evaluation of salivary zinc level in chronic periodontitis

يمبسم الله الرحمن الرح

Al-Neelain University

Faculty of Graduate Studying

The Master of Physiology Program

Evaluation of Salivary Zinc Level in Chronic Periodontitis Patients with Type II Diabetes Mellitus and Non-Diabetics in

Khartoum state 2019.

A Thesis Submitted for Partial Fulfillment to Award Master Degree in Medical Physiology

By:

Eman AbdElmoniem M.Elamin Alhnnan.

BDS(Al-Neelain University)

Supervisor:

Dr/Azza O Alawad

MBBS(UofK), MSc(UofK), PhD(UofK)

Assistant Professor of Physiology

Al-Neelain University

2019

I

Abstract:

Introduction: Zinc micronutrient is essential for human health, there is

accumulating data that zinc level is altered in both diabetes mellitus and

chronic periodontitis, but the exact role of zinc in the pathogenesis of those

conditions stay unclear. Recent innovative advances have made saliva as a

diagnostic tool for numerous pathological conditions.

Objectives: The present study was done to evaluate the salivary zinc level in

chronic periodontitis patients with type II diabetes mellitus and non-diabetics.

Materials and Methods: A Case-control study was conducted in Khartoum

state during June to December 2019 at Khartoum dental teaching hospital,

Jaber Abo-alezz diabetic center and Al-Neelain University, chronic

periodontitis was diagnosed with CAL ≥ 3mm, controlled type II diabetes

mellitus was diagnosed with Hemoglobin A1c ≤ 6.5%, the level of zinc was

assessed in 64 subjects, 26 systemically healthy subjects without chronic

periodontitis (Group A), 19 patients with chronic periodontitis otherwise

systemically healthy (Group B) and 19 diabetic patients with chronic

periodontitis (Group C). Atomic absorption spectrophotometry method was

utilized to measure the salivary zinc level. The data were statistically analyzed

by analysis of variance, Scheffe multiple comparison tests and Pearson’s

correlation utilizing statistical software SPSS version 20.

Results: The results showed that salivary zinc level decreased in periodontitis

patients with and without type II diabetes mellitus compared to healthy control,

whereas there was no statistically significant difference between salivary zinc

level in periodontitis patients and diabetic patients with periodontitis.

Conclusion: Decreased salivary zinc level may predispose an individual to the

risk of developing chronic periodontitis.

II

مستخلص البحث:

مستوى :المقدمة بأن متراكمة بیانات وھناك ، الإنسان لصحة الضروریھ العناصر من عنصرالزنك یعتبر

اللثة المزمن ، ولكن دور الزنك في الاصابھ بتلك الامراض لا الزنك یتغیر في كل من داء السكري والتھاب

ة الأخیرة اللعاب یزال غیر واضح. من الممكن استخدام اللعاب للتاكد من ذلك حیث جعلت التطورات المبتكر

لعدید من الحالات المرضیة. لكأداة تشخیصیة

ا الھدف: في مرضى اللعابي الزنك مستوى لتقییم الدراسة ھذه اللثة أجریت بداء المصابو المزمن لتھاب ن

.دراسة للحالات والشواھد .الثاني و غیر المصابین بالسكري السكري من النوع

واھد اجریت في ولایة الخرطوم في الفترة من یونیو الى دیسمبر للحالات و الش في دراسھ المواد والطرق:

ابو العز للسكري, وجامعة في الاماكن التالیة ذكرھا: مستشفى الاسنان التعلیمي, مركز جابر 2019من سنة

داء تشخیص تم المریضھ,كما الانسجھ معدلات قیاس باستخدام المزمن اللثھ التھاب تشخیص تم النیلین,

الھیموغلوبین السكري السكري من للرقابھ باستخدام التاني الخاضع الزنك . %6.5≤النمط تقییم مستوى تم

، 64في اللثة 26شخصًا التھاب یتمتعون بصحة جیدة بدون ا)(شخصًا یعانون من 19، مجموعھ مریضًا

مریضًا بالسكري یعانون من التھاب 19و (مجموعھ ب)التھاب اللثة المزمن وإلا فإنھم یتمتعون بصحة جیدة

. تم استخدام طریقة القیاس الطیفي للامتصاص الذري لقیاس مستوى الزنك اللعابي. (مجموعھ ج)اللثة المزمن

الإصدار باستخدام المتعددة، المقارنة واختبارات التباین تحلیل طریق عن إحصائیًا النتائج تحلیل تم

للبرنامج. 20الإحصائي

أظھرت نتائج ھذه الدراسة أن مستوى الزنك اللعابي انخفض في مرضى التھاب اللثة المصابین بداء ئج:النتا

النوع السكري مقارن 2من السكري داء لدیھم اي مشاكل صحیوبدون لیس باشخاص یكن ةة لم ، في حین

ى السكري الذین ھناك فروق ذات دلالة إحصائیة بین مستوى الزنك اللعاب في مرضى التھاب اللثة ومرض

یعانون من التھاب اللثة.

للثة المزمن. قد یؤھب الفرد لخطر الإصابة بالتھاب ا اللعابي انخفاض مستوى الزنك لاستنتاج:ا

III

Dedication

I would like to dedicate this research to my parents for their endless love, support,

and encouragement, to my sisters and brothers who never left my side. I will

continuously appreciate all they have done, which are exceptionally extraordinary.

I love you all.

IV

Acknowledgement

In the beginning, I would like to thank God for much success and the ability to

complete this work. For he who doesn’t thank people, he doesn’t thank God, so

i would like to thank my supervisor Dr.Azza O Alawad, assistant professor of

physiology, faculty of medicine, Al-Neelain University for her continuous

guidance and close supervision to get this work out the best way.

Thanks also to Dr. Lamis Kaddam, assistant professor of physiology, faculty of

medicine, Al-Neelain University for advice and assistance and for her constant

support despite the distance. I am extremely grateful to the Environment and

Natural Resources and Desertification Research Institute represented by the

person of Dr. Omar for their scientific honesty in the work and informed us the

results of samples in a short period.

I must not also forget to thank all doctors in the Department of Periodontology,

Al-Neelain University For their help and constant encouragement. I also place on

record my sense of appreciation to all doctors and staff at the Jaber abo alizz

diabetic centre in Khartoum, for their help and facilitation to meet the patients.

Thanks also extended to my colleagues in the Department of Physiology,

especially synerty Dr.Nour for helping me with her experience in research.

