Category Archives: Anemia

Injectable Iron


Iron deficiency, the commonest cause of anaemia, is treated by iron supplementation. Oral iron, introduced by the French physician Pierre Blaud in the 19th century is the mainstay of therapy of iron deficiency. Oral iron is inexpensive and safe but is poorly absorbed and causes abdominal adverse effects in 35-59% of the patients. Injectable iron preparations were initially developed for the treatment of iron deficiency in patients intolerant to iron. Their use became more prevalent with it became clear that iron deficiency was a common cause of failure of erythropoiesis stimulating agent therapy and that oral iron was insufficient for this indication.

 

Is it possible to inject an iron salt, say ferric chloride, for iron deficiency, like it is to inject calcium chloride for hypocalcaemia? Iron, unlike calcium or potassium,  produces free radicals (oxyradicals) that can damage macromolecules and result in cellular injury. Ferric hydroxide,  the first injectable iron preparation used, caused an immediate release of iron in circulation resulting in severe reactions.  When an iron oxide, hydroxide or an iron salt is used, only a small dose can be safely administered. One estimate calculated the maximum iron permissible as 8mg/day (this is equivalent of the unbound iron binding capacity of transferrin). It would take about 6 months to bring up haemoglobin at this dose.

 

About  25mg of iron is delivered each day to the erythroid precursors for haemoglobin synthesis. Iron is a poorly absorbed micronutrient and stores are needed to  provide for sudden increase in demand as may be seen in patients with acute blood loss. Given the propensity of iron to cause free radical induced cellular injury, transport and storage systems capable of protecting the body from iron have evolved. Iron is transported bound to transferrin and stored as ferritin and haemosidin. In both the situations iron is bound to apoproteins (apotrasferrin and apoferritin) and this binding does not allow free radical generation.

Erythrocyte destruction takes place in the macrophages and macrophages have systems for the handling, storage and recycling of iron. Injectable iron preparations have a carbohydrate shell that prevents exposure of the plasma to free iron.  Injectable iron preparations are taken up by macrophages of the reticuloendothelial system by endocytosis. The iron is released and enters the macrophages iron pool. This iron has a fate similar to iron reaching the macrophage from other sources viz. intestinal absorption and erythrocyte destruction. Binding iron to carbohydrate shell has made it possible to administer as high as dose as 1000mg over as short a time as 15 minutes. This is a 125 fold increase over the amount of iron that can be administered without the carbohydrate shell.

 

Injectable iron preparations share a common structure. They have a core of iron oxide/hydroxide that is associated with a carbohydrate shell. The carbohydrate shell  keeps free iron from entering the plasma and in a way performs the same function as transferrin. The antigenicity of the shell and the strength of binding between the iron core and the carbohydrate shell determines the side effects and the maximum dose per injection.

 

Preparations of Injectable Iron

The preperations of injectable iron include

  1. Preparations with a strong association between iron core and the carbohydrate shell
    1. High-Molecular weight iron dextran
    2. Low -molecular weight iron dextran
    3. Ferric carboxymaltose
    4. Ferumoxytol
  2. Perperations with a weak association between iron core and carbohydrate shell
    1. Iron Sucrose
  3. Preperations that have a labile low molecular weight components
    1. Ferric Gluconate
    2. Iron-Sorbitol-Citric Acid Complex, Dextrin-Stabilized

 

