HEME SYNTHESIS & DISORDERS M.Prasad Naidu MSc Medical Biochemistry, Ph.D.Research Scholar

HEME SYNTHESIS Heme is the most important porphyrin containing compound. Heme is a derivative of the porphyrin. Porphyrins are cyclic compounds formed by fusion of 4 pyrrole rings linked by methenyl (=CH-) bridges.

Metal ions can bind with nitrogen atoms of pyrrole rings to form complexes. Since an atom of iron is present, heme is a ferroprotoporphyrin. The pyrrole rings are named as l, ll, lll, lV and the bridges as alpha, beta, gamma and delta.

Naturally occurring porphyrins contain substituent groups replacing the 8 hydrogen atoms of the porphyrin nucleus. When the substituent groups have a symmetrical arrangement (1, 3, 5, 7 and 2, 4, 6, 8) they are called the I series –type l porphyrins. The lll series have an asymmetrical distribution of substituent groups (1, 3, 5, 8 and 2, 4, 6, 7)-type ll porphyrins.

Type lll is the most predominant in biological systems. It is also called series 9, because fischer, the pioneer in porphyrin chemistry has placed it as the 9th in a series of 15 possible isomers. Hans Fischer, the father of porphyrin chemistry, proposed a short hand model for presentation of porphyrin structures.

Hans Fischer synthesised heme in laboratory in 1920(Nobel prize, 1930). The usual substitutions are : a.propionyl (-CH2-CH2-COOH) group b. acetyl (-CH2-COOH) group c. methyl (-CH3) group d. vinyl (-CH=CH2) group

Biosynthesis of Heme Heme can be synthesised by almost all the tissues in the body. Heme is primarily synthesised in the liver and the erythrocyte-producing cells of bone marrow (erythroid cells). Heme is synthesised in the normoblasts, but not in the matured ones.

The pathway is partly cytoplasmic and partly mitochondrial. Step 1: ALA synthesis The synthesis starts with the condensation of succinyl CoA and glycine in the presence of pyridoxal phosphate to form delta amino levulinic acid (ALA). The enzyme ALA synthase is located in the mitochondria and is the rate-limiting enzyme of the pathway.

Step 2: Formation of PBG Next few reactions occur in the cytoplasm. Two molecules of ALA are condensed to form porphobilinogen (PBG). The condensation involves removal of 2 molecules of water and the enzyme is ALA dehydratase. Porphobilinogen is a monopyrrole. The enzyme contains zinc and is inhibited by Lead.

Step 3: Formation of UPG Condensation of 4 molecules of the PBG, results in the formation of the first porphyrin of the pathway, namely uroporphyrinogen(UPG). The pyrrole rings are joined together by methylene bridges, which are derived from alpha carbon of glycine.

When the fusion occurs, the lll series of isomers are predominantly formed; and only the lll series are further used. This needs 2 enzymes which catalyse the reactions; PBG-deaminase (Uroporphyrinogen-l-synthase) and Uroporphyrinogen-lll-cosynthase. During this deamination reation 4 molecules of ammonia are removed.

Step 4: synthesis of CPG The UPG-lll is next convertedto coproporphyrinogen (CPG-lll) by decarboxylation. Four molecules of CO2 are eliminated by uroporphyrinogen decarboxylase. The acetate groups (CH2-COOH) are decarboxylated to methyl (CH3) groups.

Step 5: synthesis of PPG Further metabolism takes place in the mitochondria. CPG is oxidised to protoporphyrinogen (PPG-lll) by coproporphyrinogen oxidase. Two propionic acid side chains are oxidatively decorboxylated to vinyl groups.

Step 6: Generation of PP The Protoporphyrinogen-lll is oxidised by the enzyme protoporphyrin-lll (PP-lll) in the mitochondria. The oxidation requires molecular oxygen. The methylene bridges (-CH2) are oxidised to methenyl bridges (-CH=) and coloured porphyrins are formed. Protoporphyrin-9 is thus formed.

Step 7: Generation of Heme The last step in the formation of heme is the attachment of ferrous iron to the protoporphyrin. The enzyme is heme synthase or ferrochelatase which is also located in mitochondria. Iron atom is co-ordinately linked with 5 nitrogen atoms (4 nitrogen of pyrrole rings of protoporphyrin and 1st nitrogen atom of a histidine residue of globin).

The remaining valency of iron atom is satisfied with water or oxygen atom. When the ferrous iron (Fe++) in heme gets oxidised to ferric (Fe+++) form, hematin is formed, which loses the property of carrying the oxygen. Heme is red in colour, but hematin is dark brown.

Regulation of Heme synthesis ALA synthase is regulated by repression mechanism. Heme inhibits the synthesis of ALA synthase by acting as a co-repressor. ALA synthase is also allosterically inhibited by hematin. When there is excess of free heme, the Fe++ is oxidised to Fe+++(ferric), thus forming hematin.

The compartmentalisation of the enzymes in the synthesis of heme makes it easier for the regulation. The rate-limiting enzyme is in the mitochondria. The steps 1,5,6, and 7 are taking place inside mitochondria, while steps 2,3 and 4 are in cytoplasm.

Drugs like barbiturates induce heme synthesis. Barbiturates require the heme containing cytochrome p450 for their metabolism. Out of the total heme synthesised, two thirds are used for cytochrome p450 production. The steps catalysed by ferrochelatase and ALA dehydratase are inhibited by lead.

