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1 Chapter 1 : HPU website - english version - What's HPU? The Porphyrin and Heme Synthesis and Bilirubin Metabolism page describes the processes of heme synthesis and degradation as well as descriptions of diseases related to defects in these pathways. In two sequential reactions a hydroxyethylfarnesyl moiety blue is added at the 2-position and an aldehyde purple is added at the 8-position. Nomenclature is shown in green. Isolated hemes are commonly designated by capital letters while hemes bound to proteins are designated by lower case letters. Cytochrome a refers to the heme A in specific combination with membrane protein forming a portion of cytochrome c oxidase. Other hemes The following carbon numbering system of porphyrins is an older numbering used by biochemists and not the 1â 24 numbering system recommended by IUPAC which is shown in the table above. Heme l is the derivative of heme B which is covalently attached to the protein of lactoperoxidase, eosinophil peroxidase, and thyroid peroxidase. The addition of peroxide with the glutamyl and aspartyl of lactoperoxidase forms ester bonds between these amino acid residues and the heme 1- and 5-methyl groups, respectively. Heme l is one important characteristic of animal peroxidases; plant peroxidases incorporate heme B. Lactoperoxidase and eosinophil peroxidase are protective enzymes responsible for the destruction of invading bacteria and virus. Thyroid peroxidase is the enzyme catalyzing the biosynthesis of the important thyroid hormones. Because lactoperoxidase destroys invading organisms in the lungs and excrement, it is thought to be an important protective enzyme. Heme m is the derivative of heme B covalently bound at the active site of myeloperoxidase. Heme m contains the two ester bonds at the heme 1- and 5-methyls as in heme l found in other mammalian peroxidases. In addition, a unique sulfonium ion linkage between the sulfur of a methionyl amino-acid residue and the heme 2-vinyl group is formed, giving this enzyme the unique capability of easily oxidizing chloride and bromide ions. Myeloperoxidase is present in mammalian neutrophils and is responsible for the destruction of invading bacteria and viruses. It also synthesizes hypobromite by "mistake" which is a known mutagenic compound. Ring III is also hydroxylated at position 5, in a conformation trans to the new lactone group. Heme S is related to heme B by the having a formyl group at position 2 in place of the 2-vinyl group. Heme S is found in the hemoglobin of marine worms. The names of cytochromes typically but not always reflect the kinds of hemes they contain: The process is highly conserved across biology. In humans, this pathway serves almost exclusively to form heme. In other species, it also produces similar substances such as cobalamin vitamin B The rate-limiting enzyme responsible for this reaction, ALA synthase, is negatively regulated by glucose and heme concentration. This mechanism is of therapeutic importance: However, due to its toxic properties, proteins such as Hemopexin Hx are required to help maintain physiological stores of iron in order for them to be used in synthesis. Defects in various enzymes in synthesis of heme can lead to group of disorder called porphyrias, these include acute intermittent porphyria, congenital erythropoetic porphyria, porphyria cutanea tarda, hereditary coproporphyria, variegate porphyria, erythropoietic protoporphyria. Degradation Heme breakdown Degradation begins inside macrophages of the spleen, which remove old and damaged senescent erythrocytes from the circulation. In the first step, heme is converted to biliverdin by the enzyme heme oxygenase HMOX. In addition, heme degradation appears to be an evolutionarily-conserved response to oxidative stress. Briefly, when cells are exposed to free radicals, there is a rapid induction of the expression of the stress-responsive heme oxygenase-1 HMOX1 isoenzyme that catabolizes heme see below. The reason why cells must increase exponentially their capability to degrade heme in response to oxidative stress remains unclear but this appears to be part of a cytoprotective response that avoids the deleterious effects of free heme. Page 1

2 Chapter 2 : Porphyria authorstream The liver catabolizes haem to bilirubin through microsomal haem oxygenase activity and excretes haem into bile along with porphyrins. Biliary excretion of porphyrins increases signiï cantly in patients with some types of porphyria. HPU can be can be described as a stress-induced double deficiency of pyridoxalphosfate and zinc. In most of the cases a deficiency of manganese is found as well. It belongs to the porphyrin diseases. This is a heterogeneous group of congenital or acquired defects in the metabolism of haem caused by abnormalities of enzymes involved in the biosynthesis. Porphyrin disease are sometimes classified in terms of clinical presentation, as acute or non-acute. HPU belongs to the non-acute porphyries. Normally in non-acute porphyries porphobilinogen deaminase is raised. Up till now this has not been investigated in HPU. In all porphyrin disease, HPL is found in the urine of the patients as well. HPL can be derived from 1 monovinyl- or mono-ethyl porphyrins, 2 bilirubin by chemical oxidoreduction or 3 intestinal bacteria Irvine and Wilson What are really porphyrin disorders? They are congenital enzyme deficiencies in the production of haem. Toxic porphyrins are deposited in body tissues. The patients are mainly female. Symptoms of porphyria such as abdominal discomfort belly pains, constipation, nausea, leg cramps, weakness of the muscles in the arm, anxiety and agitation, skin complaints that increase during exposure to the sun are found in HPU to. Other symptoms like a slightly rapid heartbeat and convulsions, are however, found much less frequent in HPU-patients. Studying thousands of patients with krytopyrroluria, porphyria and HPU we found the following structure in complaints. Symptoms caused by a decrease of the haem production In the case of HPU less haem is produced than normally. Only in severe cases this causes complaints. Haem is the building block of haemoglobin, myoglobulin and cytochrome C. So in these severe