Hemoglobin Synthesis and Metabolism PDF

Heamoglobin Synthesisis and Metabolism DR.HAROON HABIB BEIGH, PH.D ASSOCIATE PROFESSOR , ALATOO INTERNATIONAL UNIVERSITY  Hemoglobin (Hb)  Major protein in erythrocytes  large complex protein: haem (3%) + globin (97%)  tetra...

Heamoglobin Synthesisis and Metabolism DR.HAROON HABIB BEIGH, PH.D ASSOCIATE PROFESSOR , ALATOO INTERNATIONAL UNIVERSITY  Hemoglobin (Hb)  Major protein in erythrocytes  large complex protein: haem (3%) + globin (97%)  tetramer: haem-bound globin chain (1:1)  haem: porphyrin ring with iron in the center  Globins: 2 pairs of globin chains  Twisted together to expose haem on the exterior of the molecule  Heme  Fe-porphyrin prosthetic group: hydrophobic  Haem in hemoglobin, myoglobin: protoporphyrin IX + ferrous (Fe2+) at the center  Haem location: hydrophobic pocket of globin (hydrophobic interaction)  Synthesis of heme  Heme can be synthesized by almost all the tissues  Major sites of synthesis is liver and bone marrow  hemoglobin in bone marrow, heme production equal to globin synthesis in bone marrow.  variable in liver dependent on heme pool balance  Substrates mainly include succinyl-CoA, glycine, Fe2+.  Cytochrome p450 in liver  Heme biosynthesis 1.Haemoglobin -Iron porphyrin linked with globin. - Tetramer, made up of four subunits. Quaternary structure of Protein. -Reversibly combine with oxygen & transports oxygen. 5. Peroxidase 2.Myoglobin -Monomer, Structure is similar to Hb. -Iron porphyrin containing enzyme. -Present in muscles. Tertiary structure of Protein. -Storage of oxygen. -Present mainly in plants. 3.Cytochromes -It acts on hydrogen peroxide. - Iron porphyrin conjugated to protein. 6. Tryptophan Pyrrolase - Components of respiratory chain in mitochondria & - Iron porphyrin containing enzyme. transport electron. - It acts on tryptophan. - Cyto- P450 , takes part in microsomal hydroxylation. 4.Catalase -Iron porphyrin containing enzyme, present in animals. -It acts on hydrogen peroxide.  Hemoglobin biosynthesis  Immature erythrocytes in bone marrow (65% in nucleated RBCs, 35% in reticulocytes)  Fe acquisition: transferrin à mitochondria  Haem synthesis: mitochondria & cytosol  Globin synthesis: cytosol  Haem + Globin à Hb (in cytosol) ALA Synthase is the committed step of the heme synthesis pathway, & is usually rate-limiting for the overall pathway. Regulation occurs through control of gene transcription. Heme functions as a feedback inhibitor, repressing transcription of the ALA Synthase gene in most cells. Step 1 In mitochondrion COOH COOH HSCoA + CO2 H2C H2C CH2NH2 CH2 CH2 + ALA synthase C¡«SCoA COOH （ pyridoxal C O phosphate ） O CH2NH2 ① Heme synthesis begins with condensation of glycine & succinyl-CoA, with decarboxylation, to form d- aminolevulinic acid (ALA).  H O O C O H2 P C OH Pyridoxal phosphate O O (PLP) serves as coenzyme for d-Aminolevulinate  N CH3 Synthase (ALA Synthase). H P yrid ox al p h o sp h ate (P L P ) Condensation with H2C COO succinyl-CoA takes place N+ while the amino group of O HC H glycine is in Schiff base O H2 linkage to the PLP aldehyde. P C O O O  N CH3 H glycine-PLP Schiff base (aldimine) Step 2 COOH O CH2 OH porphobilinoge CH2 n HO O C ALA dehydratase O H C H 2H2O H N H N H2N H ★ The succeeding few reactions occur in the cytoplasm. one ALA condenses with another molecule of ALA to form porphobilinogen(PBG). ★the condensation involves removal of 2 molecules of water and the enzyme is ALA dehydratase. the enzyme contains zinc and is very sensitive to lead and other heavy metals. Inhibition of Porphobilinogen Synthase by Pb++ results in elevated blood ALA. High ALA is thought to cause some of the neurological effects of lead poisoning, although