Research Proposal

Research Title:

QUANTITATIVE ESTIMATION OF SALIVARY ALKALINE

PHOSPHATASE (ALP) AND LACTATE DEHYDROGENASE (LDH) IN
PERIODONTITIS

NOBEL COLEGE
AFFILIATED TO POKHARA UNIVERSITY
SINAMANGAL, KATHMANDU
E-MAIL: info@nobel.edu.np
www.nobelcollege.edu.np
 Passport size
photograph
Administrative Information

1. Research Title: QUANTITATIVE ESTIMATION OF SALIVARY ALKALINE PHOSPHATASE

(ALP) AND LACTATE DEHYDROGENASE (LDH) IN PERIODONTITIS.

2. Name and Title of Investigator responsible for the proposed research:

Maharjan saru
Last (Surname) Middle (if any) First name
Nationality:
Citizenship Number with district name from where it was obtained (only for
Nepali)

Signature: Date:
Postal Address:
Telephone No.:
Mobile No.:

e-mail:
Alternate e-mail:
3. Full name of the Institution associated with the Principal Investigator (if
applicable) :
Designation:

2
4. Name and Title of investigators responsible for the proposed research (Use
the similar format if more than one): Passport size
photograph

Last (Surname) Middle (if any) First name

Nationality:
Citizenship Number with district name from where it was obtained (only for
Nepali)
Passport Number (only for non Nepali citizen):
Affiliated Institution (if applicable):
Designation:
Signature: Date:
Postal Address (if different from the address given above):

Telephone No.:

e-mail:

(Use additional sheet if necessary)

5. Name and Title of investigators responsible for the proposed research (Use
the similar format if more than one): Passport size
photograph

Last (Surname) Middle (if any) First name

Nationality:
Citizenship Number with district name from where it was obtained (only for
Nepali)
Passport Number (only for non Nepali citizen):

3
 Affiliated Institution (if applicable):
Designation:
Signature: Date:
Postal Address (if different from the address given above):

Telephone No.:

e-mail:

6. Name and Title of investigators responsible for the proposed research (Use
the similar format if more than one): Passport size
photograph

Last (Surname) Middle (if any) First name

Nationality:
Citizenship Number with district name from where it was obtained (only for
Nepali)
Passport Number (only for non Nepali citizen):
Affiliated Institution (if applicable):
Designation:
Signature: Date:
Postal Address (if different from the address given above):

Telephone No.:

e-mail:

4
7. List the name(s) and institutional affiliation to the researcher(s) (other than
co-investigator) to assist your project in Nepal and abroad (if any)

Name Institution and Address

(a)
(b)
(Use additional sheet if necessary)

8. List the name(s) of Nepali researcher(s) (other than co-investigator) or

Nepalese Institution/hospital/NGO(s) etc. from whom you may seek co-
operation (if any)
(a)
(b)
(Use additional sheet if necessary)

