RESEARCH PROJECT
STATUS OF IRON AND OXIDATIVE STRESS IN DIABETIC
PATIENTS WITH IRON DEFICIENCY ANEMIA
Dr. Mala Dharmalingam and Dr Sara Rani Marcus
Dept. of Endocrinology and Biochemistry,
M.S.Ramaiah Medical College,
Bangalore 560 054
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INTRODUCTION
There is a close relationship between Diabetes mellitus, iron metabolism and oxidative
stress. Excess of free catalytic iron in circulation is known to cause diabetes mellitus as
seen in cases of systemic iron overload like Hemochromatosis (1). The removal of this
catalytic iron from the circulation by phlebotomy or by using iron chelators like
desferroxamine has been shown to reverse the diabetic state (1).
The uptake of iron into cells is influenced by insulin. In diabetes mellitus, the uptake
of iron is reduced as a result of insulin deficiency or insulin resistance and the pool of
catalytic iron in the circulation is increased (2). Normally, this catalytic iron is
sequestered by oxidation followed by binding to transferrin.
In diabetes mellitus, there is an increase in the oxidative stress due to autooxidation of
glucose, formation of advanced glycation end products and decreased antioxidant
defence mechanisms.
Transition metals, like iron, are also known to promote oxidative stress. Catalytic iron
in plasma catalyses the generation of highly reactive hydroxyl radical and superoxide
anion that can initiate and propagate the cascades leading to oxidative damage(2).
Several studies have shown that there is increased oxidative stress in diabetic patients
with iron overload (2). However, there is a paucity of studies relating to oxidative stress
in diabetic patients with iron deficiency anemia.
Iron deficiency anemia is a common nutritional disorder predominant in developing
countries like India. In iron deficiency anemia, circulatory iron is found to be normal or
low, together with enhanced oxidative stress (3). The hyperglycemia in diabetes leads to
glycation of transferrin and other proteins and this decreases the binding of iron and
increases the pool of catalytic iron in the circulation and the extent of oxidative stress
(2).
Hence, a comparative study of oxidative stress in type 2 diabetic patients with and
without iron deficiency anemia together with normal healthy controls would help to
elucidate the role of iron as a pro-oxidant in the pathogenesis of diabetes mellitus and its
complications and contribute to improve the management of diabetic patients with iron
deficiency anemia.
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OBJECTIVES
1. To study the role of iron in type 2 diabetes with iron deficiency anemia and
compare it with type 2 diabetics with normal iron levels.
2. To measure the levels of MDA (index of lipid peroxidation) and uric acid
(antioxidant) in diabetic patients with iron deficiency anemia, in diabetic patients
with normal iron levels.
MATERIALS AND METHODS
The study will be conducted on patients attending the Endocrinology department, MSR
Hospitals, Bangalore.
The subjects will be grouped as follows:
Group I – 30 adult diabetic patients with iron deficiency anemia.
Group II – 30 adult diabetic patients without iron deficiency anemia.
Statistical Analysis :
Student ‘t’ test and analysis of variance (ANOVA) will be used to compare the data
between the two groups.
Method of collection of data
Hospital diagnosed cases of diabetes as per American Diabetic Association criteria based
on the following inclusion and exclusion criteria will be chosen for the study. Patients
who volunteer for the study after informed consent will be taken and there will be no
financial liability on them. Ethical clearance from the Ethical Review Board of the
Institution will be obtained.
Inclusion criteria:
1 .Patients of either sex diagnosed as per American Diabetic Association criteria
(4) with good diabetic control(HbA1C 6-8%).
2. Patients aged between 25 and 60 years.
3. Patients of either sex diagnosed as iron deficiency anemia with hemoglobin levels
<10 g% in females and 11 g% in males (Group II) (5).
Exclusion criteria:
1. Patients taking any other medications like antioxidants, steroids, vitamins etc.,
which will affect the oxidative stress.
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2. Patients with secondary complications of diabetes like micro- or macrovascular
disorders.
3. Patients who are tobacco users, smokers and alcoholics.
4. Patients with secondary causes of diabetes.
5. Patients with dyslipidemias.
Method of collection of data
Blood samples will be collected from the subjects after informed consent. The
following clinical parameters will be recorded: Age, family history, height, weight, waist
and hip circumferences and blood pressure.
The following biochemical parameters will be estimated:
1. Estimation of hemoglobin levels by cyanomethhaemoglobin colorimetric assay.
2. Estimation of serum iron by photometric test using chromogen ferrozine kit.
3. Estimation of total iron binding capacity by photometric test using chromogen
ferrozine kit .
