|
Note: Tables and figures
of the article can be access and seen in the PDF file.
Introduction
Iron deficiency anemia is a major
public health problem in many developing countries. The
vulnerable segments of the population are pregnant women
and children and one of the common short term measure to
control Iron deficiency anemia in them is oral iron
supplementation (Srigiridhar, Nair KM 2000).The
prevalence of iron deficiency anemia is still on the
high in developing countries like india though a lot is
known about it and many treatment strategies are
available. The increasing prevalence could be due to
poor compliance which is attributed to the side effects
that arise during oral iron supplementation.
Absorption of iron is a highly regulated process. Only
10-15% of orally administered iron gets absorbed in
small intestine and in iron deficiency the percentage
may even increase to a maximal of 40% (Madhavan Nair
2001). Iron absorption is
increased in iron deficiency, i.e. when iron stores are
low, and in anemia, i.e. when tissue oxygen supply is
compromised. Conversely, iron absorption decreases when
iron stores are high. There are two forms of iron namely
haem and non haem iron. Haem iron gets directly absorbed
into the intestinal cells. In the duodenal brush border,
Dcyt b reduces ferric (trivalent) non-haem iron to the
ferrous (divalent) state, which is taken up from the
lumen by the ‘‘Divalent Metal Transporter 1’’ ( ¼
DMT-1), the expression of which is related to body iron
status. The ‘‘mucosa block’’ mechanism reduces
iron absorption after a preceding high iron exposure,
presumably by diminishing the number of DMT-1 receptors.
Ferrous
iron is the form that is mostly used for correction of
iron deficiency. Acidic milieu facilitates the
absorption by keeping iron in the ferrous form. Most of
the ferrous iron gets converted into ferric in the
basolateral membrane and enters the circulation through
transferrin and the amount of iron entering the
circulation is regulated by body iron demands. A part of
ferrous iron is converted to ferric iron and stored in
intestinal mucosal cells as ferritin .The iron which is
stored in the intestinal mucosal cells as ferritin will
be lost after 2-3 days. Ferrous iron is a central
pro-oxidant that propagates free radical reactions
through Fenton chemistry during conversion of ferrous to
ferric iron both locally (in the gastrointestinal tract)
and systemically. An excess of pro-oxidants over
antioxidants results in oxidative stress (OS).
Fe(II)
+ H2O2
àFe(III)
+ OH_ + OH+ (Fenton)
Fe(III) + O2 2-à
Fe(II) + O2
Intake of oral
iron preparations at therapeutic dose levels frequently
causes nausea, vomiting and epigastric discomfort. These
effects seem to be due to mucosal irritation and altered
gastrointestinal motility probably caused by ROS.
Iron and reactive Oxygen species (ROS)
can enhance mucosal injury by several mechanisms. One is
by initiating lipid peroxidation either by iron itself
or by the ROS produced during the Fenton reaction. Lipid
peroxidation of cell membranes, including mitochondrial
membranes, can in turn compromise cell integrity and
function and affect its energy status, thereby causing
further tissue injury. Iron and ROS can also amplify
intestinal inflammation by such mechanisms as increasing
mucosal permeability, recruiting and activating more
neutrophils and activating NF-κB, thereby upregulating
the production of proinflammatory cytokines (Carrier et
al 2002).
Unabsorbed dietary iron enters the colon and in
conjunction with intraluminal bacteria may become
available for participation in a combination of
Haber-Weiss and Fenton type reactions that generate
hydrogen peroxide and hydroxyl radicals at the mucosal
surface. Moreover continuous exposure of iron to
intestine is believed to decrease iron absorption from
subsequent doses (Lund et al 1999).
Oxidative stress as shown to play an important role in
pathogenesis of IDA (Vives et al; 1995).The results of a
recent study confirmed that antioxidant enzymes activity
like GSH-Px is decreased in children with IDA. These are
indicators of increased lipid peroxidation (Tekin et al
2001). Furthermore, it has been shown that the addition
of synthetic antioxidants in the treatment of children
with IDA results in decrease of lipid peroxidation,
prevention of pathologic progression and rapid
improvement of clinical manifestations (shved et al;
1995). This confirms iron deficiency anemia is a state
of oxidative stress.
The
effects of antioxidants with oral iron to combat the
stress and side effects have been tried in both human
subjects (Carier et al 2002) and animals (Srigiridhar,
Madhavan Nair 2000). Studies of this kind in children
are scanty (Tekin et al 2001; Panchenko et al 1979).
Commonly used antioxidants are vitamin C and vitamin E.