Table 1 – parentral iron preparations
Drug Shell MW T1/2 Labile
Iron
Maximum
Dose and administration
HMW ID High moleculer weight dextran 265 60 1-2% Should be administered 1 hr after an intravenous test dose (0.5ml over at least 5 minutes) Maximum dose 20mg/kg. Administered as intravenous bolus 100mg/day slowly. A total dose infusion may be given over a prolonged period.
LMW ID  Low molecular weight dextan  165  20  1-2% Should be administered 1 hr after an intravenous test dose (0.5ml over at least 30 seconds). It may be administered as daily iv bolus of 100mg over 2 mins or a a total dose infusion over 3-4 hours may be administered 20mg/kg
Iron Sucrose  Sucrose 30-60  6  4-5% up to 300mg in a single dose, higher doses have been administered over as a prolonged infusion. May be administered undiluted as an iv bolus 200mg over at least 10 mins or as an infusion in 0.9% saline over 15-60 minutes
Ferric Gluconate  Gluconate  289-440  1  5-6% 125mg, iv bolus at 12.5mg/min or as a 1 hr infusion in 0.9% saline
Ferric Carboxy-maltose Carboxy-maltose  150  16  1-2% The maximum dose is 20mg/kg (max 1000mg). Doses of 200-500mg should be administered at 100mg/min, doses between 500mg and 1000mg should be administered over 15 minutes. It may be administered as an iv infusion diluted in 0.9% saline
Ferric Isomaltoside Isomaltoside 150  20  <1% May be administered as an intravenous bolus dose of 500mg un to three times a week at the rate of 50mg/min or 20mg/kg (maximum 1000mg) in 0.9% saline over 1 hour
Ferumoxytol Carboxy-methyl Dextran 750 15 <1% 510mg as a iv bolus at the rate of 30mg/sec. A second dose may be administered after 3-8 days

The parental iron preparations differ in the carbohydrate that surrounds the iron core. The therapeutic implications of these differences are:

  1. Antigenicity: Dextran is antigenic. Patinets may have performed antibodies to dextran or may develop antibodies during therapy. All iron preparations  containing dextran can give rise to anaphylaxis. The risk is greatest with high molecular weight iron dextran. Anaphylaxis may also be seen with low molecular weight iron dextran though the risk is lower and has also been reported with ferumoxytol that contains carboxymethyl dextran.
  2. Maximum dose per injection: All intravenous iron preparations tend to release labile iron. There is an inverse relationship between the amount of labile iron released and the strength of association between the iron core and carbohydrate shell. Higher the molecular weight of the iron preparation, stronger is the association. Labile iron is taken up by transferrin. Non-transferrin bound iron (NTBI) appears when the binding capacity of transferrin is overwhelmed. NTBI is responsible for tissue damage and restricts the dose of a injectable preparation that can be given in a single infusion. Iron preparations may be classified on the basis of strength of the associations between iron core and the carbohydrate shell. Iron dextran, ferric carboxymaltose and ferumoxytol, that have a high molecular weights have a tight binding between iron core and the carbohydrate shell, release up to 2% labile iron. These can be given in a large single dose and are suitable for total dose infusion. Iron sucrose has a weaker association between iron core and carbohydrate shell and has 4-5% labile iron. This limits the amount of drug that can be administered at one time to 300mg. Ferric gluconate is a large molecule but has two types of polymers. Lower weight polymers (about 18,000) have a weaker association between iron and carbohydrate limiting the dose to 125mg per dose.

Dose and Administration

The total dose of parenteral iron is calculated as follows

Total iron dose (mg) = weight (kg) X Hemoglobin deficit  X 0.24 + 500

Haemoglobin deficit (g/L) = Target Haemoglobin (g/L) – Actual Haemoglobin (g/L)

 

Adverse Effects

  1. Infusion Related Events: the risk of infusion related events (per million) is 0.6 for iron sucrose, 0.9 for ferric gluconate, 3.3 for low molecular weight dextran iron and 11.3 for high molecular weight iron dextran.  Iron sucrose has been safely administered to patients who having sensitivity to iron dextran.  Despite the fact that some studies suggest that low molecular weight iron dextran in as safe as iron sucrose, a test dose must be administered before any iron dextran preparation.
  2. Delayed Reactions to Intravenous Iron: A syndrome charecterized by fever, arthralgia and lymphadenopathy may be seen as a delayed consequence of intravenous iron therapy. Premedication with steroids may reduce the risk of this manifestation.

Further Reading

  1. Rodolfo Delfini Cançado, Manuel Muñoz. Intarvenous iron therapy: How far have we come? Rev Bras Hematol Hemoter. 2011; 33(6): 461–469.
  2. Hayat A. Safety Issues With Intravenous Iron Products in the Management of Anemia in Chronic Kidney Disease. Clin Med Res. Dec 2008; 6(3-4): 93–102.
  3. Peter Geisser and Susanna Burckhardt The Pharmacokinetics and Pharmacodynamics of Iron Preparations. Pharmaceutics. Mar 2011; 3(1): 12–33.

