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Year : 2015  |  Volume : 11  |  Issue : 1  |  Page : 146-149

Status of trace elements in saliva of oral precancer and oral cancer patients

1 Department of Oral Medicine and Radiology, AB Shetty memorial Institute of Dental Science, Mangalore, Karnataka, India
2 Department of Biochemistry, KS Hegde Medical Academy, Nitte University, Mangalore, Karnataka, India
3 Department of Oral Pathology, AB Shetty memorial Institute of Dental Science, Mangalore, Karnataka, India
4 Department of Oral Surgery, AB Shetty memorial Institute of Dental Science, Mangalore, Karnataka, India

Date of Web Publication16-Apr-2015

Correspondence Address:
Shishir Ram Shetty
Department of Oral Medicine and Radiology, AB Shetty Memorial Institute of Dental, Sciences, Nitte University, Mangalore
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0973-1482.137973

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 > Abstract 

Background: Several studies in recent years have linked association between micronutrient levels and various forms of cancer. Copper and zinc have been the most researched micronutrients.
Aim: The aim of the study was to evaluate the levels of copper, zinc and iron in saliva of patients with oral leukoplakia, oral submucous fibrosis and oral squamous cell carcinoma.
Results: There was a highly significant increase in the level of salivary copper in oral submucous fibrosis patients when compared to controls (P = 0.001). Salivary copper levels were also elevated in oral leukoplakia and oral cancer patients (P = 0.01). There was a significant decrease in the salivary zinc levels in all three study groups when compared to controls (P = 0.001). A highly significant reduction in salivary iron levels was noticed oral submucous fibrosis group. The copper to zinc ratio significantly increased in all the study groups when compared to controls.
Conclusion: Results suggest that salivary copper zinc and iron could be used as biomarkers for oral precancer and cancer.

Keywords: Copper, iron, leukoplakia, oral squamous cell carcinoma, saliva, zinc

How to cite this article:
Shetty SR, Babu S, Kumari S, Shetty P, Hegde S, Karikal A. Status of trace elements in saliva of oral precancer and oral cancer patients. J Can Res Ther 2015;11:146-9

How to cite this URL:
Shetty SR, Babu S, Kumari S, Shetty P, Hegde S, Karikal A. Status of trace elements in saliva of oral precancer and oral cancer patients. J Can Res Ther [serial online] 2015 [cited 2021 Nov 28];11:146-9. Available from: https://www.cancerjournal.net/text.asp?2015/11/1/146/137973

 > Introduction Top

0 India has one of the highest incidences of oral cancer in the world. [1] The development of cancer is a multistep process arising from pre-existing potentially malignant lesions. Oral leukoplakia (OL) is the most common precancer representing 85% of such lesions. [2] Alcohol, viruses, genetic mechanisms, candida and chronic irritation have modifying effects in the etiology of oral cancer. [2] Trace elements are regarded as versatile anti-cancer agents that regulate various biological mechanisms. [3] Many researchers have observed association between trace elements and cancer mortality. [4] Decrease in contents of Copper (Cu) and Zinc (Zn) in the blood of patients with head and neck cancer. [5],[6] Most of the molecules that are found in the body fluids like blood and urine are also found in the saliva, although in lesser concentration thus making saliva an important diagnostic tool. [7] The present study was conducted to evaluate the levels of Cu Zn and Iron (Fe) in oral potentially malignant disorders and oral squamous cell carcinoma.

 > Materials and methods Top

0 Two hundred patients reporting to the department of Oral medicine and Radiology were enrolled into a cross sectional case control study over a period 3 years from August 2009 to August 2012. Ethical clearance for the study was obtained from the institutional ethical committee. They were divided into four equal study groups with 50 subjects each. The first study group OL consisted of 50 patients of histopathologically confirmed cases of oral leukoplakia (45 cases of homogenous leukoplakia and 5 cases of speckled leukoplakia). The second study group consisted of 50 histopathologically confirmed patients with oral submucous fibrosis (OSMF). The third study group consisted of 50 histopathologically confirmed cases of oral squamous cell carcinoma (OSSC). The fourth study group consisted of 50 age and gender matched healthy controls (HC). Detailed history and clinical examination was done for each of the study subjects. After informed consent 5 ml of unstimulated saliva was collected from each of the patient using the spit method. Standard pre-investigative protocol (no consumption of food/no smoking for an hour before saliva collection) for saliva collection was followed which was carried out between 9 and 11 A.M. [8] The saliva was centrifuged at 2500 rpm for 10 min at 0-5°C. The supernatants were stored frozen at −20°C until time of analysis. For the analysis of salivary Cu and Zn levels a series of standard Cu solutions were made raging from 1 to 10 ppm. The unknown samples (study samples) were analysed in comparison to the standardized Cu solutions using the GBC Avanta atom absorption spectrophotometer [Figure 1]. Similar procedure was followed for estimation of salivary Zn. Salivary Fe was measured by the Bathophenanthroline method. 100 μL of the saliva sample was taken in a clean microfuge tube and made upto 250μL with the addition of deionised water. 500 μL of protein precipitating solution was added to the diluted sample. The mixture was then centrifuged at 2000 rpm for 10 min. 500 μL of the supernatant was then added to 500 μL of chromogen solution (25 mg Bathophenanthroline sulfonate dissolved in 100 mL of sodium acetate (2M). The optical density of the pink colour formed was read immediately (within 10 min) at 535 nm against a blank treated in a similar method as the test wherein the sample was replaced with deionised water. The concentration of iron in the sample was deduced by plotting the optical densities of the test against the standard graph. The concentration that obtained was multiplied by the dilution factor (2.5). The data were statically analyzed by the ANOVA and HSD Turkey's test using the SPSS version 17 software.
Figure 1: Laboratory procedures (atom absorption spectrophotometry)

