Iron Deficiency Anaemia

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 age 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 blood 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-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, cytochromes, 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 Pantopoulos, 2005). This property is largely responsible for the biologic significance of iron. When the uptake and metabolism of iron is interrupted, this call result in iron loss and consequently, Iron deficiency will ensue. Infection 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 reduced 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 is capable of binding and 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 appear 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 infection and its relationship with iron deficiency.

1.3 Aim of the Study

The aim of this study is to investigate iron deficiency in post menopausal women in accident and emergency unit of University of Benin teaching hospital, Edo state.

1.4 Specific Objectives 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 among 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 (premenopausal 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.

1.6 Informed Consent/questionnaire

Individuals who participated in this study include those who gave personal informed consent; having adequately understood the aim and objectives of the study and has completed a questionnaire to this effect

CHAPTER TWO

Literature Review

2.0 Iron as an essential Nutrient

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-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, cytochromes, 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 Pantopoulos, 2005). This property is largely 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 excess of labile iron. This tendency 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 H2O2, 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 production 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 for organismal iron requirements. Likewise, an 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 have 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 Transports

Proteins involved in the transport of iron are; transferrin, ferritin, hemosiderin and hepcidin.

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 total iron binding capacity (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 levels 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, and 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).

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 endocytosis 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 bloodstream. 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 N1, 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 circulating insulin-like growth factors (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

With respect to the mechanism of absorption, there are two kinds of dietary iron: heme iron and non-heme iron (Hallberg, 1981). In the human diet the primary sources of heme iron are the haemoglobin and myoglobin from consumption of meat, poultry, and fish whereas non-heme iron is obtained from cereals, pulses, legumes, fruits, and vegetables. The average absorption of heme iron from meat-containing meals is about 25 percent (Hallberg, 1979) The absorption of heme iron can vary from about 40 percent during iron deficiency to about 10 percent during iron repletion (Hallberg et al., 1997). Heme iron can be degraded and converted to non-heme iron if foods are cooked at a high temperature for too long. Calcium is the only dietary factor that negatively influences the absorption of heme iron and does so to the same extent that it influences non-heme iron.Absorption of iron in the proximal duodenum is tightly regulated ,as there is no physiologic pathway for its excretion from the body. 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 the food also help to reduce it to ferric iron to ferrous form. (Robert et al 206).

Factors influencing dietary iron absorption

Heme Iron Absorption

Iron status of subject

Amount of dietary heme iron, especially as meat

Content of calcium in meal (e.g., milk, cheese)

Food preparation (time, temperature)

Non-heme Iron Absorption

Iron status of subjects

Amount of potentially available non-heme iron

Balance between enhancing and inhibiting factors

Enhancing factors

Ascorbic acid (e.g., certain fruit juices, fruits, potatoes, and certain vegetables) Meat, chicken, fish and other seafood, fermented vegetables.

Inhibition of iron absorption

Phytates are found in all kinds of grains, seeds, nuts, vegetables, roots (e.g., potatoes), and fruits. Chemically, phytates are inositol hexaphosphate salts and are a storage form of phosphates and minerals. Other phosphates have not been shown to inhibit non-heme iron absorption. Phytates strongly inhibit iron absorption in a dose-dependent fashion and even small amounts of phytates have a marked effect (Gillooly, 1983). Calcium, consumed as a salt or in dairy products interferes significantly with the absorption of both heme and non-heme iron (Gleerup et al., 1993). Because calcium and iron are both essential nutrients, calcium cannot be considered to be an inhibitor in the same way as phytates or phenolic compounds. The practical solution for this competition is to increase iron intake, increase its bio-availability, or avoid the intake of foods rich in calcium and foods rich in iron at the same meal (Gleerup, A. (1995).

Enhancement of Iron absorption

Ascorbic acid is the most potent enhancer of non-heme iron absorption (Siegenberg, 1991). Synthetic vitamin C increases the absorption of iron to the same extent as the native ascorbic acid in fruits, vegetables, and juices. The effect of ascorbic acid on iron absorption is so marked and essential that this effect could be considered as one of vitamin C physiologic roles (Hallberg et al., 1987). Meat, fish, and seafood all promote the absorption of non-heme iron (Cook and Monsen, 1976).

