A STUDY OF IRON DEFICIENCY ANAEMIA IN POST [600046]

`
A STUDY OF IRON DEFICIENCY ANAEMIA IN POST
MENOPAUSAL WOMEN ATTENDING ACCIDENT AND
EMERGENCY DEPARTMENT OF UNIVERSITY OF BENIN
TEACHING HOSPITAL (UBTH)

BY

IGBINUWEN OSAMWONYI
PG/BMS/1312257

DEPARTMENT OF MEDICAL LABORATORY SCIENCE
SCHOOL OF BASI C MEDICAL SCIENCES
COLLEGE OF MEDICAL SCIENCES
UNIVERSITY OF BENIN
BENIN CITY

OCTOBER, 2015

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A STUDY OF IRON DEFICIENCY ANAEMIA IN POST
MENOPAUSAL WOMEN ATTENDING ACCIDENT AND
EMERGENCY DEPARTMENT OF UNIVERSITY OF BENIN
TEACHING HOSPITAL (UBTH)

BY

IGBINUWEN OSAMWONYI
PG/BMS/1312257

THIS PROJECT IS SUBMITTED TO THE DEPARTMENT OF
MEDICAL LABORATORY SCIENCES, UNIVERSITY OF BENIN
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE AWARD OF MASTER OF ME DICAL LABORATORY
SCIENCE DEGREE

DEPARTMENT OF ME DICAL LABORATORY SCIENCE
SCHOOL OF BASIC MEDICAL SCIENCES
COLLEGE OF MEDICAL SCIENCES
UNIVERSITY OF BENIN
BENIN CITY

OCTOBER, 2015

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CERTIFICATE
This is to certify that the work in this project was carried out by
Igbinuwen Osamwoyi under my supervis ion in partial fulfillment for the
award of master of medical science laboratory.

______________________ ____________________
Prof Ukoh Emmanuel Date
(Project Supervis or)

____ __________________ ____ ________ ___
Dr. M. A. Emokp ae Date
(Head of Department and Co -Supervis or)

_________________________ ___________________
ETERNAL EXAMINATION Date

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DEDICATION
This project work is dedicated to the Almighty God.

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ACKNOWLEDGEMENT
My sincere appreciation goes to the Lord Almighty for his grace and
wisdom bestowed upon me in the course of my stay in this University.
My profound gratitude goes to my supervisor Prof Ukoh Emmanue l
and Dr. M.A. Emokpae who has been more than a supervisor to me for
the care and fatherly advice they gave me during the course of this project
work. I will forever be grateful Sir.
I want to say a big thank you to my mum, Mrs. Deborah Igbinu wen,
my wif e, Mrs. Igbinu wen Na omi and my children who have always been
my source of encouragement.
I also use this medium to appreciate my uncle, Associate Prof.
Osadolor H.B. All my Lecturers in the department of medical laboratory,
Dr. M.A. Emokpae, Dr. B. Adeju mo, Dr. M.A. Okungbowa, Dr. F. Akinbo
and others for the roles they played in the course of my training.
I say a big thank to Mr. and Mrs. Ebengho and family for the role they
played in the course of my training. Also I want to thank all my classmate
especially my class rep. Mr. OZ.

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TABLE OF CONTENT
Title page . . . . . . . i
Certification . . . . . . . ii
Dedication . . . . . . . iii
Acknowledgement . . . . . . iv
Table of Content . . . . . . v
Abstrac t . . . . . . . vii
CHAPTER ONE
1.0 Introduction . . . . . . 1
1.1 Background to the Study . . . . 1
1.2 Justification . . . . . . 3
1.3 Aim of the Study . . . . . 3
1.4 Specific Objectives of the Study . . . . 4
1.5 Scope of Study . . . . . 4
1.6 Informed Consent/questionnaire . . . 4
CHAPTER TWO: LITERATURE REVIEW
2.0 Iron as an essential Nutrient . . . . 5
2.1 Iron Transport . . . . . 7
2.2 Iron Absorption . . . . . 10
2.3 The role of iron in human metabolic processes . . 15
2.4 Iron storage and F erritin . . . . . 16
2.5 Iron Deficiency . . . . . 18
2.6 Helicobac ter pyloric as a cause of iron deficiency . 24

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2.7 Prevalence of Ir on deficiency . . . . 32
2.8 Management of Iron Deficiency . . . . 38
2.9 Menopause . . . . . . . 40
2.10 Sign and Symptom Associated with Menopause . . 42
2.11 Causes of Iron deficiencies in Post Menopause . . 49
CHAPTER THREE : MATERIALS AND METHOD
3.0 Study Area . . . . . . 52
3.1 Inclusion criteria . . . . . . 52
3.2 Specimen Collection . . . . . 52
3.3 Determination of Serum iron . . . . 53
3.4 Total iron binding capacity . . . . 54
3.5 Determination of Serum F erritin . . . . 56
3.6 Determination of Transfererin Saturation . . 57
3.7 Determi nation of H. pyloric antibody . . . 57
CHAPTER FOUR
4.0 Result s and Analysis . . . . . 58
CHAPTER FIVE
5.0 Discussion . . . . . . 61
5.1 Conclusion /Recommendation . . . . 62
References . . . . . . 64
Appendix I . . . . . . 75

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ABSRACT
Iron deficiency anemia is a common disorder which occurs mostly in
women of pre -menopausal age and can also be seen in post -menopausal
women. It occurs mostly in women undergoing malnutrition, which is a
primary cause of development of iron deficiency anemia. The secondary
cause of iron deficiency anemia could be due to excess blood loss in the
case of H. Pylori infection. This research was aimed at assessing the
impact of H. Pylori infection on iron bioavailability, as well as, ferritin,
transferin and total iron binding capacity (TIBC) in post -menopausal
women in accident and emergency unit of University of Benin Teaching
Hospital, Edo state. A questionnaire written in English was first
administered to subjects to know the actual subjects suitable for this
research (post -menopausal women) within the ages of fifty (50) years and
above who have not seen thei r menses for one year , who are also not on
oral contraceptive drugs. The control groups were premenopausal women
of age 20 -35years. A total number of 250 (subjects, n = 150 and control
group, n = 100), included in the study were all female. Serologic testin g
for H pylori –specific antibodies was carried out using the HM -CAP
enzyme immunoassay (E -Z-EM, Lake Success, NY) to detect
immunoglobulin G (IgG) antibodies ,serumIron was analysed
byspectrophometricmethod. Transferrin saturation (Tsat) was calculated as
a percentage of Serum Iron to TIBC ,Ferritin was done by MEIA
technique . Results obtained from this research revealed that, plasma iron
increased significantly (82 µg/dl) compared to the control (51 µg/dl). The
increase in plasma iron may be attributed to r educed menstrual blood loss
in the post -menopausal women (Mast et al., 2002) and possible increased
iron storage capacity in the form of ferritin and transferring; ferritin
increased (74 µg/dl) compared to the control (52 µg/dl) depicting

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increased storage capacity in the post -menopausal women while TIBC
did not show any change between the tests and the control group, 259
µg/dl and 257 µg/dl, respectively (p>0.05). In support of this claim,
transferrin increased significantly for the test group (32 µg/dl) c ompared
to the control group (19 µg/dl).This present research however, showed
that the 97% of the pre -menopausal women were negative for H. Pylori
thus accounting for the previous data obtained. Post menopausal women
should constan tly check their iron statu s to avoid problem of iron
overload which is associated with post -menopausal era in women.

