Hemoglobin Structure & Function PDF

Field of Medicine Medicine Program Hemoglobin Structure & Function Prof. George N.B. Morcos Date: 30 / 09 / 2024 Objectives At the end of this lecture, the students will be able to: 1. Outline the structure of hemoglobin. 2. Describe the different types of normal hemoglobin. 3. Recognize the fu...

Field of Medicine Medicine Program Hemoglobin Structure & Function Prof. George N.B. Morcos Date: 30 / 09 / 2024 Objectives At the end of this lecture, the students will be able to: 1. Outline the structure of hemoglobin. 2. Describe the different types of normal hemoglobin. 3. Recognize the function of hemoglobin as oxygen transporter. 4. List the different factors affecting hemoglobin function. 5. Recognize the difference between fetal and adult hemoglobin regarding oxygen carrier.  Hemoglobin structure Hemoglobin is a globular protein (hemoprotein) found in the cytoplasm of erythrocytes. Its primary function is the transport of O2 and CO2 between the lungs and the tissues. Normal values: In fetus - just before birth = 16.5 to 18.5 gm/dl. At the end of 1 year = 12.5 gm/dl. Adult male = 14-18 gm/dl. Adult female = 12-15.5 gm/dl.  Hemoglobin structure 1. Hemoglobin has different variants (forms) but they all share the same basic structure. 4 Heme group + 4 globin polypeptide chains (1 heme/ 1 chain) Heme (also called Iron protoporphyrin) is formed of a cyclic tetrapyrrole ring (A, B, C, D) “porphin ring”. D It contains 1 ferrous iron (Fe2+) → bound to the 4 nitrogen of tetrapyrrole ring by 4 coordination bonds. C B Iron in heme can form up to 6 coordination bonds: 4 bonds with the tetrapyrrole ring (A, B, C, D) A 1 with the imidazole ring of histidine amino acid of the polypeptide chain pyrrole ring 1 with the oxygen (it is unoccupied in absence of O2)  Hemoglobin structure 4 Heme groups + 4 globin polypeptide chains The α -chain contains 141 amino acids and β-chain (δ or  chains) contains 146 amino acids. The secondary structure of all globin chain types is formed of 8 α helices identified by the letters A to H. The tertiary folding of each globin chain forms an Globin chain approximate sphere.  Hemoglobin structure Each globin subunit contains a heme-binding pocket. In this pocket, two important histidine residues (His) have a major role in hemoglobin function: The proximal histidine residue in the F helix of the globin chain [His F8] that binds to the 5th coordination site of Fe2+ The distal histidine in the E helix [His E7] that stabilizes oxygen binding.  Hemoglobin structure Role of the globin chain: The globin surrounds the heme. This helps as follows: 1. Globin provides proximal histidine that binds to Fe2+ and the distal histidine that stabilizes oxygen binding. 2. The heme alone interacts with oxygen so that the Fe2+ becomes oxidized to Fe3+ and no longer binds oxygen. The presence of the globin chain helps in reversible oxygen binding to Hb.  Hemoglobin structure The quaternary structure of hemoglobin consists of four subunits tetramer (α2β2 in Hb A). The hemoglobin tetramer is composed of two identical dimers (αβ)1 and (αβ)2. The two chains within each dimer are held together tightly by the ionic bonds and the hydrophobic Hb Globin chains interactions, which prevent their movement relative to each other. However, the two dimers are linked with each other by weaker hydrogen and ionic bonds, so that movement at the interface of these two dimers occurs more freely during oxygenation and deoxygenation. So, 2 forms of Hb can be recognized.  Hemoglobin Function Hb is an important buffer in the erythrocytes and blood. Hb is a carrier of O2 and CO2 Hb binds O2 weakly at low oxygen pressures and tightly at high pressures. The binding of the first O2 to Hb enhances the binding of further O2 molecules. Process of O2 binding to Hb 1st. Hb must be able to bind oxygen in the lungs. 2nd. Hb must be able to release oxygen in the tissues. In these two conditions, Hb exists in 2 different forms: T-form (T = tense) and R-form (R= Relaxed).  Hemoglobin Function Difference between T and R forms of Hb deoxy- & oxyhemoglobin ▪ Following oxygenation, a considerable structural conformational change occurs in Hb. ▪ Oxygenation rotates the 11 dimer in relation to 22 dimer about 15°. ▪ Hb changes from the T → R states. T-state (Taut) R-state (relax) Deoxyhemoglobin Oxyhemoglobin  Hemoglobin Function Differences in the conformation of T and R states help in oxygen transport. The Fe is about 0.6 Å out of the heme plane in the deoxy state. When oxygen binds, it pulls the Fe back into the heme plane. Since the proximal histidine is attached to the Fe2+, this pulls the complete F helix. Oxygenation of one hemoglobin molecule produces Oxy Hb rupture of some week noncovalent hydrogen and DeoOxy Hb ionic bonds and rotation of one dimer ()1 15 degrees relative to the other dimer ()2.  