Vitamin E is the most potent liposoluble antioxidant and
has the potential to improve tolerance of iron
supplementation and prevent further tissue damage
(Burton et al 1986). Vitamin C helps in the absorption
of iron by reducing non haem ferric to ferrous iron. By
facilitating iron absorption, vitamin c makes more
ferrous iron available to participate in Fenton reaction
leading to oxidative damage. Addition of vitamin c with
iron has proved to be a toxic cocktail rather being an
advantage as an antioxidant (Anna EO Fisher† and Declan
P Naughton; 2004). The role of vitamin C as an
antioxidant or pro-oxidant is still not clear. Therefore
a study was undertaken to evaluate the oxidative stress
in IDA in children and the effect of antioxidants during
iron supplementation and to evolve an optimal suitable
therapeutic strategy to minimize oxidative stress and
there by adverse clinical effects
Methodology
Study Sample Population
All the children attending the
pediatrics OPD in IRT PMCH during JULY/AUGUST 2008 were
evaluated clinically for anemia.
Inclusion Criteria
Clinically suspected anemic
children were subjected to hemoglobin evaluation to
confirm their anemic status.
Operational Definition for Anemia
The children were termed anemic
keeping the WHOs criteria
<11g/dl
for children aging 6 months to 6 years
<12g/dl for children >6 years
Later a
written consent form was obtained from the parents of
all children. Everyone were given a brief counseling
regarding iron deficiency anemia and the present study.
Health education was also imparted to the parents
regarding best dietary practices for good iron stores.
In
order to exclude other causes for anemia a complete
blood count (CBC) with ESR including hematological
indices like MCV, MCH, MCHC, and RDW were done for all
children. CBC and other indices were done using an
automated counter.
Iron Profile
Iron profile including serum
iron, feritin , percentage saturation of transferrin and
TIBC were done for each subject to confirm iron
deficiency anemia and included in the study. Serum iron
was measured by Ferrozine method without
deproteinisation (micro g/dl) and serum ferritin by
fully automated bidirectionally interfaced
chemiluminescent immunoassay (ng/ml). With respect to
iron profile, children were graded into 3 grades namely
grade 1- negative iron balance, Grade 2 – stage of iron
deficient ineffective erythropoiesis, grade 3 – iron
deficiency anemia.(Harrison internal medicine 17th
edition; volume 1; page 630; fig 98.2)
Exclusion Criteria
Children who were already
diagnosed as anemic and were on hematinics were excluded
for the study. Children with other systemic illnesses
were also excluded.
Nutritional Assessment
The nutritional assessment of the
children was done by using a twenty four hour dietary
recall survey method and a food frequency questionnaire.
The survey aimed at evaluating the general dietary
pattern and the number of meals consumed per day. The
questionnaire included the frequency of various food
items like staple foods (rice/wheat/bajra/ragi/maize),
gram (green/Bengal/black/peas), greenleaves (amaranth/sirukeerai/cauliflower),
oil and nuts(coconut/groundnut/cashew nuts), fruits,
milk, spices and animal sources
(egg/beef/crab/prawn/chicken) and iron rich snacks.
The
recommended dietary allowance (RDA) for each of the
nutrient in terms of calories (Kcal), Protein (g), Fat
(g), iron (mg), and calcium (mg) was calculated for each
child and compared to the standard RDA for each age
group as laid down by the National Institute Of
Nutrition (NIN), ICMR, Newdelhi, India.
The
questionnaire also included demographic factors like
height, weight and were expressed in terms of percentile
for height (cm) by age and weight (kg) by age as
specified by CDC 2000 standards.
Oxidative Stress Profile
The level of oxidative stress was
assessed by measuring plasma lipid peroxides and plasma
lipid hydroperoxides).Lipid peroxides were measured in
plasma by the thiobarbituric acid method
as described by BUEGE and AUST, 1978 (micro
moles/L) and lipid hydroperoxides were measured with FOX
Reagent II as described by JIANG et al
1990 (micro moles/L).
Sampling Techniques and Study Groups
Children in the study were
assigned numbers starting form 1,2,3 and so on as they
were diagnosed sequentially as anemics (first diagnosed
was assigned 1, second one as 2 and so on). The children
in the study were randomly assigned into three groups in
the following manner. Children assigned numbers 1,4,7,10
etc were in group 1, children numbered 2,5,8,11 etc were
put in group 2, children numbered 3,6,912 etc were put
in group 3.The three groups were different in terms of
oral iron supplementation.
