Vitamin B12 and Folic Acid Lecture Notes PDF

## Learning Objectives - Discuss the factors regulating Erythropoiesis - Discuss Vitamin B12 and Folic acid metabolism in the body - Describe the biochemical functions, dietary sources and recommended daily intake of Vitamin B12 and Folic acid - Discuss the clinical significance of Vitamin B12 and...

## Learning Objectives - Discuss the factors regulating Erythropoiesis - Discuss Vitamin B12 and Folic acid metabolism in the body - Describe the biochemical functions, dietary sources and recommended daily intake of Vitamin B12 and Folic acid - Discuss the clinical significance of Vitamin B12 and Folic acid deficiency ## Erythropoiesis The process which produces red blood cells ### Regulation of Erythropoiesis - Tissue oxygenation is basic regulator of RBC production - Low blood vol., low blood flow, low Hb, anemia, failure of O2 absorption in pulmonary disease, dec. Tissue oxygenation - Secretion of hormone "Erythropoietin (glycoprotein)" from 80 to 90% from kidneys and rest from liver and macrophages, - It stimulates production of pro-erythroblast from hemopoietic stem cell which in turn inc. RBC production. ## Classification of Vitamins | | | | | |------------ |------------- |------------ |------------ | | **Vitamins** | | | | | **Water-soluble** | | **Fat-soluble** | | | **Non-B-Complex** | **B-Complex** | Vitamin A (retinol, (-carotenes) | | | Ascorbic acid (vitamin C) | | Vitamin D (cholecalciferol) | | | | | Vitamin K (phylloquinones, menaquinones) | | | | | Vitamin E (tocopherols) | | | | **Energy-releasing** | | **Other** | | | Thiamine (vitamin B₁) | **Hematopoietic** | Pyridoxine (vitamin B₂) | | | Riboflavin (vitamin B₂) | Folic acid | Pyridoxal | | | Niacin (vitamin B₃) | Vitamin B₁₂ | Pyridoxamine | | | Biotin | | | | | Pantothenic acid | | | - Present in small quantities in different types of food - Important for growth and good health - Help in various biochemical processes in cell - Function as coenzymes ## Vitamin B₁₂ - Plays an essential role RBC formation, cell metabolism, nerve function and the production of DNA. - Deficiency may cause weak muscles, numbness, trouble walking, nausea, weight loss, irritability, fatigue, and increased heart rate ## Vitamin B₁₂ = Cobalamin - **Function:** Healthy red blood cell & marrow, nervous system function, sperm production, strong immune system. Improves mental energy, memory, protects against allergies and toxins (cyanide in cigarettes) - **Sources:** Milk, eggs, liver, dairy products, fish, and shellfish - **Prevents:** Pernicious Anemia, intestinal malabsorption - **TOXICITY:** slight stomach upset ## Forms of vitamin B₁₂ - Cyanocobalamin (commercial preparation) - Hydroxycobalamin - Adenosylcobalamin (major storage form in the liver) - Methylcobalamin (mostly found in blood circulation) ## VITAMIN B₁₂ (cobalamin) - Vitamin B₁₂, is also called cobalamin, cyanocobalamin and hydroxycobalamin. - It is built from: - A nucleotide and - A complex tetrapyrrol ring structure (corrin ring) - A cobalt ion in the center. - A R-group - When R is cyanide (CN), vitamin B₁₂ takes the form of cyanocobalamin. - In hydroxycobalamin, R equals the hydroxyl group (-OH). - In the coenzyme forms of vitamin B₁₂, - R equals an adenosyl group in adenosylcobalamin. - R equals a methyl (-CH₃) group in methylcobalamin. ## Coenzyme forms of B₁₂ - Adenosylcobalamin and Methylcobalamin - Coenzymes for metabolic reactions - Liver is the good sourse, yeast - Dietary recommendations 2.4 ugm/day for men and women ## Vitamin B₁₂ - Essential for normal nervous system function and red blood cell maturation - Not synthesized in the body and must be supplied in the diet - Binds to intrinsic factor and absorbed by the ileum - Intrinsic factor is a protein secreted by cells in the stomach ## Absorption of vitamin B₁₂ - Vitamin B₁₂ binds a glycoprotein (intrinsic factor) in