Abstract
Aim:
To evaluate and compare the magnesium levels in serum and saliva of individuals with tobacco habits with and without potentially malignant disorders (PMDs).
Materials and Methods:
A total number of 60 patients were taken in the study, which included group A: 20 healthy patients without any tobacco or alcohol habits, without any PMDs, and without any systemic diseases; group B: 20 patients with tobacco habits and without PMDs; and group C: 20 patients with tobacco habits and with PMDs. The serum and salivary samples were collected from the patients and were subjected to magnesium level estimation using the calmagite method. Statistical analysis was performed using the Shapiro–Wilk W test and Mann–Whitney test.
Results:
The mean salivary magnesium levels were increased in group B (1.442 mg/dl), followed by group C (0.551 mg/dl), and then group A (0.463 mg/dl). The mean serum magnesium levels were increased in group B (1.891 mg/dl), followed by group C (1.608 mg/dl), and then group A (1.589 mg/dl). The salivary and serum magnesium levels were significantly high in patients with tobacco habits and without PMDs compared to the control group. The salivary and serum magnesium levels were significantly decreased in patients with tobacco habits with PMDs compared to the patients with tobacco habits and without PMDs.
Conclusion:
The study established a significant correlation between the salivary and serum magnesium levels in patients with tobacco habits, with or without PMDs, and showed that as the disease progressed, the salivary and serum magnesium levels decreased.
Keywords: Calmagite method, magnesium, oral potentially malignant disorders
INTRODUCTION
Oral squamous cell carcinoma (OSCC) refers to a subgroup of head and neck malignancies that may develop at any intraoral location. It is the third most common form of cancer in India and accounts for more than 90% of all oral malignant manifestations. Most of these cancers develop from pre-existing potentially malignant disorders (PMDs), such as leukoplakia, erythroleukoplakia, verrucous leukoplakia, etc.[1] Often these PMDs are associated with the habit of tobacco in various forms, such as smoking tobacco or smokeless tobacco.[2] There are various other risk factors also that are responsible for the development of PMDs such as human papilloma virus, radiation, and sun exposure.
Tobacco smoking poses a risk to health because of the inhalation of poisonous carcinogens in tobacco smoke. It has been observed that many tobacco users often have a habit of alcohol also. Therefore, when an individual's daily alcohol intake is higher than 45 ml and smokes more than 20 cigarettes a day, it causes a synergistic effect and increases the risk of carcinogenesis by around 15 times.[3] The alcohol disrupts the normal order of epithelial lipid molecules, resulting in gaps between epithelial cells and increasing oral mucosal permeability. Alcohol also causes DNA damage by active oxidation. All this results in spaces between the oral epithelial cells and, therefore, increases the permeability of tobacco carcinogens through the oral mucosa. Tobacco and alcohol use also poses a health risk and contributes to various morbidities such as generalised impairment of neurocognitive functions, dizziness, brain damage, coronary heart disease, lung infections, liver damage, kidney failure, and disorders of the endocrine system. These are the major causes of mortality in India and account for more than 1.35 million deaths per annum. The carcinogens present in both tobacco and alcohol interfere with the nutritional intake also and, hence, are responsible for the alterations in the levels of trace elements present in the blood. Therefore, the primary focus has shifted to early detection of oral PMDs, so that early intervention can be started.[1,4,5]
Trace elements are minerals required in minute amounts. They are found in abundance in the body and are involved in various physiological and metabolic processes. They play a major role in the living body by maintaining osmotic pressure and membrane potentials.[6]
Magnesium is one of the most abundant cations present in the living cells and is a cofactor in multitudinous intracellular processes. It is an essential element that is required for a broad variety of physiological and biological functions that may range from structural roles by intricating negatively charged groups such as phosphates in nucleic acids, a control role in enzyme activation or inhibition, and regulatory role by modulating cell proliferation, cell cycle progression, and differentiation.[7] Serum magnesium levels are remarkably constant in healthy participants and protect the body against various diseases. Any imbalances with magnesium ion concentration have often been associated with the occurrence of a pathological condition, such as pre-cancerous cell transformation.[7] It has been insinuated that magnesium insufficiency may actuate carcinogenesis by altering the fidelity of DNA replication and augmenting membrane permeability.[8]
Researchers have often observed a low magnesium level in patients with tobacco habits because of increased demand for magnesium by smokers owing to increased release of adrenaline and the thermogenic effect of nicotine, which is the main component of tobacco. Low levels could also result from a decreased supply in the diet and reduced absorption caused by disturbances in the digestive system functions.[9]
Limited studies have been done to evaluate magnesium levels in serum and saliva of patients with habits. Hence, the purpose of this study was to evaluate and compare the magnesium levels in serum and saliva of patients with tobacco habits to serve as a simple, reliable, and potential biomarker in the process of carcinogenesis of oral cancer.
