W1 Iron Folate B12 (Damuni).pdf

Iron, Vitamin B12, and Folate Metabolism Zahi Damuni Ph.D. Email: [email protected] Point Solutions Session ID: damuni 1 Office hours: Typically, online via https://teach.webex.com/meet/zahi.damuni and will be announced in advance Email for questions or to set up a one-on-one meeting: zahi.dam...

Iron, Vitamin B12, and Folate Metabolism Zahi Damuni Ph.D. Email: [email protected] Point Solutions Session ID: damuni 1 Office hours: Typically, online via https://teach.webex.com/meet/zahi.damuni and will be announced in advance Email for questions or to set up a one-on-one meeting: [email protected] Lippincott Illustrated Reviews in Biochemistry. 8th Ed. Chapter 28, sections on folate and vitamin B12. Chapter 29, section III.B on Iron. And, Chapter 21, section on Heme degradation. 1 Objectives 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. List the important types of anemia and identify their mechanisms in general terms. Describe the functions of iron in the human body, the way it is bound to proteins, and its distribution in tissues. Draw a schematic of intestinal iron absorption, and identify the main site of absorption, the carriers involved, and storage sites. Explain iron transport in the blood, and how cells regulate their acquisition of iron from the blood. Explain the role of hepcidin in the regulation of iron absorption, in iron overload, and in anemia of chronic disease. List important dietary sources of iron, and the situations in which iron deficiency is likely to occur. Identify iron overload and the different types of anemia based on diagnostic signs, symptoms and lab values. Describe the cause of hemochromatosis and its epidemiology. Explain why folate is required for hematopoiesis, and why its absence causes anemia. List some dietary sources of folate, and the situations in which folate deficiency is likely to occur. Give a reason why folate deficiency is a special concern during pregnancy. Give an example of how folate antagonism can be used pharmacologically. Describe dietary sources of cobalamin, its intestinal absorption, and transport in the blood. Identify the metabolites that are elevated in the blood in deficiencies of folate and B12. Describe the mechanism of B12 malabsorption, and the kinds of people who are most likely to have it. 2 2 Lecture Workflow • • • • Why Iron and B12 and Folate? – Anemia and Erythropoiesis Iron Metabolism – Uses, dietary sources – Absorption and distribution – Regulation – Related disorders: • Anemia, hyperchromia • Heme regulation and jaundice • Iron storage Folate and B12 Metabolism - Anemia – Folate Dietary needs and biochemistry – Folate Deficiency – Chemotherapy – B12 – Dietary needs – B12 biochemistry and relation to folate metabolism – Pernicious Anemia Discussion and questions https://en.wikipedia.org/wiki/Vitamin_B12_total_synthesis https://en.wikipedia.org/wiki/Iron B12 Folate 3 3 Anemia and Erythropoiesis • • • • Erythropoiesis requires proliferation of cells, heme synthesis, and DNA synthesis and therefore requires: – Iron – Folate – B12 Causes of Anemia: – Blood loss – Decreased erythrocyte production • Iron deficiency • Folate or B12 deficiency • Thalassemia • Cancer – Hemolysis Microcytic – small or immature erythrocytes – lack of iron, heme, or hemoglobin – Lead poisoning Macrocytic – Megaloblastic Anemia (folate) – Pernicious Anemia (B12) http://www.meddean.luc.edu/lumen/m eded/mech/cases/case7/image_f.htm Harvey and Ferrier, 5th Ed. 4 Anemia: Top panel shows pale erythrocytes on the left – these are iron deficient and hemoglobin deficient. The right-hand panel is normal. The bottom panels A and B: A – normal bone marrow B – Megaloblastic cells – enlarged cells and nuclei. This is indicative of megaloblastic anemia – a failure to make sufficient number of normal erythrocytes because cells do not divide normally. 4 Iron Anemia • Iron Insufficiency: Anemia – More common in women, women have higher dietary requirements. – Impairs hemoglobin synthesis and causes microcytic hypochromic anemia: Pallor, weakness, lassitude. – Caused by massive hemorrhage, chronic blood loss, menstruation, growth, or pregnancy. Häggström, Mikael (2014). "Medical gallery of Mikael Häggström 2014". WikiJournal of Medicine 1 (2). DOI:10.15347/wjm/2014.008. ISSN 2002-4436. Public Domain. 