Pb++ also may directly affect the nervous system. COO COO ALA is toxic to the brain, perhaps due to: Similar ALA & neurotransmitter GABACH2 CH2 (g-aminobutyric acid) structures. CH2 CH2 ALA autoxidation generates reactive C O CH2 oxygen species (oxygen radicals). CH2 NH3+ NH3+ ALA GABA  COO COO CH2 Porphobilinogen CH2 CH2 (PBG) is the first pathway intermediate N H2C that includes a pyrrole H pyrrole N ring. NH3+ H Porphobilinogen (PBG) The porphyrin ring is formed by condensation of 4 molecules of porphobilinogen. Porphobilinogen Deaminase catalyzes successive PBG condensations, initiated in each case by elimination of the amino group.  COO- CH2 COO- COO- COO- COO-CH CH2 2 COO- CH2 COO- CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 COO- NH HN Enz S N N NH HN H H CH2 COO- CH2 CH2 CH2 CH2 COO-COO- CH2 COO- PBG units are added to the dipyrromethane until a linear hexapyrrole has been formed. hydroxy- COO - COO - uroporphyrinogen methylbilane III CH 2 COO - CH 2 COO - CH 2 CH 2 CH 2 CH 2 - OOC CH 2 CH 2 CH 2 COO - - OOC CH 2 CH 2 CH 2 COO - NH HN NH HN HO C NH HN C NH HN CH 2 COO - - OOC CH 2 CH 2 COO - C C CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 COO -COO - CH 2 Uroporphyrinogen III CH 2 CH 2 COO - Synthase COO - COO - Uroporphyrinogen III Synthase converts the linear tetrapyrrole hydroxymethylbilane to the macrocyclic uroporphyrinogen III.  COO - uroporphyrinogenIII protoporphyrinIX CH COO - CH2 2 CH CH CH CH3 2 2 - OOC CH2 CH2 CH2 COO - H3C CHCH2 NH HN NH N NH HN N HN - - OOCCH2 CH2 COO H3C CH3 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 - - - - COO COO COO COO - uroporphyrinogen III converted to coproporphyrinogen III - transported back into mitochondria - converted to protoporphyrinogen IX, then to protoporphyrin IX  URO COPR PROTO O  Summary 1. Major sites of heme synthesis liver and bone marrow (erythroblasts). 2. Matured red blood cells have no mitochondria, so can’t synthesize heme. 3. The substrates mainly include succinyl-CoA, glycine, Fe2+. 3. First and last 3 reactions take place in mitochondria, others in cytoplasm  Regulation of heme synthesis 1. ALA synthase Major site of regulation is at the level of ALA synthase. ① It is regulated by repression mechanism. Heme inhibits the synthesis of ALA synthesis by acting as a corepressor. The feedback regulatory effect is a typical example of end- product inhibition.  ② ALA synthase is also allosterically inhibited by hematin. When there is excss of free heme without globin chains to bind with, the Fe++ is oxidized to Fe+++ forming hematin. Hematin will inhibit ALA synthase to prevent excessive unwanted production of heme. Hematin will also inhibit the translocation of ALA synthase from the cytoplasm into the mitochondria where its substrate, succinyl CoA is formed. thus heme synthesis is inhibited till there are sufficient globin chains to bind with. ③ Lack of Vit B6 will decrease the synthesis of ALA. Drugs like INH (isonicotinic acid hydrazide) that decrease the availability of pyridoxal phosphate may also affect heme synthesis. 2. Heme synthesis may be inhibited by heavy metals. the steps catalyzed by ALA dehydratase and ferrochelatase are inhibited by lead. 3. -Erythropoietin, EPOa hormone called Kidneys also secrete erythropoietin. - Function of erythropoietin is to stimulate the production of RBC. - The kidney produces 85~95% of the body's erythropoietin. - During kidney disease or failure, not enough erythropoietin is produced to maintain normal red  Porphyria  Porphyria is a name given to a group of metabolic disorders. These disorders cause the individual to accumulate "porphyrins" or "porphyrin precursors" in their body. which in turn causes an abundance of the porphyrins.  