Estimation of salivary and serum alkaline phosphatase level as a diagnostic marker in type-2 diabetes mellitus with periodontal health and disease: A clinico-biochemical study.

INTRODUCTION: Chronic periodontitis is a multifactorial disease resulting in the inflammation and destruction of the supporting structures around the teeth, leading to tooth mobility and subsequent loss of tooth. Metabolic disorders, such as diabetes mellitus, play a crucial role in the progression of periodontal inflammatory conditions. Alkaline phosphatase (ALP) enzyme plays a key role in gingival inflammation and bone resorption. Hence, the aim of the present study is to compare the serum and salivary alkaline phosphatase levels in chronic periodontitis patients with or without type-2 diabetes mellitus.
MATERIALS AND METHODS: A total of 45 individuals were included in the study and divided into three groups: Group I (healthy individual), Group II (Chronic periodontitis without diabetes mellitus type-2) and Group III (Chronic periodontitis with type-2 diabetes mellitus) on the basis of clinical, radiographic and blood sugar examination. The serum and unstimulated saliva were collected from all patients in aseptic condition and samples were analyzed for alkaline phosphatase level using AVANTOR™ Benesphera ALP Kit by fully automated analyzer.
RESULTS: The result showed that the concentration of serum and salivary alkaline phosphatase increases significantly in patients with chronic periodontitis with type-2 diabetes mellitus than chronic periodontitis without diabetes mellitus and healthy patients.
CONCLUSION: We can conclude that alkaline phosphatase can be used as a key inflammatory diagnostic biomarker in periodontal diseases.

INTRODUCTION

Periodontal pathogens and their products in the dental plaque are the primary cause of periodontal disease. Along with direct destructive effects of periodontal pathogens, inflammatory and immune responses in host cause most of the tissue destruction. Current clinical diagnostic methods are not precisely accurate and only allow retrospective diagnosis of attachment loss. Conventional diagnostic methods of periodontitis are based on the measurement of clinical attachment loss and radiographic evaluation of alveolar bone loss.[1]

The term biomarkers refer to a measurable indicator of some biological state or condition, which evaluates normal biological mechanisms, pathogenic process or pharmacological therapeutic interventions. Biomarkers can play an important role in life sciences and have begun to assume a greater role in diagnosis, monitoring of therapy outcome and drug discovery.[2]

Biomarkers are released by the inflammatory and immune cells in the tissues and blood during the disease process and many of these pass into the gingival crevicular fluid, blood, saliva and are thus easily available for analysis. Human saliva is a body fluid secreted by major and minor salivary glands which contain locally derived and systemically derived markers of periodontal disease,[3] hence, may offer the basis for a patient's specific diagnostics test for periodontitis. Various enzymes are released from host cells during the initiation and progression of periodontal disease.

Alkaline phosphatase is a host derived, hydrolytic enzyme acting optimally at high pH.[4] It plays a key role in gingival inflammation and bone homeostasis.[5] The main source of alkaline phosphatase is liver, kidney, bone, intestine and placenta as also it is found in many cells of the periodontium including neutrophils, osteoblasts, fibroblasts.[6] It is mainly released from polymorphonuclear neutrophils during their migration to the site of infection, from osteoblast during bone formation and from fibroblast in periodontal ligament during periodontal regeneration.[7]

Environmental factors, such as diabetes mellitus, are also associated with severe periodontal destruction and progression of disease. Diabetes is a condition of prolonged sustained increased blood glycemic levels which when left untreated leads to multiple acute and chronic complications.

The purpose of the present study was to evaluate alkaline phosphatase levels in saliva and serum of patients with chronic periodontitis and chronic periodontitis with type II diabetes mellitus as compared to healthy controls.

MATERIALS AND METHODS

A total 45 patients were selected from the Department of Periodontology, Haldia Institute of Dental Sciences and Research, Haldia, for this study and they were divided into three groups on the basis of clinical and radiographic examination.

Group 1: Healthy patients

Group 2: Patients with chronic periodontitis

Group 3: Patients with chronic periodontitis co affected by diabetes mellitus.

All individuals underwent a basic periodontal examination by one examiner, who had been calibrated for reproducibility before the study was conducted.(kappa value 0.85) The clinical parameters recorded by the trained professional were as follows: (a) plaque Index (Silness and Loe, 1964).[8] (b) gingival Index (Loe and Silness 1963).[9] (c) probing pocket depth ≥5 mm which were measured using a graduated periodontal probe. Patients were also subjected to blood sugar analysis and a radiographic evaluation through orthopantomogram.

Study technique

After taking informed written consent, 5 ml of blood was collected from the antecubital vein by venipuncture using 20G needle and immediately transferred to the laboratory [Figure 1]. Then, 1 ml of unstimulated whole saliva sample was collected in a sterile disposable plastic container [Figure 2]. Patients were instructed not to brush or eat 8 h before collection of the samples. No dental examination and treatment were carried out 48 h before saliva collection. Samples were stored at 4°C and sent to the Department of Biochemistry, ICARE Institute of Medical Sciences and Research. The blood samples were centrifuged at 3000 rpm for 5 min [Figure 3]. The serum was separated from blood. Then whole saliva samples were centrifuged. All serum samples were subjected to Auto Analyzer (BeneSphera) for analysis [Figure 4], and all saliva samples were sent to Semi Autoanalyzer [Figure 5]. Human AVANTOR Benesphera alkaline phosphatase (ALP) Kit [Figure 6] was used to estimate the alkaline phosphatase concentration in saliva and serum.

