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Porphyrins and the porphyrias


Abstract

Background

The porphyrias are a group of rare, mainly inherited metabolic disorders that result from decreased or, in one rare form of erythropoietic protoporphyria, increased, activities of the enzymes of heme biosynthesis. Each porphyria is defined by the association of characteristic clinical features with a specific pattern of heme precursor accumulation that reflect the buildup of substrates upstream of the enzyme that is partially deficient, or of a secondarily rate-limiting enzyme.

Content

This chapter describes the metabolic pathway and regulation of heme biosynthesis, the excretion of heme precursors, the different porphyrias, and abnormalities of porphyrin metabolism not caused by porphyria. Porphyrias can be classified as hepatic or erythropoietic according to the main site of overproduction of heme precursors. From a clinical viewpoint, porphyrias are usually classified as acute, in which acute neurovisceral attacks occur, or as nonacute. Acute porphyrias include 5-aminolevulinate dehydratase deficiency porphyria (ADP), acute intermittent porphyria (AIP), variegate porphyria (VP), and hereditary coproporphyria (HCP). The nonacute porphyrias encompass porphyria cutanea tarda (PCT), congenital erythropoietic porphyria (CEP), erythropoietic protoporphyria (EPP), and X-linked erythropoietic protoporphyria (XLEPP). This chapter also covers the diagnostic approaches, with detailed information on biochemical and genetic analysis, and clinical management of the various forms of porphyrias.

The porphyrias are a group of uncommon, inherited disorders of heme biosynthesis. , Each porphyria results from a partial deficiency of one of the enzymes of the pathway converting 5-aminolevulinate (ALA) to heme, or in one disorder, increased activity of the rate-controlling enzyme of erythroid heme biosynthesis. Each functional abnormality is associated with a specific pattern of overproduction, accumulation, and excretion of pathway intermediates, which are excreted in excessive amounts in urine, feces, or both. The clinical consequences depend on the nature of the heme precursors that accumulate. In the acute porphyrias, excess porphyrin precursors (ALA and porphobilinogen [PBG]) are associated with potentially fatal acute neurovisceral attacks that most often are provoked by various commonly prescribed drugs or hormonal factors. In the nonacute porphyrias, and in those acute porphyrias in which skin lesions occur, accumulation of porphyrins results in photosensitization of sun-exposed skin. Diagnosis depends on laboratory investigations to demonstrate the pattern of heme precursor accumulation and excretion specific for each type of porphyria, and it requires examination of appropriate specimens for the key metabolites using adequately sensitive and specific methods. DNA analysis is rarely necessary for diagnosis of symptomatic cases, but it is the method of choice when investigating healthy at-risk relatives. Molecular analysis also continues to provide new information about the underlying pathophysiology. Abnormalities of porphyrin accumulation and excretion also occur in a wide variety of other disorders that are collectively more common than the porphyrias. Recognition of secondary porphyrin disorders is important to avoid diagnostic errors.

Historical perspective

The word porphyrin stems from porphuros, the Greek word for purple, and describes the color of the oxidized tetrapyrroles which are byproducts of the heme biosynthesis pathway. The history relating to identification of porphyrins, and the unraveling of the heme biosynthesis pathway together with identification and classification of its associated disorders, the porphyrias, is a long and illustrious one. Heme together with chlorophyll were characterized by Hans Fischer, who in 1930 was awarded the Nobel Prize for Chemistry for this work. The first porphyria case, pemphigus leprosis (now recognized as congenital erythropoietic porphyria [CEP]), was reported in 1874 by JH Schultz while he was a medical student. He described a patient with skin sensitivity, splenomegaly, and wine-red urine. However, this characterization was dependent on work by Hoppe-Seyler, who had previously identified iron free hematin and named it hematoporphyrin, a pigment that was similar to that excreted in the urine of Schultz’s patient.