Last but not least, I would like to thank everyone who had a direct or indirect role

in completing this research.

V

List of tables:

Table Page Table of contents. IX

Table I : Criteria for diagnoses of DM. 7

Table1: Age distribution in the study population. 24

Table2: Sex distribution in the study population. 25

Table3: Comparing Means and standard deviations of salivary Zinc levels in Group A (control), Group B (Periodontitis), and Group C (Periodontitis and diabetes).

26

Table4: Comparing mean levels of clinical periodontal parameters (CAL) in Group A (control), Group B (Periodontitis), and Group C (Periodontitis and diabetes).

28

Table5: Comparing means of CAL in Group B (periodontitis) Vs Group C (Diabetic patients with periodontitis).

30

Table6: Comparing the means of salivary zinc level of Group A (control), Group B (Periodontitis), and Group C (Periodontitis and diabetes).

31

Table7: Inter Group comparison with Scheffe test. 32

Table8: Test the correlation of salivary zinc level and CAL between Group A (control), Group B (Periodontitis), and Group C (Periodontitis and diabetes).

33

VI

List of Figures:

Figure Page Figure1: Comparing Mean of salivary Zinc levels in Group A (control), Group B (Periodontitis), and Group C (Periodontitis and diabetes).

27

Figure2: Comparing mean levels of clinical periodontal parameter (CAL) in Group A (control),Group B (Periodontitis), and Group C (Periodontitis and diabetes).

29

VII

List of Illustrations:

Illustration Page Illustration I: Estimated number of adults with DM. 5

Illustration II: Properties of adolescents with and without periodontal conditions.

9

Illustration III: Properties of adults with and without periodontal conditions.

9

Illustration IV: Periodontitis may lead to degradation of soft tissue and alveolar bone which may lead to tooth loss.

10

Illustration V: Shows the concept of continued exposure to bacteria overtime which may lead to severe periodontitis.

13

Illustration VI: Progression of chronic periodontitis and intervention therapy.

15

VIII

List of Abbreviations:

ACC Actinobacillus Actinomycetemcomitans ANOVA Analysis of Variances B.forsythus Bacteroids forsythus BOP Bleeding On Probing CAL Clinical Attachment Loss CEJ Cemento Enamel Junction CPI Community Periodontal Index DM Diabetes Mellitus FPG Fasting Plsma Glugose GCF Gingival Crevicular Fluid GDM Gestational Diabetes Mellitus GI Gingival Index GTT Glucose Tolerance Test HbA1c Hemoglobin A1c MMPs Matrix Metaloprotienases PDL Periodontal Ligament P.gingivalis Porphyromonus gingivalis PI Paque Index PMNs Polymorphonuclear Neutrophils PPD Probeable Pocket Depth TIDM Type I Diabetes Mellitus TIIDM TypeII Diabetes Mellitus WHO World Health Organization Zn Zinc ZnT8 Islet-restricted Zinc Transpoter

IX

Table of contents Chapter Page English abstract I Arabic abstract II Dedication III Acknowledgement IV List of tables V List of figures VI List of illustrations VII List of abbreviations VIII Table of contents IX

Chapter I : Introduction 1.1 Introduction 1 1.2 Problem statement 2 1.3Research questions 2 1.4 Research hypothesis 2 1.5 Justification 2 1.6 Objectives 3 1.6.1 General Objectives 3 1.6.2 Specific Objectives 3

Chapter II: Literature Review 2.1 Diabetes Mellitus(DM) 4 2.1.1 Definition of DM 4 2.1.2 Longterm complications of DM 4 2.1.3 Periodontitis as an oral manifestation 4 2.1.4 Prevelance of DM 4 2.1.5 Pathophysiology of DM 6 2.1.6 Criteria for diagnosis of DM 6 2.1.7 Classification and Treatment 7 2.2 Periodontitis 7 2.2.1 Definition 7 2.2.2 Prevalence of Periodontitis 8

X

2.2.3 Pathophysiology of Periodontitis 10 2.2.4 Risk factors for Periodontitis 11 2.2.5 DM as a risk factor for Periodontitis 11 2.2.6 Criteria for diagnosis of periodontitis 12 2.2.7 Saliva as diagnostic tool for Periodontitis 12 2.2.8 Prevention of periodontitis 13 2.2.9 Treatment of Periodontitis 14 2.3 Zinc 15 2.3.1 Zinc and human health 15 2.3.2 Zinc and Diabetes Mellitus 16 2.3.3 Zinc and chronic Periodontitis 17

Chapter III: Materials and Methods 3. Methodology 18 3.1 Study design 18 3.2 Study area 18 3.3 Study duration 18 3.4 Study population 18 3.4.1 Inclusion criteria 18 3.4.2 Exclusion criteria 18 3.5 Sample size and sampling technique 19 3.6 Methods of data collection 20 3.6.1 Questionnaire and history taking 20 3.6.2 Clinical examination 20 3.6.3 Saliva collection 21 3.7 Data analysis 21 3.8 Data presentation 21 3.9 Ethical considerations 22

Chapter IV: Results Results 23

Chapter V: Discussion 5.1 Discussion 35 5.2 Limitations 36

XI

Chapter VI: 6.1 Conclusion 37 6.2 Recommendations 38 6.3 References 39 ANNEXES

1

1.1 Introduction: Diabetes Mellitus (DM) can be defined as a metabolic syndrome with hyperglycemia, which

is the major finding in DM. Both type1 (insulin-dependent) and type2 (non-insulin

dependent) are the two major types. Worldwide, people with diabetes are expected to

increase from 180 million in 2000 to 320 million in 2025. It is characterized by many

complications, including nephropathy, retinopathy, neuropathy and cardiovascular problems.

In addition to the oral findings like xerostomia, tooth loss, odontogenic abscesses and

periodontitis.(1)

Periodontitis is a microbial inflammatory disease that causes tissue destruction due to altered

immunoinflammatory processes.(2) It is one of the major dental problems all over the world.

