n)
Female (n)
Group 1 (40)
Uncontrolled
Diabetic
group
21
19
Group 2 (40)
Controlled
Diabetic
group
23
17
Group 3 (40)
Healthy nondiabetic
group
20
20
P value, probability value; SD, standard deviation; NS, not statistically significant
According to Age
(ANOVA test P
value=0.32-NS)
Group 1 (40)
Uncontrolled
Diabetic
group
Mean SD
48.507.86
(Age range in
years)
Group 2 (40)
Controlled
Diabetic
group
Group 3 (40)
Healthy nondiabetic
group
49.5010.88
46.1210.25
P value, probability value; SD, standard deviation; NS, not statistically significant
Duration of
diabetes mellitus
Group 1 (40)
Uncontrolled
Diabetic
group
08
Long duration
diabetics
Short duration 32
diabetics
Group 2 (40)
Controlled
Diabetic
group
07
Group 3 (40)
Healthy nondiabetic
group
-
33
P value, probability value; SD, standard deviation; NS, not statistically significant
49
Group
Mean Sd
Uncontrolled
group
Controlled group
Healthy group
8.096.45
7.646.44
1.891.44
Group
Mean Sd
Uncontrolled
group
Controlled group
Healthy group
108.486.37
100.8360.77
146.7210.70
Group
Mean Sd
Uncontrolled
group
Controlled group
Healthy group
90.0144.22
50
88.9149.71
98.2549.59
Group
Mean Sd
Uncontrolled
group
Controlled group
Healthy group
193.8
189.0
9.71.4
Intergroup
comparisons
Salivary glucose
(mg/dl)
Uncontrolled
group
With healthy
group
Controlled group
With healthy
group
Dunnett
D test P
value
0.00
S
Intergroup
comparisons
Z-test
P value
Uncontrolled group
0.75
With controlled
group
NS
0.00
S
Intergroup
comparisons
Salivary amylase
(mg/dI)
Uncontrolled
group
With healthy
group
Controlled group
With healthy
group
Dunnett
D test P
value
0.10
NS
0.04
S
Intergroup
comparisons
Z-test
P value
Uncontrolled group
0.65
With controlled
group
NS
Intergroup
comparisons
Salivary total
protein (mg/dl)
Uncontrolled
group
With healthy
group
Controlled group
With healthy
group
Dunnett
D test P
value
0.66
NS
Intergroup
comparisons
Z-test P
value
Uncontrolled
group
With controlled
group
0.91
NS
0.59
NS
Intergroup
comparisons
Salivary urea
(mg/dl)
Uncontrolled
group
With healthy
group
Controlled group
With healthy
group
Dunnett
D test P
value
0.03
S
0.035
S
52
Intergroup
comparisons
Z-test
P value
Uncontrolled group
0.70
With controlled
group
NS
Group
G1 (n 40)
G2 (n 40)
G3 (n 40)
MeanSD
CFU
8,257 935.0
1,610250.0
41075.0
P value
.000
.000
.000
53
Fig (3.1): Positive correlation between candidal forming units and salivary
Glucose level.
54
CHAPTER FOUR
Discussion
4.1 THE BIOCHEMICAL ANALYSIS OF SALIVA
Diabetes mellitus is a group of metabolic disorders that share the common
underlying feature of hyperglycemia. Hyperglycemia in diabetes results from
defects in insulin secretion, insulin action, or most commonly from both f them.
Chronic hyperglycemia and the attendant metabolic dysregulation may be
associated with secondary damage in multiple organ systems, especially the
kidneys, eyes, nerves, and blood vessels (158).
The disease is the fifth most common cause of death in the world and it is
estimated that one in eight deaths (12.2%) among adult individuals were
attributable to the condition (4).
Reports in 2011, showed that 366 million people worldwide were affected by
diabetes and the number is continuing to rise steeply and by the year 2030,
predictions suggest that the number of people with diabetes will reach 552 million
(159) . In Sudan the prevalence of the disease has risen dramatically from 3.4% at
55
the Northern part of the country in1996 (16) to 14.5% with a prevalence rate of
19.2% at Khartoum state in 2006 (17).