5 Main Symptoms of iron deficiency: Fatigue Weakness Pale skin (pallor) Chest pain, shortness of breath, rapid heart rate, palpitations. Headache, lightheadedness, dizziness Cold hands and feet Sore or inflamed, swollen tongue Brittle nails Restless leg syndrome Hair loss https://www.mayoclinic.org/diseases-conditions/iron-deficiencyanemia/symptoms-causes/syc-20355034 5 Lecture Workflow • • • • Why Iron and B12 and Folate? – Erythropoiesis and Anemia Iron Metabolism – Uses, dietary sources – Absorption and distribution – Regulation – Related disorders: • Anemia, hyperchromia • Heme regulation and jaundice • Iron storage Folate and B12 Metabolism - Anemia – Folate Dietary needs and biochemistry – Folate Deficiency – Chemotherapy – B12 – Dietary needs – B12 biochemistry and relation to folate metabolism – Pernicious Anemia Discussion and questions https://en.wikipedia.org/wiki/Vitamin_B12_total_synthesis https://en.wikipedia.org/wiki/Iron B12 Folate 6 6 Iron has two important properties: 1. Electron carrier in redox reactions: eFe3+ Fe2+ e- 2. Reversible oxygen binding: Fe2+ + O2 Fe2+…O2 Only ferrous iron (Fe2+) binds O2, ferric iron (Fe3+) does not. 7 7 Biochemical Roles and Function of Iron • Role of Iron – Present in many proteins as part of hemes. • Hemes: Hemoglobin, myoglobin, cytochromes – Iron-sulfur clusters; redox centers in electron transport; active sites of various proteins. – Can switch between ferrous and ferric states to facilitate reduction/oxidation and electron transfer. Myoglobin 8 Role of Iron in metabolism: Some proteins contain iron, either bound to Cys side chains (FeS proteins) or as heme (globins, cytochromes, catalase…). For example: Hemoglobin: Binds O2 in RBCs Myoglobin: Binds O2 in muscle Respiratory cytochromes: Electron carriers in respiratory chain Cytochrome P450: Enzymes of drug metabolism and steroid synthesis Many other enzymes contain iron – e.g., electron transport proteins. Heme is a tightly bound prosthetic group of hemoglobin, myoglobin, the 8 cytochrome P450, catalase, and many other proteins Heme comprises: One ferrous ion (Fe2+) in the center. Protoporphyrin IX (a tetrapyrrole ring). Thus heme + globin protein = hemoglobin Iron-sulfur complex: Often cubic or rhomboid forms of iron and sulfur (Fe4S4 or Fe2S2), though others are possible as well. These are redox centers in electron transport, for example. 8 Dietary Needs and Sources of Iron • • • • Iron from dietary sources. Recommended intake: 8-18 mg daily (m/f) Absorption and distribution is tightly regulated About 1 mg/day is absorbed. – Free (unbound or un-chelated) iron is dangerous due to catalyzing free radical formation. • Sources: – Liver, and lean meat are excellent sources of heme iron. – Beans and spinach are rich in non-heme iron. – Non-heme iron absorbed with < 10% efficiency through the DMT-1 intestinal transporter. – Heme iron is absorbed with 30% efficiency. – Heme iron is mainly in meat; non-heme iron is in vegetables. – Absorption of non-heme iron is facilitated by gastric acid and vitamin C, and reduced by phytate, tannins, and antacids. – Low iron status can occur with vegetarian diets because vegetable iron is more poorly absorbed. – Some alcohol abuse patients are at risk of iron overload. https://ridhelp.com/foods-with-iron/ 9 1. Recall the reactive oxygen species lecture where the role of iron in the Haber-Weiss reaction was described. Free iron can catalyze the formation of hydroxyl radical, the hyper-reactive ROS species. 2. We distinguish between heme-iron and non-heme iron because they are absorbed through distinct mechanisms and have distinct sources. Heme iron is absorbed more efficiently. 9 Lecture Workflow • • • • Why Iron and B12 and Folate? – Erythropoiesis and Anemia Iron Metabolism – Uses, dietary sources – Absorption and distribution – Regulation – Related disorders: • Anemia, hyperchromia • Heme regulation and jaundice • Iron storage Folate and B12 Metabolism - Anemia – Folate Dietary needs and biochemistry – Folate Deficiency – Chemotherapy – B12 – Dietary needs – B12 biochemistry and relation to folate metabolism – Pernicious Anemia Discussion and questions https://en.wikipedia.org/wiki/Vitamin_B12_total_synthesis https://en.wikipedia.org/wiki/Iron B12 Folate 10 10 Distribution and Storage of Iron • • • • • • • Iron Distribution (mg) in humans: Free iron (Fe3+ and Fe2+) is toxic because it causes hydroxyl radical formation. Fe3+ is safer. Almost all iron in the body is tightly bound to proteins. Functional iron in proteins may be either ferrous (Fe2+) or ferric (Fe3+), depending on its role or redox state. Transport across membranes is in the ferrous state (divalent cation transporters). Redox enzymes called ferroxidases cooperate with membrane transporters to change the oxidation state. Stored in cells as ferric iron in ferritin and hemosiderin. Transport in the blood as ferric iron bound to transferrin. 