Hemoglobin metabolism and porphyrias

Hemoglobin Metabolism and Porphyrias

Heme Metabolism and PorphyriasDr. Abhishek RoyJunior Resident (3rd Year)Dept. of Biochemistry,Grant Govt. Medical College andSir J.J. Group of Hospitals, MumbaiEmail: [email protected]: @abhishekroy

1

ObjectivesTo study Heme Synthesis in detailTo understand the Heme CatabolismTo study the Biochemical Basis and Clinical Implications of Porphyrias2nd August, 2015Dr. A Roy2

Biosynthesis of -Aminolevulinate

2nd August, 2015Dr. A Roy3

2nd August, 2015Dr. A Roy4Heme Synthesis, enzymes involved and the site of synthesis at different steps.

4

Spectral Properties of Hematoporphyrins



Heme Catabolism

Since 1 g of hemoglobin yields about 35 mg of bilirubin,human adults form 250 to 350 mg of bilirubin per day

Bilirubin Metabolism in brief


Bilirubin Diglucuronide

Enzymes of Heme Biosynthesis2nd August, 2015Dr. A Roy8

5-Aminolevulinate Synthase (EC 2.3.1.37) (ALAS)Catalyzes the formation of ALA from succinyl-CoA and glycineMitochondrial and requires a cofactor of pyridoxal phosphate, which forms a Schif base with the amino group of glycine at the enzyme surface.The carbanion of the Schiff base displaces CoA from succinyl-CoA with the formation of -amino--ketoadipic acid, which is then decarboxylated to ALAThe activity of ALAS is rate limiting as long as the catalytic capacities of other enzymes in the pathway are normal.2nd August, 2015Dr. A Roy9

5-Aminolevulinic Acid Dehydratase (EC 4.2.1.24) (ALAD)ALAD (also known as porphobilinogen synthase) is a cytoplasmic enzymeCatalyzes the formation of the monopyrrole PBG from two molecules of ALA with elimination of two molecules of water.Requires zinc ions as a cofactor and reduced sulfhydryl groups at the active site and therefore is susceptible to inhibition by lead.2nd August, 2015Dr. A Roy10

Hydroxymethylbilane Synthase (EC 2.5.1.61) (HMBS)HMBS (also known as PBG deaminase) is a cytoplasmic enzymeCatalyzes the formation of one molecule of the linear tetrapyrrole 1-hydroxymethylbilane from four molecules of PBG with the release of four molecules of ammonia.Has two molecules of its own substrate: PBG, attached covalently to the apoenzyme as a prosthetic groupIs susceptible to allosteric inhibition by intermediates farther down the heme biosynthetic pathway, notably coproporphyrinogen-III and protoporphyrinogen-IX.


Uroporphyrinogen-III Synthase (EC 4.2.1.75) (UROS)Cytoplasmic enzyme that rearranges and cyclizes HMB to form uroporphyrinogen-IIIBy the rotation of zero, one, or two alternate or two adjacent pyrrole rings, it is possible to arrive at four different isomers.The enzyme rotates the D-ring via a spirane intermediate,7 producing the type III isomeran essential reaction because only this isomer contributes to heme biosynthesis.HMB is unstable, and in those porphyrias in which excess HMB accumulates, cyclization occurs nonenzymatically with the formation of the type I isomer.2nd August, 2015Dr. A Roy12

Uroporphyrinogen Decarboxylase (EC 4.1.1.37) (UROD)Last cytoplasmic enzyme in the pathwayCatalyzes the decarboxylation of all four carboxymethyl groups to form the tetracarboxylic coproporphyrinogen.Uses I and III isomers of uroporphyrinogen as substrateDecarboxylation commences on ring D and proceeds stepwise through rings A, B, and C with formation of heptacarboxylate, hexacarboxylate, and pentacarboxylate intermediates at a single active site.At high substrate concentrations, decarboxylation occurs by a random mechanism2nd August, 2015Dr. A Roy13

Coproporphyrinogen Oxidase (EC 1.3.3.3) (CPOX)CPOX, which is located in the intermembrane space of mitochondriaCatalyzes the sequential oxidative decarboxylation of the 2- and 4-carboxyethyl groups to vinyl groups to produce the more lipophilic protoporphyrinogen-IXRequires sulfhydryl groups for activity, making it a target for inhibition by metalsThe enzyme is specific for the type III isomer, so that metabolism of the I-series of porphyrins does not occur beyond coproporphyrinogen-I2nd August, 2015Dr. A Roy14

Protoporphyrinogen Oxidase (EC 1.3.3.4) (PPOX)Flavoprotein located in the inner mitochondrial membrane, catalyzes the removal of six hydrogens to form protoporphyrin-IX.This involves a three-step, six-electron flavin adenine dinucleotide (FAD)-dependent oxidation that consumes molecular oxygen.The protoporphyrin produced is the only porphyrin that functions in the heme pathway.Other porphyrins are produced by nonenzymatic oxidation and represent porphyrinogens that have irreversibly escaped from the pathway.


