Comparative Study on Saliva Proteins in Patients of Brain Tumors and Healthy Individuals
In the setting of neoplastic conditions, many biochemical
substances deviate from their normal values; these may include proteins
of body fluid. To verify this claim a prospective study was carried out
to investigate the values of plasma proteins in salivary fluids in patients
with primary brain tumors. This study had been conducted between September
2008 and July 2009 on 160 individuals. The qualified subjects for the
study included, 80 patients (40 from each sex) with primary brain tumor
and eighty age and sex normal subjects were used as control for salivary
and serum proteins measurements. Concentrations of proteins were found
higher in saliva and blood of male and female patients as compared with
normal individuals. These results might suggest possible preliminary detection
of primary brain tumor by observing the increase of protein concentration
in the saliva of the patient which is easily carried out in clinical laboratory.
Proteins play a central role in cell function and cell structure, in this respect serum contains a mixture of proteins differing in origin and function, the amount of proteins in the vascular compartment depends on the balance between the rate of synthesis and the rate of catabolism or loss. It is a well established and evidence-based fact that plasma proteins levels may suffer changes during a neoplastic disease process (Suzuki, 2006).
Saliva is a glandular secretion, which constantly bathes the teeth and oral
mucosa to ensure the stability of the oral cavity environment. The volume of
saliva is secreted by the major salivary glands; the rest is secreted by the
minor salivary glands, which are scattered in the sub-mucosa in different parts
of the oral cavity. Saliva is constituted by secretions of three paired major
salivary glands: submandibular (70%), parotid (25%) and sublingual (5%), together
with minor salivary glands (Rosen and Bailey, 2001). The daily secretion of
saliva ranges normally between 800-1500 mL day-1, about 0.5 mL is
secreted each minute all the time except during sleep where it become very little
(Guyton and Hall, 2000). Saliva is a readily available specimen, which can be
collected by noninvasive procedures, the scientific and biomedical research
have provided improved methods to measure and monitor the levels of ions, chemicals
and molecules in saliva as well as all body fluids, in this respect the value
of saliva as an indicator for systemic disease had been explored and found application
in clinical diagnosis (Hofman, 2001; Ginzkey et al., 2009). It is worth
to mention that saliva had been used to monitor levels of endogenous material
as antibodies, e.g., IgA in hepatitis A virus infection, immunoglobulins in
lymphomas (Kazmi et al., 2006; Pink et al., 2009), also DNA and
nitrites levels had been studied in saliva of patients with cancer of gastrointestinal
tract (Tenovou, 1986); moreover hormones were also analyzed in saliva (Chang
et al., 2005).
Primary Brain Tumors (PBT) originate in the brain and can be benign (slow growing) or malignant (fast growing), these tumors can vary in malignancy but even so-called benign tumors are commonly lethal because of their infiltrating properties and their tendency to undergo malignant transformation over time (Behin et al., 2003; Doolittle, 2004). Brain tumors are the second leading cause of cancer death in children under 15 years and young adults up to the age of 34 years. These tumors are also the second fastest growing cause of cancer death among humans older than 65 years (Xie and Zhu, 2009). Recent reports published by Jordans Ministry of Health indicated that 196 new brain cancer cases were recorded during 2006, of these 138 cases were Jordanians and 58 cases for non Jordanians; during 2007 the number of new cases of brain tumors increased to 237, of these 142 Jordanians and 95 non Jordanians (Taraweneh and Nimri, 2006, 2007). Primary brain tumors have devastating consequences and induced clinical signs and symptoms can result from the mechanical effect of the tumor and/or chemical stimulation/signaling induced by the tumor. Thus, the aim of the study is to investigate changes which might occur in the level of salivary proteins in the setting of PBT.
MATERIALS AND METHODS
Patients and Normal Individuals
This study had been carried out between September 2008 and July 2009 at
both Public and Private Hospitals in Amman, Jordan. Patients were evaluated
by full medical history to exclude any existing systemic disease that may affect
the parameters to be diagnosed, particularly diabetes, liver disease, renal
disease and chronic drug intake, such a patient was excluded from the study.