Based on the documentation, it affects (20-50%) of the world wide population.(3)

Diagnoses of periodontitis are made by several periodontal parameters, including Plaque

Index(PI), Gingival Index(GI), and Clinical Attachment Level(CAL).(4) Moreover, it is

mainly treated by the removal of the causative agents (dental plaque) by utilizing the scaling

and root planning.(5)

Diet is a very important factor in the maintenance of the tooth and periodontium, as well as

systemic health. So any nutritional deficiency is a risk factor for periodontal and systemic

problems.(2)

The health of periodontal tissues is based on an adequate supply of proteins, carbohydrates

and minerals. Many researches revealed that alterations in the minerals level have both

systemic and oral consequences.(2)

Zinc (Zn) is one of the micronutrients that is very important for normal growth, maintaining

the integrity of the cell membrane, and for insulin action .(2,6) It is found mainly in foods of

animal products.(7)

Zn has a role in bone metabolism by stimulating osteoblastic bone synthesis. In addition to

its antioxidant properties.(2) It comes the second in a list that contains the most abundant

trace elements found in the human body.(5)

In a another study made by Sprren kiilerich et al. Observed that Zn absorption is decreased

in diabetic patients, causing intracellular Zn depletion .(8) Another study concluded that Zn

level was increased in type2 DM without periodontitis, while its level decreased in patients

with both DM and periodontitis. (6)

2

In 2013, Biju Thomas and his team conducted a case- control study that showed a low

serum level of Zn in patients with both periodontitis and type II diabetes mellitus.(9)

Although a role of Zn in periodontitis is not fully understood.(2)

Al-Maroof et al. Also conducted a study that investigated the serum level of zinc in diabetic

patients. They concluded that DM affects Zn homeostasis by producing hypozincemia as a

result of hyperzincuria, or decreased intestinal absorption, or both.(7)

Different results and diversity in Zn level among researches may be due to differences in

the study design and sample collection criteria as well as the stage and progression of the

disease .(1)

In the present study, whole saliva rather than blood is used because its non-invasive

method,as well as it can be used for diagnosis of different systemic diseases with oral

manifestations.(1)

1.2 Problem statement: The effects of both DM and chronic periodontitis on the salivary Zn level are not conclusive.

1.3 Research questions: − Do Patients with chronic periodontitis with or without DM suffer from decreased

salivary level Of Zn?

− Does DM lead to worsening periodontal diseases through a further reduction in

the salivary Zn level?

1.4 Research hypothesis:

It is hypothesized that the salivary Zn level is altered in both diabetic and chronic

periodontitis patients, compared to healthy individuals.

1.5 Justification: Due to the lack of enough data, it is essential to study the salivary Zn level in both chronic

periodontitis and diabetes, because any alteration in Zn level may affect the severity and

progression of the disease. Zn supplements may have a good role in improving the glycemic

3

status and chronic periodontitis. (7) Zn also prevents diabetes-induced chronic periodontitis.

(10)

1.6 Objectives:

1.6.1 General objective:

To study salivary Zn level in chronic periodontitis patients with type II diabetes mellitus

and non- diabetics.

1.6.2 Specific objectives:

− To measure salivary Zn level in chronic periodontitis patients with type II

diabetes mellitus and non-diabetics.

− To compare salivary Zn level in type II DM patients and healthy individuals

with, and without chronic periodontitis.

4

2.1 Diabetes Mellitus (DM) :

2.1.1 Definition: DM is defined as a group of metabolic diseases with hyperglycemia. The hyperglycemia is a

result of either insufficient insulin secretion, or defects in insulin action, or both with

disturbances of carbohydrate, fat, and protein metabolism.(11)

2.1.2 Longterm complications of DM:

DM is associated with longterm damage to several organs including, retinopathy that may

lead to blindness, nephropathy and renal failure, neuropathy with potential amputation,

autonomic dysfunction (sexual dysfunction), and cardiovascular diseases(1,11). In addition to

oral complications, like tooth loss, odontogenic abscesses, periodontitis, salivary dysfunction

that cause xerostomia, taste dysfunction, fissured tongue, traumatic tongue, angular cheilitis,

and delayed wound healing.(1,12)

2.1.3 Periodontitis as an oral manifestation of DM:

The way by which hyperglycemia can cause periodontal diseases( the sixth complication of

DM)(13) is not well known.(12) Theories suggest many factors like: impaired immune

functions, which facilitate bacterial accumulation in periodontal tissues, as well as increased

pro-inflammatory cytokines.(12)Concerning collagen destruction, it is most probably due to

increasing collagenase, as well as a reduction in collagen synthesis.(12)

Many researchers concluded that DM and periodontitis influence each other, that is to say;

poor periodontal conditions may worsen the glycemic status. Also, DM is a well known risk

factor for periodontitis.(12)

2.1.4 Prevalence of DM :

DM is a major health problem, its prevalence worldwide is expected to rise from 2.8%

(affecting 117 million) in the year 2000, to 4.4% ( affecting 360 million) in the year

2030.(14)In the developing countries, most of the cases in the age group between 45 to 64,

While in developed countries the majority of cases are above 64 years old.(14) DM is higher

5

in men than women, although there are more women with DM than men, this may be due to a

higher number of elderly women than men, and it is well known that the risk of DM

increases with age.(14) All these data indicate that, the number of DM patients is growing,

especially in developing countries.(15)

Illustration I: Estimated number of adults with DM by age, year, countries

For developed and developing categories and for the world.(15)

There is a limited data concerning the prevalence of DM in Africa. In Sudan, Elbagir et al. made

a study to estimate the prevalence of DM in Northern Sudan, and they found that; the crude

prevalence was (7.9% - 8.3%). They noticed that DM is more common in Danagla community,

most probably due to different environmental, nutritional, and genetic factors.(16)

6

2.1.5 Pathophysiology of DM:

The main problem in DM is deficient insulin, either due to autoimmune destruction of beta cells

(type I), or resistance to insulin action (type II). With subsequent hyperglycemia and abnormal

fat, protein, and carbohydrate metabolism.(11)

Type II Diabetes Mellitus (T2DM) results from peripheral insulin resistance( the main cause), in

addition to a decrease in insulin secretion and inadequate inhibition of glucagon after glucose

ingestion.(17) Symptoms of DM include polyuria, polydipsia, weight loss, polyphagia, blurred

vision, increased susceptibility to infections, increased fatigue, and irritability. In general,

symptoms are the same for both type1 and type2 DM, except for sexual dysfunction, which was

related more to type2 DM.(18) Uncontrolled DM is associated with life-threatening consequences

like hyperglycemia, ketotic, and nonketotic coma .(11)

2.1.6 Criteria for diagnosis of DM:

In general diagnostic tests should be repeated to exclude the laboratory error.(11) Hemoglobin