The first description of acute porphyria, paralysis associated with red urine, was reported in 1889 by Barend Stokvis, and coincided with the introduction of the sedative drugs sulfonmethane and barbiturates into medical usage. These are now recognized to be potent inducers of heme biosynthesis, and in acute porphyria patients, acute attacks. Jan Waldenstrom, who coined the term porphyria, was the first to recognize that acute porphyrias were autosomal dominant. He also contributed to identifying and naming PBG, the monopyrrole precursor of porphyrinogens, and showed that by using the Ehrlich aldehyde test, increased urine PBG could confirm a diagnosis of acute porphyria. The potential of porphyrins to cause phototoxicity was proven in 1912 by Friederich Meyer-Benz, who injected himself with hematoporphyrin and showed that the pain, erythema, and edema developed only on the side of his body exposed to sunlight.

Hans Günther was the first to recognize these conditions as inborn errors of metabolism and proposed a classification of hematoporphyria congenita and hematoporphyria acuta. This was later amended by Waldenstrom to include porphyria cutanea tarda (PCT), as a distinct condition, that presented later in life under the group hematoporphyria congenita. Classification was further revised in the 1950s into erythropoietic porphyrias and hepatic porphyrias, based on the source of the excess porphyrins and included the “mixed type” (photosensitivity and acute symptoms) which were later characterized as variegate porphyria (VP; South African porphyria) by Barnes and Dean and hereditary coproporphyria (HCP) by Berger and Goldberg. The last two porphyrias described were erythropoietic protoporphyria (EPP) by Magnus et al. in 1961 13 and ALA dehydratase deficiency porphyria (ADP) by Doss et al. in 1979.

Porphyrin chemistry

Before porphyrin synthesis and disorders of porphyrin metabolism are discussed, porphyrin structure, nomenclature, and chemical characteristics are reviewed.

Structure and nomenclature

The basic porphyrin structure consists of four monopyrrole rings connected by methene bridges to form a tetrapyrrole ring ( Fig. 41.1 ). Many porphyrin compounds are known, but only a limited number are of clinical interest. The porphyrin compounds of relevance to the porphyrias ( Table 41.1 ) differ in the substituents occupying peripheral positions 1 through 8. Variation in the distribution of the same substituents around the peripheral positions of the tetrapyrrole ring gives rise to porphyrin isomers, which are usually depicted by Roman numerals (e.g., I, II, III). The reduced form of a porphyrin, known as a porphyrinogen (see Fig. 41.1 ), differs by the presence of six additional hydrogens (four on the methylene bridges and two on ring nitrogens). Porphyrinogens are unstable in vitro and are spontaneously oxidized to the corresponding porphyrins. Under the lower oxygen tension of the cell, porphyrinogens are sufficiently stable to act as intermediates of the heme biosynthetic pathway; aromatization to protoporphyrin at the penultimate step requires an enzyme.

FIGURE 41.1, Porphyrin and porphyrinogen structures. Numbers 1 to 8 represent various substituents, the nature and order of which determine the type of porphyrin or porphyrinogen (see Table 41.1 ). The numbering system and ring designations are based on the Fischer system. A revised system formulated by the International Union of Pure and Applied Chemistry–International Union of Biochemistry Joint Commission on Biochemical Nomenclature is appropriate for more complex needs. Because of increasing complexity, this Roman numeral designation is no longer recommended above IV.

TABLE 41.1
Substituents Around the Macrocycle in Porphyrins of Clinical Importance
Position 1 2 3 4 5 6 7 8
Uroporphyrin-I C m C et C m C et C m C et C m C et
Uroporphyrin-III C m C et C m C et C m C et C et C m
Heptacarboxylate porphyrin-III C m C et C m C et C m C et C et Me
Hexacarboxylate porphyrin-III Me C et C m C et C m C et C et Me
Pentacarboxylate porphyrin-III Me C et Me C et C m C et C et Me
Coproporphyrin-I Me C et Me C et Me C et Me C et
Coproporphyrin-III Me C et Me C et Me C et C et Me
Isocoproporphyrin Me Et Me C et C m C et C et Me
Dehydroisocoproporphyrin Me Vn Me C et C m C et C et Me
Deethylisocoproporphyrin Me H Me C et C m C et C et Me
Protoporphyrin Me Vn Me Vn Me C et C et Me
Pemptoporphyrin Me H Me Vn Me C et C et Me
Deuteroporphyrin Me H Me H Me C et C et Me
Mesoporphyrin Me Et Me Et Me C et C et Me
C et , Carboxyethyl (–CH 2 CH 2 COOH); C m , Carboxymethyl (–CH 2 COOH); Et, ethyl (–CH 2 CH 3 ); Me, methyl (–CH 3 ); Vn, vinyl (–CH=CH 2 ).

