Iron Metabolism and Hemoglobin

4.3.1 Iron Metabolism

Iron is an integral component of hemoglobin, myoglobin, and other substances such as cytochrome, cytochrome oxidase, peroxidase, and catalase. In addition to this, iron is involved at all stages of energy metabolism as it is necessary for appropriate functioning of enzymes of the electron transport chain (cytochrome oxidase, ferredoxin, myeloperoxidase, catalase, succinate dehydrogenase, and the cytochrome P-450).

4.3.1.1 AbsorptionofIron

The primary sites for iron absorption are duodenum and jejunum; however, in ileum, slow uptake of iron occurs. The rate of iron absorption depends upon the iron reserve and is reported to be increased during iron deficiencies. The maximum absorption of iron requires around 24 h. In carnivores and omnivores, heme iron is an important dietary source of iron which absorbs more readily than non-heme iron of vegetables and grain. Several iron transporters are expressed in enterocytes namely divalent metal transporter-1 (DMT-1) and heme carrier protein (HCP1), respectively, for non-heme and heme iron. At acidic pH in the stomach, the feed iron is released in ferric form (Fe³⁺). But DMT1 is the most important transporter of ferrous iron. Therefore, addi­tional machinery is required to convert Fe³⁺ to its stable form Fe²⁺ for absorption.

4.3.1.2 Exportation of Iron from Intestine into Circulation

The iron in the enterocytes is exported to circulation by ferroportin (FPN1), situated either in the basolateral mem­brane of the enterocytes or in the macrophages. The expres­sion of FPN1 is stimulated by cellular iron and suppressed by the hormone hepcidin. Hepcidin causes internalization of FPN1 and degradation by lysosomal enzymes thus decreases iron absorption in enterocytes. After iron exportation, Fe²⁺ needs to be converted to Fe³⁺ for its binding with apotransferrin, an iron carrier protein in plasma. Ferroxidases such as ceruloplasmin (Cp) helps in this conversion. The internalization of FPN1 accelerates in the absence of ferroxidase.

4.3.1.3 Iron Transportation in Blood

In circulation, iron is mainly transported by transferrin (Tf). Under normal condition, 20-40% of the total iron-binding sites in transferrin is occupied by Fe³+. The saturation of iron- binding capacity of transferrin occurs as a result of iron overload with the generation of non-Tf-bound iron (NTBI). Presence of transferrin indicates iron status in animals, and thus used as diagnostic tool to measure iron deficiency or iron overload. Erythroid precursors generally use iron of transfer­rin whereas macrophages and hepatocytes are able to use both transferrin bound iron and NTBI. The internalization of transferrin bound iron inside erythroid precursors of macrophages is facilitated by transferrin receptor (Tfr). Tf-Tfr complex then internalize within endosomes. In acidic pH, endosomes release iron and Tf-Tfr complex localized to cell surface where Tfr dissociates from Tf-Tfr at neutral pH and get recycled.

4.3.1.4 Tissue Storage of Iron

Iron is stored mostly in the liver (50-60 mg/100 g), followed by brain (40-50 mg/100 g), spleen (15-20 mg/100 g), heart (10-20 mg/100 g), and bone marrow (4-5 mg/100 g) in the form of ferritin or hemosiderin. The lowest iron content is reported in stomach, pancreas, jejunum, colon, and urinary bladder (1-2 mg/100 g). When the renal threshold of hemo­globin exceeds, a substantial amount of iron gets accumulated in the epithelium of the convoluted tubules of nephron (20-30 mg/100 g of kidney tissue), smooth muscle and mucous membranes (3-4 mg/100 g). In healthy subjects, iron accumulates in lungs (6-7 mg/100 g) which rapidly declined during anemia (3-4 mg/100 g). However, the iron content in the tissues stated above depends on the status of the animals and the storage rapidly declined on iron defi­ciency within 3-4 months.

4.3.1.5 Regulation of Iron Absorption

The regulation of iron metabolism is controlled by iron reserve in the body, hypoxia, and rate of erythropoiesis. Heme iron is more efficiently absorbed (25-30%) when compared to non-heme iron (5-15%). The regulation of iron absorption can be explained by two proposed model.

Crypt programming model: This model proposes that the crypt cells of the duodenum sense body iron levels and determines the level of iron to be absorbed. Cytosolic iron regulatory proteins (IRPs) 1 and 2 are the intracellular iron censors and stimulate the expression of TfR1 or DMT1 under iron deficient state.

