|Year : 2022 | Volume
| Issue : 2 | Page : 107-112
Use of saliva aspartate aminotransferase in diagnosing periodontal disease: A clinical and biochemical study
Harshada Shinde, Reetika Gaddale, Karthikeyan Ilangovan
Department of Periodontology, H. K. E Society's S. Nijalingappa Institute of Dental Sciences and Research, Sedam Road, Kalaburagi, Karnataka, India
|Date of Submission||12-Jun-2020|
|Date of Decision||31-Mar-2021|
|Date of Acceptance||31-Jan-2022|
|Date of Web Publication||3-Aug-2022|
Dr. Harshada Shinde
Reader, Department of Periodontology, Maratha Mandal's Nathajirao G. Halgekar Institute of Dental Sciences and Research Center, Near APMC Police Station, R. S. No. 47 A/2, Bauxite Road, Belagavi - 590 010, Karnataka
Source of Support: None, Conflict of Interest: None
Background and Aims: Periodontal disease is one of the common inflammatory diseases. Several enzymes in saliva and gingival crevicular fluid are evaluated for early diagnosis of periodontal disease. The objective of our study was to evaluate the relationship between aspartate aminotransferase (AST) levels in saliva and periodontal status indicated by the Community Periodontal Index of Treatment Needs (CPITN).
Methods: Thirty patients were assigned to each one of four groups C0, C1, C3, and C4, based on their largest CPITN code among the examined sites, totaling 120 participants. Un-stimulated saliva was collected from the individuals. Biochemical analysis of saliva samples was carried out with an RA-XT auto analyzer by using the International Federation of Clinical Chemistry method in order to quantify their AST concentration.
Results: There were statistically significant differences between levels of AST (IU/L) from groups C0 (24.07 ± 15.16 IU/L), C1 (39.73 ± 21.46 IU/L), C3 (68.27 ± 44.41 IU/L), and C4 (175.93 ± 137.39 IU/L). There was a significant positive correlation between clinical parameters and AST concentration in saliva in each group.
Conclusion: AST levels in saliva increased with an increase in the CPITN score, Group C0 had the least and Group C4 had the highest AST levels. Periodontal destruction such as periodontal pockets, gingival bleeding seems to be related to AST levels in saliva. Salivary AST levels represent useful adjuncts to the diagnostic of screening periodontal diseases.
Keywords: Aspartate aminotransferase, diagnosis, enzymes, periodontitis, saliva
|How to cite this article:|
Shinde H, Gaddale R, Ilangovan K. Use of saliva aspartate aminotransferase in diagnosing periodontal disease: A clinical and biochemical study. J NTR Univ Health Sci 2022;11:107-12
|How to cite this URL:|
Shinde H, Gaddale R, Ilangovan K. Use of saliva aspartate aminotransferase in diagnosing periodontal disease: A clinical and biochemical study. J NTR Univ Health Sci [serial online] 2022 [cited 2022 Dec 10];11:107-12. Available from: https://www.jdrntruhs.org/text.asp?2022/11/2/107/353224
| Introduction|| |
Periodontal disease is one of the common inflammatory diseases that has a complex etiology and multifactorial origin. The right diagnosis of periodontal disease is the key to prognosis, treatment plan, and maintenance of periodontal health. The study of methods for periodontal diagnosis is influenced by the knowledge of pathogenesis and progression of the disease.
The progression of periodontal disease is episodic in nature. Hence, periodontal disease sites can be active or inactive. Diagnosis of periodontal disease has been mainly based upon clinical and radiographic measures of periodontal tissue destruction. These parameters do not distinguish between disease active and inactive sites and are of limited use in early diagnosis as they measure past destruction. Enzymes assessed for the diagnosis of periodontal disease can differentiate between disease active and inactive sites, predict future tissue destruction in particular individuals and sites. These markers can also be helpful in the early diagnosis of periodontal disease.
A lot of evidence shows the utilization of gingival crevicular fluid (GCF) for the diagnosis of periodontitis, but a special technique is required for sampling and it is difficult to obtain GCF from all sites of dentition.,, Saliva can be used for the diagnosis of periodontal disease. Whole saliva sampling is simpler, non-invasive, and cheaper than the GCF collection.
