Sudan Journal of Medical Sciences
Volume 14, Issue no. 3, DOI 10.18502/sjms.v14i3.5215
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Research Article

Quantitative Analysis of Serum Levels of
Trace Elements in Sudanese Snuff Dippers
Abdel Azim Alsanousi1, Abdel Raouf Ahmed1, 2, and GadAllah Modawe1

1Omdurman Islamic University, Faculty of Medicine and Health sciences, Biochemistry
Department, Omdurman,Sudan
2Omdurman Islamic University, Faculty of Sciences and Technology

Abstract
Background: Oral cancer in Sudan has high incidence rate due to the use of Toombak,
a home- made smokeless tobacco, rich in tobacco specific nitrosamines.There have
been calls for continuous monitoring on toombak users to discover very early the
carcinogenic changes to avoid mortality, and morbidity.
Objective: The purpose of this study was to assess technically, and financially
affordable methodologies, that are reliable, reproducible, sensitive, specific and cost
effective valid for use in mass screening amongst the high risk groups. (The toombak
users). The present study was done to evaluate the levels of copper, zinc and
magnesium in serum of toombak dippers.
Materials and Methods: The levels of copper, zinc, and magnesium were estimated
in the serum of 150 toombak dippers. The values were compared with 50 normal age
matched, healthy control subjects and 50 patients with oral squamous cell carcinoma,
using Atomic absorption spectrophotometry.
Results: There was significant difference of the mean serum copper, zinc, and
magnesium levels of toombak dippers when compared to the normal controls. In oral
cancer patients there was significant difference in the copper, zinc, and magnesium
levels. Copper level was found increased gradually starting from control through
toombak dippers to oral squamous cell carcinoma patients, while the level of zinc
and magnesium was found decreased in the same sequence.
Conclusion: Serum may be used as a potential diagnostic tool, which can be efficiently
employed to evaluate the copper, zinc and magnesium levels in Sudanese snuff
dippers for population based mass screening.Serum trace elements levels could be
used as potential diagnostic markers for early changes caused by toombak dipping.

Keywords: Atomic absorption spectrophotometry, copper, magnesiumm, zinc, oral
squamous cell carcinoma, tobacco specific nitrosamines, serum, toombak.

1. Introduction

Oral Cancer is the 6𝑡ℎ most common Cancer in the world, and the most common cancer
in head and neck.500,000 new cases are added annually worldwide, with increasing

How to cite this article: Abdel Azim Alsanousi, Abdel Raouf Ahmed, and GadAllah Modawe (2019) “Quantitative Analysis of Serum Levels of Trace
Elements in Sudanese Snuff Dippers,” Sudan Journal of Medical Sciences, vol. 14, issue no. 3, pages 152–161. DOI 10.18502/sjms.v14i3.5215 Page 152

Corresponding Author:

Abdel Azim Alsanousi;

email:

aalsanoocy@yahoo.com

Received 21 February 2019

Accepted 12 May 2019

Published 30 September 2019

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Sudan Journal of Medical Sciences Abdel Azim Alsanousi et al

