Salivary enzymes as diagnostic markers for detection of gingival/periodontal disease and their correlation with the severity of the disease.

CONTEXT: Host responses to periodontal disease include the production of different enzymes released by stromal, epithelial or inflammatory cells. Important enzymes associated with cell injury and cell death are aspartate aminotransferase, alanine aminotransferase (AST, ALT), alkaline phosphatase, acidic phosphatase (ALP, ACP), and gama glutamyl transferase (GGT). Changes in enzymatic activity reflect metabolic changes in the gingiva and periodontium, in the inflammation. AIMS: In this article we examined the activity of AST, ALT, GGT, ALP, and ACP in the saliva from patients with periodontal disease, before and after periodontal treatment (experimental group - 20 gingivitis patients and 20 periodontitis patients), and in the saliva from healthy subjects (control group - 20 samples). SETTINGS AND
DESIGN: Periodontal disease was determined based on the clinical parameters (gingival index (GI), probing depth (PD), and clinical attachment loss (CAL)). Patients with periodontal disease were under conventional periodontal treatment.
MATERIALS AND METHODS: The stimulated saliva of the patient was collected in a sterile test tube and analyzed using the Automatic Analyzer.
RESULTS: The obtained results showed statistically significant increased activity of AST, ALT, GGT, ALP, and ACP in the saliva from patients with periodontal disease, in relation to the control group. A significant reduction in the enzyme levels was seen after conventional periodontal therapy.
CONCLUSIONS: Based on these results, it can be assumed that the salivary enzymes (AST, ALT, GGT, ALP, and ACP) can be considered as biochemical markers for evaluating the diagnosis and prognosis of the functional condition of periodontal tissues in disease and health, and in the evaluation of the therapy effects in periodontal disease.

INTRODUCTION

The ability to correctly diagnose and assess the periodontal disease has received considerable attention in the last decade. The traditional diagnostic methods using clinical scores, such as, probing pocket depth, clinical attachment loss, bleeding on probing, and radiographic assessment of alveolar bone loss, give information of the past damage, and hence, have been considered inefficient to distinguish the disease activity with accuracy.[1] Newer methods have been expected to facilitate the diagnosis of active periodontal disease and they help to determine the risk of an inactive site from becoming active. Analysis of the various gingival crevicular fluid biochemical markers has been proposed as a means to predict clinical attachment loss. Among the important gingival crevicular fluid components, are the various enzymes. A response of an organism to the periodontal infection includes production of several enzyme families, which are released from stromal, epithelial, inflammatory or bacterial cells. The enzymes of tissue degradation are AST (Asparatate Aminotransferase), ALT (Alanine Aminotransferase), GGT (Gamma Glutamyl Transferase), ALP (Alkaline Phosphatase), and ACP (Acidic Phosphatase).[23] There are a number of studies available in literature correlating the levels of these enzymes in gingival crevicular fluid (GCF) with the severity of periodontal disease.[45] However, there are inherent problems in the collection of GCF in a routine office dental setting. The sampling technique is not easy, as a long time is required for sample collection and it only reflects gingival inflammation at each specific site sampled. Thus, GCF is not suitable for mass screening.[6] Recent studies have shown that these enzymes can be quantified easily in a saliva sample.[378] The use of saliva to measure these biomarkers (enzymes) offers several advantages over GCF. As collection of saliva requires no specialized equipment or techniques, it is faster and more convenient for the patient and the practitioner to collect. In addition, whole saliva represents a pooled sample with contributions from all periodontal sites, and analysis of the biomarkers in the saliva may provide an overall assessment of the disease status as opposed to site-specific GCF analysis.[9]

MATERIALS AND METHODS

Examination included 20 patients with gingivitis; 20 patients with periodontitis, and 20 healthy adult volunteers. The categorization of the patients was done based on the following evaluation parameters: Gingival index (Loe and Sillness), probing depth, and clinical attachment loss. Patients with at least four teeth with ≥ 5 mm clinical attachment loss, having moderate bone loss on an orthopantomogram (OPG) were categorized as the periodontitis group. Patients with a history of smoking, alcohol abuse or suffering from any systemic diseases, who had taken antibiotics in the past six months or who had undergone periodontal treatment in the past six months were excluded from the study. Pregnant and lactating mothers were also excluded.