cases anaemia, tiredness, muscular weakness and functional liver problems are present. In the haem formed in most of the cases more zinc is linked than normal. So part of the haemoglobin is zinc-haemoglobin. Symptoms caused by deficiencies of Pyridoxalphosphate and zinc Much more complaints are however caused by the deficiencies of pyridoxalphosphate, zinc and manganese. To these complaints belong also muscle weakness, joint complaints hypermobility, problems carbohydrate intolerance, allergy due by a bad protein digestion, problems around menstrual cycle, pregnancy and childbirth miscarriage instability of the pelvis, hypoglycaemia or diabetes. Symptoms caused by down regulation Other symptoms are caused by down regulation. Alcohol and certain drugs show an increase of porphyria complaints, while vitamin B6 and zinc show an improvement. Case One of the most severe cases of kryptopyrroluria was described in literature Pfeiffer, Her history included insomnia, episodic loss of reality, attempted suicide by hanging, partial seizures, nausea, vomiting and loss of periods. Here knees were so painful and her mind so disperceptive that she walked slowly with her feet wide apart like a peasant following a hand plough drawn by tired oxen. Her brain wave and standard blood tests were within normal limited. At times her lift side of the body went into spasms with foot clawed and fist doubled up. Both arm and leg had a wild flying motion. Restraints were needed at these times. Psychotherapy was ineffective and most tranquillisers accentuated the muscle symptoms. She was diagnosed as B6 and zinc deficient and treatment started. In children only specified complaints can be seen. The following signs can be an indications for HPU: Page 2

3 Chapter 3 : Table of contents for Medical biochemistry at a glance All neonates have a tendency toward elevated bilirubin levels because bilirubin glucuronyl transferase is low at birth. -Kernicterus is caused by unconjugated hyperbilirubinemia either from haemolytic disease or the inability of the liver to conjugate bilirubin. Pathogenesis[ edit ] Heme synthesisâ note that some reactions occur in the cytoplasm and some in the mitochondrion yellow In humans, porphyrins are the main precursors of heme, an essential constituent of hemoglobin, myoglobin, catalase, peroxidase, and P liver cytochromes. The body requires porphyrins to produce heme, which is used to carry oxygen in the blood among other things, but in the porphyrias there is a deficiency inherited or acquired of the enzymes that transform the various porphyrins into others, leading to abnormally high levels of one or more of these substances. Porphyrias are classified in two ways, by symptoms and by pathophysiology. Physiologically, porphyrias are classified as liver or erythropoietic based on the sites of accumulation of heme precursors, either in the liver or in the bone marrow and red blood cells. Heme function plays a central role in cellular metabolism. This is not the main problem in the porphyrias; most heme synthesis enzymes â even dysfunctional enzymes â have enough residual activity to assist in heme biosynthesis. The principal problem in these deficiencies is the accumulation of porphyrins, the heme precursors, which are toxic to tissue in high concentrations. The chemical properties of these intermediates determine the location of accumulation, whether they induce photosensitivity, and whether the intermediate is excreted in the urine or feces. There are eight enzymes in the heme biosynthetic pathway, four of whichâ the first one and the last threeâ are in the mitochondria, while the other four are in the cytosol. Defects in any of these can lead to some form of porphyria. The hepatic porphyrias are characterized by acute neurological attacks seizures, psychosis, extreme back and abdominal pain, and an acute polyneuropathy, while the erythropoietic forms present with skin problems, usually a light-sensitive blistering rash and increased hair growth. Variegate porphyria also porphyria variegata or mixed porphyria, which results from a partial deficiency in PROTO oxidase, manifests itself with skin lesions similar to those of porphyria cutanea tarda combined with acute neurologic attacks. All other porphyrias are either skin- or nerve-predominant. Porphyrin studies[ edit ] Porphyria is diagnosed through biochemical analysis of blood, urine, and stool. As a result of feedback, the decreased production of heme leads to increased production of precursors, PBG being one of the first substances in the porphyrin synthesis pathway. The urine screening test has been known to fail in the initial stages of a severe, life-threatening attack of acute intermittent porphyria. The exception to this may be latent post-puberty genetic carriers of hereditary coproporphyria. In general, testing involves sending samples of blood, stool, and urine to a reference laboratory. Samples should be taken during an acute attack; otherwise a false negative result may occur. Samples must be protected from light and either refrigerated or preserved. A careful medication review often will find the cause of pseudoporphyria. Additional tests[ edit ] Further diagnostic tests of affected organs may be required, such as nerve conduction studies for neuropathy or an ultrasound of the liver. Basic biochemical tests may assist in identifying liver disease, hepatocellular carcinoma, and other organ problems. Acute porphyria[ edit ] Carbohydrates and heme[ edit ] Often, empirical treatment is required if the diagnostic suspicion of a porphyria is high since acute attacks can be fatal. However, this can worsen hyponatraemia and should be done with extreme caution as it can prove fatal. These drugs need to be given very early in an attack to be effective; effectiveness varies amongst individuals. They are not curative drugs but can shorten attacks and reduce the intensity of an attack. Side effects are rare but can be serious. These heme-like substances theoretically inhibit ALA synthase and hence the accumulation of toxic precursors. Pain should be treated as early as medically possible. Nausea can be severe; it may respond to phenothiazine drugs but is sometimes intractable. Hot baths and showers may lessen nausea temporarily, though caution should be used to avoid burns or falls. Early identification[ edit ] It is recommended that patients with a history of acute porphyria, and even genetic carriers, wear an alert bracelet or other identification at all times. This is in case they develop severe symptoms, or in case of accidents where there is a potential for drug exposure, and as a result they are unable to explain their condition to healthcare Page 3

