To estimate salivary aspartate aminotransferase levels in chronic gingivitis and chronic periodontitis patients prior to and following non-surgical periodontal therapy: A clinico-biochemical study.

CONTEXT: Saliva can be used as a diagnostic fluid in dentistry. Various enzymes have been proposed as markers for periodontal destruction. One of them is aspartate aminotransferase, for which salivary analysis can offer a cost-effective approach for monitoring the disease. Changes in enzymatic activity reflect metabolic changes in the gingiva and periodontium in inflammation. AIMS: The purpose of this study was to assess the aspartate aminotransferase levels in saliva prior to and following scaling and root planning (SRP) at 1 month and 3 month interval and correlating it with the clinical parameters in generalized chronic gingivitis and chronic periodontitis patients.
MATERIALS AND METHODS: Thirty patients with generalized chronic gingivitis and 30 with generalized chronic periodontitis were selected. The activity of aspartate aminotransferase levels in saliva were assessed biochemically before and after SRP at 1 month and 3 months. The aspartate aminotransferase levels were correlated with clinical parameters (gingival index and probing depth). STATISTICAL ANALYSIS USED: A paired t test was done.
RESULTS: A decrease in gingival index, probing depth, and aspartate aminotransferase levels were seen in both the groups at 1 and 3 months which was found to be statistically highly significant (P value 0.00). Aspartate aminotransferase levels were statistically significantly correlated with the clinical parameters at baseline (P < 0.05) but at 3 months, a positive correlation was seen in both the groups which was statistically insignificant (P > 0.05).
CONCLUSIONS: Elevated salivary aspartate aminotransferase levels were seen in generalized chronic gingivitis and chronic periodontitis patients, with higher values recorded in generalized chronic periodontitis correlating to the tissue destruction taking place in these conditions.

INTRODUCTION

Traditionally, periodontal diseases have been diagnosed based on detailed history, clinical, and radiographic assessment.[1] Progression of periodontal destruction is episodic and periodontal pockets can be disease active or disease inactive. Unfortunately, none of the above diagnostic procedures distinguishes between disease active and inactive sites.[2] One approach to the development of an objective diagnostic test is to measure the presence and levels of molecules present in gingival crevicular fluid (GCF) that may correlate with measures of disease activity. Components of interest include prostaglandin E2 , hydroxyproline, hydrolytic enzymes, and other substances released by neutrophillic granulocytes, collagenases, matrix degradation products, and enzymes that are released only upon cell death like lactate dehydrogenase and aspartate aminotransferase (AST). Among most promising markers of tissue destruction is AST. It is found in serum and erythrocytes of healthy individuals but its concentrations in active periodontal pockets of patients with periodontitis are higher. It is released into extracellular fluid upon cell death; it seems that higher concentrations in GCF result from local tissue destruction. AST activity in periodontal pockets can thus be a useful marker of periodontal disease activity.[3] AST levels can be assessed in the saliva and GCF biochemically. Saliva offers advantages over GCF in that it is cost effective, easier method of collection, storage and transport, and the same enzymes which are found in GCF can be detected in saliva also. It is a safe, and noninvasive approach for periodontal disease detection, and possesses a high potential to revolutionize the next generation of diagnostics.[4]

MATERIALS AND METHODS

The study included 60 patients in the age group of 20-65 yrs who were selected from the Outpatient Department of Periodontics. The following criteria were observed in the enrolment of the patients: (1) who had a minimum of 20 teeth present and had not undergone any dental treatment for the past 1 yr; (2) with good systemic health; (3) no use of medications such as antibiotics, anticoagulants, steroids, hormonal therapy in the last 6 months; (4) pregnant women and lactating mothers were excluded; (5) no history of allergy; (6) no history of tobacco and alcohol use. An ethical committee approval was sought before commencement of the study.

Subjects

Patients were screened for gingival bleeding and pocket depth. Subjects were explained about the nature of the study and informed consent was taken.