In the initial examination, each subject completed a detailed medical questionnaire and received a complete periodontal examination, which included: gingival index (GI): Loe and Sillness, probing depth (PD), and clinical attachment loss (CAL). Patients with periodontal disease were under conventional periodontal treatment consisting of oral hygiene instructions, scaling, and root planing. The saliva sample was collected at baseline and three weeks post treatment.

The patient was asked to rinse with 15 ml of water (to wash out exfoliated cells) and then asked to chew paraffin wax for five minutes [Figure 1a]. The stimulated saliva (10 ml) of the patient was collected in a sterile test tube. The saliva sample was stored in a refrigerator (2-8°C) until transported in an ice bag to the laboratory, where the activities of the following salivary enzymes were determined spectrophotometrically, with the help of an autoanalyzer — Asparatate aminotransferase, Alanine aminotransferase, Gamma glutamyl transferase, Alkaline phosphatase, and Acidic phosphatase. The saliva samples were centrifuged at 10000 rpm for 10 minutes. The activity of the enzymes in the saliva was determined spectrometrically by the International Federation of Clinical Chemistry (IFCC) method on the Hitachi Automatic Analyzer [Figure 1b]. Group 1 (Healthy Subjects) was provided no treatment. Group 2 (Gingivitis Patients) and Group 3 (Periodontitis Patients) were provided Oral Prophylaxis, which included scaling and root planing. The following were the applied statistical analyses: Mean value, standard deviation, standard error, and Student's t-test. Probabilities less than 0.05 (P<0.05) were considered statistically significant.

Figure 1

(a) Chewable paraffin wax (b) Hitachi's clinical automatic analyzer

Laboratory procedure for assessment of various enzyme activities

Asparatate aminotransferase

The test for AST is based on the following principle.

Principle

AST

L - Asparatate + 2 - Oxoglutarate → Oxaloacetate + L-Glutamate.

MDH

Oxaloacetate + NADH → Malate + NAD +

LDH

Sample pyruvate + NADH → L - Lactate + NAD +

AST = Asparatate aminotransferase

MDH = Malate dehydrogenase

LDH = Lactate dehydrogenase

Aspartate aminotransferase catalyzes the transfer of the amino group from L-aspartate to 2-Oxoglutarate to yield oxalacetate and L-glutamate. The oxalacetate undergoes reduction with simultaneous oxidation of nicotinamide adenine dinucleotide plus Hydrogen (NADH) to the nicotinamide adenine dinucleotide radical (NAD) in the malate dehydrogenase (MDH) catalyzed indicator reaction. The resulting rate of decrease in absorbance at 340 nm is directly proportional to the AST activity. Lactate dehydrogenase (LDH) is added to prevent interference from the endogenous pyruvate, which is normally present in the serum.

Reagent composition (when reconstituted as directed)

Reagent reconstitution

Allow the reagent bottle and Aqua 4 (supplied in the kit) to attain room temperature. Add the amount of Aqua 4 indicated on the label to the contents of each vial. Swirl to dissolve. Do not shake vigorously.

Storage and stability

Prior to reconstitution:

Unopened reagents are stable till the expiry date stated on the label when stored at 2-8°C.

After reconstitution:

The working reagent is stable for 30 days at 2°-8°C. Discard if the reagent turns turbid or if the absorbance is less than 0.8 at 340 nm, against distilled water.

