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Estimation of salivary lactate dehydrogenase in oral squamous cell carcinoma, oral leukoplakia, and smokers


 Department of Oral and Maxillofacial Pathology, KSR Institute of Dental Science and Research, Tiruchengode, Tamil Nadu, India

Date of Submission14-Jul-2020
Date of Decision02-Aug-2020
Date of Acceptance21-Dec-2020
Date of Web Publication15-Oct-2021

Correspondence Address:
Mahalingam Bhuvaneswari,
Department of Oral and Maxillofacial Pathology, KSR Institute of Dental Science and Research, Tiruchengode, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_969_20

 > Abstract 


Introduction: Lactate dehydrogenase (LDH) is an enzyme seen within every cell during their normal metabolic function. It is always confined within the cell cytoplasm and it becomes extracellular only when a cell dies. The extracellular presence of LDH is related to cell necrosis and tissue breakdown. Therefore, we designed a study to estimate and compare LDH levels in the saliva of patients with oral cancer, oral leukoplakia (OL), and smokers without lesions and in controls.
Materials and Methods: A total of 81 subjects of both genders, between the ages of 20 and 70 years, were included in the study. The study group was divided into four: group I-controls (n = 20), Group II-smokers (n = 20), Group III-subjects with OL (n = 20), and Group IV-subjects with oral squamous cell carcinoma (OSCC) (n = 21). Five milliliters of unstimulated salivary sample was collected from each participant, and salivary LDH level was measured. The obtained values were tabulated and statistically analyzed. P < 0.05 was considered to be statistically significant.
Results: The difference in LDH levels among the four groups was found to “BE” statistically significant. LDH values showed a marked increase in the leukoplakia group (49.79 ± 19.88 IU/L) and OSCC group (106.97 ± 32.75 IU/L) when compared to controls and smokers.
Conclusion: We found that salivary LDH was increased in patients with leukoplakia and OSCC. Smoking alone did not produce any alterations in salivary LDH. It is possible that salivary LDH could be a potential biomarker to identify early premalignant or malignant changes in smokers.

Keywords: Lactate dehydrogenase, oral leukoplakia, oral squamous cell carcinoma, saliva, smokers



How to cite this URL:
Bhuvaneswari M, Prasad H, Rajmohan M, Sri Chinthu KK, Prema P, Mahalakshmi L, Kumar GS. Estimation of salivary lactate dehydrogenase in oral squamous cell carcinoma, oral leukoplakia, and smokers. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 6]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=328264




 > Introduction Top


India has one-third of oral cancer cases in the world. Oral cancer accounts for around 30% of all cancers in India. Estimations by Globocan in 2018 revealed 119,992 new cases and about 72,616 deaths are due to oral cancer. Habits such as chewing tobacco with betel nut, reverse smoking, and heavy alcohol usage are common. The mortality rate due to oral squamous cell carcinoma (OSCC) is also high mainly due to delayed diagnosis.[1] Potentially malignant disorders of the oral cavity (now called as potentially premalignant oral epithelial lesions) are also commonly noticed in the Indian population. Oral leukoplakia (OL) is the most commonly occurring oral precancerous lesion of the oral cavity representing 85% of such lesions. Significant dysplastic features are noted in the epithelium in this lesion.[2] However, lack of awareness usually results in a delay in diagnosis of even these potentially malignant lesions. Saliva is a readily available diagnostic medium, collected by noninvasive procedures, and contains many hormones, drugs, and antibodies in screening and diagnosis.[3] Salivary diagnostics has advanced and serves as a subset of the larger field of molecular diagnostics, which is recognized as the main component in a wide variety of biomedical basic and clinical areas.[4] In spite of these advances, salivary diagnostics are rarely being used in oral cancer detection.

Lactate dehydrogenase (LDH), a tetrameric enzyme, plays a major role in anaerobic glycolysis. It is always confined within the cell cytoplasm and the extracellular presence of LDH is related to cell necrosis and tissue breakdown.[5] LDH concentration in saliva, as an expression of cellular necrosis, could be a specific indicator for oral lesions that affect the integrity of the oral mucosa.[6] Therefore, we designed a study to assess the levels of salivary LDH in OL, OSCC, and in smokers, and to determine if there was any difference between the various study groups.


