|Year : 2010 | Volume
| Issue : 3 | Page : 278-281
Estimation of salivary protein thiols and total antioxidant power of saliva in brain tumor patients
HR Suma1, Krishnananda Prabhu1, Revathi P Shenoy1, Raja Annaswamy2, Suryanarayana Rao2, Anjali Rao1
1 Department of Biochemistry, Kasturba Medical College and Hospital, Manipal University, Manipal - 576 104, Karnataka, India
2 Department of Neurology and Neurosurgery, Kasturba Medical College and Hospital, Manipal University, Manipal - 576 104, Karnataka, India
|Date of Web Publication||29-Nov-2010|
Department of Biochemistry, Kasturba Medical College, Manipal - 576104, Karnataka
Source of Support: None, Conflict of Interest: None
Background: The role of oxidative stress in the genesis of various types of cancers is well established. Several chemical, cell culture and animal studies also indicate that antioxidants may slow or even prevent the development of cancer. Brain is considered abnormally sensitive to oxidative damage as brain tissue has high rate of oxygen consumption, high lipid content and relatively low antioxidant defenses, compared to other tissues.
Materials and Methods: The study design chosen for the present study was cross sectional. The biochemical parameters that were estimated in saliva manually using spectrophotometric methods were ferric reducing antioxidant power (FRAP) assay - a direct measure of total antioxidant activity of biological fluids and protein thiols. The physical parameters of saliva that were also assessed were salivary flow rate, pH of the saliva and the osmolality of the saliva.
Results: The mean values of salivary flow rate and pH were significantly decreased among malignant and benign tumor patients whereas the salivary osmolality was significantly increased in both the groups of patients. The mean values of salivary FRAP were significantly reduced among malignant and benign tumor patients. However, the salivary protein thiols were significantly increased in these patients.
Conclusion: Hence with these observations it can be concluded that in saliva, besides the physical characteristics, salivary FRAP and protein thiol levels are appropriate indicators of the antioxidant status in brain tumor patients.
Keywords: Brain tumors, protein thiols, saliva, total antioxidant power
|How to cite this article:|
Suma H R, Prabhu K, Shenoy RP, Annaswamy R, Rao S, Rao A. Estimation of salivary protein thiols and total antioxidant power of saliva in brain tumor patients. J Can Res Ther 2010;6:278-81
|How to cite this URL:|
Suma H R, Prabhu K, Shenoy RP, Annaswamy R, Rao S, Rao A. Estimation of salivary protein thiols and total antioxidant power of saliva in brain tumor patients. J Can Res Ther [serial online] 2010 [cited 2021 Jan 25];6:278-81. Available from: https://www.cancerjournal.net/text.asp?2010/6/3/278/73357
| > Introduction|| |
Various studies have been conducted to emphasize the role of oxidative stress among the patients of different cancers, including brain tumors. ,,, Many different oxidative stress markers have been analyzed, both in serum and saliva of cancer patients. ,, Repeated analysis of these markers of oxidative stress may be needed at various stages viz. preoperative, post-operative and during the follow up, to establish the exact role of oxidative stress and antioxidants in these patients. Many of these analytes have been studied in serum, repeated collection of which is an invasive technique. Thus, salivary estimation of these parameters may be helpful as the repeated collection of saliva is easy, cost effective and noninvasive. , Moreover, determination of saliva constituent levels represents a putatively reasonable approach to the evaluation of a patient's risk for disease occurrence, intensity and prognosis. ,
Hence, the main aim of the present study has been to know whether the estimation of total antioxidant capacity and protein thiol levels in saliva can be used for assessing oxidative status and to predict the prognosis of the disease in patients suffering from brain tumors.
