Porphyrin Chemistry and Metabolism PDF

BIOCHEMISTRY Porphyrin Chemistry and Metabolism Dr. Lorenzo PORPHYRIN This just shows the  F...

BIOCHEMISTRY Porphyrin Chemistry and Metabolism Dr. Lorenzo PORPHYRIN This just shows the  From the Greek word which means “purple” absorption curve of  Has the ability to bind other atoms via porphyrin. The Nitrogen distinguishing feature  Heterocyclic macrocycles of porphyrin ring in the  Composed of four modified pyrrole subunits curve is called the interconnected at their alpha carbon atoms via Soret Band which is methyne bridges (=CH-) around 400 nm  Picture on the left is a porphyrin Picture on the Right:  This is due to the double bonds joining the Porphyrins have 4 pyrrole pyrrole rings subunits (numbered) and 4  Often used to detect small amounts of free Fluoresce porphyrins methyne bridges (boxed in  Porphyrins dissolved in strong mineral red) which are designated as α, acids/organic solvents illuminated by UV light ß, γ, and δ. The pyrrole emit STRONG RED FLUORESCENE subunits have a common  Fe-containing: center which is Nitrogen o Hemoglobin o Myoglobin Ability to form o Cytochrome complexes with  Heme Ability to form o Catalase METAL IONS bound to o One of the best known complexes o Peroxidase NITROGEN atom of porphyrins  Mg-containing: pyrrole rings o It is the pigment in red o Chlorophyll blood cells  Co-containing: o Cofactor of the protein o Vitamin B12 hemoglobin HEMOPROTEINS/METALLOPROTEINS: CLASSIFICATION/TYPES OF PORPHYRINS Type Description With completely symmetric arrangement of Acetate (A), Type I Porphyrin Propionate (P), and Methylene (M) substituents With asymmetric arrangement of A, P, and M substituents Type III Porphyrin Most abundant type Also known as Series 9 (Hemoglobin is known as Protoporphyrin IX) GENERAL CHARACTERISTICS OF PORPHYRINS Property Characteristic Example  Chlorophyll  Absorption curve for o Responsible for the green pigment seen in leaves a solution of o Contains Magnesium bound to the nitrogen porphyrin in 5% HCl Absorption spectrum  There will be a sharp  Heme in both the visible and absorption band o Positioned at the center of the structure is Iron Colored the ultraviolet near 400 nm spectrum is of great o In this Iron atom is where the oxygen is attached as an (distinguishing value feature of the axial ligand porphyrin ring; the o Component of the enzyme called cytochrome P450 band is termed Soret Band) #GrindNation “Strength In Knowledge” BESHYWAP 1  BIOCHEMISTRY Porphyrin Chemistry and Metabolism Dr. Lorenzo Picture below is the difference of Chlorophyll and Heme: BIOSYNTHESIS OF PORPHYRIN AND HEME  Cytochrome P450 (CYP) o An enzyme found in the liver o Unlike hemoglobin, to which a molecule of oxygen binds, it is the highly reactive single atom of oxygen that binds to the iron located in the center of CYP o When a CYP sees a potential target compound, the Picture on Above: Overview of the Synthesis of Porphyrin and Heme enzyme oxidizes it by transferring its oxygen atom to it  Generally it happens in the Mitochondria and Cytosol  The “committed step” in porphyrin biosynthesis is the formation of δ-aminolevulinate (ALA)  85% of heme synthesis occurs in the erythroid precursor cells in the bone marrow, the rest occur in hepatocytes Succinyl CoA + Glycine  α-amino-ß ketoadipate  This happens in the Mitochondria  Enzyme: ALA Synthase which is the rate-limiting enzyme  Cofactor: Pyridoxal phosphate which is needed to “activate” glycine  This is a condensation reaction  Vitamin B12 o Contains a structure called Corrin o Corrin is an essential compound in the synthesis of nucleic acids o At the core of the Corrin skeleton sits a cobalt atom, a