MATERIALS AND METHODS
The study was aimed to evaluate and compare the magnesium levels in serum and saliva of individuals with tobacco habits, with and without PMDs. Before starting the study, proper case history and informed written consent of the patients were taken in his/her language. A total number of 60 patients were taken in the present study, which included group A: 20 healthy patients without any tobacco and alcohol habits, without any PMDs, and without any systemic diseases; group B: 20 patients with tobacco habits and without PMDs; and group C: 20 patients with tobacco habits and with PMDs. The serum and salivary samples were collected from the patients and subjected to magnesium level estimation using the calmagite method in the Department of Biochemistry.
Procedure
Serum collection
Two millilitres of blood was collected in plain red vacutainers by venipuncture using aseptic measures. At 3500 rpm, the serum was separated by centrifugation for 10 min.
Unstimulated saliva collection
Three millilitres of unstimulated whole saliva sample was collected from the mouth directly into the Eppendorf on a single occasion by asking the patient to press their tongue against their palate so that freshly secreted saliva was collected in the sublingual region for 5 min. Saliva was centrifuged at 3500 rpm for 10 min to remove the excess mucus and unwanted particles.
Serum and saliva estimation
The estimation of magnesium in serum and saliva was done using a Coral Magnesium Kit (Tulip Diagnostics Pvt Ltd. (a PerkinElmer company), Goa, India) by calmagite method. The results were recorded in mg/dl.
RESULTS
The collected data were analysed using SPSS 21 (Chicago, USA) and IBM Inc. Descriptive data were reported for each variable and presented using Tables 1 and 2 and Graphs 1 and 2. Data were not normally distributed as tested using the Shapiro–Wilk W test (P-value was less than 0.05). Mann–Whitney U test was used for the comparison of two independent data. A level of P value less than 0.05 was considered statistically significant.
Table 1.
Comparison of salivary and serum magnesium levels among healthy patients (group A) versus patients with tobacco habits and without potentially malignant disorders (group B)
| Group | n | Mean (mg/dl) | Standard Deviation | Mean Difference | Z | P |
|---|---|---|---|---|---|---|
| Mg Saliva | ||||||
| Group A | 20 | 0.4630 | 0.30145 | −0.97900 | −3.837 | 0.001*, |
| Group B | 20 | 1.4420 | 0.82723 | sig | ||
| Mg Serum | ||||||
| Group A | 20 | 1.5890 | 0.60638 | 0.30250 | −0.920 | 0.369, |
| Group B | 20 | 1.8915 | 0.39769 | ns |
Values are expressed as mean ± SD. SIG: significant, NS: Non-significant; Differences were considered significant at P<0.05 in groups A and B in salivary magnesium, and non-significant in groups A and B in serum magnesium
Table 2.
Comparison of salivary and serum magnesium levels among healthy patients (group A) versus patients with tobacco habits and with potentially malignant disorders (group C)
| Group | n | Mean (mg/dl) | Standard Deviation | Mean Difference | Z | P |
|---|---|---|---|---|---|---|
| Mg Saliva | ||||||
| Group A | 20 | 0.4630 | 0.30145 | −0.8800 | −1.194 | 0.242, |
| Group C | 20 | 0.5510 | 0.22136 | ns | ||
| Mg Serum | ||||||
| Group A | 20 | 1.5890 | 0.60638 | −0.19000 | −0.772 | 0.445, |
| Group C | 20 | 1.6080 | 0.37966 | ns |
Values are expressed as mean ± SD. NS – Non-significant; Differences were considered non-significant in groups A and B in both serum and salivary magnesium
Graph 1.
Evaluation of salivary magnesium levels in various groups
Graph 2.