4 professionals. Some drugs are absolutely contraindicated for patients with any form of porphyria. This is thought to be due to axonal nerve deterioration in affected areas of the nervous system and vagal nerve dysfunction. Pain treatment with long-acting opioids, such as morphine, is often indicated, and, in cases where seizure or neuropathy is present, Gabapentin is known to improve outcome. Some psychotropic drugs are porphyrinogenic, limiting the therapeutic scope. Other psychiatric symptoms such as anxiety, restlessness, insomnia, depression, mania, hallucinations, delusions, confusion, catatonia, and psychosis may occur. Most seizure medications exacerbate this condition. Treatment can be problematic: Some benzodiazepines are safe and, when used in conjunction with newer anti-seizure medications such as gabapentin, offer a possible regimen for seizure control. Gabapentin has the additional feature of aiding in the treatment of some kinds of neuropathic pain. The addition of hematin or heme arginate has been used during status epilepticus. These include hemochromatosis and hepatitis C. Treatment of iron overload may be required. Other typical risk factors for liver cancer need not be present. However, oral contraceptives have also triggered photosensitivity and withdrawal of oral contraceptives has triggered attacks. Androgens and fertility hormones have also triggered attacks. The pain, burning, swelling, and itching that occur in erythropoietic porphyrias generally require avoidance of bright sunlight. Most kinds of sunscreen are not effective, but SPF-rated long-sleeve shirts, hats, bandanas, and gloves can help. Chloroquine may be used to increase porphyrin secretion in some EPs. The rarest is congenital erythropoietic porphyria C. The signs may present from birth and include severe photosensitivity, brown teeth that fluoresce in ultraviolet light due to deposition of Type 1 porphyrins, and later hypertrichosis. Hemolytic anemia usually develops. Pharmaceutical-grade beta carotene may be used in its treatment. More than genetic variants of AIP are known, some of which are specific to families, although some strains have proven to be repeated mutations. The underlying mechanism was first described by Felix Hoppe-Seyler in, [38] and acute porphyrias were described by the Dutch physician Barend Stokvis in In the early s, patients with porphyrias occasionally referred to as "porphyric hemophilia" [41] and severe symptoms of depression or catatonia were treated with electroshock therapy. Vampires and werewolves[ edit ] Porphyria has been suggested as an explanation for the origin of vampire and werewolf legends, based upon certain perceived similarities between the condition and the folklore. In January, L. Later, Nancy Garden argued for a connection between porphyria and the vampire belief in her book, Vampires. The Aetiology of European Metamorphosis Legends," gained widespread media coverage, popularizing the idea. The theory has been rejected by a few folklorists and researchers as not accurately describing the characteristics of the original werewolf and vampire legends or the disease, and as potentially stigmatizing people with porphyria. As it was believed that the folkloric vampire could move about freely in daylight hours, as opposed to the 20th century variant, congenital erythropoietic porphyria cannot readily explain the folkloric vampire but may be an explanation of the vampire as we know it in the 20th century. In addition, the folkloric vampire, when unearthed, was always described as looking quite healthy "as they were in life", while due to disfiguring aspects of the disease, sufferers would not have passed the exhumation test. Individuals with congenital erythropoietic porphyria do not crave blood. The enzyme hematin necessary to alleviate symptoms is not absorbed intact on oral ingestion, and drinking blood would have no beneficial effect on the sufferer. Finally, and most important, the fact that vampire reports were literally rampant in the 18th century, and that congenital erythropoietic porphyria is an extremely rare manifestation of a rare disease, makes it an unlikely explanation of the folkloric vampire. The first, written in, thirty-five years after his death, concluded that he had acute mania. Guttmacher, in, suggested manic-depressive psychosis as a more likely diagnosis. Many psychiatrists disagreed with the diagnosis, suggesting bipolar disorder as far more probable. The theory is treated in Purple Secret, [47] which documents the ultimately unsuccessful search for genetic evidence of porphyria in the remains of royals suspected to have had it. In, an exhaustive analysis of historical records concluded that the porphyria claim was based on spurious and selective interpretation of contemporary medical and historical sources. The closing credits of the film include the comment that the illness that King George had has been attributed to porphyria and that it is hereditary. It is assumed she inherited the disorder, if indeed she had it, from her father, James V of Scotland. Both father and daughter endured well-documented attacks that could fall within the constellation of symptoms of porphyria. Maria I of Portugal in a c. Maria I of Page 4

5 Portugal â known as "Maria the Pious" or "Maria the Mad" because of both her religious fervor and her acute mental illness, which made her incapable of handling state affairs after â is also thought to have had porphyria. Francis Willis, the same physician who treated George III, was even summoned by the Portuguese court but returned to England after the court limited the treatments he could oversee. Page 5