9. Is this research part of your Thesis?

Yes ■ No

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 QUANTITATIVE ESTIMATION OF SALIVARY ALKALINE
PHOSPHATASE (ALP) AND LACTATE DEHYDROGENASE (LDH)
IN PERIODONTITIS.
1. INTRODUCTION:
Periodontitis is an inflammatory disease of the supporting tissues of the teeth caused by
specific microorganisms or a group of specific microorganisms, resulting in progressive
destruction of the periodontal ligament and alveolar bone with increased probing depth
formation, gingival recession or both.(Miricescu et al., 2014)
The enzymes released from host cells can be easily obtained within the oral cavity either from
gingival crevicular fluid or from whole saliva. The whole saliva is composed of secretions
from salivary gland as well as from gingival crevicular fluid, desquamated epithelial cells,
microorganisms, and leucocytes. (Dawes, 1978)
Alkaline phosphatase (ALP) is a glycoprotein and a membrane-bound enzyme. It hydrolyzes
monophosphate ester bonds of nonorganic pyrophosphate at alkaline pH, increasing the local
concentratons of phosphate ions.(Perinetti et al., 2002)
ALP is a very important enzyme as it is a part of the normal turnover of periodontal ligament,
cementum, and bone homeostasis.(Nakashima, Roehrich, & Cimasoni, 1994)
It is found in many cells of periodontium including neutrophils, osteoblasts and fibroblasts.
ALP is released from polymorphonuclear neutrophils during inflammation, osteoblasts during
bone formation and periodontal ligament fibroblasts during periodontal regeneration.
(Kunjappu, Mathew, Hegde, Kashyap, & Hosadurga, 2012)
LDH is known to catalyze the oxidative conversion of the substrate pyruvate to lactate and
has been used as an inflammatory marker. LDH is routinely present in the cytoplasm of the
cell that gets released into the extracellular environment upon cellular lysis and cell death.
Thus, LDH represents a marker to cell death and tissue breakdown and its raised level often
signifies a disease process.(Gandolfo et al., 2006)
Human saliva is an easily accessible biochemical fluid, which is similar to blood in various
biological aspects. Besides, it possesses a simple and non-invasive collection with low-cost
storage and easily storage nature.(Paknjad & Rezaei, 2013)

2. PREVALENCE:

3. Periodontal diseases are the most

common and
4. widespread chronic dental diseases
worldwide.

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 5. Periodontal diseases are the most
common and
6. widespread chronic dental diseases
worldwide.
7. Periodontal diseases are the most
common and
8. widespread chronic dental diseases
worldwide.
9. Periodontal diseases are the most
common and
10. widespread chronic dental diseases
worldwide.
Periodontal disease are the most common and widespread chronic dental disorder worldwide.
The prevalence of periodontitis has been reported by the World Health Organization (WHO)
which showed a prevalence of severe periodontitis in around 8-10% of the Population.
Poor oral hygiene, use of tobacco products, malnutrition, psychosocial factors, compromised
immune system and poverty are some of the risk factors for periodontal diseases.
Approximately 31% of Nepali age 35-44 years surveyed in a study had developed deep
periodontal pockets. This ranks Nepal as one of the top 15% of the countries in the world
where this age group suffers from deep periodontal pocketing.(Pradhan & Bhat, 2009)

3. RESEARCH OBJECTIVE:
 To evaluate the differences in the Salivary Alkaline Phosphatase and Lactate
Dehydrogenase levels between patients with and without periodontal disease.

4. NEED OF THE STUDY:

Traditional periodontal diagnostic procedures include probing depths, bleeding on probing,
clinical attachment levels, plaque index and radiographs assessing alveolar bone level. All

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these clinical and radiographic parameters are difficult in identifying and measuring the
progression of the disease.(Fine, 1992)
The early-stages of periodontitis is known as gingivitis and, in most cases, it has no signs and is not
painful. Therefore, it is very important with clinical benefits to diagnose the disease at early stages
using a sensitive and reliable method. Research evidence suggests that altered activities of
various enzymes, both at cellular and sub-cellular levels, can influence the disease process.
Thus, measurements of enzyme activities can furnish valuable diagnostic and prognostic
information.(Fine & Mandel, 1986; Todorovic et al., 2006)

