Document Type : Original Research Article

Authors

1 Department of Basic Sciences, College of Dentistry, University of Basrah, Iraq

2 College of Medicine, University of Al-Ameed, Karbala, Iraq

3 Department of Chemical Engineering, Faculty of Engineering, Universiti Kebangsaan Malaysia, Malaysia

Abstract

Periodontal disease is an inflammatory disorder affecting the oral cavity, caused by Streptococcus mutans. Diagnostic tests for periodontal disease involve analysis of biomarkers in saliva, such as lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and arginase (AR) levels. This study aimed to assess salivary enzyme levels and isolate S. mutans, in smoking and non-smoking patients with periodontal disease. A sample between 18 and 60 years old who suffering from Periodontitis due to bacterial infection was involved and divided into three groups: Group A consisted of 25 smokers with periodontitis, Group B included 25 non-smokers with periodontitis, and Group C (control) comprised 25 healthy non-smokers without periodontitis. Saliva samples were collected from each participant and analysed using auto-analyser to measure enzyme levels. S. mutans was identified using biochemical, and polymerase reaction (PCR). Salivary enzyme levels were found higher in Groups A and B compared to the control Group C. LDH levels were 323.21 ± 22.31 UI/L (Group A), 138.02 ± 13.22 UI/L (Group B), and 104.21 ± 12.33 UI/L (Group C). ALP levels were 67.77 ± 5.34 UI/L (Group A), 53.36 ± 4.24 UI/L (Group B), and 21.88 ± 1.79 UI/L (Group C). AR levels were 21.58 ± 1.21 UI/L (Group A), 17.89 ± 1.35 UI/L (Group B), and 15.31 ± 1.45 UI/L (Group C). Approximately 80% of bacterial isolates were cultured on MSB from smokers with periodontal and approximately 60% from non-smokers with periodontal disease identified.  S. mutans was in 90% of smokers with periodontal disease and 80% non-smokers with periodontal disease.

Graphical Abstract

Analysis of salivary levels of lactate dehydrogenase, alkaline phosphatase and arginase as well as detection of streptococcus mutans in smoking and non-smoking periodontitis patients

Keywords

Main Subjects

Introduction

The salivary glands are exocrine organs responsible for producing saliva. Major salivary glands, including the parotid, submandibular, and sublingual glands, along with other minor salivary glands, contribute to saliva production. Saliva primarily consists of water, along with salivary proteins, electrolytes, antibodies, and other substances important for dental health [1]. When conducting salivary testing to assess underlying issues, the entire saliva sample is typically considered. Saliva is a complex fluid, with water constituting the majority of its composition and organic and inorganic materials making up around 1% [2]. The major salivary glands account for approximately 93% of saliva volume, while the minor glands contribute the remaining 7%. Saliva's pH typically ranges from 5.3 to 7.8 [2]. Periodontitis is a chronic bacterial infection characterised by the loss of connective tissue and surrounding bone in the teeth, as well as the formation of periodontal pockets resulting from the apical migration

of the epithelial junction which leads to the formation of periodontal pockets and the loss of bone surrounding the teeth [3]. Bacteria play a significant role in the development of periodontitis, and the complex microbial community, along with the host's immune response, determines the outcome of the infection, making it challenging to define precise markers for the disease and to determine the level of infection among smokers. The virulence factors produced by bacteria directly degrade target tissues and induce the release of biological mediators from host tissue cells, leading to tissue destruction [4].

Host immune response generates cytokines, prostaglandins, and proteinases, which contribute to tissue destruction, along with various enzymes derived from bacteria such as collagenases, trypsin-like proteases, elastase-like enzymes, aminopeptidases, dipeptidyl peptidases, and other inflammatory mediators [5]. S. mutans is a common constituent of the human oral microbiota, alongside 25 different types of oral streptococci [6]. Different regions of the oral cavity harbour distinct biological characteristics, and each species has specific properties for colonizing different oral sites oral sites. S. mutans is predominantly found in pits and fissures, constituting 39% of the total streptococci in the oral cavity. On the buccal surface, the presence of S. mutans microorganisms is relatively lower, ranging from 2% to 9% [7]. Extensive research has been conducted on S. mutans due to its significant role in extracellular matrix production and its ability to rapidly form cariogenic biofilms in the presence of fermentable sugars in the diet. Among sugars, sucrose is considered the most cariogenic as it serves as an excellent substrate for the synthesis of extracellular (EPS) and intracellular [8].