Chelation of metals

The arrangement of four nitrogen atoms in the center of the porphyrin ring enables porphyrins to chelate various metal ions. Protoporphyrin that contains iron (Fe) is known as heme; ferroheme refers specifically to the Fe 2+ complex and ferriheme to Fe 3+ . Ferriheme associated with a chloride counter ion is known as hemin or as hematin when the counter ion is hydroxide.

Spectral properties

Porphyrins owe their color to the conjugated double-bond structure of the tetrapyrrole ring. The porphyrinogens have no conjugated double bonds and are therefore colorless. Porphyrins show particularly strong absorbance near 400 nm, often called the Soret band. When exposed to light in the 400-nm region, porphyrins display a characteristic orange-red fluorescence in the range of 550 to 650 nm. Absorbance and fluorescence are altered by substituents around the porphyrin ring and by metal binding. Zinc (Zn) chelation shifts the fluorescence emission peak of protoporphyrin to shorter wavelengths and reduces the fluorescence intensity. The strong binding of iron alters the character of protoporphyrin to the extent that heme lacks significant fluorescence.

Solubility

Porphyrins are only marginally soluble in water. The differing solubilities of individual porphyrins are of importance not only in determining the route of excretion from the body but in the design of analytical methods for their extraction and fractionation. At pH 7, the carboxyl groups are ionized, and the molecule has a net negative charge. Below pH 2, the pyrrole nitrogens and the carboxyl groups become protonated so that the molecule has a net positive charge. At physiologic pH, the solubility of a given porphyrin is determined by the number of substituent carboxyl groups. Uroporphyrin has eight carboxylate groups and is the most soluble porphyrin in aqueous media. Protoporphyrin has only two carboxylate groups and is essentially insoluble in water, but it dissolves readily in lipid environments and binds readily to the hydrophobic regions of proteins (e.g., albumin). Coproporphyrin, with four carboxylate groups, has intermediate solubility.

Traditional extraction methods for porphyrins involve two steps, extraction into an acidified organic solvent, followed by a second or back extraction into aqueous acid. The initial extraction takes advantage of the fact that at pH 3 to 5 (near to their isoelectric point) porphyrins are less soluble in aqueous media and move into the organic phase. Coproporphyrin and protoporphyrin are readily extracted into diethyl ether, but the more carboxylated porphyrins (uroporphyrin and heptacarboxylate porphyrin) require a more hydrophilic solvent such as cyclohexanone or butanol. The back extraction induces porphyrin compounds to move back into the aqueous solution by decreasing the pH to less than 2, which causes protonation of the pyrrolenine nitrogen and carboxylate groups, thereby reversing the solubility characteristics of porphyrins. Compounds such as heme and chlorophyll, in which the pyrrole nitrogens are tightly bound to Fe and magnesium, respectively, remain uncharged at low pH and trapped in the organic layer.