stomach. I.F. is secreted by the parietal cells. - Vitamin-intrinsic factor complex recognizes surface receptors of mucosal cells in ileum and is absorbed there. - It is stored mainly in the liver in amounts (3-5mg) sufficient to last a couple of years. - Increased need in pregnancy, lactation, growth - One molecule of IF can combine with 2 nolecules of B₁₂. This IF-B₁₂ complex is attached with specific receptors on nucosal cells. The whole IF-B₁₂ complex s internalized ## Sources & absorption of vitamin B₁₂ - Vitamin B₁₂ is Not synthesized in the body (synthesized only by microorganism) - Humans obtain vitamin B₁₂: - performed by natural bacterial flora - or/supplied in the diet (animal sources of diet - not in plants) - Vitamin B₁₂ binds to intrinsic factor and absorbed by intestine. - Intrinsic factor is a protein secreted by cells in the stomach ## Functions of Vitamin B₁₂ ### Two reactions require B₁₂ - D-Methyl malonyl CoA is formed in the body from propionyl CoA. It is then converted to L form by a racemase and then isomerized by methyl malonyl CoA mutase (containing Ado-B₁₂) to succinyl CoA, which enters into citric acid cycle - In B₁₂ deficiency, methyl malonyl CoA is excreted in urine (methyl malonic aciduria). Myelin synthesized with these abnormal fatty acids is unstable and degraded causing neuropathy ## Homocysteine Methyl Transferase - Step 2 is catalyzed by the enzyme methionine synthase or homocysteine methyl transferase. The steps marked as 1 and 2 in need the activity of vitamin 312 (methylcobalamin). ## Methyl Folate Trap and Folate Deficiency - The production of methyl THFA is an irreversible step, Therefore, the only way for generation of free THFA is step No. 1 - WhenB₁₂ is deficient, this reaction cannot take place. This is called the methyl folate trap. This leads to the associated folic acid scarcity in B₁₂ deficiency. ## Neurological manifestations of vitamin B₁₂deficiency - **Demyelination** - Myelin sheath of neurons is chemically unstable and damaged - **Neuropathy** - Peripheral nerve damage - **Causes of neurological manifestations:** - Deficiency of vitamin B₁₂ leads to accumulation of methylmalonyl CoA - High levels of methylomalonyl CoA is used instead of acetyl CoA for fatty acid synthesis resulting in synthesis of abnormal fatty acids. - Myelin sheath is synthesized with these abnormal fatty acids is unstable and degraded causing neuropathy ## Functions of vitamin B₁₂ (cont.) ### Reaction 2: - Conversion of homocysteine to methionine - Methionine synthase requires B₁₂ in converting homocysteine to methionine. - When vitamin B₁₂ is deficient, homocysteine accumulates leading to neurological manifestations. - Also, tetrahydrofolate will not be available for formation of purine & TMP leading to megaloblastic anemia. ## Functions of Vitamin B₁₂ - Homocystinuria and Pernicious anemia occur due to deficiency of Vit B₁₂. - Absorption of Vit B₁₂ is prevented by lack of Intrinsic factor. - Folate is trapped. This leads to folate deficiency and deficiency of other TH₄ derivatives ## Clinical manifestations of vitamin B₁₂ deficiency ### Block of reaction 1 & 2: - B₁₂ deficiency causes accumulation of homocysteine and methylmalonic acid which are harmful for nervous tissue leading to neurological manifestations ### Block of reaction 1: - Methyl tetrahydrofolate cannot be converted to tetrahydrofolate - Hence folate is trapped as N-methyltetrahydrofolate (folate trap) - This leads to folate deficiency (not available for purine synthesis). - So, vitamin B₁₂ deficiency(indirectly) causes megaloblastic anemia. - TREATMENT OF THIS CASE BY FOLIC ACID ONLY CURES ANAEMIA ONLY BUT NERVOUS MANIFESTATIONS ARE NOT CURED (masking of B₁₂ def.) ## VITAMIN B₁₂ - VITAMIN B₁₂ (along with folic acid) - DNA SYNTHESIS - MATURATION OF ERYTHROCYTES - HEME SYNTHESIS - HEMOGLOBIN - ERYTHROPOIESIS ## Vitamin B₁₂ in Heme Synthesis - Vitamin B₁₂, in the form of 5-deoxyadenosylcobalamin, acts as coenzyme for enzyme Methylmalonyl CoA mutase for the conversion of Methylmalonyl CoA to Succinyl CoA. ## Vitamin B₁₂ Deficiency ### Causes of vitamin B₁₂ deficiency: - Deficiency of vitamin B₁₂ in diet (rare) - Deficiency of absorption of vitamin B₁₂ from intestine, called pernicious anemia (more common) - Autoimmune destruction of gastric parietal cells (that synthesizes intr. f). - Partial or total gastrectomy - N.B. As liver stores 4-5 mg of vitamin B₁₂ (in contrast to other water soluble vitamins), clinical symptoms develop in several years after gastrectomy. | **Cause** | **Particulars** | |---------- |----------------- | | Inadequate intake | Alcohol consumption | | | Vegetarian diet | | Malabsorption | Food vitamin B₁₂ malabsorption | | | Lack of intrinsic factor or parietal cell | | | Pernicious anaemia | | | Atrophic gastritis | | | Post-gastrectomy | | | Ileal malabsorption | | | Ileal resection | | | Crohn's disease | | | Bacterial overgrowth | | Defective transport | Transcobalamin deficiency (genetic) | ## Disorders of Vitamin B₁₂ Deficiency ### Neuropathy - Deficiency of vitamin B₁₂ leads to accumulation of methylmalonyl CoA - High levels of methylomalonyl CoA is used instead of malonyl CoA for fatty acid synthesis - Myelin synthesized with these abnormal fatty acids is unstable and degraded causing neuropathy - Pernicious anemia - Vitamin B₁₂ deficiency is mainly due to the deficiency of intrinsic factor ## VITAMIN B₁₂ DEFICIENCY - PERNICIOUS AΝΕΜΙΑ (ADDISONIAN ANEΜΙΑ): - It is a type of Vitamin B₁₂ Deficiency resulting from impaired uptake due to lack of intrinsic factor(IF), secreted from parietal cells of stomach. - Vitamin B₁₂ deficiency is most commonly seen in malabsorption - MAJOR CAUSES: - AUTO-IMMUNE DESTRUCTION OF PARIETAL CELLS: - Parietal cells are responsible for synthesis of intrinsic factor. Lack of IF prevents Vitamin B₁₂ absorption. - Patients who have/had gastrectomy become IF deficient and therefore Vitamin B₁₂ deficient. - GENETIC DEFECTI CONGENITAL PERNICIOUS AΝΕΜΙΑI Very rare ## MEGALOBLASTIC ANEMΙΑ: - Occurs due to the inhibition of DNA Synthesis during RBC Production. - The Cell Cycle cannot progress from G2 phase to Mitotic Phase - As a result, MEGALOBLASTS & IMMATURE RBCs are formed - These RBCs have fragile membrane and get broken down more rapidy leadiing to short life span of RBCS. - ASSESMENT OF VITAMIN B₁₂ - Methylmalonic aciduria - Serum B₁₂: quantitated by radioimmunoassay or by ELISA - Peripheral smear : Bone marrow morphology shows megaloblastic anemia - Homocystinuria ## Treatment of vitamin B₁₂ deficiency ### Caution - Administration of high levels of folic acid can mask vitamin B₁₂ deficiency - So, therapy is initiated with folic acid and vitamin B₁₂ until the cause of the anemia can be determined (either due to folic acid def. or vit. B₁₂ def.) ### Therapy with vitamin B₁₂: - **Route:** - Oral: High doses - (or/ IM injection of cyanocobalamin - **Duration must be continued life-long:** ## FOLIC ACID - The word folic acid is derived from latin word Folium means leaf & it is also isolated from the leafy vegetable spinach - Folic acid mainly consists of three components - Pteridine ring - PABA (p-amino benzoic acid) - Glutamic acid residue (1 to 7 residues) - Hence it is known as Pteroyl-glutamic acid ## Folate in foods - Fortified ready to eat cereals - Dark green leafy vegetables - Asparagus - Broccoli - Orange juice - Wheat germ - Legumes ## BIOCHEMICAL FUNCTION OF FOLIC ACID - Folic acid is not biologically active - The active coenzyme forms of folic acid are - Tetrahydrofolic acid (FH₄) - N5 methyl tetrahydrofolic acid (N5FH₄) - N5.N10 methylene tetrahydrofolic acid - N10 formyl tetrahydrofolate (N10 formyl FH₄) - N5 formimino tetrahydrofolate (N5 formimino FH₄) ## Folate requirements - Folate equivalents are