 > Materials and Methods Top


Patients visiting the Department of Oral Medicine and Radiology in our institution and from dental clinics in and around our area were included in our study. Institutional Ethical Committee clearance and Institutional Review Board approval were obtained before the study was initiated. After receiving informed consent, eighty-one subjects of both genders in the age range of 20-70 years were selected as per specific inclusion and exclusion criteria and categorized into 4 groups:

  • Group-I: Normal healthy individuals (controls) with clinically normal oral mucosa (n = 20)
  • Group-II: Smokers (history of smoking for minimum of 3 years) with clinically normal oral mucosa (n = 20)
  • Group-III: Patients clinically diagnosed with OL (n = 20)
  • Group-IV: Patients with histopathologically confirmed OSCC (n = 21).


Subjects with systemic diseases like myocardial infarction, liver disease, renal disease, muscular dystrophy, diabetes mellitus, previous history of malignancy, those under chemotherapy or antioxidant therapy, were excluded from the study.

Each participant's complete medical, dental history, and clinical photographs were recorded. Unstimulated saliva sample (5 ml) was collected from subjects of all four groups between eight and eleven in the morning to avoid circadian variations, immediately transported and deep frozen at minus 80°C for storage until final analysis.

At the time of analysis, samples were thawed and then centrifuged (1000 thousand rotations) at 4°C for 10 min to remove cell debris. LDH was assayed using the LDH enzyme kit from Biosystems Laboratories Ltd. (Chennai) using the standard biochemical method with an ultraviolet-visible spectrophotometer, as per the manufacturer's recommended protocols.


 > Results Top


A box and whisker plot of the obtained values was made [Figure 1]. We noticed three extreme outliers (shown as an asterisk in the box plot), which were excluded from any statistical analysis to avoid bias. Out of these three, two belonged to the smokers' group, and one was in the control group. The reason for these extreme values could not be ascertained. The final sample size, therefore, was 19 in controls, 18 in smokers, 20 in leukoplakia, and 21 in the carcinoma group. All statistical analysis was carried out in this sample size only. The mean and standard deviation in each of these groups is shown in [Table 1].
Figure 1: Box plot showing salivary lactate dehydrogenase levels in different groups. *Extreme outliers

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Table 1: Levels of salivary lactate dehydrogenase enzyme in all the groups

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Salivary LDH enzyme levels among the different groups were compared using one-way ANOVA. We found that LDH levels in controls (28.76 ± 21.42 IU/L) and smokers (27.21 ± 24.08 IU/L) were almost similar. However, LDH values showed a marked increase as we progress to leukoplakia group (49.79 ± 19.88 IU/L) and OSCC group (106.97 ± 32.75 IU/L). This difference was highly significant statistically (F - 44.56; P < 0.001) [Table 2].
Table 2: Comparison of salivary lactate dehydrogenase within the groups and between groups

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Tukey post hoc test was done after ANOVA to determine which pair of groups showed significantly different values. We found that the LDH level in the OSCC group was higher than in the control group and this difference was highly significant statistically (P < 0.001) [Table 3]. However, we did not find any significant difference in LDH when comparing controls with smokers or the leukoplakia group.
Table 3: Multiple comparison of salivary lactate dehydrogenase levels in controls with smokers, leukoplakia, and oral squamous cell carcinoma

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When LDH values in smokers were compared with leukoplakia group and OSCC group, we found that LDH levels were higher in both leukoplakia and OSCC groups. In the leukoplakia group, the mean LDH level was 49.78 IU/L compared to 27.21 IU/L in smokers and this difference was statistically significant (P = 0.036). Similarly, the mean LDH level in OSCC was 106.97 IU/L, which was significantly higher than smokers 27.21 IU/L (P < 0.001) [Table 3].

The mean LDH levels of leukoplakia were compared with OSCC. LDH level in leukoplakia was 49.78 IU/L compared to 106.97 IU/L in OSCC. This difference was highly significant statistically (P < 0.001) [Table 3].