Heme biosynthesis

The complex tetrapyrrole ring structure of heme is built up in a stepwise fashion from the very simple precursors succinyl–coenzyme A (CoA) and glycine ( Fig. 41.2 ). The pathway is present in all nucleated cells. From measurements of total bilirubin production, it has been estimated that daily synthesis of heme in humans is 5 to 8 mmol/kg body weight. Of this, 70 to 80% occurs in the bone marrow and is used for hemoglobin synthesis. The pathway is compartmentalized, with some steps occurring in the mitochondrion and others in the cytoplasm. Several carriers that transfer intermediates across the mitochondrial membrane have now been identified. Although all are potential sites for pathogenic mutations, so far only one such mutation has been identified: a mutation in the erythroid-specific mitochondrial glycine transporter SLC25A38 that causes nonsyndromic autosomal recessive sideroblastic anemia.

FIGURE 41.2, Biosynthetic pathway of porphyrins and heme. C et , Carboxyethyl (–CH 2 CH 2 COOH); C m , carboxymethyl (–CH 2 COOH); CoA , coenzyme A; Fe , iron; Me, methyl (–CH 3 ); Vn, vinyl (–CH=CH 2 ); ALA , 5-aminolevulinic acid; PBG, porphobilinogen.

Enzymes of heme biosynthesis

The genes for all enzymes of human heme biosynthesis have been characterized ( Table 41.2 ). The eight enzymes of the heme synthesis pathway listed in pathway order and by name and Enzyme Commission (EC) number are described in the following paragraph. The structures of human 5-aminolevulinic acid dehydratase (ALAD), hydroxymethylbilane synthase (HMBS), uroporphyrinogen-III synthase (UROS), uroporphyrinogen decarboxylase (UROD), coproporphyrinogen oxidase (CPOX), protoporphyrinogen oxidase (PPOX), ferrochelatase (FECH), and yeast 5-aminolevulinate synthase (ALAS) have been determined by x-ray crystallography.

TABLE 41.2
Human Enzymes and Genes of Heme Biosynthesis
Enzyme Monomer Mol Mass a , b (kDa) Chromosomal Location of Gene Gene Size (kb) No. of Exons Expression
ALAS1 70.6 3p21.2 17 12 Ubiquitous
ALAS2 64.6 Xp11.21 22 11 Erythroid cells
ALAD 36.3 9q32 13 13 Ubiquitous and erythroid-specific mRNAs
HMBS 37.0 11q23.3 10 15 Ubiquitous and erythroid-specific isoforms
UROS 29.5 10q26.1-q26.2 34 10 Ubiquitous and erythroid-specific mRNAs
UROD 40.8 1p34.1 3 10 Ubiquitous
CPOX 40.3 3q11.2-q12.1 14 7 Ubiquitous
PPOX 50.8 1q23.3 5 13 Ubiquitous
FECH 47.8 18q21.3 45 11 Ubiquitous
PPOX , Protoporphyrinogen oxidase; UROD , uroporphyrinogen decarboxylase.

a 5-Aminolevulinic acid dehydratase (ALAD) is a homo-octamer, and hydroxymethylbilane synthase (HMBS) and uroporphyrinogen-III synthase (UROS) are monomers; all other enzymes are homodimers.

b Molecular masses for 5-aminolevulinate synthase (ALAS)1, ALAS2, coproporphyrinogen oxidase (CPOX), and ferrochelatase (FECH) include presequences that are cleaved during mitochondrial import.

5-Aminolevulinate synthase (EC 2.3.1.37)

ALAS, the initial enzyme of the pathway, catalyzes the formation of ALA from succinyl-CoA and glycine. The enzyme is mitochondrial and requires a cofactor of pyridoxal phosphate, which forms a Schiff 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 α-amino-β-ketoadipic acid, which is then decarboxylated to ALA. The activity of ALAS is rate limiting as long as the catalytic capacities of other enzymes in the pathway are normal.

5-Aminolevulinic acid dehydratase (EC 4.2.1.24)

5-Aminolevulinic acid dehydratase (also known as PBG synthase) is a cytoplasmic enzyme that catalyzes the formation of the monopyrrole PBG from two molecules of ALA with elimination of two molecules of water. The enzyme requires Zn ions as a cofactor and reduced sulfhydryl groups at the active site; therefore it is susceptible to inhibition by lead.