2500 mg Iron Key: male (blue) female (red) 1700 800 500 300 3 3 150 11 11 Distribution and Storage of Iron • Iron Trafficking – Absorbed from intestine – Circulated via transferrin – Stored in liver in ferritin – Used in the bone marrow and all other cells – Marrow uses ~ 20 mg/day – Senescent RBCs are degraded in macrophages – Iron stored there is returned to the marrow or liver Pharmaceutics 2011, 3, 12-33; doi:10.3390/pharmaceutics3010012 https://www.researchgate.net/figure/Schematic-representation-of-iron-metabolism-Under-normalconditions-the-iron-in-the_fig1_259206402 12 Iron Distribution The major stores of iron are: 1. Liver - ~ 1g 2. Reticuloendothelial macrophages – ~ 0.6 g Iron in circulation or use: 1. erythrocytes - ~ 1.8 g 2. Bone marrow ~ 0.3 g 3. Myoglobin – 0.3-0.5 g Iron Circulation: 1. A small percentage of dietary iron is taken up (typically < 10 %; about 1 mg) 2. A similar amount is lost to excretion, loss of skin, etc. 3. Iron is transported using transferrin 4. Iron is stored as ferritin (mostly) 5. Only a few mgs (~3 mg) is in circulation. 6. Iron from senescent red blood cells is stored in the reticuloendothelial macrophages in a rapidly deliverable form 7. The Bone marrow takes up ~20 mgs a day for use in making red blood cells. 12 Iron absorption and use can be regulated by erythropoietin, but hepcidin is the major regulatory hormone (not shown here) 12 Uptake of Iron from the Intestine • • • • • • • Dietary Iron is generally liberated as the Fe3+ form This is converted to the Fe2+ form for transport through the dmt-1 divalent metal transporter Stored in enterocytes in ferritin (Fe3+) Released as needed and converted to Fe2+ Fe2+ exits through ferroportin Is converted back to Fe3+ by hephaestin or by ceruloplasmin and bound to transferrin (Tf) Excess dietary iron in ferritin lost as enterocytes are sloughed off (~1 mg/day) 13 Iron Uptake in the Duodenum: Facilitated by: 1. Ascorbate 2. Amino acids 3. Citrate 4. Iron deficiency Inhibited by: 1. Phytates 2. Tannins 3. Soil 4. Iron overload 5. Antacids Competed by: 1. Lead 2. Cobalt 3. Strontium 4. Manganese 13 5. Zinc DMT1 absorbs non-heme iron as Fe2+. - Ferritin stores Fe3+ in the cell. Also, hemosiderin, a partially denatured form of ferritin. - Ferroportin brings Fe2+ across basolateral membrane - Transferrin (Tf) binds Fe3+ in the blood Duodenal Cytochrome b (DCYTB) 13 Transferrin – Iron Carrier • Transferrin • Carries 2 Fe3+ • Main iron carrier in circulation. https://cdn.rcsb.org/images/rutgers/qy/3qyt/3qyt.pdb1500.jpg 14 Transferrin Most iron in the blood is bound tightly to transferrin. Transferrin carries 2 iron atoms Transferrin is rarely fully occupied – normally around 30% of sites have iron (normal range is 20-55%). Transferrin concentration is measured as total iron binding capacity (or TIBC): how much iron can be bound by transferrin. Serum Iron is also measured. By dividing that by the total binding capacity, you get the transferrin saturation. These numbers, together, are useful for screening chronic iron overload or deficiency. https://www.mayomedicallaboratories.com/testcatalog/Clinical+and+Interpretive/34623 14 Cellular Absorption of Iron • • • • • Transferrin (Tf)-bound iron binds to receptors. Receptor-mediated transferrin endocytosis. Free iron is released, reduced, and transported to cytoplasm (DMT). Re-oxidized Iron can be stored in ferritin (as Fe3+). Iron can be translocated to mitochondria for incorporation into: – Heme – Iron-sulfur clusters THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 35, pp. 26753–26759, August 27, 2010 © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. 15 Cells acquire iron by receptor-mediated endocytosis. Note that heme and iron-containing proteins are synthesized in individual cells, so all cells require that iron be delivered to them. Iron, Cellular Absorption • Transferrin (Tf)-bound iron binds to receptors. • Receptor-mediated transferrin endocytosis. • Free iron is released, reduced, and transported to cytoplasm (DMT). • Re-oxidized Iron can be stored in ferritin (as Fe3+). • Iron can be translocated to mitochondria for incorporation into: – Heme – Iron-sulfur clusters 15 Cellular Storage of Iron: Ferritin • Ferritin • Major storage form of iron in cells • Regulated to maintain iron levels appropriately • Measured clinically for anemia and hemochromatosis Basic iron storage unit: Mineral form Iron: Brown Oxygen: Red http://www.chemistry.wu stl.edu/~edudev/LabTutor ials/Ferritin/Ferritin.html# Figure%209 https://cdn.rcsb.org/images/rutgers /mf/1mfr/1mfr.pdb1-500.jpg 16 Ferritin: Ferritin can oxidize iron to Fe3+ and stores it in a mineral form (ferric hydroxyphosphate FeO(OH) with some FeO(H2PO4)). This is enclosed by many (24) subunits in a shell around the mineral core. The red/brown structure shows a repeating unit for iron storage – there are many of these inside the shell. Release of iron from ferritin occurs by transport of ferritin to lysosomes (along with DMT1 transporters) where it is degraded, and the iron reduced and exported. Partially denatured ferritin is called hemosiderin. It is abundant when iron stores are high. The normal range for blood ferritin is: For men, 24 to 336 micrograms per liter. For women, 11 to 307 micrograms per liter. Normal values: Men 18–270 nanograms per milliliter (ng/mL) 16 Women 18–160 ng/mL Children (6 months to 15 years) 7–140 ng/mL Infants (1 to 5 months) 50–200 ng/mL Neonates 25–200 ng/mL > 1000 ng/ml indicates hemochromatosis. Note: ng/ml = mcg/L Ferritin can carry up to 4500 iron molecules in its core. The ferritin complex contains two types of chains, heavy and light. The heavy chains gather iron quickly and retain it through enzymatic ferroxidase activity, while light chains lack this enzyme activity but facilitate iron storage. 