 

 

Clinical Manifestation of Iron Deficiency


The manifestations of Iron deficiency evolve insidiously and are multi-systemic. They include

Anaemia

Iron deficiency causes hypochromic microcytic anaemia. The anaemia has an insidious onset and the patient may become severely anaemic before symptoms of anaemia become evident.

Non-Specific Symptoms

Iron deficiency can cause fatigue, irritability, dizziness, breathlessness, and headache. One may attribute these symptoms to anaemia but  these symptoms are seen in patients with iron deficiency even in the absence of anaemia. Iron therapy has been shown to correct many of these manifestations.

Skin and Hair

Iron deficiency results in brittle nails that have longitudinal ridges. The nails become thin, brittle and become flat (platonychia) or concave (koilonychia). Hair may become thin and brittle.

Gastrointestinal System

Next to blood the effects of iron deficiency are most pronounced on the gastrointestinal system

  1. Tongue: Iron deficiency causes atrophy of the papillae of the tongue. The filiform papillae of the anterior part of the tongue are the first to be affected. The fungiform papillae of the posterior tongue atrophy later resulting in a smooth red tongue.  Papillary atrophy results in soreness of the tongue. The changes reverse after about 1-2 weeks of iron therapy.
  2. Mouth: Iron deficiency results in angular stomatitis. This anomalies is not specific to iron deficiency may be seen in pyridoxine and riboflavin deficiency.
  3. Dysphagia: Iron deficiency results in the formation of webs at the junction of hypopharynx and oesophagus. These manifest as dysphagia that has an insidious onset. The patients complains of discomfort on swallowing, initially for solids, at the level of the cricoid cartilage. Mild dysphagia may be corrected by iron replenishment but dysphagia persists in most patients despite iron replenishment and dilatation may be needed. Oesophageal webs predispose to carcinoma. (See Plummer-Vinson Syndrome/Peterson-Kelly Syndrome)
  4. Pica: Pica is craving for non-nutritive substances like chalk, paint and ice. Pagophagia, the craving for ice is a common form of pica.

Neuromuscular Symptoms

Iron deficiency impairs performance of muscles. Children with iron deficiency show irritability, are disruptive, have impaired attention span and may show scholastic impairment. Iron deficiency has also been shown to be associated with developmental delay, ischaemic stroke and raised intracranial pressure.

Effect on Immunity and Infections

Iron deficiency decreases the number of T lymphocytes and impairs phagocytosis. This however does not appear to result in an increased risk of infections.

Menstrual Anomalies

While menstrual anomalies can be a cause of iron deficiency, they can often be a consequence of iron deficiency. Only response to treatment can tell which of the two is the case in a given patient.

Skeletal Expansion

Young patients who have prolonged iron deficiency may develop bone changes line haemolytic anaemia or thalassaemia because of bone marrow expansion.

The Erythrocyte Membrane


The erythrocyte membrane is subject a great deal of physical stress in circulation. It needs to withstand the high sheering stress in the arteries, it needs to squeeze past capillaries that ma be as small as 7.5µm and need to withstand the ionic changes. It has a well-developed network of proteins known as the erythrocyte cytoskeleton below the lipid bilayer of the plasma membrane to meet these needs. Inherited defects in these proteins have been associated with disorders of erythrocyte shape including hereditary spherocytosis, hereditary elliptocytosis, hereditary pyropoikilocytosis, Southeast Asian stomatocytosis and hereditary acanthocytosis (see table below).

Protein Gene Chromosome Disorders
α-Spectrin SPTA1 1q22-q23 Hereditary ElliptocytosisHereditary PyropoikilocytosisHereditary Spherocytosis
β-Spectrin SPTB 14q23-q24.1 Hereditary ElliptocytosisHereditary PyropoikilocytosisHereditary Spherocytosis
Ankyrin-1 ANK1 8p11.2 Hereditary Spherocytosis
Band 3 SLC4A1 17q21 Hereditary SpherocytosisHereditary AcanthocytosisSoutheast Asian OvalocytosisHereditary Stomatocytosis
Protein 4.1R EPB41 1p33-p34.2 Hereditary Spherocytosis
Protein 4.2 EPB42 15q15-q21 Hereditary Spherocytosis
Stomatin STOM 9q33.1 Hereditary Stomatocytocytosis
Glycophorin C GYPC 2q14-q21 Hereditary Elliptocytosis
Glycophorin D GYPD 2q14-q21 Hereditary Elliptocytosis