A1C (Hb A1C) is recommended for diagnosing of DM since 2010. The value of 5.7% or less

excludes the DM, while 6.5% or above is enough for diagnosis of DM,(19) but the problem is that

the Hb A1C can be misleading in case of hemoglobinopathies, and it is very costly and not

always available especially in developing countries. For decades, Fasting Plasma Glucose (FPG),

and Glucose Tolerance Test (GTT) have been used for the diagnosis of DM.(11)

According to the World Health Organization (WHO), GTT is performed by asking the patient to

drink 75g glucose dissolved in 250-300 ml water within 5 minutes, and then blood sample at

zero time and then two hours later is taken.(19)

Table I: Criteria for the diagnosis of Diabetes Mellites.(11)

2.1.7 Classification of Diabetes Mellitus:

The major types of Diabetes Mellitus include:

1. Type I Diabetes Mellitus (TIDM): Which results from destruction of pancreatic beta cells

causing absolute insulin deficiency, it is mainly treated by insulin injections.(20,21)

7

2. Type II Diabetes Mellitus (TIIDM): It is mainly due to insulin resistance and relative

insulin deficiency.(20,21) This type may require several medications(Diet control, oral

hypoglycemic drugs, insulin).(22)

3. Gestational Diabetes Mellitus (GDM): Which recognized in the second or third trimesters

of pregnancy.(20,21)

4. Other causes like: Genetic causes, diseases of exocrine pancreas, endocrinopathies, drugs

and chemicals infections, or sometimes related to specific syndromes.(20,21)

2.2 Periodontitis:

2.2.1 Definition:

Periodontitis, from a pathological point, can be defined as the destruction of collagen fibers

and apical relocation of the junctional epithelium. The inflammatory processes additionally

cause alveolar bone loss found in the radiograph.(23) It is also defined as a chronic

inflammatory disease (to gram-negative and anaerobic bacteria) that influences the tooth-

supporting structures, which comprise the gingiva, periodontal ligament (PDL) and alveolar

bone.(24,25) Ongoing definition showed the importance of the presence of pocket probing

depth (PPD) of more than or equal 4 mm. Clinical signs and symptoms incorporate,

gingivitis, bleeding on probing (BOP), periodontal pocket, clinical attachment loss (CAL)

and bone loss.(23)

2.2.2 Prevalence of Periodontitis:

Periodontitis is more likely to exist in older individuals, male, of lower educational level,

have lower financial resources, and who, smoke, hypertensive and socially segregated.(25)

Periodontitis is prevalent both in developed and developing countries, It influences 20-

50% of the world wide population.(3)

The World Health Organization (WHO) utilized the Community Periodontal Index (CPI) for

the estimation of periodontal diseases. CPI score ranges from 0 to 4, it depicts the periodontal

conditions at population level. CPI score 0 implies no periodontal disease, score 1 means

gingival bleeding on probing ,score 2 demonstrates the presence of calculus and BOP, score

3 shows shallow PPD about 4-5mm, score 4 represents deep PPD of 6mm or above.(3)

8

Compared with developed nations, developing nations have a higher prevalence of calculus

and BOP among young people peaking to 35%-70%, while in developed countries, it is 4-

34%.(3)

Illustration II: Proportions of adolescents (15-19) years with, and without

periodontal conditions using CPI score. Pd: Pocket depth.

9

Illustration III: Proportion of adults (35-44) with and without periodontal

conditions using CPI score. Pd: Pocket depth.

In 2015 Mihee et al. Made a study to investigate the prevalence of periodontitis in diabetic

patients, they found that the prevalence of periodontitis was significantly higher in adults

with DM (43.7%) than in those without DM(25%). Poor oral hygiene and smoking are the

major risk factors for periodontitis in adults with DM.(26)

2.2.3 Pathophysiology of Periodontitis:

In view of histopathological characteristics, the pathogenesis of periodontitis includes the

interaction between the bacterial plaque, and the host immunoinflammatory responses(27-30)

i.e. bacteria cause periodontal disease.(30) In spite of the fact that it is modified by

environmental and genetic factors.(30) Porphyromonas gingivalis (P.gingivalis),

Actionbacillus actinomycetemcomitans (AAC) and Bacteroids forsythus (B.forsythus) are

10

implicated in the initiation and progression of periodontitis, by discharging substances like

lipopolysaccharides and other virulence factors to start immunoinflammatory responses.(28)

Illustration IV: Periodontitis is characterized by degradation of the soft

connective tissue and alveolar bone which may lead to tooth loss.(27)

Bacterial infection usually occurs in stages, firstly attachment to host cells is initiated, at that

point the proliferation of the bacteria takes place, and then damaging the host cells. From the

opposite side the host cells produce antibodies to destroy the bacteria, in addition to

activating the polymorphonuclear neutrophils (PMNs), as well as matrix metaloprotienases

(MMPs), interlukin-1, and prostaglandins.(28,30)

Recently it is well known that, the periodontitis is not simply as the interaction between

bacteria and host immunoinflamatory processes. There are some other determinant factors

like smoking and DM(Known as risk factors), they lead to impaired host responses, which

result in more destructive periodontititis.(30)

2.2.4 Risk factors for Periodontitis:

11

Lately, it has turned out to be evident that pathogenesis of Periodontitis is more complex than

the presence of bacteria, because the bacteria sometimes is insufficient to develop the

Periodontitis.the presence of risk factors may change the host response toward the

disease.(31,32) The risk factors may be associated with the periodontitis, however do not

necessarily cause the disease; they has been classified in to modifiable and non-

modifiable(31). The modifiable risk factors are smoking and alcohol, DM, cardiovascular

diseases, obesity, osteoporosis, and the microorganisms.(31-34) Unlike the modifiable factors,

the non-modifiable factors are not easily changed like genetic susceptibility, defective

neutrophils function and the host response.(31,33,35)

2.2.5 DM as a risk factor for Periodontitis:

Diabetes is a risk factor for development of periodontal diseases, especially in those with

poor glycemic control. The exact role of diabetes in Periodontitis is obscure, perhaps it is due

to alteration in vascular, cellular and repair mechanisms. (36,38) Diabetes-related vascular

changes include, endothelial dysfunction due to oxidative stress, reduced nitric oxide

production and increased nitric oxide inactivation, all these will prompt decrease the blood

flow to the gingival. (36,38) Imperfect neutrophils function in diabetic patients, plus

dysregulation of both innate and adaptive immunity as a result of glycation end products, all

are reasons for cellular defects found in DM. When glycation end products bind to receptors

in the surface of monocytes, this will lead to increase the levels of tumor necrosis factor-

alpha and interlukin6, consequently they will lead to more tissue destruction(36,38)