5. LITERATURE REVIEW:
Saliva is a specific bio-fluid, in many aspects similar to blood samples, which is composed of
various important biomarkers. A number of phosphoproteins, naturally present in saliva, are
quite important for the maintenance of enamel homeostasis.(Actis, Perovic, Defagó,
Beccacece, & Eynard, 2005)
The absence of cells in enamel differentiates it from bone structure. Therefore, enamel is not
able to perform biological repair in the case of any injury or eruption without the aid of other
systems, such as the salivary glands. In health conditions, salivary glands can provide a
variety of phosphorylated proteins, including salivary proteins and enzymes.(Armitage &
Douglass, 2003)
A tendency towards the use of salivary proteomic technology for diagnosis purposes in oral
cavity internal problems and Sjogren’s syndrome is found in the recent literature.
Quantitatively measurement of salivary proteins can provide a specific way of diagnosis and
the possibility to follow the success of treatment.(Wu, Shu, Luo, Ge, & Xie, 2009)
It is suggested that whole saliva may contain simply measured indicators of effect of
thiocyanate and aspartate aminotransferase (AST), alanine aminotransferase (ALT) and
lactate dehydrogenase (LDH) activity, and may provide a reliable and important body fluid
for monitoring and treating periodontitis. Rai et al. showed that salivary AST, ALT and LDH
levels reflected inflammation and destruction of periodontal tissue, suggesting their role as
clinically useful markers.(Rai, Kharb, & Anand, 2007)
The existence of dehydrogenases is rarely reported in saliva. However, the important member
of the family, lactate dehydrogenase (LDH), is present in the salivary fluid. An increase of
salivary LDH activity has been observed in the saliva of periodontitis sufferers. In this aspect,
it has been reported that LDH activity is directly proportional to the severity of periodontitis.
(De La Peña, Dios, & Sierra, 2007)
Numabe et al., 2004 stated that LDH activity was reduced after an individual undergoes the
periodontal therapy (with significant reduction with just phase 1 therapy). Thus, LDH in
saliva has been a suitable indicator and diagnostic tool to assess the periodontal health.
(NUMABE et al., 2004)
Todorovic et al., 2006 in their study observed that the activities of LDH enzyme in saliva
which was significantly increased in the patients with periodontal disease in comparison to
those healthy patients.(Todorovic et al., 2006)

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It has been observed in several recent studies that in patients with untreated chronic
periodontitis the level of AP in whole saliva was higher when compared to healthy
individuals.(Nakamura & Slots, 1983)
Researchers have also discovered that a positive correlation exists between pocket depth and
AP in periodontitis patients. Researchers have stated that AP is released by secondary
granules of neutrophils and its concentration increases considerably with increase in
inflammation and plaque accumulation.(Kumar & Sharma, 2011)
Dabra and Singh, 2012 examined the activities of salivary ALP and acid phosphatase (ACP)
in patients with periodontal disease, before and after periodontal treatment. The results
obtained in their study showed statistically signifcant increased activities of ALP and ACP in
saliva from patients with periodontal disease in relation to controls. It was also observed that
signifcant reduction in the enzyme levels was seen after conventional periodontal therapy.
(Dabra & Singh, 2012)
Ramesh et al. in 2013 conducted a study which included 40 female subjects, aged 50-60
years, divided in two groups: the frst included 20 postmenopausal women without chronic
periodontitis and the second 20 postmenopausal women with chronic periodontitis. When the
salivary ALP levels were recorded, they found a signifcant increase in postmenopausal
women with periodontitis. Based on these results, they concluded that salivary ALP levels
could be used as additional diagnostic aid in diagnosing periodontitis in post-menopausal
women.(Ramesh, Bhandary, Thomas, D'Souza, & Kumari, 2013)
Kishore and Yuvarajparmar in 2014 examined the activities of salivary ALP and other
salivary markers in pre and postperiodontal therapy patients and found that these were
signifcantly higher in patients with periodontal disease in relation to controls. However the
authors also noticed that there was a signifcant reduction in the enzyme levels after
conventional periodontal therapy.(Kishore & Yuvarajparmar, 2014)

6. MATERIALS AND METHODS:

6.1. Methodology :
6.1.2. Study Design :
 This is a prospective type of study.
6.1.3. Study setting :

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  The research will be conducted in Medical Biochemistry Research
Laboratory, Nobel College, Sinamangal, Kathmandu.

