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 Table of Contents  
Year : 2019  |  Volume : 31  |  Issue : 4  |  Page : 333-338

Estimation of salivary copper, zinc, iron and copper to zinc ratio in oral submucous fibrosis patients and its comparison with healthy individuals

1 Department of Oral Medicine and Radiology, ACPM Dental College, Dhule, Maharashtra, India
2 Department of Oral Medicine and Radiology, Maratha Mandal's NGH Institute of Dental Sciences, Belgaum, Karnataka, India

Date of Submission09-Apr-2019
Date of Acceptance01-Aug-2019
Date of Web Publication03-Mar-2020

Correspondence Address:
Dr. Gitanjali Khulbe
Department of Oral Medicine and Radiology, ACPM Dental College, Post Box No. 145, Sakri Road, Dhule - 424 001, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jiaomr.jiaomr_81_19

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Background: Trace elements have been found to be altered in oral submucous fibrosis (OSMF), an oral potentially malignant disorder which occurs as a result of a derangement in collagen metabolism. The literature on variation observed in the parameters of trace elements, in the saliva of OSMF patients and its correlation with the staging of OSMF, is limited. Hence, a case-control study was carried out to estimate the salivary concentrations of copper, zinc, and iron in OSMF patients. Materials and Methods: Out of the 120 patients enrolled, 60 OSMF cases and 60 healthy individuals were studied. Unstimulated whole saliva from each subject was centrifuged and the supernatant stored at -80°C. Laboratory analysis was done to estimate copper, zinc, and iron and the values were statistically analyzed. Results: The mean values of salivary copper, zinc, and iron were lower in OSMF cases than the controls and the mean copper to zinc ratio was higher in the cases, the differences being statistically insignificant. Conclusion: The chance differences that occurred in our study might have been due to lifestyle variations among the different subjects. Saliva may or may not be a representative of the changes seen in the trace element levels of OSMF patients.

Keywords: Areca nut, copper, iron, OSMF, saliva, zinc

How to cite this article:
Khulbe G, Tantradi P, Ammanagi R, Byahatti S. Estimation of salivary copper, zinc, iron and copper to zinc ratio in oral submucous fibrosis patients and its comparison with healthy individuals. J Indian Acad Oral Med Radiol 2019;31:333-8

How to cite this URL:
Khulbe G, Tantradi P, Ammanagi R, Byahatti S. Estimation of salivary copper, zinc, iron and copper to zinc ratio in oral submucous fibrosis patients and its comparison with healthy individuals. J Indian Acad Oral Med Radiol [serial online] 2019 [cited 2022 May 26];31:333-8. Available from: https://www.jiaomr.in/text.asp?2019/31/4/333/279866

   Introduction Top

Oral submucous fibrosis (OSMF) is a potentially malignant disorder of the oral mucosa which was first described by Schwartz in 1952 who coined the term “atrophicaidiopathica mucosae oris”. Later, Pindborg in 1966 defined OSMF as an insidious chronic disease affecting any part of the oral cavity and sometimes pharynx, although occasionally preceded by and\or associated with vesicle formation, it is always associated with juxtaepithelial inflammatory reaction followed by fibroelastic changes in the lamina propria, with epithelial atrophy leading to stiffness of the oral mucosa causing trismus and difficulty in eating. Epidemiologically, areca nut has been found to be strongly associated with the development of OSMF. Trace elements, as contents of areca nut, have been extensively studied considering its role in cancerous conditions. Copper, zinc, iron, and magnesium are biologically essential for normal development, growth, and function.[1]

Previous studies have highlighted the role of trace elements in the pathogenesis of OSMF.[2] The levels of trace elements (TEs) have shown to be altered in oral potentially malignant disorders (OPMDs) and malignant diseases but the exact role of these elements is unknown.[3]

