Evaluation of Salivary and Plasma Levels of Salivary α-Amylase in Patients of Oral Squamous Cell Carcinoma.

Aim: The aim of the study was to evaluate levels of salivary alpha-amylase (sAA) in plasma and saliva of patients with oral squamous cell carcinoma (SCC). The diurnal patterns of sAA and its response to stress were also determined. Materials and
Methods: A randomized clinical study was conducted to evaluate the salivary and plasma levels of sAA in three study groups, containing ten subjects each. sAA concentration in plasma and saliva samples was measured using Bioassay Technology Laboratory human alpha-amylase kits, and the levels were compared among control and test groups.
Results: In all groups, the mean plasma α-amylase level and mean saliva α-amylase level show an increasing trend with time, i.e., from morning to night, and among the groups, it was highest in Group III followed by Group II and Group I the least (Group III, Group II, and Group I) at all times with significantly higher mean values in Group III subjects at all times of a day.
Conclusion: Alpha-amylase is one of the principal salivary proteins and its secretion is regulated by the sympathetic nervous system. The measurement of salivary alpha-amylase activity has been proposed to reflect stress-related changes in the autonomic nervous system, and it may be a good choice for monitoring sympathetic nervous system activity in specialized subjects. Hence, it can be concluded that salivary sAA levels can be taken as a predictable as well as reproducible marker for oral SCC or premalignant lesions. Copyright:

INTRODUCTION

Malignancy is a treatable pathology, if detected at an early stage, but is still associated with high morbidity and mortality. Owing to lack of awareness and unavailability of amenities, malignancy generally remains undetected at early stage and majority of the people seek medical advice at an advanced stage when their chances of survival reduce considerably. Worldwide quotients of people diagnosed with cancer account up to 14.1 million. The reported incidence of cancer is 12.6 per 100,000 people. Malignancies most commonly affecting human beings are lung cancer and breast cancer followed by colorectal cancer.[1]

Out of the above, carcinoma of head and neck accounts for 10% of all the malignancies and oral squamous cell carcinoma (SCC) comprises approximately 90% of all the head-and-neck carcinomas. Worldwide oral SCC of head and neck is the 6th most frequent form of cancer, and it is the 3rd most common cancer in Indian women and the 2nd most common cancer in men. Due to the delay in diagnosis of the disease, there is high morbidity and mortality rate in oral carcinoma. This underscores the importance of understanding the biological process of cells causing malignancy. Although continuous research for the early diagnosis and better treatment for cancer is carried on, it still has a poor prognosis and survival rate of 5 years. Oral SCC is said to be the disease of the developing countries with prevalence of 30% of all cancers in India.[2]

To reduce the mortality and morbidity associated with it, early diagnosis by histopathological examination of tissue is advocated. A better understanding of carcinogenesis at molecular level is essential to prevent the progression of cancer. Identifying the carcinogenic cells which are already dysplastic and tag them through various tumor markers to depict either the presence of cancer or provide information about the chances of that cell to turn carcinogenic in future, is an essential step. There is a wide range of tumor markers which can be broadly classified based on the type of tissues such as epithelial markers, connective tissue markers, and salivary markers.[3]

Saliva, the so-called mirror of the body, reflects virtually the entire spectrum of normal and diseased state of body. Since time immemorial saliva has been portrayed as unique yet complex body fluid and salivary gland markers prove to be a better option being noninvasive, easily available, and a cheaper alternative to serum testing.[4]

Saliva, when examined at molecular level, comprises a protein, amylase, a heterogeneous calcium-dependent metalloenzyme of molecular weight 54–62 kDa. It exists as two isoenzymes: pancreatic (P-type) and nonpancreatic (S-type). S-type is being found in the exocrine pancreas and salivary glands and one of the plentiful components in saliva. After birth, S-type amylase activity increases in serum steadily with age and reaches normal adult values at 2 years of age. Various reasons can be sited for the increase in salivary amylase levels such as pancreatic disease, salivary gland disease, tumors, HIV, ketoacidosis, and so on.[5] Since amylase is seen in acinic cell tumors, it serves as a “tumor marker” and further it can be used to identify the malignant cells and cells turning into malignancies.[6]

This study intends to compare the plasma and salivary levels of salivary alpha-amylase (sAA) in oral SCC patients and premalignant patients with that of the control group which comprises healthy patients. The diurnal patterns of sAA and its response to stress were also determined in all the three groups.