Group I- oral iron only,
in the form of ferric ammonium citrate (syrup
ferrochelate), 6mg/kg/day in two divided doses; Group
II- oral iron with vitamin C (tablet Celin) at the
dose of 250 mg once daily; Group III- oral iron
with vitamin E (capsule Evion) at the dose of 200 μg
once daily. The parents and the biochemical analyst were
blinded regarding the groups.
All the
children in the study were dewormed before therapeutic
intervention.
Follow-up
All the children in the study
were followed up continuously up to 30 days after
initiation of drug treatment. The parents were
instructed to contact at any time if they face any
adverse effects during the treatment. All the parents
were requested to attend the OPD for 2 follow ups:
-
First Follow up: 10 days after initiation of
treatment
-
Second Follow up: 30 days after initiation of
treatment
Adverse
effects (if any) were asked for during each follow ups.
Indices of oxidative stress namely plasma lipid
peroxides and lipid hydroperoxides were measured again
during both follow up. Iron profile (serum iron and
ferritin) was measured in the second follow up to look
for improvement due to therapeutic intervention.
Institutional committee approval was obtained prior to
the study
Statistical Analysis and Study Type
The statistical methods employed
in the study were Analysis Of Variance (ANOVA) and
Paired-t test. The software used for analysis is SPSS.
Type of study is Randomised clinical trial.
Results
Majority of the subjects were
male (16) and majority of them (12) belong to the age
group of 1-5 years. Only two were less than 2 years and
four in the age group of 5-10 years and three more than
10 years. Fourteen out of twenty one subjects had their
weight less than third percentile, eleven had their
height less than third percentile. When height and
weight were put together, nine were less than third
percentile. Most of the subjects were of low
socioeconomic status.
With
respect to the nutritional profile, majority of the
subjects (17) were nonvegetarian. Majority of them (13)
had three meals pattern per day. The food composition
showed that inclusion of iron rich sources in their diet
like green leafy vegetables, chicken, egg and fish was
meager in majority of the subjects. The deficit profile
comparing the RDA of each nutrient with the standard RDA
showed that most of the subjects consumed less than
their required RDA with respect to all nutrients.
By
virtue of iron profile, subjects fall under three
categories (Table I). Majority of them were in Grade III
(iron deficiency anemia) whereas six subjects were in
grade I (stage of negative iron balance), and four were
in grade II (Stage of ineffective erythropoiesis). In
terms of response to oral iron therapy as measured by
serum iron and ferritin, all the subjects in three
groups showed significant improvement by Paired T
test(Table II & III) but there is no significant
difference between the three groups by ANOVA (Table IV).
Oxidative stress indices namely lipid peroxide and lipid
hydroperoxides though showed a decreasing trend in all
the three groups was not statistically significant by
ANOVA (Table V&VI).The basal level of lipid
hydroperoxide was statistically significant than lipid
peroxide by ANOVA (Table V&VI). The parents of the
subjects reported improvement in them during I Follow
up. Adverse effects were not reported from any of the
subjects during treatment.
Discussion
Iron deficiency anemia presents
with a spectrum of oxidative stress and altered
antioxidant activity. The view of treating IDA has been
changing for past few years. The fact that the ability
of iron in generating free radicals and hence the
adverse effect lead to the role of antioxidant as
adjuvant and also lead to modification of iron dosage
particularly in adults. Lipid peroxidation is a well
known example of oxidative damage in cell membranes,
lipoproteins, and other lipid containing structures.
Peroxidative modification of unsaturated phospholipids,
glycolipids, and cholesterol can occur in reactions
triggered by free radical species such as hydroxyl
radicals derived from iron-mediated reduction of
hydrogen peroxide. ROS like HO. generated
by Fenton chemistry (H2O2/iron)
gives rise to primary stage LOOHs (lipid hydroperoxides).
These LOOHs may undergo iron-mediated one-electron
reduction and oxygenation to give epoxyallylic peroxyl
radicals (OLOO.), which trigger exacerbating
rounds of free radical-mediated lipid peroxidation.
These free radicals get neutralized by antioxidant
defense system. When there is excess iron available as
in case of maintaining the same dose of iron throughout
the course of treatment for restoring the iron stores,
there would be generation of more free radicals beyond
the ability of antioxidant defense system. These leads
to oxidative damage which could be responsible for the
adverse clinical effects encountered during oral iron
therapy. Studies (Neeta Kumar et al 2009; Jansson et al
1985; Chen et al 2007; Yang et al 1999; Rehema et al
2004; Schumann 2001; Srigiridhar, Nair KM 1998) also
found that supplementation of iron leads to oxidative
stress.
