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Laxmi Shruthi et al. Int. Res. J. Pharm. 2013, 4 (5)
Page 219
INTERNATIONAL RESEARCH JOURNAL OF PHARMACY
www.irjponline.com ISSN 2230 – 8407
Research Article
ROLE OF COPPER AND IRON DEFICIENCIES IN PATHOGENESIS OF RECURRENT APHTHOUS ULCER
Laxmi Shruthi1*, Shetty Pushparaja1, Pandey Bhavna2
1Department of Oral Pathology & Microbiology, A.B. Shetty Memorial Institute of Dental Sciences, Mangalore, Karnataka,
India
2Department of Oral Pathology & Microbiology, Vydehi Institute of Dental Sciences, Bangalore, Karnataka, India
*Corresponding Author Email: laxmigbhat@gmail.com
Article Received on: 20/03/13 Revised on: 11/04/13 Approved for publication: 11/05/13
DOI: 10.7897/2230-8407.04546
IRJP is an official publication of Moksha Publishing House. Website: www.mokshaph.com
© All rights reserved.
ABSTRACT
The aim of the study was to esti mate the serum levels of copper and i ron in recurrent aphthous ulcer (RAU) pat ients and to c ompare these levels with normal
healthy individuals. A total of 60 patients were examined with 30 recurrent aphthous ulcer (RAU) patients and 30 heal thy controls. Serum samples obtained
from the pat ients were digested using nitric acid and subjected to atomic absorption spectrophotometer for copper estimation. Serum i ron was esti mated using
Bathophenanthroline method. All parameters were statistically analysed using student’s t- test. A highly significant decrease in serum level s of copper and iron
were seen in recurrent aphthous ulcer patients than in healt hy control group. Copper and iron deficiencies may play a key role in etio-pathogenesis of recurrent
aphthous ulcer.
Keywords: Recurrent aphthous ulcer, copper, iron.
INTRODUCTION
Recurrent aphthous stomatitis (RAS) is recognized as the
most common oral mucosal disease. Epidemiologic studies
indicate that prevalence of recurrent aphthous stomatitis is
between 25%-30% in general population. Recurrence is the
hallmark of recurrent aphthous ulcer and three month
recurrence rates are as high as 50%1. RAS is characterised by
recurrent, small, round, or ovoid ulcers often multiple with
circumscribed margins, erythematous haloes, and yellow or
grey floors that present first in childhood or adolescence2.
Stanley has classified RAU into three variants namely minor
aphthous ulcer, major aphthous ulcer and herpetiform ulcer3,4.
Local, systemic conditions, genetic, immunologic, and
infectious microbial factors have been proposed as
contributory factors in RAU5. Nutrition as a major
contributing factor in recurrent aphthous ulcer is debatable.
Haematinic deficiency is found in up to 20% of patients1.
Supplementation of aphthous ulcer with zinc and copper
sulphate has known to cause fast recovery6. Thus numerous
attempts have been made to prove the role of nutrition in
aetiology and management of RAU. However no conclusive
results are available on the role of copper and iron in
aetiology and management of RAU, especially in Indian
population. Hence this study was planned to assess the role of
copper and iron in etio-pathogenesis of RAU.
MATERIALS AND METHODS
A total of 60 patients in the age range of 20 to 40 years were
examined with 30 recurrent aphthous ulcer patients and 30
healthy controls from Out Patient Department of a private
dental college in Mangalore. The study group consisted of
patients diagnosed with recurrent aphthous ulcer. Diagnosis
of recurrent aphthous ulcer was made in the Out Patient
Department by a single examiner in order to avoid any inter-
examiner variability. Inclusion criteria included those patients
who have experienced at least 3 recurrences of aphthous ulcer
in past one year and were examined between third and fifth
day of ulcer period. The control group consisted of healthy
individuals with clinically normal oral mucosa who reported
to the dental hospital for routine diagnostic and therapeutic
purposes. Exclusion criteria of the study included patients
with systemic diseases, traumatic ulcers, ulcers due to
hormonal changes, ulcers as a part of ulcerative colitis,
Chron’s disease, Behcet’s syndrome, and Reiter’s syndrome,
ulcers related to food allergies, drug induced ulcers and those
on copper and/or iron supplementations. Ethical clearance
was obtained from an institutional review board to carry out
the study and written informed consent was obtained from the
patients. Performa inventory was completed detailing name,
age, gender, and relevant medical history. Clinical history
related to site, frequency, number, and type of recurrent
aphthous ulcer was recorded. Preliminary blood
investigations were carried out for RBC count, total
leukocyte count, hemoglobin, hematocrit and red blood cell
indices in both control group and recurrent aphthous ulcer
patients. Both the groups in the study were subjected for
copper and iron analysis. Approximately 5.0ml of blood was
drawn from the patients by venipuncture using a sterile
disposable syringe. Blood samples were subjected to
centrifugation at 3,000rpm for 10minutes and serum was
separated. These samples were stored at -20◦c until shortly
before assay.