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CHAPTER ONE
1.0 Introduction
1.1 Backgound to the study
Iron deficiency anaemia is a common disorder which occurs mostly in
women of pre -menopausal a ge and can also be seen in post menopausal
women (Haseena et al ., 2014). It occurs in women undergoing
malnutrition, which is a primary cause of development of iron deficiency
anaemia. The secondary cause of iron deficiency anaemia could be due to
excess b lood loss and in the case of H. Pylori infection, which impair iron
metabolism. Iron is a trace element of nutritional importance to living
cells and it exists in a divalent state (Elizabeth et al., 2008). The human
body stores iron in the form of ferritin and hemosiderin in liver, spleen,
marrow, duodenum, skeletal muscle and other tissues. Hemosiderin and
ferritin are iron -containing proteins with magnetic susceptibility.
Hemosiderin is water -insoluble and thermally denatured, but ferritin is
water -solubl e and heat -resistant up to 75°C. The total amount of body
iron stores is around 600 to1000 mg in the normal adult male and around
200 to 300 mg in the normal adult female (Abboud and Haile, 2000).
Because of its divalent nature, iron may act as a redox com ponent of
proteins, and therefore is integral to vital biologic processes that require
the transfer of electrons. It is intimately involved in numerous vital
biologic processes, including oxygen transport, oxidative phosphorylation,

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DNA biosynthesis, and x enobiotic metabolism (Hentze et al., 2004). Iron
is a constituent of such important proteins as hemoglobin, cytochromes,
oxygenases, flavoproteins, and redoxins. The transition metal participates
in the transfer of electrons via oxidation -reduction reactio ns that result in
the fluctuation of iron between its ferric (3+) and ferrous (2+) states
(Papanikolaou and Pantopoulos, 2005). This property is largely
responsible for the biologic significance of iron. When the uptake and
metabolism of iron is interrupte d, this call result in iron loss and
consequently, Iron deficiency will ensue. Infections with H. Pylori have
been reportedly been associated with iron deficiency (Helena et al., 2013).
Although chronic infection with H. pylori does not appear to produce a
hemorrhagic gastritis, the infection may impair iron absorption. The
absorption of non -heme iron is highly dependent on gastric acid secretion
(an acidic pH <3) and ascorbic acid (either secretory or dietary sources)
for the reduction of ferric to ferrous iron and subsequent intestinal uptake.
Ascorbic acid also promotes formation of a soluble chelate with ferrous
iron for absorption (Annibale et al ., 2003). H. pylori -induced gastritis
reduces gastric acid secretion and is therefore a potential cause of red uced
iron absorption and increased risk of IDA (Annibale et al ., 2003). The
relationship between refractory IDA and H. pylori infection may be
explained by several hypotheses. H. pylori is a bacterium which requires
iron as an essential growth factor and i s capable of binding and

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transporting iron across the cell membrane (Annibale et al., 2003). Thus,
the bacterium may compete for iron within the GI tract. IDA is also
commonly present in chronic inflammatory disorders such as celiac
disease (Annibale et al ., 2003). In these conditions, IDA results from
sequestration of iron in the antral mucosa, as a result of inflammation
(Annibale et al., 2003) and inflammation from H. pylori infection could
result in similar changes. A low serum iron and ferritin with an elevated
TIBC are diagnostic of iron deficiency. While a low serum ferritin is
virtually diagnostic of iron deficiency, a normal serum ferritin can be seen
in patients who are deficient in iron and have co existent diseases
(Haseena et al., 104). There ap pear to be dearth of information on Iron
status of post menopausal women in Benin City, Edo State, Nigeria.
Hence, this study was designed to bridge this gap.
1.2 Justification for the study
Worldwide, Iron deficiency is a common disorder among women of
reproductive age. This disorder may also be seen among post menopausal
women who are undergoing malnutrition. H. pylori infection have also
been implicated to be associated with this disorder. Hence, this work was
designed to investigate the H. Pylori infec tion and its relationship with
iron deficiency.

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1.3 Aim of the Study
The aim of this study is to investigate iron defi ciency in post menopausal
women in accident and emergency unit of University of Benin teaching
hospital, Edo state.
1.4 Specific Objectiv es of the Study
The specific objectives of this study includes:
 To investigate iron status in post menopausal women
 To determine the proportion of post menopausal women with iron
deficiency anaemia.
 To investigate the prevalence of H. Pylori infection amon g post
menopausal women.
 To determine the level of Total iron binding capacity in post
menopausal women
 To determine the level of serum transferin and serum ferritin in post
menopausal women
 To compare the levels of these parameters with the control
(preme nopausal women)
1.5 Scope of Study
This work was designed to cover the investigation and prevalence of iron
deficiency and H. Pylori infection among post menopausal women.

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1.6 Informed Consent/question naire
Individuals who participated in this study includ e those who gave
personal informed consent; having adequately understood the aim and
objectives of the study and has completed a questionnaire to this effect

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CHAPTER TWO
Literature Review
2.0 Iron as an essential Nutrient
Iron is a trace element of nutriti onal importance to living cells and it
exists in a divalent state (Elizabeth et al., 2008). The human body stores
iron in the form of ferritin and hemosiderin in liver, spleen, marrow,
duodenum, skeletal muscle and other tissues. Hemosiderin and ferritin a re
iron-containing proteins with magnetic susceptibility. Hemosiderin is
water -insoluble and thermally denatured, but ferritin is water -soluble and
heat-resistant up to 75°C. The total amount of body iron stores is around
600 to1000 mg in the normal adult male and around 200 to 300 mg in the
normal adult female (Abboud and Haile, (2000). Because of its divalent
nature, iron may act as a redox component of proteins, and therefore is
integral to vital biologic processes that require the transfer of electrons. It
is intimately involved in numerous vital biologic processes, including
oxygen transport, oxidative phosphorylation, DNA biosynthesis, and
xenobiotic metabolism (Hentze et al., 2004). Iron is a constituent of such
important proteins as hemoglobin, cytoc hromes, oxygenases,
flavoproteins, and redoxins. The transition metal participates in the
transfer of electrons via oxidation -reduction reactions that result in the
fluctuation of iron between its ferric (3+) and ferrous (2+) states
(Papanikolaou and Panto poulos, 2005). This property is largely

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responsible for the biologic significance of iron. The same property that
allows iron to participate in energy production by electron transfer also
causes the toxicity resulting from an ex cess of labile iron. This te nden cy
to undergo oxidation -reduction reactions is also responsible for the
toxicity of iron (Papanikolaou and Pantopoulos, 2005). Most cytoplasmic
iron is in its reduced form, meaning that it is an excellent substrate for
oxidation. Donation of electrons leads to the formation of reactive free
radicals; when ferrous iron interacts with H 2O2, it undergoes the Fenton
reaction (Papanikolaou and Pantopoulos, 2005). The Fenton reaction
produces ferric iron,−OH, and the hydroxyl radical. It may also result in
the peroxidation of adjacent lipids and lead to oxidative damage of DNA
and other macromolecules.
In conjunction with this dichromatic nature, both severe iron overload and
iron deficiency may be deleterious. Because iron is intimately involved in
the produc tion of energy and oxygen transport, iron deficiency is a
serious problem that causes cell damage, reduction of cell growth and
proliferation, hypoxia, and death (Elizabeth et al. , 2008). Each day
about 25 mg of iron is needed for erythropoiesis and other vital functions.
Only 1 to 2 mg of iron comes from intestinal iron sources; thus, other
mechanisms for iron regulation, including release of iron from cellular
storage depots and recycling of iron from protein sources, are critically
important to provide f or organismal iron requirements. Likewise, an

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excess of iron systemically and at the cellular level leads to deleterious
effects including free radical -induced damage to cells, cellular
components, tissues, and organs. Deviations from normal iron levels ha ve
been indicated in the pathogenesis of aging, neurodegenerative disease,
cancer, and infection (Tsuji et al., 1993; Trinder et al., 2002; Lee et al.,
2006 ).

Fig. 1: Heme molecule containing Iron in ferrous state (Trinder et al., 2002)
2.1 Iron Transport s
Proteins involved in the transport of iron are; transferrin, ferritin,
hemosiderin and hepcidin.

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Transferrin
Tranferrin is a plasma glycoprotein responsible for transporting iron from
one organ to another and is accomplished by the reversible binding of
iron to the transport protein, transferrin, which will then form a complex
with a highly specific transferrin receptor (TfR) located on the plasma
membrane surfaces of cells. Transferrin is normally 30% saturated with
iron.
The t otal iron binding capac ity (TIBC ) reflects the status of iron in the
body and is defined as the amount of iron needed for 100% transferrin
saturation (Shazia et al., 2012). Transferrin levels are typically used for
diagnosis of iron overload rather than iron deficiency. Serum le vels of
soluble transferrin receptor (sTfR) reflect transferrin receptor not bound
to transferrin, with low levels of sTfR reflecting higher levels of receptor
saturation with iron and lower erythropoiesis. As sTfR is increased in iron
deficiency anemia, a nd ferritin is low in iron deficiency anemia and
elevated in anemia of chronic disease, the sTfR: ferritin ratio can be
useful for further distinguishing women with elevated ferritin levels due
to lack of iron as opposed to the inflammation associated with chronic
disease (Shazia et al., 2012).