In porphyria, the cells do not convert porphyrins to heme in a normal manner.  Porphyrias Porphyrias are genetic diseases in which activity of one of the enzymes involved in heme synthesis is decreased (e.g., PBG Synthase, Porphobilinogen Deaminase, etc…). Symptoms vary depending on ★ the enzyme ★ the severity of the deficiency ★ whether heme synthesis is affected primarily in liver or in developing erythrocytes. Biochemical causes of major sign and symptoms of porphyria 2. Photosensitivity is another common symptom. ◆formation of superoxide radicals. ◆ Skin damage may result from exposure to light. This is attributable to elevated levels of light- absorbing pathway intermediates and their degradation products.  3. porphyrins build up in the body and are excreted in the urine and stool in excessive amounts. When present in very high levels, they cause the urine to have a port wine color.  Porphyrias can be grouped into erythropoietic porphyria and hepatic porphyria - hepatic can be acute or chronic caused by hereditary or acquired defects in heme synthesis — genetic diseases: the enzymes of heme synthesis — Liver dysfunction, lead posioning  common symptom of 1. Porphyrias Occasional episodes of severe neurological symptoms ◆had acute bouts of abdominal pain and mental confusion Permanent nerve damage and even death can result, if not treated promptly.  Acute hepatic -Acute Intermittent porphyria is a result of a porphyrias deficiency of one of the enzymes(Uroporphyrinogen I synthase) in the heme biosynthesis pathway. -These deficiencies result in an accumulation of the precursors of porphyrins in the liver (delta- aminolevulinic acid, ALA and porphobilinogen, PBG).. - variagate porphyria(Protoporphyrinogen oxidase) -Hereditary coproporphyria(Coproporphyrinogen oxidase), an accumulation of porphyrins resulting in cutaneous manifestations.  -When an acute attack is confirmed, urgent treatment with an injection of human hemin. - Management includes the prevention of attacks (by avoiding causal factors) and the protection of skin from the light in cases of cutaneous manifestations  Porphyria cutanea tarda - a chronic porphyria - liver and erythroid tissues - deficiencey in uroporphyrinogen decarboxylase - often no symptoms until 4th or 5th decade clinical expression determined by many factors: - hepatic iron overload - exposure to sunlight - hepatitis B or C symptoms include: - cutaneous rashes, blisters - urine that is red to brown in natural light, or pink to red in UV light  Erythropoietic porphyrias - congenital erythropoietic porphyria (uroporphyrinogen III - synthase) erythropoietic protoporphyria (ferrochelatase) symptoms include: - skin rashes and blisters early in childhood - cholestatic liver cirrhosis and progressive liver failure Treatment for Porphyrias - Medical support for vomiting and pain - Hemin, decreases ALA synthase synthesis - Avoidance of sunlight and precipitating drugs, factors  Acquired Porphyrias - Hexochlorobenzene used as a fungicide in Turkey in 1950s - thousands of children ate bread from treated wheat - they acquired porphyria cutanea tarda due to inhibition of uroporphyrinogen decarboxylase - due to hypertrichosis - referred to locally as the “monkey children”  Acquired Porphyrias lead poisoning -inhibition of ferrochelatase, ALA dehydratase - displaces Zn+2 at enzyme active site children - developmental defects - drop in IQ - hyperactivity - insomnia - many other health problems adults - severe abdominal pain - mental confusion - many other symptoms  FATE OF RED BLOOD CELLS  Life span in blood stream is 60-120 days  RBCs are phagocytosed and/or lysed ·Normally, lysis occurs extravascularly