In the
present study, there was no discernible clinical adverse
effect in all the three groups. However, all the three
groups showed good improvement in response to oral iron
as evident from the iron profile. There was a decreasing
trend in oxidative stress indices (lipid peroxides and
lipid hydroperoxides) in all the three groups although
there was no difference between the groups. So
supplementation of antioxidants with iron in children
has no added advantage. A significant level of lipid
hydroperoxides was observed in children with the
diseased state. These indicate that iron deficiency is a
state of oxidative stress. As we know iron is required
as a structural and functional component of various
compounds (catalase, peroxidase, cytochrome oxidase,
NADPH reductase, iron sulfur complex), it plays a vital
role in maintaining the antioxidant defense system of
our body. Normally there exists a balance between free
radical production and antioxidant defense system. In
iron deficiency anemia the enzymes involved in the
antioxidant defense system will be functionally
defective. So the balance gets tilted towards free
radicals triggering oxidative damage. Further iron
dependent mitochondrial oxidative phosporylation also
gets affected in iron deficiency causing decreased ATP
production and ultimately leading to loss of structural
and functional integrity of cell. Studies (Isler et al
2002; Moriarty et al 1995, Kumerova et al 1998;
Srigiridhar, Madhavan Nair 2000; King et al 2008) are on
par with the fact that antioxidant status is lowered in
iron deficiency.
The
correction of iron deficiency with oral iron
supplementation leads to rejuvenation of defective
antioxidant system and brings back the balance between
ROS and antioxidant system. This could explain the
absence of any adverse clinical side effect seen during
oral iron supplementation in the present study. Due to
rejuvenation of antioxidant defense system and metabolic
functions of the cell, supplementing antioxidants with
iron to combat free radicals is not needed. Antioxidants
could play a role once hemoglobin and serum iron reaches
normal level during later part of iron therapy where
therpaeutic iron acts as a pro-oxidant.
Conclusion
Oxidative stress as in adults
does exist in children with Iron deficiency. Among the
indices measured (lipid peroxides and hydroperoxides),
lipid hydroperoxides may be an earlier and sensitive
indicator of oxidative stress. But unlike in adults,
aggravation of oxidative stress and its consequent
adverse effects do not occur with oral iron
supplementation in children. There was no significant
difference in therapeutic response between the groups-
iron only, iron supplemented with antioxidants.
Therefore, oral iron alone is safe and efficacious in
children with iron deficiency anemia. However, results
have to be confirmed by a larger sample size and follow
up for a longer period of time with varying doses.
Acknowledgments
We dedicate this study to Dr.
N.Ramachandran, MBBS, DCH, M.D (Ped.), Professor
Emeritus, Department of Pediatrics, IRT PMCH. We are
also indebted to our Dean Dr. A. Celestine Rajmanohar,
M.D. (GEN MED) who gave us the ethical committee
approval for this study. There was no conflict of
interest between the authors. We should thank our
Professor Dr. Balasubramanian MBBS, DCH, M.D. (Ped.) who
was a constant source of support for us throughout the
study. We should thank Dr. Baskaran, Msc, PhD, Professor
and Head, Department Of Biochemistry, Perunthalaivar
Kamaraj Government Medical College, Pondicherry, India.
We should also thank Dr. Chelladurai, MBBS, DCH and Dr.
Bama, MBBS, DCH, faculty members of the department of
Pediatrics, IRT PMCH for their constant support
throughout the study. All the authors played very
important role in this study. We also extend our thanks
to the funding agencies which were very much needed for
the study.
Dr Parasuram & Dr Natesh Prabu
References
Anna EO Fisher† and Declan P
Naughton. Iron supplements: the quick fix with long-term
consequences. Nutrition Journal 2004; Jan 16;3:2.
Buege
JA, Aust SD. Microsomal lipid peroxidation,Methods in
Enzymology.1978; 52:302-10.
Burton,
G. W. & Ingold, K. U. (1986) Vitamin E: application of
the principles of physical organic chemistry to the
exploration of structure and function. Acc. Chem. Res.
19: 194–201.
Carrier
Julie, Elaheh Aghdassi, Jim Cullen and Johane P. Allard.
Iron Supplementation Increases Disease Activity and
Vitamin E ameliorates the Effect in Rats with Dextran
Sulfate Sodium-Induced Colitis. J. Nutr. 132: 3146–3150,
2002.
Chen Q,
Le GW, Shi YH, Zhang SM, Jin X. Effect of iron
supplementation on intestinal function and oxidative
stress in piglets with induced colitis. J Animal Feed
Sciences 2007;16: 205–13.