6 Chapter 4 : Heme - Wikipedia Goresky CA, Gordon ER, Shaffer EA, Parà P, Carassavas D, Aronoff A. Definition of a conjugation of dysfunction in Gilbert's syndrome: studies of the handling of bilirubin loads and of the pattern of bilirubin conjugates secreted in bile. Bibliographic record and links to related information available from the Library of Congress catalog. Contents data are machine generated based on pre-publication provided by the publisher. Contents may have variations from the printed book or be incomplete or contain other coding. Acids, bases and hydrogen ions protons 2. Production and removal of protons into and from the blood 4. Metabolic acidosis and metabolic alkalosis 5. Respiratory acidosis and respiratory alkalosis 6. Amino acids and the primary structure of proteins 7. Secondary structure of proteins 8. Tertiary and quaternary structures;collagen 9. Regulation of enzyme activity Absorption of carbohydrates and metabolism of galactose Anaerobic production of ATP by substrate-level phosphorylation, phosphocreatine, adenylate kinase Aerobic production of ATP Biosynthesis of ATP by oxidative phosphorylation I What happens when protons or electrons leak from the respiratory chain? Free radicals, reactive oxygen species and oxidative damage Aerobic oxidation of glucose to provide energy as ATP Anaerobic oxidation of glucose by glycolysis to form ATP and lactate Anaerobic glycolysis in red blood cell, 2,3 BPG and the Bohr effect The fate of glucose in liver: Glucose-stimulated secretion of insulin from the beta-cell Regulation of glycogen metabolism Glycogen breakdown glycogenolysis and glycogen storage diseases Insulin signal transduction and Diabetes mellitus Regulation of glycolysis and Krebs cycle Oxidation of fatty acids to produce ATP in muscle and ketone bodies in liver Regulation of lipolysis, beta-oxidation, ketogenesis and gluconeogenesis Structure of lipids Metabolism of carbohydrate and fat to cholesterol HDL metabolism "reverse" cholesterol transport Digestion and disposal of dietary triacylglycerol and cholesterol by chylomicrons Urea cycle and overview of amino acid catabolism Nonessential and essential amino acids Phenylalanine and tyrosine metabolism in health and disease The products of tryptophan and histidine metabolism Haem, bilirubin and porphyries Fat-soluble vitamins I Fat-soluble vitamins II Water-soluble vitamins 1 thiamin. Water-soluble vitamins 2 pyridoxal phosphate Water-soluble vitamins 3 folate and vitamin B12 Water-soluble vitamins 4 biotin and vitamin C Library of Congress Subject Headings for this publication: Biochemistry -- Outlines, syllabi, etc. Clinical biochemistry -- Outlines, syllabi, etc. Page 6

7 Chapter 5 : Hans Fischer racedaydvl.com Heme or haem is a coordination complex "consisting of an iron ion coordinated to a porphyrin acting as a tetradentate ligand, and to one or two axial ligands." The definition is loose, and many depictions omit the axial ligands. Structure of Fe-porphyrin subunit of heme A. In two sequential reactions a hydroxyethylfarnesyl moiety is added at the 2-position and an aldehyde is added at the 8-position. Isolated hemes are commonly designated by capital letters while hemes bound to proteins are designated by lower case letters. Cytochrome a refers to the heme A in specific combination with membrane protein forming a portion of cytochrome c oxidase. Heme l is the derivative of heme B which is covalently attached to the protein of lactoperoxidase, eosinophil peroxidase, and thyroid peroxidase. The addition of peroxide with the glutamyl and aspartyl of lactoperoxidase forms ester bonds between these amino acid residues and the heme 1- and 5-methyl groups, respectively. Heme l is one important characteristic of animal peroxidases; plant peroxidases incorporate heme B. Lactoperoxidase and eosinophil peroxidase are protective enzymes responsible for the destruction of invading bacteria and virus. Thyroid peroxidase is the enzyme catalyzing the biosynthesis of the important thyroid hormones. Because lactoperoxidase destroys invading organisms in the lungs and excrement, it is thought to be an important protective enzyme. Heme m contains the two ester bonds at the heme 1- and 5-methyls as in heme l found in other mammalian peroxides. In addition, a unique sulfonamide ion linkage between the sulfur of a methionyl amino-acid residue and the heme 2-vinyl group is formed, giving this enzyme the unique capability of easily oxidizing chloride and bromide ions. Myeloperoxidase is present in mammalian neutrophils and is responsible for the destruction of invading bacteria and viruses. It also synthesizes hypobromite by "mistake" which is a known mutagenic compound. Ring III is also hydroxylated at position 5, in a conformation trans to the new lactone group. Heme S is found in the hemoglobin of marine worms. This convention may have been first introduced with the publication of the structure of heme A. Lowercase letters may then be freely used for cytochromes and enzymes, as well as to describe individual protein-bound heme groups for example, cytochrome bc, and aa3 complexes, cytochrome b5, heme c1 of the bc1 complex, heme a3 of the aa3 complex, etc. Thus cytochrome oxidase, which has two A hemes heme a and heme a3 in its structure, contains two moles of heme A per mole protein. Cytochrome bc1, with hemes bh, bl, and c1; contains heme B and heme C in a 2: The practice seems to have originated in a paper by Caughey and York in which the product of a new isolation procedure for the heme of cytochrome aa3 was designated heme A to differentiate it from previous preparations: For this reason, we shall designate our product heme A until the apparent differences can be rationalized. The process is highly conserved across biology. In humans, this pathway serves almost exclusively to form heme. In other species, it also produces similar substances such as cobalamin vitamin B The rate-limiting enzyme responsible for this reaction, ALA synthase, is negatively regulated by glucose and heme concentration. This mechanism is of therapeutic importance: However, due to its toxic properties, proteins such as Hemopexin Hx are required to help maintain physiological stores of iron in order for them to be used in synthesis. Defects in various enzymes in synthesis of heme can lead to group of disorder called porphyrias, these include acute intermittent porphyria, congenital erythropoetic porphyria, porphyria cutanea tarda, hereditary coproporphyria, variegate porphyria, erythropoietic protoporphyria. In the first step, heme is converted to biliverdin by the enzyme heme oxygenase HMOX. Briefly, when cells are exposed to free radicals, there is a rapid induction of the expression of the stress-responsive heme oxygenase-1 HMOX1 isoenzyme that catabolizes heme see below. Page 7