Hepcidin model: Hepcidin (HAMP, LEAP 1) is a cysteine- rich peptide containing 25 amino acids synthesized mainly from hepatocytes and cleared through kidney. There is an inverse relationship between hepcidin expression and iron state in animals. Hepcidin binds with ferroportin (FPN1) of enterocytes, hepatocytes, and macrophages and causes the internalization and degradation of FPN1 to block the exportation of iron.

4.3.1.6 Factors Affecting Iron Absorption

4.3.1.6.1 Factors Enhancing Iron Absorption

Ascorbic acid (Vit-C) increases the absorption of dietary non-heme iron either by preventing the formation of insolu­ble non-absorbable iron compounds or by the reduction of ferric to ferrous iron.

Citric acid chelates metal ions that interfere iron absorp­tion. The beneficial effect of citric acid is maximum at acidic pH and the effect decreases at neutral or higher pH.

Studies have shown that consumption of alcohol at low level enhances iron absorption by increasing ferritin or TfR 1.

Carotene forms a soluble complex with iron and prevents the inhibitory effects of phytate and polyphenols on iron absorption.

Animal proteins such as meat, chicken and fish stimulate gastric HCl secretion thus promotes iron absorption.

4.3.1.6.2 Factors Inhibiting Iron Absorption

Calcium decreases luminal iron absorption affecting DMT 1 and iron exportation by inhibiting ferroportin (FPN).

Phytic acids present in grains and cereals form a complex with positively charged iron (phytic acid is negatively charged) that decreases bioavailability of iron.

Phenolic compounds present in vegetables, seed, or beverages (coffee, tea, and wine) combine with iron in lumen of intestine, making it unavailable for absorption.

4.3.2 Hemoglobin

Hemoglobin is an iron containing conjugated protein exclu­sively found in erythrocytes and transports the oxygen from lungs to tissues and carbon-dioxide from tissues to lungs.

4.3.2.1 Structure of Hemoglobin

Hemoglobin is one of the most exclusively studied proteins in nature. It is the first oligomeric protein (composed of two different polypeptide chains), and its complete tertiary and quaternary structures were identified by X-ray crystallogra­phy by M. F. Perutz and coworkers, awarded Nobel Prize for Chemistry in 1962. Normal adult hemoglobin molecules (HbA) have a molecular weight of 64,458 Da and are com­posed of four subunits, each having one polypeptide chain globin (apoprotein) and one heme group (non-protein pros­thetic group).

Structure of heme '. Heme molecule is composed of porphy­rin molecule (Protoporphyrin-IX) with iron (Fe²⁺) at its center.

Structure of globin chains: The globin chains of the hemo­globin are tetrameric polypeptides composed primarily of two α and two non-α (β, γ, and δ) chains. Each α and β chain are having 141 and 146 amino acids, respectively, and thus total 574 amino acids are present in hemoglobin. However, the polypeptide chains are different in different forms of hemoglobin. The β chain starts with amino acid valine and histidine at N terminal end and the C terminal residues are tyrosine (145) and histidine (146). The δ chain differs from β chain in 10 residues. The first 8 residues and C terminal 21 residues (127-146) are same in β and δ chains. The γ chains are exclusively found in fetal hemoglobin (HbF) and are differed from β chain at 39 residues. The proximal N terminal amino acids of δ chain are glycine and valine whereas C terminal amino acids are same as β and δ chains. The polypeptide chains of hemoglobin molecule are held together by hydrogen bond, hydrophobic and ionic interactions and folded in such a way that the polar residues face the external surface and the non-polar residues are internal making the hemoglobin water soluble.

4.3.2.2 Biosynthesis of Hemoglobin

The synthesis of hemoglobin begins in the pro-erythroblast stage and continues till the reticulocyte stage. Synthesis of hemoglobin comprises two distinct steps, namely synthesis of heme and production of globin.

4.3.2.2.1 Heme Synthesis

The heme synthesis is a multistep process occurring both at mitochondria and cytosol of erythrocytes, involving at least eight enzymes out of which four works in the mitochondria and four in the cytosol.

1. Succinyl-CoA, formed in the Krebs metabolic cycle binds with glycine to form aminolevulinic acid (ALA) with the help of enzyme ALA synthase.

