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Table of Contents   
ORIGINAL RESEARCH  
Year : 2010  |  Volume : 21  |  Issue : 4  |  Page : 531-536
Alkaline phosphatase as a periodontal disease marker


Department of Periodontology and Oral Implantology, National Dental College and Hospital, Gulabgarh, Derabassi, Distt. SAS Nagar, Mohali, Punjab, India

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Date of Submission06-Aug-2009
Date of Decision24-Feb-2010
Date of Acceptance16-Aug-2010
Date of Web Publication24-Dec-2010
 

   Abstract 

Background: The potential of alkaline phosphatase (ALP) as an important diagnostic marker of gingival crevicular fluid (GCF) has been the subject to investigation since 1970. ALP is stored in specific granules and secretory vesicles of the neutrophils and is mainly released during their migration to the site of infection. It is also present in bacteria within dental plaque, osteoblasts and fibroblasts. It has, thus, become important to elucidate whether GCF levels of ALP are potential measures of the inflammatory activity occurring in the adjacent periodontal tissues.
Objective: The aim of this study was to assess the total activity of ALP in the GCF collected from healthy sites, sites with gingivitis and with chronic adult periodontitis. An attempt was also made to establish the correlation of ALP activity with plaque index, gingival index, bleeding index and probing depth.
Materials and Methods: A total of 18 patients were divided into three groups: viz., healthy sites, Group I; gingivitis, Group II; chronic periodontitis, Group III. Clinical parameters like plaque index, bleeding index, gingival index and probing depth were recorded. The ALP level in GCF of all three groups was determined by spectrophotometric analysis.
Results: Total enzyme activity of ALP was significantly higher in periodontitis as compared with that in healthy and gingivitis sites, and was significantly and positively correlated with probing depth.
Conclusion: ALP can be considered as a periodontal disease marker as it can distinguish between healthy and inflamed sites. However, to better define its capacity for periodontal diagnosis, additional longitudinal studies are required.

Keywords: Alkaline phosphatase, chronic periodontitis, gingival crevicular fluid

How to cite this article:
Malhotra R, Grover V, Kapoor A, Kapur R. Alkaline phosphatase as a periodontal disease marker. Indian J Dent Res 2010;21:531-6

How to cite this URL:
Malhotra R, Grover V, Kapoor A, Kapur R. Alkaline phosphatase as a periodontal disease marker. Indian J Dent Res [serial online] 2010 [cited 2020 Jan 26];21:531-6. Available from: http://www.ijdr.in/text.asp?2010/21/4/531/74209
Periodontal diseases consist of a group of inflammatory diseases initiated by bacteria that colonize the teeth and infect their surrounding soft tissues. The end result of this infection is the clinical manifestation of disease, which results in several distinct signs and symptoms. A majority of these clinical signs and symptoms, including radiographic evidence of bone loss and increasing pocket depth, have been used in a semiquantitative manner to evaluate patients afflicted with periodontal diseases. [1] Conventional methods of evaluating periodontal tissues are usually sufficient to arrive at a diagnosis and enable effective clinical treatment, but they are associated with the following limitations [2] :

  • They do not detect the presence of disease before it has caused destruction.
  • It is difficult to determine when the periodontal disease is active and calls for treatment.
  • Inability to reflect even marked histological changes.
An era of "enlightened diagnosis" has resulted that has spawned many new methodologies designed to improve patient care. These include genetic susceptibility analysis, microbiologic analysis and biochemical analysis.

Biochemical methods in periodontal diagnosis have centered primarily on gingival crevicular fluid (GCF) and its constituents. [2]

GCF is formed when fluid exudes from the vessels of the microcirculation into the inflamed periodontal tissue and into the sulcus or pocket. As the fluid traverses the inflamed tissue, it is thought to pick up enzymes and other molecules that participate in the destructive process as well as products of cell and tissue degradation. A few of the choice contenders found in GCF are products of polymorphonuclear leukocytes (PMNs), macrophages and plasma cells. [1]

The fluid, therefore, offers great potential as a source of inflammatory and immune markers, tissue breakdown products, enzymes of bacterial origin, host-derived enzymes and their inhibitors. The biochemical markers in GCF have been related to gingivitis, periodontitis and, most recently, to periodontal disease activity. [2] Analysis of GCF therefore represents the most practical approach for the biochemical analysis of the host response in periodontal disease. [2] A meaningful host parameter that can be measured in a quantitative and reproducible manner should provide an even earlier look at tissue destruction. [1]

Among the host enzymes in GCF, alkaline phosphatase (ALP) (orthophosphoric-monoester phosphohydrolase) was one of the first to be identified. It is a membrane-bound glycoprotein produced by many cells within the area of periodontium and gingival crevice. It is released from polymorphonuclear neutrophils during inflammation, osteoblasts during bone formation and periodontal ligament fibroblasts during periodontal regeneration. Thus, it has dual involvement in the process of periodontal inflammation and healing/regeneration. [3] ALP is stored in specific granules and secretory vesicles of neutrophils and is mainly released during their migration to the site of infection. ALP allows bone mineralization by releasing an organic phosphate and by hydrolyzing inorganic pyrophosphate, a potent inhibitor of hydroxy apatite crystal formation and dissolution. [4]

ALP activity is valuable to clinicians because enzymatic modifications occur at the GCF level earlier than clinically evident modifications associated with tissue destruction.