The study involves the measurement of the enzymatic concentration at the lactate dehydrogenase, alkaline phosphatase, and arginase in saliva. The enzymes analysis in the salivary and gingival crevicular fluid can enhance the understanding of periodontitis aetiology and improve the accuracy of diagnosis. Lactate dehydrogenase (LDH) is a widely distributed enzyme found in the cytoplasm of nearly all human cells, catalysing the anaerobic reduction of pyruvate to lactate [9,10]. Alkaline phosphatase (ALP) is an intracellular glycoprotein enzyme produced throughout the periodontium and gingival crevice, secreted by polymorphonuclear neutrophils, osteoblasts, and periodontal ligament fibroblasts during bone inflammation, development, and periodontal regeneration, respectively [11,12].

Arginase (AR) is a predominantly liver-based enzyme involved in various physiological processes, such as protein and urea synthesis [13]. Cigarette smoking has been shown to cause an oxidative imbalance in the periodontium, leading to increased production of free radicals and oxidants, resulting in cell necrosis and tissue degradation. This smoking-induced tissue injury leads to the release of enzymes such as LDH, ALP, and AR, and subsequently, their elevation in saliva [10]. In addition, individuals with periodontitis have exhibited higher salivary levels of these enzymes compared to their healthy counterparts [12]. The current study aimed to assess the salivary levels of LDH, ALP, and AR in both smokers and non-smokers, while also conducting molecular detection of Streptococcus mutans bacteria in patients with periodontitis. The study was conducted in the Microbiology Laboratory and the Central Unit Laboratory in the College of Medicine, as well as in the College of Dentistry. 

Experimental

Study population

Seventy-five individuals, aged 18 to 60 years, participated in this study. The participants were divided into three groups: group A consisted of 25 smokers with periodontitis, group B included 25 non-smokers with periodontitis, and group C comprised 25 healthy non-smokers without periodontitis. The selection criteria for smokers included a minimum of 10 cigarettes per day for at least 10 years. Participants with chronic illnesses or recent medication use within the last 6 months were excluded. Ethical approval was obtained, and informed consent was obtained from all participants before their inclusion in the study.

Saliva sampling

Participants in the study were instructed to rinse their mouths thoroughly with water and chew on a standardized paraffin wax to stimulate saliva production. Saliva samples were collected every 60 seconds, resulting in a total volume of 1.5 ml for each sample.

A known sample size was determined using graduated test tubes. The collection process occurred at approximately 9 a.m. To remove any particulate matter, the samples were centrifuged at 15,000 g for 10 minutes at 4 °C. The samples were then stored at -20 °C until further analysis.

LDH estimation

Salivary LDH levels were assessed by measuring the conversion of reduced nicotinamide adenine dinucleotide (NADH) to oxidized NADH at 340 nm in the presence of sodium pyruvate. A 100 μL aliquot of saliva was mixed with a reaction mixture containing 0.05 M tris buffer (pH 7.4), 3.33 mM magnesium chloride, and 1.6 mM sodium pyruvate. After the addition of 100 μL of NADH (final concentration: 0.08 mM), the change in absorbance was monitored for 3 minutes using a U-2910 spectrophotometer [14-16].

ALP estimation

For the estimation of salivary ALP, ALP IFFCC mod. Kits® were utilized following the manufacturer's instructions (Huma Star Company, Germany). Salivary samples (20 μL) were mixed with the working reagent. The absorbance was recorded at regular intervals of time (e.g., every 30 seconds) at 405 nm using a colourimeter. The mean absorbance change per minute was calculated.

Estimation of AR

To determine the AR activity, the urea produced by the hydrolysis of L-arginine was measured spectrophotometrically using the Diaacetylomonoxime method. Under strongly acidic conditions, diacetylmonoxime reacts with the urea generated by arginase in the presence of ferric ion and thiosemicarbazide, resulting in the formation of a pink diacetyl complex that can be measured spectrophotometrically at 540 nm. The intensity of the colour complex was directly proportional to the arginase activity [17].

Isolation and identification of S. mutans

The saliva samples were homogenized for 2 minutes using a vortex mixer. To obtain tenfold serial dilutions, normal saline was used. Two dilutions were selected for each type of microorganism and inoculated onto Mitis Salivarius Bacitracin Agar (MSB agar), which is a selective medium for streptococcus mutans. From the dilutions (10^3-10^5), 0.1 mL of the sample was withdrawn and plated in duplicate on MSB agar plates using a sterile microbiological spreader. The plates were then incubated anaerobically at 37 °C for 48 hours [18]. After quantifying the positive samples on the MSB medium, small colonies were subcultured onto the surface of blood-agar plates for further purification. These plates were incubated anaerobically at 37 °C for two days. The isolates were initially characterized based on the colony morphology on MSB agar, Gram staining, microscopic examination, catalase test, and carbohydrate fermentation.