rate of incidence in under developed countries [1]. The world cancer report of 2008
ranked oral cancer as the fifth most common cancer type among males in the less
developed countries [2]. Oral cancer is the 5𝑡ℎ most common cancer in the Sudan with
incidence rate of (920/year) comprising 9 % of the cases reported annually in Africa.
This is strongly attributed to the use of local type of snuff known as Toombak, a very
popular material in the Sudanese community [3]. toombak, is home-made from finely
ground leaves of Nicotiana rustica, a tobacco species with a particularly high content
of nicotine and minor alkaloids. This tobacco is mixed with Natron or atron (sodium
bicarbonate) (about 4:1), to raise the PH for rapid absorption of nicotine then water is
added to the mixture, and after a period of about 2 hours or longer the mixture is
ready for use [4]. Snuff is habitually used orally by insertion between lower gum and
cheek or lip (dipping) [5]. Nicotine ranges (8-102) mg/g. dry weight. Tobacco specific
nitrosamines. TSNA ranges (420-550) μg/g.for N-nitroso nor nicotine.NNN, and (620-
780) μg/gm. for 4- (N –Nitrosomethylamino)- 1- (3- pyridyl)- 1- butanone.NNK, dry weight
respectively, the highest ever reported TSNA concentration in a tobacco product [6].
The toombak habituate is exposed minimally to (0.12-0.44 mg) of NNK per day, the
highest documented uptake of a non occupational carcinogen [7]. The World Health
Organization WHO. recommended less than (2.0 μg /g) dry weight combined NNN
and NNK,in smokeless tobacco [8]. Toombak contains 100-fold higher levels of tobacco
specific nitrosamines TSNA than commercial snuff in the U.S. and Sweden [9]. The TSNA
content in the saliva of toombak dippers is at least ten times higher than that reported
in the saliva of dippers of commercial snuff in USA or Sweden [10]. The abnormally
high concentration of Tobacco specific nitrosamines in toombak is attributed first to
the use of species Nicotiana - Rustica that contains Nicotine (8–10) times the nicotine
in the nowadays universally used species Nicotiana – Tabaccum, as well as a high
level of alkaloids [11]. Curing of snuff by fermentation is another major factor as it
accelerates production of TSNA out of the tobacco leaves alkaloids, as well as the
high temperature during storing [12] in addition to contamination, and poor hygiene
environment during, processing, and the highly alkaline PH [13]. Toomback trade is
legal in the Sudan, without any sort of control, or supervision on manufacturing process
by health care authorities. Attempts should be made to reduce exposure to TSNA in
oral snuff users in Sudan [14]. Toombak dipping is considered social stigma in Sudan
for females, 95% of toombak users are males, [15] Although oral cancer is associated
with documented risk factors, yet no comprehensive screening program [16, 17]. There
is need for screening and continuous monitoring for toomback users, as a high risk
group [14]. The short in budget hinders the National cancer control plans for oral

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Sudan Journal of Medical Sciences Abdel Azim Alsanousi et al

cancer screening [18]. Taking biopsy is not suitable for screening purposes of high
risk individuals for early oral cancer detection due to its invasive nature, high cost,
need for specially trained medical person, and equipment [19]. Biopsy for detection of
early oral cancerous signs, suffers from the reliability for the appropriate site for the
obtainment of specimen to be conclusive, that leads to multiple follow up biopsies
while repeated surgical biopsies can discourage the habituate from agreeing to further
diagnostic biopsies as it causes fear, and stress, pain, and damage to healthy tissues
in addition to risk of infection, and temporary disability, and discomfort [19]. considering
that the habituate is not motivated to co operate with the test idea as he believes no
need for it as long as there is no pain or swelling.Toludine blue, Rose Bengal, Iodine
stain, 6 tolonium chloride stain have been used as detectors for early carcinogenic
changes, but inexperienced practitoners can not use these techniques to diagnose such
changes, besides low specificity [20–23]. The imaging diagnostic techniques such as
radiographic techniques, nuclear medicine, magnetic resonance, or ultrasonography,
need equipments, and trained staff, which is hard to provide by the health care system,
suffering already from limited resources, and escalating costs. The WHO guidelines
for screening programs in developing countries, put the cost of any test as a very
important consideration.The national screening programs should avoid imposing the
high technology of the developed world on countries that lack the infrastructure, and
resources to use the technology appropriately, or to achieve adequate coverage of the
population [18]. The trace elements have been extensively studied in recent years, to
assess whether they had any role in the aetiology of cancer. Magnesium and zinc are
the elements which have essential roles in the regulation of the cell growth, division and
differentiation. High levels of copper have been observed to protect against a chemical
induction of tumours [24]. The present study was done to evaluate the levels of copper,
zinc and magnesium in serum of toombak dippers.

2. Materials and Methods

Randomized cross sectional study was conducted involving 250 subjects (all males)
from Khartoum state,Sudan.This study was approved by concerned institution’s ethics
Committees. Study subjects were divided into 3 groups. first group 150 toombak dippers
in an age range 27 to 79 years mean age (50 ± 11.85).Years with history of toomback use
for more than 10 years, non smokers or alcohol drinkers. with no diagnosable change
in oral mucousa. The second group 50 healthy non tobacco users control with the
same age range.The third group oral squamous cell carcinoma OSCC. Patients with

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Sudan Journal of Medical Sciences Abdel Azim Alsanousi et al

age ranges between (30 to 82 years), with history of toombak use, and no previous
history of malignancy. The study protocol was explained for the subjects involved, and a
consent was obtained.5 ml of venous blood were collected from each subject in a plain
polyethylene tube, the tubes were left for 30 minutes at room temperature. samples
were centrifuged at 3,000 rpm for10 minutes to separate serum.2 ml of serum from
each sample was put in a labeled cryo- tube kept in liquid nitrogen tank (-196 c), till the
time of estimation.