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Non Intestinal Iron Transport by Transferrin
All cells require iron to maintain normal function. In non intestinal cells,
circulating iron is bound to transferrin (Tf) and is imported via
receptor -mediated endocyto sis after binding to the transferrin receptor
(TfR) (Ponka et al. , 1998). Because Tf and TfR are absent from
enterocytes, Tf binds iron and plays an essential role in the transport of
iron only once it is exported from duodenal enterocytes into the
bloodst ream. Tf is also involved in the transport of iron from
reticuloendocytic cells [red blood cell (RBC) recycling] and the liver to
proliferative cells throughout the body, thereby controlling the levels
of“labile iron”(Thorstensen and Romslo, 1990). In this sense, Tf serves as
a storage sink for sequestering iron extracellularly until iron is needed,
and then allowing it to reach target tissues. Tf is composed of single
chains that are bilobal, containing N – and C -lobes, each with two domains,
referred to as the N1, N2, C1, and C2 domains. The lobes are connected
by a hinge, which creates a cleft that contains the iron -binding domains.
Iron binding and release are coordinated by a conformational change in
which the two sub domains of each lobe open, and the N 1, N2, C1, and
C2 domains twist (Grossmann et al ., 1993). Each of the homologous
amino domains binds one atom of Fe3+. Tf is an insulin -like growth
factor -binding protein 3 (IGFBP3) -binding protein (Gutteridge and
Quinlan, 1992). IGFBP3 binds to circulatin g insulin -like growth factors

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(IGFs) and has growth -enhancing or inhibitory effects on cells, which are
modulated by IGFBP3 -binding protein. Although Tf has been shown to
bind IGFs and IGFBP3, it does not contain the conserved
GCGCCXXC -motif found in other IGFBPs (Storch et al., 2001). The role
of Tf in the IGF -IGFBP pathway is unclear; however, treatment with
exogenous Tf abrogated IGFBP3 -mediated proliferation, and in prostate
cancer cells, Tf inhibited apoptosis caused by IGFBP3 action (Weinzimer
et al ., 2001). Tf bound with iron releases iron at acidic pH because of
major conformational changes including a 54 – to 63 -degree rotation
between the two domains on each lobe.
2.2 Iron Absorption
As regards mechanism of absorption, two kinds of dietary iron are known
which are : the heme iron and the non-heme iron (Hallberg, 1981). In
humans, haemoglobin and myoglobin are the primary sources of heme
iron in the human diet which is derived from the consumption of meat,
poultry, and fish while the non -heme iron is usually obtained from cereals,
pulses, fruits, legumes and vegetables. The average amount of heme
gotten from animal diets is about 25% (Hallberg, 1979). Heme absorption
varies from about 10% during iron repletion to 40% during iron (Hallberg
et al., 1997) . When heated for long period (during prolonged cooking),
heme is degraded and converted to a non -haem iron. Calcium influences
the absorption of heme iron negatively as the only dietary factor . There

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are no known mechanisms on the excretion of iron from t he body where
absorption is in the proximal duodenum and is tightly regulated. When
the dietary iron in ferric state (fe3) enters the duodenum, the enzyme
ferroreductase present in the surface of enterocytes , reduce it to ferrous
form (fe2). Vitamin c in t he food also help s to reduce it to ferric iron to
ferrous form (Robert et al., 2006).
Factors that influence the absorption of dietary iron
Heme Iron Absorption
 The iron status of the subject
 The a mount of the dietary heme iron
 The calcium contents in the meal
 The preparation of the meal (i.e. temperature and time)
Absorption of non -haem iron
 The iron status of the subject
 The potential amount of available non -haem iron
 The balance between enhancement and inhibition factors
Enhancing factors
Meals such a s animal products (meat, chicken, sea foods), fruits and fruit
juices, potatoes and vegetables.
Inhibition of iron absorption
Phytates are chemical found in all kinds of grains, vegetables, nuts, roots,
seeds and fruits. They contain inosito l hexaphosphat e salts and are the

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storage form of minerals and phosphates . Most o ther phosphates have not
been observed to inhibit non -heme iron absorption. Phytates have been
shown be able to strongly inhibits the absorption of iron in a dose
dependently (Gillooly, 198 3). Calcium, also have been observed to alter
the absorption of haem and non -haem iron (Gleerup et al ., 1993).
However, due to the fact that calcium and iron are both essential nutrients
it cannot be considered as an inhibitor when compared to phytates whi ch
are not . Increased bioavailability of iron and avoiding eating meal with
high calcium -rich food together with those containing iron can be used to
solve the absorption problem (Gleerup , 1995 ).
Enhancement of Iron absorption
Non-haem iron absorption is strongly enhanced by ascorbic acid Ascorbic
acid and it is the most potent enhancer (Siegenberg, 1991). Absorption of
iron by vitamin C can be considered as its main physiologic roles
(Hallberg et al., 1987). Other food stuffs such as meat, sea foods al so
enhance non -iron absorption (Cook and Monsen, 1976).
Iron balance and regulation of Iron absorption
There are three unique mechanisms by which the body uses in
maintaining iron balance so as to prevent iron deficiency and iron
overload.
1. The c ontin uous re-utilisation of iron catabolised from erythrocytes in
the body. At 120 days, erythrocytes are normally degraded by the

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macrophages in the reticular endothelial system where iron is been
released and transferred to transferring where it is taken to the r ed cell
precursor cells in the bone marrow. The absorption and distribution of
iron is controlled by the transferring receptors on the surface of the
cells and thus controlling the rate of iron distribution amongst
different tissues according to their iron needs and preventing the
formation of free iron radicals in the circulation.
2. The assess ment of ferritin (the specific storage protein ) which stores
and release iron to meet iron demands of the body . Thus serving as a
reservoir for iron which is specifica lly helpful in cases where the
body‟s iron stores are prone to depletion as in the third trimester of
pregnancy.
3. The regulation of iron absorption from the intestines: a decreased iron
store is usually accompanied with increased iron absorption and then
absorption decreases as soon as the iron stores are replenished thus
maintaining equilibrium . However, this regulation can be limited for a
given diet which can only balance losses up to a certain critical point
beyond which iron deficiency may develop (Hall berg, 1995).
Primarily, basal iron is lost through the gastrointestinal tract and the blood
where these losses are dependent on the haaemoglobin levels (Hallberg et
al., 1998). With increasing iron requirements or decreasing
bio-availability, the regulato ry capacity to prevent iron deficiency is

15
limited (Hallberg, 1995).
2.3 The role of iron in human metabolic processes
Several functions are been played by iron in the body. Amongst these
functions are that it serves as a transport medium for electrons in the body,
transports oxygen in the red blood cells from the lungs. It also helps to
integrate important enzymes system in various tissues in the body
(Hallberg, 1982; Dallman, 1986).
The erythrocytes contain most of the body‟s and are stored as
haemaoglob in which is composed of four units each with one heme and
protein chain. The morphological characteristics of haemoglobin enable it
to be loaded completely with oxygen molecules in the lungs and the
partial off loading to the tissues . The myoglobin, an iro n-containing
molecules found in the muscles are also similar to haemoglobin
structurally but however, have only one heme and globin chains (Mascotti,
et al. , 1995). Other iron -containing compound such as cytochromes, also
have a similar heme -globin structu re of myoglobin where they act as
electron transporters and do not permit reversible loading/unloading of
oxygen in the mitochondria of cells . The functions of other key
iron-containing enzymes are:
a) Synthesis of steroid hormones and bile acids
b) Detoxificat ion of foreign and toxic substances in the liver
c) Controlling signaling of some neurotransmitters (dopamine and

16
serotonin ) systems in the brain.
Iron is transported in the body via the protein transferring and stored
reversibly as ferritin and haemosiderin .
2.4 Iron Storage and Ferritin
Ferritin is the major iron -storage protein at the cellular and organismal
level. It is responsible for the sequestration of potentially harmful,
reactive iron. Ferritin stores iron in its unreactive Fe3+form inside its
shell as a result of a strong equilibrium between ferritin -bound iron (Fe3+)
and the labile iron pool in the cells (Fe2+), by which ferritin prevents the
formation of ROS mediated by Fenton reaction (Elizabeth et al., 2008).
Because of its important function in the storage of iron, ferritin is
ubiquitous in tissues, serum, and in other multiple locations within the
cell. It is regulated at the transcriptional and posttranscriptional level by
various pathways in response to diverse stimuli (Elizabeth et al., 2008 ).