Figure 1

Collection of blood

Figure 2

Collection of unstimulated whole saliva

Figure 3

Centrifuge machine

Figure 4

Serum analysis by Auto Analyzer (BeneSphera)

Figure 5

Semi Autoanalyzer for saliva sample analysis

Figure 6

Human AVANTOR BeneSphera alkaline phosphatase Kit for alkaline phosphatase estimation

Data were entered in Microsoft excel sheets and were statistically analyzed using “paired Student's t-test”. P < 0.05 was considered as statistically significant.

RESULTS

Quantitative analysis revealed salivary alkaline phosphatase levels to be 18.9647 ± 4.3301, 100.0907 ± 18.2945, 119.0767 ± 20.8691 (mean value ± standard deviation) in Groups 1, 2 and 3, respectively. The groups statistically differed from each other (P < 0.05). Results revealed that salivary alkaline phosphatase levels exhibit significantly higher concentration in chronic periodontitis patients and highest in chronic periodontitis patients with type II diabetes mellitus patients than the healthy periodontium group [Tables 1–3 and Graph 1].

Table 1

Comparison of alkaline phosphatase level in the saliva of Group-A (healthy) and Group-B (chronic periodontitis)

Table 3

Comparison of alkaline phosphatase level in saliva of Group-B (chronic periodontitis) and Group-C (chronic periodontitis with type-2 diabetes mellitus)

Graph 1

The alkaline phosphatase levels in saliva in different study group

Serum alkaline phosphatase levels were found to be 127.4820 ± 15.5674, 211.2833 ± 27.1612, 245.3047 ± 31.8110 (mean value ± standard deviation), respectively. The groups statistically differed from each other (P < 0.05). Results show that the serum alkaline phosphatase levels exhibit significantly higher in chronic periodontitis patients and highest in chronic periodontitis patients with type II diabetes mellitus patients than the healthy periodontium group [Tables 4–6 and Graph 2].

Table 4

Comparison of serum alkaline phosphatase level of Group-A (healthy) and Group-B (chronic periodontitis)

Table 6

Comparison of serum alkaline phosphatase level of group-B (Periodontitis) and group-C (chronic periodontitis with type-2 diabetes mellitus)

Graph 2

The Serum Alkaline Phosphatase levels in different study group

DISCUSSION

The present study was a cross-sectional observational study which attempted to focus on the changes in the total alkaline phosphate level based on the changes in the bone caused due to chronic periodontitis and type 2 diabetes mellitus.

Both physiologic and pathologic conditions have been associated with rise in serum alkaline phosphatase levels, possibly like bone growth, bone pathologies, hepatobiliary diseases, etc.[4] Diabetes mellitus on the other hand renders the patient susceptible to further infections and diseases because of compromised immunological status.[10] Thus, a significant number of other pathologies develop in the body, which may lead to bone destruction activities. Chronic periodontitis is well known for the changes it induces in bone activity.[11] Keeping these in mind it was hypothesized that total (serum plus salivary) alkaline phosphatase levels will show a rise in diabetic patients suffering from chronic periodontitis.

The ALP levels in blood serum in the present study showed an increase in chronic periodontitis patients as compared to the control group. The results of the present study are in accordance with the study done by Shaheen et al. in 2009.[4] ALP level of serum showed a significant rise in both the groups i.e., in patients with periodontitis and in patients with periodontitis coaffected by type 2 diabetes mellitus when compared to the healthy controls. Similar results were observed in the study of Armitage in 1992.[12] The increase in levels of alkaline phosphatase in diabetic individuals could be attributed to the sustained raised blood glycemic levels which render the patient highly susceptible to infection due to compromised immune status.

In the present study, the total amount of salivary alkaline phosphatase levels was also found to be significantly higher in chronic periodontitis as compared to healthy individuals. Similar result was also reported by Kumar and Sharma in 2011[13] and Nakamura and Slots[14] in their respective studies. In the present study, it was found that the salivary ALP was higher in chronic periodontitis with and without diabetes mellitus than the control group. Ishikawa and Cimasoni[15] showed a positive correlation of alkaline phosphatase in periodontitis patients with increase pocket depth. The results of our study are well in accordance with this study. Thus, it may be deduced that a significant amount of alkaline phosphatase levels present in saliva is produced locally by diseased periodontal tissues. The alkaline phosphatase levels can be used as a pro-inflammatory marker for monitoring periodontal diseases.

CONCLUSION

The present study was conducted to estimate and to compare the saliva and serum ALP levels in chronic periodontitis with and without type 2 diabetes mellitus patients with periodontally healthy individuals. A highly significant correlation of increased levels of serum and salivary ALP in chronic periodontitis with or without type 2 diabetes mellitus was observed as compared to healthy individuals. Thus, it can be safely deduced that salivary alkaline phosphatase levels could be used as a useful biomarker for monitoring periodontal disease.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Table 2

Comparison of alkaline phosphatase level in the saliva of Group-A (healthy) and Group-C (chronic periodontitis with type-2 diabetes mellitus)

Table 5

Comparison of serum alkaline phosphatase level of Group-A (healthy) and Group-C (chronic periodontitis with type-2 diabetes mellitus)