Evaluation of serum magnesium levels in various groups
Evaluation of salivary magnesium levels
The mean salivary magnesium levels in patients with tobacco habits and without PMDs were highest, i.e., 1.442 mg/dl, followed by patients with tobacco habits and with PMDs, i.e., 0.551 mg/dl, and the control group showed the least with a mean value of 0.463 mg/dl.
Evaluation of serum magnesium levels
The mean serum magnesium levels in patients with tobacco habits and without PMDs were highest, i.e., 1.8915 mg/dl, followed by patients with tobacco habits and with PMDs, i.e., 1.6080 mg/dl, and the control group showed the least with a mean value of 1.5890 mg/dl.
Comparison of salivary and serum magnesium levels in group A and group B
The mean salivary magnesium levels were found to be increased in patients with tobacco habits and without PMDs (group B) when compared with the control group (group A). Results were statistically significant with a P value of 0.001. The mean serum magnesium levels were found to be increased in patients with tobacco habits and without PMDs (group B) when compared with the control group (group A). Results were statistically non-significant with a P value of 0.369.
Comparison of salivary and serum magnesium levels in group A and group C
The mean salivary magnesium levels were found to be increased in patients with tobacco habits and with PMDs (group C) when compared with the control group (group A). Results were statistically non-significant with a P value of 0.242. The mean serum magnesium levels were found to be increased in patients with tobacco habits and with PMDs (group C) when compared with the control group (group A). Results were statistically non-significant with a P value of 0.445.
DISCUSSION
Oral mucosa is an active part of the oral cavity that mirrors the general health of the human body. Any insult such as radiation, sun exposure, viruses, or in the form of multiple habits such as the use of any form of tobacco (smoking or smokeless) or drinking alcohol can result in severe damage to the oral tissues.[10] Constant irritation to the mucosa by these can cause PMDs of the oral cavity, which can further lead to OSCC. Hence, an early diagnosis can be achieved by assessing the alterations in the trace elements in the body fluids. The purpose of this study was to evaluate and compare the magnesium levels in serum and saliva of patients with habits to serve as a simple, reliable, and potential biomarker in the process of carcinogenesis of oral cancer.
Magnesium is an essential cation that plays a crucial role in many physiological and cellular functions. In biological systems, magnesium ions exist in three different states: Bound to proteins, complexed to anions, and free magnesium ions. Only free magnesium has biological activity. The adult human body contains approximately 24 g of magnesium in cells versus 280 mg in extracellular fluids. The mean serum magnesium concentration is about 0.8 mmol/l.[11]
Magnesium is critical in energy-requiring metabolic processes, protein synthesis, membrane integrity, nervous tissue conduction, neuromuscular excitability, muscle contraction, hormone secretion, and intermediary metabolism. It is also involved in more than 300 enzymatic reactions in the body, participating in the metabolism of glucids, lipids, proteins, and nucleic acids, in the synthesis of H2 transporters, and particularly in all reactions involving the formation and use of ATP.[11] In a cell, magnesium forms complexes with the phospholipids and stabilises the cell membrane by reducing its fluidity and permeability. It also alters the receptor sites and ion movements across the cell membrane. It has a structural role, a control role in enzyme activity, and a regulatory role in the cell cycle.[7]
Modifications in the cell membrane are principal triggering factors in cell transformation leading to cancer. And these cancer cells showed that there is much less magnesium binding to the membrane phospholipids of cancer cells than to normal cell membranes, which might be involved in precancerous changes. In the pre-neoplastic phase, the binding of magnesium to membranes is decreased, and at the same time, the cytosolic magnesium increases. Both the defective membranes of cancer cells and those of magnesium deficiency show a marked alteration in the ionic flux from the outside and inner cell membranes (higher Ca and Na, lower Mg and K levels). It has been suggested that magnesium deficiency may trigger carcinogenesis by altering the fidelity of DNA replication and increasing membrane permeability. Magnesium deficiency depresses cell-mediated immunity and impairs phagocytic activity as well as lymphocytic function. Magnesium also causes an increase in DNA fragmentation, cleavage of substrates associated with caspase activation, and tissue shrinkage.[8,12,13]
In the present study, it was found that the mean salivary magnesium levels in patients with tobacco habits without PMDs were highly increased when compared with the control group. And in patients with tobacco habits with PMDs, the mean salivary magnesium levels were slightly raised than the control group. Similar observations were made by Kolte et al.[14] and Manea et al.[15] They justified that this could be because of the presence of magnesium in tobacco that could be possibly responsible for an increased level of magnesium content in the saliva. The presence of magnesium in slaked lime and smokeless tobacco was also observed by Bhisey[2] and Kode et al.[16] Aziz et al.[7] and Bagulkar et al.[8] found salivary magnesium levels to be decreased in patients with PMDs and further decreased in OSCC compared to the control group. They stated that the decrease in salivary magnesium levels when the disease progressed was because of the participation of magnesium in the process of carcinogenesis. Our results were found to be inconsistent with the findings of Dziewulska et al.[17] and Shpitzer et al.[18] They observed that individuals with OSCC exhibited increased levels of salivary magnesium when compared with the control group.