| > Materials and Methods|| |
The study design chosen for the present study was cross sectional. Patients with brain tumors getting admitted to a tertiary care hospital were taken as the study group during the period July 2007 to August 2008 (convenient sampling). Preoperative patients with provisionally diagnosed brain tumor (age group between 18 and 80 years) constituted the study group. No drugs had been administered to the patients at the time of collection of saliva samples. Age and sex matched healthy persons who came for a routine health check-up to the hospital were included in the study. Both cases and controls with unhealthy oral cavity were excluded. The study was carried out on 60 brain tumor patients preoperatively confirmed by diagnosis based on the clinical features and neuroimaging diagnostic techniques and 42 normal controls. Postoperatively, brain tumor patients were categorized later as benign and malignant based on the histopathology report of the tumor [Table 1]. Consent form and questionnaire was filled by all the subjects. The protocol of the study was approved by the Institutional ethical committee.
The subjects were instructed not to eat, drink or smoke for 1 h before sampling. Unstimulated whole saliva  was collected, after allowing saliva to pool in the floor of the mouth for 5 min by leaning forward and letting saliva drain into a graded sampling tube. Since the salivary composition, flow rate, viscosity and pH show diurnal variations,  a specific time was fixed for sample collection - 9.00 am to 12.00 am. Immediately after collection, the samples were analyzed (within 4-6 h, meanwhile it was stored on crushed ice). After the total volume of saliva collected in 5 min was estimated using calibrated tube, the sample was taken in eppendorf tube and centrifuged for 10 min at 8000 rpm. The supernatant, free of debris was used for analysis of the parameters.
The following biochemical parameters were estimated in saliva using spectrophotometric methods :
- Ferric reducing antioxidant power (FRAP) assay - a direct measure of total antioxidant activity of biological fluids.
- Protein thiols.
The following physical characteristics of saliva were also assessed:
- Salivary flow rate. ,
- pH of saliva.
- Osmolality of saliva.
The entire quantity of saliva collected for 5 min duration was transferred into the small calibrated tube and the volume was measured. The flow rate was estimated by calculating saliva volume per minute and expressed as ml/min. pH of the saliva was determined by dipping the pH paper strips and comparing the color change with that of the standard color bar provided. Fifty microliter saliva supernatant was taken in a small eppendorf tube and the osmolality was measured using cryoscopic osmometer (Osmomat 030, Gonotec GmBH, Germany) and expressed as mOsmol/kg of saliva.
At low pH, reduction of a ferric tripyridyltriazine (Fe III -TPTZ) complex to the ferrous form, which has an intense blue color, can be monitored by measuring the change in absorption at 593nm. The change in absorbance is directly related to the combined or "total" reducing power of the electron donating antioxidants present in the reaction mixture.  Salivary protein thiol was measured by spectrophotometric method using di thionitrobenzene (DTNB)-Ellman's method.  Ellman's reagent or 5,5'-dithiobis (2-nitrobenzoate, DTNB) is a symmetrical aryl disulfide which readily undergoes the thiol-disulfide interchange reaction in the presence of a free thiol.  The TNB dianion has a relatively intense absorbance at 412nm compared to both disulfides. The protein thiol concentration in saliva was determined by using the molar extinction coefficient of the TNB complex in the assay mixture at 412nm obtained after using known standard concentrations and their absorbance values.
Statistical analysis for comparison of means among the groups was done by ANOVA. Except FRAP, with respect to other parameters there was no homogeneity of variance and hence inter-group comparison of means (ANOVA) was done using post hoc test in case of FRAP and Tamhane test for rest of the parameters (SPSS version 16).
| > Results|| |
The study included 102 individuals of whom, 42 were healthy controls, 32 were patients of benign brain tumor and 28 were patients of malignant brain tumor. The demographic parameters and their distribution within the groups are shown in [Table 1]. The mean values of salivary flow rate were significantly decreased among malignant (P< 0.0001) and benign (P < 0.0001) tumor patients. But there was no significant difference between benign and malignant groups, though the levels were slightly lower in malignant group [Table 2]. Similarly, the mean values of salivary pH were significantly decreased among malignant (P < 0.0001) and benign (P = 0.002) tumor patients. But there was no significant difference between benign and malignant groups, though the levels were slightly lower in malignant group [Table 2]. The mean values of salivary osmolality were significantly increased among malignant (P= 0.003) and benign (P < 0.0001) tumor patients. But there was no significant difference between benign and malignant groups [Table 2].