Organization of the Erythrocyte Membrane

The erythrocyte membrane consists of a lipid layer on protein scaffolding known as the cytoskeleton. The relationship between erythrocyte membrane proteins and lipid membrane bilayer is shown in the figure below. The main component of cytoskeleton is spectrin. Spectrin is tethered to the cell membrane by vertical interactions with band 3 proteins via ankyrin and protein 4.2. Spectrin also has horizontal interactions with protein 4.1R, actin, tropomodulin, tropomyosin and adducin. Protein 4.1R interacts with glycophorin C, a trans membrane protein.

Red Cell Membrane-600px

Erythrocyte Membrane Proteins

Spectrin

Spectrin has three functions

  1. Supporting the lipid layer
  2. Maintaining cell shape
  3. Regulating the lateral movement of integral membrane proteins.

It is made of two chains α and β that interwine to form dimers. Two dimers associate to form a tetramer which is the functional subunit. The α-chain is encoded by the gene SPTA1 at 1q22-q23 and the β-spectrin is encoded by the gene SPTB at 14q23-q24.1.

Ankyrin

Ankyrins are ubiquitous adapter proteins thattarget diverse proteins to specialized membrane domains of smooth muscles and endoplasmic reticulum. The erythrocyte ankyrin, ankyrin-R is encoded by the gene ANK1 located at 8p11.

Band 3

Band 3 glycoprotein of the erythrocyte membrane that is coded by the gene is SLC4A1 at 17q21. The membrane domain transports anions across the cell membrane and the cytoplasmic domain provide binds the lipid membrane to spectrin via ankyrin.  major integal

Protein 4.2

Protein 4.2 regulates the interactiom of band 3 with ankyrin. It is encoded by the gene EPB42 at 1p33-p34.2.

Protein 4.1R

Protein 4.1R stabilized the spectrin-actin interactions. It is encoded by the EPB42 gene at 1p33-p34.2.

Disorders of Erythrocyte shape

Disruption in the cytoskeleton is the basis of in a viariey erythrocyte disorders charecterzid by alterations in erythrcoyte shape (see tabel and figure above). Disruption in vertical interactions results in instability of lipid layer resulting in loss of lipid layer and spherocytosis. Disruptions in horizontal interactions results in hereditary elliptocytosis.

Changes in the lipid layes also results in changes in erythrocyte shape. Unlike disorders ofthe cytskeleton, most of these disorders are accquired.

  1. Target Cells: Traget cells or codocytes are cells that have an appearance of a shooting target with a central bulls eye. Reletive increase in the membrane lipids results in the formation of target cells. This is seen in severe microcytis anaemias like severe iron deficiency, thalassaemia, haemoglobin C disease and haemoglobin E disease where the intracellular contents decrease. It may aslo bee seen in obstructive liver disease where the lipid and cholesterol content of the membrena increase.
  2. Stomatocytes: Stomatocytes are erythrocytes with a central elongated mouth-like area of pallor. Expansion of the inner layer results in stomatocytosis. This may be seen in alcoholism and with the use of vinca alkaloids.
  3. Echinocytes Ecchinocytes are cells that are no longer disc shaped and are covered by 10-30 short projections. The change is because of expansion of outer lipid layer. Ecchinocytes are seen in uraemia, pyruvate kinase deficiency or may be a fixing/staining artefact.
  4. Acanthocytes: Acanthocytes are cells with a few spiny projections on the surface (from acanthus, The Greek word for thorn). The result from accumulation of cholesterol (liver disease) or sphingomyelin (abetalipoproteinaemia) in the outer lipid layer results in acanthocytosis.