2. Two molecules of ALA form porphobilinogen (PBG) with the help of ALA.

3. Four molecules of PBG and produce hydroxymethylbilane by the enzyme porphobilinogen deaminase.

4. Hydroxymethylbilane produces uroporphyrinogen III with the help of enzyme uroporphyrinogen III cosynthase.

5. Conversion of uroporphyrinogen III to coproporphyrinogen III by uroporphyrinogen decarboxylase.

6. Coproporphyrinogen III is converted to protoporphyrinogen IX by coproporphyrinogen oxidase.

7. Protoporphyrinogen oxidase catalyzes the conversion of protoporphyrinogen IX to protoporphyrin IX.

8. Finally, heme molecule is produced after the incorporation

of Fe in protoporphyrin IX by ferrochelatase.

Step 1 occurs in the mitochondria, steps 2-5 occur in cytosol and the final three steps (6-8) occur in mitochondria.

4.3.2.2.2 Globin Synthesis

Production of globin chain occurs in the cytosol of erythrocytes by genetic transcription and translation. Gene encoded α chains are situated in chromosome 16 and gene for the β chain are on chromosome 11.

4.3.2.2.3 Alpha Globin Locus

Two identical α globin genes namely α1 and α2 are situated in each chromosome 16. Thus, four α globin genes are present in each cell (due to paired chromosome). Zeta, a substitute for α globin gene, expressed transiently during early embryonic development in alpha globin locus.

4.3.2.2.4 Beta Globin Locus

The β globin gene cluster in chromosome 11 containing the genes are arranged sequentially from 5' to 3'. In the first sequence, epsilon genes are situated (expressed during embryonic life), following the epsilon genes there are gamma (two copies) and delta genes (single copy). The β globin locus ends with adult beta globin gene (single copy). The protein expressed by these two beta globin genes (due to paired chromosome) matches precisely with four alpha glo­bin genes.

4.3.2.3 Switching of Fetal to Adult Hemoglobin: Ontogeny of Hemoglobin Synthesis

During the first trimester of pregnancy, the zeta gene of the α globin gene cluster is expressed along with the ε-globin in β globin gene cluster, forming hemoglobin grower 1 (or HbE Gower-1). The primitive erythrocytes developed in the yolk

Fig. 4.4 Ontogeny of Hemoglobin Synthesis: In primitive erythroblasts, hemoglobin is synthesized in primitive erythroblasts of the embryonic yolk sac. In definitive erythropoiesis, α and β globin gene clusters express α and β globins, respectively, leading to the formation of fetal hemoglobin (HbF, α2γ2) in the fetal liver. The production of γ-globin declines at the time of birth together with the higher expression of β-globin to form adult hemoglobin (HbA, α2β2). The delta gene produces small amount of delta globin to form adult hemoglobin A2 (HbA2, α2δ2) comprising less than 5% of the total adult hemoglobin. (Source: Wienert et al. 2018)

4.3.2.4 Types of Hemoglobin

Different types of hemoglobin together with their composi­tion and abundance are summarized in Table 4.15.

The normal range of hemoglobin in different species have been presented in Table 4.16.

4.3.2.5 Cooperative Binding of Oxygen with Hemoglobin: T and R States of Hemoglobin

The affinity of hemoglobin for oxygen is proportional to the quantity of oxygen that binds with the hemoglobin at a given time. In other words, binding of one oxygen to heme increases the affinity of hemoglobin for oxygen. Thus, affin­ity for first oxygen to bind with heme is 100 times more than the last oxygen. This is called cooperative binding or heme­heme interaction. This interaction is achieved due to the conformational changes in the hemoglobin structure after binding with oxygen in such a manner that favors further oxygen binding. The deoxygenated form of hemoglobin is called T-state (tense) which has less affinity for oxygen. In T state, the iron is bound to nitrogen of Histidine side chain (His E8) which pulls the iron out of the porphyrin plans but when oxygen binds with iron, the new bond pulls the iron back to heme plan. The movement of iron facilitates the movement of alpha- and beta-helix and creates a favorable condition to bind with oxygen. This state of hemoglobin is called relaxed state or R state.

sac which is replaced with hemoglobin grower 2 comprises alpha (2) expressed by α globin gene cluster, epsilon (2) by β globin gene cluster. During the production of first enucleated definitive erythrocytes in the fetal liver, α and β globin gene clusters express α and β globins, respectively, leading to the formation of fetal hemoglobin (HbF, α2γ2). The production of γ-globin declines at the time of birth along with the increased expression of β-globin by β-globin gene cluster and forms adult hemoglobin (HbA, α2β2). The delta gene located in β globin gene cluster on chromosome 11 between the gamma and beta genes produces small amount of delta globin which forms adult hemoglobin A2 (HbA2, α2δ2) comprising less than 5% of the total adult hemoglobin. Ontogeny of hemoglobin synthesis is depicted in Fig. 4.4.