2.1. Methodology for trace Elements estimation

Trace Elements were estimated using full automatic device. (Buck Scientific 210 /211 VGP
atomic absorption spectrophotometer.USA) at National center for research. Khartoum,
Sudan. The estimation of copper was done according to the method of weinstock,
and Uhleman, 1981 [25]. The estimation of Zinc was done according to the method of
Butrimovtz, and Purdy, 1977 [26]. Estimation of magnesium was done according to the
method of Amino, 1964 [27].

2.2. Statistical analysis

Statistical analysis was performed using the Statistical Package for the Social Sciences
(SPSS version 21) software. Values are expressed as (means ± SD) and P (probability)
values of 0.05 were considered to be significant with a 95% confidence interval. Serum
levels of different parameters were statistically analyzed in all the three groups included
in the study using analysis of variance (ANOVA) test, and intergroup relationship was
analyzed using t- test. Relationships between variables were evaluated by Pearson
correlation coefficient. P value of 0.05was considered to be statistically significant.

3. Results

The trace elements in the present study were found to be related to toomback dipping,
as well as to duration of habit, frequency of dipping/day, and consumption of toombak in
grams/day. The present study showed that the mean serum copper level in control group
was:0.78 0 ± 0.27 mg/L. (mean ± SD).The serum copper level in toombak dippers group
was 0.83 ±0. 22 mg / L (mean ± SD), while it was found to be 0.69 ± 0.23 mg / L. (mean
± SD) in the OSCC patients group. (Table 2). The difference between serum copper
level in the toombak dippers group, and the level in the control group was significant (p

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Sudan Journal of Medical Sciences Abdel Azim Alsanousi et al

< 0.002).The mean serum copper level increased in toombak dippers group. toombak
in gram/day (r=0.221 p=0.006) (Figure 1). The mean serum zinc level in control group
was 0.83 ±0.33 mg / L (mean ± SD).The serum zinc level in toombak dippers group
was 0.79 ± 0.27 (mean ± SD), while it was found to be 0.69 ± 0.25 mg / L. (mean ± SD)
in the OSCC patients group. (Table 3). The difference between serum zinc level in the
toombak dippers group, and the level in the control group was significant (p 039).The
mean serum zinc level showed a decreasing trend from control group through toombak
dippers group to OSCC patients group. the mean serum magnesium level in control
group was 26.1 ± 5.10 mg / L (mean ± SD). The serum magnesium level in toombak
dippers group was 24.8 ± 4.56 mg / L. (mean ± SD), while it was found to be 21.7 ±
5.60 mg / L. (mean ± SD) in the OSCCpatients group. (Table 4). The difference between
serum magnesium level in the toombak dippers group, and the level in the control
group was highly significant (p < 0001). The mean serum magnesium level showed a
decreasing trend from control group through toombak dippers group to OSCC patients
group.

Table 1: General characteristics of toombak users.

Study variable Minimum Maximum Mean Std. Deviation

Duration of habit (year) 10.0 55.0 26.63 10.98

Consumption in grams/day 30.00 70.00 46.50 9.65

Frequency of dipping/day 10.0 30.0 14.18 3.065

Time the quid kept in the mouth
(minute)

5.00 25.0 9.373 2.539

Age (year) 27.0 79.0 50.90 11.86

Table 2: Shows copper results of subjects with OSCC, and toombak dippers as compared to healthy control.

Parameter Group Mean S±D P-Value
Serum Copper mg / L 1-Toombak Dippers 0.83 ± 0.22

2-Healthy control 0.78 ± 0.27
3-OSCC Patients 0.69 ± 0.23

0.002

Table 3: Shows serum Zinc results of subjects with OSCC., and toombak dippers as compared to healthy
control.