Intestinal Iron Absorption


Intestinal absorption of iron (figure 1)

Iron absorption occurs in two steps

  1. Absorption of iron into the enterocyte at the luminal surface of the enterocytes
  2. Transport of iron to the lamina propria at the basilateral surface of the enterocyte

click here for fig 1

Absorption of iron into the enterocyte

Dietary iron is in two forms, heme and non-heme. Animal foods have 40% iron as heme iron and 60% as non-heme iron. Plant foods contain only non-heme iron. Most of the iron available for absorption comes from non-meat sources even in meat eaters. The widely held perception that vegetarian diets contain less iron than meat eaters is not true. Vegetarian diets contain as much or more iron than meat diets (MJA Open 2012; 1 Suppl 2: 11-16). Vegetarian foods have less heme iron. Different pathways absorb heme and non-heme iron.

Absorption of heme iron

Digestion of proteins releases heme which is oxidized to haemin and absorbed by the enterocytes. A receptor for the absorption of heme has been postulated but not identified. Once within the cell the haemin is acted upon by heme oxygenase 1 to release the iron. The heme iron is added to the enterocyte iron pool and follows the same path out of the cell as non-heme iron. Food does not affect the absorption of heme iron. Some heme may be absorbed directly and is bound to hemoprixin.

Non-Heme Iron

Dietary non-Heme iron is present in the ferric state. Ferric iron is insoluble and needs to be converted to the more soluble ferrous state for absorption. The reduction of ferric iron is aided by the acidic environment of the stomach, dietary components like ascorbic acid and duodenal cytochrome b (Dcytb). Dcytb does not appear to be essential for iron absorption. Ferrous iron is absorbed by the divalent metal ion transporter (DMT1). In addition to  Fe2+,DMT 1 also transports Mn2+, Co2+, Ca2+, Zn2+, Cd2+ and Pb2+. DMT1 is expressed at the brush-border of the enterocyte near the tips of small intestinal villi. DMT1 needs protons to co-transport with iron. Protons come from the gastric juice and are most abundant in the duodenum. Most of the iron is absorbed in the duodenum. Antacid, H2 antagonists and proton pump inhibitors hamper iron absorption by reducing hydrogen ion availability. All these drugs have been shown to impair the efficacy of medicinal iron.

The absorption of non-heme iron, unlike heme iron, is affected by food.

  1. Foods enhancing iron absorption: Ascorbate, animal proteins, human milk, keto sugars, organics, amino acids that form soluble chelates with iron enhance absorption of non-heme iron.
  2. Inhibiting iron absorption: `Inhibitors of absorption of non-heme iron include
    1. Phytates present in grains and vegetables
    2. Dietary fibre
    3. Polypohenols present in tea, coffee and wine,
    4. Phosphates and phosphoproteins present in egg yolk, bovine milk
    5. Calcium and zinc.

Transport of iron to the lamina propria

Iron is transported to the lamina propria by an iron transporter ferroportin. Ferroportin transports iron in the ferrous form. This needs to be oxidized to the ferric form for binding to transferrin. Hephaestin oxidizes ferrous iron to ferric iron.

Ferroportin is the key to controlling body iron. In iron deficient state ferroportin expression at the basolateral surface of the enterocyte is increased and more iron is transferred to the blood. When the body is iron repleted ferroportin expression is low and the iron remains in the enterocyte as ferritin.  Iron stored as ferritin is lost with the enterocytes when it is shed at the end of it’s lifespan of 5-6days.

Ferroportin levels are controlled by enhancing it’s degradation when iron stores are adequate. The liver, on sensing adequate iron stores, secretes a peptide hepcidin that binds to ferroportin. Binding of ferroportin to hepcidin causes internalisation and degradation of ferroportin preventing iron transport. Mutations ferroportin, hepcidin and molecules that promote the secretion of hepcidin  (haemachromatosis protein (HFE),  haemojuvelin (HJV), transferrin receptor 2 (TFR2)) result in increased iron absorption and haemachromatosis.  Mutations in TMPRSS6 a negative regulator of hepcidin results in iron refractory iron deficiency anaemia.

 

Hepcidin and Ferroportin


Iron is an essential micronutrient that is toxic because of its ability to form oxygen free radicals that can damage macromolecules. It is this property of iron that makes it essential for total body iron content be controlled and tissue be protected while iron is being transported from the sources of iron (macrophages and enterocytes) to consumers of iron (erythroid cells and syncytitrophoblast). Iron has no significant excretory pathways controlling absorption is the only way to check against iron overload. Ferroportin the only known transporter of iron and hepcidin a peptide that binds and degrades ferroportin are central to control body iron content. Mutations in both have been implicated in haemochromatosis.