Hemoglobin Function  Hemoglobin Function ✓ Factors affecting Hemoglobin function: Factors affecting the ability of Hb to bind and transport oxygen include: 1) Oxygen 2) 2,3-bisphosphoglycerate (BPG). 3) pH [H+ and CO2] and 4) Carbon monoxide (CO)  Hemoglobin Function ✓ Factors affecting Hemoglobin function: 1) Oxygen The oxygen dissociation curve for hemoglobin is sigmoidal in shape, indicating that the subunits cooperate in binding oxygen. This effect is referred to as heme-heme interaction.  Hemoglobin Function ✓ Factors affecting Hemoglobin function: Although it is more difficult for the first oxygen molecule to bind to hemoglobin, the subsequent binding of oxygen occurs with high affinity, as shown by the steep upward curve in the region near 20 to 30 mmHg. The net effect is that the affinity of hemoglobin for the last oxygen bound is approximately 300 times greater than its affinity for the first oxygen bound. The cooperative binding of oxygen enhances the efficiency of hemoglobin as an oxygen transporter.  Hemoglobin Function ✓ Factors affecting Hemoglobin function: 2) 2,3-bisphosphoglycerate (BPG) is a three-carbon molecule formed during glycolysis [Breakdown of glucose]. - It is formed in human red blood cells. - 2,3 BPG binds with greater affinity to deoxygenated hemoglobin decreasing their affinity for oxygen. This enhances the ability of RBCs to release oxygen near the tissues.  Hemoglobin Function ✓ Factors affecting Hemoglobin function: Hemoglobin in blood is bound to BPG Interaction is electrostatic, between negative charges on BPG and positive side chains of basic amino acid of globin chain (e.g., histidine, Lysine, Arginine) If Hb doesn’t bind to BPG, it remains saturated with O2. BPG binds specifically to the deoxy state and stabilizes it in the T state. In the R state, the central cavity is too narrow for BPG to fit or bind.  Hemoglobin Function ✓ Factors affecting Hemoglobin function: 3) Effect of pH: - High pH= low [H+] or alkaline; this promotes tighter binding of oxygen to hemoglobin. - Low pH= high [H+] or acidic; this permits the easier release of oxygen from hemoglobin. Hb O2 + H + Hb H+ + O 2 The effect of pH on the oxygen binding ability to Hb is called the Bohr effect.  Hemoglobin Function ✓ Factors affecting Hemoglobin function: 3) Effect of pH: The effect of pH on the oxygen binding ability to Hb is called the Bohr effect. The Bohr effect is the reciprocal coupling of protons and O2 binding to Hb. Also, it explains why red blood cells unload oxygen in tissues. More oxygen is released in those tissues that have higher CO2 values. As the pH decreases (H+ increases), the oxygen binding decreases. The opposite effect occurs when the pH increases.  Hemoglobin Function ✓ Factors affecting Hemoglobin function: As oxygen is consumed → CO2 is released (in tissues). CO2 promotes the release of O2 from oxyHb. Carbonic anhydrase catalyzes this reaction in red blood cells. carbonic anhydrase CO 2 + H 2 O H2 CO 3 H2 CO 3 H+ + HCO 3 - The H+ generated from this reaction is taken up by Hb and causes it to release more oxygen. This proton uptake facilitates the transport of CO2 by stimulating bicarbonate formation.  Hemoglobin Function Summary of Bohr Effect: 1. In peripheral tissues, Hb affinity for oxygen is decreased in the presence of carbon dioxide and at lower pH. 2. Carbon dioxide reacts with water to give carbonic acid which dissociate into bicarbonate & free protons via the reaction: CO2 + H2O ---> H2CO3 ---> H+ + HCO3- 3. In addition, hemoglobin transports CO2 from peripheral tissues to the lungs. Hemoglobin carries CO2 as carbamates formed with the amino terminal of the globin chains.  Hemoglobin Function Summary of Bohr Effect: 4. Conversely, in the lung the CO2 levels decrease as it is continuously exhaled, this increases the oxygen affinity of Hb.  Hemoglobin Function ✓ Factors affecting Hemoglobin function: 4) Carbon monoxide CO CO binds tightly to the hemoglobin iron, forming carboxyhemoglobin (HbCO). When CO binds to one or more of the four heme sites, Hb shifts to the relaxed conformation, which causes the remaining heme sites to bind oxygen with high affinity. As a result, the affected hemoglobin is unable to release oxygen to the tissues. Since, the affinity of hemoglobin for CO is about 200 times greater than for oxygen, as a result even minute concentrations of carbon monoxide in the environment can produce toxic concentrations of HbCO in the blood. Carbon monoxide poisoning is treated with 100 percent oxygen therapy, which facilitates the dissociation of CO from hemoglobin.  