Porphyrin metabolism & porphyrias HMIM224

Objectives Understand the structure of metalloprphyrins Apply that the role of heme is dictated by the environment created by the three dimensional structure relation to protein Identify the rate limiting step and the iso-enzymes sites of production Describe the site of effect of certain drugs on heme biosynthesis and its clinical importance Identify how blocking in one of the enzyme involved in heme biosynthesis will affect the mode of presentation of the disease Identify the most common type of porphyrias & its cause Identify the heme degradation product

What are porphyrins ? Porphyrins are cyclic compounds that bind metal ions (usually Fe2+ or Fe3+) Porphyrin + Metal = Metalloporphyrin Most prevalent metalloporphyrin in humans is heme (metal here is iron ion) Heme consists of: One ferrous ion (Fe2+) in the centre Protoporphyrin IX (a tetrapyrrole ring) The role of heme is dictated by the enviroment created by the three dimensional structure of protein Heme is the prosthetic group of hemoglobin, myoglobin, cytochromes, catalase, tryptophan pyrrolase So, heme + globin protein = hemoglobin

Structure of porphyrins Porphyrins are cyclic molecules formed by 4 pyrrole (tetrapyrrole) rings linked by methenyl bridges. Different porphyrins vary in the nature of side chains that are attached to each of the 4 pyrrole rings Protoporphyrin IX contains vinyl, methyl & propionate Distribution of side chains - Side chains can be ordered around tetrapyrrole nucleus in 4 different ways designated I, II, III & IV series. Only type III porphyrins are physiologically important in humans - Protoporphyrin IX is a member of type III series Porphyrinogens are porphyrin precursors intermediate between porphobilinogen & protoporphyrin Porphobilinogen Porphyrinogens Protoporphyrins

Structure of Porphyrin Pyrrole ring Methenyl bridge Side Chains Structure of Porphyrin Determine the type of porphyrin

Structure and Properties of Iron Protoporphyrin IX propionate methyl vinyl pyrrole ring Derived from protoporphyrin IX Pattern of side chains defines isomer Binds metals: Heme- Fe2+ (ferrous) Hemin- Fe3+ (ferric) Zinc protoporphyrin (ZnPP)- Zn2+ Extended conjugation across ring system

Overview of Heme Synthesis Succinyl CoA + Glycine Protoporphyrin IX ALA synthase Protoporphyrinogen IX -aminolevulinic acid Coproporphyrinogen III mitochondrial matrix cytoplasm -aminolevulinic acid Uroporphyrinogen III Coproporphyrinogen III Porphobilinogen 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.

First & second steps of heme synthesis Biosynthesis of heme (cont.) First & second steps of heme synthesis

Biosynthesis of heme Steps: Site of biosynthesis: Liver & Bone Marrow (erythryoid producing cells) There are 2 isozymes ALA S1 (liver) & ALAS2 (erythroid) Steps: Step 1: Formation of d-amino levulinic acid (ALA): in mitochondria (Rate Controlling Step) Glycine + Succinyl CoA (nonessential amino acid) (intermediate of citric acid cycle) Enzyme: ALA Synthase Coenzyme: Pyridoxal Phosphate (PLP) d-Amino levulinic acid (ALA)

Cytochrome P450 Monooxygenase System Cytochrome P450s (CYPs) are actually a superfamily of related, heme-containing monooxygenase enzymes that participate in abroad variety of reactions.This system performs different functions in two separate locations in cells. The over-all reaction catalyzed by a cytochrome P450 enzyme is: R-H + O2+ NADPH + H+→R-OH + H2O NADP+ where R may be a steroid, drug, or other chemical. The name P450 reflects the absorbance at 450 nm by the protein. These modifications is two-fold: First, it may itself activate or inactivate a drug or Second, make a toxic compound more soluble, thus facilitating its excretion in the urine or feces.