Ferroportin

Ferroportin, the product of the SLC40A1 (Solute carrier family 40 (iron-regulated transporter) member 1) gene (OMIM: 604653) situated at chromosome 2q32.2,  is the only known iron transporter. It is a 571 amino acid peptide with a yet to be defined. It is believed to contain between 9-12 transmembrane domains. Ferroportin is expressed on the macrophages, Küpffer cells, hepatocytes, intestinal enterocytes and placental cells. All these cells are involved in iron transport.

Regulation of expression
  1. Transcriptional level: IRE in the untranslated 5′ region. The effect or ion loading is tissue specific. Iron loading increases the expression of ferroportin in liver cells and macrophages . Iron deficiency increases the expression of ferroportin the duodenum. These findings are consistent with the functions of the hepatocytes and macrophages on one hand (releasing iron at a rate dictated by iron deficiency) and the duodenum on the other (transporting iron to the lamina propria in the presence of iron deficiency).
  2. Post-translational Modification: Hepcidin binds to and phosphorylation ferroportin leading to internalisation and  degradation of ferroportin.
Mutations in Ferroportin Gene

Mutations in the Ferroportin Gene result in haemochromatosis type 4 or ferroportin disease.  Mutations result in synthesis of ferroportin molecules that can not bind hepcidin

Hepcidin

Hepcidin (Hepcidin Antimicrobial Peptide, HAMP) is an 25 amino acid peptide secreted predominantly by the liver. It is encoded by the HAMP (OMIM 60464)gene located in chromosome 19q13.12. It is synthesised as an 84 amino acid precursor. It has 8 cysteine residues and four disulphide bond. It circulates in the plasma associated with α2-macroglobulin and albumin.

Function of Hepcidin

Hepcidin  was firs identified as an antimicrobial peptide but is now known to the main regulator of iron metabolism. Hepcidin binds ferroportin to bring about its internalisation and degradation. Hepcidin impairs release or iron from the cells.  Hepcidin levels are decreased by depletion of iron stores, phlebotomy, haemolysis and elevated erythropoietin levels. Hepcidin regulates the absorption and distribution of iron

  1. Absorption of Iron: Hepcidin levels increase iron overload resulting in decreased ferroportin expression on the basilateral aspect of the duodenal cell.  The cell is not able to transport absorbed iron to the plasma and addition of more iron to the body iron pool is controlled.
  2. Distribution of Iron: Increased hepcidin in states of iron overload decreases the ferroportin expression of macrophages and hepatocytes. Hepcidin prevents entry of recycled iron from the macrophages and stored iron from the hepatocytes into the plasma.

Mice with disruption id the USF2 gene have a complete suppression of hepcidin levels and develop an illness like human haemochromatosis. Inducing over expression of hepcidin by placing the gene under transgenic control of the liver-specific transthyretin promoter resulted in a mice that were severely anaemic with hypochromia and microcytosis that survived only a few hours (Proc Natl Acad Sci USA2002;99(7):4596-4601).

Regulation of Hepcidin
  1. Positive Regulators: Bone morphogenic Proteins 6 (BPM6), hemojuvelin (HJV), mothers against decapentaplegic homolog 4 (SMAD4), Transferrin receptor 2 (TFR2) and haemochromatois protein (HFE)
  2. Negative Regulators: TMPRSS6

Distribution of Body Iron


Body Iron Stores

Iron is normally stored in the reticuloendothelial system and the hepatocyte. The total body iron varies with age and gender.

Iron Stores at birth

The total body iron content of neonates born to mothers without iron deficiency has been estimated to be 75mg/kg. About 60% of this is acquired in the third trimester. Neonates born to iron deficient mothers have lower iron stores. A fall in iron stores is evident when the haemoglobin falls below 8.5g/dL and the depletion becomes more severe below maternal haemoglobins of 6.0g/dL.

Postnatal period to adolescence

The rapid growth between birth and adolescence needs expansion in blood volume. The iron supply remains precarious for most of this period and children frequently run the risk of developing iron deficiency. Children born to iron deficient mothers are at a higher risk of iron deficiency.