A short-term clinical investigation has been carried out to assess the total activity of ALP in GCF collected from healthy sites, sites with gingivitis and chronic adult periodontitis, and to determine its correlation with clinical parameters, viz. plaque index, gingival index, bleeding index and pocket depth.


   Materials and Methods Top


Subject and site selection

A total of 18 subjects (eight females and 10 males) in the age group of 20-55 years were selected among the patients visiting the Department of Periodontology and Oral Implantology of National Dental College and Hospital, Derabassi, Punjab.

The patients selected for the study were categorized into the following three groups:

Group I consisted of six subjects with clinically healthy periodontium (probing depth not exceeding 3 mm, no bleeding on probing and no evidence of bone loss).

Group II consisted of six patients with gingivitis (probing depth not exceeding 4 mm, presence of bleeding on probing and no radiographic evidence of bone loss).

Group III consisted of six patients with chronic adult periodontitis having pocket depth exceeding 4 mm, presence of bleeding on probing and radiographic evidence of bone loss.

All patients were systemically healthy. The patients who were on any systemic antimicrobial and/or anti-inflammatory drug therapy within 3 months prior to this study, who had any history of periodontal treatment within 6 months prior to this study, involvement of furcation sites and who had teeth with a fixed or removable prosthesis were excluded.

GCF sample collection was performed from three randomly selected sites of each patient in each group.

Clinical parameters

  • Plaque index (P.I.): Silness and Loe
  • Gingival index (G.I.): Loe and Silness
  • Gingival sulcus bleeding index (B.I.): Muhlemann and Son
  • Probing pocket depth (P.D.): Measured with William's periodontal probe


Method of study

Before the crevicular fluid collection, supragingival plaque was scored using the P.I. After the supragingival plaque was removed from each tooth with cotton pellets, isolation was carried out with cotton rolls and the individual tooth site was gently air dried.

GCF samples were collected with calibrated capillary tubes, and the sampling time for each site was 30s [Figure 1]. GCF was transferred with a jet of air pressure from the capillary tube into the eppendorf tube containing 250 μl of normal saline and stored at -20°C until it was analyzed with a semiautomated analyzer. Gingival inflammation was scored following crevicular fluid collection using the G.I. Bleeding on probing was measured dichotomously. Probing depth measures were obtained from the sample sites using a William's periodontal probe (Hu-Friedy, Chicago, IL, USA).
Figure 1: Gingival crevicular fluid collection with a microcapillary tube

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Enzyme assay

ALP levels were determined by using a commercially available diagnostic kit (Accurex Biomedical Pvt. Ltd., Mumbai, India) and the sample was assayed according to the kit's instructions.

The components and concentration of the working solution are:

  • Diethanolamine buffer, pH 9.8 1 Mol/L
  • p -nitrophenyl phosphate 10 mMol/L
  • Magnesium chloride 0.5 mMol/L
When samples were added to the ALP kit, the following reaction occurred:



ALP in a sample hydrolyzes para-nitrophenyl phosphate into para-nitrophenol and phosphate in the presence of magnesium ions. Under alkaline conditions, a colorless p-nitrophenol is converted to 4-nitrophenoxide, which develops a very intense yellow color. The rate of increase of absorbance of the reaction mixture at 405 nm due to liberation of para-nitrophenol is proportional to the ALP activity in the sample. The spectrophotometric analysis was performed using a semiautoanalyzer.

Alkaline phosphatase (IU/L) = ∆Absorbance/min×2,720

Statistical analysis

Data were expressed as means and standard deviations. Data analysis was performed using SPSS as software for statistics. For comparison between the groups, ANOVA test including Bonferrini-adjusted Wilcoxon signed rank test was used. In addition, Pearson's rank correlation coefficient was used in order to examine the relationship between the clinical and the biochemical parameters. The null hypothesis was rejected at P<0.05.


