Metabolism of Porphyrins Biochemistry notes PDF

Metabolism of porphyrins  Hemoglobin (Hb) is a hemoprotein only found in the cytoplasm of erythrocytes (ery) transports O2 and CO2 between lungs and various tissues normal concentration of Hb in the blood: adult males 135 – 175 g/L adult females 120 – 168 g/L  S...

Metabolism of porphyrins  Hemoglobin (Hb) is a hemoprotein only found in the cytoplasm of erythrocytes (ery) transports O2 and CO2 between lungs and various tissues normal concentration of Hb in the blood: adult males 135 – 175 g/L adult females 120 – 168 g/L  Structure of hemoglobin Hb is a spherical molecule consisting of 4 peptide subunits (globins) = quartenary structure Hb of adults (Hb A) is a tetramer consisting of 2 α- and 2 β-globins → each globin contains 1 heme group with a central Fe2+ ion (ferrous ion)  Heme structure Heme is a metaloporphyrine (cyclic tetrapyrrole) Heme contains: conjugated system of double bonds → red colour 4 nitrogen (N) atoms 1 iron cation (Fe2+) → bound in the middle of tetrapyrrole skelet by coordination covalent bonds methine bridge pyrrole ring  Properties of iron in heme Coordination number of iron in heme = 6 6 bonds: 4x pyrrole ring (A,B,C,D) 1x link to a protein 1x link to an oxygen In which compounds can we find a heme group? Hemoproteins Hemoglobin (Hb) Myoglobin (Mb) Cytochromes Catalases (decomposition of 2 H2O2 to 2 H2O and O2) Peroxidases  Myoglobin (Mb) is a single-chain globular protein of 153 AA, containing 1 heme group transports O2 in skeletal and heart muscle is found in cytosol within cells is a marker of myocard damage  Heme biosynthesis in bone marrow (85% of Hb) and liver (cytochromes) cell location: mitochondria / cytoplasm / mitochondria substrates: succinyl-CoA + glycine important intermediates: δ-aminolevulinic acid (= 5-aminolevulinic acid, ALA) porphobilinogen (PBG = pyrrole derivate) uroporphyrinogen III (= porphyrinogen – heme precursor) protoporphyrin IX (= direct heme precursor) key regulatory enzyme: ALA synthase  δ-aminolevulinic acid (ALA) synthesis of heme starts in mitochondria succinyl-CoA and Gly undergo a condensation → ALA reaction is catalyzed by enzyme ALA synthase  Porphobilinogen (PBG) ALA leaves the mitochondria → cytoplasm 2x ALA condense together to form porphobilinogen reaction is catalyzed by porphobilinogen synthase (ALA dehydratase)  Uroporphyrinogen → coproporphyrinogen III enzyme hydroxymethylbilane synthase catalyzes the linkage of four PBG molecules to yield uroporphyrinogen III 4 acetate residues are decarboxylated into methyl groups → coproporphyrinogen III returns to the mitochondria again  Protoporphyrinogen IX → protoporphyrin IX oxidation of protoporphyrinogen IX produces the conjugated π-electrone system of protoporphyrin IX  Final formation of heme Fe2+ is incorporated into protoporphyrin IX reaction is catalyzed by enzyme ferrochelatase  Regulation of heme biosynthesis ALA synthase is a key regulatory enzyme it is an allosteric enzyme that is inhibited by an end product - heme (feedback inhibition) requires pyridoxal phosphate as a coenzyme certain drugs and steroid hormones can increase heme synthesis Porphobilinogen synthase is inhibited by lead ions Pb2+ in case of lead poisoning. Ferrochelatase (heme synthase) can be also inhibited by Pb2+. Its activity is influenced by the availability of Fe2+ and ascorbic acid.  Summary of heme biosynthesis 1. Heme biosynthesis begins with simple molecules, succinyl CoA and glycine. 2. The first and last steps are mitochondrial; the other steps are cytosolic. 3. The first step, catalyzed by δ-ALA synthase, is rate-limiting. 4. The condensation of 2 moles of δ-ALA produces a substituted pyrrole. 5. As four pyrroles condense to form a porphyrin ring, the ordering of the side chains is regulated by a protein cofactor of uroporphyrinogen synthase, called cosynthase. 6. Subsequent modification of the side-chains gives rise to uroporphyrins first and then coproporphyrins. The distinction between these forms relates to the number of carboxyl groups on the periphery of the porphyrin ring. 7. The last step is the insertion of Fe+2 atom into the other center of the porphyrin ring. 8. The average, healthy adult produces from 5 to 6 g/day of hemoglobin, the most abundant protoporphyrin-containing protein.  