  1. Men:The iron stores in men after adolescence gradually increase with age. Men have about 35-45mg/kg of iron
  2. Women: Premenopausal women have lower iron store than men because of iron loss associated with menstruation and pregnancy. The normal iron content is women 35mg/kg. The iron content gradually rises after menopause.

Normal erythropoiesis needs 25mg of iron about 1-2mg comes from absorption and the rest from macrophage iron recycling. Macrophage iron recycling is an important source of iron for haematopoiesis and as discussed later, more sensitive to hepcidin.

Figure 1. The Iron Cycle
Figure 1. The Iron Cycle

Body iron has four compartments. The movement of iron between these compartments in know as the iron cycle (figure 1)

  1. Heme containing oxygen transport proteins: Haemoglobin, which has about two third of the total body iron and myoglobin
  2. Storage compartment: Ferritin and Hemosiderin have about one fourth of the body iron. About half the storage compartment is ferritin and half hemosiderin. Ferritin iron is more readily available.
  3. Transport compartment: Plasma contains a small fraction of the body iron stores. Plasma iron is bound to transferrin. Non-transferrin bound iron is toxic and seen in states of iron overload when the iron binding capacity of transferrin is exceeded. Though this compartment is small it is turned over six to eight times a day.
  4. Iron Containing Enzymes: Iron Enzymes like cytochromes, catalase, peroxidase and ribonuclease reductase

Sickle Cells


Sickle Cell - 100X - IMG_0542

Sickle Cells 40X

Sickle Cell 100X - IMG_0540

Sickle Cells – 100X

Sickle Cell 100X - IMG_0538

Sickle Cells – 100X


The three photomicrographs above show sickle cells from a patients with sickle cell anemia. Sickle cell anaemia occurs because an A→T substitution in codon 6 of the β globin chain of haemoglobin. The single nucleotide polymorphism results in valine substituting for glutamic acid resulting in the formation of haemoglobin S (HbS). HbS crystallizes in hypoxic conditions resulting in sickling of erythrocytes.

Related articles
Sickle Haemoglobin and Variants

Erythrocyte Metabolism


GlucoseMetaboloisRBC As erythrocytes lack mitochondria they are not able to use fats or generate energy from Krebs cycle. Though they have enzymes to synthesize glycogen the balance between synthesis and breakdown favours breakdown. Normal erythrocytes do not have glycogen and depend on a continuous supply of glucose to meet their energy requirements.  Glucose enters the erythrocyte freely by facilitated diffusion. Insulin does not have any effect on the entry of glucose into the erythrocyte.

They erythrocyte metabolism needs ATP as a source of energy and NADH and NADPH cofactors. The erythrocyte does not synthesize nucleic acids but it has a small requirement for ribose to synthesize nucleosides for energy transfer The metabolic needs of erythrocytes are met by metabolism of glucose through three pathways glycolysis, the hexose monophosphate shunt and Rapport-Luebering glycolytic shunt.

Glycolysis in Erythrocytes
Glycolysis is a process in which one molecule of glucose is converted to two molecules of pyruvate with the a net formation of two ATP and two NADH molecules. The energy released in the process stored as ATP. The electron released in the conversion of glyceraldehyde-3-phosophate to 1,3-bisphosphoglycerate is accepted by NAD+. Glycolysis can not proceed in the absence of an electron acceptor. NADH produced by glycolysis is transported to the mitochondria where it generates a net of 2 ATPs (3 ATP are generated but one is consumed to transport NADH into the mitochondria). Erythrocytes do not have mitochondria. Methaemoglobin reductase is the only sink for NADH. Glycolysis can proceed only if NAD+ is regenerated. Conversion of pyruvate to lactate by lactate dehydrogenase regenerates NAD+ allowing glycolysis. The end product of glycolysis in erythrocytes is lactate.