 > Discussion Top


Saliva is emerging as a valuable biomarker for diagnosing various systemic and oral diseases. Several salivary enzyme levels are altered during pathological states involving the oral mucosa.[6] LDH is one such enzyme present in saliva. It is an important indicator of cellular injury as well as a by-product of anaerobic glycolysis. This enzyme is present in almost all tissues but is mainly concentrated in the heart, liver, red blood cells, kidneys, muscles, brain, and lungs. It can also be detected in various body fluids. It is found in five isoforms-LDH-1, LDH-2, LDH-3, LDH-4, and LDH-5.[7]

We found insignificant variation in salivary LDH levels in smokers when compared to healthy controls. Rao et al. noticed that salivary LDH in smokers was significantly elevated when compared to nonsmokers.[8] Sometimes, increased serum levels of LDH in smokers or tobacco habituated persons are seen, which could be due to the production of a large number of free radicals that cause cell damage.[9] The cell membrane, which is usually made of lipids, is attacked by excessive reactive oxygen species from cigarette smoke leading to membrane peroxidation, thereby resulting in increased LDH liberation extracellularly.[10]

Joshi et al., in their study, found that levels of salivary LDH were significantly elevated in subjects of leukoplakia when compared to controls. They also analyzed the distribution pattern of different LDH isoenzymes in leukoplakia patients and correlated it with histopathological grading of leukoplakia and found LDH-5> LDH-4> LDH-3> LDH-2 with increasing grades of dysplasia. They concluded that the major source of salivary LDH is probably the exfoliating epithelial cells.[11] Although in our study, the salivary LDH in leukoplakia patients was higher than that in controls or smokers, the difference was not statistically significant. However, when compared with smokers, leukoplakia patients had significantly elevated salivary LDH.

Salivary LDH was significantly elevated in OSCC subjects when compared to controls and the levels were also raised among different grades of OSCC, as per the findings of D'Cruz and Pathiyil.[12] Similar results were also observed by Lokesh et al.[13] Our study findings are similar to these studies, wherein the OSCC group had significantly elevated salivary LDH levels when compared to all the other groups.

In cancer, the metabolism of the cell is modified by a process called the Warburg effect/aerobic glycolysis. This was first discovered by Otto Warburg in the 1920s. Cancer cells utilize LDH to increase the rate of glycolysis which in turn increases adenosine triphosphate (ATP) production and increased lactate production even in the presence of oxygen. This is adopted by most of the tumor cells to increase the cellular proliferation as well as to utilize energy for the functioning of the cell.[9] This could also be a contributing factor that results in increase in levels of salivary LDH in premalignant lesions and OSCC.

It is not clearly known whether there is a direct correlation between serum and salivary LDH levels. However, Rao et al. established that serum LDH level does not show an increase in level with increase in the duration of the habit but the salivary LDH levels are increased markedly for patients who gave history of areca use of more than 30 years.[14] This suggests that salivary LDH levels are more likely to be influenced by local factors within the oral cavity, although these changes may not necessarily affect serum levels of LDH. Various studies also have shown that there is an increase in levels of salivary LDH in potentially malignant conditions like oral submucous fibrosis and lichen planus.[1],[15]

Studies in the past have been able to establish the role of salivary LDH level as a diagnostic aid in oral pathologies. However, the normal range of salivary LDH in health has to be established first. Wide variations in LDH of saliva have been noted in numerous studies even among healthy individuals. One reason could be due to the lack of standardization of the protocol for the salivary LDH level estimation. Salivary LDH levels are also affected by various other factors including gingival and periodontal status, age, etc., To the best of our knowledge, no other studies assessing salivary LDH levels in smokers without any local/systemic pathologies have been reported.