16 Lecture Workflow • • • • Why Iron and B12 and Folate? – Erythropoiesis and Anemia Iron Metabolism – Uses, dietary sources – Absorption and distribution – Regulation – Related disorders: • Anemia, hyperchromia • Heme regulation and jaundice • Iron storage Folate and B12 Metabolism - Anemia – Folate Dietary needs and biochemistry – Folate Deficiency – Chemotherapy – B12 – Dietary needs – B12 biochemistry and relation to folate metabolism – Pernicious Anemia Discussion and questions https://en.wikipedia.org/wiki/Vitamin_B12_total_synthesis https://en.wikipedia.org/wiki/Iron B12 Folate 17 17 Intracellular Regulation • Cellular Iron Regulation • In iron deficiency, cells make more transferrin receptor and less apoferritin • This is achieved by iron regulatory proteins (IRPs) that bind to iron response elements in the mRNAs when iron is scarce • These are forms of translational regulation. 18 Note – The IRP is a cytosolic form of aconitase. - IRP is Iron Regulatory Protein - the IRP is active with low intracellular iron 18 Systemic Regulation of Iron by Hepcidin • • • • • • Iron increases hepcidin Some pathological conditions (cancer or inflammation) also increase hepcidin Hepcidin is regulated transcriptionally (increased hepcidin mRNA levels with higher iron levels) Hepcidin acts to inhibit the iron exporter ferroportin in: – Intestine – Macrophages (which phagocytose senescent RBCs) – Liver Hepcidin net effect is to lower circulating iron and dietary absorption Effectively acts as feedback inhibition Iron Inflammation Cancer http://www.bloodjournal.org/content/117/17/4425?sso-checked=true 19 The liver hormone hepcidin is released when iron is high. So, hepcidin levels track iron levels. Hepcidin acts to lower circulating iron by feedback inhibition mechanisms. It limits circulating iron and iron absorption by blocking the iron exporter ferroportin. This block occurs in intestinal enterocytes, in liver stores, and in macrophages that process senescent RBCs and release the iron from the cells to the circulation. Other functions: Iron can be rate-limiting for growth of bacteria in blood. Increased hepcidin restricts iron supply in some infections and some cancers. This effect can cause anemia of chronic disease, because long-term-increased hepcidin causes iron to be trapped in macrophages and not released for use in the bone marrow. In this case, iron stores will be adequate, but transferrin saturation will be low, as will be other markers of anemia. 19 Iron Transport in the Body Iron from worn-out RBCs is recycled from macrophages in spleen, liver and elsewhere to the bone marrow. Net intestinal absorption and excretion is near zero in adults. 20 Maintenance of Iron Homeostasis: On a daily basis, as much iron is lost as is absorbed. About 1 mg out of the 10-20 mg daily requirement of iron is absorbed. A similar amount is lost to excretion along with excess dietary iron. The bone marrow uses about 20 mg a day for making fresh erythrocytes. This is delivered via transferrin from liver and spleen macrophages. A similar amount of iron (20 mg total) is delivered to the spleen and liver from senescent erythrocytes taken up by macrophages. 20 Lecture Workflow • • • • Why Iron and B12 and Folate? – Erythropoiesis and Anemia Iron Metabolism – Uses, dietary sources – Absorption and distribution – Regulation – Related disorders: • Anemia, hyperchromia • Heme regulation and jaundice • Iron storage Folate and B12 Metabolism - Anemia – Folate Dietary needs and biochemistry – Folate Deficiency – Chemotherapy – B12 – Dietary needs – B12 biochemistry and relation to folate metabolism – Pernicious Anemia Discussion and questions https://en.wikipedia.org/wiki/Vitamin_B12_total_synthesis https://en.wikipedia.org/wiki/Iron B12 Folate 21 21 Iron Deficiency: Anemia • • • • 20-25% of world’s babies have iron deficiency anemia. 