Structure, Tissue Distribution, and importance of Cytoplasmic
Ferritin
Ferritin is found in the cytoplasm, nucleus, and mitochondria of cells. In
vertebrates, cytoplasmic ferritin is expressed in almost all tissues. This
ubiquitous protein consists of 24 subunits of heavy (H) and light (L)
chains in various ratios and can sequester 4,500 iron atoms (Harrison and
Arosio, 1996). The H subunit has ferroxidase activity, which converts

17
Fe2+to Fe3+for storage inside the shell (Lawson et al., 1989). In contras t,
the ferritin L subunit stabilizes ferritin structure and facilitates the uptake
of iron into the shell. Ferritin H and L subunits are encoded by two
different genes. The ratio of H and L subunits in the ferritin protein is not
fixed and is tissue depend ent (Arosio et al ., 1976). For example, H
expression is abundant in the heart, whereas the L subunit is predominant
in the liver and spleen. In the brain, the oligodendrocytes, microglia, and
neurons express ferritin. Oligodendrocytes have equal amounts of both H
and L subunits, whereas microglia expresses L-rich ferritin, and neurons
have H -subunit abundant ferritin (Zecca et al., 2004).

Fig. 2: Body Iron Distribution and storage (Zecca et al., 2004).

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Hepcidin Regulate Iron Distribution
Hepcidin is a pept ide hormone synthesized in the liver and is the principal
regulator of systemic iron homeostasis. Hepcidin controls plasma iron
concentration and tissue iron distribution by inhibiting intestinal iron
absorption, iron recycling by macrophages, and iron mob ilization from
hepatic stores (Andrews and Schmidt, 2007). Hepcidin influences iron
absorption through direct binding to ferroportin at the basolateral
membrane, leading to decreased export of iron to the circulation system
(Nemeth et al., 2004).
2.5 Iron Deficiency
The iron deficiency is a condition in which the disposed amount of iron is
more than the adsorbed amount. The first sign of this situation is the
negative balance of iron (Hoseinzadeha et al., 2010). In such a situation,
the stored amount of iro n is decreased, depleting the plasma ferritin
resulting in an increase in TIBC, which is called iron deficiency
(Hoseinzadeha et al. , 2010). Iron deficiency implies the state of storage
iron exhaustion. It can be classified into 3 stages: iron deficiency w ithout
anemia, iron deficiency anemia without tissue damage, and iron
deficiency anemia with tissue damage (Umbreit, 2005). Iron deficiency
tissue damage, so -called Paterson -Kelly or Plummer -Vinson syndrome,
occurs mostly in middle -aged female patients wit h chronic iron
deficiency anemia. Iron deficiency in the developing brain in the early

19
stage of life seems to be a risk factor (Beard, (2003.) Iron deficiency
limits hemoglobin synthesis and this knowledge is applied for the
treatment of polycythemia vera. Hayashi et al., (1994) revealed that iron
deficiency sedated chronic hepatitis C. Furthermore, Kato et al., (2007)
observed that iron deficiency inhibited the incidence of hepatoma in
chronic hepatitis C, and Saito (1977) found the inhibition of
hyperthyroi dism by iron deficiency. Finberg (2009) discovered iron
refractory iron deficiency anemia with high hepcidin levels and
suppressed iron absorption resulted from a defect in the TMPRSS6 gene.
Moreover, the utilization of intravenously infused iron in iron r efractory
iron deficiency anemia is inferior to that of ordinary iron deficiency
anemia.

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Fig. 3: Blood film of Iron deficiency anaemia (Finberg, 2009)

21

Fig. 4: Blood film of iron deficient anaemia, Normal and anaemic (Finberg, 200 9)

Functional Iron Deficiency
“Functional iron deficiency” occurs where there is an inadequate iron
supply to the bone marrow in the presence of storage iron in
reticuloendothelial cells. Perhaps the most important clinical setting for

22
this is in patients with renal failure who will require parenteral iron
therapy to respond to administered erythropoietin to correct anaemia.
None of the currently available tests have more than fair utility for
deciding which patients will benefit from parenteral iron in th is setting
(Fernandez -Rodriguez et al., 1999). Low reticulocyte haemoglobin
content provides an early indication of functional iron deficiency (Mast et
al., 2002), whilst a reduced percentage of hypochromic erythrocytes is a
good predictor of response (Col e and Williams, 1992).
Causes of Iron Deficiencies
Iron deficiency develops when the quantity of absorbed iron from die t is
lower than the amount of iron needed to cover the physiological iron
requirements. Consequently, the population groups most at risk to
develop ID are those with the highest iron requirements. These are infants,
children, adolescents, and pregnant women who have additional iron
needs due to growth as well as women of childbearing age who have
higher iron losses due to menstrual b lood l oss (Mast et al., 2002).

23

Fig. 5:Predisposing factors and population at risk of Iron deficiency (Mast et al .,
2002)

Dietary Causes of Iron Deficiency
Inadequate iron absorption can be caused by multiple factors. Low dietary
iron intake is one of the reasons and can for example result from energy
restriction or a diet low in iron, for example a diet based on white rice.
Low iron absorption can further be a result of poor dietary quality rather
than low iron intake (Bothwell et al. 1989). Dietary quali ty, based on the

24
content of enhancers and inhibitors of iron absorption in the diet, is
frequently dependent on socio -economic factors. For example, in
Venezuela the diets consumed by the different socio -economical classes
were compared for iron content as well as their content of enhancing and
inhibiting factors.
The results showed that while iron content was higher in the diet of the
lower social classes, the intake of meat and ascorbic acid was
substantially lower and that of phytic acid substantially hi gher than in
higher social classes (Taylor et al., 1995). This can be explained by the
high cost of meat and fresh fruits which limits their consumption in
underprivileged population groups, in developing as well as industrialized
countries (Marx, 1997; Ham braeus, 1999).

2.6 Helicobacter pylori as a cause of Iron Deficiency
Helicobacter pylori is a small, curved, highly motile Gram negative
bacillus from the family, spirallacea, that colonises only the mucus layer
of the human stomach (Hoseinzadeha et al ., 2010). There are some
species of helicobacter genus recognized in the gastrit of mammalians
amongst which only H. Pylori can infect humans (Dufour et al. , 1993). H.
pylori is a non -spore bearing organism, having 4 -6 shielded flagella and
motile, with a sm ooth external cell wall together with glycocalix
(Kostrzynska et al ., 1991). It has a somatic antigen, heat resistant

25
lipopolysacharide, flagellum -related heat sensitive antigen and urease
antigen at the external surface and periplasmic space (Buck and Smi th,
1987). Initially, the bacterium was sensitive to metronidazole while at
present 40% of cases are resistant. This bacterium secrets different
enzymes such as catalase, protease, phospholypase C, A2 gastric acid
inhibitor protein, leukotactic factors, ha emolysine, HSP (Heat Shock
Proteins), cag A, urease, alcohol hydrogenase, PAF (Platelet Activity
Factor) , and gastric ulcer – induced factor (Lee, 1994). The role of this
bacterium in acute and chronic gastritis peptic ulcer and gastric
adenocarcinoma has been investigated (Cellini et al., 1992). H. pylori can
inhibit gastric acid secretion and increases the pH by producing alkaline
substances and receiving iron nutrients from lactoferrin. Helicobacter
pylori colonisation in gastric mucosa may impair iron u ptake and increase
iron loss, potentially leading to Iron deficiency Anaemia. Many
researchers analyzed the relationship of H. pylori infection and iron
deficiency anemia. The first case report was by Dofour et al. in 1993 on a
child with H. pylori infection and anemia who was cured after treatment
of H. pylori and without using any complementary foods and iron
(Dufour et al., 1993).

26

Fig. 6: H. pylori (Dufour et al., 1993).

27

Fig. 7: Stomach infection of H. pylori (Dufour et al., 1993).

28

Fig. 8: Gastritis induced by H. Pylori (Blaster, 1994)

29

Fig. 9: Antigenic and enzyme system of H. pylori (Blaster, 1994)

30

fig. 10: Symptoms of H. Pylori infection (Blaster, 1994)

Epidemiology of H. Pylori Induced Iron deficiency
Epid emiologic studies have shown that persons seropositive for H. pylori
infection have a significantly lower serum ferritin level (Rabieipoor et al.,
2011). In a population -based study (n=2794) from Denmark, H. pylori
-seropositive persons were at 40% increas ed risk of having reduced serum
ferritin level (<30 mg/L) compared to seronegative individuals (after
adjustment for age, gender, menopausal status, socioeconomic status,
blood donation, and alcohol consumption). Analysis of a cross -sectional
national heal th survey from Germany (n=1806) revealed that persons
with H. pylori infection had 17% decrease (95% CI 9.8 -23.6) in serum

31
ferritin concentration, after adjustment for age and sex (Rabieipoor et al.,
2011). A study of Alaskan natives (n=2080) also showed a n increased risk
of low serum ferritin for persons sero positive for H. pylori infection
(relative risk 1.13, p=0.013). Positive findings were also reported in a
study of Korean children aged 6 -12 (n=753), in whom H. pylori
seropositivity was associated wi th lower mean serum ferritin level (24
ng/mL vs. 39 ng/mL, p<0.001), and a significantly increased prevalence
of iron deficiency (serum ferritin <15 ng/mL). Another study of Korean
adolescents (n=937) confirmed a significant association between H.
Pylori s eropositivity and anemia, hypoferritinemia and iron deficiency.
An epidemiologic study of Australian women showed significantly lower
ferritin levels in women with H. pylori infection compared to
non-infected controls despite similar dietary iron intake (R abieipoor et al.,
2011).
While the above studies support an association between H. pylori
infection and indices of iron stores, a few (usually smaller) epidemiologic
studies have not found significant association between H. pylori infection
and iron indic es. In a study of 1060 adults from New Zealand, there was
no significant differences in serum ferritin level according to H. pylori
status, and a study of 693 Korean children found no significant difference
in the prevalence of H. pylori infection for chil dren with and without IDA
(Rabieipoor et al., 2011).

