Are salivary amylase and pH – Prognostic indicators of cancers?

Abstract

Background

Saliva, “Mirror of body's health” has long been of particular interest as a substitute for blood for disease diagnosis and monitoring. The radiation effects on salivary glands are of particular interest in which salivary amylase is a good indicator of salivary glands function. Thus, estimation of these parameters represents a reasonable approach in evaluation of patient's risk for disease occurrence, intensity and prognosis.

Aim of study

To evaluate and compare the pH and amylase levels in saliva of cancer patients prior to treatment, patients during treatment.

Materials and methods

Saliva samples of 90 individuals were taken which were divided into 3 groups - 30 individuals without cancer, 30 cancer patients prior treatment and 30 cancer patients during treatment. Materials used were pH strips and pH meter, Salivary Amylase assay.

Results

Statistical analysis – ANOVA with post-hoc Tukey's test.

1) Significant decrease in salivary amylase levels – in cancer patients, during treatment when compared to others.

2) Significant decrease in salivary pH levels in newly diagnosed cancer patients prior to treatment.

Conclusion

To conclude, pH strips and pH meter showed to be a useful tool in the measurement of pH of saliva in individuals with and without cancer. This study showed that cancer patients without treatment have a lower pH of saliva. Treatment increased the pH of the saliva to a more alkaline level whereas amylase levels decreased in those subjects. Therefore those parameters can be an area of further research with an increased sample size, which in-turn may help in opening the doors for new dimension in non invasive prognostic markers.

1. Introduction

Cancer is one of the most common causes of morbidity and mortality today with about 6 million deaths reported each year worldwide.¹ Oral cancer, the sixth most common human cancer, is a serious and growing problem in many parts of the globe with a five year mortality rate of approximately 50%.² The high morbidity rate in oral cancer can be attributed to the delay in the diagnosis of the disease.³ This underscores the importance of understanding the biological process of cancer for early detection of cancer risk and to predict its prognosis. In responding to the call for early detection of cancer, several studies have led to discovery of many cancer biomarkers, including salivary biomarkers.

Since time immemorial, saliva has been portrayed as a unique yet complex body fluid.⁴ Saliva, the so called ‘mirror of the body’ is a non invasive and an easily accessible oral fluid, that reflects virtually the entire spectrum of normal and disease states of the body.⁵ It has long been of particular interest as a substitute for blood for disease diagnosis and monitoring.⁴ Saliva is an ideal translational research tool and diagnostic medium which is being used in novel ways to provide molecular biomarkers for a variety of oral and systemic diseases and conditions.⁵

Saliva is made up water (99%), many organic and inorganic components which are responsible for the roles exhibited by itself. Most of the potential salivary biomarkers include salivary pH levels and salivary proteins like amylase & lysozyme.⁶

Saliva has a pH range of 6.7–7.3 in healthy individuals.⁷ A saliva pH below 7.0 usually indicates acidemia (increase acid production in the body). In tumor conditions there is a shift in pH towards acidic, this is due to anaerobic metabolism of glucose in hypoxic conditions created by the tumor burden.⁸ This acidic condition acts as a favorable factor for the tumor cells to survive and flourish.⁷

Salivary α-Amylase (sAA) is one of the most plentiful components in saliva, accounting for 10–20% of the total protein content which is known to facilitate digestion.^9,10 Saliva is responsible for the initial digestion of starch, mainly by the presence of salivary amylase (or ptyalin).¹¹

Many patients with cancer undergo radiation therapy as a main or adjuvant treatment. All or part of the major and minor salivary glands are often included within the radiation field owing to the site and extension of primary tumors.¹² Exposing the salivary glands to radiation often results in severe salivary gland hypo function and changes in saliva composition, leading to a number of acute and long-term oral complications like decreased salivary flow rates.¹² The radiation effects on salivary glands are of particular interest as the fluctuation of the salivary flow rate coincides with the fluctuation in the amylase concentration.^10,13

This study is aimed at evaluating and comparing the salivary pH and amylase levels in cancer patients prior to treatment and of patients during the treatment (mainly radiotherapy).