Hemoglobin Function Tissues Lung 1.High CO2 Low CO2 in lungs 2.Higher H+ Lower H+ 3.Lower pH Higher pH 4.Affinity for O2 decreases Affinity for O2 increases 5.O2 released to tissues O2 binds hemoglobin 6.T state favored R state favored  Fetal Hemoglobin as O2 carrier Fetal hemoglobin has a different subunit (than the β subunit of the adult hemoglobin) called a γ subunit or α2γ2. The γ chain differs from the chain β in that, there is a change in a single amino acid found in the 2,3-BPG 'binding pocket': from Histidine to serine. 2,3 BPG binds less well to HbF than to HbA. This gives HbF a higher affinity for O2 than HbA. In Fetal hemoglobin, BPG does not affect O2 binding and the baby’s blood will get its oxygen from the mother’s hemoglobin. The transfer of oxygen is from the mother (less tightly bonded) to the baby (more tightly bonded).  Hemoglobin Derivatives Oxyhemoglobin (oxyHb) = Hb with O2 Deoxyhemoglobin (deoxyHb) = Hb without O2 Carbaminohemoglobin (HbCO2) - CO2 is non-covalently bound to globin chain of Hb. HbCO2 transports CO2 in blood. Carboxyhemoglobin (HbCO) – carbon monoxide (CO) binds to Fe2+ in heme in case of CO poisoning or smoking. CO has more than 200x higher affinity to Fe2+ than O2.. ### Key Points on Hemoglobin Structure and Function. Hemoglobin / \ / \ Structure Function | 1. **Structure of Hemoglobin** | ---------------------- ----------------------- - Globular protein (hemoprotein) | | | | | | - Composed of: - 4 heme groups Heme Globin Tetramer O2 transport - 4 globin polypeptide chains (2 α and 2 β chains in adult hemoglobin) CO2 transport Buffering - Heme structure: cyclic tetrapyrrole ring with ferrous iron (Fe2 +) | | | | | - Secondary structure: 8 α-helices (A-H) Iron Binding (Fe2+) 4 Chains α2β2 Affinity - Tertiary structure: globin chains form a sphere changes Influence of pH - Quaternary structure: tetramer (α2β2), two dimers \_____/ 2. **Function of Hemoglobin** - Primary function: transport O2 and CO2 | - Acts as a buffer in erythrocytes Regulation by: - Cooperative binding of oxygen (sigmoidal dissociation curve) - BPG - Oxyhemoglobin (R-state) vs. Deoxyhemoglobin (T-state) - CO - Factors affecting function: ``` - 2,3-bisphosphoglycerate (BPG) This outline provides an organized view of - pH (Bohr effect) - Carbon monoxide (CO) hemoglobin's 3. **Variations in Hemoglobin** structure, function, and its variations, along with a - Fetal hemoglobin (α2γ2) has higher affinity for O2 than adult hemoglobin (HbA) simplified diagram to represent its key components 4. **Hemoglobin Derivatives** and interactions. - Oxyhemoglobin (HbO2) - Deoxyhemoglobin (deoxyHb) - Carbaminohemoglobin (HbCO2) - Carboxyhemoglobin (HbCO) ### Diagram of Hemoglobin Structure and Function Field of Medicine Medicine Program Hemoglobin Synthesis Ass. Prof. George N. B. Morcos Date: 30 / 09 / 2024 Objectives At the end of the lecture, the students will be able to: 1. Review the heme structure. 2. Recognize the steps of heme synthesis. 3. Identify the different causes of porphyrias.  Heme Structure  Heme Structure ▪ Porphin ring: It is formed of four pyrrole rings (I, II, III & IV) united by four methenyl (=CH-) bridges (α, β, γ & δ). A' It has 8 hydrogen atoms at the corners of the pyrrole rings (1, 2, 3, …..8) ▪ Substitution of the 8 hydrogen atoms with chemical groups - 's ‫ــــ‬porphyrin prophyrin will change the name of the ring into (or ‫ــــ‬porphyrinogen if pyrrole rings are united by 233. methylene (-CH2-) bridges if porphipronger÷". "%? porto  Heme Structure ▪ Types of the chemical groups attached determine the name of the porphyrin ▪ 4 acetate (-CH2.COOH) & 4 propionate (-CH2.CH2.COOH) Uroporphyrin ▪ 4 methyl (-CH3) & 4 propionate (-CH2.CH2.COOH) Coproporphyrin ▪ 4 methyl (-CH3) & 2 vinyl (-CH=CH2) & 2 propionate (-CH2.CH2.COOH) Protoporphyrin IN 2hydrogynation oxidation  Heme Structure ▪ Types of the chemical groups attached determine the name of the porphyrin ▪ Positions of the chemical groups determine the isomer (number) of porphyrin. ▪ if the 4 acetate are attached to positions 1, 3, 5 & 7 -24£'II & propionate are attached to positions 2, 4, 6 & 8 Uroporphyrin I ▪ if the 4 acetate are attached to positions 1, 3, 5 & 8 & propionate are attached to positions 2, 4, 6 & 7 Uroporphyrin III  Hemoproteins = Heme + protein Hemoproteins include many important compounds I- Myoglobin - muscle Q: compliment home protein? II- Hemoglobin heme p III- Cytochromes s I 1.RI Ñ IV- Catalase and peroxidase t.