Heme Metabolism PDF

Heme Metabolism Dr. Zahi Damuni [email protected] Recommended Reading: Lippincott Illustrated Reviews: Biochemistry (Lippincott Illustrated Reviews Series) by Denise Ferrier 8th edition Chapter 21 1 1 Learning Objectives 1. 2. 3. 4. Describe the pathway of heme synthesis with emphasis on the po...

Heme Metabolism Dr. Zahi Damuni [email protected] Recommended Reading: Lippincott Illustrated Reviews: Biochemistry (Lippincott Illustrated Reviews Series) by Denise Ferrier 8th edition Chapter 21 1 1 Learning Objectives 1. 2. 3. 4. Describe the pathway of heme synthesis with emphasis on the porphyrias Explain the types of porphyrias & their causes Know which intermediates accumulate in the porphyrias Describe the effects of various drugs on heme biosynthesis and their clinical importance 5. Identify how blocking of one of the enzymes involved in heme biosynthesis will affect the mode of presentation of the disease, e.g., Lead Poisoning 6. Describe the pathway of heme degradation and the main types of jaundice 7. Know the differences between conjugated (direct) and unconjugated (indirect) hyperbilirubinemias (Crigler-Najjar, Gilberts, Dubin-Johnson, Rotor) 2 2 Heme Metabolism: Lecture Summary • • • • • What are hemes? Heme synthesis and its regulation Disorders related to heme synthesis: Porphyrias Heme degradation Disorders of Heme degradation: – Jaundice – Hyperbilirubinemias 3 3 Heme Heme is a tightly bound prosthetic group of hemoglobin, myoglobin, the 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 Porphyrins • • • • A group of macrocyclic organic compounds Composed of 4 modified pyrrole subunits The pyrroles are interconnected through methine/methylene bridges (=C-) The pyrroles carry side chains that vary in structure and disposition on the macrocycle 4 4 Heme https://publications.nigms.nih.gov/findings/sept13/hooked-on-heme.asp Myoglobin: An oxygen carrying protein; Journal of Experimental Biology 2004 207: 3441-3446 5 Heme and hemin are porphyrin protein molecules. The key difference between heme and hemin is that heme contains ferrous ion, whereas hemin contains ferric ion. Furthermore, hemin molecules contain chloride atoms in the chemical structure, whereas heme does not contain chloride atoms. Heme Heme is a tightly bound prosthetic group of hemoglobin, myoglobin, the 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 Porphyrins A group of macrocyclic organic compounds Composed of 4 modified pyrrole subunits The pyrroles are interconnected through methine/methylene bridges ( =C-) 5 The pyrroles carry side chains that vary in structure and disposition on the macrocycle 5 Heme Side Chains • Heme contains: – Pyrrole – Methyl – Vinyl – Propionate position is variable 6 Porphyrins A group of macrocyclic organic compounds Composed of 4 modified pyrrole subunits The pyrroles are interconnected through methine bridges ( =C-) The pyrroles carry side chains that vary in structure and disposition on the macrocycle 6 Porphyrin & Heme Synthesis • Heme is synthesized principally in the: – Bone marrow for erythrocytes – Liver for Cytochrome P-450 – Other locales are minor in amount, but support mitochondrial and other enzymes • Heme is ultimately excreted and not recycled 7 7 Heme is made where it is needed Hemoglobin 1. 2. 3. 4. 5. The body contains 800-900 gm This amount includes about 30-35 gm heme About 6 gm of hemoglobin is synthesized daily (2.4 million erythrocytes) This translates to about 250-300 mg of heme synthesized daily in the bone marrow This amount is ~ 70-80% of bodily heme synthesis Note: Erythropoiesis is the process which produces red blood cells (erythrocytes). It is stimulated by decreased O2 in circulation, which is detected by the kidneys, which then secrete the hormone erythropoietin. This hormone stimulates proliferation and differentiation of red cell precursors, in the red bone marrow. Cytochrome P-450 1. This major liver enzyme contains heme and accounts for 65% of the total liver heme synthesis 2. This protein is responsible for inactivation of drugs and other xenobiotics 3. Cytochrome P-450 has a faster turnover rate than does hemoglobin, and therefore