Porphyrias Rare inherited defects in heme synthesis, (occasionally it’s acquired). This results in the accumulation and increased excretion of porphyrins or their precursors. Signs and symptoms result from a deficiency of the metabolic products beyond the enzymatic block or from the accumulation of metabolites behind the block. If the enzyme lesion occurs early in the enzymatic pathway before the formation of hydroxymethyl-bilane, this leads to the accumulation of ALA and PBG. Patients will complain of neuropsychiatric symptoms.  Porphyrias If the enzyme block leads to the accumulation of hydroxymethylbilane, patients will complain of photosensitivity. There are skin itches and burns (pruritis) when exposed to visible light. These symptoms are thought to be a result of the porphyrin-mediated formation of superoxide radicals from oxygen. These reactive oxygen species can oxidatively damage membranes. Biochemical Causes of Major Signs & Symptoms of Porphyrias  Treatment During acute porphyria, patients require medical support, particularly for treatment of pain and vomiting. The severity of symptoms of the porphyria can be diminished by intravenous injection of hemin and glucose, which decreases the synthesis of ALA synthase. Avoidance of sunlight and ingestion of β-carotene (a free-radical scavenger) are also helpful in porphyrias with photosensitivity.  Hemoglobin degradation In the human body approx. 100 – 200 million ery are broken down every hour. Degradation of Hb begins in ER of reticuloendothelial system (RES) of the liver, spleen, bone marrow and skin. Hb is degraded to: globins → AAs → metabolism heme → bilirubin Fe2+ → transported with transferrin and used in the next heme biosynthesis Not only Hb but other hemoproteins also contain heme groups which are degraded by the same pathway.  Properties of the free bilirubin Free bilirubin names Non- direct bilirubin Properties:1. Toxic, there is detoxication in liver. 2. It is not solved in water. 3. It is not filtrated into urine. 4. Does not react with reagent on bilirubin. 5. It is transported by blood albumin.  Futher fate of bilirubin Bilirubin (Bil) is released from RES into the blood. BUT! Bil is only poorly soluble in plasma, and therefore during transport it is bound to albumin („nonconjugated Bil“). ↓ LIVER In the hepatocytes, Bil is conjugated by 2 molecules of glucuronic acid → bilirubin diglucuronide (soluble in water, „conjugated Bil“) ↓ BILE ↓ INTESTINE Bil is reduced to urobilinogen and stercobilinogen Transport of free bilirubin by blood albumin N NH N NH  Detoxication of free bilirubin by UDP-glucuronic acid: СООН О 2 ОН О + N NH N NH ОН ОН UDP UDP-glucuronid free bilirubin  СООН СООН О О ОН ОН ОН ОН О О ОН ОН N NH N NH Bilirubin- diglucuronid (bilirubin-2) Bilirubin metabolism Heme released from senescent red blood cells can be readily oxidized by heme oxygenase to biliverdin, which is subsequently reduced to bilirubin by biliverdin reductase. Biliverdin reductase is found in all tissues under physiological conditions, but especially in reticuloendothelial macrophages of the kidney, spleen, liver and brain. Bilirubin binds to albumin and is then transported to the liver, where it is conjugated with UDP-glucuronic acid to increase its solubility in water.  Properties of conjugated bilirubin Conjugated bilirubin Names: Direct bilirubin Properties:1. Non- toxic. 2. Well solved in water 3. It is filtrated into urine, providing brown colour. 4. It is interacted with reagent on bilirubin.  In intestine: Urobilinogen and stercobilinogen can be: reabsorbed and returned to the liver (= enterohepatic circulation) b) oxidized (in the presence of O2) to pigments urobilin (orange) and stercobilin (yellow) → they are excreted in the stool Urobilinogen also appears in the urine.  