Ethical Clearance Number: Absm/Ec/80/2010 on 22/10/10
Copper Estimation
Serum samples were digested using nitric acid and serum
copper concentration was determined using atomic
absorption spectrophotometer. About 20µl of each sample
was introduced through sample port into the Nebulizer, where
sample gets converted into tiny aerosols. These aerosols were
fed into the air-acetylene flame where the atomization of the
sample occurs and copper present gets excited. These excited
copper atoms absorb energy from the spectrum produced by
the copper hallow cathode lamp. The decrease in energy is
measured which is directly proportional to the concentration
of the copper atoms. Absorbance signal was measured and
the concentration of copper in each sample was determined
by comparison of its absorbance signal with that of the
standard solutions with known copper concentrations.
Laxmi Shruthi et al. Int. Res. J. Pharm. 2013, 4 (5)
Page 220
Iron Estimation
Serum iron was estimated using Bathophenanthroline
method. An average of 100µl of serum sample was taken in
a clean microfuge tube and made up to 250µl with deionised
water. To this diluted sample, 500µl of protein precipitating
solution was added. The mixture is then centrifuged at
2000rpm for 10 minutes. An average of 500µl of supernatant
was taken and added to 500µL of the chromogen solution.
The optical density of the pink colour formed was read
immediately at 535nm against a blank treated in a similar
method as the test where in the sample was replaced with the
deionised water. Comparison between means of study and
control groups was done using student’s t-test in SPSS
software version 13.0. Probability values, p < 0.05 were
considered as significant (s) and p < 0.01 were considered as
highly significant (HS).
Table 1: Comparison of Mean Serum Copper Levels in Study and Control Groups
Group
N
Minimum
Maximum
Mean
Std. Deviation
Median
t value
p value
Study group
30
70.00
84.00
75.95
4.55
75.00
12.785
.000
Control group
30
86.00
113.00
93.90
6.19
93.00
HS*
Total
60
70.00
113.00
84.93
10.53
85.00
*Highly significant at 1% level of significance (p< 0. 01)
Table 2: Comparison of M ean Serum Iron Levels i n Study and Control Groups
Group
N
Minimum
Maximum
Mean
Std. Deviation
Median
t value
p value
Study group
30
8.83
9.57
9.25
.15
9.27
9.391
.000
Control group
30
9.40
10.32
9.78
.27
9.72
HS*
Total
60
8.83
10.32
9.52
.35
9.42
*Highly significant at 1% level of significance (p< 0. 01)
Figure 1: Comparison between mean se rum copper levels of st udy group
and control group
Figure 2: Comparison bet ween mean serum iron levels of study group
and control
RESULTS
The age of patients in control and study group ranged
between 20 and 40 years. In the present study all the 30
recurrent aphthous ulcer patients showed minor type of
recurrent aphthous ulcer. Among recurrent aphthous ulcer
(RAU) patients, 60% were females and 40% male. Among
control group 60% were males and 40% females. Most
common site of RAU was labial mucosa (46%) followed by
buccal mucosa (27%), tongue (17%), and floor of the mouth
(10%). Most patients gave the history of frequency of
occurrence of RAU to be once in three months (50%). There
was statistically highly significant decrease in serum levels of
copper (Table 1, Figure 1) as well as iron in recurrent
aphthous ulcer patients when compared to control group
(Table 2, Figure 2).
DISCUSSION
Recurrent aphthous stomatitis (RAS) is a disease
characterized by appearance of small round to oval recurring
ulcers in the oral mucosa without any sign of other diseases.