High amounts of copper, zinc, iron, and magnesium have been found in various brands of gutkha.[1],[4] Copper, zinc, and iron are essential for numerous enzymes, functioning in the body and it is reasonable to assume that variations of these biochemical markers may be associated with the pathogenesis of oral cancer and OPMDs.[5],[6] The ratio of copper to zinc has been found to be significantly decreased in OSMF patients. The Cu/Zn ratio is considered to be a more specific and reliable indicator in assessing progression to malignancy, compared to the individual parameters.[7] A good correlation of concentrations of certain constituents has been observed between saliva and plasma. Saliva comes into direct contact with the oral cavity and reflects the tissue composition and its collection is relatively easy and noninvasive.[8]

Hence, this study was carried out to estimate the salivary concentrations of copper, zinc, iron, and copper to zinc ratio in OSMF patients and compare them with healthy individuals.

   Materials and Methods Top

The study was conducted in the Department of Oral Medicine and Radiology in the period between August 2016 and May 2017. Approval from the ethical committee was obtained from the institution. A total of 120 subjects aged between 18 to 56 years from the outpatient's department—60 OSMF patients and 60 healthy subjects were taken. Written informed consent was obtained from all the subjects.

OSMF cases clinically diagnosed according to the international consensus and having a current habit of areca nut chewing were included. Various types of habit among the cases are shown [Table 1]. Clinically, More et al. classification was used to ascertain the staging of OSMF [Table 2]. Patients who had any systemic disease and were taking treatment or have been treated for OSMF or those on any kind of antioxidants or supplements (vitamins, minerals, etc.) were excluded. Healthy subjects matched for age and gender and without any habit of tobacco, areca nut, and/or alcohol were taken as controls.
Table 1: Types of habit among the cases

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Table 2: Clinical and functional staging of OSMF patients

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Demographic and other details specified in the case-history proforma with emphasis on habit history were recorded. Saliva was collected in the period between 10:00 am to 1:00 pm to avoid diurnal variation. The subjects were told to minimize tongue movements and given distilled water to rinse the mouth thoroughly. They had to sit in an erect posture with the neck bent slightly forwards and downward and were asked to accumulate saliva in the mouth for about 1 min or till they felt the urge to swallow. About 5 ml of unstimulated whole saliva was collected from all the subjects by spitting method. The samples were centrifuged at 3000 rpm for 15 min to obtain a supernatant. The “supernatant” samples [Figure 1] were stored at −80°C in the ultra-low-temperature freezer, and later analyzed biochemically. Salivary copper, zinc, and iron were estimated using semiautomated analyzer (ERBA CHEM-5 Plus V2) based on “absorption photometry”. The reagent used for each of the elements was of TULIP Diagnostics, Crest Biosystems (Goa). Copper to Zinc (Cu/Zn) ratio was also calculated. The data were analyzed using SPSSv20.0 software.
Figure 1: Eppendorf tubes containing “supernatant” saliva of 120 subjects

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   Result Top

A total of 60 OSMF cases, 58 males and 2 females, were enrolled in the study. The control group also had 58 males and 2 females. Mean salivary copper and zinc level was lower in OSMF cases than healthy subjects. However, the differences were not statistically significant according to unpaired t-test [Table 3]. The mean salivary iron level was lower in the OSMF cases than normal healthy subjects but the difference was statistically insignificant according to Mann-Whitney U-test [Table 3]. The mean salivary “Cu to Zn” ratio was higher in the OSMF cases than normal healthy subjects but the difference was statistically insignificant, according to Mann-Whitney U-test [Table 4].
Table 3: Comparison of salivary Cu (μg/dL), Zn (μg/dL) and Fe (μg/dL) scores in OSMF cases and controls

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Table 4: Comparison of salivary “Cu to Zn” ratio scores in OSMF cases and controls

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Copper, zinc, and iron contents, as well as copper to zinc ratio, were maximum in patients with only supari-paan habit. However, one-way ANOVA test showed no statistical significance [Table 5].
Table 5: Comparison of Type of Habit with Cu (μg/dL), Zn (μg/dL), Fe (μg/dL) and “Cu to Zn” ratio scores