Iron balance and regulation of Iron absorption

The body has three unique mechanisms for maintaining iron balance and preventing iron deficiency and iron overload. The first is the continuous re-utilisation of iron from catabolised erythrocytes in the body. When an erythrocyte dies after about 120 days, it is usually degraded by the macrophages of the reticular endothelium. The iron is released and delivered to transferrin in the plasma, which brings the iron back to red blood cell precursors in the bone marrow or to other cells in different tissues. Uptake and distribution of iron in the body is regulated by the synthesis of transferrin receptors on the cell surface. This system for internal iron transport not only controls the rate of flow of iron to different tissues according to their needs but also effectively prevents the appearance of free iron and the formation of free radicals in the circulation.

Thes second mechanism is the access of the specific storage protein, ferritin, which can store and release iron to meet excessive iron demands. This iron reservoir is especially important in the third trimester of pregnancy.

The third mechanism involves the regulation of absorption of iron from the intestines, with an increased iron absorption in the presence of decreasing body iron stores and a decreased iron absorption when iron stores increase. Iron absorption decreases until an equilibrium is established between absorption and requirements. For a given diet this regulation of iron absorption, however, can only balance losses up to a certain critical point beyond which iron deficiency will develop (Hallberg, 1995). About half of the basal iron losses are from blood, primarily in the gastrointestinal tract. Both these losses and the menstrual iron losses are influenced by the haemoglobin level; during the development of an iron deficiency, menstrual and basal iron losses will successively decrease when the haemoglobin level decreases. In a state of more severe iron deficiency, skin iron losses may also decrease. Iron balance (absorption equals losses) may be present not only in normal subjects but also during iron deficiency and iron overload.

The three main factors that affect iron balance are absorption (intake and bio-availability of iron), losses, and amount in stores. The interrelationship among these factors was recently been described in mathematical terms, making it possible to predict, for example, the amount of stored iron when iron losses and bio-availability of dietary iron are known (Hallberg et al., 1998). With increasing iron requirements or decreasing bio-availability, the regulatory capacity to prevent iron deficiency is limited (Hallberg, 1995). However, to prevent iron overload with increasing dietary iron intake or bio-availability, the regulatory capacity seems to be extremely good.

2.3 The role of iron in human metabolic processes

Iron has several vital functions in the body. It serves as a carrier of oxygen to the tissues from the lungs by red blood cell haemoglobin, as a transport medium for electrons within cells, and as an integrated part of important enzyme systems in various tissues. The physiology of iron has been extensively reviewed (Hallberg, 1982; Dallman, 1986).

Most of the iron in the body is present in the erythrocytes as haemoglobin, a molecule composed of four units, each containing one heme group and one protein chain. The structure of haemoglobin allows it to be fully loaded with oxygen in the lungs and partially unloaded in the tissues (e.g., in the muscles). The iron-containing oxygen storage protein in the muscles, myoglobin, is similar in structure to haemoglobin but has only one heme unit and one globin chain (Mascotti, et al., 1995). Several iron-containing enzymes, the cytochromes, also have one heme group and one globin protein chain. These enzymes act as electron carriers within the cell and their structures do not permit reversible loading and unloading of oxygen. Their role in the oxidative metabolism is to transfer energy within the cell and specifically in the mitochondria. Other key functions for the iron-containing enzymes (e.g., cytochrome P450) include the synthesis of steroid hormones and bile acids; detoxification of foreign substances in the liver; and signal controlling in some neurotransmitters, such as the dopamine and serotonin systems in the brain. Iron is reversibly stored within the liver as ferritin and hemosiderin whereas it is transported between different compartments in the body by the protein transferrin.

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 Fe2+to Fe3+for storage inside the shell (Lawson et al., 1989). In contrast, 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 dependent (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 express 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).

Hepcidin Regulate Iron Distribution

Hepcidin is a peptide 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 mobilization 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 iron 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 without 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 with chronic iron deficiency anemia. Iron deficiency in the developing brain in the early 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 hyperthyroidism 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 refractory iron deficiency anemia is inferior to that of ordinary iron deficiency anemia.

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

Fig. 4: Blood film of iron deficient anaemia, Normal and anaemic (Finberg, 2009)

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 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 this 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 (Cole and Williams, 1992).

