Heme Metabolism & Porphyrias Lecture Notes PDF

Rafat khrais Heme Metabolism & Porphyrias Prof Mohammed Hamad  Learning Objectives At the end of the lecture each student should be able to: 1. Understand the steps of heme synthesis. 3. Understand the steps of heme degradation. 4. Differentiate between direct & indirect bil...

Rafat khrais Heme Metabolism & Porphyrias Prof Mohammed Hamad  Learning Objectives At the end of the lecture each student should be able to: 1. Understand the steps of heme synthesis. 3. Understand the steps of heme degradation. 4. Differentiate between direct & indirect bilirubin. 5. Know the definition of jaundice and differentiate between the types of jaundice. 6. Types of Porphyrias  Heme Heme consist of ferrous iron (Fe2+) complexed with four nitrogens of the specific porphyrin molecule identified as protoporphyrin IX. There are three structurally distinct hemes in humans identified as heme a, heme b, and heme c Hemes are critical as the prosthetic group of: Hemoglobin used for oxygen transport Biological function enzymes as cytochromes of oxidative phosphorylation Biological function enzymes as the xenobiotic metabolizing enzymes of the cytochrome P450 family (CYP) The catalase, and tryptophan pyrrolase  Heme The iron is held in the center of the heme molecule by bonds to the four nitrogens of the porphyrin ring. A The heme (iron) can form two additional bonds, one on each side of the planar A porphyrin ring. For example, in myoglobin and hemoglobin, one of these positions is coordinated to the - side chain of a histidine residue of the globin molecule, whereas the other position is available to bind oxygen.  Heme Heme is clinically significant because a number of genetic disease states are related with deficiencies of the enzymes used in its biosynthesis and degradation. An important feature of the intermediates in heme biosynthesis and degradation, is their chromophoric character, some are colored while others are not. All heme intermediates and degradation products that end in -ogen (e.g. porphobilinogen) will be colorless -in (e.g. bilirubin) will be colored. These heme proteins are rapidly synthesized and degraded. For example, 6 to 7g of hemoglobin are synthesized each day to replace heme lost through the normal turnover of erythrocytes. Coordinated with the turnover of heme proteins is the simultaneous synthesis and degradation of the associated porphyrins, and recycling of the bound iron ions.  Biosynthesis of heme The major sites of heme biosynthesis are: The liver, which synthesizes a number of heme proteins (particularly, cytochrome P450), In the liver, the rate of heme synthesis is highly variable, responding to alterations in the cellular heme pool caused by fluctuating demands for heme proteins. The erythrocyte-producing cells of the bone marrow, which are active in hemoglobin synthesis. Heme synthesis in erythroid cells relatively constant, and is matched to the rate of globin synthesis. The initial reaction and the last three steps in the formation of porphyrins occur in mitochondria, where as the intermediate steps of the biosynthetic pathway occur in the cytosol.  Structure of Porphyrins Porphyrins are cyclic molecules formed by the linkage of four pyrrole rings through methenyl bridges. Different porphyrins vary in the nature of the side chains that are attached to each of the four pyrrole rings. For example, Uroporphyrin contains acetate (-CH2-C00-) and propionate (-CH2-CH2-COO"), Coproporphyrin is substituted with methyl (-CH3) and propionate groups. The side chains of porphyrins can be ordered around the tetrapyrrole nucleus in four different ways, designated by Roman numerals I to IV. Only type III porphyrins, which contain an asymmetric substitution on ring D are physiologically important in humans. [Note: in congenital erythropoietic porphyria , type I porphyrins, which contain a symmetric arrangement of substituents are synthesized in appreciable quantities.  Structure of Porphyrins Porphyrin precursors exist in the chemically reduced form called porphyrinogens. In contrast to the porphyrins, which are colored, the porphyrinogens, such as uroporphyrinogen, are colorless. Porphyrinogens serve as intermediates between porphobilinogen and protoporphyrin in the biosynthesis of heme.  