32
2.7 THE PREV ALENCE OF IRON DEFICIENCY
Iron deficiency (ID) is one of the most common and widespread
nutritional disorders worldwide . It is however difficult to estimate how
many people are affected and m ostly the prevalence of anemia is used as
an indicator for IDA and an indirect indicator of ID. In 1985 DeMaeyer
and Adiels -Tegman estimated, based on the evaluation of 523 studies, that
30% of the world's population was anemic. Estimates of the prevalence of
anemia from 1990 -1995 were similar with approximately 2 billion people
being anemic (over 30% of the world population) (WHO/UNICEF/UNU,
2001). However, as anemia can be due to multiple causes, such as other
nutritional deficiencies as well as infection s, inflammation, and malaria, it
is obvious that the prevalence of IDA is not equal to the prevalence of
anemia. It has been predicted that approximately 50% of all anemia is
caused by ID (DeMaeyer and Adiels -Tegman, 1985). Thus, world -wide
IDA affects app roximately 1 billion individuals. This number can further
be used to calculate the number of people affected by ID. It has been
estimated that the prevalence of ID is approximately 2.5 -times that of
IDA, based on data from US women and children showing tha t 30-40%
of those with ID were also anemic (Yip, 1994). This factor may however
only be valid in industrialized countries, in developing coun tries, for
example the Ivory Coast, prevalence of ID was shown to be twice that of
IDA (Asobayire et al . 2001). Th us, world -wide ID may affect 2 to 2.5

33
billion people.
Markers of Iron Deficiency
Iron deficiency can be diagnosed by no stainable iron in the
reticuloendothelial cells on bone mar row smears and quantitatively by a
low serum ferritin of less than 15 µg/l . depletion of iron store is a good
indicator of iron deficiency though it does not necessarily mean an
immediate effect of the deficiency.
Iron-deficient erythropoiesis sets in during iron deficiency even prior to a
complete depletion of the iron stores in the bone marrow (Hallberg, 1993).
This can be explained by the fact that there is a decreased rate of iron
release from the stores whose rate is influenced by amount left present .
Serum markers of iron deficiency include low ferritin, low iron, raised
total iron binding capacity, raised red cell protoporhyrin and increased
transferrin binding receptors (sTfR). Serum ferritin is the most powerful
test for iron deficien cy. The cut -off level of ferritin which is diagnostic
varies between 12 –15 μmg/L (Cook et al., 1992). This value only holds
for patients without co -existent disease. In such settings, a cut -off value
of <50 μmg/L is still consistent with iron deficiency1. The sTfR level is
said to be a good marker of iron deficiency in healthy subjects (Cook,
1999) but its utility in the clinical setting remains to be proven. Several
studies show that the sTfR/log10 serum ferritin ratio provides superior
discrimination to e ither test on its own, particularly in chronic disease

34
(Cook et al., 2003).
Further tests to confirm iron deficiency are occasionally necessary.
Estimation of iron concentration in bone marrow by the histochemical
method14 may distinguish between „true‟ ir on deficiency and other
chronic disorder s in which there is impaired release of iron from
reticuloendothelial cells, but is subjective. A therapeutic trial of oral iron
for three weeks is less invasive and may aid diagnosis, but depends on
compliance. A tr ial of parenteral iron may be more reliable, and a
measurable change in MCH should occur within 7 days when there is iron
deficiency anaemia.
Effects of Iron Deficiency
In animals, studies have shown that there are negative effects of iron
deficiency in the normal functioning of the body (Dallman, 1986). Finch
(1976), revealed tha t there was a reduction in the physical working
capacity in rats especially in the endurance activities during iron
deficiency where the effect is more related to impa ired muscular
oxidative metabolism (leading to lactic acid acidiosis) , and lack of
iron-containing enzymes (which serves as rate limiting enzymes of
oxidative metabolism) than to the degree of anaemia (Scrimshaw, 1984).
Pollitt (1993) revealed that the brain functioning is related to iron
deficiency and hence should be of great importance in combating iron
deficiency because many brain structures were observed to contain high

35
iron content where 10% was observed to be present in human infant ‟s
brain and only reaches half of its content at 10 years and to its optimal
amount at about ages 20-30 years.
It has been shown in several populations with iron deficiency that after
the administration of iron therapy, there were improvements in the
working cap acity in those population group (Scrimshaw, 1984). Iron
deficiency has been shown to be associated with impa irment of normal
defense systems of the body against infections as it was observed that
T-cell mediated immune response was specifically impaired due to the
reduction of cell formation as well as other defense cells such as
neutrophils, leuk ocytes , e.t.c. (Brock, 1994).
Immunologic impairment was also found in murine studies associated
with iron deficiency however, with the administration of iron supplements
was found to normalize these chang es between 4 -7 days of therapy .
Studies have shown that there is no effect of iron deficiency or the
administration of iron therapy on mental behavior such as memory,
attention, and learning in children , pregnant women, adolescence and
infants as well as in animals . However , Bruner (1996 ) showed that there
were memory and verbal improvements in non-anaemic, iron-deficient
girls because this condition is associated with reduced physical endurance ,
ability to conce ntrate, and mood changes (Ballin, 1992). Reduction in the
maximum oxygen consumption has been observed in women with

36
non-anaemic, iron-deficien cy.

Fig. 11: Effects of Iron deficiency (Zhu and Haas, 1997)

2.8 Management of Iron Deficiency
Aim of treatment
The aim of treatment should be to restore haemoglobin levels and red cell
indices to normal, and replenish iron stores. I f this cannot be achieved,
consideration should be given to further evaluation.
Iron therapy
Treatment of an underlying cause should prevent further iron loss but all
patients should have iron supplementation both to correct anaemia and
replenish body stor es (Smith, 1997). This is achieved most simply and

37
cheaply with ferrous sulphate 200 mg twice daily. Lower doses may be as
effective and better tolerated and could be considered in patients not
tolerating traditional doses. Other iron compounds (e.g. ferro us fumarate,
ferrous gluconate) or formulations (iron suspensions) may also be
tolerated better then ferrous sulphate. Ascorbic acid (250 –500 mg twice
daily with the iron preparation) may enhance iron absorption43.
Parenteral iron may be used when there is intolerance or noncompliance
with oral preparations. Intravenous iron sucrose, when given according to
the manufacturers‟ instructions, is reasonably well tolerated (35% of
patients have mild side effects) with a low incidence of serious adverse
reactions (0.03 –0.04%) (Fishbane, 2003). Bolus intravenous dosing of
iron sucrose (200mg iron) over 10 minutes is licensed and more
convenient than a two -hour infusion. Intravenous iron dextran can
replenish iron and haemoglobin levels in a single infusion. But serious
reactions can occur (0.6 –0.7%) and there have been fatalities associated
with infusion (31 reported between 1976 –1996) (Silverstein and Rodgers,
2004). However, it can be given via the intramuscular route when venous
access is problematic. Blood trans fusions should be reserved for patients
with, or a risk of, cardiovascular instability due to their degree of anaemia,
particularly if they are due to have endoscopic investigations before a
response from iron treatment is expected (BCSH, 2001). Transfusio ns
should aim to restore haemoglobin to a safe level, but not necessarily