2. Materials and methods

The study was conducted in the outpatient department of GITAM Dental College, Vishakhapatnam for a span of 18 months. Histopathologically diagnosed cancer patients were included in the study. Study population comprised of 90 subjects within age group of 10–50 years, in which 51 were females and 39 were males (Fig. 1).

Fig. 1.

Sample size.

All patients were verbally explained the nature of the study and an informed written consent was obtained prior to the study. The study protocol was approved by the Ethical committee of the institution.

2.1. Saliva sampling

• (1)The subject is informed about the early morning collection of saliva sample and is ask to refrain from eating or drinking for at least 1 h prior to the collection.
• (2)The subject is asked to rinse their mouth with water 5 min before the collection of saliva sample.
• (3)After Five minutes of oral rinse, the subject is asked to spit into a sterile saliva collection tube and about 5 mL volume of saliva is collected.
• (4)Immediately after the collection of saliva sample pH of the sample is measured using pH strip by placing 2–3 drops of sample on the test zone of indicator strip. (MERCK SERENO – PH INDICATOR STRIPS, USA).
• (5)The samples are stored at 3 °C for further pH meter and amylase analysis.⁵

2.2. Salivary pH analysis

The pH meter (ELICO- pH meter, Hyderabad, India) is initially standardized and calibrated using distilled water. The 5 ml samples are diluted upto 30 ml and pH of the sample is measured using the pH meter. The pH meter readings of the diluted samples were tabulated.

2.3. Salivary amylase analysis

Saliva samples are stored at 3 °C for the amylase assay using calorimetric method. Salivary α-amylase was assayed by the enzymatic hydrolysis of the Di-nitro salicylic acid reagent and amylase levels were analyzed using calorimeter or spectrophotometer at 520 nm.¹⁰

2.4. Statistical analysis

Statistical analysis of the data was performed using ANOVA with post-hoc Tukey's test and the p value was <0.001 was considered statistically significant.

3. Results

Salivary pH and α-amylase activity were analyzed. A decrease in salivary pH was observed in group B & C when compared to group A, when measured using pH strip (Fig. 2).

Fig. 2.

pH variation in groups (pH Strip).

A significant decrease in salivary pH was observed in Group B than Group A & C when measured using pH meter. Group B and C showed an acidic pH. Among group B and C, group C exhibited a lesser acidic pH than group B (Fig. 3).

Fig. 3.

pH variation in groups (pH meter).

Significant decrease in salivary amylase levels was observed in Group C when compared to Group A & B(Fig. 4).

Fig. 4.

Amylase variation in groups.

4. Discussion

The decreased amylase levels and pH values can be linked to the cancer pathogenesis and complications after the radiotherapy. It is known that apart from blood, other body fluids also have a potential to become a diagnostic medium, in which saliva offers some distinctive advantages.^14–16 Saliva is a unique fluid, and interest in it as a diagnostic medium has advanced exponentially in the last 10 years.¹⁷

Salivary alpha-amylase which is synthesized and secreted by acinar cells and cells of the intercalated ducts, which make up more than 80% of the cells in the major salivary glands.¹⁸

Since salivary alpha amylase secretion is regulated by the sympathetic nervous system which stimulates acinar cells of the salivary glands via beta-adrenergic receptors, the measurement of salivary alpha-amylase activity has been proposed to reflect the general health of the salivary glands.¹⁹ Salivary amylase enzyme is considered as a reliable enzyme to determine the serous cell functionality.²⁰ The difference between serous and mucous cells in radio-sensitivity may be attributed to the presence of heavy metals in the serous secretory granules, which upon absorption of radiation energy promote the release and action of free radicals.¹²

