Biochemistry of Iron, Folate & Vitamin B12 Metabolism PDF

BIOCHEMISTRY OF IRON, FOLATE & VITAMIN B12 METABOLISM BBM1233 MEDICAL BIOCHEMISTRY 2  Learning Objectives At the end of this lecture, the student will be able to: Describe the metabolism of iron: Absorption, storage, transportation and excretion Explain the disorders of...

BIOCHEMISTRY OF IRON, FOLATE & VITAMIN B12 METABOLISM BBM1233 MEDICAL BIOCHEMISTRY 2  Learning Objectives At the end of this lecture, the student will be able to: Describe the metabolism of iron: Absorption, storage, transportation and excretion Explain the disorders of iron metabolism: Iron deficiency and iron overload  IRON Iron is an essential nutrient for the human body. About 3-5g of iron is stored in the body (75% is in blood and the rest is stored in bone marrow, liver & muscles) Iron is principally found in heme ▪ Heme is found mostly in hemoglobin, myoglobin and cytochromes. Iron plays a role in oxygen transportation and basic metabolic oxidative and reductive reactions  Dietary sources of iron include organ meats, fish, oysters, egg yolks, dried beans, dried figs and dates, green vegetables. ▪ Heme: iron in animal products ▪ Non-heme: iron in vegetables Daily requirement: ▪ Male: 5-10 mg/day; Female: 20 mg/day; ▪ Pregnant women: 40mg/day  Iron Absorption Iron absorption occurs mainly in the duodenum and upper jejunum Only Fe2+ (ferrous) form is absorbed. Fe3+ (ferric) form is not absorbed Fe3+ are reduced to Fe2+ with the help of gastric HCl, ascorbic acid and cysteine Divalent metal transporter (DMT-1) facilitates transfer of iron across the intestinal epithelial cell  Storage of Iron Inside the mucosal cell, Fe2+ is oxidized to Fe3 + and is complexed with apoferritin to form ferritin Major storage form of iron: Ferritin Free iron can promote the production of free radicals that damage cellular constituents Such undesirable reactions are minimized when iron is stored inside ferrintin The largest amounts of ferritin iron are found in the liver, the spleen, and the bone marrow Ferritin in plasma level is elevated in iron overload conditions  Storage form of iron: Hemosiderin Membrane-enclosed, insoluble degradation product of ferritin that is normally found in the liver, bone marrow and spleen Hemosiderin accumulates when iron levels are increased  Iron Transport Iron in the ferritin is released, then crosses the mucosal cell with the help of a transport protein, called ferroportin Iron crosses cell membrane in ferrous form (Fe2+) In blood, it is re-oxidized to ferric state (Fe3+) and transported by transferrin Transferrin is a plasma protein for iron transport. It is a glycoprotein, synthesized in the liver. It transport iron in the circulation to sites where iron is required Total iron binding capacity (TIBC) represents the capacity of transferrin to bind iron  Divalent metal transporter (DMT-1) facilitates transfer of iron across the intestinal epithelial cell Iron within the enterocytes is release via ferroportin into the bloodstream Iron is then bound with transferrin in the bloodstream  Iron Excretion There is NO physiological regulated mechanism for iron excretion About 1-2 mg of iron per day is lost by: Exfoliated epithelial cells of the GI tract Exfoliated cells of the skin Menstruation  Regulation of Iron Absorption 1. Mucosal Regulation Iron metabolism is unique because homeostasis is maintained by regulation at the level of absorption & NOT by excretion When iron stores in the body are depleted, absorption is enhanced Mucosal block When adequate quantity of iron is stored, absorption is decreased 2. Stores regulation As body iron stores fall, the mucosa is signaled to increase absorption 3. Erythropoietic regulation In response to anemia, the RBCs will signal the mucosa to increase iron absorption. This signal may be erythropoietin from kidney 4. Control of ferritin and transferrin receptor (TfR) synthesis When the concentration of iron is high, mRNA for ferritin is translated and ferritin is synthesized (to store iron). But mRNA for TfR is degraded, resulting in reduced TfR protein synthesis (no requirement for further uptake of iron) Factor Affecting