- V- Nitric oxide synthase VI- Tryptophan dioxygenase  Biosynthesis of Heme Sites of synthesis Organ site: ▪ The major sites of heme biosynthesis are the liver (13%) which synthesizes a number of heme proteins (particularly, cytochrome P450), and the erythrocyte-producing cells of the bone marrow (85%) which are active in hemoglobin synthesis. ▪ In the liver, the rate of heme synthesis is highly variable, depending on the fluctuating demands for heme proteins. ▪ Heme synthesis in erythroid cells is relatively constant and is matched to the rate of globin synthesis.  Biosynthesis of Heme Sites of synthesis Intracellular site: The initial reaction and the last three steps in the formation of porphyrins occur in mitochondria, whereas the intermediate steps of the biosynthetic pathway occur in the cytosol. Biosynthesis of Heme Steps: Q mutation enzymat and what Bonemarno product defency?  Biosynthesis of Heme Steps: 1-Formation of -aminolevulinate (ALA) in mitochondria: l essinthal 2step  Biosynthesis of Heme Steps: 1-Formation of -aminolevulinate (ALA) in mitochondria: ▪ Glycine (a nonessential a.a) and succinyl CoA (an intermediate in the Krebs cycle) condense, then decarboxylation occurs to form ALA. ▪ This reaction is catalyzed by -aminolevulinate synthase (ALA synthase), in the mitochondria. Ad d (active Be) ▪ This reaction requires pyridoxal phosphate (PLP; the active form of iI vitamin B6) as a coenzyme. B- Sidroblastic animea ▪ -aminolevulinate synthase is the rate-controlling step in porphyrin ♂' EI DNA I biosynthesis. Ñ1 && ▪ It is allosterically inhibited by heme (end product feedback inhibition). %aH il IS, Heme, also represses the synthesis of  -aminolevulinate synthase gene.  Biosynthesis of Heme Steps: Subsequent Reactions Occur in Cytosol 2- Formation of porphobilinogen (PBG): ¥" on porphobylongin synthytass 2 y☁#☆  AA ← c- Biosynthesis of Heme ALA day ew I.m AA e Initiation § Steps: by homes Subsequent Reactions Occur in Cytosol 2- Formation of porphobilinogen (PBG): Two molecules of -aminolevulinate then condense to form porphobilinogen (PBG). This dehydration reaction is catalyzed by - aminolevulinate dehydratase (ALA dehydratase or PBG synthase). ALA dehydratase is extremely sensitive to inhibition by heavy metal ions (e.g. lead). III.IT & ♂{ This inhibition is responsible for the elevation in ALA and the 923 anemia seen in lead poisoning.◦½:*  Biosynthesis of Heme Steps: 3- Formation of hydroxymethylbilane: 4 Hydroxymethylbilane synthase Porphobilinogen deaminase  Biosynthesis of Heme Steps: 3- Formation of hydroxymethylbilane: Four porphobilinogens condense head-to-tail to form a linear 1 tetrapyrrole (hydroxymethylbilane), by the enzyme uroporphyrinogen I synthase (also called hydroxymethylbilane synthase, or porphobilinogen deaminase). An ammonium ion is released for each methylene bridge formed.  Biosynthesis of Heme Steps: 4- Formation of uroporphyrinogen: ◦ Éuro prom, I synthis hydrome"." I isomer yacith. d upro g 02 *TIN a acil.ir  Biosynthesis of Heme Steps: 4- Formation of uroporphyrinogen: Hydroxymethylbilane cyclizes by removal of H2O to form uroporphyrinogen III, in the presence of uroporphyrinogen III synthase, which is essential for isomerizing one of the rings to yield asymmetric uroporphyrinogen III. Normally, little uroporphyrinogen I is formed spontaneously. However, if uroporphyrinogen III synthase is deficient, large quantities of uroporphyrinogen I are formed.  Biosynthesis of Heme Steps: 5- Formation of coproporphyrinogen III: 0 ¥3s 0 0  Biosynthesis of Heme Steps: 5- Formation of coproporphyrinogen III: It is formed by decarboxylation of the four acetate side chains (A) (by uroporphyrinogen decarboxylase) to form four methyl groups (M). This enzyme acts on both uroporphyrinogen I and uroporphyrinogen III. n  Biosynthesis of Heme Steps: Subsequent Reactions Occur in Mitochondria 6- Formation of protoporphyrinogen IX: amethet 2 uprop amethel 20:L v8 ☁ - ♂. 2  Biosynthesis of Heme Steps: Subsequent Reactions Occur in Mitochondria 6- Formation of protoporphyrinogen IX: Conversion of two of the propionate side chains into vinyl groups by coproporphyrinogen oxidase leads to formation of protoporphyrinogen IX. (removal of 4 hydrogen atoms and two CO2 molecules). This enzyme acts only on type III coproporphyrinogen.  