8 Chapter 6 : Porphyria and kidney diseases Clinical Kidney Journal Oxford Academic Following haem injection (6 4 μmol/kg) the flow of protoporphyrin but not of the other porphyrins was reduced, and the bile haem flow increased ( pl  5 nmol/h), while the flow of bilirubin did not increase significantly. Eijnden Eur J Pediatr Despite phototherapy and correction of polycythaemia by partial exchange transfusion, coagulopathy, hypoglycaemia and conjugated hyperbilirubinaemia persisted, suggesting hepatic failure. Metabolic work-up led to the diagnosis of tyrosinaemia type 1 on day 4. Severe skin purpuric lesions occurred in areas exposed to phototherapy. These resolved slowly after its discontinuation. Urine analysis sampled just before and 6 days after starting NTBC treatment showed high levels of type 1 coproporphyrin isomers. Conclusion We describe a cutaneous form of porphyria in a neonate presenting with severe liver failure due to tyrosinaemia type 1. This porphyria is tentatively attributed to a secondary accumulation of coproporphyrins due to cholestasis, as reported in the bronze baby syndrome and recently described in neonates with purpuric phototherapy-induced eruption, rather than to a primary defect of porphyrin metabolism. Crocq, Brussels, Belgium C. Yet conjugated bilirubin was still increased at 9. The coproporphyrins with minimal uroporphyrinuria. On day 10, a same day, despite blue-light phototherapy and correction of poly- second urine sample showed again a high excretion of copropor- cythaemia by partial exchange transfusion, severe coagulopathy, phyrins, mainly type 1 isomers, whereas uroporphyrins were nor- hypoglycaemia and conjugated hyperbilirubinaemia conjugated mal Table 2. Neither plasma porphyrin nor harderoporphyrin bilirubin 6. The cutaneous lesions slowly resolved after phototherapy in a sick newborn persisted, suggesting liver failure. The child was presumably from hypoglycaemia. The post-mortem liver biopsy treated with ampicillin and netilmicin. Metabolic work-up showed high urinary succinylacetone establishing the diagnosis of Discussion tyrosinaemia type 1 on day 4. This case report seems to illustrate the following NTBC therapy, indicating a possible causative need for early recognition and treatment of the disease. The presence of the isomers from the biliary to the urinary route is well rash prior to NTBC makes the hypothesis of an adverse known in cholestatic jaundice [4, 6], but an associated reaction to this drug unlikely. Congenital erythropoietic photosensitivity is more rarely described. Skin toxicity porphyria was excluded by the measurement of normal due to secondary porphyria in the neonate is well uroporphyrinogen III cosynthase activity in the patient documented in the bronze baby syndrome but its pre- and both parents. An hereditary All had conjugated hyperbilirubinaemia peak total bi- coproporphyria is also very unlikely in view of the lirubin ranging from As observed in this newborn, succinyla- erythroblastosis fetalis due to Rhesus iso-immunisation, cetone inhibits d-ala dehydratase, but in this second- which is also the most frequent condition associated ary porphyria the accumulation of d-ala results with the bronze baby syndrome. All had mainly an in- exclusively in the neurotoxicity associated with tyrosi- crease in plasma coproporphyrins and normal urinary naemia type 1, without photosensitivity [8] Fig. The observed skin Fig. Relevant changes of porphyrins due to cholestatic liver failure caused by with NTBC treatment abstract. J Inher Metab Dis 21[Suppl 2]: Labbe RF, Lamon JM Porphyrins and disorders of association with severe photosensitivity needs further porphyrin metabolism. Tietz NN ed Textbook of clinical investigation. It could result either from a direct enzy- chemistry. Mallon E, Wojnarowska F, Hope P, Elder G Neonatal bullous eruption as a result of transient porphyrinemia in a porphyrin accumulation due to cholestatic liver failure. Eur J Clin Chem Clin picture likely to occur with the wide use of phototherapy. Marsdens and Dr A. Brachet Laboratoire de Physiopathologie, U. Page 8

9 Chapter 7 : Porphyrias Laurent Gouya and J. Deybach - racedaydvl.com Heme or haem (from Greek αἠμα haima meaning blood) is a cofactor consisting of an Fe 2+ ion contained in the centre of a large heterocyclic organic ring called a porphyrin, made up of four pyrrolic groups joined together by methine bridges. Hans Fischer The German organic chemist Hans Fischer was awarded the Nobel Prize in Chemistry in for his researches into the constitution of hemin and chlorophyll and especially for his synthesis of hemin. Hans Fischer, the son of Dr. He entered the University of Lausanne in, read chemistry and medicine, and subsequently transferred to the University of Marburg, where he graduated in chemistry in Two years later he qualified in medicine at Munich. In he graduated as a doctor of medicine at Munich. He was assistant to the chemist Emil Fischer at Berlin and did some early work on bile pigments at Munich After a year as a teacher of internal medicine and three as lecturer in physiology at Munich, Fischer held the chair of medical chemistry at Innsbruck and then at Vienna From until his death he was professor of organic chemistry at the Technische Hochschule in Munich. From Fischer studied the pyrrole group of the heterocyclic compounds. In he showed that the urine and feces of a case of congenital porphyria, a disease then recently discovered, contained uroporphyrin and coproporphyrin. Structure of Hemoglobin Fischer then began to study hemoglobin, a very important member of the pyrrole group. Its molecule consists of the pigment heme combined with the protein globlin. Heme contains iron, and its chloride, which is easier to work with, is hemin. Hemoglobin possesses the unique property of forming a loose reversible combination