One hundred sixty individuals qualified for the study, they include: 80 patients
(40 for each sex) diagnosed by clinical and histological means as having a primary
brain tumor, their age ranged between 2-75 years; gender: 40 males and 40 females.
Control groups composed of age- and sex- matched normal subjects (40 for each
Serum and Saliva Collection and Preparation
Five to ten milliliters of venous blood were aspirated through venepuncture
following an overnight (about 10 h) of fasting. Unstimulated whole saliva was
collected after the patient and normal individuals have rinsed their mouths
several times with deionized water, then the accumulated saliva in the floor
of the mouth under the tongue (Nishanian et al., 1998) was drawn by a
plastic disposable pipette, collection time was always between 8.00-9.00 am
(before surgery). The collected saliva was cold centrifuged at 2500 rpm for
10 min at 5°C; this was done within one hour after collection to eliminate
debris and cellular matter. The centrifuged supernatants were divided into 5
equal parts. All sample were stored frozen at (-20°C) in polyethylene tubes
till assayed at the laboratories of Research Centre at Zarka University College.
Total Protein Determination
The total protein was measured by using kit which was obtained from TECO
Diagnostics Laboratory (Anaheim, CA 92807, USA). The determination of total
protein in serum makes use of the Biuret color reaction. The present method
for quantitative determination of total protein in serum is based on the method
proposed by the American Association for Clinical Chemistry (Doumas et al.,
1981a, b) and National Committee for Clinical Laboratory Standards (1979). The
principle of the method is depended on the enzymatic reaction sequence employed
in the assay of total protein.
Data were calculated and interred into a computerized data base structure.
Statistical analyses were done by using SPSS (Statistical Package for Social
Sciences). The mean and standard deviations (±SD) were calculated. The
difference in mean of normally distributed variables between two groups was
assessed by independent samples t-test.
In this study patients were subjected to clinical and histological diagnosis, the obtained results confirmed that 80 patients as having a PBT, their age ranged between 2-75 years; gender of patients were 40 males and 40 females. Protein concentrations were measured in each samples of saliva and serum obtained from patients and age-sex matched samples which were obtained from normal subjects as controls.
Protein Concentrations in Saliva of Male Patients
The measurements of saliva proteins (g dL-1) in both male patients
and male normal individual showed consistent increase in protein concentrations
in saliva of male patients in comparison with normal individuals. The range
of protein concentrations in PBT patients were between 0.89 and 2.8 g dL-1,
whereas in controls protein concentrations were in the range 0.24 to 0.57 g
dL-1 (Fig. 1).
Protein Concentrations in Serum of Male Patients
The obtained results indicated that the values of protein concentrations
in serum of male patients and control group (Fig. 2), were
higher than the values of protein concentrations in saliva of male patients
and control group (Fig. 1). But the results indicated that
concentrations of proteins were still higher in patients as compared with control
group in both saliva and serum. The range of protein concentrations in PBT were
between 7.75 and 11.5 g dL-1, whereas in controls protein concentrations
were in the range 5.88 to 7.15 g dL-1.
||Concentrations of saliva proteins in both groups of males,
PBT patients and normal individuals
||Serum protein concentrations in both groups of males PBT patients
and normal individuals
||Saliva protein concentrations in both groups of females PBT
patients and normal individuals
Protein Concentration in Saliva of Female Patients
The values of protein concentrations in saliva obtained from female normal
individuals and patients were comparable to that obtained in normal male individuals
and PBT male patients. The range of protein concentrations in PBT patients were
between 0.77 and 2.18 g dL-1, whereas in controls were in the range
0.29 to 0.58 g dL-1 (Fig. 3).
Protein Concentration in Female Serum
The results of the protein concentrations in the serum of female PBT patients
and normal subjects indicated almost similar pattern of protein concentrations
obtained in male PBT patients and controls. The range of protein concentrations
in PBT patients were between 7.5 and 10.5 g dL-1, whereas in controls
protein concentrations were in the range 4.89 to 6.4 g dL-1 (Fig.