Parameter Group Mean ± SD P-Value
Serum Zinc mg / L 1-Toombak Dippers 0.79 ± 0.27

2-Healthy control 0.83 ± 0.33
2-OSCC Patients 0.69 ± 0.25

0.039

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Table 4: Shows Magnesium results of subjects with OSCC., and toombak dippers as compared to healthy
control.

Parameter Group Mean ± SD P-Value
Serum Magnesium mg / L 1-Toombak Dippers 24.8 ± 4.56

2-Healthy control 26.1 ± 5.10
3-OSCC Patients 21.7 ± 5.60

0.000

Table 5: Descriptive statistics of serum Copper level in toombak dippers compared to healthy control.

Serum copper Mg/L Minimum Maximum Mean Std. Deviation

1-Toombk dippers 0.10 1.65 0.87 0.22

2-Healthy Control 0.35 1.38 0.78 0.27

Table 6: Descriptive statistics of serum zinc level in toombak dippers compared to healthy control.

Serum Zinc Mg/L Minimum Maximum Mean Std. Deviation

1-Toombk dippers 0.22 1.70 0.79 0.28

2-Healthy Control 0.39 1.61 0.83 0.33

Table 7: Descriptive statistics of Magnesium level in toombak dippers compared to healthy control.

Serum Magnesium Mg/L Minimum Maximum Mean Std. Deviation

1-Toombk dippers 15.3 37.5 24.8 4.56

2-Healthy Control 15.8 35.5 26.1 5.10

4. Discussion

serum copper level of toombak dippers group in the present study was high compared
to the level of control group serum copper (Table 2) the difference was statistically
significant. The serum copper level of squamous cell carcinoma was found to be
high compared to healthy control.The results agreed with the findings of Tudek, et al
[28] Darna, et al [29] and with Sreelatha, et al [30] who reported increased level of
serum copper in oral squamous cell carcinoma patients group compared to control
group. Varhgese et al. [31], studied the serum copper level in leukoplakia, they found
high level of copper in leukoplakia group compared to control group with a statistical
significance Silverman, and Thompson. [32] reported significant increase in serum
copper in head, and neck squamous cell carcinoma. Serum Zinc in the present study
had shown lower level in toombak dippers, and squamous cell carcinoma patients
compared to control group. (Table 3). There was statistically significant difference.
Darna, etal [29] reported statistically significant difference between low serum zinc
of oral squamous cell carcinoma patients group, and the higher level of zinc of the
control group Sreelatha,et al. [30] reported similar results. The results of the present
study disagreed with the study of Boliniarz, et al [33], who had found higher zinc level in

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oral squamous cell carcinoma patients group compared to healthy control group, and
also contradicted with the results of Silver,and Thompson [32] who had found higher
level of zinc in cancer patients compared with controls. Magnesium levels in serum of
the groups of the present study had shown statistically significant variation (Table 4).
The level in toombak dippers group was found to be (21.6 ± 4.56 mg/l),while it was
(26. ± 15.1 mg /l), in healthy control group.These results agreed with what was reported
by (Deheinzelin, et al [34] who had found hypo magnesemia in oral cancer patients
confirming the earlier results of (Chernow, et al (1995), and (Guern, et al (1996) who
both found low magnesium level in oral squamous cell carcinoma patients compared to
healthy control group. Sreelatha, et al [30] found decreased serum magnesium level in
oral squamous cell carcinoma patients group compared to healthy control group. Taysi
S, etal [34] also reported lower serum magnesium level in cancer patients compared
to controls. Vyas RK et al [36] concluded that the zinc, and serum magnesium levels
were found to be low in the patients with malignancies. The results of the present study
disagreed with the findings of Akinmoladunm, etal [37] as they reported higher serum
magnesium level in cancer patients compared to controls. Trace elements assessment
in serum, is a highly appreciated parameter in diagnosis, and prognosis processes
concerning oral squamous cell carcinoma.

5. Conclusion

The present study is a trial to assess the effects of toombak on dippers depending on
biochemical markers, with the advantage of detecting the anticipated changes before
being displayed by the affected cells. The results of the three groups studied (toombak
dippers, control group, OSCC group) had shown clear variation between the 3 groups
members. The change in the level of these elements can be used as marker that reflects
susceptibility towards cancer.

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	Introduction
	Materials and Methods
	Methodology for trace Elements estimation 
	Statistical analysis

	Results
	Discussion
	Conclusion
	References