4.3.2.6 Derivatives of Hemoglobin

4.3.2.6.1 Oxyhemoglobin

Combination of oxygen with hemoglobin during physical respiration forms oxyhemoglobin. One molecule of hemo­globin can bind with four molecules of oxygen. 1 g of Hb can bind with 1.34 mL of oxygen.

• Gram molecular weight of hemoglobin is 64,500 g.

• One mole of any gas at NTP occupies 22,400 mL.

• So, 4 mol of gas occupy 22,400 ? 4 = 89,600 mL.

• 64,500 g hemoglobin contains 89,600 mL of oxygen.

• 1 g hemoglobin contains = 89,600/64,500 = 1.39 mL of oxygen.

Table 4.15 Different types of hemoglobin

• 0.05 mL/g of oxygen is not capable of binding with hemoglobin as small amounts of hemoglobin exist as met-hemoglobin unable to carry oxygen.

• So, 1 g of hemoglobin carries 1.39 — 0.05 = 1.34 mL of oxygen.

Oxygenated hemoglobin is bright red in color and deoxy­genated hemoglobin is purplish red in color.

4.3.2.6.2 Myoglobin

It is a hemoprotein found in muscle tissue. Unlike hemoglo­bin, myoglobin has only one heme molecule; hence, it can bind with only one molecule of oxygen and a single polypep­tide chain of 154 amino acids. The molecular weight of myoglobin is 17,000. Myoglobin has higher affinity for oxy­gen compared to hemoglobin. The main function of myoglo­bin is to store oxygen in muscle tissue and maintain a steady supply during hypoxia.

4.3.2.6.3 Carboxyhemoglobin

Binding of hemoglobin with carbon monoxide yields carboxyhemoglobin. The affinity of carbon monoxide with hemoglobin is 200 times greater than oxygen and carboxy hemoglobin is about 250 times more stable than oxyhemo­globin. Carboxyhemoglobin interferes with cellular respira­tion either by preventing the oxygen transport or by inhibiting the enzyme cytochrome oxidase. The color of carboxyhemoglobin is bright cherry red. High pressure oxy­gen therapy is applied as medication in carbon monoxide poisoning.

4.3.2.6.4 Methemoglobin

It is the true oxide of hemoglobin as ferrous iron is converted to ferric iron during the formation of methemoglobin. The oxidizing agents such as ferricyanide and nitrites react with hemoglobin to form methemoglobin. Like carboxyhemoglobin, methemoglobin is also unable to carry oxygen. Under normal condition, a small amount of methe­moglobin is formed in the circulation but, the reducing agents such as glutathione and ascorbic acid decrease its accumula­tion. About 1.5% of total hemoglobin in the body remains as methemoglobin. Other than ferricyanide and nitrites; chlorates, peroxides, hydroquinone, iodine, and sulfonamides also cause the formation of methemoglobin.

The reduction of methemoglobin back to hemoglobin is mediated mainly by NADH-cytochrome b5-methemoglobin reductase. Methemoglobin is also prone to direct reduction by intracellular ascorbate and glutathione.

The deficiency of methemoglobin reductase leads to type- I congenital methemoglobinemia whereas type-II congenital methemoglobinemia occurs due to generalized reductase deficiency. Methemoglobinemia is common in horses, dogs, and cats.

Met-hemoglobinemia is common in animals exposed to nitrate (fertilizers) and chlorate (herbicides) poisoning. In rumen, nitrate is converted to nitrite and leads to the forma­tion of methemoglobin. Methylene blue and ascorbic acid is used as medication of methemoglobinemia.

Methemoglobin itself is used as medication in cyanide poisoning. Excessive ingestion of sorghum (hydrocyanic acid) causes cyanide poisoning in animals due to formation of thiocyanate. Under these circumstances, sodium nitrite is applied to form methemoglobin which in turn combines with thiocyanate to form cyan-met hemoglobin and rapidly eliminated from the body.