relatively rare entry for biological molecules α-amino-ß ketoadipate  ALA  Enzyme: ALA Synthase which is the rate-limiting enzyme  This is a decarboxylation reaction  This is the committed step Continued next page….. #GrindNation “Strength In Knowledge” BESHYWAP 2  BIOCHEMISTRY Porphyrin Chemistry and Metabolism Dr. Lorenzo 2 ALA  Porphobilinogen (PBG) Coproporphyrinogen III  Protoporphyrinogen III  2 molecules of ALA are condensed by ALA dehydratase to form 2 H2O  This conversion happens in the mitochondria & PBG  Enzyme: Coproporphyrinogen oxidase o ALA dehydratase is a zinc-containing enzyme o Catalyzes the decarboxylation and oxidation of two o Inhibited by Lead propionic side chains  Reaction occurs in the cytosol o Is only able to act on coproporphyrinogen III that’s why type I do not generally occur in nature  Protoporphyrinogens are colorless, containing 6 extra hydrogen atoms as compared with the corresponding colored porphyrins Protoporphyrinogen III  Protoporphyrin III  Enzyme: Protoporphyrin oxidase 4 Porphobilinogen  Hydroxymethylbilane (HMB)  Condensation of 4 molecules of PBG in a head-to-tail manner to form cyclic tetrapyrrole (HMB) by uroporphyrinogen I synthase (other names: PBG deaminase or HMB synthase) Protoporphyrinogen III  Heme  Enzyme: Ferrochelatase (other name: heme synthase) o Action: incorporates ferrous iron into protoporphyrin  Heme acts as a negative regulator of ALAS I Porphobilinogen – cyclic tetrapyrrole Hydroxymethylbilane – linear tetrapyrrole HMB  Uroporphyrinogen I or HMB  Uroporphyrinogen III Summary:  HMB can cyclize spontaneously to form uroporphyrinogen I or…  It can be converted to uroporphyrinogen III o Enzyme: Uroporphyrinogen III Synthase Uroporphyrinogen I  Coproporphyrinogen I or Uroporphyrinogen III  Coproporphyrinogen III  Enzyme: Uroporphyrinogen decarboxylase  Via decarboxylation of all the acetate groups which change them to methyl (M) substituents #GrindNation “Strength In Knowledge” BESHYWAP 3  BIOCHEMISTRY Porphyrin Chemistry and Metabolism Dr. Lorenzo Mitochondrial Enzymes Cytosolic Enzymes  Effects of Drugs and Steroids ALA Synthase ALA Dehydratase o Most drugs which are metabolized by liver utilizes the Coproporphyrinogen Oxidase Uroporphyrinogen I Synthase enzyme cytochrome p450 Protoporphyrinogen Oxidase Uroporphyrinogen III Synthase o Because cytochrome p450 uses Heme; there would be ↓ Ferrochelatase Uroporphyrinogen Decarboxylase Heme = ↑ ALA Synthesis (De-repression signal) Exercise: PORPHYRIA 1. What is the rate-limiting enzyme? ALA Synthase  Collection of disorders of Heme synthesis 2. Where is the rate-limiting reaction happening? Mitochondria  Results from separate enzyme deficiency in the pathway 3. What is the precursor pyrrole? Porphobilinogen (PBG)  Not prevalent and can be genetic or acquired 4. How many PBG do you need to form a linear tetrapyrrole? 4 5. What is your linear tetrapyrrole called? Hydroxymethylbilane (HMB)  Are inherited in autosomal dominant manner except Congenital 6. HMB cyclizes but it also uses an enzyme, what is it called? erythropoietic porphyria (inherited in recessive manner) Uroporphyrinogen Synthase  Can be classified on the basis of the organs or cells that are most 7. What is the direct precursor of heme? Protoporphyrin affected, particularly on organs or cells in which synthesis of heme 8. If you lack ALA synthase, will you have porphyria? No particular 9. What will you be having if you lack ALA synthase? Anemia, because you o Bone marrow – synthesizes hemoglobin would not be able to form heme o Liver – synthesizes cytochrome P450  One classification of Porphyrias is to designate them as HEME SYNTHESIS predominantly either