 > Conclusion Top


Based on the findings of our study, we conclude that there is a definite increase in salivary LDH in OL and OSCC patients. Salivary LDH in smokers was not significantly different when compared with controls. This suggests that salivary LDH levels are positively influenced by the premalignant and malignant changes in the oral mucosa. Smoking, on its own, does not seem to result in elevated LDH levels in saliva. Therefore, it is possible that an increase in salivary LDH points toward a change to dysplasia and/or carcinoma. A well-designed cohort study can help determine whether premalignant or malignant changes at a very early stage can be identified in high-risk individuals by monitoring salivary LDH levels.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

1.
Kallalli BN, Rawson K, Muzammil, Singh A, Awati MA, Shivhare P, et al. Lactate dehydrogenase as a biomarker in oral cancer and oral submucous fibrosis. J Oral Pathol Med 2016;45:687-90.  Back to cited text no. 1
    
2.
Warnakulasuriya S. Clinical features and presentation of oral potentially malignant disorders. Oral Surg Oral Med Oral Pathol Oral Radiol 2018;125:582-90.  Back to cited text no. 2
    
3.
Bavle RM. Salivary Glands. In: Kumar GS, editor. Orban's Oral Histology & Embryology. India: St. Louis: Reed Elseiver Pvt. Ltd; 2015. p. 241-65.  Back to cited text no. 3
    
4.
Shah S. Salivaomics: The current scenario. J Oral Maxillofac Pathol 2018;22:375-81.  Back to cited text no. 4
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5.
Sivaramakrishnan M, Sivapathasundharam B, Jananni M. Evaluation of lactate dehydrogenase enzyme activity in saliva and serum of oral submucous fibrosis patients. J Oral Pathol Med 2015;44:449-52.  Back to cited text no. 5
    
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Malathi K, Usha R, Dable S. Salivary enzymes-a diagnostic marker for periodontal disease. Intl J Sci Res Rev 2013;2:68-80.  Back to cited text no. 6
    
7.
Valvona CJ, Fillmore H, Nunn PB, Pilkington GJ. The regulation and function of lactate dehydrogenase a therapeutic potential in brain tumor. Brain Pathol 2016;26:3-17.  Back to cited text no. 7
    
8.
Rao K, Babu SG, Shetty UA, Castelino RL, Shetty SR. Serum and salivary lactate dehydrogenase levels as biomarkers of tissue damage among cigarette smokers. A biochemical study. Stomatologija 2017;19:91-6.  Back to cited text no. 8
    
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Raddam QN, Moafaq MZ, Mostafa AA, Nahla KA. Smoking effects on blood antioxidants Level: Lactate dehydrogenase, catalase, superoxide dismutase and glutathione peroxidase in university students J Clin Exp Pathol 2017;7:331.  Back to cited text no. 9
    
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Anbarasi K, Sabitha KE, Devi CS. Lactate dehydrogenase isoenzyme patterns upon chronic exposure to cigarette smoke: Protective effect of bacoside A. Environ Toxicol Pharmacol 2005;20:345-50.  Back to cited text no. 10
    
11.
Joshi PS and Golgire S. A study of salivary lactate dehydrogenase isoenzyme levels in patients with oral leukoplakia and squamous cell carcinoma by gel electrophoresis method. J Oral Maxillofac Pathol 2014;18:S39-44.  Back to cited text no. 11
    
12.
D'Cruz AM, Pathiyil V. Histopathological differentiation of oral squamous cell carcinoma and salivary lactate dehydrogenase: A biochemical study. South Asian J Cancer 2015;4:58-60.  Back to cited text no. 12
    
13.
Lokesh K, Kannabiran J, Rao MD. Salivary lactate dehydrogenase (LDH)- A novel technique in oral cancer detection and diagnosis. J Clin Diagn Res 2016;10:ZC34-7.  Back to cited text no. 13
    
14.
Rao K, Gogineni S, Kumari S. Estimation of serum and salivary lactate dehydrogenase levels among healthy individuals and oral cancer patients-A clinical and biochemical study. Int J Dent Res 2017;2:31-5.  Back to cited text no. 14
    
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Vasavikadiyala S. A study of salivary lactate dehydrogenase (LDH) levels in oral cancer and oral submucosal fibrosis patients among the normal individuals. J Pharm Sci & Res 2015;7:455-7.  Back to cited text no. 15
    


    Figures

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    Tables

  [Table 1], [Table 2], [Table 3]



 

 
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