Biosynthesis of Heme Steps: 7- Formation of protoporphyrin IX: m  Biosynthesis of Heme Steps: 7- Formation of protoporphyrin IX: The oxidation of protoporphyrinogen to protoporphyrin IX is catalyzed by the mitochondrial enzyme, protoporphyrinogen oxidase. (removal of 6 hydrogen atoms)  Biosynthesis of Heme Steps: 8- Formation of heme: Heme synthase Yots '♂  Biosynthesis of Heme Steps: 8- Formation of heme: The insertion of the ferrous form of iron into the center of the protoporphyrin ring is catalyzed by ferrochelatase (heme synthase) to form heme. Ferrochelatase is inhibited by lead. atass pAdg' £310 geo  Biosynthesis of Heme Steps: Biosynthesis of Heme Steps: Biosynthesis of Heme Steps:  Regulation of Heme Synthesis ALA synthase is a key regulatory enzyme ▪ It is an allosteric enzyme that is inhibited by the end product (heme) (feedback inhibition). ▪ Regulation occurs at the level of enzyme synthesis. Increased levels of heme repress the synthesis of the enzyme ALA synthase. ▪ The iron atom may regulate the heme synthesis (in bone marrow). ▪ Several substances induce the synthesis of hepatic ALA synthase (called microsomal inducers). e.g. steroid hormones, ethanol, certain drugs like barbiturates. Most of the drugs are metabolized in the liver by cytochrome P450. So, maximum amount of heme is utilized for formation of cytochrome P450, which in turn diminishes the intracellular heme concentration. Regulation of Heme Synthesis End product inhibition by heme When porphyrin production exceeds the availability of globin, heme accumulates and is converted to hemin by the oxidation of Fe2+ to Fe3+. Hemin decreases the activity of ALA synthase by causing decreased synthesis of the enzyme. In erythroid cells, heme synthesis is under the control of 1) erythropoietin and 2) the availability of intracellular iron.  Porphyrias Definition Porphyrias are a group of rare inherited diseases. They are caused by defects in heme synthesis, resulting in the accumulation of porphyrins or porphyrin precursors (ALA & PBG) in tissues & their increased excretion. Being water-soluble, ALA, PBG & uroporphyrinogen are excreted in urine. Being water-insoluble, protoporphyrin is excreted in stools. Being moderately water-soluble, coproporphyrins III are excreted both in urine and stools. The various porphyrinogens are colorless. They can undergo spontaneous oxidation, especially in presence of light, to the corresponding porphyrins which are all colored.  Porphyrias Porphyria directly affects 1) the nervous system and 2) the skin; but is possible to only affect one of the two. This disease is often inherited from one parent (Autosomal dominant), with some exceptions, e.g., congenital erythropoietic porphyria - ( autosomal recessive); erythropoietic protoporphyria (X-linked).  Porphyrias According to the clinical manifestations , two groups can be observed in porphyrias, these are: I- Neuropsychiatric manifestation (Acute Porphyria): These are characterized by the accumulation of ALA or PBG, as a result of relative or absolute inhibition of the enzyme uroporphyrinogen I synthase together with increased activity of the enzyme ALA synthase. Porphyrias  Porphyrias According to the clinical manifestations , two groups can be observed in porphyrias, these are: I- Neuropsychiatric manifestation (Acute Porphyria): ALA and PBG are neurotoxic; they cause injury to sympathetic nerves (abdominal pain) as well as to somatic nerves (peripheral neuritis, skeletal muscle paralysis, and neuropsychiatric symptoms (anxiety to delirium). This explains the neuropsychiatric symptoms that occur in acute porphyria.  Porphyrias According to the clinical manifestations , two groups can be observed in porphyrias, these are: I- Neuropsychiatric manifestation (Acute Porphyria): Porphyrias  Porphyrias According to the clinical manifestations, two groups can be observed in porphyrias, these are: II- Photosensitivity This results from the accumulation of porphyrinogens in the skin. Upon exposure to light the colorless porphyrinogens are oxidized to colorful porphyrins which are photosensitizing molecules. These activated porphyrins cause the release of free radicals that cause damage to lysosomes, leading to the release of lysosomal enzymes, which destroy the skin. Thus, exposure to light leads to skin damage and scarring.  Porphyrias According to the clinical manifestations, two groups can be observed in porphyrias, these are: II- Photosensitivity The most common porphyria - Appear in early childhood - Complication: cholestatic liver cirrhosis and progressive hepatic failure. Porphyrias Porphyrias  Porphyrias The porphyrias are classified as erythropoietic or hepatic, depending on whether the enzyme deficiency occurs in erythropoietic cells of bone marrow or in the liver.  Porphyrias Hepatic porphyrias They are genetically determined, and the attack may be precipitated if the requirement for heme is increased due to drugs that require cytochrome P450. They include: ▪Acute intermittent porphyria, ▪Porphyria cutanea tarda, ▪Hereditary coproporphyria & ▪Variegate porphyria.  