Biosynthesis of heme (cont.) Clinical importance of first step: When heme (end product) is produced in excessive amounts, heme is converted to hemin. Hemin decreases action of ALA synthase in liver. (end product inhibition). The reverse occurs when heme biosynthesis is reduced. Drugs as grisofulvin (antifungal), hydantoin & phenobarbital (anticonvulsant) increase ALA synthase activity: as these drugs are metabolized by cytochrome p450 in liver resulting in more consumption of heme (component of cytochrome). Accordingly, heme concentration is reduced resulting in stimulation of action of ALA synthase.

First & second steps of heme synthesis Biosynthesis of heme (cont.) First & second steps of heme synthesis

Biosynthesis of heme (cont.) Step 2: Formation of porphobilinogen: 2 molecules of d-Amino levulinic acid (ALA) condense to form porphobilinogen by the enzyme ALA dehydratase. Clinical importance: ALA dehydratase enzyme is inhibited by heavy metals as lead that results in anemia. (lead poisoning). In this case: ALA in blood is elevated (lab investigation)

Biosynthesis of heme (cont.) Further steps of heme synthesis

Biosynthesis of heme (cont.) Further steps: (in mitochondria) Formation of protoporphyrin IX Then, ferrous ions (Fe2+) are introduced into protoporphyrin IX, either: simultaneously or: enhanced by ferrochelatase Clinical importance: Ferrochelatase enzyme is inhibited by lead

Summary of biosynthesis of heme Glycine + Succinyl CoA Enzyme: ALA Synthase STEP 1 PLP d-Amino levulinic acid (ALA) Enzyme: ALA dehydratase. STEP 2 porphobilinogen FURTHER STEPS Protoporphyrin IX Ferrous ion (Fe2+ ) introduction of iron Enzyme: ferrochelatase heme

Porphyrias Porphyria are rare inherited defects in heme synthesis. An inherited defect in an enzyme of heme synthesis results in accumulation of one or more of porphyrin precursors depending on location of block of the heme synthesis pathway. These precursors increase in blood & appear in urine of patients. Porphyria means purple colour caused by pigment-like porphyrins in urine of patients. (Diagnosed by lab investigation) Most porphyrias show a prevalent autosomal dominant pattern, except congenital eythropoietic porphyria, which is recessive

Porphyrias (cont.) Clinical manifestations of porphyrias: Two types of porphyrias: erythropoietic (bone marrow) & hepatic Hepatic porphyrias are: acute & chronic porrphyrias Generally, individuals with an enzyme defect prior to the synthesis of the tetrapyrroles manifest abdominal and neuropsychiatric signs Those with tetrapyrrole intermediates show photosensitivity with formation of reactive oxygen species (ROS) that damage membranes by oxidation resulting in the following effects: - Skin blisters, itches (pruritis) - Skin may darken, grow hair (hypertrichosis)

Porphyria Cutanea Tarda Chronic hepatic porphyria The most common type of porphyria a deficiency in uroporphyrinogen decarboxylase Clinical expression of the enzyme deficiency is influenced by various factors, such as exposure to sunlight, the presence of hepatitis B or C Clinical onset is 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

Porphyrias (cont.)

Acute Hepatic Porphyrias e.g. Acute Intermittent Porphyria Porphyrias leading to accumulation of ALA and porphobilinogen cause abdominal pain and neuropsychiatric disturbances, ranging from anxiety to delirium. Symptoms of the acute hepatic porphyrias are often precipitated by administration of drugs such as barbiturates and ethanol.

Types of Porphyrias

Degradation of Heme

CO has a vasodilator effect while bilirubin has an antioxidant effect RBCs last 120 days, degraded by reticuloendothelial (RE) system [liver and spleen]. Most heme from RBCs (85%) - rest from turnover of cytochromes, p450s, immature erythrocytes. Microsomal heme oxygenase hydroxylates methenyl bridge carbon and oxidizes Fe2+ to Fe3+. Second reaction open ring and release methenyl carbon as CO. Serum albumin carries bilirubin in circulation, ligandin in hepatocytes. CO has a vasodilator effect while bilirubin has an antioxidant effect Bilirubin & its derivative is collectively known as bile pigments

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Metabolised by CYP 450