Calculation

The general formula for converting absorbance change into international units (IU) of activity is:

where:

T.V = Total reaction volume (μl)

S.V = Sample volume (μl)

Absorptivity = millimolar Absorptivity of NADH at 340 nm = 6.22

P = Cuvette lightpath (cm) = 1 cm

Activity of AST = ∆ Abs / minute × 1768

Alanine Aminotransferase

The test for ALT is based on the following principle.

ALT

L - Alanine + 2 - Oxoglutarate → Pyruvate + L - Glutamate.

LDH

Pyruvate + NADH → L - Lactate + NAD +

ALT = Alanine aminotransferase

LDH = Lactate dehydrogenase

Alanine aminotransferase catalyzes the transfer of the amino group from L-alanine to 2-oxoglutarate resulting in the formation of pyruvate and L-glutamate. Lactate dehydrogenase catalyzes the reduction of pyruvate and the simultaneous oxidation of NADH to NAD. The resulting rate of decrease in the absorbance is directly proportional to the ALT activity.

Allow the reagent bottle and Aqua 4 (supplied in the kit) to attain room temperature. Add the amount of Aqua 4 indicated on the label to the contents of each vial. Swirl to dissolve. Do not shake vigorously.

Storage and stability

Prior to reconstitution:

Unopened reagents are stable till the expiry date stated on the label when stored at 2-8°C.

After reconstitution:

The working reagent is stable for 30 days at 2°-8°C. Discard if the reagent turns turbid or if the absorbance is less than 0.8 at 340 nm against distilled water.

The general formula for converting absorbance change into international units (IU) of activity is:

(∆ A / minute ) × T.V × 103

IU / L = S.V × Absorptivity × P

where:

T.V = Total reaction volume (μl)

S.V = Sample volume (μl)

Absorptivity = millimolar Absorptivity of NADH at 340 nm = 6.22

P = Cuvette lightpath (cm) = 1 cm

Activity of ALT = ∆ Abs / minute × 1768

Alkaline phosphatase

Alkaline phosphatase is a hydrolase enzyme responsible for removing phosphate groups from the 5- and 3- positions, from many types of molecules, including nucleotides, proteins, and alkaloids.

The determination of ALP is based upon the IFCC recommendations and according to the following principle:

ALP

p-nitrophenyl phosphate + water ↔ phosphate + p-nitrophenol

ρ-Nitrophenyl phosphate is hydrolyzed to ρ-nitrophenol and inorganic phosphate. The rate at which p-NPP is hydrolyzed and measured at 405 nm, is directly proportional to the alkaline phosphatase activity.

Preservative

When stored at 2°-8°C and protected from light these reagents are stable until the expiry date.

One international Unit (IU / L) is defined as the amount of enzyme that

catalyzes the transformation of one micromole of substrate per minute under specified conditions.

(IU / L) = ΔAbs. / minute × 2187

Acid phosphatase

Acid phosphatase catalyses the hydrolysis of a-naftylphosphate to a-naphtol and a phosphate, in an acid medium. a naphtol reacts with diazo 2 chloro 5 toluene (Fast Red TR) to form a diazo dye, which has a strong absorbance at 405 nm and increases its absorption at 405 nm proportional to the total acid phosphatase activity (ACP-T) present in the sample.

ACP α-naphthylphosphate + H2O ----> α-naphthol + PO4

α-naphthol + Fast Red TR ----> Diazonium Dye

Package: collection and storage

Store at room temperature (+ 15 / 25°C).

Stable till the expiration date reported on the package.

After unsealing and removing the reagent, it is advised to close the bottle immediately in order to avoid evaporation, direct exposure to light, and bacterial contamination.

ACP (IU/L) = ∆ Absorption / minute × 750

Gamma glutamyl transferase

The Gamma Glutamyl Transferase present in the sample catalyses the transfer of the glutamyl group from the substrate to glycylglycine, forming glutamylglycylglycine and 5-amino 2-nitrobenzoate.