Clinical correlations Determination of bilirubin (Bil) in serum Blood tests Bil reacts directly when reagents are added to the blood sample → conjugated bilirubin = direct Bil (up to 3.4 µmol/L) free Bil does not react to the reagents until alcohol (methanol) or caffeine is added to the solution. Therefore, the measurement of this type of bilirubin is indirect → unconjugated bilirubin = indirect Bil (up to 13.6 µmol/L) total bilirubin measures both unconjugated and conjugated Bil (normal value up to 17 µmol/L).  Jaundice: 1. Haemolytic 2. Hepatic 3. Obstructive 34 * Causes of Jaundice: Caused by hyperbilirubinemia (increased levels of bilirubin in the blood). Hyperbilirubinemia subsequently causes increased levels of bilirubin in the extracellular fluid. 35  * Pathophysiologic classification of Jaundice: 1. Hemolytic Jaundice. 2. Hepatic Jaundice. 3. Obstructive Jaundice (Cholestasis). 36  Pathologic Neonatal jaundice In cases where bilirubin rises higher and so jaundice persists for more than 3 weeks after birth. This neonatal hyperbilirubinemia may be due to the congenital absence of the enzymes required for bilirubin conjugation inside the liver. The most dangerous complication is brain-damage due to the deposition of bilirubin, a condition known as kernicterus leading to significant lifelong disability. 37  Hemolytic Jaundice Overproduction of unconjugated bilirubin can result from excessive hemolysis Lab investigations of hemolytic jaundice: ↑ Unconjugated bilirubin. ↑ Urobilinogen in feces and urine Hemoglobinuria (in acute intravascular hemolysis) ↑Reticulocyte counts  Increase in free bilirubin at haemolytic jaundice Blood and urine analyses at haemolytic jaundice Intensive hemolysis Blood analysis: Urine analysis: Free Bile acids - N Conjuga Free bilirubin - elevated ted bilirubin bilirubin Conjugated bilirubin- N Bile pigments- N UDP-glucur onide Diazo reaction- N ALT, AST- N Conjugated Alkaline phosphatase- N Urobilin---elevated bilirubin 5-nucleotidase- N BILE hepatocytes Gamma-GTPT -N Conjugate Blood LDH - slightly increased d bilirubin intestine  Obstructive Jaundice It is due to extrahepatic Symptoms of Obstructive Jaundice obstruction of bile ducts so conjugated bilirubin can not Patients have GI pain, reach the intestine. and nausea, and * Causes of obstruction of bile produce stools that duct: are a pale, clay color. 1. Gall bladder stones. The liver 2. Fibrous stricture of the "regurgitates" biliary ducts following conjugated bilirubin inflammatory lesions. into the blood, which 3. Tumors of Ampulla of Vater is excreted in the can compress the bile duct urine. from outside. 40  Dynamics of the increasing of the enzymes at cholestasis Enzyme activity Alkaline Phosphatase ALT AST 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Days of the disease Increase in conjugated bilirubin in blood during cholestasis Blood and urine analyses at obstructive jaundice Bone marrow Blood analysis: Free bilirubin –N Conjugated Free Conjugated bilirubin-elevated bilirubin bilirubin ALT, AST-N UDP- Alkaline phosphatase--elevated glucuron id Conjugated bilirubin 5-Nucleotidase-elevated Gamma- GTP-elevated Alkaline phosphatase LDH--N γ-GTP Urine analysis 5-nucleotidase Bile acids- elevated hepatocytes BILE Bile pigments-elevated BLOOD Diazo reaction - positive Urobilin- NONE  The causes of hepatic jaundice Cytolysis - irreversible cell injury 1. Viral hepatitis with membrane disruption and 2. Drug hepatitis exit of the enzymes to blood. 3. Toxic hepatitis Blood vessel 4. Alcohol hepatitis ALT AST Entry to the vessel of the Hepatocyte enzymes from injured cells  Dynamics of elevation of enzymes activity during hepatitis Activity of enzymes ALT AST Alkaline Phosphatase 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Days of disease  Increase of all fractions of bilirubin at hepatic jaundice Blood analysis and urine analysis at hepatic jaundice Blood analysis: Bone marrow Free bilirubin –elevated Conjugated bilirubin- elevated Conjugated Free bilirubin АLT, AST-elevated bilirubin Alkaline phosphatase- N UDP- glucuronide 5-nucleotidase- N Conjugated bilirubin Gamma-GTP -N LDH-N ALT B Bile L Urea analysis Conjugated AST O bilirubin hepatocytes Bile acids- elevated O D Bile pigments- elevated Diazo reaction ----- positive Intestine Urobilin- normal or elevated