Ferrochelatase (EC 4.99.1.1) (FECH)FECH (also known as heme synthase) is an iron-sulfur protein located in the inner mitochondrial membrane.Inserts ferrous iron into protoporphyrin to form heme.In iron-deficient states, Zn2+ successfully competes with Fe2+ in developing red cells, so that the concentration of zinc protoporphyrin in erythrocytes increases.Integration of the final stages of erythroid hemeBiosynthesis may be facilitated by interaction between FEC and proteins involved in iron import.2nd August, 2015Dr. A Roy16

Heme biosynthesis is regulated differently in erythroid and liver cellsIn liver, heme biosynthesis must really be controlled, whereas in erythroid cells, the process is more like breaking a dam.In liver, the main control target in heme biosynthesis is ALA synthase.Heme, or its Fe(III) oxidation product hemin, controls this enzymes activity through three mechanisms:feedback inhibition, inhibition of the transport of ALA synthase (ALAS) from its site of synthesis in the cytosol to its reaction site in the mitochondrionrepression of ALAS synthesis


PorphyriaThe termporphyriais derived from theGreek,porphyra,meaning "purplepigment".Original descriptions are attributed toHippocratesThe disease was first explained biochemically byFelix Hoppe-Seylerin 1871Acute porphyrias were described by the Dutch physicianBarend Stokvisin 1889.


Porphyrinogen and Porphyrins2nd August, 2015Dr. A Roy19Porphyrinogens are reduced form of PorphyrinsPorphyrinogens are reduced forms of porphyrinsDifference- 6 hydrogensThey are unstable in-vitroSpontaneously oxidised to respective porphyrinsThey can serve as intermediates of heme synthesis in situations of low oxygen tension

Heme synthetic pathway and various porphyrias


Genetic Basis of development of Porphyrias2nd August, 2015Dr. A Roy21All Porphyrias are autosomal dominant except CEP and ADADP which are autosomal recessive.

Summary of Major findings in Porphyrias


Porphyria EpidemiologyBased on European studies, the prevalence of the most common porphyria, porphyria cutanea tarda (PCT), is 1 in 10,000The most common acute porphyria, acute intermittent porphyria (AlP), is about 1 in 20,000The most common erythropoietic porphyria, erythropoietic protoporphyria (EPP), is estimated at 1 in 50,000 to 75,000Congenital erythropoietic porphyria (CEP) is extremely rare with prevalence estimates of 1 in 1,000,000 or lessOnly 6 cases of ALAD-deficiency porphyria (ADP) are documented2nd August, 2015Dr. A Roy23

Myths and Legends explainedVampires- Blood Sucking, Intolerance to night and Act weird.Werewolves- Hairy individuals with super human strength and human killer.Porphyrias and explanation- Porphyria Cutanea Tarda- The hallmark is photosensitivity causing photomutilations and thus loss of skin around mouth lips and excessive sensitivity to light.Photomutilation can also cause disfigurement of faces with hypertrichosis in later stages. Initially it might be less, later on it might lead to hirsutism and excessive body hair.Photosensitivity may also explain scarring of cornea and skin at various parts of the body.2nd August, 2015Dr. A Roy24

Acute PorphyriasAcute Intermittent Porphyria (AIP)Alanine Deaminase Deficiency Porphyria (ADP)Hereditary Coproporphyria (HCP)Variegate Porphyria (VP)


Clinical Signs and Symptoms2nd August, 2015Dr. A Roy26

Do drugs and environment play a role?EthanolExcessive consumption of IronSmokingSulfonamidesBarbituratesTranquilizersOral ContraceptivesStressEven in predisposed individuals- Sun Exposure


Non-Acute PophyriasPorphyria Cutanea TardaPhotosensitivity, Skin Burns, Hypertrichosis/Hirsutism, Disfigurement, Scleroderma, Often associated HCVCongenital Erythropoietic porphyriaMuch more severe than PCT, often leading to death in Utero, Hydrops fetalis, Severe skin blistering and extensive scarringShortened RBC life span, HemolysisErythropoietic protoporphyriaMost common porphyria in childhood (3rd most common overall)Caused by mutation in FECH or ALAS2 gene. When ALAS2 (rarely) involved, its called X-Linked Protoporphyria.Painful photosensitivity to sunlight including light that passes through glasses.Swelling, scarring, impaired quality of life.Hallmark: PBG and ALA not produced in excess.


Adult Lab Parameters2nd August, 2015Dr. A Roy29


Types and Epidemiology2nd August, 2015Dr. A Roy31

Patterns of overproductions of Porphyrins2nd August, 2015Dr. A Roy32

A Simple SOP for Acute Porphyrias Work Up2nd August, 2015Dr. A Roy33Urine PBG+_Urine ALATotal Fecal PorphyrinsNo PorphyriaADAPAIPPlasma HPLCHCPVPProtoporphyrin

Coproporphyrin+__+


Treatment of Acute PorphyriaPrecipitating agents are to be identified and are to be withdrawnPain killers including opioids need to be administered.Anti histaminics at times may balance the excessive requirement of opioidsElectrolyte correction to counter hyponatremiaAnti emetics and dextrose if required.IV Heme (very specific)Orthotopic Hepatic Transplant Best Treatment Modality


A Simple SOP for Cutaneous Porphyria2nd August, 2015Dr. A Roy36Total Plasma Porphyrins+_Plasma HPLCNo PorphyriaPCT/HEPEPPCEP7 and 8 carboxyl Porphyrin

Protoporphyrin

Uroporphyrin ICoproporphyrin I


Treatment of Cutaneous PorphyriaPhotoprotectionHigh SPF Creams/GelsProtection from Infections(Secondary)Blood Transfusion also helps but temporarilyIron Chelators may be beneficial like Desferrioxamine, Defereprime.Oral Activated CharcoalGene Therapy and Allogenic BMT- Best Treatment Modality



Thank You

Health & Medicine

Hemoglobin metabolism and porphyrias