Episodes of recurrent aphthous ulcers may be infrequent
resulting in mild or only transient symptoms or may be
continuous causing persistent pain and disability over
extended period of time. RAS occurs in men and women of
all ages, races and geographic regions. The peak age of onset
of RAS is the second decade of life7. Attacks of recurrent
aphthous ulcer may be precipitated by local trauma, stress,
food intake, drugs, hormonal changes, vitamin and trace
element deficiencies4. Recently more emphasis is being laid
on micronutrient deficiencies in recurrent aphthous ulcer
patients. Adequate intakes of micronutrients like copper, iron,
and zinc are required for the immune system to function
efficiently. Micronutrients contribute to the body’s natural
defences on three levels by supporting physical barriers-
skin/mucosa, cellular immunity and antibody production.
Micronutrient deficiency suppresses immunity by affecting
innate, T cell mediated and adaptive antibody responses,
leading to dysregulation of the balanced host response8.
Micronutrients in the body have known to show interactions.
Copper and iron levels are interdependent as iron requires
copper dependent enzymes. The best-characterised link
between copper and iron is provided by ceruloplasmin, a
multi-copper binding protein that acts as a serum ferrioxidase
and is essential for the mobilisation of iron from storage
tissues. Decreased copper status is known to reduce
ceruloplasmin production and impair ferrioxidase activity
thus leading to decreased tissue iron release resulting in iron
deficiency anaemia. Dietary iron absorption also requires the
presence of a multi-copper ferrioxidase9. Treatment of
recurrent aphthous ulcer patients with copper sulphate is
known to reduce the severity and incidence of ulcers. In the
present study serum copper were significantly lower in
recurrent aphthous patients than in healthy control group.
These findings are in accordance to study conducted by Bor
Laxmi Shruthi et al. Int. Res. J. Pharm. 2013, 4 (5)
Page 221
N M et al. in 19906 where copper sulphate treatment of 67
patients revealed complete recovery of ulceration in 40
patients and 22 showed definite improvement after 3 months
of treatment. But according to the study conducted by Pang
JF in 199210 copper levels were within the normal range in
recurrent aphthous ulcer patients. Iron is essential regulation
of cell differentiation and cell growth8. Cytochrome oxidase
is an iron-dependent enzyme which is required for the normal
maturation of the epithelium. In iron deficiency state, the
levels of cytochrome oxidase are low, consequently leading
to epithelial atrophy. An atrophic epithelium makes the oral
mucosa vulnerable to the soluble irritants. Lack of iron in
tissues causes improper vascular channel formation resulting
in decreased vascularity. This leads to derangement in the
inflammatory- reparative response of the lamina propria
resulting in defective healing11. Iron deficiency is known to
cause recurrent aphthous ulcer. Relationship of recurrent
aphthous ulcer to iron deficiency which in itself may be
associated an underlying gastrointestinal disorder or a poor
diet. In the present study serum iron level was determined by
Bathophenanthroline method. Present study shows that serum
iron levels are significantly lower in recurrent aphthous ulcer
patients compared to normal healthy controls which is in
accordance to studies conducted by Wray D et al. in 197812,
Hutcheon A W in 197813, Porter S R et al. 198814, Barnadas
MA et al. in 199715, Burgan SZ et al. in 200616 and Ogura M
et al. in 199817. But according to studies conducted by
Koybasi S et al. in 2006 7, Olson A et al. in 198218, and Piskin
S et al. in 200219 iron deficiency was not found in recurrent
aphthous ulcer patients. There was no significant difference
between iron levels in study and control groups. Also
according to studies conducted by Wray D et al. in 197520
and Challacombe SJ et al. in 197521 replacement therapy with
iron for recurrent aphthous ulcer patients has shown less or
no response. This inconsistent response to iron replacement
therapy was because patients in study group had low serum
iron levels secondary to chronic disease and not due to
primary iron deficiency anaemia.
CONCLUSION
Recurrent aphthous ulcer is a multi-factorial disease. Present
study suggests that combined nutritional deficiencies,
especially copper and iron can occur in recurrent aphthous
ulcer. Hence routine haematological investigations of copper
and iron may be carried out in recurrent aphthous ulcer
patients. Also supplementations of copper and iron in
recurrent aphthous ulcer patients may prevent or decrease the
frequency of recurrent aphthous ulcers.
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Cite this article as:
Laxmi Shruthi, Shetty Pushparaja, Pandey Bhavna. Role of copper and iron
deficiencies in pat hogenesis of recurrent aphthous ulcer. Int. Res. J. Pharm.
2013; 4(5):219-221
Source of support: Nil, Conflict of interest: None Decl ared
