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Table of Contents
Year : 2021  |  Volume : 14  |  Issue : 3  |  Page : 124-128

Salivary enzymes and oxidant-antioxidant parameters as biomarkers of head and neck cancer: Correlation between blood and saliva

1 Department of Biochemistry, Father Muller Medical College, Mangalore, Karnataka, India
2 Father Muller Research Centre, Mangalore, Karnataka, India
3 Department of Radiation Oncology, Father Muller Medical College, Mangalore, Karnataka, India
4 Department of Community Medicine, Father Muller Medical College, Mangalore, Karnataka, India

Date of Submission22-Feb-2021
Date of Decision31-May-2021
Date of Acceptance31-May-2021
Date of Web Publication01-Oct-2021

Correspondence Address:
Manjeshwar Shrinath Baliga
Mangalore Institute of Oncology, Pumpwell, Mangalore, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/hmj.hmj_5_21

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Background: Studies with saliva are of immense advantage as the process of it collection from humans is a noninvasive method and can be repeated with least compliance problems. In this study, the levels of lactate dehydrogenase (LDH), amylase, reduced glutathione (GSH), glutathione S-transferase (GST), protein-bound sialic acid (PBSA), lipid peroxidation (LPO) and total antioxidant capacity (TAC) were analysed in the blood and saliva samples of head and neck cancer (HNC) patients and compared it with age-matched healthy controls (N = 30) to explore the usefulness of the parameters as an endpoint biomarkers. Materials and Methods: HNC patients visiting hospital for treatment (n = 31) were the participants of the study; age- and sex-matched healthy controls were included. Levels of LDH, amylase, PBSA, LPO (as malondialdehyde), GSH, GST and TAC were analysed in whole, unstimulated saliva samples. Results: The levels of LDH, amylase, PBSA and LPO were significantly higher, and levels of GSH, GST and TAC were significantly lower in blood and saliva of HNC patients when compared to healthy controls (P < 0.001). There was statistically significant correlation between blood and saliva with regard to the levels of amylase and GSH (P < 0.001). Conclusions: This study demonstrated significant changes in the biochemical parameters of blood and saliva in HNC patients and correlation of salivary GSH and amylase with corresponding levels of blood. Salivary biochemical parameters could serve as sensitive and convenient biomarkers of HNC.

Keywords: Amylase, biomarker, glutathione, head and neck cancer, saliva

How to cite this article:
Shivashankara AR, Simon P, Pais S, Suresh S, George T, Baliga MS. Salivary enzymes and oxidant-antioxidant parameters as biomarkers of head and neck cancer: Correlation between blood and saliva. Hamdan Med J 2021;14:124-8

How to cite this URL:
Shivashankara AR, Simon P, Pais S, Suresh S, George T, Baliga MS. Salivary enzymes and oxidant-antioxidant parameters as biomarkers of head and neck cancer: Correlation between blood and saliva. Hamdan Med J [serial online] 2021 [cited 2021 Dec 7];14:124-8. Available from: http://www.hamdanjournal.org/text.asp?2021/14/3/124/327426

  Introduction Top

Globally, in tobacco habituals the cancer in the head and neck cancers (HNCs) region, especially in the oral cavity, tongue, lip, nasopharynx, and pharynx are very common.[1],[2] Recent reports from the World Health Organisation (WHO) and International Agency for Research on Cancer reports indicate that the global incidence of HNC is high in Asia Pacific countries,[3],[4],[5] and attempts are on to develop a prognostic marker.

Saliva that contains nucleic acids, cytokines, growth factors, oxidant-antioxidant molecules, enzymes and glycoconjugates is being considered to be an ideal body fluid to develop prognostic markers principally because its collection is non-invasive and does not have any compliance problems.[6],[7] Global reports also suggest that when compared to healthy control, the levels of various salivary constituents significantly vary at premalignant and cancer stages.[8],[9],[10],[11],[12],[13],[14]

In the present study, an attempt to assess the correlation between blood and saliva with regard to the levels of major intracellular thiol glutathione (GSH), enzymatic oxidant defence and detoxification enzyme glutathione S-transferase (GST), indicator of non-enzymatic antioxidant defence total antioxidant capacity (TAC), marker of lipid peroxidation (LPO) malondialdehyde (MDA), and enzymatic markers of cell health amylase and lactate dehydrogenase (LDH) in blood and saliva in HNC.