6.1.4. Study Population :

 Sample of diagnosed case of Periodontitis will be collected and transported to
Nobel College from Himal Dental Hospital.
6.1.5. Basic criteria :
Inclusion criteria
1. Patients suffering from generalized chronic periodontitis (minimum four sites
of probing PD of 5 to 7 mm)
2. Smokers who smoked 15–20 cigarettes/beedi per day.
3. Systemically healthy individuals.
4. Patients should not have undergone any periodontal treatment within 6 months
prior to this study.
5. No antibiotic use in the previous 3 months and no history of chronic antibiotic
use.
6. Cooperative patients showing acceptable oral hygiene.
Exclusion criteria
1. Patients having any history of chronic systemic disease.
2. Presence of removable prosthesis.
3. Furcation sites.
4. Patients on any antimicrobial drug in the previous 3 months.
5. Patients on nonsteroidal anti-inflammatory drugs in the past 1 month.
6. Patients on regular medication for any systemic disease that might alter the
ALP and LDH concentration.
7. Patients with physical or mental disability.

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 6.1.6. Study Period :
month month month
month
Working plan / Month

Literature review, proposal

writing and proposal defense

Experimental design and

Laboratory analysis

Sample collection and

processing

Data collection, Statistical

analysis, Result interpretation

6.1.7. Sample Size :

 Approximately seventy to hundred samples will be taken for this study.
7. Lab Methodology:
7.1. Sample Collection and processing:
7.1.1. Salivary Sample collection:
The whole saliva samples could be collected by stimulation using paraffin for
mastication, or sour drops or without stimulation. It is worth noting that, when
stimulated, it may be more diluted of biomarkers and therefore, not suitable for
detection. For this reason, unstimulated whole saliva samples are often used in
most cases of diagnostic applications. A sample will be collected after an
individual was asked to rinse his/her mouth thoroughly with water to insure the
removal of any possible debris or contaminating materials and waiting for 1-2
min for water clearance. The samples will be collected at least 1 h after the last
meal. Each one of the groups’ subjects will be asked to spit saliva into the
polyethylene tubes until 5 ml was collected. The collected saliva will be

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 centrifuged and then the centrifuged clear supernatant saliva will be collected
by micropipette into eppendorf tubes and kept frozen and store at -20 degree
Centigrade until biochemical analysis of salivary enzymes.

7.1.2. Sample processing:

 After sample collection, it will be carried out in the laboratory for
further procedure using the Spectrophotometric Analysis.

8.Ethical Clearance:
 Ethical approval for the study protocol will be obtained from Nobel
Research Council (NIRC).

9.Flow chart of Methodology:

10. REFERENCES

Actis, A. B., Perovic, N. R., Defagó, D., Beccacece, C., & Eynard, A. R. (2005). Fatty acid profile of human
saliva: a possible indicator of dietary fat intake. Archives of oral biology, 50(1), 1-6.
Armitage, G., & Douglass, C. (2003). Supplemental diagnostics for the assessment of periodontal diseases.
Fundamentals of periodontitis. Chicago: Quintessence Publishing Co, 284-299.
Dabra, S., & Singh, P. (2012). Evaluating the levels of salivary alkaline and acid phosphatase activities as
biochemical markers for periodontal disease: A case series. Dental research journal, 9(1), 41.