In the present study, it was found that the mean serum magnesium levels in patients with tobacco habits without PMDs were significantly increased when compared with the control group. And in patients with tobacco habits with PMDs, the mean serum magnesium levels were slightly raised than the control group. Our results were found to be consistent with the observations made by Hassan et al.[19] They found serum magnesium levels to be high in the smoker's group compared with the non-smoker's group. In addition, Bagulkar et al.[8] and Aziz et al.[7] found serum magnesium levels to be decreased in patients with PMDs and further decreased in OSCC compared to the control group. They noted a significant decrease in the concentration of magnesium levels in serum in PMDs and OSCC compared with the control group. They concluded that these findings could be possibly because of the regulatory role of magnesium in the cell cycle that affected the transphorylation and synthesis of DNA. The results obtained by Mudawi et al.,[9] Khand et al.,[20] and Ata et al.[21] were inconsistent with our findings. They established that serum magnesium levels significantly decreased in smokers because of the poor appetite and reported an increase in the demand for magnesium by smokers owing to increased release of adrenaline and the thermogenic effect of nicotine, which is the main constituent of tobacco. In addition, Nechifor[22] proposed that magnesium ions were involved in different functions of the central nervous system and were also found to be associated with nicotine addiction. He stated that magnesium was generally seen to inhibit the synthesis of certain substances and some neurotransmitters that were involved in tobacco addiction. Magnesium was also known to decrease the dopamine release and the N-methyl-D-aspartate receptor stimulation by glutamate. Thus, he concluded that hypomagnesemia could favor the development of tobacco addiction in an individual.
In the present study, it was found that the mean salivary and serum magnesium levels were increased in patients with tobacco habits without PMDs compared to the control group. And in patients with tobacco habits with PMDs, the mean salivary and serum magnesium levels were decreased compared to the patients with tobacco habits without PMDs. The possible reason behind this could be the active participation of magnesium in the cell cycle and cancer exacerbation, which could affect the transphorylation and synthesis of DNA and promotes angiogenesis.[23,24]
In this study, a significant correlation could be established between the salivary and serum magnesium levels in patients with tobacco habits, with or without PMDs. But, further studies with a larger sample size can be done to assess the levels of salivary and serum magnesium in patients with tobacco habits and with PMDs.
CONCLUSION
In the present study, the salivary and serum magnesium levels were significantly high in patients with tobacco habits and without PMDs compared to the patients with tobacco habits with PMDs. The study established a significant correlation between the salivary and serum magnesium levels in patients with tobacco habits, with or without PMDs, and showed that as the disease progressed, the salivary and serum magnesium levels decreased. Hence, magnesium can serve as a simple, reliable, and potential biomarker in the process of carcinogenesis of oral cancer. However, further studies with a larger sample size can be done to assess the levels of salivary and serum magnesium in patients with tobacco habits and with PMDs.
Ethics
This study was approved by the institutional ethical review board reference number SGTU/FDS/MDS/24/1/547 dated 11.11.2019. Informed consent was obtained from all patients for being included in the study.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
Acknowledgements
We would like to express our gratitude and appreciation to the laboratory technicians Miss. Seema in the Department of Oral Pathology, Mr. Lokesh, and Mrs. Monika in the Department of Biochemistry for their help in offering us the resources for the study and for helping in the estimation at every step.
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