with oxygen, so that oxygen taken up by it in the lungs can be given off in the tissues. In this loose combination the active part is heme, and heme combined with any other protein except globin does not possess the property of giving up combined oxygen. It was already known, partly through the work of Fischer himself, that hemin has the formula C34H32O4N4FeCl, and his problem now was to determine the steric arrangement of the 76 atoms in this molecule of hemin. When Fischer started his work it was known that, when the iron atom is removed from hemin, porphyrins are formed. His first problem was therefore to clarify the structure of the porphyrins. At a later stage of this investigation he synthesized the substance "porphin," which is not known to occur naturally. Porphin consists of four pyrrole rings, arranged as at the four points of the compass. Fischer then showed that all porphyrins contain this porphin nucleus, in which, in each of the four pyrrole rings, the two carbon atoms most distant from the nitrogen atom are each linked with an atom of hydrogen. He also showed that, in all naturally occurring porphyrins, these hydrogen atoms are replaced by substitution groups methyl, ethyl, vinyl, and so on. The possible permutations are therefore considerable. In Fischer discovered porphyrin syntheses, and he synthesized over isomers. It was recognized that the iron atom in hemin was situated at the center of the porphin nucleus. He studied the substance ooporphyrin, a constituent of the spots on the eggs of certain birds. By a short-term putrefaction of hemoglobin he produced protoporphyrin; and by putrefaction carried on for several months he produced deuterohemin, from which he obtained deuteroporphyrin by splitting off the iron. By the mild reduction of hemin the substance called mesoporphyrin had previously been obtained. Fischer found that, by decarboxylating mesoporphyrin, etioporphyrin was formed, and he determined that etioporphyrin contained four methyl and four ethyl groups. He then found that, by introducing four methyl and four ethyl groups into the porphin nucleus, etioporphyrin was obtained. But, depending on the position assumed by these residues as side chains in the porphin nucleus, this introduction could be effected in four different ways. He synthesized these four etioporphyrins and related them to the ooporphyrins. He then turned to a study of the mesoporphyrins and found that there were 15 possible isomers. He synthesized 12 of the He found also that mesoporphyrin IX was identical with the mesoporphyrin obtained from hemin. Further, mesoporphyrin IX was derived from etioporphyrin III, and the arrangement of the side chains in hemin was thus determined. Fischer next determined the formula for deuteroporphyrin and synthesized that substance. By the introduction of iron, deuterohemin was obtained, and by a complex process two acetyl residues were introduced into the latter, producing diacetyl-deuterohemin. Partial reduction of diacetyl-deuterohemin yielded hematoporphyrin, and from it protoporphyrin was produced by the removal of two molecules of water. On the Page 9

10 introduction of iron into protoporphyrin, a synthetic hemin was produced that was indistinguishable from natural hemin obtained from hemoglobin. Fischer completed this synthesis in Structure of Chlorophyll Meanwhile Fischer had also been working on chlorophyll. During his career he wrote nearly papers on that subject. From chlorophyll he obtained three different porphyrins, and from these etioporphyrin, which he considered identical with the etioporphyrin obtained from hemoglobin. Fischer started his researches here, and from the three porphyrins he obtained two distinct etioporphyrins. He synthesized very many isomers, and he converted pyroporphyrin into his mesoporphyrin IX. He then worked on the substance phylloerythrin, found in the gastrointestinal tract of ruminants. He showed that it is a porphyrin and that it exhibited an atypical linkage of two of the pyrrole nuclei. It was known that chlorophyll contains magnesium, and Fischer determined that the magnesium atom was situated at the center of the porphin nucleus. Even with the lead that phylloerythrin gave Fischer, it required many years to enable him to put forward his formula for the structure of chlorophyll a, a formula which seems to be correct. This formula is notable not only for the atypical linkage of two of the pyrrole nuclei, but also for the presence of two surplus hydrogen atoms in 7-and 8-positions, and of a very complex phytyl group in the 7-position. He found that the formula for chlorophyll b is the same as that for chlorophyll a, except that in the former a formyl group replaces the methyl group in pyrrole ring II of the latter. Although the pigments biliverdin and bilirubin had been known for nearly a century, when Fischer began his investigations practically nothing was known of their chemical composition. He showed that, whereas the molecule of the porphyrins consists of four pyrrole rings linked by four carbon linkages to form a closed ring the porphin nucleus, the molecule of a bile pigment is the same except that it lacks one of the carbon linkages. The pigment molecule can therefore be regarded either as an open ring or as a linear chain of four pyrrole nuclei linked by three carbon linkages. Fischer worked out the structural formulas for both biliverdin and bilirubin. In he synthesized biliverdin, and in he effected the even more difficult synthesis of bilirubin. Later Life Fischer was not only a superb research chemist but also a very fine administrator of a research institute, and he was extremely popular with his staff and students. He was a keen mountaineer, skier, and motorist, despite the fact that as a young man he had suffered from serious surgical tuberculosis. In addition to innumerable scientific papers Fischer was the author, with two colleagues, of a standard work on pyrrole chemistry, Die Chemie des Pyrrols 3 vols. The title of Privy Councilor Geheimrat was conferred on Fischer in In Harvard University conferred on him an honorary doctorate. He died in Munich on March 31, Karrer, Organic Chemistry trans. Thompson, Biochemistry in Relation to Medicine 3d ed. Smith, Principles of Biochemistry 3d ed. Page 10