|Table 2: Comparison of physical characteristics of saliva in brain tumor patients as compared to controls#, Mean ± SEM|
Click here to view
The mean values of salivary FRAP were significantly reduced among malignant (P = 0.004) and benign (P < 0.0001) tumor patients. But there was no significant difference between benign and malignant groups [Table 4]. The mean values of salivary protein thiols were significantly increased among malignant (P = 0.002) and benign (P = 0.011) tumor patients. However, there was no significant difference between benign and malignant groups [Table 3].
|Table 3: Comparison of FRAP and protein thiol levels in saliva of brain tumor patients and controls,# Mean ± SEM|
Click here to view
| > Discussion|| |
Brain tumors are uncommon but represent lethal form of cancers. Generation of reactive oxygen species (ROS) plays an important role in maintaining cancer phenotype due to their stimulating effects on cell growth and proliferation. Formation of brain tumors involves an accumulation of lesions in genes important for the regulation of cell proliferation, differentiation and death. ,,
In our laboratory, various antioxidant levels have been assessed ,,,, previously and altered levels have been noticed. Altered levels of RBC glutathione and serum glutathione-S transferase  and significant reduction in the RBC glutathione reductase activity was observed in most type of brain tumor patients.  Significantly elevated concentration of lipid peroxidation products had also been noticed in most type of intracranial neoplasms indicating increased susceptibility to free radical attack in these cases.  Plasma antioxidant vitamin levels were also studied but no significant difference had been observed.  Levels of protein thiols and protein carbonyls in serum of brain tumor patients have been studied as an indicator of protein damage due to oxidative stress and significant alterations in their levels were observed in these patients.  As an indicator of the burden of tumor growth (altered cellular antioxidant status, higher susceptibility of DNA to ROS), the in vitro susceptibility to radiation-induced DNA damage of peripheral lymphocytes had been studied in the brain tumor patients and significant results have been noticed. 
Salivary flow rate has been observed to influence composition of saliva by affecting secretion and absorption in the ducts of the gland, including concentration of electrolytes, mucins etc, which in turn affect the osmolality and pH of saliva.  The salivary flow rate itself is affected by various factors like drugs, therapeutic irradiation, systemic diseases, psychogenic disorders like depression, old age. 
In the present study, salivary flow rate in the tumor patients was significantly decreased when compared to controls. It could be due to the diseased condition itself. Salivary pH was observed to be significantly less among the tumor patients when compared to healthy controls which could be because of decreased flow rate among the patients which in turn affects the various ion concentrations of the saliva. It has been observed in various studies, that the salivary pH increases with the increase in the flow rate (because of increased bicarbonate levels, probably due to influence of metabolically active gland cells. , Salivary osmolality depends on composition of the saliva including presence of various electrolytes and mucins. It has been observed in various studies that with the altered flow rates, the composition of the saliva also gets altered. , In the present study, there is a significant increase in the osmolality observed in brain tumor patients as the salivary flow rate is also markedly decreased.
FRAP levels indicate the total antioxidant capacity except the antioxidant activity contributed by the protein thiols.  FRAP levels have exhibited a significant decrease in periodontitis (salivary FRAP),  thyroid cancer (plasma FRAP)  and colorectal cancer (urinary FRAP)  indicating reduced total antioxidant capacity and increased oxidative stress in such cases. In the present study, the salivary FRAP levels are significantly decreased in the brain tumor patients, though there is no significant difference among the benign and malignant groups. This indicates that salivary FRAP is an effective indicator of the total antioxidant power. Hence, it could be used for repeated analysis of oxidative stress in brain tumor patients.