Hydroxymethylbilane synthase (EC 2.5.1.61)

Hydroxymethylbilane synthase (also known as PBG deaminase) is a cytoplasmic enzyme that catalyzes the formation of one molecule of the linear tetrapyrrole 1-hydroxymethylbilane (HMB; also known as preuroporphyrinogen) from four molecules of PBG with the release of four molecules of ammonia. The enzyme has two molecules of its own substrate: PBG, which is attached covalently to the apoenzyme as a prosthetic group. The enzyme is 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)

Uroporphyrinogen-III synthase is a cytoplasmic enzyme that rearranges and cyclizes HMB to form uroporphyrinogen-III. Each pyrrole ring of HMB contains a methylcarboxylate and an ethylcarboxylate substituent, which are in the same orientation. By the rotation of zero, one, or two alternate or two adjacent pyrrole rings, it is possible to arrive at four different isomers. Apart from closing the ring structure, the enzyme rotates the d-ring via a spirane intermediate, producing the type III isomer, which is an 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. Normally, only minimal amounts of uroporphyrinogen-I are formed.

Uroporphyrinogen decarboxylase (EC 4.1.1.37)

This is the last cytoplasmic enzyme in the pathway, and it catalyzes the decarboxylation of all four carboxymethyl groups to form the tetracarboxylic coproporphyrinogen. The enzyme will use I and III isomers of uroporphyrinogen as substrate. Decarboxylation 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. Decreased UROD activity causes accumulation of these intermediates in addition to its substrate, uroporphyrinogen. At high substrate concentrations, decarboxylation occurs by a random mechanism.

Coproporphyrinogen oxidase (EC 1.3.3.3)

Coproporphyrinogen oxidase, which is located in the intermembrane space of the mitochondria, catalyzes the sequential oxidative decarboxylation of the 2- and 4-carboxyethyl groups to vinyl groups to produce the more lipophilic protoporphyrinogen-IX, with formation of a tricarboxylic intermediate, harderoporphyrinogen. Oxygen is required as the oxidant. The enzyme requires sulfhydryl groups for activity, making it a target for inhibition by metals. The enzyme is specific for the type III isomer, so that metabolism of the I series of porphyrins does not occur beyond coproporphyrinogen-I. The product of the enzyme differs from the substrate in that replacement of two of the carboxyethyl groups by vinyl groups introduces a third substituent into the molecule. Therefore the number of possible isomeric forms is increased, and conventionally the numbering system changes, so that the III isomer becomes the IX isomer. In UROD-deficient states, one of the ethylcarboxylate groups of the accumulated pentacarboxylate porphyrinogen is decarboxylated by CPOX to form the isocoproporphyrin series of porphyrins.

Protoporphyrinogen oxidase (EC 1.3.3.4)

Protoporphyrinogen oxidase, a flavoprotein located in the inner mitochondrial membrane, catalyzes the removal of six hydrogens (four from methylene bridges and two from ring nitrogens) to form protoporphyrin-IX. This involves a three-step, six-electron flavin adenine dinucleotide–dependent oxidation that consumes molecular oxygen. Nonenzymatic oxidation also occurs in vitro. However, under the low oxygen tension in the cell, PPOX is essential for oxidation to occur. The protoporphyrin produced is the only porphyrin that functions in the heme pathway. Other porphyrins are produced by nonenzymatic oxidation and originate from porphyrinogens that have irreversibly escaped from the pathway.

Ferrochelatase (EC 4.99.1.1)

Ferrochelatase (also known as heme synthase) is an Fe-sulfur protein located in the inner mitochondrial membrane. This enzyme inserts ferrous Fe into protoporphyrin to form heme. During this process, two hydrogens are displaced from the ring nitrogens. Other metals in the divalent state also act as substrates, yielding the corresponding chelate (e.g., incorporation of zinc [Zn 2+ ] into protoporphyrin to yield Zn protoporphyrin). In Fe-deficient states, Zn 2+ successfully competes with Fe 2+ in developing red cells, so that the concentration of Zn protoporphyrin in erythrocytes increases. Some other dicarboxylic porphyrins also serve as substrates (e.g., mesoporphyrin). Integration of the final stages of erythroid heme biosynthesis may be facilitated by interaction between FECH and proteins involved in Fe import.