MATERIALS AND METHODS

A randomized controlled clinical study was conducted in the Department of Oral and Maxillofacial Surgery, Saraswati Dental College, Lucknow, to evaluate the salivary and plasma levels of sAA among test and control groups. Histopathologically proven 10 subjects suffering from oral SCC along with equal number of patients suffering from oral premalignant conditions were randomly selected and 10 healthy patients were also included in the study as control group. The ethical clearance was obtained from the Institutional Ethical Committee, and it was monitored in accordance with Declaration of Helsinki and Good Clinical Practice (ICH) guidelines. Patients with acute pancreatitis, salivary gland inflammation, perforated duodenal ulcer, pregnant and lactating women, persons using any drugs that might interfere with hypothalamic–pituitary–adrenal axis function, individuals with history of cancer, and premalignant lesions are excluded. The control group (Group 1, Figure 1) had healthy patients with no history of oral SCC, premalignant conditions, systemic disease, or other immunocompromised conditions. The test group (Group 2, Figure 2) had patients with oral premalignant conditions and Group 3 [Figure 3] had patients with oral SCC. Prior to initiating the study, the patients were informed of the purpose and design of the study and were requested to sign an informed consent, detailed history was recorded, and thorough clinical examination was done. Both test and control groups were subjected to hematological examination such as complete blood count, blood sugar levels, and radiographic and ultrasound investigations if necessary. Incisional/excisional biopsy was performed under local anesthesia, and tissue was sent for the histopathological examination. Based on the results obtained from histopathological examinations, the subjects were divided among the group.

Figure 1

(Group I) clinical picture of the patient in healthy group

Figure 2

(Group II) Clinical picture of the patient in premalignant group

Figure 3

(Group III) Clinical picture of the patient in oral squamous cell carcinoma group

Plasma and saliva collection

Peripheral venous blood and saliva samples were collected from each patient in calm, stress-free mental, and physical state with quiet and peaceful environment [Figures 4-7]. Samples were obtained from each patient at about 7–10 AM, at 2–4 PM and between 11 PM and 12 midnight to evaluate daily pattern (diurnal variation) of sAA secretion, adjusted according to their own comfort levels and sleep/awake pattern. Peripheral venous blood was collected in sterile plain tubes using disposable sterile syringe and needle and centrifuged, and the plasma is stored. About 5 mL unstimulated saliva samples were collected using passive drool method. Both plasma and salivary samples were stored at −20°C but if the storage warranted preservation of samples for a longer time at a temperature of −75°C were maintained. Further blood contamination in saliva samples were assessed by Hemastix Reagent strip test. sAA concentration in plasma and saliva samples was measured using Bioassay Technology Laboratory human alpha-amylase kits following the manufacturer's instructions [Figure 8].

Figure 4

Armamentarium for clinical examination

Figure 7

Collection of blood sample

Figure 8

(a) Centrifuge machine. (b) Reagent for analysis of salivary alpha-amylase. (c) ELISA plate with wells showing processing of salivary samples. (d) ELISA plate with wells showing processing of plasma samples. (e) ELISA plate reader

Armamentarium for sample collection

Collection of saliva sample

Statistical analysis

Groups were compared by one-way analysis of variance, and the significance (inter) of mean difference between the groups was done by Tukey's honestly significant difference post hoc test. Analyses were performed on SPSS software (Windows version 17.0, Bioassay Technology Laboratory, Shanghai, China).

RESULTS

The present study evaluates and compares the salivary and plasma sAA levels in normal healthy subjects (Group 1), premalignant subjects (Group 2), and oral SCC patients (Group 3). Total 30 patients were included in the study with 10 patients in each group. The outcome measure of the study was based on sAA levels assessed at morning (7–10 AM), afternoon (2–4 PM), and night (11 PM–12 midnight). The sAA levels measured in unit/liter were assessed in blood (plasma) and saliva separately. Comparing the mean age and the gender proportion of three groups, the subjects in the three groups were age and gender matched and hence they did not influence the study outcome measures. On evaluating the plasma sAA level (U/L) of three groups, the mean plasma α-amylase level shows an increasing trend with time, i.e., from morning to night, and among the groups, it was highest in Group III followed by Group II and Group I the least (Group III, Group II, and Group I) at all times. The mean saliva sAA level in both Group I and Group II shows a trend with time, i.e., increase from day to night, and among the groups, it was highest in Group III followed by Group II and Group I with the least value. On comparing the plasma and saliva sAA at morning, afternoon, and night, the mean sAA level in saliva was comparatively higher than the plasma levels in all the three groups during different time periods in a day [Graphs 1-11].