S-thiolated proteins (mixed disulfides of proteins and low molecular weight thiols) are very early products of protein oxidation during oxidative stress, occurring within few seconds after generation of oxygen radicals. As a result, assessment of the extent and specificity of this process during oxidative stress is one of the best measures of primary effects of oxygen radicals. S-thiolation has been correlated with an alteration in protein function in many cases. Hence, the estimation of salivary protein thiol levels indicates the oxidative stress status. ,, Many studies have observed altered protein thiols including alterations in the glutathione levels in various disease conditions with oxidative stress. , In this study, the salivary protein thiols were significantly increased in the brain tumor patients when compared to healthy controls, though the levels did not show any difference between benign and malignant brain tumor patients. Similar reports were available in studies on salivary and serum antioxidants levels (peroxidase, superoxide dismutase, salivary total antioxidant status) in type 1 diabetes mellitus patients where increased antioxidant levels were observed indicating oxidative stress.  In the oral cavity, albumin is regarded as a serum ultra filtrate to the mouth and it may also diffuse into the mucosal secretions.  Thus, an increased turnover of thiols probably counteracts the ROS milieu prevalent in the above cases in this study.
Hence, with this observation it can be concluded that in saliva besides the physical characteristics, salivary FRAP and protein thiol levels are appropriate indicators of oxidative stress in brain tumor patients. Further, salivary analysis could prove to be a noninvasive, patient friendly technique to assess the antioxidant status in these cases.
| > References|| |
|1.||Smita M, Naidu K, Suryakar AN, Swami SC, Katkam RV, Kumbar KM. Oxidative stress and antioxidant status in cervical cancer patients. Indian J Clin Biochem 2007;22:140-4. |
|2.||Rao GM, Rao AV, Raja A, Rao S, Rao A. Role of antioxidant enzymes in brain tumours. Clin Chim Acta 2000;296:203-12. |
|3.||Kwon D, Yoon S, Carter O, Bailey G, Dashwood RH. Antioxidant and antigenotoxic activities of Angelica keiskei, Oenanthe javanica and Brassica oleracea in the salmonella mutagenicity assay and in HCT 116 human colon cancer cells. Biofactors 2006;26:231-44. |
|4.||Rao GM, Rao AV, Raja A, Rao S, Rao A. Lipid peroxidation in brain tumours. Clin Chim Acta 2000;302:205-11. |
|5.||Chittiprol S, Arathi, Bhat S, Nair S, Ponduri M, Rao A. Erythrocyte glutathione and serum glutathione-S-transferase in patients with brain tumors. Clin Chim Acta 2003;333:97-9. |
|6.||Bahar G, Feinmesser R, Shpitzer T, Popovtzer A, Nagler RM. Salivary analysis in oral cancer patients - DNA and protein oxidation, reactive nitrogen species and antioxidant profile. Cancer 2007;109:54-9. |
|7.||Alturfan EE, Demir G, Kasikel E, Akbay TT, Pisiriciler R, Caliskan E, et al. Altered biochemical parameters in the saliva of patients with breast cancer. Tohoku J Exp Med 2008;214:89-96. |
|8.||Wong DT. Salivary diagnostics powered by nanotechnologies, proteomics and genomics. J Am Dent Assoc 2006;137:313-21. |
|9.||Hofman LF. Human saliva as a diagnostic specimen. J Nutr 2001;131:1621S-5S. |
|10.||Rantonen P. Salivary flow and composition in healthy and diseased adults [dissertation]. Helsinki: University of Helsinki; 2003. |