Mean Protein Concentrations in Serum and Saliva
The study revealed that the mean values of protein concentrations of saliva
in the brain tumor group were higher than those in the healthy subjects with
statistical significance (p<0.1). The mean values of protein concentrations
in saliva of male and female patients were other hand, the mean values of protein
concentrations in serum of male and female 1.76±0.56 and 1.64±0.42
g dL-1, respectively.
||Serum protein concentrations in both groups of females PBT
patients and normal individuals
||Mean concentrations of proteins for both healthy and PBT patients
groups. ♂P, ♀P, ♂N ♀N indicate male patients , female
patients, normal male and normal female, respectively
The values obtained in normal male and female individuals which were 0.46±0.11
and 0.44±0.08 g dL-1, respectively. On the individuals were
9.4±1.01 and 8.94±0.93 g dL-1, respectively. The values
obtained in normal male and female individuals which were 6.39±0.41 and
5.7±0.46 g dL-1, respectively (Fig. 5).
Several types of cancers of the brain and Central Nervous System (CNS) have been reported among Jordanian patients. Brain tumors are the most frequent type of these tumors, there were 96.7 and 93.3% of all brain and nervous system tumors in males and females, respectively. The median age at diagnosis is 35 years. In 2007, there were 142 cases accounting 3.3% of all newly diagnosed malignant cancers among Jordanians. These cancers ranked the 8th in males and females and affected 82 males ( 4.0%) and 60 females (2.6%) with a male to female ratio of 1.4:1. Cancer incidence in Jordan is increasing at substantial rate, in this respect new recorded cases of brain tumors in 2007 increased by 17.3% as compared with new cases registered during 2006 (Taraweneh and Nimri, 2006, 2007).
Brain tumors are currently classified according to their grade (how quickly they may grow) and the type of cells they are composed of. This system, however, is not always accurate and sometimes two tumors that appear to be identical under the microscope will have very different growth patterns and responses to treatment. On the other hand the molecular classification system is based on molecular genomics and proteomics may be used to better predict a given tumor's behavior and response to therapy. Research in the field of molecular diagnosis is directed toward exploring the possibility of using genomic biomarkers (Hainfellner, 2009) and cytogenetic analysis which included interphase fluorescence in situ hybridization (Korshunov et al., 2008); others explored mass spectrometry and expression microarray profiling to identify candidate protein and mRNA biomarkers of various tumors (Hu et al., 2007). These methods are time consuming and expensive. Thus, it would useful to ascertain simple basic inexpensive test for diagnosis of PBT. In this study it was possible to show that estimation of protein concentrations in saliva can give preliminary indication of PBT. A highly significant increase in total protein levels in serum and saliva of primary brain tumor patients was noticed when compared to that of normal subjects. This increase could be explained on the basis that the whole body of cancer patients is engaged in protein synthesis of various forms like: C-reactive, proteins, tumor markers, enzymes and immunoglobulin and other proteins material. Present results are in agreement with the results of patients with oral cancers (Al-Rawi et al., 2005; Kashmoola et al., 2001), their results had shown that patients with oral squamous cell carcinoma had also markedly increased salivary total protein concentrations; furthermore they indicated that elevation in saliva total protein, may be due to increasing of salivary IgG and IgM. Other study grouped several plasma proteins as Acute Phase Reactant (APR), which significantly rises during inflammation and neoplasm (Schreiber et al., 1982).
It is worth mentioned that in comparison with the reported results in present work, molecular detection of cancer requires more sophisticated proteomic and genomic diagnostic assays as well as trained and skilled personnel, beside specialized equipments. In this respect DNA/RNA analysis is one possible way to develop effective molecular diagnostic assay (Hilton and Melling, 2004), on the other hand proteomics assays are considered promising to elucidate protein changes between healthy and diseased states (Colantonio and Chan, 2005).
The measurement of higher protein values in saliva of PBT patients as compared with healthy individuals might be considered a preliminary indication of diagnosis of PBT, which is easily carried out in clinical laboratory.
The authors are grateful and express their appreciation to Al-Balqa Applied University, Zarka University College and to Royal Scientific Society for facilitating and supporting this research.
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