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 > Results Top

0 The mean salivary Cu levels in group HC, OSMF, OL and OSCC were 46.07 ± 4.56 μg/dL, 87.45 ± 2.67 μg/dL, 55.54 ± 2.57 μg/dL and 57.87 ± 4.98 μg/dL, respectively [Figure 2]. There was a highly significant (P = 0.001) increase in the levels of Cu in the OSMF group when compared to controls. The Cu levels were significantly elevated (P = 0.01) in the OL and OSCC groups when compared to HC. However there was no statistically significant difference (P > 0.05) when the groups OL and OSCC were intercompared. The mean salivary Zn levels in group HC, OSMF, OL and OSCC were 35.78 ± 3.97 μg/dL, 24.67 ± 4.86 μg/dL, 27.33 ± 3.05 μg/dL and 15.23 ± 2.93 μg/dL, respectively. There was a highly significant reduction (P = 0.001) in the salivary Zn levels in all three study groups OSMF, OL and OSCC when compared to HC [Figure 3]. There was no significant difference (P > 0.05) in the mean zinc levels between the OL and OSMF groups. The Cu/Zn ratio was higher in all three study groups OSMF, OL and OSCC when compared to the HC [Figure 4]. The mean salivary Fe levels in group HC, OSMF, OL and OSCC were 76.07 ± 3.65 μg/dL, 35.67 ± 1.34 μg/dL, 47.08 ± 3.33 μg/dL and 36.78 ± 2.23 μg/dL, respectively [Figure 5]. The salivary Fe levels were least in the OSMF group.
Figure 2: Graph showing salivary Copper levels in study groups

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Figure 3: Graph showing mean salivary Zinc levels in study groups

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Figure 4: Graph showing Copper to zinc ratio in study groups

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Figure 5: Graph showing mean salivary iron levels in study groups

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 > Discussion Top

0 Trace elements like Cu and Zn have a role in the anti-carcinogen defense system of the human body. Cu is involved in the cell metabolism, as a part of various enzymes tyrosinase, uricase and cytochrome oxidase, which are mainly concerned with oxidation reactions. [8] It was observed that mean serum Cu levels were significantly higher in the sera of patients with oral premalignant and malignant lesions. [9] In our study there was an elevation of the salivary Cu in the OL and OSCC group. Although few serum studies have been carried out in the recent past only one salivary study regarding status of Cu in saliva has been published recently wherein they reported an increase in the salivary Cu levels in the saliva of subjects with OSCC and OL when compared to controls. [10] In our study we used a larger sample size in each of the study groups and also intercompared the salivary Cu levels between group OSMF, OL and OSCC wherein we found a significant increase in the levels of salivary Cu levels in group OSMF. The reason for the increase in Cu levels in serum is due to its ability to induce an intrinsic protein based enzyme (lysl-oxidase) in the connective tissue that crosslinks collagen and elastin which is an important factor in etiopathogenesis of OSMF. [11]

The role of Cu in carcinogenesis is highlighted by its involvement in Fenton reaction → H 2 O 2 + Cu (I)→Cu (II) OH + OH . Cu also activates angiogenic factors like vascular endothelial growth factor (VEGF), Tumour Necrosis Factor (TNF) alpha and Interleukin-1(IL-1). [12] The levels of cerruloplasmin which is a Cu transporting protein, increases 4-8 times when tumours become palpable. [13]

Zn acts as a cofactor for Cu-Zn superoxide dismutase enzyme that is a part of the primary antioxidant system of all vertebrates. [16] Some studies have revealed lower Zn level in serum of patients with potentially premalignant disorders like in OL. [15],[16] This might be because of the consumption of Zn in counter reacting to oxidants which are generated due to tobacco or high Cu of areca quid metabolism. [17] In our study the salivary levels of Zn was significantly reduced in all three study groups when compared to controls. Although there was no statistically significant difference between the salivary Zn levels in OL and OSMF groups. Researchers also believe that Zn inhibits the invasive/migration activities in malignant prostate cells. [18] Further Zn is said to have an antioxidant role in prevention of carcinogenesis by two mechanisms. Firstly, the protection of sulfhydryl groups against oxidation and the inhibition of the production of reactive oxygen species by transition metals. Secondly, supra-physiological concentration of Zn has antioxidant-like effects in organelle-based systems and isolated cell-based systems. Thus, a decrease in the Zn levels and Zn dependent SOD is said to be an indicator of early mucosal changes preceding carcinogenesis. [19]