in the reticuloendothelial system subsequent to RBC phagocytosis · Lysis can also occur intravascularly (in blood stream) Extravascular Pathway for RBC Destruction (Liver, Bone marrow, & Spleen) Phagocytosis & Lysis Hemoglobin Globin Heme Bilirubin Amino acids Fe2+ Amino acid pool Excreted  · Uptake of bilirubin by the liver is NORMAL BILIRUBIN mediated by a carrier protein (receptor) METABOLISM · On the smooth ER, bilirubin is conjugated with glucoronic acid · Glucoronic acid is the major conjugate - catalyzed by UDP glucuronyl tranferase ·“Conjugated” bilirubin is water soluble and is secreted by the hepatocytes into the biliary canaliculi · Converted to stercobilinogen (urobilinogen) (colorless) by bacteria in the gut · Oxidized to stercobilin which is colored · Excreted in feces · Some stercobilin may be re-adsorbed Prehepatic (hemolytic) jaundice  Results from excess production of bilirubin (beyond the livers ability to conjugate it) following hemolysis  Excess RBC lysis is commonly the result of autoimmune disease; hemolytic disease of the newborn (Rh- or ABO- incompatibility); structurally abnormal RBCs (Sickle cell disease).  High plasma concentrations of unconjugated bilirubin (normal concentration ~0.5 mg/dL) Intrahepatic jaundice  Impaired uptake, conjugation, or secretion of bilirubin  Reflects a generalized liver (hepatocyte) dysfunction  In this case, hyperbilirubinemia is usually accompanied by other abnormalities in biochemical markers of liver function Posthepatic jaundice  Caused by an obstruction of the biliary tree  Plasma bilirubin is conjugated, and other biliary metabolites, such as bile acids accumulate in the plasma  Characterized by pale colored stools (absence of fecal bilirubin or urobilin), and dark urine (increased conjugated bilirubin)  In a complete obstruction, urobilin is absent from the urine  Neonatal Jaundice  Common, particularly in premature infants  Transient (resolves in the first 10 days)  Due to immaturity of the enzymes involved in bilirubin conjugation  High levels of unconjugated bilirubin are toxic to the newborn – due to its hydrophobicity it can cross the blood-brain barrier and cause a type of mental retardation known as kernicterus  If bilirubin levels are judged to be too high, then phototherapy with UV light is used to convert it to a water soluble, non-toxic form If necessary, exchange blood transfusion is used to remove excess bilirubin Phenobarbital is often administered to Mom prior to an induced labor of a premature infant – crosses the placenta and induces the synthesis of UDP glucuronyl transferase Jaundice within the first 24 hrs of life or which takes longer then 10 days to resolve is usually pathological and needs to be further investigated Causes of Hyperbilirubinemia  Gilbert’s Syndrome ·Benign liver disorder · ½ of the affected individuals inherited it  Characterized by mild, fluctuating increases in unconjugated bilirubin caused by decreased ability of the liver to conjugate bilirubin ·Onset of symptoms in teens, early 20’s or 30’s · Can be treated with small doses of phenobarbital to stimulate UDP glucuronyl transferase activity  Crigler-Najjar Syndrome · Autosomal recessive · Extremely rare < 200 cases worldwide · Characterized by a complete absence or marked reduction in bilirubin conjugation · Present with a severe unconjugated hyperbilirubinemia that usually presents at birth · Condition is fatal when the enzyme is completely absent · Treated by phototherapy (10-12 hrs/day) and liver transplant by age 5 Dubin-Johnson and Rotor’s Syndromes  Characterized by impaired biliary secretion of conjugated bilirubin  Present with a conjugated hyperbilirubinemia that is usually mild