Absorption of Iron Enhance absorption Inhibit absorption Acidity (gastric HCl) Phosphates Ascorbic acid Calcium Iron deficiency (anemia) Phytic acid (in cereals) Amino acids Oxalic acid (in leafy vegetables) Tannic acid (in tea & coffee) Antacids  Diagnostic Tests for Iron Status Tests Reference range Serum iron Free iron circulating in Men: 55-160 µg/dL bloodstream Women: 40-155 µg/dL Total iron binding Measure the ability of 255-450 µg/dL capacity (TIBC) transferrin to carry iron in the blood Ferritin The amount of stored iron in Men: 12-300 ng/mL the body Women: 12-150 ng/mL Transferrin Uptake of iron into cells is Men: 2.2-5 mg/L receptor (TfR) facilitated by this receptor Women: 1.9-4.4 mg/L present on the surface of all iron-requiring cells in the body TfR synthesis is controlled by the concentration of iron  Disorders of Iron Metabolism: Iron Deficiency Anemia (IDA) Causes: Nutritional deficiency of iron Inability to absorb iron E.g. Celiac disease, chronic inflammatory bowel disease, colonisation of stomach with Helicobacter pylori Bleeding causes: Excessive menstrual blood flow (menorrhagia) Hookworm infection Hemorrhoids, peptic ulcer Pregnancy Iron Deficiency Anemia: Symptoms  Spoon shaped nail (koilonychias) Glossitis Angular cheilitis Laboratory Findings of IDA Tests Results Serum iron Decreased Total iron binding capacity Increased (TIBC) Serum ferritin Greatly reduced Soluble transferrin receptor Increased level (TfR)  Treatment of Iron Deficiency Oral iron supplementation is the treatment of choice 100 mg iron + 500 µg folic acid are given to pregnant women 20 mg iron + 100 µg folic acid to children Iron tablets are usually given along with vitamin C to enhance absorption of iron Unabsorbed iron may generate free radicals and so it is advisable to give vitamin E (to prevent free radical generation) along with iron Parenteral Iron therapy Often effective when oral iron supplementation is unsuccessful or inappropriate  Disorders of Iron Storage Hemosiderosis Deposition of excess iron within the body tissues Occurs in person receiving repeated blood transfusion E.g. patients suffering from thalassaemia & sickle cell anemia particularly at risk for iron overload Hereditary hemochromatosis Iron absorption is increased and transferrin level in serum is elevated due to mutation in HFE1 gene The low hepcidin level relative to ferritin in these patients leads to increased intestinal iron absorption and body iron overload  Hepcidin (iron regulatory hormone) regulate iron transport, thereby preventing excess iron absorption and maintaining normal iron level Production of hepcidin is regulated by iron  Treatment of Hemosiderosis Phlebotomy Desferoxamine (Desferrioxamine), a chelating agent, forms an iron chelate with Fe3+ to form ferroxamine which is excreted in urine  Folic Acid/Folate (Vitamin B9) The word folic acid is derived from the Latin word folium, which means leaf of vegetable It is composed of three components: 1. Pteridine ring Pteroyl 2. Para amino benzoic acid (PABA) glutamic acid/ 3. Glutamic acid residues Folic acid  Folic acid is soluble in water. When exposed to light, it is rapidly destroyed Recommended Daily Allowance (RDA) of folic acid: 400 µg/day Pregnant women: 600 µg/day; lactation: 500 µg/day  Absorption of Folic Acid Folic acid is readily absorbed by the upper part of jejunum. In the blood, it is transported by beta globulins. It is taken up by the liver where the co-enzymes are produced When stored in the liver or ingested, folic acid exists in a polyglutamate form Intestinal mucosal cells remove some of the glutamate residues through the action of the folate conjugase The removal of glutamate residues makes folate less negatively charged and therefore more capable of passing through the basal luminal membrane of the intestinal epithelial cells and into the bloodstream  Folic acid is reduced within cells to 7,8-dihydrofolic acid and further reduced to 5,6,7,8-tetrahydrofolic acid (THFA). Both reactions are catalysed by NADPH dependent folate reductase The THFA is the carrier of one-carbon groups Formyl (-CHO) Attached Formimino (-CH=NH) either to the 5th or to the 10th