Regulation of heme biosynthesis

Heme supply in all tissues is controlled by the activity of mitochondrial ALAS, the first enzyme of the pathway. Two isoforms of ALAS are known. The ubiquitous isoform, ALAS1, is encoded by a gene on chromosome 3p21 and is expressed in all tissues. Because it has a half-life of approximately 1 hour, changes in its rate of synthesis produce short-term alterations in enzyme concentration and cellular ALAS activity. Synthesis of ALAS1 is under negative feedback control by heme. In the liver, but not most other tissues, ALAS1 is induced by a wide variety of drugs and chemicals that induce microsomal cytochrome P450–dependent oxidases (CYPs). This effect is thought to be mediated mainly by direct transcriptional activation by drug-responsive nuclear receptors, rather than occurring secondary to depletion of an intracellular regulatory heme pool as a consequence of use of heme for CYP assembly. Induction of ALAS1 is prevented by heme, which acts by destabilizing messenger RNA (mRNA) for ALAS1, by blocking mitochondrial import of pre-ALAS1, by increased proteolysis by Lon peptidase 1, and possibly by inhibiting transcription. In addition, ALAS1 activity is regulated by a transcriptional co-activator, PGC-1α, an effect that forms a link between the rate of hepatic heme synthesis and nutritional status.

The erythroid isoform, ALAS2, is encoded by a gene on chromosome Xq11.21 and is expressed only in erythroid cells. Its activity is regulated by two distinct mechanisms. Transcription is enhanced during erythroid differentiation by the action of erythroid-specific transcription factors, and mRNA concentrations are regulated by Fe. Fe deficiency in erythroid cells promotes specific binding of iron regulatory proteins (IRP) to an iron-responsive element (IRE) in the 5′ untranslated region of ALAS2 mRNA with consequent inhibition of translation.

Function of heme

Heme functions as a prosthetic group in various proteins in which, depending on the function of the protein, the iron shifts freely between the 2 + and 3 + valency states. About 70 to 80% of heme biosynthesis occurs in the bone marrow and approximately a further 15% in the liver. Heme-containing proteins (also called hemoproteins) participate in a variety of redox reactions, including:

  • 1.

    Oxygen transport (by hemoglobin in the blood) and storage (by myoglobin in muscle)

  • 2.

    Mitochondrial respiration (by cytochromes b 1 , c 1 , and a 3 )

  • 3.

    Enzymatic destruction of peroxides (by catalase and peroxidase)

  • 4.

    Drug metabolism (by microsomal cytochrome P-450 mixed function oxidases)

  • 5.

    Desaturation of fatty acids (by microsomal cytochrome b 5 )

  • 6.

    Tryptophan metabolism (by tryptophan oxygenase)

Reactions of nitric oxide (NO) are often mediated by the reaction of heme with NO in control enzymes such as guanylate cyclase.

Other naturally occurring tetrapyrrole derivatives include vitamin B 12 and chlorophyll, each of which contains an atom of chelated cobalt and magnesium, respectively.

Excretion of heme precursors

Typically, only minute quantities of heme precursors accumulate in the body. The route of excretion largely depends on solubility. The porphyrin precursors ALA and PBG are water soluble and are excreted almost exclusively in urine. Uroporphyrinogen, with eight carboxylate groups, is readily water soluble and is also excreted via the kidney. The last intermediate of the pathway, protoporphyrin (and also protoporphyrinogen), which has only two carboxylate groups, is insoluble in water and is excreted in the feces via the biliary tract. The other porphyrins are of intermediate solubility and appear in both urine and feces. Coproporphyrinogen-I is taken up and excreted by the liver in preference to the III isomer, so that coproporphyrinogen-I predominates in feces and coproporphyrinogen-III in urine. All porphyrinogens in the urine or feces are slowly oxidized to the corresponding porphyrins. Reference intervals for porphyrins and their precursors in urine, feces, and blood are given in Table 41.3 and in the Appendix on Reference Intervals. For additional discussion on reference intervals, refer to Chapter 9 .