DNA extraction and PCR analysis

To confirm the presence or absence of the 16S rRNA genes in the bacterial isolates, PCR (Polymerase Chain Reaction) was employed. Genomic DNA was extracted from Streptococcus mutans bacterial isolates using the Genomic DNA Mini Bacteria Kit, and specific primers for the detection of the 16S rRNA genes were designed by the Bioneer Company (Korea) as described by Miyoshi et al. (2005) [19]. The PCR reaction was carried out using 5μL of template DNA in a total volume of 20 μL, which included 1.5 μL of the forward primer, 1.5 μL of the reverse primer, and 12 μL of nuclease-free water. The PCR program consisted of 30 cycles of denaturation (95 °C for 30 seconds), annealing (58 °C for 30 seconds), and extension (72 °C for 1 minute), followed by a final extension step at 72 °C for 5 minutes to amplify the 16S rRNA gene. The presence of the amplified product was confirmed by running the PCR products on a 1% agarose gel using electrophoresis at 100 volts and 80 amperes for 1 hour. The PCR products were visualized using a UV transilluminator [20].

Statistical analysis

The data were expressed as mean ± standard deviation. To determine the statistical significance between the study groups and control groups, this group had healthy gums and was not infected with bacteria. The test result was performed and shows a significance level set at p < 0.05.

Results

The relative concentrations of salivary enzymes of groups in this study namely, Group A (smokers with periodontitis) exhibited the highest LDH level, with a mean LDH value of 323.21 ± 22.31 UI/L as presented in Table 1, which was significantly higher compared to group B (non-smokers with periodontitis) with a mean LDH value of 138.02 ± 13.22 UI/L, as well as group C (control) with a mean LDH value of 104.21 ± 12.33 UI/L (p < 0.05). Group B also showed significantly higher LDH values compared to group C (138.02 ± 13.22 UI/L vs. 104.21 ± 12.33 UI/L) at p < 0.05. Similar patterns were observed for ALP levels, where group A exhibited the highest ALP level with a mean ALP value of 67.77 ± 5.34 UI/L, significantly higher compared to group B (53.36 ± 4.24 UI/L) and group C (21.88 ± 1.79 UI/L) (p < 0.05). Additionally, group B showed significantly higher ALP values compared to group C (53.36 ± 4.24 UI/L vs. 21.88 ± 1.79 UI/L) at p < 0.05. Regarding AR levels, group A had significantly higher AR levels compared to groups B and C (21.58 ± 1.21 UI/L (group A) vs. 17.89 ± 1.35 UI/L (group B) and 21.58 ± 1.21 UI/L. (group A) vs. 15.31 ± 1.45 UI/L (group C). However, there was no statistically significant difference in AR levels between groups B and C (p < 0.05). The relative concentration of the salivary enzymes in the different study groups is graphically presented in Figure 1. 

 

 

In the samples collected from patients with periodontitis (group A and group B), S. mutans colonies were observed. These colonies exhibited colour variations, ranging from light blue, and their diameter was approximately 2-3 mm. The colonies appeared either round or irregular in shape with raised or curved surfaces, firmly attached to the medium. Some colonies displayed a rough or iridescent glass-like appearance, while others had a smooth surface. Many of the S. mutans colonies exhibited a depressed centre containing polysaccharide droplets, and in some cases, the entire colony was immersed in a pool of polysaccharides (for visual representation, see Figure 2). The existence of Streptococcus mutans on the MSB medium was taken from smokers with periodontitis.

   

 

As presented in Table 2, Streptococcus mutans was detected on the MSB medium in 20 patients (80%) in group A and 15 patients (60%) in group B. Other bacterial species were also identified in 5 patients (20%) in group A and 10 patients (40%) in group B.

Subsequently, DNA extraction and PCR analysis were performed on the bacteria isolated from the MSB medium to confirm the presence of the 16S rRNA gene. The results revealed the presence of a 1500 base pair (bp) band, indicating the presence of the gene, as demonstrated in Figure 3. The PCR analysis further demonstrated that 18 patients from group A (90% of the 20 patients with S. mutans in group A) and 12 patients from group B (80% of the 15 patients with S. mutans in group B) tested positive for the gene, Table 2.

 

 

Discussion

The severity of periodontitis is influenced by various factors including microbial, immunological, ecological, and genetic risks, as well as age and sex. Smoking is responsible on 85% [21] a major source of risk factors that contribute to the occurrence and progression of periodontal disease. Moreover, non-smokers who are passive smokers are also more susceptible to developing oral and systemic disorders [22].