erythropoietic or hepatic  Heme synthesis occurs in most mammalian cells with the exception (Summary of Porphyrias next page….) of mature erythrocytes  Signs and Symptoms of Porphyria may result from a deficiency of o It does not occur on mature RBC because it has no metabolic products beyond the enzymatic block or an accumulation mitochondria, hence no mitochondrial enzymes of metabolites behind the enzymatic block  Approximately 85% occurs in erythroid precursor cells in the bone marrow Enzyme Involved Class  Majority of the remainder in hepatocytes ALA Synthase Erythropoietic ALA Dehydratase Hepatic ALA Synthase is the rate-limiting enzyme in hepatic biosynthesis of heme. PBG Deaminase Hepatic It has 2 types: Uroporphobilinogen III Synthase Erythropoietic Uroporphyrinogen Decarboxylase Hepatic Types of ALA Synthase Coproporphyrinogen Oxidase Hepatic Hepatic form, a regulatory enzyme that catalyzes the Protoporphyrinogen Oxidase Hepatic ALAS1 rate-limiting reaction in the synthesis of heme in the liver Ferrochelatase Erythropoietic ALAS2 Erythroid form DIAGNOSIS AND TREATMENT OF PORPHYRIAS Diagnosis Physiologic Regulation of Heme Synthesis  Clinical History  Substrate Availability  Family History o Iron (II) must be available for ferrochelatase  Physical Examination o Ferrochelatase is the enzyme that incorporates Iron to  Laboratory Tests (Plasma Fluorescence included) Porphyrin in the final step of Heme synthesis, because of Treatment that, iron must be always available for the reaction to occur  Hematin (a hydroxide of heme, given IV to repress ALAS1)  Feedback Regulation  Pain medication o Heme act as a negative regulator of the synthesis of ALAS1  Propranolol (to control palpitations) o When there is sufficient or increased amount of Heme,  Sedatives (to lessen anxiety) there would be a decrease in the enzyme needed for the Other Treatment synthesis of Heme in the liver (Repression signal)  Chloroquine o ↑ Heme = ↓ ALAS1 Synthesis  Fluids & Glucose (to boost carbohydrate levels, which helps limit  Subcellular Regulation the production of porphyrins) o ALA Synthase  synthesized in the cytoplasm   Removal of blood (phlebotomy) transported to mitochondria  ß-carotene supplements (lessen production of free radicals) o When the enyzyme ALA Synthase is already in the Depending on the Type of Porphyria, the ff. can be observed: mitochondria, the substrate Succinyl CoA is produced  Avoidance of alcohol o The transport of ALA Synthase from cytoplasm to  Avoidance of drugs that may trigger an attack mitochondria may be regulated  Avoidance of skin injury o Remember that we need Succinyl CoA in the formation of  Use sunscreen that filter out visible light ALA, in the committed step of Porphyrin and Heme  Eating a high carbohydrate diet synthesis Prognosis  Porphyrias are life-long diseases with symptoms that come and go #GrindNation “Strength In Knowledge” BESHYWAP 4  BIOCHEMISTRY Porphyrin Chemistry and Metabolism Dr. Lorenzo TYPES OF PORPHYRIAS Enzyme Involved Porphyria Results of Laboratory Tests Major Signs and Symptoms ALA Synthase None ↓ RBC count Anemia Abdominal pain ALA Dehydratase ALA Dehydratase deficiency ↑ Urinary ALA/Coproporphyrin Neuropsychiatric Abdominal pain PBG Deaminase Acute Intermittent Porphyria ↑ Urinary ALA/PBG Neuropsychiatric Congenital Erythropoietic Porphyria - patients with this kind of Uroporphyrinogen III Synthase ↑ Urinary, Fecal & RBC Uroporphyrin I Photosensitivity condition exhibits photosensitivity and disfigurement Uroporphyrinogen Decarboxylase Porphyria Cutanea Tarda ↑ Urinary Porphyrin Photosensitivity Fecal Coproporphyrin III Coproporphyrinogen Oxidase Hereditary Coproporphyria