42% of women worldwide and 26% of men are ‘anemic’ according to WHO. In the US, 9-11% of toddlers, adolescent girls and women of childbearing age are ‘iron deficient’. Breast milk has more bio-available iron in lactoferrin than cow's milk. • Causes – Poor nutrition – Rapid growth – Menorrhagia – Pregnancy – Acute blood loss – Blood-sucking parasites – Occult bleeding: Colon cancer? By James Heilman, MD - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?cur id=10313974 22 Normal in infants: Hematocrit of 50-60% at birth, 35-40% at 6 months. Daily allowance in US is 8 mg/day for men, 18 mg/day for menstruating women. Iron supplements are given routinely during pregnancy. Other forms of Anemia (not directly an iron deficiency) 1. Aplastic anemia: Bone marrow failure 2. Hemolytic anemia: Destruction of RBCs 3. Thalassemia: Inability to make hemoglobin α or β chains 4. Sideroblastic anemia: Inability to use iron for heme synthesis, for example in deficiency of vitamin B6 5. Megaloblastic (macrocytic) anemia: Caused by impairment of DNA synthesis 6. Anemia of renal disease: Lack of the kidney hormone erythropoietin, which is needed to stimulate the bone marrow 22 Requirements for Iron in Pregnancy • Pregnancy requires increase iron • This is a simple calculation of increased iron requirements. 23 23 Iron Overload: Hemochromatosis • • Hemochromatosis – Genetic Iron overload – Cumulative: 10-40 g, (2.5 g is typical, male) – More prevalent in older men, from northern Europe • Is exacerbated by mutations in HFE gene, which senses iron in the liver. – Affects liver, heart and endocrine glands – Increases risk for liver cirrhosis, diabetes, cardiomyopathy, and arthritis. Overload can also be due to: – Alcoholism: Alcohol increases iron absorption, frequent iron excess in liver of patients with alcoholic liver disease. – Anemia: Even if not caused by iron deficiency, anemia increases iron absorption. – Blood transfusions: Iron introduced by blood transfusions ends up as storage iron. Frequency of the C282Y HFE Mutation in Europe 24 Common genetic form in Europeans: Hemochromatosis. -Homozygosity for the C282Y mutation in the HFE gene. - The HFE gene codes for an iron sensing protein expressed on the cell surface of some cells. -The mutation ends up reducing hepcidin secretion. - More iron absorption through ferroportin. - Causes liver damage, diabetes mellitus, cardiomyopathy, anterior pituitary malfunction, arthritis, darkening of skin. - Treatment is by repeated phlebotomy. - Only 5% of homozygotes develop symptoms. - “Bantu hemosiderosis” in South Africa is caused by a mutation in the ferroportin gene 24 What does this suggest about the history of this mutation? The data suggest a recent origin with positive selection. 24 Laboratory Values for Iron Deficiency and Overload: Transferrin saturation Measure Normal Iron Deficiency Iron Load Hematocrit 41-49% Decreased Normal Hemoglobin 12-18% Decreased Normal Transferrin Saturation 20-55% Decreased Increased Serum Ferritin 1.2-30 mcg/dL Decreased Increased Transferrin Saturation: 1. Measure serum iron concentration (this is mostly transferrin-bound iron) 2. Measure Total Iron Binding Capacity (TIBC; amount of transferrin carrying capacity) 3. Calculate Transferrin Saturation = (serum iron concentration)/TIBC 25 Measurements: Hemoglobin: g/dL or percent. Transferrin saturation – this is the good indicator of iron deficiency or overload. It is determined from two measurements – total iron binding capacity (TIBC), which essentially measures the total transferrin in the blood (times 2). - the second measurement is the amount of serum iron. - the ratio of serum iron:TIBC gives the transferrin saturation. 25 Laboratory Values for Iron Deficiency and Overload: Serum Ferritin Values • Serum ferritin is an indicator of tissue iron stores. • It leaks out of dying cells in liver and elsewhere and can be measured using a radioimmunoassay. Iron Stores, mg 0 1–300 300–800 800–1000 1000–2000 Iron overload Serum Ferritin, mcg/dL <1.5 1.5–3.0 3.0–6.0 6.0–15.0 >15.0 >50–100 26 Serum ferritin – This absolute value is much lower than serum iron, which is mostly on transferrin. Serum ferritin is measured separately. Ferritin comes from sloughed off cells which release ferritin. Serum ferritin is a good indicator of iron stores and excess iron. It is used to confirm iron storage deficiency and iron storage overload. 