Saliva plays a crucial role in maintaining oral health. Alterations in its composition and production can hurt oral health and also enhance tooth decay. Dry mouth patients are prone to dental cavities and struggle to maintain proper oral hygiene. Interestingly, both saliva and Gingival Crevicular Fluid (GCF) have a dual role in preventing periodontal disease and promoting periodontal pathology [23]. LDH, a widely distributed enzyme, plays a significant role in clinical diagnostics for pathological processes. The high concentration of LDH in saliva indicated cellular necrosis can be utilised as a marker of the oral lesion affecting mucosa of oral, as presented in Table 1 [24].  The results showed increased salivary LDH, ALP and AR levels in smokers with periodontal and non-smokers with periodontal patients compared to healthy controls as indicated through a statistical study using SPSS assay. Our findings are consistent with a study that examined LDH activity in whole saliva of individuals with and without periodontal disease. The study revealed elevated LDH levels in smokers and passive smokers with periodontal disease compared to healthy individuals, suggesting LDH is a potential epidemiological biomarker for this condition [25].

ALP, an intracellular enzyme released from neutrophil granules, significantly increases with inflammation and plaque accumulation. The presence of periodontitis may be a consequence of destructive processes in the alveolar bone and metabolic changes in inflamed gingival tissues. High levels of ALP indicate increased cellular damage, and its concentration is elevated in both smokers and non-smokers with periodontitis [26,27]. In the present study, the levels of salivary AR were found to be elevated in both smoking and non-smoking patients with periodontitis, compared to the healthy control group. This observation supports previous research findings [28,29] that also reported increased salivary AR levels in individuals with the disease. The pathogenesis of periodontal diseases can manifest in various ways. One common cause of periodontitis is the accumulation of a bacterial biofilm on the tooth's surface, which triggers an excessive inflammatory response [30]. Dental caries and decay, on the other hand, are primarily caused by Streptococcus mutans, as listed in Table 2, besides some other parameters that allowed S. mutans to thrive [31].

The presence of sucrose promotes the production of insoluble glucans by S. mutans, leading to the formation of dental plaque or oral biofilm and initiating the caries process [32]. Our findings support the hypothesis proposed by multiple authors that alterations in oral microbiota interspecies competition occur before visible tooth lesions [33]. These results were further substantiated by a local study conducted by Chayan, Sekhi et al. (2021) [34], which reported a higher prevalence of S. mutans isolates. Numerous studies have consistently identified S. mutans as the primary etiological bacterium responsible for human dental caries and the formation of biofilms on the hard tissues of the oral cavity [35].

Conclusion

Periodontitis is a common oral disorder that can have significant implications for both oral and overall health. Bacterial infection, particularly by S. mutans, has been identified as the primary cause of this disease. S. mutans possesses several unique virulence factors that contribute to its role in the development of periodontitis. Early detection and assessment of periodontal health are crucial for preventing disease progression. Our study findings demonstrated elevated levels of salivary flow enzymes, including LDH, ALP, and AR, in both smokers and non-smokers with periodontitis compared to healthy individuals. These enzymes serve as reliable markers for evaluating the status of periodontal health and can indicate the presence of tissue damage or defects in the oral cavity.

Acknowledgements 

The authors would like to extend their thanks to the University of Basrah.

Funding

This work is not funded by any institution.

Authors' Contributions

Hazim  & Rana: data collection

Abdul Amir: Article formulation and approval

Ahmed: Graphic design

Conflict of Interest

All authors declare that there is no conflict of interest in this study.

Consent for Publication

The authors consent to the publication of the article.

Orcid:

Hazim Saad: https://orcid.org/0000-0003-0779-2023

Rana Adnan: https://orcid.org/0000-0002-3119-3154

Abdul Amir H. Kadhum: https://orcid.org/0000-0003-4074-9123

Ahmed A. Al-Amiery: https://orcid.org/0000-0003-1033-4904

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How to cite this article: Rana Adnan Fayez Al-jaberi , Hazim Saad Jabbar Al -Maliki , Abdul Amir H. Kadhum, Ahmed A. Al-Amiery,  Analysis of salivary levels of lactate dehydrogenase, alkaline phosphatase and arginase as well as detection of streptococcus mutans in smoking and non-smoking periodontitis patients. Journal of Medicinal and Pharmaceutical Chemistry Research, 2024, 6(10), 1547-1557. Link: https://jmpcr.samipubco.com/article_195510.html 

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Copyright © 2024 by SPC (Sami Publishing Company) + is an open access article distributed under the Creative Commons Attribution License(CC BY)  license  (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


  

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