It has been shown that a wide spectrum of oral manifestations of DM has been
reported. These manifestations included: Xerostomia , Periodontal diseases ,
Dental caries , Taste impairment , Sialosis , Fungal infections, Lichen planus
,Geographic & Fissured tongue (160).
According to our knowledge there is only one study carried on diabetes mellitus in
relation to oral health in Sudan published by Kamil and Ghandour in 2013 (161).
This study investigated the periodontal health of diabetics compared to nondiabetics at Khartoum University Clinic. The authors found diabetics were at a
higher risk to develop periodontitis compared to non-diabetics.
The present study was the first one in the country investigating saliva composition
and candidal counts among type- 2 diabetic patients. The study was conducted at
Jabir Abu EL Izz Diabetic Centre Khartoum in the period May to August 2010.
4.1.1 SALIVARY GLUCOSE:
In the present study, it was found that the salivary glucose values were higher
among diabetics than in the controls. (Mean level among controlled diabetics:
56
pathways have been proposed (171), but this is still a hypothesis rather than an
established theory. Many authors have tried to explain the increased glucose
content in the salivary secretion of diabetic patients. Lopez et al (172) tried to show
that the salivary glands act as filters of blood glucose that are altered by hormonal
or neural regulation. And according to Qureshi et al (173) persistent hyperglycemia
leads to microvascular changes in the blood vessels, as well as basement
membrane alteration in the salivary glands. This leads to increased leakage of
glucose from the ductal cells of the salivary glands, thereby increasing the glucose
content in saliva. Sreedevi et al (168) showed that glucose is a small molecule that
easily diffuses through semi permeable membranes. Thus, large amounts of
glucose become available to saliva when blood glucose levels are elevated, as in
diabetes. Alterations in the permeability, occurring as a result of basement
membrane changes in diabetes, may be an a additional explanation for the
increased concentration of glucose in saliva.
On the other hand, Belazi et al (166) proposed that the increased permeability of
basement membrane in patients with diabetes mellitus may lead to enhanced
leakage of serum-derived components into whole saliva via gingival crevices. The
small glucose molecule can easily diffuse via the semi permeable basement
membrane. Therefore the authors blamed the gingival crevicular fluid as the culprit
for increased glucose levels in salivary secretion.
58
59
level
among
uncontrolled
diabetics:108.48u/ml.controlled
60
With regard to salivary total protein, the present study results revealed similarity to
many previous studies (136, 179, 177), which showed no significant differences
were detected between diabetics & non diabetics. [Mean level among controlled
diabetics:88.91mg/dl., uncontrolled diabetics: 90.01mg/dl., non- diabetics:
98.25mg/dl. P.>0.05]. But recent studies have reported higher salivary total protein
levels in diabetics (176, 180) ,while Streckfus et al. (181) estimated significant
lower protein concentrations in diabetics and emphasized protein utilization by other
biochemical metabolic pathways as an overall systemic response to glucose intolerance.
Also Insulin is known to have the potential to alter protein metabolism (177).
diabetics had a low grade infection of their parotid glands. Although it was not
evident that diabetics had elevated concentrations of albumin in their saliva,
albumin is substantially increased, like lactoferrin, with acute inflammation of the
salivary glands as reported by Mandle et al (185) & Tabak et al (183), suggesting
that low grade infection of the salivary glands causing increased leakage of serum
proteins into saliva might be a common finding in diabetics.
4.1.3 SALIVARY UREA:
The average value of urea in the saliva of the non-diabetic group was 9.7 mg/dl.
The mean value of urea in the saliva of patients with controlled diabetes was 19.0
mg/dl, and the mean value of urea in the saliva of the uncontrolled diabetic patients
was 19.0 mg/dl ,showing a significant difference.( P < 0.05) between diabetics &
non-diabetics.
This result is in consistent with Carda et al (165) & Ivanoviski et al (186) studies.