Group divisions

The selected patients were grouped as:

Group I: 30 patients with generalized chronic gingivitis

Group II: 30 patients with generalized chronic periodontitis

Criteria for group division

Group I: Plaque-associated gingivitis or marginal gingiva was erythematous and edematous with bleeding on probing. Sulcus depth was ≤3 mm. There was no clinical loss of attachment and gingival index score (Loe and Silness index, 1963) was ≥2.0 [Figures 1 and 2]

Figure 1

Gingival index scoring at baseline for group I

Figure 2

Probing depth at baseline for group I

Group II: Sites from patients diagnosed with chronic periodontitis with a probing depth of >4 mm Figure 3 and Figure 4.

Figure 3

Gingival index scoring at baseline for group II

Figure 4

Probing depth at baseline for group II

Each group was assessed for the following clinical parameters at baseline, 1 month, and 3 months: Gingival Index (using Loe and Silness index 1963) and Pocket depth (using UNC 15 probe).

Method

Subjects were seated comfortably in a semireclined position on the dental chair and instructed to rinse mouth thoroughly with 15 ml of water. Subjects were then instructed to be seated for 5 min and directed not to speak, eat/rinse and minimize orofacial movements during the 5 min period. Following this subjects were instructed to spit the pooled saliva from the floor of the mouth into sterile plastic vials (Spitting method[4] ). The plastic vials were then closed and sent to the biochemistry lab for assaying the AST levels.

After this, the subjects were subjected to through scaling and root planning and saliva sampling was done at 1 month and 3 months.

Biochemical estimation of AST levels

Biochemical assay for salivary AST levels was done using semi automatic analyzer (Protonic Proto 99). This analyzer works on optimal UV test as per IFCC (International federation of clinical chemistry and laboratory medicine) recommendations. AST formerly called glutamate oxaloacetate catalyzes the reversible transfer of an amino group from aspartate to α ketoglutarate forming glutamate and oxaloacetate. The oxaloacetate produced is reduced to malate by malate dehydrogenase (MDH) and NADH. The oxidation of NADH is measured at 340 nm:

The rate of decrease in the concentration of NADH measured photometrically is proportional to the catalytic concentration of AST present in saliva sample.

RESULTS

Comparison between group I and group II for all the measures of periodontal parameters and AST levels were analyzed by applying Student paired “t” test. Karl Pearson's correlation coefficient (“r”) was calculated among different parameters of periodontal disease and AST concentration. Five patients in the group I and 1 patient in group II didn't report at the specified intervals for follow up. Hence, for statistical assessment 25 patients in each group were considered.

In group I: At baseline, the mean GI was 1.55 ± 0.19, the mean PD was 1.53 ± 0.13 mm, and the mean AST level was 51.4 ± 5.94 μL [Table 1]. At 1 month, the mean GI was 0.48 ± 0.19, the mean PD was 1.5 ± 0.13 mm, and the mean AST level was 41.84 ± 3.58 μL [Table 1]. At 3 month, the mean GI was 0.37 ± 0.06, the mean PD was 1.48 ± 0.14 mm, and the mean AST level was 36.64 ± 2.02 μL [Table 1, Graphs 1 and 3].

Table 1

Test of significance (paired ‘t’ test) among various parameters in Group I

Graph 1

Bar diagram showing mean values of GI & PD values of group I

Graph 3

Line graph showing AST levels in group I at baseline, 1 month, and 3 month

In group II: At baseline, the mean GI was 1.75 ± 0.37, the mean PD was 3.86 ± 0.45, and the mean AST level was 76.8 ± 7.56 μL [Table 2]. At 1 month, the mean GI was 0.45 ± 0.52, the mean PD was 1.63 ± 0.26 mm, and the mean AST level was 46.36 ± 3.78 μL [Table 2]. At 3 month, the mean GI was 0.42 ± 0.53, the mean PD was 1.43 ± 0.14 mm, and the mean AST level was 37.16 ± 1.77 μL [Table 2, Graphs 2 and 4].

Table 2

Test of significance (paired ‘t’ test) among various parameters in Group II

Graph 2

Bar diagram showing mean values of GI & PD values of group II

Graph 4

Line graph showing AST levels in group II at baseline, 1 month, and 3 month

In the group I: Comparison of clinical and biochemical parameters at baseline and at 3 months exhibited a reduction in GI and AST levels which were found to be statistically highly significant (P value 0.0000) [Table 1].