L - γ-glutamyl-3-carboxy-4-nitroanilide + glycylglycine

↓ GGT

L- γ- Glutamylglycylglycine + 5-amino-2-nitrobenzoate.

The rate of formation of 5-amino-2-nitrobenzoate is proportional to the activity of the GGT present in the sample and can be measured kinetically at 410 nm.

Reagent preparation

The reagent is supplied ready to use. Transfer the contents of Reagent A and Reagent B into the appropriate compartments of the User Defined Reagent (UDR) cartridge included in the kit. Use care to avoid contamination.

Stability and storage

The unopened reagents are stable until the expiration date when stored at 2-8 °C.

Testing procedures

Load the reagent onto the system as directed. Program the samples and controls for analyses as directed.

RESULTS

The obtained results show that the activity of the examined enzymes in the saliva of the patients with periodontal disease was significantly higher in relation to the control group. The established differences showed a statistical significance of a high level [P<0.001, Table 1]. After conventional periodontal treatment the activity of all salivary enzymes along with various evaluation parameters decreased significantly [Table 2 and Figures 2–4].

Table 1

Differences between AST, ALT, GGT, ALP, ACP activities (U / L±SD) in saliva of healthy controls and patients with periodontal disease (gingivitis patients and periodontitis patients), and before & after periodontal treatment

Table 2

Mean values of various parameters at baseline and three weeks after periodontal therapy in all the groups

Figure 2

Mean comparison of pre-treatment and post-treatment salivary enzymes in gingivitis patients

Figure 4

Mean percentage reduction in evaluation parameters after periodontal therapy (Ω GI – Percentage change In values of GI after periodontal therapy. Ω PD – Percentage change in values of PD after periodontal therapy. Ω CAL – Percentage change in values of CAL after periodontal therapy)

Mean comparison of pre-treatment and post-treatment salivary enzymes in periodontitis patients

DISCUSSION

Diagnostic laboratory tests of the serum are routinely used in the evaluation of many systemic disorders. In contrast, the diagnosis of many periodontal diseases relies primarily on the clinical (GI, PD, BOP) and radiographic parameters (alveolar bone loss).[4] The strengths of these traditional tools are their ease of use, their cost effectiveness, and that they are relatively noninvasive, but these traditional diagnostic procedures are inherently limited, in that, only the disease history and not the current disease status and the sites at risk for future periodontal breakdown, can be assessed. Clinical attachment loss readings by the periodontal probe and radiographic evaluations of alveolar bone loss measure the damage from the past episodes of destruction and require a 2 to 3 mm threshold change before a site can be identified as having experienced a significant anatomic event. Advances in oral and periodontal disease diagnostic research are, therefore, moving toward methods whereby periodontal risk can be identified and quantified by objective measures such as biomarkers.[10] Numerous biomarkers in GCF have been proposed as diagnostic tests for periodontal disease. Among the intracellular enzymes that have received considerable attention as possible biomarkers of active periodontal destruction are Asparatate aminotransferase (AST), Alanine aminotransferase (ALT), Gamma glutamyl transferase (GGT), β-glucuronidase, Elastase, Lactate dehydrogenase (LDH), Creatinine kinase (CK), Alkaline phosphatase (ALP), and Acid phosphatase (ACP).[4]

Enzymes AST, ALT, and GGT are intracellular enzymes included in the metabolic processes of cells and they are mostly present in the cells of soft tissues. These enzymes are indicators of higher levels of cellular damage and their increased activity in GCF is a consequence of their increased release from the damaged cells of the soft tissues of the periodontium, and is a reflection of the metabolic changes in the inflamed gingiva. There are enough studies available in literature correlating the levels of these enzymes in GCF with the severity of periodontal disease.[45]

Alkaline Phosphatase and ACP are intracellular enzymes present in most of the tissues and organs, particularly in bones. Their increased activity is probably the consequence of the destructive processes in the alveolar bone, in the advanced stages of the development of periodontal disease.[3] Some studies have shown a remarkably increased activity of ALP in the acute phase of periodontal disease, and after periodontal therapy, the activity of these enzymes was restored to the value found in healthy persons.[11] Here again, these studies referred to the gingival crevicular fluid.[1213]