Several enzymes are assessed for the early diagnosis of periodontal disease namely aspartate and alanine aminotransferase (AST and ALT, respectively), lactate dehydrogenase (LDH), creatine kinase (CK), alkaline and acid phosphatase (ALP and ACP, respectively), and gamma glutamyl transferase (GGT)., AST is a ubiquitous enzyme found in all cells, and its levels vary in different cell types. Significant AST levels have also been found in human gingival fibroblasts, human gingival epithelial cells, and human periodontal ligament fibroblasts (60 KU/1,000 cells, 100 KU/1,000 cells, and 20 KU/1,000 cells, respectively). During periods of tissue destruction, these cells release AST into the extracellular space. As periodontal disease is a destructive process, dead and damaged periodontal tissue releases AST into the GCF and saliva where its activity can be measured. Hence AST can be a biochemical marker of the functional condition of periodontal tissues, and periodontal disease activity.
Atici et al. evaluated the GCF intracytoplasmic enzyme activity in patients with adult periodontitis (chronic periodontitis) and rapidly progressive periodontitis (aggressive periodontitis). Levels of AST, LDH, and CK were studied in saliva and serum before and after treatment. This study suggested that AST and LDH have a significant relationship with periodontal disease status. Nomura et al. determined the usefulness of salivary biomarkers like LDH, AST, and blood urea nitrogen for screening of periodontal disease. The sensitivity and specificity of AST were more than 0.60. They suggested that salivary AST may also be a candidate for screening of periodontitis, possibly due to the fact that salivary AST may reflect tissue necrosis. Barbosa e Silva et al. determined the use of AST in diagnosing periodontal disease by conducting a comparative study of clinical and microbiological parameters. The correlation between AST levels and any of the analyzed parameters was not statistically significant. He concluded that the lack of any association between the factors studied does not indicate, however, that the latter cannot be used in diagnosing the actual periodontal condition of patients and/or sites. As the results are conflict, further studies are required to assess the diagnostic role of salivary AST in periodontitis.
The purpose of this study was to evaluate the relationship between aspartate aminotransferase levels in saliva and different periodontal conditions, to assess the usefulness of salivary AST in the diagnosis of periodontal disease, and to compare the clinical indices with the saliva AST levels.
| Methods|| |
The present study was conducted on 120 patients visiting the Out patient Department of Periodontics, to whom the study design was explained and informed consent was obtained. The study was approved by the Institutional Ethical Committee. Patients were screened and classified according to Community Periodontal Index of Treatment Needs (CPITN) scores, by a single professional, using a WHO 621 periodontal probe. This index was primarily designed for rapid and practical assessments of periodontal treatment needs rather than periodontal status, but it is based on various disease indicators such as gingival bleeding on probing and pocket depth, which are related to tissue destruction.
Patients of both genders between 20 to 65 years of age were included in the study. Individuals, who participated in the study, were assigned to one of the following four groups based on their largest CPITN score found among all the examined sites:
- Group C0: No periodontal disease (Healthy periodontium).
- Group C1: Bleeding observed during or after probing.
- Group C3: Pocket 4 to 5 mm in depth. Gingival margin situated on the black band of the Probe.
- Group C4: Pocket 6 mm or more in depth. Black band of the probe is not visible.
Other clinical parameters recorded were Gingival index (Loe and Silness), Turesky - Gilmore - Glickman modification of Quigley - Hein plaque index and Modified Sulcular bleeding index. Patients were interviewed to obtain their medical and dental histories. Patients with systemic diseases associated with an increase in AST like chronic hepatitis, myocardial infarction, inflammatory skeletal muscle diseases, progressive muscular dystrophy. Patients with lesions of oral mucosa other than gingivitis and periodontitis, pregnant, lactating, post-menopausal females, patients with removable prosthesis and/or caries, smokers, patients with disturbances in salivary flow related with systemic processes or therapeutic procedures, patients with a history of any periodontal treatment in previous 6 months, patients with a history of antibiotic therapy in previous 3 months were excluded from the study.
Various techniques can be used for collection of saliva like the draining, spitting, suction, and absorbent method. In our study, 1 ml of whole saliva sample was collected by unstimulated passive drool in a sterile disposable plastic container. Patients were instructed not to brush or eat 8 hours before the collection of the sample. Dental examination was not carried out 48 hours before saliva collection. Samples were stored at 4°C and sent to the biochemistry laboratory. AST activity remains stable at room temperature for several hours or up to 3 days at 4°C. Freezing may result in loss of enzyme activity and is not recommended. The level of AST was estimated with an RA-XT Auto analyzer (Technicon) by using the International Federation of Clinical Chemistry method.