This elevation in salivary urea among diabetic patients could be due to the nature
of diabetes as a metabolic disease causing disruption of metabolic processes in the
human body, so it is likely that serum levels of urea in patients with diabetes are
elevated. These increased levels of serum urea in patients with diabetes may also
be due to the dietary regimen and the increased intake of protein among those
patients. Another factor to be considered is that the increased permeability of
62
acinar cells in the salivary glands of patients with diabetes may allow enhanced
ultra filtration of blood.
4.1.4 DURATION OF DIABETES MELLITUS:
In this study, there was no significant finding with regard to the duration of
diabetes mellitus and salivary glucose level. (P >0.05). No correlations were
previously found between salivary glucose levels and disease duration, according
to many studies (187, 129), Although few studies reported that with increased
duration of diabetes, glucose values in saliva decreased as a result of fatty
infiltration in the acini and diabetic microangiopathy of salivary glands (176, 188).
It is of interest to notice that the alterations in saliva composition in diabetic
subjects seen in the present study might be due to the presence of diabetes induced impairment of salivary gland function. Similar findings have also been
previously described in the literature and were associated with diabetes inducedneuropathic changes in the salivary parenchyma with Iymphocythic gland infiltrate
similar to the one occurring in the pancreas of these diabetic patients (189).
The above mentioned alterations in saliva composition may well explain the
pathogenesis of oral lesions seen among
the
development
Of diabetic complications. Proteins and lipids exposed to aldose sugars go through
reactions which are not enzyme-dependent reactions . Later, irreversible advanced
glycosylation end products (AGEs) are formed. This process also takes place
during normal ageing, but in diabetes their formation is accelerated to an extent
related to the level and duration of
This interaction results in oxidant stress of the target cells, inducing production of
different patterns of cytokines and growth factors depending on the type of cells
involved. Excess production of growth factors and cytokines plays an essential
role in both micro- and macrovascular alterations. AGEs, by themselves, appear to
generate reactive oxygen intermediates and the interaction between AGE and
RAGE further induces production of intra- and extracellular oxidants.
Oxidative modifications of lipoproteins, in turn, accelerate atherogenesis (196).
Free oxygen radicals cause tissue destruction directly and exaggerate the
inflammation related tissue destruction because activated monocytes produce
proinflammatory cytokines, such as IL-1a, IL-6 and TNF-a.
In conclusion, hyperglycaemia, either directly or through AGE formation, causes
various structural and functional modifications of cells as well as quantitative and
qualitative alterations of the extracellular matrix, which may modify the host
response and alter all tissues homeostasis including the oral tissues.
65
CHAPTER FOUR
Discussion
4.2 SALIVARY CANDIDAL COUNTS
Candidosis is a superficial opportunistic infection, essentially facilitated by local
and systemic predisposing factors. It is the most common mycosis of the oral
cavity in both healthy & Immunosuppresed persons (197).
One reason for commonality of this disease is probably because 4060% of
healthy adults harbor commensal Candida in the oral cavity, without any signs or
symptoms of candidosis (198). Candida species have been frequently isolated from
the oral cavities of patients with diabetes mellitus (DM) (199) and it has been
reported that up to 77% of diabetic patients harbor oral Candida (200). A number
of candidal species could be recovered from the oral cavities of DM patients, with
C. albicans representing the most commonly recovered species, in more than 80%
of diabetic patients (155).
Most of the previous studies on salivary microbial counts focused on the bacterial
counts and controversial results were reported. Sandholm et al (201) reported more
gram-negative rods and a higher proportion of total gram-negative bacteria in
samples of patients with diabetes than in control samples. Later studies have not
conclusively supported this, as they have revealed no significant differences in
66
mutans
the need for gel electrophoresis to detect amplification products. Appropriate data
analysis and/or use of opposite chemistries also eliminate the need for Southern
blotting or DNA sequencing for amplicon identification.
According to Stephen (207) the technique is simple, specific & sensitive. Its
reliable instrumentation, and improved protocols, has made it the benchmark
technology for the detection of DNA.