38
normal values. Iron treatment should follow transfusion to replenish
stores.
2.9 Menopause
Menopause is permanent cessation of ovulation and menses. As defined
by Stages of Reproduct ive Aging Workshop (STRAW), menopause (ie,
“spontaneous” or “natural” menopause) is said to have occurred after 12
months of amenorrhea with no obvious pathologic cause. Menstruation is
a unique physiological phenomenon in young women, characterized by
the periodic high levels of estrogen and the shedding of the endometrium.
Because of this monthly blood loss, iron deficiency is prevalent in
premenopausal women.
There is no adequate independent biological marker for menopause. This
cesation often begins in the late 30s, and most women experience
near-complete loss of production of estrogen by their mid -50s. During
perimenopause, fewer eggs exist for the ovaries to stimulate, and
menstrual periods become irregular. This period of fluctuation can last up
to 1 0 years. Cessation of menstruation marks the later stage of
perimenopause. Because iron is no longer lost through menstruation, it
accumulates in the body. The transition from normal ovarian function to
ovarian failure is described as the menopausal transi tion. Although some
of these women may be asymptomatic, estrogen deficiency is associated
with hot flashes, sweating, insomnia, and vaginal dryness and discomfort

39
in up to 85% of menopausal women. Most women with menopausal
symptoms will experience spontan eous cessation of them within 5 years
after onset; a substantial proportion of women, however, continue to
experience symptoms beyond 5 years. Menopausal hormone therapy
(MHT) is the most effective intervention for management of these
symptoms that diminis h the quality of life. The goal of MHT, defined as
estrogen therapy alone or a combination of estrogen and a progestational
agent (E+P), is to alleviate the quality -of-life symptoms in menopausal
and perimenopausal women. In addition, chronic disorders ass ociated
with both aging and the menopausal state affect the brain, skeleton,
integument, and urogenital and cardiovascular systems. The role of MHT
in the prevention of such disorders remains controversial.
Postmenopause
Postmenopause refers to the years after the final menopausal period (FMP)
resulting from natural (spontaneous) or premature menopause. An
estimated 75% of women ages 50 to 55 are assumed to be
postmenopausal. These estimates include women who may have had
induced or premature natural meno pause earlier in life. Among women
ages 40 to 45, an estimated 5% have experienced natural menopause,
based primarily on data from the Study of Women‟s Health Across the
Nation (SWAN) (Johnston et al., 2006).

40
2.10 Signs and symptoms associated with Menopa use
Certainly, for many women, the transition to, through and beyond
menopause is filled with many biological, biochemical, physical, and
emotional changes (Hunter, 1990). The body, i.e., the ovaries gradually
produce less progesterone and estrogen until ovarian function ceases,
which then is accompanied by a cascade of effects on a woman‟s body.
The following are some signs and symptoms of menopause:
 Irregular mentruation
 Decreased fertility
 Hot flashes
 Sleep disorder (Insomnia)
 Mood swing
 night sweat s
 vaginal dryness.
 osteoporosis, arteriosclerosis, dyslipidaemia, depressed mood,
irritability,
 headache,
 forgetfulness,
 dry eyes, dry mouth, reduced skin elasticity, restless legs, and muscle
and joint pain) have also been implicated as associated with the
menopause (Dennerstein et al., 1978)

41
Vasomotor symptoms
Hot flushes are defined as transient, recurrent periods of heat sensation
and redness, often concomitant with sweats. An increase in peripheral
vasodilatation, skin temperature and skin moist ure has been demonstrated
during such episodes by the registration of skin conductance,
thermograms or plethysmography in the affected areas of the face, neck,
head or breast (Sturdee and Reece, 1979). The duration is often 2 to 3
minutes but with a range from a few seconds up to one hour and there is a
wide variety in frequency.Vasomotor symptoms are probably caused by
changes in the temperature centre in the hypothalamus via different
neurotransmitter systems as a result of fluctuations in oestrogen level s.
Vasomotor symptoms have been reported as occurring among women
from different countries and societies but with varying prevalence. For
example Mayan women35 experience no hot flushes whereas in most
western countries there is general agreement from both cross -sectional
and longitudinal studies that 50 –75% of postmenopausal women report
hot flushes and night sweats of varying severity (Thunell et al ., 2004).
This difference may be explained by genetic differences, different ways
of identifying symptoms, d ifferent lifestyles and dietary habits (Sharma
and Saxena, 1981). Vasomotor symptoms have been reported as most
frequently experienced around the menopause but even 30 –50% of
women over 60 years of age experience symptoms (Stadberg et al., 1997).

42
Women wit h a surgically induced menopause often have more severe
symptoms compared to women with a natural menopause (Sherwin and
Gelfand, 1985).
Urogenital symptoms
Vaginal atrophy and urogenital complaints such as vaginal discomfort,
dysuria, dyspareunia and recu rrent lower urinary tract infections are more
common in women after the menopause (Milsom and Molander, 1998).
Epidemiological studies have demonstrated that more than 50% of
postmenopausal women suffer from at least one of these symptoms.
Symptomatic and cytological changes have been demonstrated in the
genitourinary tract during the menstrual cycle, in pregnancy and
following the menopause. In addition, factors influencing vaginal
cytology, vaginal pH and the vaginal bacterial flora in elderly women
have been identified (Milsom et al., 1993). Several features of the vaginal
microenvironment change with increasing age, mostly in response to
alterations in oestrogen and progesterone concentrations.
Treatment of menopausal symptoms
Menopause hormone therapy (MHT) is prescribed during the
perimenopausal period and early menopause for relief of menopausal
symptoms and for treatment of vulvovaginal atrophy. Oestradiol and
conjugated oestrogens are effective in treating the vasomotor symptoms,
urogenital atrophy symptoms and irregular menstrual bleeding that occur

43
in the perimenopausal period. Conjugated oestrogens are given orally and
oestradiol may be given orally as tablets, transdermally as a patch or gel,
or intranasally for a period of 3 weeks. Subdermal pel lets or long -acting
oestrogen injections have also been used. Perimenopausal women and
women during the first 1 to 2 years after the menopause who have an
intact uterus must be treated with a gestagen for at least 12 to 14 days
every month in order to prev ent endometrial hyperplasia and possible
endometrial cancer. There is a wealth of evidence to support the efficacy
of hormone replacement therapy (HRT) in the treatment of climacteric
symptoms such as vasomotor symptoms, urogenital symptoms and
irregular b leeding in perimenopausal women. During the last two decades
a debate has continued regarding the possible pros and cons of HRT. A
large number of observational studies have shown that long -term use of
oestrogens has prophylactic effects against coronary h eart disease (CHD)
and osteoporosis (CDC, 1993).
Benefit -versus -risk analysis of MHT
Menopause and aging are associated with the onset and progression of
many chronic illnesses, including CHD, stroke, osteoporosis, dementia,
and cancer. Physicians who are responsible for the care of women must
consider the potential benefit and risk of therapy for both treating
symptoms and potentially preventing disease with MHT. The timing of
therapy may be critical because it has been shown that disease prevention

44
may be possible only if therapy is initiated early in menopause, whereas
the same treatment may prove more deleterious later.
Endometrial Cancer
The use of unopposed estrogen in menopausal women with an intact
uterus has been associated with development of endom etrial cancer (Neil
et al., 2011).
Breast Cancer
The weight of evidence from years of basic research indicates that
exposure to estrogen is an important determinant of the risk of breast
cancer. The proposed mechanism of estrogen -induced carcinogenesis is
the transformation of estrogen into genotoxic, mutagenic metabolites that
initiate and promote the development of breast cancer cells (Neil et al.,
2011).
Effect of MHT on nonreproductive organ systems
Prevention of the consequences of aging and menopause in
nonreproductive organs by the use of estrogen has been evaluated in
many studies, including observational, case -controlled, and interventional
trials (Neil et al., 2011).
Osteoporosis
Postmenopausal osteoporosis causing spine and hip fractures is asso ciated
with considerable morbidity and mortality. Data from randomized
contriol trials substantiate the efficacy of estrogens in preserving bone

45
mass and, less consistently, preventing fractures. The beneficial effects of
MHT on bone protection (Speroff et al., 1996) persist even with doses of
estrogen below those commonly used for relief of symptoms, although
the benefit may decrease with lower doses of estrogen. In some women,
the skeleton may not respond to conventional doses, and a lower dosage
may be e ffective (Neil et al., 2011).
Dementia
After age 80 years, women have an increased risk of Alzheimer disease in
comparison with men (possibly attributable to postmenopausal depletion
of endogenous estrogen). The prospective, longitudinal Cache County
[Utah ] Study (Zandi et al., 2002) investigated the prevalence and
incidence of Alzheimer disease in a cohort of 5,677 elderly adults. Study
results showed that the risk of this disorder varied with the duration of
self-selected use of MHT. A longer duration of MHT use was associated
with a greater reduction in the risk of Alzheimer disease. Prior MHT use
was associated with a decreased risk in comparison with nonusers, and
women‟s higher risk in comparison with men was virtually eliminated
after more than 10 yea rs of exposure to MHT. In addition, there was no
apparent benefit with current use of MHT unless that use exceeded 10
years (Zandi et al., 2002).
Iron Status in post menopause
Estrogen and iron are two of the most important growth nutrients in a