liver heme synthesis accounts for ~15% of the bodily total 8 8 Porphyrin Synthesis • • • • Porphyrins are macrocycles of 4 smaller cycles Contain side chains in various patterns (types I, II, III, and IV). Heme uses type III, which is the only physiologically relevant pattern for people Chelate metals in the center Porphyrinogens are porphyrin precursors and are intermediate between porphobilinogen & protoporphyrin 9 9 Porphyrin Synthesis • -aminolevulinate synthase – ALAS1, liver – ALAS2, bone marrow – Both are mitochondrial – Rate-limiting step of heme synthesis • Inhibited by hemin and low iron: – Hemin decreases expression of ALAS1 in liver – Low iron inhibits ALAS2 expression in erythroid cells 10 10 Hemin is the Fe3+ Form of Heme (Fe2+) Hemin is effectively a feedback inhibitor acting on ALAS1 in the liver by repression. ALAS1 supplies heme principally for Cytochrome P450, which is inducible. ALAS1 is readily repressed and induced and has a relatively short 1-3 hr half life. Thus, its levels are readily regulated through changes in expression. Cytochrome P-450 is often induced during drug administration to metabolize the drugs and this induction leads to an increase in ALAS1 activity to supply heme. Phenobarbital, griseofulvin or hydantoins, by causing ALA synthase activity increase, induce high levels of ALA and porphobilinogen in urine and an accumulation of protoporphyrin in liver, leading to regressive processes and cirrhosis. The cause is a production of cytochrome P450, induced by the drugs, consuming heme. When the amount of heme in the liver decreases, its hemin form no longer acts as a repressor for its synthesis. 11 11 ALAS2 in the bone marrow is regulated by the availability of iron. Low iron inhibits ALAS2 production. The enzyme is produced only if there is an adequate supply of iron. Transcription of ALA synthase is tightly controlled by the presence of Fe2+‐binding elements, to prevent accumulation of porphyrin intermediates in the absence of iron. X-linked sideroblastic anemia is due to an ALAS2 deficiency. In this disorder, iron is available, but cannot be used for heme. The marrow produces ringed sideroblasts that are atypical, nucleated erythroblasts with iron granules accumulated in their mitochondria. Expression of ALAS2 is also regulated by erythropoietin. Both ALAS1 and ALAS2 are regulated by expression. [Note: The inhibition shown in the figure is not allosteric.] 12 Erythropoietin (Epo) was found to act as a concentration-dependent inducer of aminolevulinic acid (ALA) synthase and porphobilinogen (PBG) deaminase in normal human bone marrow in culture. 12 Porphyrin Synthesis • Next steps are cytosolic • 2 ALA are condensed to porphobilinogen by ALA dehydratase • This generates the characteristic side chains and the linkers that will be used to form the macrocyclic porphyrin – Note side chains and linkers 13 13 Porphyrin Synthesis • Condensation • Macrocycle Closure • Side-chain modification: – Decarboxylation to make methyl groups – Decarboxylation and oxidation to make vinyl groups • Oxidation of linkers from methyl to methine groups • Note: The oxidation steps (last two arrows in the right figure) take place in the mitochondria. 14 Hydroxymethylbilane Synthase is the same as Porphobilinogen Deaminase 14 Heme Synthesis from Protoporphyrin • Ferrochelatase – Adds Fe2+ – Mitochondrial – Sensitive to lead – one source of lead poisoning pathophysiology – Lead poisoning results in formation of zinc protoporphyrin (ZnPP) resulting in anemia 15 15 Porphyrias: Defects in Heme Synthesis • Lead poisoning • Congenital defects in Porphyrin and heme synthesis – Can be liver or bonemarrow specific • Porphyrias are generally rare • Diagram also shows which steps are mitochondrial vs cytosolic Porphobilinogen Deaminase 16 16 Porphyrias Rare, usually inherited defects in one of the enzymes of heme synthesis. One or more porphyrin precursors accumulate and increase in the blood and in the urine. Most are autosomal dominant, except erythropoietic porphyria, which is recessive. Neurological abnormalities are seen in many porphyrias because some intermediates of heme biosynthesis are neurotoxic. Abdominal