Harrisons Textbook of Internal Medicine 17th
edition; volume 1; Page 630; Fig. 98.2.
Jansson
LT, Perkkiö MV, Willis WT, Refino CJ, Dallman PR. Red
cell superoxide dismutase is increased in iron
deficiency anemia. Acta Haematol 1985; 74(4): 218-21.
Jiang
ZY, Hunt JV, Wolff SP. Ferrous ion oxidation in the
presence of xylenol orange for detection of lipid
hydroperoxide in low density lipoprotein; Analytical
Biochemistry.1992 May 1;202(2):384-9.
King
Sarah, Carmen M. Donangelo,Mitchell D. Knutson, Patrick
B, Walter, Bruce N. Ames, Fernando E. Viteri, and Janet
C.King. Daily Supplementation with Iron Increases Lipid
Peroxidation in Young Women with Low Iron Stores Exp
Biol Med 233:701–707, 2008.
Kumerova A, Lece A, Skesters A, Silova A, Petuhovs V:
Anaemia and antioxidant defense of the red blood cells.
Materia Medica Polona 30:12–15, 1998.
Lund
Elizabeth K, S Gabrielle Wharf, Susan J Fairweather-Tait,
and Ian T Johnson. Oral ferrous sulfate supplements
increase the free radical–generating capacity of feces
from healthy volunteers. Am J Clin Nutr 1999;69:250–5.
Madhavan Nair. Alternate strategies for improving iron
nutrition: lessons from recent research. British Journal
of Nutrition (2001), 85, Suppl. 2, 187-191.
Mehmet
Isler, Namik Delibas, Muhittin Guclu, Fatih Gultekin,
Recep Sutcu, Mehmet Bahceci, Ali Kosar. Superoxide
Dismutase and Glutathione Peroxidase in Erythrocytes of
Patients with Iron Deficiency Anemia: Effects of
Different Treatment Modalities.Croatian Medical Journal,
43(1):16-19, 2002.
Moriarty PM, Picciano MF, Beard JL: Classical selenium
dependent glutathione peroxidase expression is decreased
secondary to iron deficiency in rats. J Nutr125:293–301,
1995.
Neeta
Kumar, Nomita Chandhiok, Balwan S Dhillon, Pratik Kumar.
Role Of Oxidative Stress While Controlling Iron
Deficiency Anemia During Pregnancy – Indian Scenario.
Ind J Of Clinic Biochem, 2009;24(1) 5-14.
Panchenko LF, Lamchingiin T, Gerasimov AM, Sukhanov IS,
Konoplina LA. Superoxide dismutase activity in the blood
of children with iron deficiency anemia. Vopr Med Khim
1979;25(2): 181-5.
Rehema
Aune, Kersti Zilmer, Ursula Klaar, Helle Karro, Tiiu
Kullisaar, Mihkel Zilmer. Ferrous iron administration
during pregnancy and adaptationaloxidative stress (Pilot
study). Medicina (Kaunas) 2004; 40(6).
Schümann K. Safety aspects of iron in food. Ann Nutr
Metab 2001;45:91-101.
Shved
MI, Palamar TO. The antioxidant effects of emoxipin in
patients with iron deficiency anemia. Lik Sprava
1995; 9: 72–5 (in Ukranian).
Srigiridhar and K. Madhavan Nair. Supplementation with
a-tocopherol or a combination of a-tocopherol and
ascorbic acid protects the gastrointestinal tract of
irondeficient rats against iron-induced oxidative damage
during iron repletion. British Journal of Nutrition
(2000), 84, 165-173.
Srigiridhar K, Nair KM. Iron-deficient intestine is more
susceptible to peroxidative damage during iron
supplementation in rats. Free Radic Biol Med 1998;
25:660–65.
Tekin
Demet, Sema Yavuzer, Mustafa Tekin, Nejat Akar and
Lukrucin. Possible effects of antioxidant status on
increased platelet aggregation in childhood
irondeficiency anemia. Pediatrics International (2001)
43, 74–77.
Vives
CJL, Miguel-Garcia A, Pujades MA et al. Increased
susceptibility of microcytic red blood cells to in
vitro oxidative stress. Eur. J. Haematol.
1995; 55:327-31.
Yang M, Collis CS, Kelly M, Diplock AT, Evans CR. Do
iron and vitamin C cosupplementation influence platelet
function or LDL oxidizability in healthy volunteers? Eur
J Clin Nutr 1999; 53(5): 367-74. |