Graph 1

Mean age of three groups

Graph 11

Comparison of α-amylase level of three groups in plasma and saliva at night

Distribution of gender of three groups

Mean plasma α-amylase level of three groups over the periods

For each group, comparison of mean difference in plasma α-amylase level between the periods

For each period, comparison of mean difference in plasma α-amylase level between the groups

Mean saliva α-amylase level of three groups over the periods

For each group, comparison of mean difference in saliva α-amylase level between the periods

For each period, comparison of mean difference in saliva α-amylase levels between the groups

Comparison of α-amylase level of three groups in blood and saliva at morning

Comparison of α-amylase level of three groups in plasma and saliva at afternoon

DISCUSSION

In recent years, components of saliva have generated increasing interest among researchers, due to the minimally invasive nature of sample collection, and it is becoming a valuable option as a biomarker for clinicians and health-care providers. Salivary alpha-amylase is currently being utilized in physiological research and clinical facilities for psychological stress testing for populations such as children, those under mental stress, and those with mental or physical disabilities.[7]

Alpha-amylase is one of the principal salivary proteins. It is a calcium-containing metalloenzyme that hydrolyzes the α1, 4 linkages of starch to glucose and maltose.[8] Since sAA secretion is regulated by the sympathetic nervous system which stimulates acinar cells of the salivary glands via beta-adrenergic receptor, the measurement of salivary alpha-amylase activity has been proposed to reflect stress-related changes in the autonomic nervous system.[9] Studies provide evidence that sAA may be a good choice for monitoring sympathetic nervous system activity to stress in specialized participants as well as in the field due to its minimally invasive nature.

sAA testing is preferable to blood testing due to the minimally invasive nature of collection, the ease at which it can be collected, and repeated collection is possible without inducing or causing any anxiety in the patients. Our result advocates salivary testing over plasma testing, as there was similar pattern of diurnal change between saliva and plasma levels of sAA during the course of day. When compared to plasma, sAA levels in saliva showed more consistent pattern of raised levels throughout the day. There are a number of studies reporting that both salivary flow rate and saliva composition vary rhythmically over a 24-h period.[10] The idea of using sAA as a stress marker was originally proposed in the 1970s by Gilman et al.[11] and since has been investigated by numerous researchers.[11]

As per various studies done by different authors, there were no differences between men and women in their average sAA levels, in the diurnal profile of men or women, nor did it differ by body mass, activity level, food, and drinks. These studies evaluated the diurnal cycle of sAA and demonstrated that there is a distinct rhythm in sAA with lowest value within an hour after waking up and rise throughout the afternoon and evening.[12]

Biochemical analyses of tumor tissue, studies of cell cultures, immunohistochemical techniques, and Northern blot analyses support the theory that amylase is produced by carcinoma cells. Alpha-amylase, which is produced by lung cancer tissue, was studied by cloning cDNA from a cell line originating from lung cancer that produces amylase. Sequencing studies with this cDNA showed that the expressing gene is of the salivary type.[13]

Ours is the first study which evaluates and compares diurnal profile of salivary alpha-amylase in patients of oral premalignant, oral SCC, and normal healthy subjects. Only a scarce number of studies have explored diurnal sAA rhythms. In this a marked diurnal profile of salivary alpha amylase activity with a pronounced decrease after awakening, and steadily rising levels toward the afternoon and evening.

In all groups, the mean plasma α-amylase level shows an increasing trend with time, i.e., from morning to night, and among the groups, it was highest in Group III followed by Group II and Group I the least. In different groups comparing the mean difference of plasma sAA level were high at night as compared to morning in both Group I and Group II. Further, at morning, it was also found to be significantly (P < 0.05) higher in Group III as compared to Group II. In contrast, at both, afternoon and night, the difference of sAA levels between Group II and Group III was found to be statistically insignificant.

The mean saliva sAA level in both Group I and Group II shows a trend with time, i.e., increase with increase from day to night, and among the groups, it was highest in Group III followed by Group II and Group I the least. Within each group, comparing the mean difference in saliva sAA level between the periods, higher saliva sAA levels are obtained at night as compared to morning in both Group I and Group II. Further, at morning, it was also found significantly (P < 0.01) higher in Group III as compared to Group II.

It has been shown that there are a reduction of salivary alpha-amylase activity at the beginning of the day and an increase at the end of the afternoon in rats.[14] The same result was obtained from three recent studies done on humans.[151617]

A recent study showed distinct changes in salivary alpha-amylase in saliva over the course of the day.[12] The data collected in this study show a great variation of readings in each individual, when all the groups are analyzed. However, if mean of each group is considered it formed to be in line with these studies demonstrating slight variation in high- and low-range values, reason may be the type of ELISA kit used.