Reduced healing responses found in DM most likely because of reduced collagen synthesis,

and imbalance in the (MMPs) system.(36,38)

2.2.6 Criteria for diagnosis of periodontitis:

Since it is very difficult to reconstruct the periodontium after bone loss, early diagnosis is the

point of the periodontal treatment. Accurate diagnosis of periodontitis needs full mouth

clinical measurements utilizing a periodontal probe, and recording the parameters at six sites

per tooth. (31,41) Periodontitis is diagnosed using radiography and clinical measurements like

PPD (of more than or equal 4), BOP and CAL. The severity of periodontitis can be assessed

12

based on CAL as follows, 1 to 2mm CAL demonstrates mild periodontitis, 3 to 4 CAL shows

moderate periodontitis, while severe periodontitis is diagnosed when the CAL is 5mm or

more. However these parameters detect evidence of past disease and not demonstrate the

current disease activity. (39,42)

2.2.7 Saliva as a diagnostic tool for periodontitis:

Saliva collection is safe, non-invasive, simple and can be collected repeatedly with minimum

discomfort to the patient. A number of biomarkers distinguished in saliva that related to

Periodontitis (metabolites from bacteria and host). So saliva could be utilized as a diagnostic

tool to diagnose Periodontitis, however unlike the gingival crevicular fluid (GCF) it can not

provide site-specific information, although it gives information about the diagnosis, disease

progression, and prediction of further destruction.(39,41)

Salivary biomarkers originate from both bacteria and host, bacteria determined biomarkers

incorporate DNA and proteins, host-related biomarkers are associated with hard and soft

tissue destructions like, alkaline phosphatase, calcium, inflammatory cytokines, MMPs,

lactate dehydrogenase, aspartate aminotransferase etc.(39)

2.2.8 Prevention of Periodontitis:

Several studies demonstrated that the basic role of bacteria in Periodontitis, so we could

prevent the disease by professional as well as patient-directed bacterial control, this is very

crucial because continued exposure to bacteria overtime would result in severe

periodontitis.(43)

13

Illustration V: Shows the concept of continued exposure to bacteria

overtime would result in severe periodontitis.(43)

The preventive measurements should be focused on the following points: early and

appropriate periodontal diagnosis is needed, so the dentist should advise their patients for

regular check up to identify early Periodontal problems,(43,44) educating mothers, children,

and young adults about oral hygiene methods(tooth brush and types of interdental kit),(43,44)

the underlying risk factors should be controlled (smoking, uncontrolled DM ,etc.),(43,44)

repeated prophylaxis with scaling and root planning in the isolated sites that show early

Periodontitis,(43) use of fluoride and antimicrobial mouthwashes both have antibacterial

properties, so they may be used to control the bacteria as well as plaque. (3,45) Because of the

strong relationship between chronic Periodontitis and oxidative stress, antioxidants such as

vitamin C, vitamin E, and coenzyme Q are very effective in preventing the disease, as well as

minerals iron, copper, and zinc which are required for the action of antioxidant enzymes.(46)

2.2.9 Treatment of Periodontitis:

Both prevention and treatment of Periodontitis have a participatory elements, so as a part of

the treatment plan, we should reduce the susceptibility of the patient to periodontitis by

modifying the risk factors (smoking cessation, control of DM, intake of calcium, nutritional

supplements).(43,47)

14

Treatment of periodontitis is a great challenge, in light of the fact that the infection is due to

bacterial biofilm which is resistant to both antibacterial and host response; subsequently the

removal of bacteria from the oral cavity is the fundamental undertaking.(47) Different

modalities applied for the treatment of Periodontitis including surgical intervention,

mechanical therapy (brushing and flossing as well as professional treatment with scaling and

root planning)(48), and use of pharmacological agents. Medications used like intra-pocket

drug delivery systems and host response modulating agents (for example: sub-anti microbial

dose of doxycycline), which are frequently used to modify the host response like reduction of

excessive enzymes level, cytokines, prostaglandins, and osteoclast activity.(47) Concerning

the surgical procedure, the laser has become a desirable choice to scalpel surgery because

lasers maintain the hemostasis as a result of heat-induced coagulation, in addition the intense

heat has a bactericidal effect. Although several researches have reported that the laser surgery

is accompanied by more pain and slower healing than scalpel surgery.(49)

15

Illustration VI: Progression of the disease and intervention therapy.(47)

2.3 Zinc :

2.3.1 Zinc and human health:

Periodontal health and regeneration are influenced by various vitamins, minerals, and trace

elements. Zinc (Zn) is second to iron as the most abundantly found trace mineral in the body,

it is mainly found in protein-rich foods.(5) The average amount of Zn in the adult body is

about 1.4-2.3g and the recommended daily dietary Zn requirement is assessed at

15mg/day.(50)

16

Zn acts as co-factor in many enzyme-controlled reactions; it has a critical effect in

homeostasis, immune function, in oxidative stress, apoptosis, and aging. In addition to it is

role in cell growth, DNA synthesis, RNA transcription, in wound healing, fetal growth,

sperm production, proper thyroid function and blood coagulation. Therefore the Zn levels

ought to be assessed, and Zn deficiency ought to be corrected.(5,50,51)

Dietary Zn may play an important role in periodontal health, it has been reported that a lack

of Zn leads to worsening the periodontal status especially in patients with type 2 diabetes. A

systemic review made by Pushparani has additionally supported the great role of Zn in

preventing diabetes-related periodontitis by exerting anti-oxidant impact.(5,52)

The most widely utilized test for diagnosing Zn deficiency in humans is the determination of

the plasma zinc level, typical plasma zinc levels in adults are 100± 10 µg/dl, values

underneath 80 µg/dl indicate Zn deficiency while plasma zinc levels of less than 50 µg/dl

demonstrate severe Zn deficiency.(51 )In our study we used saliva for evaluation of the Zn

levels, because salivary sample is non-invasive and easy to collect for research purposes. (53,54)

2.3.2 Zinc and Diabetes mellitus:

Zn assumes an important job in the physiology of insulin, as well as in the pathogenesis of

type II diabetes mellitus. (55) In the pancreatic beta cells Zn is required for crystallization and

storage of insulin, with two Zn ions lying at the focal point of each hexameric unit of the

hormone, several studies also identified a type of transporter called islet-restricted Zinc

transporter( ZnT8) which is most likely play a role in control of insulin secretion,(55)moreover

