Title : Salivary Lactate Dehydrogenase, Lipid Peroxidation and Glutathione As Prognostic

Salivary Lactate Dehydrogenase, Lipid Peroxidation and Glutathione as Prognostic Markers of Cell Death in Oral Cancer Patients Undergoing Radiation Therapy: an Investigative Study Introduction Despite recent advances in treatment oral cancer is reported as having one of the highest mortality ratios and this can much be attributed to the late diagnosis of the disease. Oral cancer refers to all malignancies that arise in the oral cavity, lips and pharynx, with 90% of all oral cancers being oral squamous cell carcinoma. In late stages radiation, including chemoiradiation is the choice of treatment in oral cancer. 1 However evaluation of the efficacy of treatment when initiated is a major problem as in advanced stages the hypoxic tumor cells, invasiveness and metastasis complicated the regular treatment evaluation. There is a search for sensitive and specific diagnostic and prognostic biomarkers of oral cancer and effects of radiation therapy in oral cancer. 1 Lactate dehydrogenase (LDH) is a ubiquitous enzyme, serum levels of which are in response to cell damage in various conditions such as cancer, systemic diseases, inflammation and infections. It is proposed that radiation treatment causes the release of LDH from the buccal mucosa cells in to the saliva, and that this can be a surrogate marker for evaluating the tumor cell kill and possible tumor regression. 2,3 Ionic irradiation exposes all cells in the involved field to high levels of oxidative stress resulting in formation of ROS, increasing DNA damage and ultimately leading to cell death . Salivary oxidant-antioxidant status could serve as sensitive marker of oral cancer.2,3 Whole saliva can be collected non-invasively and no special equipment or skilled person is needed for saliva collection. Salivary chemical constituents have been analyzed in autoimmune diseases, systemic diseases, cancer and infectious conditions. 4 Saliva analysis is not given due thrust in India. There is paucity of studies on salivary enzymes and oxidative stress markers to assess cell damage in oral cancer patients undergoing radiation therapy. Objectives 1. To assay the salivary levels of lactate dehydrogenase, malondialdehyde and glutathione in oral cancer patients, in comparison to healthy controls 2. To assess the changes in salivary levels of lactate dehydrogenase, malondialdehyde and glutathione in oral cancer patients as prognostic markers of radiation therapy in oral cancer patients Methodology This is a hospital-based study involving the departments of Oncology and Biochemistry of the Medical College Hospital. The study subjects comprise of patients who visit the oncology department and diagnosed of oral cancer, based on clinical examination and histopathological study (Group-1A). These patients are then followed up with radiation therapy (Group-1B). Thirty age- and sex-matched, apparently healthy volunteers will be included as controls in the study (Group-2). Patients with cancer of regions other than oral cavity, any systemic diseases, infections, and alcoholics, smokers and tobacco chewers without diagnosed cancer, will be excluded from the study. Detailed history of habits (smoking, alcohol abuse, tobacco chewing), chronic illness and clinical history, will be collected from all the subjects. Unstimulated saliva samples will be collected from oral cancer patients before and after 2, 4 and 6 weeks of radiation therapy. The samples will be collected between 9-11 AM, according to the method of Navazesh. 5 This is to ensure that the variability in salivary flow rate and composition, be minimized. The subject will asked to rinse the mouth with distilled water thoroughly to remove any food debris and then after 10 minutes, directed to spit into a sterile plastic container. Forcible spitting will be avoided. The saliva samples will be centrifuged at 3000 rpm for 10 minutes and the supernatants will be taken for analysis of malondialdehyde (MDA), glutathione (GSH) and LDH in subjects of group-1A, group-1B and group-2. Levels of malondialdehyde, the marker of lipid peroxidation will be estimated as thiobarbituric acid-reactive substances (TBARS). Levels of GSH, the antioxidant tripeptide, will be estimated based on reduction of 5,5/ -dithiobis-(2 nitrobenzoic acid) by GSH. LDH activity in saliva will be assayed by standard kinetic spectrophotometric method, measuring absorbance change at 340 nm. The statistical significance of the results will be evaluated by Student’s “t” test and Karl Pearson’s correlation analysis. Implications of the Study The present study for the first time aims at understanding the prognostic value of salivary LDH, LPX and GSH as a surrogate marker to the cell kill and tumor regression of the oral cancer being subjected to external beam radiatiotherapy either alone or in combination to chemotherapy. The results obtained from the study will help in validating these salivary in evaluating marker for cell death and remission. It is expected that it may also help the physicians modify the treatment modality and the dose for better tumor remission and possible cure. References; 1. Park K. Park’s Text Book of Preventive and Social Medicine, 19th edition, M/s B. Bhanot; 2007: 313 – 315. 2. Chitra S, Shyamaladevi CS. Effect of α-tocopherol on pro-oxidant and antioxidant enzyme status in radiation-treated oral squamous cell carcinoma. Indian J Med Sci 2008; 62: 141-148. 3. Nagler R, Bahar G, Shpitzer T, Feinmesser R. Concomitant analysis of salivary tumor markers- a new diagnostic tool for oral cancer. Clinical Cancer Research 2006; 12: 3979-3984. 4. Ziang L, Xiao H, Wong DT. Salivary biomarkers for clinical application. Mol Diagn Ther 2009; 13 : 245- 259. 5. Navazesh M. Methods for collecting saliva. Ann Ny Acad Sci 1993; 20: 72-73.