Porphyrias Erythropoietic porphyrias They are either genetically determined ▪congenital erythropoietic porphyria & ▪protoporphyria or due to lead poisoning (inhibition of PBG synthase and ferrochelatase).  Porphyrias Lead Poisoning: High levels of lead can affect heme metabolism by combining with the SH groups in enzymes like ferrochelatase and ALA dehydratase. Elevated levels of protoporphyrin are found in red cells, and elevated levels of ALA and of coproporphyrin are found in urine.  Summary ▪ Structure of heme. ▪ Heme Biosynthesis. ▪ Porphyrias.  References – Lippincott's Illustrated Reviews: Biochemistry, 5th Edition. Chapter 21. pp277-281. Field of Medicine Medicine Program Iron metabolism & Vitamin K Ass. Prof. George N. B. Morcos Date : 01 / 10 / 2024  Objectives At the end of the lecture, the students will be able to: 1. Identify iron importance, sources and requirements. 2. Recognize the iron absorption and factors affecting it. 3. Recognize the iron transport, storage, regulation and effect of its deficiency and overload. 4. Describe the role of vitamin K in hemostasis.  Iron Metabolism Iron is one of the most important essential trace elements. Dietary Sources: ▪ Iron is present in food as ferric hydroxide and ferric organic compounds. ▪ The liver, heart, kidney, spleen, meat and egg yolk are very good sources of iron. ▪ Molasses, dates, legumes, vegetables and whole cereals are good sources. Daily requirement: ✓ The RDA /day for iron is as follows: ✓ Infants, children and adult males: 10-15 mg. ✓ Adult females: 15-20 mg.  Iron Metabolism Iron absorption: ▪ Iron absorption occurs predominantly in the duodenum and upper jejunum. Only the ferrous form (Fe++) is absorbed. ▪ The absorption of dietary iron is a variable and dynamic process, depending on the following main factors: - Total iron stores - The rate of erythropoiesis - The oxygen content of the blood and various other factors. - A feedback mechanism exists that enhances iron absorption in people who are iron deficient. In contrast, people with iron overload decrease iron absorption.  Iron Metabolism Several dietary factors influence iron absorption: Factors increasing iron absorption: ✓ Vit C (Ascorbic acid) and SH-containing proteins and Gastric HCL favors the reduction of the ferric form of iron to the ferrous form. Factors decreasing iron absorption: ✓ Plant phytates, phosphates, oxalates, and tannins decrease the solubility of iron by forming insoluble complexes; thus inhibiting its absorption. ✓ Phytates are prominent in wheat and some other cereals, while tannins are prevalent in teas.  Iron Metabolism Iron absorption:  Iron Metabolism Iron absorption: ▪ A ferric reductase enzyme on the enterocytes' brush border, Duodenal cytochrome b (Dcytb), reduces ferric Fe3+ to Fe2+. ▪ Ferrous ions are transported across the enterocyte apical membrane by proton-coupled divalent metal transporter (DMT1). ▪ The amount of DMT1 in the apical membrane is regulated by body iron requirements. ▪ Once inside, iron can either ▪ After being taken up by the intestinal mucosa, iron is either stored in the form of ferritin in the mucosal cells or transported across the mucosal cells to the plasma in the form of transferrin.  Iron Metabolism Iron absorption: ▪ Iron effluxes from the enterocyte basolateral membrane through ferroportin and is oxidized by a membrane-bound ferroxidase, hephaestin, yielding ferric ions that are then bound by plasma transferrin (glycoprotein, synthesized in the liver) for distribution around the body via the blood. ▪ Heme is transported into the enterocyte by a separate heme transporter (HT).  Iron Metabolism Regulation of Iron absorption: ▪ The principle regulatory mechanism involves sensing of high body iron stores or low erythroid iron requirements by the liver which produces an inhibitory peptide, hepcidin, that acts on the intestine to decrease iron absorption. ▪ When erythroid iron requirements are increased or body iron stores are low, hepcidin production by liver is decreased, leading to increased iron absorption. ▪ Hepcidin is a 25 amino acid peptide that binds to ferroportin and induces its internalization and degradation, thereby reducing iron export from enterocytes.  Iron Metabolism Regulation of Iron absorption: ▪ Dcytb and DMT1 levels are also affected, most likely in response to altered iron levels in enterocytes caused by hepcidin's action on iron efflux through ferroportin. ▪ Inappropriate decreases in iron absorption are seen in chronic diseases with increased inflammation, as hepcidin levels are increased in these conditions leading to anemia of chronic disease.  