11 Chapter 8 : Porphyrin and Heme Synthesis and Bilirubin Metabolism L Berglund, B Angelin, R Blomstrand, GS Drummond, A KappasSn-Protoporphyrin lowers serum bilirubin levels, decreases biliary bilirubin output, enhances biliary heme excretion and otently inhibits microsomal heme oxygenase activity in normal human subjects. Porphyrins are complex chemical compounds that are large heterocyclic organic ring structures. The complex ring structures of porphyrins are composed of four modified pyrrole 5-membered organic ring subunits connected by methine carbon with one double bond and two single bonds: The naturally occurring porphyrins of biological significance are the hemes. The major function of heme in humans is its role in the coordination of O2 molecules in hemoglobin. There are three structurally distinct hemes in humans identified as heme a, heme b, and heme c discussed below. In addition to the heme b of hemoglobin and its role in oxygen transport, hemes are critical for the biological functions of several enzymes such as the cytochromes of oxidative phosphorylation and the xenobiotic metabolizing enzymes of the cytochrome P family CYP. Aside from its importance as the prosthetic group of hemoglobin and the cytochromes, heme is clinically significant because a number of genetic disease states are associated with deficiencies of the enzymes used in its biosynthesis and catabolism. Some disorders of heme biosynthesis are more insidious such as the various porphyrias, a list of which can be found below and in the Inborn Errors Page. In addition to the clinical consequences of defects in heme biosynthesis, defects in heme catabolism can lead to potentially lethal elevations in the primary catabolic byproduct, bilirubin see below An important feature of the intermediates in heme biosynthesis, as well as in heme degradation, is their chromophoric character, some are colored while others are not. An easy way to distinguish which will have a color and which will not is to look at the suffix of the compound name. All heme intermediates and degradation products that end in -ogen e. Delta-aminolevulinic acid ALA is also called 5-aminolevulinic acid. This reaction is both the rate-limiting reaction of heme biosynthesis, and the most highly regulated reaction see Regulation below. Due to the loss of ALAS activity in vitamin B6 deficiency there is an associated microcytic hypochromic anemia where the erythroblasts are histopathologically associated with siderosomes. There are two forms of ALAS encoded by two different genes. ALAS1 is considered a house-keeping gene and is expressed in all cells. ALAS2 is an erythroid-specific form of the enzyme and is expressed only in fetal liver and adult bone marrow. The ALAS1 gene is located on chromosome 3p Sideroblasts are erythroblasts with non-heme iron-containing conjugates predominantly associated with the mitochondria, called siderosomes. XLSA has also been called congenital sideroblastic anemia, hereditary sideroblastic anemia, hereditary iron-loading anemia, X-linked hypochromic anemia, hereditary hypochromic anemia, and hereditary anemia. Following synthesis, the mitochondrial ALA is transported to the cytosol, where ALA dehydratase also called porphobilinogen synthase dimerizes two molecules of ALA to produce the pyrrole ring compound porphobilinogen. The ALA dehydratase gene symbol: ALAD is located on chromosome 9q32 and is composed of 14 exons that generate three alternatively spliced mrnas, each of which encode a distinct protein isoform. The next step in the pathway involves the head-to-tail condensation of four molecules of porphobilinogen to produce the linear tetrapyrrole intermediate, hydroxymethylbilane. The enzyme for this condensation is porphobilinogen deaminase PBG deaminase. This enzyme is also called hydroxymethylbilane synthase or uroporphyrinogen I synthase. The PBG deaminase gene official symbol: HMBS, for hydroxymethylbilane synthase is located on chromosome 11q PBG deaminase isoform 1 is composed of amino acids, PBG deaminase isoform 2 is composed of amino acids, PBG deaminase isoform 3 is composed of amino acids, and PBG deaminase isoform 4 is composed of amino acids. PBG deaminase isoform 2 is an erythroid cell-specific form of the enzyme. Once produced, hydroxymethylbilane has two main fates, one is due to enzymatic action, the other is non-enzymatic. Non-enzymatic alteration in hydroxymethylbilane is a cyclization to the compound called uroporphyrinogen I. The latter fate of hydroxymethylbilane is of significance only in patients with defects in enzymes downstream of PBG deaminase. Of significance to patients harboring a defective heme biosynthetic enzyme is the fact that defects prior to hydroxymethylbilane synthesis ARE NOT associated with photosensitivity, whereas, defects Page 11

12 from this point on ARE associated with photosensitivity. Defects in the PBG deaminase gene result in the autosomal dominant hepatic porphyria called acute intermittent porphyria, AIP. The most important fate of hydroxymethylbilane is the regulated, enzymatic conversion to uroporphyrinogen III, the next intermediate on the pathway to heme synthesis. This step is mediated by the enzyme, uroporphyrinogen-iii synthase. The uroporphyrinogen-iii synthase gene symbol: UROS is located on chromosome 10q Defects in the UROS gene are associated with the autosomal recessive erythroid porphyria called congenital erythropoietic porphyria, CEP In the Figure below you can place your mouse over intermediate names to see their structures. Clicking on enzyme names will take you to a descriptive page of the porphyria resulting from deficiency in that enzyme. Pathway of Heme Biosynthesis. Heme biosynthesis begins in the mitochondria from glycine and succinyl-coa, continues in the cytosol, and ultimately is completed within the mitochondria. The heme that it produced by this biosynthetic pathway is identified as heme b. The resultant products have methyl groups in place of acetate and are known as coproporphyrinogens, with coproporphyrinogen III being the important normal intermediate in heme synthesis. The UROD gene is located on chromosome 1p Following its synthesis, coproporphyrinogen III is transported to the interior of the