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Dawes, C. (1978). The chemistry and physiology of saliva. Shaw JH, Sweeney EA, Cappuccino CC, et al, Text
book of Oral biology, Philadelphia.
De La Peña, V. A., Dios, P. D., & Sierra, R. T. (2007). Relationship between lactate dehydrogenase activity in
saliva and oral health status. Archives of oral biology, 52(10), 911-915.
Fine, D. H. (1992). Incorporating new technologies in periodontal diagnosis into training programs and patient
care: A critical assessment and a plan for the future. Journal of periodontology, 63, 383-393.
Fine, D. H., & Mandel, I. D. (1986). Indicators of periodontal disease activity: an evaluation. Journal of Clinical
Periodontology, 13(5), 533-546.
Gandolfo, S., Pentenero, M., Broccoletti, R., Pagano, M., Carrozzo, M., & Scully, C. (2006). Toluidine blue
uptake in potentially malignant oral lesions in vivo: clinical and histological assessment. Oral oncology,
42(1), 88-94.
Kishore, P., & Yuvarajparmar, S. S. (2014). Evaluating the levels of salivary enzymes as biochemical markers in
periodontal disease. International J. of Healthcare and Biomedical Research, 2(3), 170-174.
Kumar, R., & Sharma, G. (2011). Salivary Alkaline Phosphatase level as Diagnostic marker for periodontal
disease. Journal of International Oral Health, 3(5).
Kunjappu, J. J., Mathew, V. B., Hegde, S., Kashyap, R., & Hosadurga, R. (2012). Assessment of the alkaline
phosphatase level in gingival crevicular fluid, as a biomarker to evaluate the effect of scaling and root
planing on chronic periodontitis: An in vivo study. Journal of oral and maxillofacial pathology:
JOMFP, 16(1), 54.
Miricescu, D., Totan, A., Calenic, B., Mocanu, B., Didilescu, A., Mohora, M., . . . Greabu, M. (2014). Salivary
biomarkers: relationship between oxidative stress and alveolar bone loss in chronic periodontitis. Acta
Odontologica Scandinavica, 72(1), 42-47.
Nakamura, M., & Slots, J. (1983). Salivary enzymes: origin and relationship to periodontal disease. Journal of
periodontal research, 18(6), 559-569.
Nakashima, K., Roehrich, N., & Cimasoni, G. (1994). Osteocalcin, prostaglandin E2 and alkaline phosphatase in
gingival crevicular fluid: their relations to periodontal status. Journal of Clinical Periodontology, 21(5),
327-333.
NUMABE, Y., HISANO, A., KAMOI, K., YOSHIE, H., ITO, K., & KURIHARA, H. (2004). Analysis of saliva
for periodontal diagnosis and monitoring. Dentistry in Japan, 40, 115-119.
Paknjad, M., & Rezaei, A. (2013). Salivary biochemical markers of periodontitis. Rom J Biochem, 50(2), 129-
146.
Perinetti, G., Paolantonio, M., D'Attilio, M., D'Archivio, D., Tripodi, D., Femminella, B., . . . Spoto, G. (2002).
Alkaline phosphatase activity in gingival crevicular fluid during human orthodontic tooth movement.
American Journal of Orthodontics and Dentofacial Orthopedics, 122(5), 548-556.
Pradhan, S., & Bhat, M. (2009). Assessment of periodontal status of rural nepalese population using the
community periodontal index. J Nepal Dent Assoc, 10(2), 97-104.
Rai, B., Kharb, S., & Anand, S. (2007). Salivary enzymes and thiocynate: Salivary markers of periodontitis
among smokers and non-smokers; a pilot study. Adv Med Dent Sci, 1(1), 1-4.
Ramesh, A., Bhandary, R., Thomas, B., D'Souza, S. R., & Kumari, S. (2013). Alkaline Phosphatase-A diagnostic
marker of periodontitis in postmenopausal women-A biochemical study. Nitte University Journal of
Health Science, 3(4), 71.
Todorovic, T., Dozic, I., Barrero, M. V., Ljuskovic, B., Pejovic, J., Marjanovic, M., & Knezevic, M. (2006).
Salivary enzymes and periodontal disease. Medicina oral, patología oral y cirugía bucal. Ed. inglesa,
11(2), 4.
Wu, Y., Shu, R., Luo, L. J., Ge, L. H., & Xie, Y. F. (2009). Initial comparison of proteomic profiles of whole
unstimulated saliva obtained from generalized aggressive periodontitis patients and healthy control
subjects. Journal of periodontal research, 44(5), 636-644.

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