pain is a frequent symptom, most likely because visceral pain fibers are stimulated by the accumulating metabolic intermediates. Cutaneous photosensitivity is typical for porphyrias in which porphyrins or porphyrinogens accumulate. Photosensitive – if a closed ring structure. Photosensitivity arises from enzyme defects that lead to accumulation of tetrapyrroles (enzymes after formation of hydroxymethylbilane). The porphyrinogens are oxidized nonenzymatically to the corresponding porphyrins. In the skin, the porphyrins become photoexcited by the action of sunlight, which leads to the formation of highly reactive and therefore toxic singlet oxygen (free radical formation). These porphyrias frequently result in unusually dark or colorful urine. 17 17 Classification of Porphyrias Hepatic Erythropoetic • Chronic • Congenital erythropoetic porphyria • Erythropoetic protoporphyria • Porphyria cutanea tarda • Acute • • • • ALA dehydratase deficiency Acute intermittent porphyria Hereditary coproporphyria Variegate porphyria 18 18 Porphyria Cutanea Tarda • • • • • Chronic, hepatic porphyria Deficiency in uroporphyrinogen decarboxylase This is the most common Porphyria Late onset – 4th or 5th decade Clinical expression is influenced by iron load, sunlight exposure, alcohol use, Hepatitis (B or C), HIV, or estrogen therapy • Symptoms: – Skin eruptions – Discolored urine normal Porphyria cutania tarda 19 19 All porphyrias result in decreased synthesis of heme • • • Therefore, ALAS activity is higher in hepatic porphyria’s because ALAS1 is regulated by heme (derepression of ALAS1) Increased enzyme synthesis and activity results in accumulation of more intermediates upstream of the deficient enzyme block. It is the accumulation of these intermediates that lead to toxicity and the major pathophysiology of the porphyrias Chronic Hepatic Porphyria • • Clinical expression dependent on hepatic iron overload, exposure to sunlight, alcohol use, and presence of hepatitis B or C or HIV Onset is late in life, in the 4th or 5th decade Acute Hepatic Porphyrias • • • • Acute attacks of GI, neuropsychiatric, and motor symptoms May be accompanied by photosensitivity Porphyrias that accumulate ALA and porphobilinogen (acute intermittent porphyria) cause abdominal pain and neuropsychiatric disturbances (anxiety to delirium) Symptoms can be precipitated by drug administration such as alcohol, barbiturates, because of p450 induction Erythropoetic Porphyrias • Skin rashes and blisters that appear early in childhood. Eventually complicated by cholestatic liver cirrhosis and progressive hepatic failure 20 20 Heme Degradation & 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 it to bile and then 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) 21 21 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 22 22 • Heme oxygenase – Release of Fe3+ – Breaking of protoporphyrin IX • Product is Biliverdin Heme Degradation: Oxidation steps Fe3+ 23 23 Heme Degradation: Oxidation steps • Biliverdin Reductase: – Reduces methine bond to methane in one place in the macrocycle • Product: Bilirubin – Carried in the blood on albumin – Delivered to the liver – Further modified in the liver 24 24 Heme Degradation: Conversion to Bilirubin Diglucuronide • After transport to liver: – Propionic acids are modified – Reacted with UDPglucuronide • Secreted as Bilirubin diglucuronide into the bile 25 25 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 • Bilirubin is readily measured • Normal serum bilirubin is 2-17 micromolar (or 1.0 mg/dL) • Jaundice occurs with > 40 micromolar serum bilirubin (or > 2.5 to 3 mg per dL) 26 26 Jaundice • Prehepatic Jaundice – Hemolytic anemia, thalassemia, transfusion • Hepatic Jaundice – Congenital disorders – Hepatitis, cirrhosis, cancer, drugs • Post-Hepatic Jaundice – obstructive (pale stools) – Blocked bile duct (e.g., tumor) – Gallstones • Neonatal – Incomplete maturation of bilirubin UDP-glucuronyl transferase – Treat with light (phototherapy) to make bilirubin isomers that are soluble. 