In the group II: Comparison of clinical and biochemical parameters at baseline and at 3 months exhibited a reduction in GI, PD, and AST levels which were found to be statistically highly significant (P value 0.0000) [Table 2].

DISCUSSION

Periodontal disease is a chronic bacterial infection characterized by persistent inflammation, connective tissue breakdown, and alveolar bone destruction.[5] Subjective symptoms are typically mild during the early phase of disease, subjects tend to ignore the condition until more severe symptoms appear, e.g. increased tooth mobility, etc., Periodontal disease may be a major cause of tooth loss. Thus, early detection of periodontal diseases may be important with respect to quality of later life.[6]

Traditional diagnostic measures such as periodontal pocket depth, attachment level, plaque index, bleeding on probing, and radiographic assessment of alveolar bone loss are informative to evaluate disease severity but provide few useful determinants of disease activity.[78] It has been a great challenge in periodontology to determine biomarkers for screening and predicting the early onset of disease (prognostic tests) or evaluating the disease activity and the efficacy of therapy (diagnostic tests).[9]

Biochemical tests are used extensively in medicine both in relation to diseases that have an obvious metabolic basis and those in which biochemical changes are a consequence of the disease. In dentistry, these tests are gaining importance in the diagnosis, prognosis, monitoring, and screening of periodontal diseases in which changes in enzymatic activity reflect metabolic changes in gingiva and periodontium in inflammation. These tests have been proposed to assess periodontal disease activity in addition to clinical assessments.[7]

The present study was designed to assess the clinical and biochemical parameters in patients with generalized chronic gingivitis and periodontitis.

At baseline, a decrease in GI was seen in both the groups at 1 month and 3 months which exhibited a statistically highly significant reduction (P < 0.01). The reduction in gingival index is attributed to the effects of SRP. SRP is considered as the gold standard therapy in the treatment of periodontal diseases. SRP improves clinical parameters by decreasing the inflammatory infilterate.[10] This is also in agreement with the studies conducted by Persson et al.,[11] Shimada et al.,[12] and Arora et al.,[13] which showed that SRP and motivation for oral hygiene helps improve the periodontal health of the subjects.

At baseline, the mean PD in group I was 1.53 ± 0.13 mm [Table 1]. Ideal PD of the gingival sulcus is 0 mm but this can be produced experimentally only in germ-free animals (Attstrom et al.,[14] Caffesse[15] ). In a clinically healthy human gingiva, some depth of the sulcus can be found as determined by histologic sections. Weski[16] and Gargiulo et al.[17] have reported a depth between 1.5 mm and 0.6 mm, respectively.

At 3 months, the PD in Group I was 1.48 ± 0.14 mm [Table 1]. The minor reduction could be attributed to the effects of SRP. However, the reduction in PD at 3 months as compared to the baseline was not found to be statistically significant (P > 0.05).

Likewise, group II showed a decrease in PD after SRP at 1 month and 3 month which was highly significant statistically (P < 0.01) [Table 2]. This goes in accordance with the studies done by Arora et al.,[13] Yoshie et al.,[18] Shimada et al.,[12] and Persson et al.[11] who supported that clinical parameters can be improved by SRP.

At baseline, AST level in saliva was 51.4 ± 5.94 μL in group I [Table 1] and 76.8 ± 3.78 μL in group II [Table 2]. AST is a cytoplasmic enzyme present in many body tissues with especially pronounced distribution in heart, liver, and skeletal muscle. It is important for the production of various aminoacids and serve as a diagnostic analyte of cellular injury. It is a ubiquitous component of saliva and is detected in periodontal tissue, GCF, enamel pellicle, and saliva.

Their activity can be proved in saliva, within some normal limits, as these enzymes are determined even in blood of healthy persons. However, if there is periodontal tissue destruction, these intracellular enzymes are increasingly being released into the GCF and saliva where their activity can be measured. Due to this, it can be a biochemical marker of the functional condition of periodontal tissues.(Numabe,[19] Mc Culloch,[20] Nakashima[21] ).

These enzymes are indicators of a higher level of cellular damage and their increased activity in GCF and saliva is a result of their increased release from the damaged cells of soft tissues of peridontium and a reflection of metabolic changes in the inflamed gingiva (Numabe,[19] Ozmeric[22] ). Studies done by Kolte et al.,[23] Golub et al.,[24] Cohen,[25] Persson and Page RC[26] have also observed AST levels to be high at diseased sites.