Zn upgrades phosphorylation of insulin receptors and encourages transporter of glucose in to

the cell.(56) The greater part of the complications of DM may be a manifestation of oxidative

stress which is related to diminished intracellular Zn and Zn-dependent anti-oxidant enzymes

(carbonic anhydrase, alkaline phosphatase, alcohol dehydrogenase, and superoxide

dismutase). (55)

In a study made by Kinlaw et al. To investigate the abnormal Zn metabolism in type 2

diabetes, they found that those patients had diminished serum Zn levels and they concluded

Zn depletion is most probably due to hyperzincuria uncompensated by increased intestinal

17

absorption, this intervened by hyperglycemia and aggravated by proteinuria.(55)Al-maroof et

al. Likewise noted hypozincemia in patients with type II diabetes mellitus alongside Zn

supplementation has positive effects in reducing their HbA1c concentration.(7) Pidduck et al.

Found hyperzincuria in diabetic patients and their prediabetic relatives they recommended

hereditary causes.(57) The literature has shown that Zn supplementation may enhance insulin

sensitivity in patients with type 2 diabetes, hence may reduce the complications.(55)

2.3.3 Zinc and chronic Periodontitis:

A lack of data exists on the possible role of trace elements in periodontal disease.(58) The

exact mechanism by which Zn deficiency mediates chronic periodontitis has not been

established, but rather it is most likely through its influence on the oral mucosa, bone

metabolism, and host response, Zn deficiency alters the thickness and keratinization of oral

mucosa, which turns out to be more susceptible to infections, Zn deficiency additionally

influences immune cells, namely monocytes, PMNs, and T lymphocytes, moreover it

increases the production of inflammatory cytokines,(59) Zn plays an important role in bone

metabolism, it has been implicated in altered coupling mechanism, reduced osteoblastic

activity and increased osteoclastic like cells. It likewise advances bone mineralization

through alkaline phosphatase enzyme.(59) Zn may prevent the progression of chronic

periodontitis through its anti-oxidant properties. Many researchers noted Zn deficiency might

worsen the periodontitis especially if it is associated with diabetes mellitus.(59) Orbak et al.

Made a study to investigate the alterations of periodontal tissues in Zn deficient rats and they

found Zn deficiency is a risk factor for periodontal disease. (60) On the other hand Freeland et

al. have provided evidence that dietary trace elements including Zn, were not related to the

periodontal disease.(58)

18

3 Methodology:

3.1 Study design: Analytic Case-control study.

3.2 Study area: The study was conducted in Khartoum state (Capital of Sudan) at Khartoum Dental

Teaching Hospital, Al-Neelain Dental Periodontology Clinics and Jaber Abo-alezz

Diabetic Center.

3.3 Study duration:

The study was conducted from June to December 2019.

3.4 Study population: Chronic Periodontitis patients with or without type II DM attending the clinic, the

subjects are classified into three main groups:

− Group A: Systemically healthy subjects without chronic periodontitis.

− Group B: Subjects with chronic periodontitis but systemically healthy.

− Group C: Subjects with type II Diabetes mellitus and chronic periodontitis.

3.4.1 Inclusion criteria:

− Gingival Index (GI) should be ≤ 2 for Group A.

− Patients with clinical attachment loss (CAL) ≥ 3mm for Groups B,C.

− Well diagnosed diabetic patients taking oral hypoglycemic drug or insulin for

Group C.

− Age group from 30 to 60.

− Subjects with a minimum 20 teeth.

3.4.2 Exclusion criteria:

− History of any systemic diseases for Group A,B.

− Patients with systemic diseases other than diabetes mellitus type2 for Group

C.

− Patients on medications other than those for diabetes for Group C including

vitamins and minerals.

19

− Patients with history of use of mouth wash within last 3 months.

− Smokers, Alcoholic, Snuffers.

− Pregnant ladies, and contraceptive pills.

− Patients with aggressive periodontitis.

3.5 Sample Size and sampling technique:

The sample was collected using simple random sampling. The total number of subjects required (n) is calculated using the formula:

n= m/√(g-1)

N = m + (g-1)*n

Where:

𝛼𝛼: 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙.

β: the power of the test.

g: overall number of groups.

z(1−𝛼𝛼/2): 𝑠𝑠𝑠𝑠 𝑠𝑠 𝑙𝑙𝑠𝑠𝑙𝑙𝑣𝑣𝑙𝑙 𝑠𝑠𝑓𝑓𝑓𝑓𝑓𝑓 𝑠𝑠ℎ𝑙𝑙 𝑠𝑠𝑓𝑓𝑓𝑓𝑓𝑓𝑠𝑠𝑙𝑙 𝑑𝑑𝑠𝑠𝑠𝑠𝑠𝑠𝑓𝑓𝑠𝑠𝑑𝑑𝑣𝑣𝑠𝑠𝑠𝑠𝑓𝑓𝑠𝑠 𝑓𝑓𝑙𝑙𝑙𝑙𝑠𝑠𝑠𝑠𝑙𝑙𝑑𝑑 𝑠𝑠𝑓𝑓 𝑠𝑠𝑠𝑠𝑑𝑑 𝑓𝑓𝑙𝑙𝑟𝑟𝑓𝑓𝑙𝑙𝑠𝑠𝑙𝑙𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 𝑠𝑠ℎ𝑙𝑙

𝑠𝑠𝑓𝑓𝑠𝑠𝑠𝑠𝑠𝑠𝑑𝑑𝑙𝑙𝑠𝑠𝑠𝑠𝑙𝑙 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑓𝑓𝑙𝑙𝑠𝑠𝑙𝑙.

(1- β): is a value from the normal distribution related to and representing the power of the test.

m: sample size of the control group.

n: sample size corresponding to each treatment group.

20

N: total sample size.

Δ: standard effect size.