INH (Isonicotinic acid hydrazide) that decreases the availability of pyridoxal phosphate may also affect heme synthesis. High cellular concentration of glucose prevents induction of ALA synthase. This is the basis of glucose to relieve the acute attack of porphyrias.

Shunt Bilirubin When 15N or 14C labelled glycine is injected, this is incorporated into heme and into RBCs. After 100-120 days, when RBCs are lysed, the radiolabelled Hb level is decreased, along with consequent rise in radioactive bilirubin. However, about 15% of radioactive bilirubin is excreted within about 10 days. This is called Shunt bilirubin.

This is the formation of bilirubin from heme in bone marrow, without being incorporated into Hb. This is the result of ineffective erythropoiesis. In porphyrias, especially in the erythropoietic varieties, the shunt biliribin will be increased.

Disorders of Heme synthesis Porphyrias are group of inborn errors of metabolism associated with the biosynthesis of heme.(Greek ‘porphyria’ means purple). These are characterised by increased production and production and excretion of porphyrins and/or their precursors (ALA + PBG). Many of the porphyrias are inherited as autosomal dominant traits.

Porphyrias may be broadly grouped into 3 types: Hepatic porphyrias b. Erythropoietic porphyrias c. porphyrias with both erythropoietic and hepatic abnormalities.

Acute intermittent porphyria This disorder occurs due to the deficiency of the enzyme uroporphyrinogen l synthase. Acute intermittent porphyria is characterised by increased excretion of porphobilinogen and δ- aminolevulinate. The urine gets darkened on exposure to air due to the conversion of porphobilinogen to porphobilin and porphyrin.

It is usually expressed after puberty in humans. Clinical features The symptoms include abdominal pain, vomiting and cardiovascular abnormalities. The neuropsychiatric disturbances observed in these patients are believed to be due to reduced activity of tryptophan pyrrolase (caused by depleted heme levels), resulting in the accumulation of tryptophan and 5-hydroxytryptamine.

These patients are not photosensitive since the enzyme defect occurs prior to the formation of uroporphyrinogen. The symptoms are more severe after administration of drugs (e.g. barbiturates) that induce the synthesis of cytochrome P450. This is due to the increased activity of ALA synthase causing accumulation of PBG and ALA.

Treatment: Acute intermittent porphyria is treated by administration of hematin which inhibits the enzyme ALA synthase and the accumulation of porphobilinogen.

Congenital erythropoietic porphyria This disorder is due to a defect in the enzyme uroporphyrinogen lll cosynthase. It is a rare congenital disorder caused by autosomal recessive mode of inheritance, mostly confined to erythropoietic tissues.

Clinical features : The patients are photosensitive (itching and burning of skin when exposed to visible light) due to the abnormal porphyrins that accumulate. Increased hemolysis is also observed in the individuals affected by this disorder. The individuals excrete uroporphyrinogen l and coproporphyrinogen l which oxidize respectively to uroporphyrin l and coproporphyrin l (red pigments).

Porphyria cutanea tarda This is a chronic disease caused by a deficiency in uroporphyrinogen decarboxylase. It is the most common porphyria. It is also known as cutaneous hepatic porphyria. It is usually associated with liver damage caused by alcohol overconsumption or iron overload.

Uroporphyrin accumulates in the urine. Clinical features: Cutaneous photosensitivity is the most important clinical manifestation of these patients. Liver exhibits flourescence due to high concentration of accumulated porphyrins.

Hereditary coproporphyria This disorder is due to a defect in the enzyme coproporphyrinogen oxidase. Coproporphyrinogen lll and other intermediates (ALA and PBG) of heme synthesis prior to the blockade are excreted in urine and feces. Patients are photosensitive. They exhibit the clinical manifestations observed in the patients of acute intermittent porphyria.

Infusion of hematin is used to control this disorder. Treatment : Infusion of hematin is used to control this disorder. Hematin inhibits ALA synthase and thus reduces the accumulation of various intermediates. E

Variegate porphyria It is an acute disease caused by a deficiency of protoporphyrinogen oxidase. Protoporphyrinogen IX and other inermediates prior to the block accumulate in the urine. The urine of these patients is coloured. Patients are photosensitive.

Protoporphyria This disorder is also known as erythropoietic protoporphyria. The disease is due to a deficiency in ferrochelatase. Protoporphyrin IX accumulates in erythrocytes, bone marrow, and plasma. Patients are photosensitive. Reticulocytes and skin biopsy exhibit red flourescence.

Acquired porphyrias The porphyrias may be acquired due to the toxicity of several compounds. Exposure of the body to heavy metals (e.g. lead ), toxic compounds (e.g. hexachlorobenzene) and drugs (e.g. griseofulvin) inhibits many enzymes in heme synthesis.

These include ALA dehydratase, uroporphyrin l synthase and ferrochelatase. Ferrochelatase and ALA dehydratase are particularly sensitive to inhibition by lead. Protoporphyrin and ALA accumulate in urine.

Diagnosis of porphyrias To demonstrate porphyrins, UV flourescence is the best technique. The presence of porphyrin precursor in urine is detected by Ehrlich’s reagent. When urine is observed under ultraviolet light; porphyrins if present, will emit strong red flourescence.

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