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Copper and iron levels are the highest in patients with a frequency of intake 6–10 times a day. Zinc and Iron levels reduced with the increasing frequency of chewing habit. Cu/Zn ratio increased with the frequency of the habit. One-way ANOVA test showed a difference in copper levels to be close to significance [Table 6].
Table 6: Comparison of Frequency of intake with Cu (μg/dL), Zn (μg/dL), Fe (μg/dL) and “Cu to Zn” ratio scores

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Copper and zinc levels reduced with increase in the duration of the habit. Iron and Cu/Zn ratio did not show a definite pattern of change with the duration of habit [Table 7]. Spearman's correlation coefficient analysis for frequency and duration of habit showed that copper level reduced with increase in the duration of the habit, with moderate statistical significance. Other correlations were not found to be significant [Table 8].
Table 7: Comparison of duration habit with Cu (μg/dL), Zn (μg/dL), Fe (μg/dL) and “Cu to Zn” ratio scores

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Table 8: Spearman's Correlation coefficient to test the correlation of frequency of intake (per day) and duration of habit (in years) with Cu, Zn, Fe and Cu: Zn ratio values

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   Discussion Top

Oral submucous fibrosis (OSMF) is an insidious chronic disease affecting the oral cavity.[9] A worldwide estimate of OSMF shows confinement to Indians and Southeast Asians. In the year 2002, statistics for OSMF from the Indian continent was about 5 million people which constitutes approximately 0.5% of the population.[10] Data from numerous studies suggest that areca nut is the main etiological factor in OSMF.[3],[11],[12],[13],[14]

In our study, we included 120 subjects—60 OSMF cases and 60 healthy controls, between 18 to 56 years of age. The mean age of cases in our study was 33.1 years with predominance seen in males. The population below 20 years of age, especially the males, are increasingly getting affected with the disease apparently due to easy and lucrative availability of gutkha and other similar products.[15],[16]

In our study, for estimating salivary trace elements we used the colorimetric method in a semiautomated analyzer. The equipment was readily available, and the method is inexpensive. For Fe estimation, Ferrozine method was used, which is a type of colorimetric method also used by Bhardwaj et al.[17] In addition, bathophenanthroline method[18] and dipyridyl method[19] have been used to estimate Fe content. The other methods used for salivary Cu, Zn, and Fe analysis are atomic absorption spectrophotometer,[6],[20],[21],[22],[23],[24] DPASV[6], and inductively coupled plasma mass spectrometry method[6] [Table 9].
Table 9: Comparison of salivary Cu, Zn and Fe levels of our study with other studies

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Mean value of salivary copper in our cases was 24.13 μg/dL and 24.42 μg/dL in controls. This can be attributed to the involvement of Copper in stimulating fibrinogenesis.[14]

Copper is involved in cell metabolism as a part of various enzymes viz. tyrosinase, uricase, cytochrome oxidase, superoxide dismutase, metallothioneine, all concerned mainly with “oxidation” reactions.[26] Most of the copper found in tissues is specifically bound, comprising an integral part of enzymes such as superoxide dismutase. A slight variation in total tissue copper content represents a considerable change in the total nonspecific, complexed copper.[27]

Lysyl oxidase is a copper-dependent extracellular enzyme critical for collagen cross-linking and organization of ECM.[22],[28] A few theories have proposed that copper levels in OSMF patients might be lower than normal individuals, due to the upregulation of LOX enzyme which occurs in excessive fibrosis.[29] In a study by Karcioglu et al., in one of the six cases of hypernephroma, an insignificant decrease in copper content was measured in kidney tissue.[27] Trivedy et al. have reported that copper-induced mutagenesis through p53 aberrations may be critical in the progression of OSMF to OSCC.[25],[30]