Causes of Iron Deficiencies

Iron deficiency develops when the amount of iron absorbed from the diet 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 blood loss (Mast et al., 2002).

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 quality, based on the 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 higher 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; Hambraeus, 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 smooth external cell wall together with glycocalix (Kostrzynska et al., 1991). It has a somatic antigen, heat resistant lipopolysacharide, flagellum-related heat sensitive antigen and urease antigen at the external surface and periplasmic space (Buck and Smith, 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, haemolysine, 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 uptake and increase iron loss, potentially leading to Iron deficiency Anaemia. Many researches 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).

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

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

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

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

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

Epidemiology of H. Pylori Induced Iron deficiency

Epidemiologic 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% increased 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 health survey from Germany (n=1806) revealed that persons with H. pylori infection had 17% decrease (95% CI 9.8-23.6) in serum ferritin concentration, after adjustment for age and sex (Rabieipoor et al., 2011). A study of Alaskan natives (n=2080) also showed an 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 with 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 seropositivity 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 (Rabieipoor 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 indices. 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 children with and without IDA (Rabieipoor et al., 2011).

2.7 Prevalence of Iron Deficiency

Iron deficiency (ID) is one of the most common and widespread nutritional disorders in the world. It is however difficult to estimate how many people are affected and mostly 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 infections, 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 approximately 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 that 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). Thus, world-wide ID may affect 2 to 2.5 billion people.

Markers of Iron Deficiency

The absence of iron stores (iron deficiency) can be diagnosed by showing that there is no stainable iron in the reticuloendothelial cells in bone marrow smears or more easily by a low concentration of ferritin in serum (15 µg/l). Even if an absence of iron stores per se may not necessarily be associated with any immediate adverse effects, it is a reliable and good indirect indicator of iron-deficient erythropoiesis and of an increased risk of a compromised supply of iron to different tissues.

Even before iron stores are completely exhausted, the supply of iron to the erythrocyte precursors in the bone marrow is compromised, leading to iron-deficient erythropoiesis (Hallberg, 1993). A possible explanation is that the rate of release of iron from stores is influenced by the amount of iron remaining. As mentioned above it can then be assumed that the supply of iron to other tissues needing iron is also insufficient because the identical transport system is used. serum markers of iron deficiency are 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 deficiency. 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 either test on its own, particularly in chronic disease (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’ iron deficiency and other chronic disorders 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 trial 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

Studies in animals have shown that iron deficiency has several negative effects on important functions in the body (Dallman, 1986). Physical working capacity in rats has been shown to be significantly reduced in iron deficiency, that is especially valid for endurance activities (Finch, 1976). This negative effect seems to be less related to the degree of anaemia than to impaired oxidative metabolism in the muscles with an increased formation of lactic acid, that in turn is due to a lack of iron-containing enzymes which are rate limiting for the oxidative metabolism (Scrimshaw, 1984).

The relationship between iron deficiency and brain function is of great importance for the choice of strategy in combating iron deficiency (Pollitt, 1993). Several structures in the brain have a high iron content of the same magnitude as observed in the liver. Of great importance is the observation that the lower iron content of the brain in iron-deficient growing rats cannot be increased by giving iron later on. This fact strongly suggests that the supply of iron to brain cells takes place during an early phase of brain development and that, as such, early iron deficiency may lead to irreparable damage to brain cells.

In humans about 10 percent of brain iron is present at birth; at the age of 10 years the brain has only reached half its normal iron content, and optimal amounts are first reached at the age of 20-30 years.

In populations with long-standing iron deficiency, a reduction of physical working capacity has been demonstrated by several groups with improvement in working capacity after iron administration (Scrimshaw, 1984). Iron deficiency also negatively influences the normal defence systems against infections. The cell-mediated immunologic response by the action of T lymphocytes is impaired as a result of a reduced formation of these cells. This in turn is due to a reduced DNA synthesis depending on the function of ribonucleotide reductase, which requires a continuous supply of iron for its function. The phagocytosis and killing of bacteria by the neutrophil leukocytes is an important component of the defence mechanism against infections. These functions are impaired in iron deficiency. The killing function is based on the formation of free hydroxyl radicals within the leukocytes, the respiratory burst, and results from the activation of the iron-sulphur enzyme NADPH oxidase and probably also cytochrome b (a heme enzyme) (Brock, 1994).