Methenyl (-CH=) or to both 5th Methylene (-CH2-) and 10th Hydroxymethyl (-CH2OH) nitrogen atom Methyl (-CH3) of THFA  Co-enzyme of Folic Acid The most common circulating form of the vitamin is 5-methyltetrahydrofolic acid The major function of the folic acid co-enzyme is the transfer of one-carbon group in a variety of synthetic reaction Methyl group in N5-methyl THFA is used for synthesis of active methionine, which takes part in transmethylation reactions. Such transmethylation reactions, where the methyl group donor is S-adenosyl methionine (SAM), are required for synthesis of choline, epinephrine, creatine, etc. Functions of folic acid  Causes for folic Acid Deficiency Increased requirement E.g. pregnancy, haemolytic anemia Defective absorption E.g. celiac disease (gluten induced enteropathy) and resection of jejunum Drugs (hydantoin, dilantin, phenytoin, phenobarbitone) The drugs will inhibit the intestinal enzyme so that folate absorption is reduced Dietary deficiency Absence of vegetables in food for prolonged periods may lead to deficiency  Folic Acid Deficiency Manifestations Reduced DNA synthesis Megaloblastic anemia/macrocytic anemia Decrease in purines and pyrimidines leads to a decrease in nucleic acid synthesis and cell division Rapidly dividing cells in bone marrow and intestinal mucosa are seriously affected Large, immature red blood cells are present Hyperhomocysteinemia Folic acid deficiency may cause increased homocysteine levels in blood since remethylation of homocysteine is affected Plasma homocysteine levels above 15 mmol/L is known to increase the risk of coronary artery diseases  Cancer Folate deficiency contributes to the etiology of bronchial carcinoma and cervical carcinoma Birth defect Folic acid deficiency during pregnancy may lead to neural tube defects in the fetus Anencephaly Spina bifida Encephalocele TS: Thymidylate synthase  Assessment of Folic Acid Deficiency Serum folic acid level Normal folic acid level in serum is about 20 ng/mL Histidine load test or FIGLU excretion test In folic acid deficiency, formiminoglutamic acid (FIGLU) is excreted in urine Histidine Formiminoglutamic acid (FIGLU) Folic acid Glutamic acid  Amino imidazole carboxamine ribonucleotide (AICAR) excretion In the purine ring synthesis, the last step is the addition of C2 with the help of N10-formyl THFA. When this is blocked, the precursor, AICAR accumulates and is excreted in urine Peripheral blood smear  Folic Acid Therapy Therapeutic dose is 1 mg of folic acid per day orally Folic acid alone should not be given in macrocytic anemia, because it may aggravate the neurological manifestation of vitamin B12 deficiency. So, folic acid and vitamin B12 are given in combination to patients Regular supplementation of folic acid may reduce the incidence of birth defects, cardiovascular diseases and cancers  Vitamin B12 (Cobalamin) Also known as anti-pernicious anemia vitamin Is a heat stable, water soluble vitamin with a key role in the normal functioning of the brain and nervous system, and for the formation of blood Involved in the DNA synthesis, fatty acid synthesis and amino acid metabolism Synthesize only by microorganisms and not by animals and plants  Structure of Cobalamin Composed of: A corrin ring (4 pyrrole rings bound to a central cobalt atom) A 5,6 dimethylbenzimidazole group is linked to the 5th valency of the cobalt The 6th valency of the cobalt is occupied by any of the following groups: cyanide, hydroxyl, adenosyl or methyl Structure of Vitamin B12  Types of Cobalamin 1. Cyanocobalamin Cyanide is added at the R position Industrially produced stable cobalamin form Used in vitamin supplements (oral preparations are in this form) 2. Hydroxocobalamin Hydroxyl group is added at the R position Denoted by Vitamin B12a It is supplied typically in water solution for injection (injectable preparations are in this form) 3. Deoxyadenosylcobalamin (Ado-B12) When taken up by the cells, the cyanide group is removed and deoxyadenosylcobalamin (Ado-B 12) is formed This is the major storage from, seen in liver Functional co-enzyme in the body 4. Methylcobalamin (Methyl B12) Methyl group is added at the