   Results Top


  • P.I., G.I., B.I. and probing depth values were significantly lower in Group I as compared with Group II and Group III patients [Table 1], [Table 2] and [Table 3], [Figure 2], [Figure 3], [Figure 4] and [Figure 5].
    Figure 2: Mean plaque scores in Groups I, II and III

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    Figure 3: Mean gingival index scores in Groups I, II and III

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    Figure 4: Mean bleeding scores in Groups I, II and III

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    Figure 5: Mean pocket depth in Groups I, II and III

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    Table 1: Plaque index, gingival index, bleeding index, probing depth and ALP total activity in Groups I, II and III

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    Table 2: Comparison of plaque index, gingival index, bleeding index, probing depth and ALP total activity between Groups I and II

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    Table 3 : Comparison of plaque index, gingival index, bleeding index, probing depth and ALP total activity between Groups I and III

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  • Difference in P.I., G.I. and B.I. between Groups II and III was statistically non-significant [Table 4] and [Table 5].
    Table 4: Comparison of plaque index, gingival index, bleeding index, probing depth and ALP total activity between Groups II and III

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    Table 5: Correlation of ALP total activity with plaque index, gingival index, bleeding index and probing depth in Groups II and III

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  • The mean probing depth was significantly higher in Group III compared with Group II [Table 4], [Figure 5].
  • In Group I, low levels of ALP activity were found [Figure 6].
    Figure 6: Mean alkaline phosphatase activity in Groups I, II and III

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  • Increased GCF ALP activity was observed in Group II (gingivitis) patients as compared with healthy sites, but this was not found to be statistically significant [Table 2], [Figure 6].
  • GCF ALP activity was significantly increased in chronic adult periodontitis as compared with that in gingivitis [Table 4], [Figure 6].
  • A statistically non-significant correlation was found between P.I., G.I. and B.I. of patients with chronic adult periodontitis and ALP activity [Table 5].
  • GCF ALP activity was found to be positively correlated with probing depth [Figure 7].
    Figure 7: Correlation between ALP activity and probing depth

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   Discussion Top


The processes responsible for the destruction of the human periodontium are highly complex, and a vast range of biological substances are involved. [5] One area that is receiving a great deal of attention nowadays is the biochemical analysis of GCF [6] for diagnosing inflammation of periodontal tissues.

The potential of ALP as an important biochemical marker of GCF was identified by Ishikawa and Cimasoni [7] in 1970. They quantified ALP in GCF using the colorimetric PNPP method and demonstrated that the levels of enzyme in GCF are three times greater than those in serum. They also showed a significant correlation between ALP concentration in GCF and pocket depth. This indicates that periodontal bone is also a possible source of enzyme. It is indeed known that ALP is highly active in this tissue. [8]

Binder et al. [9] carried out a longitudinal study and demonstrated that GCF ALP concentrations showed a positive correlation with clinical attachment loss, with 73% sensitivity and 64% specificity, for active disease. Because of the poor sensitivity of the PNPP assay used, they had to sample sites that were already inflamed and leave the sample papers in place until they were "visibly wet." ALP concentration (c) in GCF varied inversely with fluid volume (v) at each site; hence, the site-specific variation in GCF volume (with lower volumes obtained in anterior regions), favoring higher GCF ALP concentrations at the front of the mouth. Total ALP (q) did not differ significantly from sample to sample.

This condition, namely

q = constant

c = K/v

can be explained by a substance that is produced at a constant rate by a local source, possibly a tissue component, and enters the periodontal pocket independent of gingival fluid, which dilutes it.

Because GCF volume is influenced by many other factors such as flow rate, gingival trauma and repeat sampling, it is logical to expect that total enzyme levels in GCF will correlate better with disease than with enzyme concentrations. [6]

The interpretation of the results obtained after statistical analysis revealed that the P.I., G.I., B.I. and probing depth values were significantly lower in healthy gingiva as compared with that in patients with gingivitis and periodontitis.

Differences in P.I., G.I. and B.I. between the gingivitis group and the periodontitis group, respectively, were statistically non-significant.

A significant correlation between G.I. and ALP could not be established in our study in contrast to the study of Chapple et al. (1994), which reported that as the gingival index values increased, the total ALP also increased, and this was significant in the early stages of gingivitis. This may be due to the difference in the sample size, GCF collection method and the method of analysis.

The mean probing depth was significantly higher in patients with periodontitis compared with that in patients with gingivitis.

Low levels of ALP activity were found in healthy gingiva. Increased GCF ALP activity in gingivitis cases as compared with that in healthy gingiva was observed, but this was not found to be statistically significant.