26 Lecture Workflow • • • • Why Iron and B12 and Folate? – Erythropoiesis and Anemia Iron Metabolism – Uses, dietary sources – Absorption and distribution – Regulation – Related disorders: • Anemia, hyperchromia • Heme regulation and jaundice Folate and B12 Metabolism - Anemia – Folate Dietary needs and biochemistry – Folate Deficiency – Chemotherapy – B12 – Dietary needs – B12 biochemistry and relation to folate metabolism – Pernicious Anemia Discussion and questions https://en.wikipedia.org/wiki/Vitamin_B12_total_synthesis https://en.wikipedia.org/wiki/Iron B12 Folate 27 27 Heme Degradation and Excretion • Senescent erythrocytes taken up by macrophages • Heme converted to bilirubin in macrophages • Bilirubin travels on albumin to the liver • Liver conjugates bilirubin and sends diglucuronide to bile and intestine • Bilirubin is de-conjugated in intestine and converted to urobilinogen • Urobilinogen is: – Mostly converted by bacteria to brown stercobilin and excreted – Some is re-absorbed, oxidized in the kidney, and excreted there as urobilin (yellow) 28 Walk through Note difference between unconjugated (indirect) and conjugated (direct) and relate to indirect vs direct bilirubin. 28 Heme Degradation • Summary – Erythrocytes last about 120 days – Most lost heme is from erythrocytes (85%) with the remainder from hepatic P450 and other cytochromes – Heme is oxidized, iron released, and the macrocycle broken – Converted to bilirubin – Sent to liver for excretion in the bile as a diglucuronide adduct Fe2+ 29 29 Heme Degradation: Conversion to Bilirubin diglucuronide • After transport to the liver: – Propionic acids are modified – – Reacted with UDPglucuronide • Secreted as Bilirubin diglucuronide into the bile 30 30 Disorders of Heme Degradation Jaundice • Also called icterus: not a disease, but a common symptom. • Yellow discoloration of sclerae, nails, and skin. • Reflects increased bilirubin levels in the blood and deposition 31 Bilirubin is readily measured in serum. Normal is 2-17 micromolar (1.0 mg/dL) Jaundice occurs with > 40 micromolar Jaundice can be caused by many things: Hemolysis (prehepatic jaundice) Biliary obstruction Neonatal jaundice Congenital defects. 31 Lecture Workflow • • • • Why Iron and B12 and Folate? – Erythropoiesis and Anemia Iron Metabolism – Uses, dietary sources – Absorption and distribution – Regulation – Related disorders: • Anemia, hyperchromia • Heme regulation and jaundice Folate and B12 Metabolism - Anemia – Folate Dietary needs and Deficiency – Folate biochemistry – Chemotherapy – B12 – Dietary needs – B12 biochemistry and relation to folate metabolism – Pernicious Anemia Discussion and questions https://en.wikipedia.org/wiki/Vitamin_B12_total_synthesis https://en.wikipedia.org/wiki/Iron B12 Folate 35 35 Vitamin B9, Folate: Source and Deficiency • • • Sources: Green leafy vegetables, yeast, liver, some fruits (heat labile). Deficiency: Pregnant women; Alcohol abuse disorder patients. – Most common vitamin deficiency in the US – Mild or borderline deficiency is common • Caused by loss (pregnancy) • Poor dietary habits • Poor absorption (alcohol abuse disorder, intestinal pathology) • Or inhibition by methotrexate – Symptoms: Megaloblastic anemia Folate supplements helps prevent Spina bifida and anencephaly, two common neural tube defects 36 Vitamin B9, Folate Note ‐ Neural tube defects are among the most serious birth defects (1:400 births). They include anencephaly (absence of the brain) and spina bifida (incomplete closure of the lumbar spine). Current recommendation is that all women who might become pregnant should consume at least 400 μg of folic acid per day. Megaloblastic Anemia: Megaloblasts are oversized RBC precursors in the bone marrow. Megaloblastic changes are also seen in other tissues with rapidly dividing cells, for example intestinal epithelium. Macrocytes are oversized RBCs in the blood. These cells are formed when DNA replication cannot keep pace with cell growth, while RBC production slows down. Symptoms: Loss of appetite; Weight Loss; Weakness; Glossitis (sore tongue); Irritability. 36 Folate Structures n 5,10N-Methylene-THF Folate 5-Methyl-THF Dihydro-Folate (DHF) TetraHydro-Folate (THF) 10N-formyl-THF 37 Folate Chemistry These structures are shown so you can see the changes that folate goes through. The principal role of folate is to act as a single methyl group donor. 1. Methylene – from 5,10N-methylene-THF. 2. Methyl – from 5N-methyl-THF. 3. Formyl – from 10N-formyl-THF. 37 Folate Biochemistry Single Carbon Metabolism Purine Synthesis Methylation Of DNA/RNA Lipids, and protein Folate His Dihydrofolate Reductase (DHFR) DHF Dihydrofolate Reductase (DHFR) THF Glu + N5-Formimino-THF Serine or Glycine 1-carbon pool donor S-adenosyl methionine Thymidylate Synthase FIGLU Glycine Methionine Methionine Synthase (B12) N10-FormylTHF Homocysteine 1-carbon pool donor NH3 + CO2 5,10-methylene THF MTHFR S-adenosyl homocysteine Modified from Springer Images: Polymorphic Variation and Risk of Colorectal Cancer by Hubner, Richard A.; Houlston, Richard S. Book: Hereditary Colorectal Cancer Chapter: 8 Published: 2010-01-01 Thymidine Synthesis 5-methyl-THF (5-methyl-THF is the folate trap: The only way to regenerate THF from here is through methionine synthase) 38 Key: SAM = S-Adenosyl methionine SAH = S-Adenosyl homocysteine THF = tetrahydrofolate DHF = dihydrofolate dUMP = deoxyUridine monophosphate dTMP = deoxyThymidine monophosphate MTHFR – Methylene-tetrahydrofolate reductase FIGLU – formimino-glutamate Serine → glycine glycine → NH3 +CO2 (glycine cleavage enzyme) FIGLU is a degradation product of Histidine, formiminoglutamate; this leads to Formimino-THF 1-Carbon pool donors: Note 1: for donating methyls, either serine or glycine is used, not both at once. Note 2: Serine and glycine can be broken down in the reaction of THF to 5,10N-methylene-THF. They can also be used for the synthesis of glycine and serine; the reactions lie near equilibrium and are reversible by mass action. 