The impairment of the immunologic defence against infections that was found in animals is also regularly found in humans. Administration of iron normalises these changes within 4-7 days. It has been difficult to demonstrate, however, that the prevalence of infections is higher or that their severity is more marked in iron-deficient subjects than in control subjects. This may well be ascribed to the difficulty in studying this problem with an adequate experimental design. A relationship between iron deficiency and behaviour such as attention, memory, and learning, has been demonstrated in infants and small children by several groups. In the most recent well-controlled studies, no effect was noted from the administration of iron. This finding is consistent with the observations in animals. Therapy-resistant behavioural impairment and the fact that there is an accumulation of iron during the whole period of brain growth should be considered strong arguments for the more active and effective combating of iron deficiency. This is valid for women, especially during pregnancy, for infants and children, and up through the period of adolescence and early adulthood. In a recent well-controlled study, administration of iron to non-anaemic but iron-deficient adolescent girls improved verbal learning and memory (Bruner, 1996). Well-controlled studies in adolescent girls show that iron-deficiency without anaemia is associated with reduced physical endurance and changes in mood and ability to concentrate (Ballin, 1992). A recent careful study showed that there was a reduction in maximum oxygen consumption in non-anaemic women with iron deficiency that was unrelated to a decreased oxygen-transport capacity of the blood.

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. If 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 stores (Smith, 1997). This is achieved most simply and 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. ferrous 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 transfusions 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). Transfusions should aim to restore haemoglobin to a safe level, but not necessarily 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 Reproductive 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 10 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 transition. Although some of these women may be asymptomatic, estrogen deficiency is associated with hot flashes, sweating, insomnia, and vaginal dryness and discomfort in up to 85% of menopausal women. Most women with menopausal symptoms will experience spontaneous 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 diminish 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 associated 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 menopause 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).

2.10 Signs and symptoms associated with Menopause

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 sweats

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)

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 moisture 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 levels. 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, different 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). Women with 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 recurrent 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 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 pellets 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 prevent 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 bleeding 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 heart 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 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 endometrial 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 associated with considerable morbidity and mortality. Data from randomized contriol trials substantiate the efficacy of estrogens in preserving bone 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 effective (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 years 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 woman's body 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 deficiency has been considered the major cause of menopausal symptoms and diseases. With the iron data obtained from the Third National Health and Nutrition Examination Survey (NHANES III), shows 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 perimenopause 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 linked 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, diabetes, infections, and neurodegenerative 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 malnutrition 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.

Helicobacter pylori infection

Helicobacter pylori (H. pylori) colonisation in gastric 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 involved 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 treatment 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

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%

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 total of 200 subjects, comprised of 150 post menopausal women and 50 pre menopausal women.

3.1 Inclusion criteria

Postmenopausal women without diseases that may lead to IDA.

Exclusion criteria; Post menopausal women with diseases that may result in IDA

Control Group

Control group included 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 (SI), total iron binding capacity (TIBC), and serum ferritin (SF). 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.

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

Fresh test tubes were set up together with a test tube labelled standard and blank

50μl of sera (test) and standard were pipetted and dispensed into the test tubes and standard test tubes respectively.

50μl of distil water was pipetted into the tube labeled blank

1000μL of reagent was pipetted into all tubes

The tubes were mixed properly and incubated in room temperature for 15 mins.

The absorbance of all tubes were read against reagent blank at 623nm

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 Tab = 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 values

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 aluminium oxide and precipitated .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

The tubes were mixed properly

One level measuring spoonful of aluminium oxide ALOX (approximately

0.25-.35g) was added to each tube after 5mins

The tubes were capped and place on a rotator or roller mixer for 10min.

The tubes were removed and centrifuged for 1 min at 5,000rpm.

Calculation of iron concentration:

From Beer-Lambert’s Law (First principle)

Tab x conc. STD

Sab 1

Where Tab = 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.

Reference values

TIBC: 274-385μg/dl.

3.5 Determination of serum Ferritin by Meia method (Ax SYM 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 specimen 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 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 enzyme 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 values 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 regression analysis.