Photosensitivity ↑ Urinary ALA, PBG, Coproporphyrin III Abdominal pain Fecal Protoporphyrin IX Protoporphyrinogen Oxidase Variegate Porphyria Neuropsychiatric ↑ Urinary ALA, PBG, Coproporphyrin III Ferrochelatase Erythropoietic Protoporphyria ↑ Fecal & RBC Protoporphyrin Photosensitivity  Enzyme lesion occurring EARLY in the pathway/prior to the formation of porphyrinogens: ↑ ALA & PBG Abdominal pain  Enzyme lesion occurring LATER in the pathway: ↑ Porphyrinogen & Porphyrin derivative Photosensitivity DEGRADATION OF HEME & HEME-CONTAINING COMPOUNDS BILIRUBIN METABOLISM  1-2 x108 per hour: amount of erythrocytes destroyed in human adult under physiologic conditions  When hemoglobin is destroyed: o Heme – iron enters the iron pool for reuse o Globin – degraded to amino acids for reuse o Porphyrin – degraded into the reticuloendothelial cells of the liver, spleen and bone marrow  The catabolism of Heme from all the Heme-proteins is carried out in the microsomal fractions of the cells  Degradation begins inside macrophages of the spleen o Remove old and damaged erythrocytes from the circulation  Heme degradation happens in response to oxidative stress  When the cells are exposed to free radicals, there is a rapid induction of the expression of the stress responsive Heme Oxygenase-1 (Hmox1), the isoenzyme that catabolizes heme 1.) Hepatic Uptake  Bilirubin – bound to serum albumin, transported into the liver 2.) Conjugation in the Liver  Bilirubin, once transported in the liver, will be conjugated with glucuronic acid to become more water soluble using the enzyme UDP-glucuronide transferase (UDPGUTF)  Certain proteins of hepatocytes such as ligandin & Y protein bind intracellular bilirubin and may prevent its efflux into the blood stream  This form of bilirubin is excreted from the liver in the bile  UDP-glucuronide transferase can be induced by drugs (e.g. Picture above: phenobarbital) and is underdeveloped in neonates which result to In the first step, heme is converted to biliverdin by the enzyme heme neonatal physiological jaundice oxygenase (HOXG) NADPH is used as the reducing agent, molecular oxygen enters the reaction, carbon monoxide (CO) is produced and the iron is released from the molecule Continued next page…. as the ferric ion (Fe3+) In the second reaction, biliverdin is converted to bilirubin by biliverdin reductase (BVR) #GrindNation “Strength In Knowledge” BESHYWAP 5  BIOCHEMISTRY Porphyrin Chemistry and Metabolism Dr. Lorenzo 3.) Secretion of Bilirubin into Bile through Bile Ductules SUMMARY OF HEME AND BILIRUBIN METABOLISM  Rate-limiting for the entire process of bilirubin metabolism  Occurs by an active transport mechanism o Via transport protein multidrug resistance-like protein 2 (MRP-2) also called multispecific organic anion transporter at plasma membrane of bile canalicular membrane and handles a number of organic anion  It is a member of the gamily of ATP- binding cassette (ABC) transporter  Transporter protein is multidrug resistance-like protein 2 (MRP-2)  Essentially ALL secreted bilirubin is conjugated  The same drugs that affect conjugation also affect secretion (coordinated functional unit) o Intestinal bacteria deconjugate bilirubin diglucuronide and convert bilirubin to urobilinogen o Some urobilinogen is absorbed by intestinal cells and transported into kidneys and excreted with urine o The remainder travels down the digestive tract & is converted to stercobilinogen; this oxidizes to stercobilin which is excreted and is responsible for color of feces Diagrammatic Representation of the 3 Major Processes Involved in the transfer of Bilirubin from Blood to Bile: UNCONJUGATED VS. CONJUGATED BILIRUBIN  Van den