Each saliva sample was centrifuged at 5000 rpm for 10 minutes. Reagents were added to about 10 μl of the supernatant sample using the auto analyzer and the value of AST was estimated in IU/L. The reagents used in the estimation of saliva AST are available in two separate bottles. The first bottle named Reagent 1 contains enzymes namely malate dehydrogenase (MDH ≥600 IU/L), LDH (≥900 IU/L), NADH (0.20 mmol/L), and α- Ketoglutarate (12 mmol/L). The second bottle named Reagent 1A contains the buffers namely Tris buffer with a pH of 7.80 (88 mmol/L) and L- Aspartate (260 mmol/L).
The test for AST is based on the following reactions:
L-Aspartate+ αKetoglutarate Oxaloacetate + L- Glutamate
Oxaloacetate + NADH + H- L- Malate + NAD-
There is a decrease in absorption at 340 nm as NADH is converted to NAD. The rate of decrease in absorbance is measured and is proportional to AST activity in the sample.
Non-parametric Kruskal Walli test was used to calculate the significant difference between more than two groups and Mann-Whitney U-test was used to compare pairs of any groups. Simple linear regression analysis was used to compare clinical indices and saliva AST levels.
| Results|| |
The 120 patients (55 men and 65 women) selected for the study were 20–65 years old (mean 31 years). The patients were divided into four groups, each group comprising of 30 patients. The mean and standard deviation of the AST concentration in saliva group C0, C1, C3, and C4 were 24.07 ± 15.16, 39.73 ± 21.46, 68.27 ± 44.41, and 175.93 ± 137.39 IU/L, respectively, as shown in [Figure 1]. The pair wise comparison between AST levels in groups C0, C1, C3, and C4 showed that there was statistically a significant difference between the groups [Table 1]. The saliva AST concentration increased with an increase in the pocket depth in groups C3 and C4. When saliva AST levels in group C1 were compared with C1, C3, and C4, the results were statistically significant.
|Figure 1: Aspartate aminotransferase levels in the saliva of four groups of patient (n=30 per group) classified according to their CPITN codes|
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The gingival index (GI), modified sulcular bleeding index (mSBI), and plaque index (PI) were compared with AST levels within each group using simple linear regression analysis. In groups C0, C1, and C3, the results were not statistically significant [Table 2]. In group C4, that is patients having pocket with a depth of 6 mm or more, the comparison between GI and AST concentration in saliva gave statistically significant results [Figure 2].
|Figure 2: Graph showing a relationship between AST values in saliva with gingival index in group C4 using simple linear regression analysis (r = 0.0440)|
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|Table 2: Relationship between aspartate aminotransferase (AST) in saliva with gingival index, msbi and plaque index scores using simple linear regression analysis|
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There was a significant positive correlation between clinical parameters and AST concentration in saliva in each group.
| Discussion|| |
Chambers et al. were the first to demonstrate that AST levels in GCF increase during the development of periodontitis in beagle dogs. In experimental gingivitis in humans, GCF AST levels were significantly associated with gingival inflammation during various stages of development and resolution.
AST enzyme in the saliva is an indicator of a higher level of cellular damage. Increased AST levels in GCF and saliva are a result of its increased release from the damaged cells of soft tissues of the periodontium and a reflection of metabolic changes in the inflamed gingiva.
CPITN has shown to be a rapid and practical method for the periodontal status assessment and determining treatment needs. The proposed method allowed establishing a quantitative relationship between AST levels in saliva and periodontal status assessed through CPITN recordings. This method simplified the clinical procedures involved in sample collection compared to the present diagnosis systems; only 1.0 ml saliva must be deposited in a sterile disposable container by the patient by unstimulated passive drool. Stimulated saliva which is collected by asking the patient to chew a gum base may get contaminated with blood as inflamed periodontal tissue may get damaged during chewing. This can cause variation in the saliva enzyme levels.
Although a lot of evidence has accumulated for the use of GCF for the diagnosis of periodontitis, this approach has a demerit, that is, a special technique is required for sampling. In addition, it is difficult to obtain GCF from all sites of the dentition. Smith et al. have shown that enzyme activities and GCF volume were different among the six sites there were sampled. Thus, it is difficult to present values representative of a subject's oral cavity or even of a tooth. On the contrary, whole saliva collection is easier, non-invasive, and cheaper than GCF collection, and the same enzymes which are found in GCF can be detected in saliva also. Because of the simple and non-invasive method of collection, salivary diagnostic tests appear to hold promise for the future. Thus saliva samples were preferred in our study instead of GCF samples.