Biosynthesis of Porphyrins The synthesis of porphyrins can be divided into three stages for understanding. Stage I: Synthesis of δ-Amino Levulenic acid (δ-ALA), which occurs in mitochondria. Stage II: Synthesis of coproporphyrinogen III (major series) and coproporphyrinogen I (minor series) which occurs in cytosol. Stage III: Synthesis of protoporphyrin IX, – which occurs in mitochondria again. Reactions of Protoporphyrin IX and Heme Synthesis  A. Mitochondria and arrangir nasi succiny) A COA r' it aim X heme limiting 1st --- Step ACA dehydration - in copropophyrinogen ↑ ⑪ synthases dehydratase ant uroporphobilinogen ALA x here decarboxylase PBGPaminase hydroxymethylbilanc CPB. the needs active site cysteine. -PB6 and rate limitings... PBGYY Step uroporphobilinogen La assymetric uroporphobilinger a re Stage 1: Synthesis of δ-aminolevulinic acid (Delta ALA) All the carbon and nitrogen atoms of the porphyrin molecule are provided by two simple building blocks: glycine and succinyl CoA. Glycine and succinyl CoA condense to form ALA in a reaction catalyzed by  - Aminolevulinate (ALA) Synthase (the rate-limiting enzyme of heme synthesis). This reaction requires pyridoxal phosphate as a coenzyme, and is the rate- limiting step in hepatic porphyrin biosynthesis. Heme and hemin allosterically inhibit ALA synthase and also, repress its transcription When porphyrin production exceeds the I availability of globin, heme accumulates and is converted to hemin by the oxidation of Fe2+ to Fe3+. > [Note: erythroid cells, heme synthesis is under the control of erythropoietin and the availability of intracellular iron.]  Stage 2: Formation of porphobilinogen The dehydration of two molecules of ALA to form porphobilinogen (the precursor of ‘pyrrole’ ring) by δ-aminolevulinate dehydratase. The enzyme is a Zn-containing enzyme and requires Cu++ as a cofactor. The enzyme active site contains a required cysteine, making the enzyme sensitive to -- inactivation by lead (Pb2+) and other heavy metals, this inhibition is, in part, responsible for the elevation in ALA and the anemia seen in lead poisoning. This is a second rate-limiting enzyme, which is inhibited by ‘feedback’ inhibition by end product heme. Stage 2:Formation of Uroporphyrinogen The condensation of four molecules of porphobilinogen I and III results in the formation of - uroporphyrinogen I and III (I ; minor, III; major) The reaction requires hydroxymethylbilane synthase and uroporphyrinogen III synthase IPBG deaminase) (which produces the asymmetric uroporphyrinogen III).  Stage 2: Formation of Coproporphyrinogen I and III Decarboxylation, catalyzed by uroporphyrinogen decarboxylase of the four acetic acid ↑ side chains of the corresponding uroporphyrinogens to “methyl groups” results in coproporphyrinogens I and III (tetramethyl tetrapropionic). Uroporphyrinogen III is converted to heme by a series of decarboxylations and oxidations. Stage 3: Formation of Protoporphyrin IX Coproporphyrinogen III enters mitochondrion. Steps between coproporphyrinogen III and protoporphyrin IX are obscure. An oxidative decarboxylase system containing flavins as coenzyme (probably the enzyme system consists of more than one enzyme) converts coproporphyrinogen III to protoporphyrinogen IX. Protoporphyrinogen IX is converted to Protoporphyrin IX by another oxidase enzyme. The above steps require the presence of molecular O2 (aerobic).  Formation of Heme The introduction of Fe2+ into the center of protoporphyrin IX occurs spontaneously, but the rate is enhanced by the enzyme heme synthase (ferrochelatase) an enzyme that is inhibited by lead. E The reaction also requires ascorbic acid and cysteine as reducing agents Lead (Pb2+) acts as a competitive inhibitor of Fe2+ but does not insert into protoporphyrin IX Iron deficiency leads to insertion of Zn2+ to yield zinc protoporphyrin (ZnPP), an important clinical indicator of iron deficiency  Overview of Heme Synthesis Heme Protoporphyrin IX Succinyl CoA + Glycine ALA synthase Protoporphyrinogen IX -aminolevulinic acid Coproporphyrinogen III mitochondrial matrix cytoplasm -aminolevulinic acid Porphobilinogen Uroporphyrinogen III Coproporphyrinogen III Uroporphyrinogen I Coproporphyrinogen I Heme synthesis occurs in all cells due to the requirement for heme as a prosthetic group on enzymes and electron transport chain. By weight, the major locations of heme synthesis are the liver and the erythroid progenitor cells of the bone marrow.  Degradation of Heme After approximately 120 days in the circulation, red blood cells are taken up and degraded by the reticuloendothelial (RE) system, particularly in the liver and spleen. Approximately 85 % of heme destined for degradation comes from red blood cells, and 15 % is from turnover of immature red blood cells and cytochromes from extra erythroid tissues. There is thus a turnover of about 6 g/day of hemoglobin, which presents 2 problems. First, the porphyrin ring is hydrophobic and must be solubilized to be excreted. Second, iron must be conserved for new heme synthesis.  1. Formation of bilirubin Fet Text The first step in the degradation of heme is catalyzed by the microsomal heme oxygenase system of the RE cells. In the presence of NADPH and O2 the enzyme adds a hydroxyl group to the methenyl bridge between two pyrrole rings, with a concomitant oxidation of ferrous iron to Fe3+ (ferric). A second oxidation by the same enzyme system results in cleavage of the porphyrin ring. Ferric iron and carbon monoxide are released, resulting in the production of the green pigment biliverdin. Biliverdin is reduced, forming the red-orange bilirubin via the action of biliverdin reductase. Bilirubin and its derivatives are collectively termed bile pigments. [Note: The changing colors of a bruise reflect the varying pattern of intermediates that occur during heme degradation.]  