Acini in the parotid glands are almost exclusively of the serous type and makes parotid glands more sensitive to radiotherapy than sublingual and submandibular glands.^21,22 The excretory and acinar cells of the salivary glands are highly differentiated with a slow mitotic rate and turnover, but they behave like acute responding tissues to radiation and are very radio-sensitive.²⁰ Therefore, an irreversible degenerative process in salivary glands leads to reduction in the number of acinar cells, incomplete tissue regeneration, selective membrane damage and also late stromal effects such as delayed vascular damage due to radiation.²³

The results of salivary amylase levels in our study are in accordance to the study conducted by Takei et al, in which they found a reduction in overall salivary amylase levels which may be caused by dysfunction of serous cells in the parotid gland. But, Takei et al, suggested that the reduction in overall salivary amylase levels may be result of both salivary gland dysfunction and reduction of food intake during RT treatment.²⁴

But Donia Sadri et al, suggests that a significant reduction in salivary amylase can be only due to salivary gland dysfunction. They also found a significant decrease in flow rate, pH and IgM in cancer patients after radiotherapy which was against the results of our study.²⁰

According to Christenson et al, a study conducted by them showed a significant decrease in salivary amylase levels after 3 weeks following radiotherapy in head and neck cancer patients which is because of the exposure of submandibular and parotid salivary glands which have more number of serous acini, making them more sensitive to ionizing radiation.²⁵

According to Grundmann et al, a consistent 14–24% reduction in amylase levels is measured in experimental mice which were irradiated with a single 5 Gy dose of radiation. They found similar findings in humans which was attributed to loss of acinar cells and glandular shrinkage after radiotherapy.²⁶

Studies conducted by Koshy et al also showed a reduced salivary flow rate and salivary mean amylase levels at 6 weeks of radiotherapy which was explained by decrease in overall functional integrity of the salivary glands after radiotherapy. Researchers also found that amylase levels increased after 3 and 6 months of radiotherapy which was attributed more to lower salivary volumes rather than a salivary gland recovery.²⁷

The results of our study showed an increase in salivary pH after radiotherapy, but according to the results of Koshy et al, they found a significant decrease in salivary pH after radiotherapy which was attributed to the reduced buffering capacity of saliva as a result of radiation injury.²⁷

Some of the studies were against the results of our study which include the studies conducted by Leslie et al, which showed a rise in salivary amylase levels in the early phase of radiotherapy in head and neck cancer patients, which then comes back to normal level. The researchers thought that this probably reflects the interphase cell death of serous cells in salivary glands.²⁸

According to the results of our study which shows a decrease in pH levels of cancer patients, which was attributed to uncontrolled growth of the tumor cells, thereby creating an imbalance in the ratio of demand and supply of the nutrition to the tumor cells. As a result of high uptake of glucose by the tumor cells and subsequent anaerobic glycolysis leads to lactic acid production, which may be the reason for the acidic environment observed in the oral cavity of cancer patients.¹⁰

Although the pH was higher in the oral cavities of radiotherapy patients (three and six weeks of treatment) than in cancer patients, it still remains in the acidic range. Higher lactic acid levels and negligible buffering capacity may be the underlying causes of the acidic pH in the oral cavities of patients who underwent radiotherapy.¹⁰

The results of our study was supported by the similar results obtained by Jensen et al, who explained that the apparent selective destruction of serous acini may be due to absorption of radiation energy which promote the release and action of free radicals, thus explaining the decrease in pH after radiotherapy.¹²

However this study needs to be carried out in greater number of cases for the exact relation between salivary analytes and cancer.

5. Conclusion

• •Saliva has the potential to become a first-line diagnostic aid of choice owing to the advancements in detection technologies coupled with combinations of biomolecules with clinical relevance.
• •To conclude the present study, pH strips and pH meter showed to be a useful tool in the measurement and comparison of the pH and amylase levels of saliva in people with and without cancer.
• •An unexpected alteration in salivary pH in cancer patients during treatment suggests an emphasis on a very important direction for future research with an increased sample size, which may in-turn help in opening the doors for a new dimension in cancer that is “Non Invasive Prognostic Indicators”.

Conflicts of interest

All authors have none to declare.