Bergh Reaction o Van den Bergh devised a method for quantitatively assaying the bilirubin content of the serum by the application of Ehrlich’s Test Ehrlich’s Test – test for the presence of bilirubin in urine  Ehrlich Reaction o Makes use of Diazotized Sulfanilic Acid (Ehrlich’s Diazo reagent) o Coupling of Ehrlich’s Diazo Reagent & Bilirubin will produce reddish purple azo compound Does not make use of Alcohol in the reaction of bilirubin and Diazo reagent Direct Reacting Form of bilirubin which would react without the addition of alcohol Makes use of Alcohol to provide a solution in which Indirect Reacting both bilirubin and Diazo reagent is soluble Conjugated Bilirubin Unconjugated Bilirubin Indirect (Free) Bilirubin Direct Bilirubin en route to the liver from the reticuloendothelial tissues Water soluble NOT water soluble Produced by the breakdown of Produced after hepatic conjugation heme porphyrins Requires addition of alcohol to Can react directly with the diazo initiate coupling with the diazo reagent reagent Can cross the BBB (Blood Brain Bilirubin glucuronide Barrier) into the CNS Can cause encephalopathy known as kernicterus #GrindNation “Strength In Knowledge” BESHYWAP 6  BIOCHEMISTRY Porphyrin Chemistry and Metabolism Dr. Lorenzo HYPERBILIRUBINEMIA  Occurs when the bilirubin in the blood exceeds 1 mg/dL (17.1 µmol)  Due to the production of more bilirubin than the normal liver can excrete  It may result from the failure of a damaged liver to excrete bilirubin  Tests that can distinguish the different types of jaundice (prehepatic, produced in normal amounts hepatic, posthepatic)  It may also be caused by obstruction of the excretory ducts of the o Serum bilirubin liver in the absence of hepatic damage o Urine urobilinogen o Urine bilirubin Classification of Hyperbilirubinemia o Fecal urobilinogen  Retention Hyperbilirubinemia  Laboratory tests on blood and serum are also important to o OVER production of Bilirubin distinguish between Prehepatic, Hepatic, and Posthepatic causes of  Regurgitation Hyperbilirubinemia Jaundice o REFLUX into the blood stream because of biliary tract obstruction Hyperbilirubinemia continued….. JAUNDICE Conjugated Hyperbilirubinemia Unconjugated Hyperbilirubinemia  Also known as Icterus Due to: Due to elevated amounts of  Occurs when bilirubin accumulates in the blood (app. 2-2.5 mg/dL) 1. Blockage of the hepatic unconjugated bilirubin in the blood bile duct or common bile  Diffuses into the tissues which then become yellow duct  Cholestatic Jaundice – refers to all cases of extra hepatic obstructive 2. Gallstone jaundice 3. Cancer of the head of the pancreas Jaundice Classification Jaundice based on Etiology  Because of the obstruction, Hemolytic – due to increased bilirubin diglucuronide cannot be production of bilirubin from excreted Choluric – occurs only in the presence of hyperbilirubinemia hemoglobin under conditions  Bilirubin diglucuronide causing accelerated degradation of regurgitates into the hepatic veins RBC and lymphatics Acholuric – occurs only in the Obstructive – due to block in flow  Conjugated bilirubin appears in presence of excess unconjugated the blood and urine (choleric of bile bilirubin jaundice)  Stool is Pale in color Some major causes of Jaundice: Examples Events in the blood stream Dublin-Johnson syndrome Hemolytic Anemia Prehepatic Major cause would be various forms Rotor syndrome Neonatal physiologic jaundice of hemolytic anemia Jaundice Crigler-Najjar Syndrome Type 1 Events in the liver such as various Crigler-Najjar Syndrome Type 2 Hepatic types of hepatitis or other forms of (Arias syndrome) liver disease like cancer Gilbert Syndrome Events in biliary