Iron Metabolism Distribution And Functions: ▪ The total iron in the body is about 4g. ▪ It is present in the body in two forms: 1- Functional forms (75%) ▪ These are mostly in the form of hemoproteins. They are responsible for cellular respiration. ▪ They include: hemoglobin forms 60-70% of total iron in the body, myoglobin forms 3-5% and enzymes as cytochromes, cytochrome oxidase, catalase, peroxidase, nitric acid synthase and tryptophan dioxygenase.  Iron Metabolism Distribution And Functions: 2- Nonfunctional forms (25%) ▪ These are transport and storage forms of iron. They are nonheme metalloproteins. Transferrin: This is the transport form of iron in the blood plasma. Ferritin: This is the chief storage form of iron in the tissues. It is present in the liver, kidneys, spleen, bone marrow, and intestinal mucosal epithelium. Hemosiderin: This is present in iron stores when the body contains excess iron. It is another iron storage protein. It is a golden-brown aggregated deposits resulting from breakdown of ferritin in lysosomes. Lactoferrin: It is present in milk & contains iron bound to a glycoprotein.  Iron Metabolism Blood Iron: 1- In red cells: ▪ The erythrocytes contain hemoglobin, which contains 3.4 mg of iron per gram.  Iron Metabolism Blood Iron: 2- In plasma a- Transferrin: ▪ The plasma iron concentration is 50-150 µg/dL. Iron is carried by a glycoprotein, transferrin, which carries 2 atoms of ferric iron/molecule. ▪ It is synthesized in the liver. Low degree of transferrin saturation by iron facilitates iron release from the mucosal cells. ▪ Transferrin may carry up to 250-450 µg of iron per dL plasma. This is known as the total iron-binding capacity (TIBC). This means that, on the average, only about 30% of the TIBC is saturated and about 60-70% of transferrin is unsaturated (UIBC). ▪ In iron deficiency anemia the plasma iron decreases while the TIBC tends to increase. ▪ TIBC is low in hemolytic anemia (Iron overload).  Iron Metabolism Blood Iron:  Iron Metabolism Blood Iron: 2- In plasma b- Ferritin: ▪ Plasma contains very low concentrations of ferritin, which is a very good index of iron storage. ▪ It decreases in iron deficiency and increases in hemosiderosis.  Iron Metabolism Abnormalities Of Iron Metabolism Both iron deficiency and iron excess can result in hazardous consequences. I- Iron deficiency anemia ▪ It is characterized by microcytic and hypochromic red blood cells. ▪ The most common causes of iron deficient anemia are excess menstrual flow or chronic gastrointestinal bleeding. Other causes include; inadequate intake, impaired absorption, and increased demand. ▪ Treatment of iron deficiency anemia includes treatment of the cause of bleeding and iron supplementation. ▪ A preparation of iron (iron dextran) for intramuscular injection has been used in patients who cannot tolerate or absorb oral iron. ▪ Caution must be taken when iron is given parenterally because of the possibility of oversaturation of tissues with resultant production of hemosiderosis.  Iron Metabolism Abnormalities Of Iron Metabolism II- Iron toxicity (Hemosiderosis or Hemochromatosis) ▪ The body is unable to excrete a large load of iron. Iron differs from most other minerals in that its quantity in the body is controlled by regulating its absorption rather than excretion. ▪ Iron toxicity is produced by one of the following causes: 1) In patients with aplastic or hemolytic anemia who have received many blood transfusions over a period of years. 2) Inherited disorder of iron absorption, some persons has excessive ability to absorb iron which produces iron accumulation after many years. 3) Overdose of parenterally administered iron. ▪ In all above conditions, hemosiderin accumulates in different tissues, a condition known as hemosiderosis.  Iron Metabolism Abnormalities of Iron Metabolism II- Iron toxicity (Hemosiderosis or Hemochromatosis) ▪ Hemochromatosis means hemosiderosis with injury to involved tissues as manifested by cellular degeneration and fibrosis. ▪ Clinical manifestations include bronzed pigmentation of the skin, liver cirrhosis and pancreatic fibrosis, leading to diabetes mellitus (bronze diabetes). ▪ Serum iron is elevated and transferrin becomes 70-90% saturated with iron.. Outline of Iron Metabolism. 