46
woman's b ody development (Jian, et al ., 2009). Estrogen affects the
growth, differentiation, and function of tissues such as breasts, skin, and
bone (Simm et al ., 2008). Iron is essential for oxygen transport, DNA
synthesis, as well as energy production. Estrogen d eficiency has been
considered the major cause of menopausal symptoms and diseases. The
3rd National Health and Nutrition Examination Survey (NHANES -3)
show ed that concurrent but inverse changes occur between iron and
estrogen levels in healthy women during menopausal transition. Whereas
estrogen decreases because of the cessation of ovarian functions, iron
increases as a result of decreasing menstrual periods (Zacharski et al.,
2000). For example, level of serum ferritin is increased by two – to
threefold during this period. It has been estimated that 1  μg/L serum
ferritin corresponds to 120  μg storage iron per kg bodyweight. This
produces an increase in body iron storage from 4.8  mg/kg bodyweight at
the beginning of perim enopause at age 45 years to 12  mg/kg bodyweight
after menopause at age 60 years. Although increased iron as a result of
menopause is considered within normal physiologic range, potential
health problems in women, as well as in men or neonates, could be lin ked
to increased iron storage, which is normal but not necessarily healthy. For
example, a role for iron has been proposed in the pathogenesis of many
diseases, such as osteoporosis, skin aging, ischemic heart disease, cancer,

47
diabetes, infections, and neu rodegenerative disorders.
2.11 Causes of Iron Deficiencies in Post Menopause
Malnutrition
Malnutrition is a major cause of iron deficiency across all population
together with socioeconomic status of individuals. Post menopause
women undergoing malnutriti on are likely to have reduce iron stores and
hence iron deficiency may ensue. The amount and quality of food taken
by individual population differs depending on their financial strenghth.
Post menopause women living in rural area tend to be undernutrished due
to their poor quality of food.
Coeliac Disease
Coeliac disease can present as iron deficiency without the typical
symptoms of gluten intolerance. Coeliac disease is characterised by
gliadin insensitivity mediated by T cells (Guha, 2009). This is
accompanied with an upregulated autoimmune response, which manifests
with the classic symptoms of abdominal pain, bloating and diarrhoea on
encountering gluten containing food. This should be further investigated
via an antitissue transglutaminase (anti TTG) and if necessary endoscopy
and subsequent duodenal biopsy (Sanders et al ., 2003). Prevalence has
been estimated at around 6% in the adult female population with anaemia.

48
Helicobacter pylori infection
Helicobacter pylori (H. pylori) colonisation in gastr ic mucosa may impair
iron uptake and increase iron loss, potentially leading to iron deficiency
anaemia (IDA) (Helena et al., 2013). The speculative mechanisms by
which H. pylori may produce IDA have recently been reviewed. H. Pylori
infection is also invo lved in the development of atrophic body gastritis14
that can in turn cause decreased gastric acid secretion and IDA (Dickey,
2002). Four meta -analyses to assess the effect of H. pylori eradication
combined with ferrous supplementation on the treatment of IDA have
been published (Yuan et al., 2010). The conclusions suggest that H. pylori
eradication therapy improves iron absorption, since H. pylori eradication
combined with iron administration was more effective than iron
administration alone for the treatm ent of IDA (Annibale et al., 2003).
Causes of iron deficiency anaemia with prevalence as percentage of
total
Occult GI Blood Loss
Common
 Aspirin/NSAID use 10 –15%
 Colonic carcinoma 5 –10%
 Gastric carcinoma 5%
 Benign gastric ulceration 5%
 Angiodysplasia

49
Uncommon causes
 Oesophagitis 2 –4%
 Oesophageal carcinoma 1 –2%
 Gastric antral vascular ectasia 1 –2%
 Small bowel tumours 1 –2%
 Ampullary carcinoma <1%
 Ancylomasta duodenale

Malabsorption
Common
 Coeliac disease 4 –6%
 Gastrectomy <5%
 H. pylori colonisation <5%
Uncommon
 Gut resection <1%
 Bacterial overgrowth <1%

50
CHAPTER THREE
Materials and Methods
3.0 Study Area
This study was conducted in the Department of Medical Laboratory
Science and the University of Benin Teaching Hospital, Benin City, Edo
State. A tota l of 200 subjects, comprised of 150 post menopausal women
and 50 pre menopausal women.
3.1 Inclusi on criteria
Postme nopausal women without diseases that may lead to IDA.
Exclusion criteria; Post menopausal women with diseases that may result
in IDA
Cont rol Group
Control group incl uded women of reproductive age (20 -35years) .
3.2 Specimen collection
10milliters of venous blood samples from subjects were collected into
vacuum tubes and sent to the laboratory for biochemical analysis after
sample collection was completed. The indicators of iron status assayed
for in this study includes : serum iron , serum ferritin, and the total iron
binding capacity . Transferrin saturation (Tsat) was calculated as a
percentage of SI to TIBC. Antibody to H. Pylori was also determed to
detect the infection in the organism.

51
3.3 Determination of Serum Iron
Photometric colorimetric test for iron with lipid clearing factor (lcf) by
chromoazural b (cab) method
Reagent stability
RGT is stable even after opening up to the stated expiry date when stored
at 2….25˚c
Contamination of the reagents was absolutely avoided.
Measurement
Against reagent blank (Rb) and only one reagent blank per series is
required.
Principle
Iron III reacts with chromoazural b (CAB) &cetyltrimethyl ammonium
bromide (CTMA) to form a coloured ternary complex with an absorbance
maximum at 623 nm. The intensity of the colour produced is directly
proportional to the concentration of iron in the sample.
Procedure
1. Fresh test tubes were set up together with a test tube labelled standard
and blank
2. 50μl of sera (test) and standard were pipetted and dispensed into the
test tubes and standard test tubes respectively.
3. 50μl of distil water was pipetted into the tube labeled blank
4. 1000μL of reagent was pipetted into all tubes

52
5. The tubes were mixed properl y and incubated in room temperature for
15 mins.
6. The absorbance of all tubes were read against reagent blank at 623nm
7. The concentration of iron in each sample was calculated using
Beer-Lambert‟s Law.
Calculation of Iron concentration
From Beer -Lambert‟s Law (First principle)
Tab x conc. STD
Sab 1
Where T ab = absorbance of sample
Sab = absorbance of standard, = 100μg/dl
Δ Ab Sample x 10
Conc of sample (μg/dl.) = Ab STD
This method is linear up to an iron concentration of 500μg/dl or
89.5μmol/l.
Reference value s
Male: 59 -148 μg/dl or 10 .6-28.3μ mol/l.
Female: 37 -145μg/dl or 6.6μ mol/l.
3.4 Total Iron Binding capacity
Principle
The iron binding protein transferrin in serum is saturated upon treatment
with excess of FE (III) ions. Unbound (excess) iron is adsorbed onto

53
aluminium oxide and pr ecipitated .The transferrin bound iron (TIBC) in
the supernatant is then determined.
Procedure
1. 1ml of Fe was pipetted into each reaction tube
2. 0.5ml of samples were added to each tube respectively
3. The tubes were mixed properly
4. One level measuring spoo nful of aluminium oxide ALOX
(approximately
0.25-.35g) was added to each tube after 5mins
5. The tubes were capped and place on a rotator or roller mixer for 10min.
6. The tubes were removed and centrifuged for 1 min at 5,000rpm.
Calculation of iron concent ration :
From Beer -Lambert‟s Law (First principle)
Tab x conc. STD
Sab 1
Where T ab = absorbance of sample
Sab = absorbance of standard, = 10μg/dl
Δ Ab Sample x 10
Conc of sample (μg/dl.) = Ab STD
Calculation for TIBC
To calculate the TIBC multiply the result of the iron determination in the
supernatant by the diluent factor 3.
TIBC = Conc. Iron X 3.