mitochondrion, where two propionate residues are decarboxylated, yielding vinyl substituents on the two pyrrole rings. The colorless product is protoporphyrinogen IX. These reactions are catalyzed by oxygen-dependent coproporphyrinogen-iii oxidase gene symbol: The CPOX gene is located on chromosome 3q Mutations in the CPOX gene result in the autosomal dominant acute hepatic porphyria called hereditary coproporphyria, HCP In the mitochondrion, protoporphyrinogen IX is converted to protoporphyrin IX structure shown below by protoporphyrinogen IX oxidase. The protoporphyrinogen IX oxidase gene symbol: PPOX is located on chromosome 1q These six mrnas encode four distinct protein isoforms. The oxidase reaction requires molecular oxygen and results in the loss of six protons and six electrons, yielding a completely conjugated ring system, which is responsible for the characteristic red color of the hemes. The enzyme catalyzing this reaction is known as ferrochelatase. The ferrochelatase gene symbol: FECH is located on chromosome 18q Ferrochelatase isoform a is composed of amino acids and isoform b is composed of amino acids. Defects in the ferrochelatase gene are associated with the autosomal dominant erythroid porphyria called erythropoietic protoporphyria, EPP. Protoporphyrin IX The enzymes ferrochelatase, ALA synthase and ALA dehydratase a sulfhydryl containing enzyme are sensitive to inhibition by heavy metal poisoning, with inhibition of ferrochelatase being the most sensitive and most significant to the clinical manifestations of heavy metal poisoning. A characteristic of lead poisoning is an increase in ALA in the circulation in the absence of an increase in heme. Indeed, due to the inhibition of ferrochelatase and the associated loss of heme synthesis, the feed-back inhibition of ALA synthase is no longer active leading to increased ALA synthesis even in the presence of a heavy metal such as lead. The consequences of heavy metal inhibition of ferrochelatase are an acquired porphyria as opposed to an inherited disease referred to as plumbism so-called because the symbol for lead is Pb. Due to the lack of enzymatic incorporation of iron into protoporphyrin IX, by heavy metal inhibited ferrochelatase, erythroblasts will acquire siderosomes. Siderosomes are histologically observable structures that result from iron deposition on mitochondria. Zn-protoporphyrin imparts a fluorescence capability that can be visualized by observing blood under appropriate wavelength light which causes the erythroid cells to "glow". This same phenomenon is observable in patients with iron deficient anemia. Indeed, the measurement of ZPP is used as a screening tool for both heavy metal lead poisoning and iron deficiency. In addition to the heme b found in hemoglobin, there are two additional forms of heme found in cytochromes such as those involved in the process of oxidative phosphorylation. Cytochromes of the c type contain a modified iron protoporphyrin IX known as heme c. Only cytochromes of the c type contain covalently bound heme. Heme a is also a modified iron protoporphyrin IX. Heme a is found in cytochromes of the a type such as those of complex IV of the oxidative phosphorylation pathway. The differences in these two tissues and their needs for heme result in quite different mechanisms for regulation of heme biosynthesis. In hepatocytes, heme is required for incorporation into the cytochromes, in particular, the P class of cytochromes CYP that are important for xenobiotic detoxification. In addition, numerous cytochromes of the oxidative-phosphorylation pathway contain heme. The rate-limiting step in hepatic heme biosynthesis occurs at the ALA synthase catalyzed step, which is the Page 12

13 committed step in heme synthesis. Heme itself functions as a co-repressor in the inhibition of ALA synthase gene expression. Heme itself, and hemin acts as a feed-back inhibitors on ALA synthase. Hemin also inhibits transport of ALA synthase from the cytosol its site of synthesis into the mitochondria its site of action. Because certain pharmaceutical drugs are metabolized by the hepatic CYP system, which requires heme, increased utilization of heme occurs upon administration of these drugs. Of particular significance are the barbiturates. Use of barbiturates should NEVER be prescribed for the pain associated with certain types of porphyrias. This is because the administration of barbiturates leads to their degradation by CYP enzymes in the liver, resulting in a reduction in overall heme levels as the heme needs to be incorporated into the CYP for their function. This results in de-repression of ALA synthase with the result being an exacerbation of the symptoms of the porphyria due to increased ALA synthesis and subsequent heme biosynthesis products upstream of the defective enzyme. In erythroid cells all of the heme is synthesized for incorporation into hemoglobin and occurs only upon differentiation when synthesis of hemoglobin proceeds. When red cells mature both heme and hemoglobin synthesis ceases. The heme and hemoglobin must, therefore, survive for the life of the erythrocyte normally this is days. In reticulocytes immature erythrocytes heme stimulates protein synthesis. The mechanism of this mode of heme-mediate regulation of protein synthesis is described in the Protein Synthesis page. Additionally, control of heme biosynthesis in erythrocytes occurs at numerous sites other than at the level of ALA synthase. Control has been shown to be exerted on ferrochelatase, the enzyme responsible for iron insertion into protoporphyrin IX, and on porphobilinogen deaminase. First, the porphyrin ring is hydrophobic and must be solubilized to be excreted. Second, iron must be conserved for new heme synthesis. Normally, senescent red blood cells and heme from other sources are engulfed by cells of the reticuloendothelial system phagocytic macrophages primarily of the spleen but also of the liver, lymph, and bone marrow. The globin is recycled or converted into amino acids, which in turn are recycled or catabolized as required. Heme is oxidized, with the heme ring being opened by the endoplasmic reticulum enzyme, heme oxygenase. This is the only reaction in the body that is known to produce CO. Page 13