Limitation and future direction

The current study relied on convenience sampling. Although the sample size is relatively small, it rivals the very few studies that have assessed sAA levels in patients of oral SCCC and premalignant diseases.

sAA levels can be affected by behavioral and lifestyle factors such as smoking, drinking, and physical exercise. Hormonal fluctuations due to menstrual cycle, the use of hormonal contraceptives, hypertension, and diabetes also may impact alpha-amylase concentrations.

Although we accounted for smoking and drinking habits in the study, we did not control for exercise habits or the use of hormonal contraception. Future studies should investigate the pattern of diurnal change in sAA in cases of oral SCC as per TNM classification, and patients should not be revealed about the diagnosis prior to sample collection. The participants must be categorized on the basis of their personality. It can be used a prognostic indicator, which may open the door for a new dimension in cancer research [Tables 1-6].

Table 1

Basic characteristics (mean±standard deviation) of three groups

Basic characteristics	Group I (n=10), n (%)	Group II (n=10), n (%)	Group III (n=10), n (%)	F/χ2	P
Age (years), mean±SD	45.90±9.49	46.50±9.22	47.90±12.08	0.10	0.907
Gender
Female	3 (30.0)	3 (30.0)	4 (40.0)	0.30	0.861
Male	7 (70.0)	7 (70.0)	6 (60.0)

SD: Standard deviation

Table 6

Comparison of α-amylase level (mean±standard deviation) of three groups in plasma and saliva at night by Student's t-test

Group	Plasma (n=10)	Saliva (n=10)	Mean difference	t	P
Group I	0.160±0.055	0.293±0.072	0.133	4.66	<0.001
Group II	0.289±0.047	0.736±0.061	0.447	18.45	<0.001
Group III	0.306±0.040	0.750±0.062	0.444	18.97	<0.001

CONCLUSION

Our result showed fluctuation in the levels of sAA in saliva. At the same time, the fluctuation in the levels of sAA in saliva was also comparable with the fluctuation in sAA levels in plasma at all time intervals. As collection of plasma sAA is an invasive method, patients may develop anxiety because of fear of withdrawing blood and may give false values, compared to saliva which is a noninvasive method and a reliable indicator of sAA. As the sample size in this study is too small, our study does not allow us to explore all the prognostic implications of sAA.

To our knowledge, this is the first evidence that oral SCC patients exhibit significant changes in the morning, afternoon, and night secretion of sAA compared with healthy controls and patients with premalignant diseases.

Hence, we can conclude that salivary sAA levels can be taken as a predictable as well as reproducible marker for oral SCC or premalignant lesions.

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

Nil.

Conflicts of interest

There are no conflicts of interest.

Table 2

Plasma α-amylase level (mean±standard deviation) of three groups over the periods

Period	Group I (n=10)	Group II (n=10)	Group III (n=10)
Morning	0.090±0.046	0.224±0.048	0.297±0.048
Afternoon	0.125±0.049	0.252±0.038	0.284±0.043
Night	0.160±0.055	0.289±0.047	0.306±0.040

Table 3

Saliva α-amylase level (mean±standard deviation) of three groups over the periods

Period	Group I (n=10)	Group II (n=10)	Group III (n=10)
Morning	0.178±0.071	0.596±0.071	0.705±0.050
Afternoon	0.238±0.068	0.695±0.067	0.758±0.043
Night	0.293±0.072	0.736±0.061	0.750±0.062

Table 4

Comparison of α-amylase level (mean±standard deviation) of three groups in blood and saliva at morning by Student's t-test

Group	Plasma (n=10)	Saliva (n=10)	Mean difference	t	P
Group I	0.090±0.046	0.178±0.071	0.088	3.27	0.004
Group II	0.224±0.048	0.596±0.071	0.372	13.74	<0.001
Group III	0.297±0.048	0.705±0.050	0.408	18.58	<0.001

Table 5

Comparison of α-amylase level (mean±standard deviation) of three groups in plasma and saliva at afternoon by Student's t-test

Group	Plasma (n=10)	Saliva (n=10)	Mean difference	t	P
Group I	0.125±0.049	0.238±0.068	0.114	4.25	<0.001
Group II	0.252±0.038	0.695±0.067	0.443	18.24	<0.001
Group III	0.284±0.043	0.758±0.043	0.473	24.83	<0.001