used in RDA for dietary folate intakes, because of differences in efficiency of folate absorbtion from foods versus folic acid (supplements and fortified products). - According to the 1998 RDAS - For adults 400 µg dietary folate requirements (DFE)/d - Pregnancy 600 μg DFE/d - Lactation 500 μg DFE/d - The center for Disease Control and Prevention (CDC) for women 400 µg synthetic folic acid /day for NTD prevention. ## Absorption of Folic Acid - 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 coenzymes are produced. Folic acid is not stored in tissues. - There are different tissue specific transporters for folic acid. These high affinity binding proteins are anchored on plasma membrane - Folate-transporter complex is internalized by a non clathrin mediated endocytosis. ## Transport and storage - Blood: - Folate is found as monoglutamate - Primarily N5-mythyle-NHF - 2/3 bound to protein albumin, 1/3 free - RBC- folate level is index of longer-term (2-3mo) folate status than dose plasma. ## Tissue distribution - Total body content: - 5-10mg (50% in liver) - In tissues with: - Rapid cell division: low 5-methyl-THF and high 10 formyl-THF - Low cell division: 5-methyl-THF dominates - Folate mainly in mitochondriah (10-formyl-THF) and cytosol (5-methyl-THF) - Stored as polyglutamates ## FUNCTIONS AND MECHANISMS OF ACTION - THF functions in the body as a coenzyme in both the mitochondria and cytoplasm to accept one-carbon groups typically generated from amino acid metabolism. - These THF derivatives then serve as donors of one-carbon units in a variety of synthetic reactions, such as dispensable amino acid synthesis. - Methyl group accepted by THF is bonded to its nitrogen in position 5 or 10 or to both. ## FOLATE IN HEME SYNTHESIS - NADH H - CO₂ NH₂ - Lipoamide - No No Methylene THEA - PLP - Glycine Synthase - Red Lipoamide - NAJ’ - THPA - GLYCINE - HEME ## Another pathway: - H₂CX - HO - NH - CH - C - L-Serine - H₁ folate - Methylene - H₁ folate - Serine Hydroxymethyl Transferase (PLP) - NH - CH₂ - C - Glycine - Porphyrins ## VITAMIN B₁₂ & FOLATE IN DNA SYNTHESIS - Undine - dUMP - Thymidine - dTMP - dTTP - dATP - dCTP - dGTP - DNA - 5.10-methylene THF - DHF - Glycine - Serine - THF - Methionine - Vitamin B₁₂ - Homocysteine - 5-methyl THF - Cytoplasm - 5-methyl THF - Plasma - METHOTRAXATE - *Polyglutamation of THF (addition of 1-6 more glutamic acid residues) is essential for es retention in cell* - ① Methionine synthase and vitamin B₁₂ (in methylcobalamin form) - ② Senne hydroxymethyl transferase and vitamin B₁₂ - ③ Thymidylate synthetase - ④ Dihydrofolate reductase ## Causes of Folic Acid Deficiency: - A FOLIC POD - Alcoholism - Folic acid antagonists (e.g.methotrexate, trimethoprim) - Oral contraceptives - Low dietary intake (e.g. excessive goat milk) - Infection with giardia - Celiac Sprue - Pregnancy/Psoriasis - Old age - Dilantin aka phenytoin ## Deficiency symptoms - Identical to those for vitamin B₁₂ deficiency: - Effect of folate deficiency on cellular processes is upon DNA synthesis. - Impairment in dTMP synthesis and purine synthesis - The result is megaloblastic anemia - Deficiency during pregnancy can cause neural tube defects. ## FOLATE DEFICIENCY - **MACROCYTIC AΝΕΜΙΑ:** - During RBC generation, DNA synthesis is delayed, but protein synthesis IS continued & hence, Hb accumulates in RBC precursors - The asynchrony or dissociation between the maturity of nucleus & cytoplasm iS manifested as IMMATURE NUCLEUS & MATURE EOSINOPHILIC CYTOPLASM in BONE MARROW - RETICULOCYTOSIS is seen - HEMOLYSIS occur due to rapid destruction of RBCs in spleen leading to their short lifespan - **ASSESSMENT OF FOLATE DEFICIENCY:** - FIGLU TEST - Serum Folate level: Normal folic acid level in serum is about 20ng/mL. Measured by radioimmunoassay - Peripheral picture: Macrocytic, Hypochromic - Homocysteinuria - RBCs & WBC are approximate same sized