In our study we observed decreased salivary Fe which was observed in OSMF, OL and OSCC patients. Decreased Fe levels in OSMF patients might be due to utilization of Fe in collagen synthesis. [20] It has been stated that the decrease in Fe content leads to decrease in epithelial vascularity which results in increased penetration of arecoline which leads to fibrosis. [21] Inadequate nutrition due to burning sensation and erosions in OSMF patients and increased tumour burden in OSCC patients are considered to be important factors for iron depletion. [22]

 > Conclusion Top

0 In the recent years a surge in number of studies involving serum levels of trace elements like Cu, Zn and Fe in various malignancies have been observed. The results of the study reveal that salivary levels Cu, Zn and Fe could be used as biological markers in oral carcinogenesis. The significant and encouraging findings of the present study thus validate and reinforce that; salivary micronutrient analysis can be used as an efficient, non-invasive, patient-friendly tool for the early diagnosis of PMD and OSCC for planning comprehensive treatment protocol.

 > References Top

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Bouquot JE, Whitaker SB. Oral Leukoplakia rationale for diagnosis and prognosis of its clinical subtypes or phases. Quintessence Int 1994;25:133-40.  Back to cited text no. 2
Koyama H. Trace elements: Mechanistic action of anticarcinogenic action action. Nippon Rinsho 1994;54:52-8.  Back to cited text no. 3
Ames BN. Micronutrients prevent cancer and delay aging. Toxicol Lett 1999; 28:102-3.  Back to cited text no. 4
Vyas RK, Gupta AP, Gupta A, Aeron AK. Serum Copper, zinc, Magnesium and cadmium levels in various human disease. Indian J Med Res 1982;76:301-4.  Back to cited text no. 5
Slavkin HC. Protecting the mouth against microbial infections. J Am Dent Assoc 1998;129:1025-30.  Back to cited text no. 6
Al rawi NH, Talabani NG. Quatitative análisis of trace elements in saliva of oral cancer patients. J Coll Dent 2005;17:32-5.  Back to cited text no. 7
Margalith EJ, Schenker JG, Chevion M. Copper and zinc levels in normal and malignant tissues. Cancer 1983;52:868-72.  Back to cited text no. 8
Khanna SS, Karjodkar FR. Circulating immune complexes and trace elements (Copper, Iron and Selenium) as markers in oral precancer and cancer: A randomised, controlled clinical trial. Head Face Med 2006;2:33.  Back to cited text no. 9
Ayinapmudi KB, Narasimhan M. Salivary Copper and zinc levels in oralpremalignant and malignant lesions. J Oral Maxillofac Pathol 2012;16:178-82.  Back to cited text no. 10
Tadakamadla J, Kumar S, GP M. Evaluation of serum copper and iron levels among oral submucous fibrosis patients. Med Oral Patol Oral Cir Bucal 2011;16:e870-3.  Back to cited text no. 11
Hannen EJ, Riediger D. The quantification of angiogenesis in relation to metastasis in oral cancer: A review. Int J Oral Maxillofac Surg 2004;33:2-7.  Back to cited text no. 12
Nasulewicz A, Mazur A, Opolski A. Role of copper in tumour angiogenesis: Clinical implications. J Trace Elem Med Biol 2004;18:1-8.  Back to cited text no. 13
Miceli MV, Tatejr DJ, Alcock NW, Newsome DA. Zinc deficiency and oxidative stress in the retina of pigmented rats. Invest Ophthalmol Vis Sci 1999;40:1238-44.  Back to cited text no. 14
Jayadeep A, Raveendran PK, Kannan S, Nalinakumari KR, Mathew B, Krishnan NM. Serum levels of copper, zinc, iron and ceruloplasmin in oral leukoplakia and Squamous cell carcinoma. J Exp Clin Can Res 1997;16:295-300.  Back to cited text no. 15
Swain N, Ray JG. Altered trace element level and antioxidant activity in whole blood of oral leukoplakia and cancer patients in comparison with healthy controls. Int J Oral Maxillofac Pathol 2011;2:2-6.  Back to cited text no. 16
Bose SC, Singh M, Vyas P, Singh M. Plasma zinc antioxidant vitamins, glutathione levels and total antioxidant activity in oral leukoplakia. Dent Res J (Isfahan) 2012;9:158-61.  Back to cited text no. 17
Costello LC, Feng P, Milon B, Tan M, Franklin RB. Role of zinc in the pathogenesis and treatment of prostate cancer: Critical issues to resolve. Prostate Cancer Prostatic Dis 2004;7:111-7.  Back to cited text no. 18
Powell SR. The antioxidant properties of Zinc. J Nutr 2000;130:1447S-54S.  Back to cited text no. 19
Anuradha CD, Devi CS. Serum protein, ascorbic acid and iron and tissue collagen in oral submucous fibrosis--a preliminary study. Indian J Med Res 1993;98:147-51.  Back to cited text no. 20
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

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