4.3.2.6.5 Sulfhemoglobin

Sulfhemoglobin is formed when reduced hemoglobin combines with hydrogen sulfide. It usually occurs in the subjects working in nitrite and coal tar factories where excess amount of sulfur is handled regularly. Sulfhemoglobin at the concentration of 3-5 g/dL of blood leads to cyanosis. Sulfhemoglobinemia also occurred with the patients suffering from bacteremia with Clostridium welchii which rapidly oxidize the hydrogen sulfide produced during diges­tion in intestinal tract to sulfate. Sulfhemoglobin is not converted back to hemoglobin and persists throughout the lifespan of erythrocytes.

4.3.2.6.6 Hemin: Reactions with Acids

The action of hydrochloric acid on hemoglobin is as follows:

Hemoglobin ^HCl → Globin + Heme(Ferriheme)

2(Ferriheme) + O₂ ^HCl → 2 Ferriheme chloride(Hemin)

This method is used in the determination of hemoglobin by Sahli’s method.

4.3.2.6.7 Hematin: Reactions with Alkali

Alkali splits hemoglobin to form globin and heme. The heme rapidly oxidized to ferriheme and combines with alkali to form ferriheme hydroxide (hematin).

Hemoglobin ^NaOH → Globin + Heme(Ferriheme)

2(Ferriheme) + O₂ ^NaOH → 2 Ferriheme Chloride(Hematin)

4.3.2.6.8 Carbaminohemoglobin

Carbaminohemoglobin is formed when carbon-dioxide reacts with free NH₂ terminal groups of the α and β chains of hemoglobin. Like oxygen, four molecules of carbon-dioxide combine with hemoglobin. About 7-10% carbon-dioxide is carried as carbaminohemoglobin by RBC and rapidly dissociates in high PO2 in the lung alveoli to form carbon­dioxide and carbamate with NH₂ groups.

4.3.2.7 Fate of Hemoglobin

During the intra vascular hemolysis mechanism, hemoglobin in released in the circulation and upon extra vascular hemo­lysis, hemoglobin is broken down by the enzymatic machin­ery of the macrophages. The “globin” portion of hemoglobin can be reutilized by the system but, the “heme” component needs further metabolic conversion before getting excreted from the body. Most of the iron of the heme are also reutilized by the body and believed to be the most important source of iron in iron homeostasis.

The heme undergoes several metabolic pathways for its excretion.

Trapping of free hemoglobin and heme: The free hemoglo­bin and its breakdown products like heme get released into the circulation and binds with plasma proteins such as haptoglobin, hemopexin, and albumin. Haptoglobin binds to free hemoglobin and hemopexin, and albumin binds with heme.

Catabolism of heme to bilirubin: Heme moiety is converted to bilirubin by two-step enzymatic breakdown in RE sys­tem. Heme oxygenase converts heme moiety to biliverdin which subsequently reduced to bilirubin by NADPH- dependent biliverdin reductase. Around 75% of the total bilirubin produced in the body comes from the senescent erythrocytes.

Transport of bilirubin to liver: Plasma albumin has strong affinity for bilirubin and acts as a vehicle to carry bilirubin from RE system to liver. The hepatocytes have organic anion transporter 2 (OATP2) which readily takes the albu­min bound bilirubin. Ligandin, a cytosolic protein inside the hepocyte carries bilirubin to endoplasmic reticulum where bilirubin conjugation occurs.

Glucuronide conjugation of bilirubin: Enzyme uridine­diphosphate (UDP)-glucuronyl transferase facilitates the conjugation of bilirubin and glucuronic acid to form bili­rubin glucuronide, which is the excretable form of bilirubin.

Excretion: The conjugated bilirubin produced in the hepatocytes is excreted through bile in the intestine where it is reduced to urobilinogen by the intestinal bacte­ria and excreted as urobilin or stercobilin, the oxidized form of urobilinogen which gives the normal color to feces. Some urobilinogen enters in the general circulation bypassing the liver and excrete in urine which subse­quently oxidized to urobilin that forms the color of urine. A part of conjugated bilirubin in the intestine is hydrolyzed by intestinal beta-glucuronidase, which releases free bilirubin that enters into enterohepatic circu­lation and again get re-excreted through bile.

4.4