GCF ALP activity was found to be positively correlated with probing depth. This finding is in accordance with the findings of Ishikawa and Cimasoni. [7] GCF ALP activity was significantly increased in chronic adult periodontitis. An increase of ALP activity has been shown by histochemical methods in inflamed gingiva, both in humans and in ferrets. [7] There are still several questions that remain to be answered, such as the exact source of the ALP and the nature of the isoenzyme(s) being measured. ALP is produced by many cells in the periodontal environment, the principal sources being PMNL, bacterial sources within supragingival and subgingival plaque and osteoblast and fibroblast activity, with a small contribution from serum. [5] There is neutrophil predominance in the pocket epithelium and the pocket itself, and the major source of ALP during inflammation has been proposed to be neutrophils. This postulate is further strengthened by the results of the plaque assays and levamisole inhibition studies, which suggested that bacterial ALP contributed <20% to total GCF ALP activity. [10]

It seems logical that all three sources would be raised in periodontal disease. If the source of GCF ALP is primarily the PMNL, it would form a potentially powerful marker of inflammation. However, if the primary source is that of host connective tissue, then the assay may be useful in monitoring tissue healing as well as destructive processes. [5]

Isoenzyme studies will greatly contribute to our knowledge of GCF ALP. [5]

GCF collection and analysis are not without problems. The amount of fluid available per site is generally below a microliter in volume, and the elution of potential biochemical markers from the sample filters used is often difficult. [6] Therefore, we preferred pre-calibrated capillary tubes to collect pooled samples of GCF from randomly selected sites.

Saliva contamination is another potential problem when sampling GCF, and this could clearly influence the results expressed as ALP concentration. ALP levels in saliva are extremely low and unlikely to influence the results for GCF ALP expressed as total enzyme in a 30 s sample. [6]


   Conclusion Top


ALP can be considered as a periodontal disease marker as it can distinguish between healthy and inflamed sites. Total activity of ALP was significantly higher in periodontitis as compared with gingivitis sites, which in turn showed levels of ALP higher than those found in healthy sites. It was found to be significantly and positively correlated with probing depth.

ALP activity is valuable to clinicians because it can quantitatively evaluate the inflammatory status of gingival and periodontal tissues. Thus, it can be used as an adjunct to clinical indices of inflammation. However, in order to better define the potential of this mediator for diagnosis, additional longitudinal studies are needed.

 
   References Top

1.Fine DH. Incorporating new technologies in periodontal diagnosis into training programs and patient care: a critical assessment and a plan for the future. J Periodontol 1992;63:383-93.  Back to cited text no. 1
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2.Zappa U. Histology of the periodontal lesion: implications for diagnosis. Periodontol 2000 1995;7:22-38.  Back to cited text no. 2
    
3.Perinetti G, Paolantonio M, Femminella B, Serra E, Spoto G. Gingival crevicular fluid alkaline phosphatase activity reflects periodontal healing / recurrent inflammation phases in chronic periodontitis patients. J Periodontol 2008;79:1200-7.  Back to cited text no. 3
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4.Daltaban O, Saygun I, Bal B, BaloΊ K, Serdar M. Gingival crevicular fluid alkaline phosphatase levels in postmenopausal women: Effects of phase I periodontal treatment. J Periodontol 2006;77:67-72.  Back to cited text no. 4
    
5.Chapple IL, Matthews JB, Thorpe GH, Glenwright HD, Smith JM, Saxby MS. A new ultrasensitive chemiluminescent assay for the site-specific quantification of alkaline phosphatase in gingival crevicular fluid. J Periodontal Res 1993;28:266-73.  Back to cited text no. 5
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6.Chapple IL, Glenwright HD, Matthews JB, Thorpe GH, Lumley PJ. Site-specific alkaline phosphatase levels in gingival crevicular fluid in health and gingivitis: cross - sectional studies. J Clin Periodontol 1994;21:409-14.  Back to cited text no. 6
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7.Ishikawa I, Cimasoni G. Alkaline phosphatase in humal gingival fluid and its relation to periodontitis. Arch Oral Biol 1970;15:1401-4.  Back to cited text no. 7
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8.Nakashima K, Roehrich N, Cimasoni G. Osteocalcin, prostaglandin E2 and alkaline phosphatase in gingival crevicular fluid: their relation to periodontal status. J Clin Periodontol 1994;21:327-33.  Back to cited text no. 8
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9.Binder TA, Goodson JM, Socransky SS. Gingival fluid levels of acid and alkaline phosphatase. J Periodont Res 1987;22:14-9.  Back to cited text no. 9
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10.Chapple IL, Socransky SS, Dibart S, Glenwright HD, Matthews JB. Chemiluminescent assay of alkaline phosphatase in human gingival crevicular fluid: investigations with an experimental gingivitis model and studies on the sorce of the enzyme within crevicular fluid. J Clin Periodontol 1996;23:587-94.  Back to cited text no. 10
[PUBMED]    

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Correspondence Address:
Ranjan Malhotra
Department of Periodontology and Oral Implantology, National Dental College and Hospital, Gulabgarh, Derabassi, Distt. SAS Nagar, Mohali, Punjab
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.74209

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]

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