R position This is the major form seen in blood circulation as well as in cytoplasm of cells Functional co-enzyme in the body  Sources of Vitamin B12 Foods of animal origin are the only sources for vitamin B12. The rich sources are liver, kidney, milk, curd, eggs, fish, pork and chicken Recommended Daily Allowance (RDA) of vitamin B12: 1-2 µg/day Pregnant women; lactation: 2 mg/day  Absorption of Vitamin B12 Absorption of vitamin B12 require two binding proteins: Intrinsic factor (IF); secreted by gastric parietal cells Cobalophilin / Haptocorrin; secreted by salivary gland Gastric pepsin release the vitamin from proteins of the food, and then B12 binds with cobalophilin. In duodenum, cobalophilin is hydrolysed by trypsin of pancreatic juice; vitamin is released, and then vitamin binds to intrinsic factor (IF). One molecule of IF can combine with 2 molecules of B 12. This IF-B12 complex is attached with specific receptor on mucosal cells of the illeum and the whole IF-B12 complex is endocytosed.  The entry of B12 into the mucosal cells is mediated by Ca2+ ions. In the mucosal cells, B12 is converted to methylcobalamin. It is then transported in the circulation in a bound form to protein namely transcobalamins (TC-l & TC-ll). Methylcobalamin is mostly bound to TC-l. Methylcobalamin which is in excess is taken up by the liver, converted to deoxyadenosylcobalamin and stored in this form. Whole liver contains about 2 mg of B 12, which is sufficient for the requirement for 2-3 years Biochemical Function of Vitamin B12 Two reactions require B12 Reaction 1: Conversion of methylmalonyl-CoA to succinyl-CoA Methylmalonyl-CoA is produced during the degradation of fatty acids with odd numbers of carbon atoms When vitamin B12 is deficient, abnormal fatty acids accumulate & become incorporated to cell membranes including nervous system leading to neurological manifestations Reaction 2: Conversion of homocysteine to methionine Methionine synthase requires vitamin B12 in converting homocysteine to methionine When vitamin B12 is deficient, homocysteine accumulates leading to neurological manifestations Tetrahydrofolate will not be available for formation of purine & thymidine monophosphate (TMP) leading to megaloblastic anemia  Causes of Vitamin B12 Deficiency Nutritional deficiency of vitamin B12 Decrease in absorption Absorptive surface is reduced by gastrectomy, resection of ileum and malabsorption syndromes Addisonian pernicious anemia An autoimmune disease Antibodies are generated against IF. So, IF becomes deficient, leading to defective absorption of B12 Pregnancy Fish tapeworm infestation Fish tapeworm (Diphyllobothrium latum) has a special affinity to B12 causing reduction in available vitamin Vitamin B12 Deficiency Manifestations Megaloblastic anemia/macrocytic anemia Abnormal homocysteine level In vitamin B12 deficiency, homocysteine is accumulated, leading to homocystinuria. Homocysteine level in blood has a positive correlation with myocardial infarction Demyelination The vitamin B12 deficiency leads to the non-availability of methionine Therefore methylation of phosphatidylethanolamine to phosphatidylcholine is not adequate. This leads to deficient formation of myelin sheaths of nerves, demyelination and neurological lesions  Subacute combine degeneration Damage to nervous system is seen in vitamin B12 deficiency Achlorhydria Absence of acid in gastric juice is associated with vitamin B12 deficiency  Assessment of Vitamin B12 Deficiency Serum B12 level Schilling test Radioactive labelled (Cobalt-60) vitamin B12 is given orally. In gastric atrophy cases, there is no absorption, hence the entire radioactivity is excreted in feces and radioactivity is not observed in liver region If the cause is nutritional deficiency, there will be enhanced absorption. Then radioactivity is noted in the liver region, with very little excretion in feces  Peripheral blood smear Serum or urinary methylmalonic acid (MMA) level In B12 deficiency, methylmalonyl-CoA accumulates and methyl malonic aciduria occurs Serum homocysteine level  Treatment Therapeutic dose of vitamin B12 is 100 to 1000 microgram by intramuscular injections