  Materials and Methods Top

The present study was prospective and was done in the Departments of Biochemistry, Research and Development and Oncology at Father Muller Medical College Hospital, Mangalore. The approval from the Institutional Ethics Committee (FMMC/FMIEC/877/2012) was obtained, and the study conducted as stipulated by the Helsinki declaration on research with humans and ICMR guidelines for Biomedical and Health Research on Human Participants. The HNC patients visiting the Oncology Department were the study participants (Group-II; n = 31). Age- and sex-matched healthy controls (n = 40; Group I) comprising of the staff and general public were included. Only the participants in the age group of 45–70 years were included. Patients with any chronic and systemic illness, and those who are already on chemotherapy or radiation therapy, and have undergone surgery for any other type of cancer in the last 6 months were excluded Group II. Chronic smokers, alcoholics and tobacco chewers were excluded from Group I. They had good oral health (as ascertained by the orodental physician of the hospital) and were having any chronic ailments and not on any medication for any ailments.

Histopathological grading

The tumor stage was performed by senior pathologists in accordance to the standard guidelines prescribed in the tenets of the 7th edition of the classification of malignant tumors of the American Joint Committee on Cancer.[15] The tumor grade was classified following the criteria suggested by the WHO.[16]

Saliva collection

During the course of the study, the investigators approached the HNC patients admitted for the proposed treatment (before initiation of the treatment). The investigator explained the objective of the study to both the patient and the attending caregiver in their mother tongue. A written informed consent was taken from the willing patient. Information regarding the patient's age and tumour details was collected from the patient file. Unstimulated saliva was collected between 9 and 10 a.m. to ensure that the variability in salivary flow rate and composition, be minimised.[17] The volunteers were asked to rinse the mouth with distilled water thoroughly to remove any food debris and then after 10 min, requested to salivate into a sterile plastic. The collected saliva was centrifuged at 3000 rpm for 10 min, and the supernatants were stored in the cold refrigerator (‒20°C).

Blood collection

Blood samples were collected taking aseptic precautions. On the day of analysis, the stored saliva was removed from cold refrigerator thawed and analyzed using appropriate blanks, controls and standards using ultraviolet-, visible spectrophotometer (Shimadzu, Japan).

Biochemical assays

The levels of protein bound sialic acid (PBSA), LDH, amylase, MDA, reduced glutathione, GST and the TAC were carried out in accordance to the standard protocol[18],[19],[20],[21],[22],[23],[24] with blood or saliva in accordance to the standard methods.

Statistical analysis

The demographic and tumour details were subjected to the frequency and percentage. The biochemical details were subjected to unpaired t-test between the control and the cancer patients. In addition to this, the correlation between each of the assayed endpoint in the serum and saliva was subjected to Pearson correlation coefficient separately for healthy controls and for oral cancer patients. The statistical analysis was performed using the SPSS 23 statistical program (IBM, Chicago, Illinois, USA). A value of P < 0.05 was considered statistically significant.

  Results Top

The demographic and other details of the study participants of Group 2 (oral cancer patients) are given in [Table 1]. The average age of the cancer patients was 53.8 years with a standard deviation (SD) of 9.98 (ranged from 45 to 68 years), whereas the average of the controls was 52 years with a SD of 6 (ranged from 45 to 62 years). The results of the analysis of biochemical parameters are represented in [Table 2], [Table 3], [Table 4]. Oral cancer patients showed significantly higher levels of LDH, amylase, MDA and PBSA in blood and saliva, when compared to healthy controls. The levels of GSH, GST and TAC in blood and saliva were significantly lower in oral cancer patients in comparison to controls. All the results were statistically significant [P < 0.001; [Table 3]]. Correlation analysis revealed significant positive correlation between blood and saliva with regard to amylase and GSH. There was no significant correlation between blood and saliva with regard to GST, LDH, TAC and MDA [Table 4].
Table 1: Demographic, tumour and habits and details of the head and neck cancer patients

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Table 2: Comparison between the various haematological, liver and renal parameters in the healthy control and in oral cancer patients

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Table 3: Comparison between the various serum and salivary parameters in the healthy control and in oral cancer patients

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Table 4: Correlation between blood verses salivary parameters in the healthy controls and in oral cancer patients

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  Discussion Top

Salivary research is gaining attention in recent years. The need for salivary biomarkers is given thrust due to distinct advantages in sample collection and anatomical proximity of saliva to the sites of cancer. We observed significantly altered levels or activities of enzymes, oxidant-antioxidant system and sialic acid in oral cancer.