|11.||Mashayekhi F, Agha HF, Rezale A, Mohammed JZ, Khorasani R, Abdollahi M. Alteration of cyclic nucleotide levels and oxidative stress in saliva of human subjects with periodontitis. J Contemp Dent Pract 2005;6:46-53. |
|12.||Dawes C. Circadian rhythms in human salivary flow rate and composition. J Physiol 1972;220:529-45. |
|13.||Sreebny LM. Saliva in health and disease: An appraisal and update. Int Dent J 2000;50:140-61. |
|14.||Benzie IF, Strain JJ. The ferric reducing ability of plasma as a measure of "antioxidant power": FRAP assay. Anal Biochem 1996;239:70-6. |
|15.||Paul AA, Motchnik B, Frei B, Ames N. Measurement of antioxidants in human blood plasma. Methods Enzymol 1994;234:269-78. |
|16.||Wilson JM, Robert, Bayer RJ, Hupe DJ. Structure-reactivity correlations for the thiol-disulfide interchange reaction. J Am Chem Soc 1977;99:7922-6. |
|17.||Droge W. Free radicals in the physiological control of cell function. Physiol Rev 2002;82:47-95. |
|18.||Dreher D, Junod AF. Role of oxygen free radicals in cancer development. Eur J Cancer 1996;32A:30-8. |
|19.||Hussain SP, Aguilar F, Amstad P, Cerutti P. Oxygen-radical induced mutagenesis of hotspot codons 248 and 249 of the human p53 gene. Oncogene 1994;9:2277-81. |
|20.||Rao GM, Rao AV, Raja A, Rao S, Rao A. Plasma antioxidant vitamins in brain tumors. Neurol India 2003;51:220-2. |
|21.||Kumar P, Devi U, Ali S, Upadhyay R, Pillai S, Raja A, et al. Plasma protein oxidation in brain tumors. Neurol Res 2008;31:270-3. |
|22.||Kalthur G, Kumar P, Devi U, Ali S, Upadhyay R, Pillai S, et al. Susceptibility of peripheral lymphocytes of brain tumor patients to in vitro radiation - induced DNA damage, a preliminary study. Clin Exp Med 2008;8:147-50. |
|23.||Edgar WM. Saliva: Its secretion, composition and function. Br Dent J 1992;172:305-12. |
|24.||Jenkins GN. The physiology and biochemistry of the mouth. 4 th ed. Philadelphia: Blackwell Scientific Publications; 1978. |
|25.||Cao G, Prior RL. Comparison of different analytical methods for assessing total antioxidant capacity of human serum. Clin Chem 1998;44:1309-15. |
|26.||Dardono A, Ghiadoni L, Plantinga Y, Caraccio N, Bemi A, Duranti E, et al. Recombinant human thyrotropin reduces endothelial - dependant vasodilatation in patients monitered for differentiated thyroid cancer. J Clin Endocrinol Metab 2006;91:4175-8. |
|27.||Chandramathi S, Suresh K, Anita ZB, Kuppuswamy UR. Comparative assessment of urinary oxidative indices in breast and colorectal cancer patients. J Cancer Res Clin Oncol 2009;135:319-23. |
|28.||Thomas JA, Chai YC, Jung CH. Protein S- thiolation and dethiolation. Methods Enzymol 1994;233:385-95. |
|29.||Hu M. Measurement of protein thiol groups and glutathione in plasma. Methods Enzymol 1994;233:381-5. |
|30.||Karthikeyan K, Sinha I, Prabhu K, Bhaskarananda N, Rao A. Plasma protein thiols and total antioxidant power in pediatric nephrotic syndrome. Nephron Clin Pract 2008;110:c10-4. |
|31.||Prakash M, Upadhyay S, Prabhu R. Protein thiol oxidation and lipid peroxidation in patients with ureamia. Scand J Clin Lab Invest 2004;64:599-604. |
|32.||Reznick AZ, Shehadeh N, Shafir Y, Nagler RM. Free radicals related effects and antioxidants in saliva and serum of adolescents with type 1 diabetes mellitus. Arch Oral Biol 2006;51:641-8. |
|33.||Oppenheim FG. Priliminary observations on the presence and origin of serum albumin in human saliva. Helv Odontol Acta 1970;14:10-7. |
[Table 1], [Table 2], [Table 3]