4. Estimation of serum ferritin by radioimmunoassay kit.
5. Estimation of serum MDA by Thiobarbituric acid reactive substances (index of
lipid peroxidation) method (6)
6. Estimation of uric acid by enzymatic kit method.
REVIEW OF THE STATE OF THE PROBLEM
There is a bi-directional association between Diabetes mellitus and iron metabolism.
Diabetes is a metabolic disorder resulting in hyperglycemia caused due to insulin
deficiency or insulin resistance. Systemic iron overload contributes to abnormal glucose
metabolism leading to type 2 diabetes and this is probably mediated by: (i) insulin
resistance caused directly by iron overload, (ii) insulin deficiency as a result of oxidative
stress on the pancreatic beta cells leading to cell death and decreased secretion of insulin
and (iii) hepatic dysfunction. (2).
Insulin affects the uptake of iron from the circulation into the cells. In diabetes, there
is a decrease in the uptake of iron and increase in the circulatory pool of catalytic iron
(Fe++). This catalytic iron causes the production of oxygen free radicals and leads to
oxidative damage.(1).
Oxidative damage is caused by an imbalance in the production of free radicals and
antioxidant defence mechanisms. Disturbance in the normal redox state causes the
production of free radicals which induce oxidative stress and damage the proteins, lipids
and DNA. The effect of the oxidative stress depends on the severity and duration of the
stress. The protective mechanisms include antioxidants like vitamins C, E, beta carotene,
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bilirubin, uric acid, glutathione and enzymes like catalase, glutathione peroxidase and
superoxide dismutase.
Insulin resistance and hyperglycemia observed in diabetes causes overproduction of
oxygen free radicals.. This is due to autooxidation of glucose, formation of advanced
glycation end products and decrease in the antioxidant defence mechanisms. Iron is a
transition metal which can be oxidized and reduced easily and serve as a potential
catalyst in many cellular reactions that produce oxygen free readicals. The catalytic iron
is normally oxidized by ceruloplasmin and bound to transferrin in the plasma and stored
as ferritin in the tissue and thereby preventing the hazardous action of free radicals.(7).
Insulin resistance is known to be closely related to the total body iron stores and
increased ferritin has been suggested as a marker for insulin resistance. An increase in
the free iron level stimulates the synthesis of ferritin. A decrease in the antioxidant
levels, as seen in diabetes, increases the reducing potential and anaerobic environment
resulting in the release of iron from ferritin. This causes the oxidative stress through
Fenton reaction leading to the formation of highly reactive free radicals like hydroxyl
radical and superoxide anion which contribute to tissue damage.(7).
In addition, glycation of transferrin and decreased ability to bind iron in diabetes also
leads to oxidative stress from the increased systemic catalytic iron. Glycated transferrin
also facilitates the production of oxygen free radicals that further amplify the oxidative
stress and damage caused by iron (2).
Iron deficiency anemia is one of the most common nutritional disorders observed in
India caused due to poor consumption of iron. In iron deficiency anemia, the levels of
iron in the blood may be normal or low and the ferritin levels are decreased. It is also
known that iron deficiency anemia enhances oxidative stress independently (3).
Therefore, the coexistence of iron deficiency anemia with diabetes mellitus would
probably enhance the catalytic iron in blood and lead to enhanced oxidative stress.
Studies on oxidative stress in iron deficient anemic patients with type 2 diabetes
mellitus are few. Hence, this investigation proposes to study the status of iron and
oxidative stress in type 2 diabetic patients with and without iron deficiency anemia in
order to understand whether iron augments the oxidative damage of type 2 diabetes and
its complications and thus provide better management of these patients.
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REFERENCES
1. Swaminathan, S., Fonseca, V.A., Alam, M.G. et.al. (2007) The role of iron in
diabetes and its complications. Diabetes Care 30: 1926-1933.
2. Fernandez-Real, J.M., Lopez-Bermejo, A ., Ricart, W (2002) Cross-talk between
iron metabolism and diabetes. Diabetes 51: 2348-2354.
3. Casnueva E., Viteri, F.E., (2003) Iron and oxidative stress in pregnancy. J. Nutr.
133: 1700S-!708S.
4. American Diabetic Association (2003) The expert committee on the diagnosis
and classificiation of diabetes mellitus: follow –up report on thew diagnosis of
diabetes mellitus. Diabetes Care 26: 3160-3167.
5. Park, K., Park’s Textbook of preventive and social medicine. 18th ed. pp450.
6. Wilbur, K.M., Bernheim F., Shapiro, O.W. (1943) The TBARS reagent as test
for the oxidation of unsaturated fatty acids by various agents. Arch. Biochem.
Biophys. 24: 305-313.
7. Galaris, D., Pantopoulos, K. (2008) Oxidative stress and iron homeostasis:
mechanistic and health aspects. Critical Rev Clin Lab Sci. 45: 1-23