27 Cholestasis is a liver disease. It occurs when the flow of bile from the liver is reduced or blocked. Bile is fluid produced by the liver that aids in the digestion of food, especially fats. When bile flow is altered, it can lead to a buildup of bilirubin. Gallstones are hardened deposits of digestive fluid that can form in the gallbladder. The gallbladder is a small, pear-shaped organ on the right side of the abdomen, just beneath the liver. The gallbladder holds the digestive fluid bile that's released into the small intestine. 27 Unconjugated Hyperbilirubinemias (Indirect Bilirubin) • • • Due to: – Hemolysis – Impaired conjugation – Hemolytic disease of the newborn Autoimmune condition where maternal antibodies cause hemolysis in the fetus Gilbert Syndrome – Mild Familial, can occur along with neonatal jaundice – Slight increase in unconjugated bilirubin – Yellow tinge of skin – 3‐12% of the population Crigler-Najjar – Reduced or no UDP-glucuronyl transferase in liver – Requires lifelong phototherapy 28 Hemolytic disease of the newborn is a severe form of anemia caused in a fetus or newborn infant by incompatibility with the mother's blood type, typically when the mother is rhesus negative and produces antibodies which attack rhesus positive fetal blood through the placenta. 28 Unconjugated Hyperbilirubinemias (indirect Bilirubin) • Neonatal jaundice – Incomplete maturation of bilirubin glucuronyl-transferase – Treat with phototherapy to make bilirubin isomers that are soluble – Can also occur from congenital disorders, hemolytic disease of the newborn, sepsis and others • Kernicterus – Bilirubin induced brain dysfunction – Can occur in newborns due to transiently low UDP-glucuronyl transferase 29 29 Conjugated Hyperbilirubinemias (Direct Bilirubin) • Dubin-Johnson syndrome – Defective secretion of bilirubin from hepatocytes into the bile – Results in dark-brown liver • Rotor syndrome – Transporter defect – Mild elevation of conjugated/direct bilirubin 30 Perivenous hepatocytes are exposed to lower oxygen tension, and lower nutrient and hormone levels, than periportal hepatocytes. Periportal hepatocytes receive both nutrientrich blood from the portal vein, and oxygen-rich blood via the hepatic artery, which contains circulating hormones. During glucose metabolism, periportal hepatocytes execute gluconeogenesis. How does Rotor syndrome cause conjugated hyperbilirubinemia? This phenomenon is a result of impaired hepatocellular storage of conjugated bilirubin that leaks into plasma causing hyperbilirubinemia. Its presenting symptom is jaundice, but Rotor Syndrome is a benign and self-limiting disorder that does not require treatment. 30 Dubin–Johnson syndrome (DJS) is a rare, autosomal recessive, benign disorder that causes an isolated increase of conjugated/ direct bilirubin in the serum. This condition is associated with a defect in the ability of hepatocytes to secrete conjugated bilirubin into the bile. Impaired biliary excretion of bilirubin glucuronides is due to a mutation in the canalicular multiple drug‐resistance protein 2 (MRP2). Usually, no treatment required. Rotor Syndrome: Autosomal recessive defect in organic anion transporting polypeptides 1B1 and 1B3 (OATP1B1/B3), which transport bilirubin and other compounds from the blood into the liver so that they can be cleared from the body. Liver cells are not pigmented. Can produce jaundice. 31 31 Summary Synthesis of heme starts from glycine and succinate and continues with a series of reactions that occur in both mitochondria and the cytosol. Defects in heme synthesis lead to porphyrias, relatively rare congenital defects in synthetic enzymes. Lead poisoning leads to anemia through effects on ferrochelatase and ALA dehydratase. Heme is oxidized to bilirubin in macrophages and conjugated in the liver for secretion into bile and excretion in the feces. Defects in heme degradation and secretion lead to jaundice, which is a symptom of hyperbilirubinemias. 32 32 Summary 33 Hepatocellular jaundice It occurs when bilirubin is unable to leave the liver cells and cannot be removed from the body by the kidneys. Hepatocellular jaundice is usually caused by liver failure, liver disease (cirrhosis), hepatitis (inflammation of the liver), or by taking certain types of medication. 33