When:

Δ = 0.5

α = 0.01

z1−α 2⁄ = 2.58

β = - 0.85

𝑧𝑧 – β = -1.04

g = 3

We obtain:

3.6 Methods of data collection:

3.6.1 Questionnaire and taking medical and dental history (Annex II).

3.6.2 Clinical examination: a) Periodontal clinical parameters (GI,CAL) were used to detect periodontal

disease using William’s periodontal probe as follow:

The Gingival Index (GI) was measured using index of lὅe and silness

(1963),(61) periodontal probe was inserted apical to the gingival margin and

the tissues are gently stroked with the instrument, scores of GI as follow:

Score 0 = Normal gingival, score 1 = sign of inflammation without bleeding

on probing, score 2 = bleeding on probing, and score 3 = Spontaneous

bleeding. (62,63)

N=64, n=19, m=26

21

Clinical Attachment Loss (CAL), which is the distance from the cemento-

enamel junction (CEJ) to the base of the proeable crevice. (62,63)

b) Chronic Periodontitis was diagnosed with CAL ≥ 3mm.

c) Controlled type II DM was diagnosed with HbA1c ≤ 6.5%.

3.6.3 Saliva collection: Saliva was collected in a quiet clinic away from any stimulation, Subjects

were instructed not to eat, or drink one hour prior to sample collection, each

subject was asked to rinse with deionized water immediately before the

procedure( Provided to the Patient), then to accumulate saliva in the mouth 2

min and to spit in sterile container. This procedure continued for 6 min to

collect unstimulated saliva .Then the samples were centrifuged at 3000 rpm at

4C for 5 min, each sample diluted five folds in 10 ml/L nitric acid. Zinc was

analyzed by Atomic Absorbance Spectrophotometer (ANNEX III), and the

results were expressed in mg/L(9,64) .

3.7 Data analysis:

− Independent square test and P-value for significance were applied

(P-value < 0.05 was found to be statistically significant).

− Data normality was tested using Kolmogorv-Smirnov and Leven’s tests.

− Intergroup comparison was done using Scheffe test.

− ANOVA test showed statistical significant (P-value < 0.05).

− Mean and SD were calculated.

− Pearson’s correlation was used for correlation at P-value < 0.05.

− The data were analyzed using SPSS version 20.

3.8 Data presentation:

The data were presented as tables and figures.

22

3.9 Ethical considerations:

− Ethical clearance was obtained from the university ethical committee

(ANNEX IV), Khartoum state ministry of health research department

(ANNEX V), and from Khartoum dental teaching hospital.

− Patients asked to sign an informed consent after full explanation of the

procedure (ANNEX I).

− Patients with periodontal problems were treated as appropriate.

23

The study was carried out on 64 subjects in the age range 30-60 years, including both gender, the subjects were divided into three groups as follow:

− Group A: 26 systemically healthy subjects without chronic periodontitis.

− Group B: 19 Subjects with chronic periodontitis but systemically healthy.

− Group C: 19 Subjects with Diabetes type2 and chronic periodontitis.

The age distribution of all subjects in the study is shown in Table 1 .Group A(control),

B(Periodontitis), and C(Periodontitis with Diabetes) were in the age group of (43.46±6.813,

47.11±7.81, and 51.53±7.81 respectively).The present study was carried out on 32M and 32F

distributed in three groups as follows(m/f: 12/14 in group A , 8/11 in group B, and 12/7 in group

C) as described in Table2. Mean and standard deviation of salivary zinc levels in different

groups are given in Table 3 and Figure 1. The salivary zinc levels in Group A (Control Group)

show mean and standard deviation of ( 0.614 ± 0.128) when compared to Group B and C, which

show mean and standard deviation of (0.241± 0.103) and (0.295 ± 0.113) respectively. With

mean value of control group shows the highest reading.The mean of CAL was found to be lower

in Group C when compared with Group B! As Shown in Table 4 and Figure2.

Independent square test was used to test difference in CAL between Group B and C it showed

significant differences in CAL means between Group B and C(P-value < 0.05), mean difference

showed that the CAL in Group B is on average 0.84 higher than Group C Table 5.

The Kolmogorv-Smirnov Test revealed that the salivary zinc levels of the study were normally

distributed (P-value >0.05), and Levene's test was used to test the homogeneity of variances

which revealed that the variances are not different.Then statistical analysis by one way analysis

of variance (ANOVA) showed statistical significance (P-value < 0.05), therefore at least one

mean was different from the others as in Table 6.

Intergroup comparison was made using Scheffe test for multiple comparisons, and it showed that

there was a difference in means difference between Group A (control) and Group B

(Periodontitis), and between Group A and Group C (Periodontitis with Diabetes). With Zinc

amount in the Group A was on average of 0.374 greater than Group B, salivary zinc amount in

group A was also higher by 0.319 than group C.

24

On the other side there was no statistically significant difference in means detected between

Group B and Group C Table 7.

To test the correlation between groups the Pearson correlation coefficient was used, we found a

strong negative correlation between salivary zinc level and one of the clinical periodontal

parameters represented by clinical attachment level (CAL) Table 8.

25

Table1: Age distribution in the study population (N=64).

Groups N Range(Y) Mean(Y) ±SD

Control 26 30-60 43.46 6.813

Periodontitis 19 30-60 47.11 7.81

Periodontitis and Diabetes

19 30-60 51.53 7.59

N=number of sample size for each group, SD=Standard Deviation.

26

Table2: Sex distribution in the study population (N=64).

Groups Frequencies Total Male(%) Female(%)

Control 12 (46%) 14 (54%) 26

Periodontitis 8 (42%) 11 (58%) 19


12 (63%) 7 (37%) 19

Total 32 32 64

27

Table3: Comparing Means and standard deviations of salivary Zinc levels in Group A (control), Group B (Periodontitis), and Group C (Periodontitis and diabetes).

Salivary Zinc concentration in Mg/L

N Minimum Maximum Mean ±SD

Control 26 0.468 0.98 0.614 0.128

Periodontitis 19 0.066 0.456 0.241 0.103


19 0.096 0.426 0.295 0.113

28

Figure1: Comparing Mean of salivary Zinc levels in Group A (control), Group B (Periodontitis), and Group C (Periodontitis and diabetes).

00.10.20.30.40.50.60.7

Control Periodontitis Periodontitisand Diabetes

0.614

0.241 0.295

Zn Mean in Mg/L

29

Table4: Comparing mean levels of clinical periodontal parameters (CAL) Group B (Periodontitis) and Group C (Periodontitis and diabetes).

Group

N

Minimum

(mm)

Maximum

(mm)

Mean (mm)

±SD

Periodontitis 19

2.375

7.833

4.044

1.302


19

2.000

4.200

3.204

0.663

CAL= Clinical Attachment Loss.