In the present study, Candidal CFUs were significantly higher in diabetic subjects
(group I & group I I) compared with non- diabetic subjects (group III). [Mean CFU
among uncontrolled diabetics: 8.200CFU/ml, controlled diabetics: 1.600CFU/ml,
non-diabetics: 400CFU/ml]. This is similar to the findings of Dodds et al & Jones
et al studies (157,208). There was a significant positive correlation(r=0.519,
P<0.05) between salivary glucose and CFU of Candida in the overall study
population, in line with the observation of other investigators that increased
Candida reflects increased salivary glucose levels (199,209).
Varying rates of oral carriage of Candida in diabetic patients ranging from 18 to
80% , were reported in literature since 1958.Some of these studies have used crude
estimates for the degree of glycosuria and the measurement of blood glucose, to
68
delineate the diabetic patients and the control subjects. Furthermore, variations in
the therapeutic regimens from one study to another were present, as some patients
were controlled by insulin whereas others were controlled by oral hypoglycaemic
agents or diet alone.
Aly et al (210) found an increased oral carriage of yeasts associated with plasma
glucose level in patients with type 2 D.M. However, Fisher et al (155), Manfredi
et al (160), failed to show such an association. Interestingly Al-Karaawi et al (211)
observed lower rates of candidal counts among diabetics.
Many reasons seem to exist behind the differences in the results of those studies,
ranging from patient selection, inappropriate control subjects, varying and
unsuitable sampling or counting methods. A number of studies have now clearly
shown that the oral rinse sampling method of Samaranayake et al (206), which is
the method adopted in this study is the most appropriate and sensitive technique for
evaluating overall oral yeast carriage compared with imprint culture, swab or
saliva sampling.
Future workers in this field should pay great attention to the sampling as well as
the counting methods and above all , appropriate selection of patient and control in
order to obtain globally comparable data.
69
Denture wearers were excluded in the present study due to the high prevalence of
oral candidal carriage associated with denture wearing, particularly in diabetic
patients. Dentures may act as an additional reservoir for these organisms.
Furthermore, denture-induced trauma may reduce tissue resistance against
infection, thus increasing the permeability of the epithelium to soluble candidal
antigens and toxins, exposing molecules such as fibronectin which act as candidal
receptors (212).
Tobacco users were also excluded because the rate of oral candidal carriage in
tobacco users was higher than in non-tobacco users. (213). In addition to that,
blood glucose levels in diabetic patients who were tobacco users were significantly
higher than in those without this habit. (209), which may be as a result of the effect
of tobacco on adrenaline levels.
Broad spectrum antimicrobial therapy may suppress oral bacteria and cause C.
albicans colonization even in non-diabetic individuals. The present study excluded
patients under medications other than those for diabetes especially those under
antimicrobial therapy.
The mechanism by which diabetes predisposes to high oral carriage of Candida is
not yet established. However, it is widely recognized that high salivary glucose
levels in diabetic patients favor yeast growth. (129).
70
72
CHAPTER FOUR
4.3 CONCLUSIONS
73
CHAPTER FOUR
4.4 RECOMMENDATIONS
1. Further studies with larger sample size and in different geographic areas are
needed to establish saliva as a diagnostic as well as a monitoring tool for
diabetes mellitus.
2. Other saliva compositions e.g.: electrolytes and hormones, and other
microbial counts e.g. bacteria and other fungal species need to be
investigated.
3. Research centers in Sudan have to focus on saliva-related researches to
highlight its significance as an important diagnostic fluid.
4. Raising public awareness including the medical staff and health workers
about the orofacial manifestations and complications of D.M is highly
recommended.
5. For all wholistic programs in the management of D.M the role of oral health
professionals must be emphasized.
6. The interaction between D.M and oral health is an area of importance that
needs to be investigated comprehensively in the future.
74
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98
Form no. ..
Name:
Gender: .
Age: ..
Address:
Occupation:Tribe
Diabetic status:
FSBHbA1c.
Diabetic
non-Diabetic
If Diabetic:
Controlled
uncontrolled
99
Type 2
Type 1
< 6 years
> 6 years
100
Other
* Antimicrobial
* Other medication (specify) .
1. Glucose level
..
2. Amylase level
..
..
5. Candida count
..
101