trees Toxic Hyperbilirubinemia Major causes are obstruction of the Posthepatic common bile duct by a gallstone or CONJUGATED HYPERBILIRUBINEMIA by cancer of the head of the  Benign autosomal recessive disorder which pancreas consists of conjugated hyperbilirubinemia in childhood or adult-life Dubin-Johnson Syndrome  Cause by mutations in the gene encoding MRP-2 (protein involved in the secretion of conjugated bilirubin into the bile)  Rare benign condition characterized by Rotor Syndrome chronic conjugated hyperbilirubinemia and normal liver histology #GrindNation “Strength In Knowledge” BESHYWAP 7  BIOCHEMISTRY Porphyrin Chemistry and Metabolism Dr. Lorenzo UNCONJUGATED HYPERBILIRUBINEMIA  Important cause of unconjugated  Hepatic bilirubin UGT activity is consistently hyperbilirubinemia decreased app. 30% of normal  Unconjugated hyperbilirubinemia is usually  Decreased bilirubin UGT activity has been Hemolytic Anemia in smaller amounts (less than 4 mg/dL; less attributed to an expansion of thymine- than 68.4 µmol/L) even in the event of adenine (TA) repeats in the promoter extensive hemolysis because of the healthy  Region of the UGT-1TA gene, the principal liver’s large capacity for handling bilirubin gene encoding this enzyme  The most common cause of unconjugated  Racial variation in the number of TA repeats hyperbilirubinemia and a correlation with enzyme activity  Results from an accelerated hemolysis suggest that these polymorphisms around the time of birth and an immature Gilbert Syndrome contribute to variations in bilirubin hepatic system for the uptake, conjugation, metabolism and secretion of bilirubin  Increased proportion of bilirubin  Bilirubin UGT (UDP-glucuronysyl monoconjugates in bile reflects reduced transferase) activity is reduced, but there is transferase activity Neonatal (Physiologic) Jaundice probably reduced synthesis of the substrate  May co-exists with other liver diseases such for that enzyme UDP-glucuronide as non-alcoholic steatohepatitis;  Capable of penetrating the BBB when its unconjugated hyperbilirubinemia in these plasma concentration exceeds that which patients may be due to Gilbert syndrome can be tightly bound by albumin (20-25 and should not always be attributed to mg/dL) underlying liver diseases  This can result in a hyperbilirubinemic toxic  Can result from toxin induced liver encephalopathy, or kernicterus, which can dysfunction such as that caused by cause mental retardation chloroform, arsphenamine, carbon  Rare autosomal recessive disorder tetrachloride, acetaminophen, hepatitis  Fatal within the first 15 months of life Toxic Hyperbilirubinemia virus, cirrhosis, and amanita mushroom  Characterized by sever congenital jaundice poisoning Crigler-Najjar Syndrome  Due to the mutations in the gene encoding  Acquired disorders which are due to Type I bilirubin UGT activity in hepatic tissues hepatic parenchymal cell damage which  Hepatic bilirubin UGT activity is impairs conjugation undetectable  Has more benign course than type I  Can respond to treatment with large doses REFERENCES of phenobarbital  OLFU|2021 D2 Trans on Porphyrin Chemistry and Metabolism  Reduction of hepatic bilirubin UGT activity  Biochemistry Manual (2018) Crigler-Najjar Syndrome is incomplete  Dr. Lorenzo Recordings Type II  Servedio et al reported patients with (Arias Syndrome) Crigler-Najjar types I & II may also possess mutations in the promoter region of the UGT1A1 gene. This gene codes for the bilirubin-UGT enzyme that is impaired in Gilbert syndrome #GrindNation “Strength In Knowledge” BESHYWAP 8  BIOCHEMISTRY Porphyrin Chemistry and Metabolism Dr. Lorenzo #GrindNation “Strength In Knowledge” BESHYWAP 9