30

Figure2: Comparing mean levels of clinical periodontal parameter (CAL) in Group A (control), Group B (Periodontitis), and Group C (Periodontitis and diabetes).

0

1

2

3

4

5

Control Periodontitis Periodontitis andDiabetes

0

4.0443.204

CAL Mean (mm)

31

Table5: Comparing means of CAL in Group B( periodontitis) Vs. Group C ( Diabetic patients with periodontitis).

P-value 0.017< 0.05.

Levene's Test for Equality of

Variances

t-test for Equality of Means

F P-value t df P-value Mean Difference

CAL Equal variances assumed

2.812

.102

2.506

36

*0.017

.84

Equal variances not assumed

2.506

26.743

0.019

.84

32

Table6: Comparing the means of salivary zinc level of Group A (control), Group B (Periodontitis), and Group C (Periodontitis and diabetes).

ANOVA

Sum of Squares

df Mean Square

F P-value

Between Groups

1.881 2 0.940 69.096 *0.000

Within Groups

0.830 61 0.014

Total 2.711 63

A significant statistical difference in means of salivary zinc levels was found.

33

Table7: Inter-Group comparison with Scheffe test.

Further statistical analysis showed that the control group has the highest mean level of salivary zinc.

(I) Group J Mean Difference (I-J) Sig.

Control Periodontitis *0.374 0.000


*0.319 0.000

Periodontitis Control *-0.374 0.000


-0.054 0.363


Control *-0.319 0.000

Periodontitis 0.054 0.363

34

Table8: Test the correlation of salivary zinc level and CAL between Group A (control), Group B (Periodontitis), and Group C (Periodontitis and diabetes).

Significant level of 0.05.

CAL

Zn Pearson Correlation

-0.779

Sig. .000

N 64

35

5.1 Discussion:

In general, zinc plays a major role in various physiological processes in humans through its role

in regeneration, for coping with oxidative stress, and for satisfactory immune response(9). While

the exact mechanism by which Zn deficiency mediates chronic periodontitis is still unclear, it is

most probably by altering the thickness and keratinization of the oral mucosa, influencing the

immune cells (monocytes, PMNs, and T lymphocytes), and increasing the osteoclastic activity.

Zn deficiency additionally increases oxidative stress and reduces regenerative capacity.(9,59) The

present study was undertaken to evaluate salivary Zn level in chronic periodontitis patients with

or without type II Diabetes mellitus, since saliva represents a useful tool in diagnosis of some

physiological and pathological conditions.(65) The unstimulated whole saliva was used in the

present study because the collection of saliva is safe, non-invasive and might be collected

repeatedly without discomfort to the patient.(66)

The age group of the participants in the present study was (30-60) years, That is because the

chronic periodontitis is expected in older age, although age alone in healthy adults doesn’t lead

to critical loss of periodontal support.(67)In addition most similar researches worked with age

group range (30-60) years.(9)

The results of the study demonstrated lower levels of zinc in the saliva of individuals with

periodontitis compared to the control group without periodontitis; the difference is found to be

statistically significant for salivary zinc levels. Frithiof et al. and Tulin et al, in separate studies,

found diminished serum zinc levels in individuals with periodontitis when compared to healthy

controls.(68,69) In contrast, Freeland et al. found no difference.(70)

The other objective of the present study, was to evaluate the salivary zinc level in diabetes typeII

mellitus patients with chronic periodontitis, the result of the study illustrated lower levels of

zinc in type II diabetes mellitus patients with chronic periodontitis when compared to healthy

individuals. Whereas there was no statistically significant difference found between diabetic

patients with chronic periodontitis and systemically healthy individuals with chronic

periodontitis. Although both animal and human studies have suggested a viable role of

decreased zinc level in the pathogenesis and complications of type II diabetes mellitus. Thomas

et al. in a similar study to the present present study found that salivary zinc levels decreased in

36

diabetic patients with chronic periodontitis compared to healthy individuals with and without

periodontitis.(9)Pushparani et al. Also reached to the similar result.(71)

Moreover,we found that the clinical attachment loss is higher in chronic Periodontitis patients

when compared to diabetic patients with chronic periodontitis, although a meta-analysis made

by Khader et al. concluded that diabetic patients had more severe periodontal disease than non-

diabetic patients.(72)

The correlation test between the Clinical Attachment Loss (CAL) and salivary zinc level proved

that the relationship is inverse, i.e. the lower the value of the salivary zinc level, the greater the

value of (CAL). Zinc deficiency may lead to loss of periodontal attachment, by depressing

immunity, increasing oxidative stress, and slowing down regenerative capacity as we pointed

earlier.(9)

The present study was conducted to evaluate salivary zinc levels in chronic periodontitis with

and without diabetes mellitus type2 confirmed that there is a negative correlation between the

salivary zinc level and chronic periodontitis as described by Thomas.B et al. in their study to

evaluate micronutrient levels in periodontitis patients with and without type II diabetes

mellitus.(9)

The present study may have important therapeutic implications in terms of the use of zinc

supplements in periodontal therapy to prevent tissue destruction. An interventional study on rat

has shown that a topical administration of zinc ion-containing compound either as a mouthwash

or toothpaste can reduce loss of alveolar bone.(9) Zinc citrate-containing dentifrices have a

remarkable effect on reducing gingivitis in humans by reducing the periodontal indices (mainly

Gingival Index, and Plague index).(73)

5.2 Limitations:

This study was limited primarily by its small sample size. Another limitation is that salivary zinc

concentration may be not the best record of micronutrient levels in periodontal tissues. Moreover

coreelation between HbA1c, FBG, and Zn was not calculated.

37

6.1 Conclusion:

− Salivary zinc level was decreased in patients with chronic periodontitis when compared

to the control Group.

− Salivary zinc level was decreased in Diabetic patients with chronic periodontitis when

compared with the control group.

− There was no statistical significant difference in the salivary zinc level in patients

with chronic periodontitis compared with diabetic patients with chronic periodontitis.

38

6.2 Recommendations:

− More longitudinal studies with a larger sample size are recommended to be carried out to

have a better understanding of the inter-relationship between zinc imbalance and chronic

diseases like periodontitis and diabetes, with a separate group of diabetic patients

without periodontitis to know the exact relation between diabetes and Zinc.

− Evaluation of salivary zinc level before and after periodontal therapy are recommended

to be carried out.

− Evaluation of salivary zinc level before and after zinc supplements in the form of

mouthwashes or tooth paste is also recommended.

39

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