Previous studies have mainly investigated the activities of these enzymes in gingival crevicular fluid, which has a much closer contact with the periodontal tissues, and due to this, it surely reflects the occurrences in them much better. However, the problem with the gingival crevicular fluid is that the technique of collecting it is rather complicated, and as a routine procedure, which possibly may be established, it will be hardly feasible in practice.

The activity of these enzymes can also be proved in saliva, as these enzymes are determined even in the blood of healthy persons. When a periodontal tissue becomes diseased, or its cells become damaged, due to edema or destruction of a cellular membrane, that is, of a cell as a whole, these intracellular enzymes are increasingly being released into the GCF and saliva, where their activity can be measured. Contrary to the gingival crevicular fluid there is plenty of saliva, and the procedure of its sampling is much easier and more bearable for the patient. On account of the simple and non-invasive method of collection, salivary diagnostic tests appear to hold a promise for the future.[1415]

This article is a study that has shown that the increased activity of certain tissue enzymes in periodontal disease can be proved with saliva, as a reflection of the pathological changes in the cells of periodontal tissues. The present study indicates that the salivary AST, ALT, GGT, ACP, and ALP levels are significantly increased in patients with periodontal disease and the salivary enzyme levels decrease in concomitance with the periodontal treatment.

The increased activities of AST, ALT, and GGT indicate the pathological changes located in the soft tissues only, primarily in the gingival tissues, which could coincide with the initial stage of periodontal disease. However, the increased activity of ACP, and especially ALP, indicates that the pathological destructive process has affected the alveolar bone, which means that the periodontal disease has significantly advanced.

In this study, analysis of salivary AST showed significantly higher levels in patients of gingivitis (P=0.001**) and chronic periodontitis (P=0.001**), as compared to the controls Table 1. Similar results were seen in the previous studies (Todorovic et al.,[4] Cesco et al.[5] ). When comparing the salivary AST levels of gingivitis and periodontitis patients, the levels were raised in periodontitis patients, as compared to the gingivitis group, but the difference of the mean was not statistically significant [P=1.000, Table 1]. Post-treatment enzymatic evaluation showed a significant decrease in salivary AST levels [P = 0.001, Table 1]. Similar readings were reported by Yoshie et al.,[3] and Todorovic et al.[4] The fall in salivary AST levels following therapy could be contributed to the resolution of inflammation and the return of the periodontal tissues toward health.

The activity of enzyme ALT reflects the levels of cellular damage and metabolic changes in the inflamed gingival tissues (Numabe et al. ).[14] The salivary ALT levels, in this study were found to be significantly raised in gingivitis and periodontitis patients [P=0.032FNx01, Table 1], as compared to those in the healthy controls, thus depicting that salivary ALT activity reflects inflammation and destruction of the periodontal tissue, suggesting clinically useful biomarkers. When compared, between the two, the salivary ALT levels were significantly higher in the gingivitis than in the periodontitis patients [P=0.026* Table 1]. The enzyme level significantly decreased following periodontal therapy (P<0.005). These findings were consistent with the readings of salivary ALT reported by Yoshie et al.[3] and Todorovic et al.[4]

Serum enzyme GGT appears to be primarily from the hepatobiliary system. Our study reported a significant increase in salivary GGT levels in patients with moderate gingivitis (P=0.019*) and chronic periodontitis (P=0.005**), as compared to the healthy controls Table 1. The levels of salivary GGT were higher in periodontitis patients when compared with the gingivitis patients, but the difference was not statistically significant [P=1.00, Table 1]. The GGT levels were found to significantly decrease following periodontal therapy. The mean difference in the pretreatment and post-treatment values of salivary GGT was found to be statistically significant [P<0.005**, Table 1]. These findings were in confirmation with the results obtained by Todorovic et al.[4]