The Rappaport-Luebering Glycolytic Shunt
The main functions of the erythrocyte to deliver oxygen to peripheral tissue. Anaemia decreases the oxygen carrying capacity of blood. Oxygen delivery is maintained in anaemic patients by increasing blood flow and increasing the oxygen extraction (see pathophysiology of anaemia). Patients with anaemia have a decrease in the oxygen affinity of haemoglobin allowing a greater amount to be offloaded for a given PO2. 2,3-Bisphosphoglycerate (2,3-BPG) and H+ ions decrease the oxygen affinity of haemoglobin. The Rappaport-Luebering glycolytic shunt synthesizes 2,3-BPG from 1,2-BPG. The shunt bypasses an ATP producing. The erythrocyte pays for increased oxygen affinity as ATP.

The Hexose Monophosphate Shunt
Erythrocytes are subjected to a high degree of oxidative stress from exposure to drugs and chemicals and from oxygen transport. The reactive oxygen species thus generated can affect cell visibility and function by conversion of ferrous iron of haemoglobin to ferric iron giving methaemoglobin. It can also damage lipid membranes shortening the erythrocyte lifespan. Glutathione is a tripeptide that scavenges reactive oxygen species and is oxidized in the process. Glutathione reductase regenerates glutathione by using NADPH as an electron donor. The only non-mitochondrial source of NADPH is the hexose monophosphate shunt. One molecule of glucose passing through the shunt gives two molecules of NADPH and is converted to ribulose-5-phosphate. The ribulose-5-phosphate can be converted to ribose-5-phosphate for nucleoside synthesis. It is also possible to generate 5 molecules of glucose-6-phosphate out of 6 molecules of ribulose-5-phosphate. This involves two enzymes transaldolase and transketolase both of which use thymine pyrophosphate as co-enzyme and are able to transfer carbon from one sugar to another. As the erythrocyte has only a small need of ribose it reconvert most of the ribulose-5-phosphate to glucose-6-phosphate. Normally about 10% of the glucose is metabolized through this shunt. When the erythrocyte is faced by an oxidizing stress almost 90% of the glucose may be metabolized through the shunt. The first and the rate limiting enzyme of the pathway is glucose-6-phosphate dehydrogenase (G6PD). G6PD deficiency is the commonest enzymatic deficiency causing haemolytic anaemia. It manifests as haemolysis in response to oxidative stress like exposure to drugs, chemicals of infection. The hexose monophosphate shunt is critical to the erythrocyte survival.

hypochromic Micro Anaemia

Microcytic Hypochromic Anaemia


hypochromic Micro Anaemia
Hypochromic Microcytic Anaemia

Shown above is an image of hypochromic microcytic anaemia. This patient had iron deficiency anaemia. Microcytosis is presence of small erythrocytes and hypochromia presence of erythrocytes that are poorly haemoglobinized. The nucleus of a small lymphocyte, the cell in the centre of the image, is a good guide to the size of erythrocytes on a peripheral smear. The nucleus has a diameter of 8.5 µm and a normal erythrocyte a diameter of 7.5 µm. The erythrocytes in in the image are substantially smaller than the lymphocyte nucleus. Erythrocytes have a central pale staining area which occupies about one third of erythrocyte diameter. As the cells get less haemoglobinized the central pale staining area increases. Many of the erythrocytes in the image above have a pale staining area occupies all but a thin rim at the periphery. In others, the pale staining area is increased. The erythrocyte sizes vary. Anisocytosis, increased variation in erythrocyte size, a feature of iron deficiency anemia, is evident in the image.

 

Iron Studies in Microcytic Anaemia


Diagnosis Serum Iron Total Iron Binding Capacity Transferrin1 Serum Ferritin2
Iron deficiency anaemis Low High or Normal <16% <12ng/mL
Anaemia of Chronic Disease Normal Low or Normal ≥16% High or normal
β-Thalasaemia Trait, HbE, HbC Normal Normal ≥16% Normal
Sideroblastic Anaemia High Normal High High
  1. Two causes of microcytic anaemia may co-exist e.g. thalassaemia trait with iron deficiency anaemia or anaemia of chronic disease with iron deficiency anaemia. When iron deficiency exists with other forms of microcytic anaemia the transferrin saturation is <16%
  2. Patients with low ferritin (<12ng/ml) always have iron deficiency. Higher values of ferritin do not exclude iron deficiency particularly in patients with anaemia of chronic disease. There are no guidelines about the ferritin levels that exclude iron deficiency in patients of anaemia of chronic disease. The reported values vary between 60-100ng/ml.