2. Uptake of bilirubin by the liver Bilirubin is only slightly soluble in plasma and, therefore, is transported to the liver by binding covalently to albumin. [Note: Certain anionic drugs, such as salicylates and sulfonamides, can displace bilirubin from albumin, permitting bilirubin to enter the central nervous system (CNS). This causes the potential for neural damage in infants.] Bilirubin dissociates from the carrier albumin molecule and enters a hepatocyte, where it binds to intracellular proteins, particularly the protein ligandin. 3. Formation of bilirubin diglucuronide In the hepatocyte, the solubility of bilirubin is increased by the addition of two molecules of glucuronic acid. [Note: This process is referred to as conjugation.] The reaction is catalyzed by bilirubin glucuronyltransferase using UDP-glucuronic acid as the glucuronate donor. [Note: Bilirubin conjugates also bind to albumin, but much more weakly than does unconjugated bilirubin.]  4. Excretion of bilirubin into bile Bilirubin diglucuronide is actively transported against a concentration gradient into the bile canaliculi and then into the bile. This energy-dependent, rate-limiting step is susceptible to impairment in liver disease. Unconjugated bilirubin is normally NOT excreted. 5. Formation of urobilins in the intestine Bilirubin diglucuronide is hydrolyzed and reduced by bacteria in the gut to yield Urobilinogen, a colorless compound. Most of the urobilinogen is oxidized by intestinal bacteria to Stercobilin, which gives feces the characteristic brown color. However, some of the urobilinogen is reabsorbed from the gut and enters the portal blood. A portion of this urobilinogen participates in the enterohepatic urobilinogen cycle in which it is taken up by the liver, and then re-excreted into the bile. [ The remainder of the urobilinogen is transported by the blood to the kidney, where it is converted to yellow Urobilin and excreted, giving urine its characteristic color.  Jaundice Jaundice (also called icterus) refers to the yellow color of skin, nail beds, and sclerae (whites of the eyes) caused by deposition of bilirubin, secondary to increased bilirubin levels in the blood (hyperbilirubinemia). Although not a disease, jaundice is usually a symptom of an underlying disorder. The accumulation of bilirubin may be a result of defects at more than one step in its metabolism Jaundice can be classified into three major forms  Types of Jaundice 1- Hemolytic jaundice - Liver can handle 3000 mg bilirubin/day (conjugate and excrete ) - normal is 300 mg/day - Massive hemolysis of heme causes production of bil. more than can be processed - cannot be conjugated - increased bilirubin excreted into bile, urobilinogen is increased in blood and urine - unconjugated bilirubin in blood increases = jaundice 2- Obstructive jaundice - resulted from Obstruction of the bile duct - tumor or bile stones - gastrointestinal pain - nausea - pale, clay-colored stools - can lead to liver damage and increased unconjugated bilirubin - The liver "regurgitates" conjugated bilirubin into the blood (hyperbilirubinemia). - The compound is eventually excreted in the urine.  Types of Jaundice 3- Hepatocellular jaundice - Liver damage (cirrhosis or hepatitis) cause increased bilirubin levels in blood due to decreased conjugation (unconjugated bilirubin levels to increase in the blood) - Conjugated bilirubin not efficiently exported to bile so diffuses into blood - Urobilinogen is increased in the urine because hepatic damage decreases the enterohepatic circulation of this compound, allowing more to enter the blood, from which it is filtered into the urine. - The urine thus becomes dark in color, whereas stools are pale, clay color. - Plasma levels of AST (SGOT) and ALT (SGPT) are elevated, and - The patient experiences nausea and anorexia.  * Jaundice in newborns Newborn infants, particularly premature babies, often accumulate bilirubin, because the activity of hepatic bilirubin glucuronyl transferase is low at birth - reaches adult levels in about four weeks. - Elevated bilirubin, in excess of the binding capacity of albumin, can diffuse into basal ganglia and cause toxic encephalopathy (kernicterus). - Thus, newborns with significantly elevated bilirubin levels are treated with blue fluorescent light , - which converts bilirubin to more polar and, hence, water-soluble isomers. - These photoisomers can be excreted into the bile without conjugation to glucuronic acid. [Note: Crigler-Najjar syndrome is caused by a genetic deficiency of hepatic bilirubin glucuronyl transferase.]  PORPHYRIAS  Porphyrias are diseases caused by inherited (or occasionally acquired) defects in heme synthesis (deficiencies of enzymes), resulting in the accumulation and increased excretion of porphyrins or porphyrin precursors.  