Alkaline and acid phosphatase are among the enzymes most commonly associated with bone metabolism.[47] ALP is enriched in the membranes of mineralizing tissue cells (e.g., osteoblasts) and is also present in polymorphonuclear leukocyte (PMN) granules. ALP is produced by some oral bacteria, including gram-negative microorganisms found in the subgingival plaque (Mc Comb, Bowers and Posen 1979, Cimasoni 1983). In our study, we could see a highly significant rise in salivary ALP levels in gingivitis (P=0.002**) and periodontitis patients (P=0.001**) as compared to the healthy controls [Table 1], thereby, indicating the pathological changes occurring in the underlying periodontal tissues.

The potential value of alkaline phosphatase as a biomarker of periodontal disease activity was identified by Ishikawa and Cimasoni, in 1970.[16] They showed a significant correlation (r=0.49; P<0.05) between the ALP concentration in GCF and pocket depth. More recently, a study with longitudinal design demonstrated a 20-fold increase of ALP activity at sites with 2 mm or more of attachment loss (Binder et al.[17] ). As per the reported results of this study, although the levels of salivary ALP were higher in periodontitis patients as compared to gingivitis patients, the difference was not statistically significant [P=1.000; Table 1]. The difference between the post-treatment and the pretreatment values of salivary ALP was statistically significant [P=0.001**; Table 1], which was inconsistent with the findings as reported by Numabe et al.,[14] wherein, a decrease of ALP activity in the saliva was observed after scaling and root planing. As per Zambon and Slots[18] the ALP activity, which was reported to be 30.0±8.9, decreased to a level of 24.0±8.0, two weeks post treatment.

Acid Phosphatase is among the enzymes associated with bone metabolism. Acid phosphatase is present in neutrophils and is considered to be a lysosomal marker.Desquamated epithelial cells, macrophages, and several bacteria, including Actinobacillus Capnocytophaga and Veillonella also produce this enzyme (Cimasoni 1983). Chauncey et al. (1954) demonstrated acid phosphatase, but not alkaline phosphatase, in parotid saliva. In our study, a statistically significant rise was seen in the activity of salivary ACP, in patients with gingivitis and chronic periodontitis when compared to healthy subjects [Table 1]. This finding of ours relates to the findings of Todorovic et al.[4] who reported a statistically significant rise in ACP activity in patients with chronic periodontitis when compared to healthy subjects. Although the levels of salivary ACP in this study were higher in periodontitis patients, as compared to gingivitis patients, the difference was not statistically significant. The activity of the salivary ACP was found to significantly decrease post treatment [P=0.001**; Table 1]. Similar results were reported by Todorovic et al.[4] where a significant decrease in the activity of ACP was seen after periodontal therapy. The results of the salivary ACP levels in this study do not correlate with the findings of Zambon and Slots J[18] who reported no change in the levels of ACP activity when pretreatment levels (40.0±0) were compared to the two-week post treatment levels (40.0±0).

CONCLUSIONS

On the basis of the results of this study, it can be concluded that the activity levels of enzymes AST, ALT, GGT, ALP, and ACP in saliva are related to periodontal destruction. The levels of these enzymes are raised statistically in the saliva of patients with gingivitis and periodontitis, as compared to the controls, and its value decreases after periodontal therapy. The raised levels in diseased individuals is probably a consequence of the pathological processes in periodontal tissues, from where these intracellular enzymes are increasingly released into the secretion that surrounds them — saliva — and the decrease in the activity of these enzymes after periodontal treatment is probably a result of periodontal tissue repair. Thus, it can be stated that the salivary enzymes can be considered as the biochemical markers of the functional condition of the periodontal tissues that provide new opportunities in making diagnoses and also evaluating the effectiveness of periodontal therapy in improving periodontal health.