54
Reference values
TIBC: 274 -385μg/dl.
3.5 Determination of serum Ferritin by Meia method (Ax S YM micro
particle enzymes (MEIA) assay technology).
Principles of the procedure
AxSYM Ferritin is based on micro particle enzyme (MEIA) technology.
Sample and all AxSYM Ferritin reagents required for one test are pipetted
in the following sequence.
Sample centre
Sample and all AXSYM Ferritin reagents were pipetted by the sampling
probe into various wells of a reaction vessel (RV).
Sample was pipetted into one well of the RV .
Anti-Ferritin coated micro particles, Anti ferritin Alkaline phosphatase
conjugate, specimen diluent and tris buffer were pipetted into another
well of RV .
The RV was immediately transferred into the processing centre.
Further pipetting was done in the processing centre with the processing
probe.
Processing Centre
An aliquot of the speci men diluent, conjugate, micro particles &Tris
buffer mixture were pipetted and mixed with the sample .The ferritin
enzyme labelled antibody and micro particles bind forming an antibody
–antigen -antibody complex.
Complex bounded to the micro particles were transferred to the matrix

55
cell. The matrix cell was washed to remove unbound materials.
The substrate, 4 -methyl umbelliferyl phosphate was added to the matrix
cell and the fluorescent product was measured by the MEIA optical
assembly.
3.6 Determination of Transferrin Saturation
Transferrin saturation (Tsat) was calculated as a percentage of Serum Iron
to TIBC (Wang and Shaw, 2005).
3.7 Determination of H. Pylori antibody
Serologic testing for H pylori –specific antibodies was carried out using
the HM -CAP en zyme immunoassay (E -Z-EM, Lake Success, NY) to
detect immunoglobulin G (IgG) antibodies against high molecular weight
cell associated proteins of H. Pylori as modified by Rabieipoor et al
(2011).
Cut-off values and Criteria for iron Deficiency
Cut-off valu es for abnormal iron indices were set at Tsat <15% and
Serum Ferritin <12μg/L (Looker et al. , 1997; Cook et al. , 1976). Elevated
iron stores were defined as Serum Ferritin >300 μg/L (Looker et al .,
1997).
Statistical Analysis
Statistical analysis was performed with SPSS Statistical Software version
16.0 to compare means between test and control. P<0.05 was considered
significant. The results were expressed as mean±standard error of mean.
The association between Serum Ferritin and other iron indices was
evaluated by Pearson correlation analysis and logistic regressi on analysis.

56
CHAPTERFOUR
RESULTS AND ANALYSIS

Table 1: Ages and Level of Helicobater pylori infection in Post -menopausal
women and control groups.
Values are represented as mean ± SEM for six determinations. Means
with ** are significantly different (p<0.05) by the independent t -test.
The table above shows the mean age of post -menopausal and
pre-menopausal women used for the study. Its revealed that H. pylorim
infection among post -menopausal women were not significantly different
from that of the pre -menopausal women (P>0.05).
Variables Post-menopausal women
Mean ± SEM (n = 100) Pre-menopausal women
Mean ± SEM (n= 50) P-value
Age (years) 54.70±0.30 29.60±0.50
Helicobacter
pylori Positive 3 (3%) 2 (4%) 0.182
Negative 97 (97% ) 48 (96%) 0.110

57

Table 2: comparison of the level serum Iron, TIBC, Transferrin, ferritin, PCV , and Hb.
Conc. Between test and cont rol subjects.
Values are represented as mean ± SEM for six determinations. Means
with ** are significantly different (p<0.05) by the independent t -test.
The table above showed a significant increase in the serum iron,
transferrin, and ferritin in post -meno pausal women when compared to
those of the pre -menopausal women at p<0.05. Whereas, the total iron
binding capacity (TIBC), PCV , and Haemoglobin levels showed no
significant change between the two groups (p>0.05).
Variables Post-menopausal women
Mean ± SEM (n = 100) Pre-menopausal women
Mean ± SEM (n= 50) P-value
Age (years) 54.70±0.30 29.60±0.50
Iron (µg/dl) 81.60±2.00 51.16±2.00 0.002
TIBC (µg/dl) 258.79±3.00 257.04±5.00 0.222
Transferrin (%) 31.83±0.70 19.26±0. 90 0.046
Ferritin (µg/dl) 73.66±2.00 51.76±2.00 0.008
PCV (%) 39.90±0.50* 40.25±0.60* 0.494
Hb conc. (g/dl) 12.79±0.20* 12.27±0.20* 0.144

58
Table 3: Correlation between age and mea sured parameters.
Parameters Correlation with age p-value
Plasma iron 0.090 0.380
TIBC 0.083 0.439
Transferrin 0.159 0.138
Ferritin 0.119 0.244
PCV -0.155 0.059
Haemoglobin concentration -0.020 0.807

The table revealed that there was no relationshi p between the age of
post-menopausal women and the parameters.

59
Table 4: Level of H. Pylori infection in post and pre menopausal women
and control groups

The table above show the mean age of post/pre menopausal women that
were positive for H. Pylori and control subject. It was revealed that H.
Pylor i infection among post/pre menopausa l women were not significant
(P>0.05).

Variables Post/Pre all positive for
H. Pylori Mean ± SEM (n= 5) P-value
Age (years) 54.70±0.30 29.60±0.50
H. Pylori (Positive) 5 (3%) 0.182
Negative 145 (97%) 0.110

60
CHAPTER FIVE
5.0 DISCUSSION
Anemia due to iron deficiency is a common feature in post -menopausal
women (Haseena et al., 2014) and blood loss due to H. Pylori infection is
one of the major causes. Thus, this research was aimed at assessing the
impact of H. Pylori infection on iron bioavailability, as well as, ferritin,
transferin and total iron binding capacity (TIBC) in post -menopausal
women. Basal Iron is lost via the blood and mainly from the GIT .
Results obtained from this research revealed that, plasma iron increased
significantly compared to the control at P<0.05. The increase in plasma
iron may be attributed to absence menstrual blood loss in the
post-menopausal women (Mast et al., 2002) and possible increased iron
storage capacity in the form of ferritin and transferrin; ferritin increased
compared to the control at P<0.05 depicting increased storage capacity in
the post -menopausal women while TIBC did not show any ch ange
between the tests and the control group, 259 µg/dl and 257 µg/dl,
respectively (p>0.05). In support of this claim, transferrin increased
significantly for the test group (P<0.05) compared to the control group.
Ferritin is the major iron -storage protei n at the cellular and organismal
level. It is responsible for the sequestration of potentially harmful,
reactive iron. Ferritin stores iron in its unreactive Fe3+form inside its
shell as a result of a strong equilibrium between ferritin -bound iron (Fe3+)

61
and the labile iron pool in the cells (Fe2+), by which ferritin prevents the
formation of ROS mediated by Fenton reaction (Elizabeth et al., 2008).
Because of its important function in the storage of iron, ferritin is
ubiquitous in tissues, serum, and in ot her multiple locations within the
cell. It is regulated at the transcriptional and posttranscriptional level by
various pathways in response to diverse stimuli (Elizabeth et al., 2008).
There was no observed correlation between the age of the
post-menopaus al women and the various parameters.
Helicobacter pylori colonization in gastric mucosa may impair iron
uptake and increase iron loss, potentially leading to Iron deficiency
Anaemia. Studies have shown that persons seropositive for H. pylori
infection have a significantly lower serum ferritin level (Rabieipoor et al.,
2011). This study however, showed that the 97% of the pre -menopausal
women were negative for H. Pylori thus accounting for the previous data
obtained.
5.1 CONCLUSION/RECOMMENDATION
From this study, iron deficiency anaemia was not observant in
post-menopausal women attending accident and emergency hospital. The
Population of the study subjects that were pulled from the
pre-menopausal and post -menopausal women was normal. The
prevalence of thi s infection in the study were relatively low, probably
may be due the low observance for iron deficiency anaemia in the study

62
group of patients.

63
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APPENDIX I

QUESTIONNAIRE INFORMATION

To investigate menopause in women attending ac cident and
Emergency unit of University of Benin Teaching hospital, Benin
city, Edo State.

Please tick where appropriate:
1. Age: 30 -40yrs 40 -50yrs above 50
2. Level of education: primary Secondary
Tertiary
3. Are you current ly on contraceptive drug? Yes No
4. Are you on chemotherapy (anticancer drug)? Yes
No
5. Do you have a child/children? Yes No
6. When was your last menstrual period?
……………………………………….
7. Is there a history of early menopause in your family? Yes
No
8. Have you ever smoked cigarette; if yes for how long
……………………..
9. Have you ever had a long term urinary tract infection? Yes
No
10. Do you often have epileptic seizure? Yes No
11. Do you often experience pain during sexual intercourse?
Yes No
12. Did you notice weight gain since your last menstrual
period? Yes No
13. Do you experience hot flashes (spontaneous feeling of
warmth all over the body lasting 30 sec)? Yes No