With the exception of congenital erythropoietic porphyria, which is a genetically recessive disease, all porphyrias are inherited as autosomal dominant disorders.  Affected individuals have an accumulation of heme precursors (porphyrins), which are toxic at high concentrations  Attacks of the disease are triggered by certain drugs, chemicals, and foods, and also by exposure to sun  PORPHYRIAS  The mutations that cause the porphyrias are heterogenous (not all are at the same DNA locus), and nearly every affected family has its own mutation.  Each porphyria results in the accumulation of a unique pattern of intermediates caused by the deficiency of an enzyme in the heme synthetic pathway.  Can be Hepatic or Erythropoietic, reflecting the two major locations of heme synthesis - hepatic can be acute or chronic  Those with tetrapyrrole intermediates show photosensitivity due to extended conjugated double bonds  - Formation of superoxide radicals  - Skin blisters, itches (pruritis)  - Skin may darken, grow hair (hypertrichosis)  Reactive oxygen species can oxidatively damage membranes, and cause the release of destructive enzymes from lysosomes. Destruction of cellular components leads to the photosensitivity]. Classification of Porphyrias Acute Porphyria 5-ALA Dehydratase Deficiency Porphyria Acute Intermittent Porphyria Hereditary Coproporphyria Variegate Porphyria Chronic Porphyria Porphyria cutanea Tarda Congenital Erythropoetic Porphyria Erythropoetic Protoporphyria Classification of Porphyrias Hepatic Porphyria 5-ALA Dehydratase Deficiency Porphyria Acute Intermittent Porphyria Porphyria cutanea Tarda Hereditary Coproporphyria Variegate Porphyria Erythropoetic Porphyria Congenital Erythropoetic Porphyria Erythropoetic Protoporphyria  Chronic Porphyrias  Porphyria cutanea tarda, - The most common porphyria, - Chronic disease of the liver and erythroid tissues. - The disease is associated with a deficiency in uroporphyrinogen Decarboxyase, but clinical expression of the enzyme deficiency is influenced by various factors such as : a. hepatic iron overload, b. exposure to sunlight, and c. the presence of hepatitis B or C, or d. HIV infections.  Clinical onset is typically during the fourth or fifth decade of life.  Porphyrin accumulation leads to cutaneous symptoms, and urine that is red to brown in natural light, and pink to red in fluorescent light.  Acute Hepatic Porphyrias  Acute hepatic porphyrias (acute intermittent porphyria, hereditary coproporphyria, and varigate porphyria) are characterized by acute attacks of gastrointestinal, neurologic/psychiatric, and cardiovascular symptoms.  Porphyrias leading to accumulation of ALA and porphobilinogens , such as acute intermittent porphyria, cause abdominal pain and neuropsychiatry disturbances. se  Symptoms of the acute hepatic porphyrias are often precipitated by administration of drugs such as barbiturates and ethanol, which induce the synthesis of the heme-containing cytochrome P450 microsomal drug oxidation system.  This further decreases the amount of available heme, which, in turn, promotes the increased synthesis of ALA synthase.  Erythropoietic Porphyrias  The erythropoietic porphyrias (congenital erythropoietic porphyria and erythropoietic protoporphyria) are characterized by skin rashes and blisters that appear in early childhood.  The diseases are complicated by : - Cholestatic liver cirrhosis and - Progressive hepatic failure.  Increased ALA synthase activity  One common feature of the porphyrias is a decreased synthesis of heme.  In the liver, heme normally functions as a repressor of ALA synthase. Therefore, the absence of this end product results in an increase in the synthesis of ALA synthase (derepression).  This causes an increased synthesis of intermediates that occur prior to the genetic block.  The accumulation of these toxic intermediates is the major pathophysiology of the porphyrias. Mitochondria PORPHYRIAS GLYCINE + SuccinylCoA Agent Orange ALA synthase -aminolevulinic acid(ALA) ALA-dehydratase ALA dehydratase Deficiency porphyria Porphobilinogen(PBG) Acute intermittent PBG deaminase porphyria hydroxymethylbilane Uroporphyrinogen III Congenital erythropoietic cosynthase porphyria uroporphyrinogen III Uroporphyrinogen Prophyria decarboxylase cutanea tarda coprophyrinogene III Coproporphyrinogen Herediatary oxidase coproporphyria Protoporphyrinogene IX Protoporphyrinogen Variegate protoporphyrin IX oxidase porphyria Ferrochelatase Erythropoietic Heme protoporphyria 33  Treatment During acute porphyria attacks, patients require medical support, particularly treatment for pain and vomiting. The severity of symptoms of the porphyrias can be diminished by : 1. Intravenous injection of hemin which decreases the synthesis of ALA synthase. 2. Avoidance of sunlight and 3. Ingestion of β-carotene (a free-radical scavenger) are also helpful.