Studies on the relationship between AST levels and periodontal diseases have focused on using spectrophotometry, and commercial chair side tests such as Pocket Watch™, and Periogard™. These kits were developed to simplify AST analysis, using a filter paper strip inserted into the periodontal sites and subsequently placed in a reagent-coated test tray. Following the chemical reaction, the resulting color is compared visually with the positive and negative controls. The RA-XT Auto analyzer (Technicon) is routinely used for biochemical analysis of blood samples, serum, or plasma. In our study, we have used it as a diagnostic method in dentistry.
Code 2 classification was not included in the study because it evaluates only the presence or absence of supra- or subgingival calculus and is not necessarily associated with tissue destruction. In our study, cut-off values, sensitivity, specificity, positive predictive value, and negative predictive value were not calculated as the patients were not randomly assigned into each one of the four groups.
Studies analyzing the association of plaque and gingival indices with AST in sites with a probing depth of ≥4 mm (Group C3), did not find any significant difference between positive and negative sites, particularly in relation to the GI. Yoshie et al. reported correlation between the GI and saliva AST levels. In cross-sectional studies, AST levels in GCF were reported to correlate with the indices of disease severity at the patient level. Atici et al. found a significant correlation between the GCF AST levels and clinical parameters. In our study, the comparison of GI and plaque index with salivary AST levels in each group did not give significant results except in group C4 (Pocket 6 mm or more in depth) where the GI was statistically significant. But the clinical parameters had a positive correlation with salivary AST levels in each group. Our results are in agreement with those previous studies reported in the literature. We noted a high incidence of gingival inflammation with higher AST scores, and this incidence increased with greater inflammation, indicating a possible relationship between diseased sites and an inflammatory condition.
In our study, AST concentrations in saliva for the groups C0, C1, C3, and C4 were statistically significant. Probing pocket depth greater than 6 mm (Group C4) and severe gingival inflammation were related to the highest AST levels. The progression of periodontal destruction is episodic in nature and does not occur in a continuous, linear manner. Furthermore, periodontal pockets can be active or inactive during the disease process. Attachment loss occurs when the periodontal pocket is active and the cells in contact with the pocket are destroyed by necrosis. Measurement of AST levels indicates the progression of periodontal destruction, and AST can be, therefore, considered a potential biomarker for differentiating between inactive and active disease sites., The results of our study demonstrated that AST levels are related to the amount of periodontal tissue destruction, which is in agreement with findings from several studies on other indicators from saliva and on AST in the GCF.,,
Barbosa e Silva et al. found no association between high AST levels and bleeding on probing. In our study, mSBI had a positive correlation with salivary AST levels in each group, although, not statistically significant. It was assumed that these subjects had active disease at periodontal sites at the time of examination. It is generally accepted that bleeding upon probing (BOP) and probing depth is critical site-specific periodontal parameters evaluating the probability of periodontal disease progression. Repeated BOP has been associated with an increased risk of loss of attachment while the absence of BOP has been shown to be an excellent indicator of periodontal stability., It is, therefore, of interest to notice that in the present study, BOP was also associated with elevated levels of AST in saliva.
It is also interesting that four individuals from group C4, that is patients with pocket 6 mm or more in depth exhibited normal levels of AST. The reason for this outcome could be related to either low disease activity or the type of tissue affected by necrosis. Mizuho et al. demonstrated that fibroblasts from the periodontal ligament produce significantly lower levels of AST than gingival epithelial cells. The occurrence of false-negative results, however, should not be excluded.
AST levels in saliva increased with an increase in the CPITN score, Group C0 (No periodontal disease) having the least and Group C4 (Pocket 6 mm or more in depth) having the highest AST levels. A statistically significant difference was observed in terms of the GI and AST level in Group C4, while in group C0, C1, and C3 the results were not statistically significant.
In a study done by Nomura Y et al. the cut-off value for salivary AST was 31.5 IU/L with a sensitivity of 0.63 and specificity of 0.65. In our study, cut-off point for sensitivity and specificity of AST in saliva was not determined. Other limitations of our study are the small sample size and cross-sectional study design.
Saliva AST estimation cannot be used as a specific test to diagnose periodontitis. Given the complex nature of the periodontal disease, it is unlikely that any single clinical or laboratory examination can address all issues concerning diagnosis and prognosis.
In conclusion, our results indicate that the use of AST levels may help to confirm clinical observations since AST levels differ significantly between diseased and healthy periodontal sites. Our study suggests that salivary AST can be considered as the biochemical marker of functional condition of periodontal tissues and is a useful adjunct to the diagnosis of different periodontal conditions. More studies are necessary to evaluate which specific clinical, microbiological, and histological characteristics of periodontal disease are associated with elevated levels of AST in saliva.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]