LDH, the sensitive and convenient enzyme marker of cell turnover was elevated in serum and saliva of HNC patients [Table 3]. Earlier, studies have observed significantly higher salivary LDH in oral squamous cell carcinoma in comparison to oral premalignant lesions and healthy controls.[10],[11] The salivary LDH has been shown to increase with the progression of oral cancer, from well differentiated to poorly differentiated cases.[11],[25] Elevated serum LDH in oral squamous cell carcinoma and oral pre malignant lesions has been reported.[26] In the present study, we did not observe any significant correlation between serum and saliva with regard to LDH activity in HNC.

Amylase, an enzyme abundantly present in saliva plays the major role in digestion of starch. Decreased activity of salivary amylase in oral cancer and oral premalignant lesions is reported by previous studies.[10] Our findings of increased activity of amylase in serum and saliva are in contradiction to the findings of previous studies.[10],[27] We also observed a significant correlation between the salivary and serum levels of amylase [Table 4].

The major intracellular thiol and the ubiquitous tripeptide GSH plays an important role in detoxification by conjugation and in the xenobiotic metabolism. This vital reducing agent of the cell keeps the thiol groups of proteins intact preventing them from oxidation and as antioxidant by virtue of being a cosubstrate for the reaction catalysed by the selenium-containing enzyme glutathione peroxidase. The present study showed significantly lower salivary and plasma GSH in HNC patients [Table 3]. Decrease in GSH could be due to utilisation of GSH in scavenging the reactive oxygen species. We also observed significant correlation between salivary and plasma GSH [Table 4]. Previous studies have observed increased salivary[2] or decreased salivary[9] or increased blood levels 30 of this thiol compound in oral cancer. GST, the major enzyme for phase 2 of detoxification, also functions as an antioxidant enzyme. We observed decreased activity of GST in saliva and plasma in oral cancer patients [Table 3]. The findings are in concordance with previous studies.[13]

We observed increased levels of MDA, the marker of LPO, in plasma and saliva of HNC patients [Table 3]. The present study observed oxidative stress indicated by increased LPO (evident by elevated malondialdehyde), and decreased levels of GSH, GST and TAC. These changes were evident both in saliva and plasma. The findings are in concordance with previous studies.[9],[12],[28] Saliva has rich content of antioxidants. The salivary antioxidant system is proposed to have a fundamental anticarcinogenic role in the oral cavity and is aimed at fighting reactive species.[29] TAC reflects mainly the non-enzymatic antioxidants in plasma or saliva. We observed decreased TAC in saliva and plasma of oral cancer patients. There was no significant correlation between saliva and plasma with regard to MDA, TAC and GST.

Glycoconjugates play an important role in malignant transformation of cells. Aberrant glycosylations are the universal phenomena in malignancy. Previously, Sanjay et al. observed the elevated levels of free sialic acid (FSA) and PBSA in the saliva in oral squamous cell carcinoma.[8] Observation of increased PBSA in saliva and serum in the present study is in agreement with the reports of previous studies.[8],[9] Some researchers have hinted at possible interactions between antioxidant enzymes and FSA in saliva.[30] They also postulated the role of sialic acid as a hydroxyl radical scavenger by virtue of its presence in salivary mucin.[30],[31]

This study had some limitations. The sample size was low, and correlations of biomarkers with grades of HNC and risk factors (habits) of HNC were not done. Our observation of significant correlation between blood and saliva with regard to the levels of amylase and GSH is of significance for the utility of salivary biomarkers. Future studies with larger sample size, correlating the salivary biomarkers with those of blood, correlation of biomarkers of grades of cancer and affect different modalities of treatment on the levels of biomarkers, are required to establish saliva as a tool supportive to blood, if not alternate tool time being, for the diagnosis and management of HNCs.

  Conclusions Top

This study demonstrated significant changes in the biochemical parameters of blood and saliva in HNC patients and correlation of salivary GSH and amylase with corresponding levels of blood. Salivary biochemical parameters could serve as sensitive and convenient biomarkers of HNC.

Ethical clearance

The study was approved by Institutional Ethics Committee of Father Muller Charitable Institution Mangalore India (FMMC/FMIEC/877/2012) and was conducted as stipulated by the Helsinki declaration on research with humans and ICMR guidelines.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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  [Table 1], [Table 2], [Table 3], [Table 4]


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