Copper has been found to be elevated in OSMF cases in most of the studies. The mean copper values in the cases ranged from 0.000338 μg/dl to 870 μg/dL in studies by other authors.[2],[4],[22],[24],[25],[26] Among the controls, it ranged from 0.0168 μg/dL to 510 μg/dL in the same studies. This can be attributed to the ingestion of copper from the areca nut products. However, in the study by Mohammed et al. a direct correlation could not be established between copper levels in areca nut and salivary copper and histological grades of OSMF.[4]

Mean value of salivary zinc in our cases was 21.80 μg/dL and 24.71 μg/dL in controls. Zinc, an integral part of biomembranes, may control membrane integrity and has also been shown to stabilize ribosomes and the DNA double helix structure.[28] Zinc is involved as a cofactor in carbonic anhydrase, superoxide dismutase, and other enzymes. It is implicated to be utilized in counteracting oxidative stress. There is an inverse relationship between copper and zinc in the body; an increase in copper intake could also lead to a reduction in zinc content.[29],[31] Its deficiency is associated with dysfunction of the zinc-dependent enzymes essential for cell replication. This might also be an important marker denoting the balance between oxidant-antioxidant status during the progression of the disease.[6],[22]

The mean zinc values ranged from 0.004472 μg/dL to 1000 μg/dL in the cases studied by other authors.[2],[22],[24],[25] In the same studies, among the controls, it varied from 0.002740 μg/dL to 1000 μg/dL. Some studies have shown an increase in zinc levels which could be due to direct transfer from gutkha consumption.

Mean value of salivary iron in our cases was 22.55μg/dL and 24.51μg/dL in controls. Iron has been recognized as an important element for the maturation of epithelium.[5] It is an essential component in DNA and collagen synthesis. Utilization of iron for hydroxylation of proline and lysine in the excessive collagen synthesis leads to a decrease in iron levels.[28],[25]

The mean iron values ranged from 0.001037 μg/dL to 1490 μg/dL in studies by other authors.[18],[19],[22],[24],[25] Among the controls, it varied from 0.1589 μg/dL to 5240 μg/dL in the same studies.

The wide variations in the mean values of trace elements in OSMF cases as well as healthy subjects are ambiguous and point towards the need for standardization of the normal salivary levels of these elements. Socio-economic status, lifestyle, and dietary factors may also be the underlying cause for the differences seen.

Different results seen in studies using similar[18],[19],[26] and dissimilar[4],[22],[24],[25] methods for estimation emphasizes an urgent need to establish a standard range of salivary trace element levels with different methods. The mode of collection of the saliva also has its impact on the constituents of saliva.

The ratio of Cu to Zn is believed to be a more reliable biomarker in the development and progression toward carcinogenesis.[8],[28] In our study, the mean salivary copper to zinc ratio in cases was found out to be 2.05 which was higher than the controls though insignificantly [Table 4]. Higher values than this, i.e. 3.78 and 3.53 are found in studies reported by Okade et al.[22] and Babu et al.[24], respectively. No studies have reported lower values as per the data available to us. Increase in Cu to Zn ratio has been noted as OSMF patients progress from premalignant state to malignancy.[9],[28],[32],[33]

   Conclusion Top

The differences that were found in our study between the cases and controls are minimal and are not statistically significant. In addition, the values of TEs in our study in both OSMF cases and controls showed wide variations from that seen in other studies. Hence, there is an urgent need to establish normal salivary levels of TEs before further studies are undertaken to establish a variety of trace element levels in saliva among OSMF patients. Furthermore, studies are also needed to standardize the methods of processing and analyzing the saliva for trace element quantification.


Dr Kishore Bhatt, Prof and HOD, Dept. of Microbiology. Mrs Rani Bansode, Lecturer, Dept. of Biochemistry. Dr Ravi Shirahatti, Prof and HOD, Dept. of Public Health Dentistry.

Financial support and sponsorship

Dept. of Hematology and Dept. of Microbiology of the college, 'Central Store' of the college.

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]


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