38 Note 3: FIGLU is formiminoglutamate. It is formed from the breakdown of histidine. The N5-formimino-THF can be converted to either 5,10 methylene THF or N10-formyl-THF MTHFR The reaction of 5,10N-methylene-THF to 5-methyl-THF is biologically irreversible it is highly exergonic. This is why the following methionine synthase reaction is so important. Without it, folate gets trapped as 5-methyl-folate and cannot be reconverted to THF. Methionine Synthase: This enzyme uses B12 as a cofactor. It catalyzes the synthesis of methionine from homocysteine. The added methyl group comes from 5-methyl-THF The net reaction: Homocysteine + 5-methyl-THF → Methionine + THF The 5-methyl-THF trap hypothesis is that a B12 deficiency fails to regenerate THF from 5-methyl-THF. 5-Methyl-THF accumulates and creates a folate insufficiency. Thus, signs and symptoms of B12 and folate deficiency are similar. Both result in macrocytic anemia. Symptoms of macrocytic anemia need to be investigated to determine whether the deficiency is folate or B12. Note: an alternative name for methionine synthase is homocysteine methyltransferase. 38 Lecture Workflow • • • • Why Iron and B12 and Folate? – Erythropoiesis and Anemia Iron Metabolism – Uses, dietary sources – Absorption and distribution – Regulation – Related disorders: • Anemia, hyperchromia • Heme regulation and jaundice Folate and B12 Metabolism - Anemia – Folate Dietary needs and Deficiency – Folate biochemistry – Chemotherapy – B12 – Dietary needs – B12 biochemistry and relation to folate metabolism – Pernicious Anemia Discussion and questions https://en.wikipedia.org/wiki/Vitamin_B12_total_synthesis https://en.wikipedia.org/wiki/Iron B12 Folate 39 39 Folate and Methotrexate • Folate requires action by the enzyme dihydrofolate reductase (DHFR) to be turned into THF • Methotrexate is a competitive inhibitor of DHFR Folate Methotrexate 40 Methotrexate (Amethopterin) Used to treat lung cancer, breast cancer, leukemia, lymphoma and osteosarcoma. It is also used to treat various autoimmune diseases. Aminopterin is another folate analogue used as a chemotherapeutic and immune suppressant. Both work by inhibiting dihydrofolate reductase (competitive inhibitor). This prevents THF formation and prevents purine and pyrimidine biosynthesis. Rapidly dividing cells require purines and pyrimidines for DNA replication. These agents therefore target dividing cells more than non-dividing cells in G0. The severe side effects are because normal cells that undergo division are also targeted. This particularly affects the immune system and erythropoiesis. 40 Lecture Workflow • • • • Why Iron and B12 and Folate? – Erythropoiesis and Anemia Iron Metabolism – Uses, dietary sources – Absorption and distribution – Regulation – Related disorders: • Anemia, hyperchromia • Heme regulation and jaundice Folate and B12 Metabolism - Anemia – Folate Dietary needs and Deficiency – Folate biochemistry – Chemotherapy – B12 – Dietary needs – B12 biochemistry and relation to folate metabolism – Pernicious Anemia Discussion and questions https://en.wikipedia.org/wiki/Iron B12 Folate https://en.wikipedia.org/wiki/Vitamin_B12_total_synthesis 41 41 Vitamin B12 - Sources and Deficiency • Sources: – Synthesized only in bacteria, not in plants. The rarest of vitamins. – Only available from animal products: Liver, red meat, eggs, dairy, and enhanced cereals. • Deficiency: results in anemia, – Malabsorption may be common in older people. – Often malabsorption due to loss of intrinsic factor (IF) resulting in pernicious anemia – The anemia is partly because of B12’s role in salvaging methyl-THF back to THF via the methionine synthase reaction. – Later stages show neuropsychiatric symptoms – RDA is 2.4 mg, but several (2-5) mg are typically stored, thus, deficiency may not be apparent for a long time 42 Vitamin B12 (cobalamin) Pernicious Anemia: Strictly defined as the loss of B12 due to loss of intrinsic factor (IF) and poor absorption. However, it is often used to described all cases of anemia from B12 deficiency. Loss of intrinsic factor can result from autoimmune disease attacking the parietal cells of the stomach or from surgical removal of part of the stomach. 