Previous studies by Silva et al.,[27] Hanioka et al.,[28] Oringer,[29] Persson,[30] Wong[31] mainly investigated the activities of these enzymes in GCF, which is in a much closer contact with periodontal tissues and due to this, it surely much better reflects the occurrences in them. However, the problem with the GCF is in that the technique of collecting is rather complicated and that in a routine procedure, which possibly might be established, it would be hardly feasible in practice. Contrary to the GCF, there is plenty of saliva, the procedure of its sampling is much easier and more bearable for the patient and however, the same enzymes as those in the GCF can be detected. Because of the simple and noninvasive method of collection, salivary diagnostic tests hold promise for the future. (Numabe,[19] Kaufman,[32] Ozmeric[22] ).

Also, improvements in clinical status were noted following periodontal therapy and there was a corresponding decrease in AST levels. So, AST levels may be a sensitive and specific enough to be a useful adjunct in the clinical assessment of periodontal disease, since AST level decrease when periodontal status improves.[12]

At baseline, when AST levels were correlated with GI scores, there was a very significant correlation (P < 0.01) suggesting that AST level is associated with severity of gingival inflammation. This is in accordance with studies of Persson et al.[1] and Kolte et al.[23] [Table 3].

Table 3

Coefficient of correlation between different parameters in group I (Karl Pearson's correlation coefficient test) ‘r’

At 3 months, following treatment in group I, a positive correlation was present between GI and AST levels [Table 4] but it was insignificant statistically (P > 0.05). In clinically healthy gingiva with PD ranging between 0 and 3 mm, small foci of inflammatory cells would be present along the sulcus. This is in accordance with studies conducted by Page and Shroeder,[13] who histologically studied inflammatory changes adjacent to junctional epithelium and gingival sulcus.

Table 4

Coefficient of correlation between different parameters in group II (Karl Pearson's correlation coefficient test) ‘r’

At baseline, in group II the correlation between GI and AST levels was positive and statistically significant (P < 0.05). This was supported by Persson et al.,[11] who suggested that increased periodontal destruction resulted by microbial activity leading to increased AST concentration [Table 4].

At 3 months, in group II a positive correlation was present between GI and AST levels following treatment [Table 4] but it was statistically insignificant. This may be attributed to persistent inflammation in deep pockets post SRP. SRP may be insufficient to completely debride the teeth with deep pockets. Hence, the concentration of AST in saliva would be higher in deep pockets. This was supported by study done by Arora et al.[13]

At baseline, in group I a positive correlation between AST levels and PD was found which was statistically insignificant. This was supported by Kolte et al.,[23] who has shown that certain inflammatory foci are seen even in healthy gingiva.

At 3 months, in group II the correlation between PD and AST levels was found to be a weak correlation (P > 0.05). This may be due to residual inflammation and ulceration in the tissues even after SRP. SRP has been found to be insufficient in completely removing the inner lining epithelium. The results go in accordance with studies done by Persson et al.,[11] who found a weak correlation between AST values and PD.

Fluctuations in AST levels in saliva varied at various sites. This may be due to the active periodontal destruction at the sites. The variations in the AST levels could also be a resultant of the episodic nature of periodontal destruction (Imrey et al.,[33] and Persson,[11] and Page.[26] ).

Periodontal destruction is associated with the anaerobic microflora resulting in tissue destruction thereby releasing AST in GCF and saliva. Studies conducted by Kuru et al.[34] concluded that Porphyromonas gingivalis, Aggregetibacter actinomycetemcomitans, and Prevotella intermedia were significantly higher in AST positive than negative sites. The study thus concludes that AST enzyme levels were significantly elevated in saliva of patients with generalized chronic gingivitis and generalized chronic periodontitis, with higher values recorded in generalized chronic periodontitis patients correlating to the tissue destruction taking place in these conditions. Importance has been given to AST activity in saliva as a diagnostic aid, and studies are still going on in order to confirm whether AST estimation can be used as a specific test to diagnose a periodontitis case and its usefulness in a clinical setting.