4. **Regulation of Iron Absorption** - Hepcidin's role in iron regulation 1. **Introduction to Iron** - Response to iron stores and erythroid needs - Importance of iron - Effects of chronic inflammation on absorption - Dietary sources 5. **Distribution and Functions of Iron** - Daily requirements - Total iron in the body (~4g) - Functional forms (hemoglobin, myoglobin, enzymes) 2. **Iron Absorption** - Nonfunctional forms (transferrin, ferritin, - Primarily in duodenum and jejunum - Forms of iron (Fe2+ absorbed, Fe3+ not) hemosiderin, lactoferrin) - Factors influencing absorption 6. **Blood Iron Levels** - Enhancers: Vitamin C, Gastric HCl - Iron in red blood cells (hemoglobin) - Inhibitors: Phytates, Oxalates, Tannins - Plasma iron transport (transferrin, ferritin) 3. **Mechanism of Iron Absorption** 7. **Abnormalities of Iron Metabolism** - Role of Dcytb (ferric reductase) - Iron deficiency anemia - DMT1 transport across enterocyte membrane - Causes and treatment - Ferritin storage vs. transferrin transport - Iron toxicity (Hemosiderosis or Hemochromatosis) - Causes and clinical manifestations. Mind Map of Iron Metabolism ```plaintext Iron Metabolism | --------------------------------------------------- | | | Dietary Sources Iron Absorption Regulation of Iron | | | RDA: 10-20 mg Duodenum, Jejunum Hepcidin action (inhibitory) Fe2+ absorption Response to iron stores | | | Enhancers: Dcytb, DMT1 transport Chronic inflammation effects Vitamin C, HCl | Inhibitors: Phytates, Tannins | --------------------------------------- | | Distribution and Functions Abnormalities | | Functional Forms: Iron Deficiency Anemia Hemoglobin, Myoglobin Causes: Bleeding, Diet | Treatment: Iron Transferrin, Ferritin supplementation | Hemosiderosis / Hemochromatosis | Causes: Blood transfusions, Inherited disorders, Overdosage | Clinical Manifestations: Bronzed skin, liver cirrhosis, diabetes  Vitamin K (Anti-Hemorrhage Vitamin) Chemistry & sources: ▪ Vitamin K is a fat-soluble vitamin. ▪ Vitamin K exists in different forms: Phylloquinone K1 Dietary Green leafy vegetables, Cabbage, spinach, liver & egg yolk. Menaquinone K2 Intestinal bacteria Menadione K3 Synthetic  Vitamin K (Anti-Hemorrhage Vitamin) Physiological functions: ▪ It helps in the post-translational modification of certain proteins that are synthesized as inactive precursors and their activation needs a carboxylation reaction in which vit K acts as coenzyme. ▪ Vitamin K serves as a coenzyme in the carboxylation of certain glutamic acid residues present in specific proteins and this reaction is catalyzed by a carboxylase enzyme. ▪ The glutamate residues are converted to γ-carboxyglutamate. ▪ The reaction requires vitamin K, O2 and CO2.  Vitamin K (Anti-Hemorrhage Vitamin) Physiological functions: A. Blood clotting factors (prothrombin (factor II) and factors VII, IX, and X). Both, the new carboxyl group together with the existing carboxyl group serve as binding sites for the divalent calcium ions. The coagulation factor-Ca2+ complex in turn binds to phospholipid membrane in platelets and this increases the proteolytic conversion of prothrombin to thrombin.  Vitamin K (Anti-Hemorrhage Vitamin)  Vitamin K (Anti-Hemorrhage Vitamin) Physiological functions: B. Osteocalcin, where γ-carboxylation helps its binding to hydroxyapatite crystals in bone. Due to this mechanism, vitamin K is involved in the regulation of bone mineralization (it helps the bones to retain calcium).  Vitamin K (Anti-Hemorrhage Vitamin) Causes of deficiency 1- Newborn infants have a great risk for vitamin K deficiency, because: The fetal intestine is sterile, so it cannot synthesize vitamin K. Very little vitamin K is crossing the placental barrier. Human milk provides only about one-fifth of the daily requirement for vitamin K. The concentrations of plasma clotting factors are low in infants due to immaturity of the liver. Vitamin K deficiency in newborns may result in fatal intracranial hemorrhage (bleeding within the skull) in the first weeks of life. So, vitamin K is routinely administered prophylactically to all newborns.  Vitamin K (Anti-Hemorrhage Vitamin) Causes of deficiency 2- Failure of synthesis by intestinal flora due to prolonged intake of antibiotics or repeated use of washing enema. 3- Failure of absorption e.g. in steatorrhea and obstructive jaundice. 4- Failure of utilization in case of liver diseases.  Vitamin K (Anti-Hemorrhage Vitamin) Causes of deficiency 5- Dicumarol and warfarin are used as anticoagulants because they are structurally similar to vitamin K (competitive inhibitors). Vitamin K will not be reduced, and carboxylation reaction of clotting factors will be interrupted. So, high doses of these anticoagulants may produce symptoms and signs of vitamin K deficiency.  Vitamin K (Anti-Hemorrhage Vitamin) Causes of deficiency  Vitamin K (Anti-Hemorrhage Vitamin) Deficiency manifestations: 1- Bruising tendency. 2- Posttraumatic bleeding and internal hemorrhage. 3- Prolonged prothrombin time and delayed clotting time.  Summary - Iron - Vitamin K