42 Biochemistry of Vitamin B12, Cobalamin • Cobalt-containing, pyrrole ring system (similar to porphyrins, but not identical) • Comes in various forms (-CN, -CH3, -deoxy-adenosyl) • Required for only two known specific reactions: – Methylmalonyl-CoA mutase – Methionine Synthase 43 Methionine synthase uses the methyl form. An alternative name for methionine synthase is 5-Methyl-THF-homocysteine methyltransferase Methylmalonyl-CoA mutase uses the deoxyadenosyl form. Cobalamin also binds well to cyanide (CN-). It is used as a treatment for potential cyanide poisoning. This can happen to people who’ve been in house fires because insulation and other construction materials release cyanide. Cyanide can be more dangerous than carbon monoxide because CO is moderated by hemoglobin while CN- doesn’t but goes more directly to complex IV. 43 Vitamin B12 - Enzyme Cofactor • Vitamin B12 is required for 2 enzymes: – Methionine Synthase – Methylmalonyl CoA mutase Branched Chain Amino Acids Threonine Methionine 44 Methyl malonyl CoA mutase (MCM) resides in the mitochondria and catalyzes the isomerization of methylmalonyl‐CoA to succinyl‐CoA. MCM is involved in the metabolism of the branched‐chain amino acids isoleucine and valine, as well as methionine, threonine, thymine and odd‐chain fatty acids. It requires the deoxyadenosyl form of B12. Methionine Synthase requires methyl-cobalamin. This enzyme uses 5methyl-THF as a substrate and ties together B12 and folate metabolism. Note: an alternative name for methionine synthase is homocysteine methyltransferase. Homocysteine is elevated in both folate deficiency and B12 deficiency, but methylmalonic acid is elevated only in B12 deficiency. Measurement of methylmalonate is diagnostic for B12 deficiency. 44 Biochemistry of Vitamin B12, Cobalamin • Absorption in the intestine and transport in the blood requires specific factors: – B12 is initially bound to transcobalamin I (TC I), secreted by the salivary glands, to protect it against stomach acid. – B12 then binds tightly to intrinsic factor, a protein secreted by the parietal cells of the stomach. – The B12/IF complex is absorbed in the ileum – In the blood, B12 is transported in complex with transcobalamin II (TC II), another protein, to be carried to the liver through the portal circulation. – Re-absorption in intestine preserves B12. – B12 lost to excretion in the bile is recovered through re-absorption in the intestine. TC I – B12 TC I from salivary secretion 45 Schilling test – Tests for absorption of vitamin B12, once normal B12 levels have been established. Transcobalamin I (TC I). This is also called haptocorrin, R-factor, or R-protein. 45 Vitamin B12 Pernicious Anemia • Pernicious Anemia: – Anemia due to poor B12 absorption by loss of intrinsic factor. – Megaloblastic with low folate – Loss of IF can be due to: • Autoimmunity • Surgical loss of stomach • Congenital deficiency • Symptoms: – Tired and weak – fatigue – Paresthesia – Glossitis – Other symptoms typically associated with anemia. 46 Pernicious Anemia: Strictly defined as the loss of B12 due to loss of intrinsic factor (IF) and poor absorption. However, it is often used to described all cases of anemia from B12 deficiency. Loss of intrinsic factor can result from autoimmune disease attacking the parietal cells of the stomach or from surgical removal of part of the stomach. Symptoms: Skin tingling – paresthesia Tongue soreness – glossitis Fatigue Can also include: Depression Low grade fever Diarrhea Dyspepsia Weight loss Neuropathy 46 Jaundice Cheilitis Eventually – cognitive impairment CNS effects may occur in absence of anemia Treatment: IM injections of cyano-cobalamin or high oral doses. Vitamin B12 Deficiency is very similar to folate, but folate does not show the neurological symptoms. 46 Summary • • • • Anemias: loss of erythrocytes from loss of Iron, vitamin B9 or vitamin B12 Iron is a major mineral constituent of the body – Poorly taken up – Carried in chelated or complexed form using carriers – Uptake and transport regulated using hepcidin-ferroportin system – Stored in ferritin, transported on transferrin – Blood loss increases dietary iron requirements Folate is an integral part of biosynthetic reactions as a methyl donor – Folate deficiency is common, particularly in pregnancy – Folate deficiency during pregnancy can cause spina bifida and anencephaly (neural tube defects) – Folate deficiency in adults causes megaloblastic anemia. Vitamin B12, cobalamin – Taken up through transcobalamin I and intrinsic factor system – Deficiency causes anemia, typically due to lack of absorption (nutritional deficiency is rare) – Loss of intrinsic factor-mediated absorption causes pernicious anemia 48 48