TABLE 41.3
Adult Reference Intervals
Specimen Analyte Reference Interval (SI Units) Reference Interval (Traditional Units)
Urine Porphobilinogen <1.5μmol/mmol creatinine
<10 μmol/L 		<3.0 mg/g creatinine
<0.23 mg/dL
5-aminolevulinic acid <3.8 μmol/mmol creatinine
<50 μmol/L 		<4.4 mg/g creatinine
<0.66 mg/dL
Total porphyrin <35 nmol/mmol creatinine
20–320 nmol/L 		<216 μg/g creatinine
14–224 μg/L
Uroporphyrin 0.8–3.1 nmol/mmol creatinine 5.9–22.8 μg/g creatinine
Heptacarboxylate porphyrin <0.9 nmol/mmol creatinine <6.3 μg/g creatinine
Coproporphyrin-I 1.2–5.7 nmol/mmol creatinine 6.9–33.0 μg/g creatinine
Coproporphyrin-III 4.8–23.8 nmol/mmol creatinine 27.8–137.7 μg/g creatinine
% Coproporphyrin-III a 68–86 68–86
Feces Total porphyrin 10–200 nmol/g dry wt 6–117 μg/g dry wt
Uroporphyrin <2%
<2.4 nmol/g dry wt 		<2 μg/g dry wt
Heptacarboxylate porphyrin <2%
<0.5 nmol/g dry wt 		<0.4 μg/g dry wt
Hexacarboxylate porphyrin <2%
<0.5 nmol/g dry wt 		<0.4 μg/g dry wt
Pentacarboxylate porphyrin <2%
<0.5 nmol/g dry wt 		<0.3 μg/g dry wt
Isocoproporphyrin <0.5%
Coproporphyrin 2–33%
14–45 nmol/g dry wt 		9–29 μg/g dry wt
Coproporphyrin-III/I ratio 0.3–1.4 0.3–1.4
Protoporphyrin 60–98%
33–170 nmol/g dry wt 		19–96 μg/g dry wt
Erythrocytes Total porphyrin 0.4–1.7 μmol/L erythrocytes 25–106 μg/dL erythrocytes
Laboratories should verify that these ranges are appropriate for use in their own settings. Further guidance is provided in Chapter 9 .
SI , Standard International.

a Percentage of total coproporphyrin.

Once in the gut, porphyrins are susceptible to modification by gut flora. The two vinyl groups of protoporphyrin are reduced to ethyl groups, hydrated to hydroxyethyl groups, or removed, giving rise to a variety of secondary porphyrins. Gut flora can also metabolize heme (whether dietary heme, as components from cells sloughed off from the lining of the gut or heme resulting from gastrointestinal bleeding) to produce a variety of dicarboxylic porphyrins. In addition, some bacteria are capable of de novo synthesis of porphyrins.

Porphyrias

The porphyrias are a group of metabolic disorders that result from decreased or, in one rare form of EPP, increased activities of the enzymes of heme biosynthesis ( Table 41.4 ). All are inherited in monogenic patterns, apart from some forms of PCT and rare erythropoietic porphyrias associated with malignant myeloid disorders. Each porphyria is defined by the association of characteristic clinical features with a specific pattern of heme precursor accumulation that reflect the buildup of substrates upstream of the enzyme that is partially deficient, or of a secondarily rate-limiting enzyme ( Table 41.5 ). Defects that cause porphyria have been identified in all enzymes of the pathway except for ALAS1. Mutations that decrease ALAS2 activity cause nonsyndromic X-linked sideroblastic anemia; those that increase activity cause X-linked erythropoietic protoporphyria (XLEPP).

TABLE 41.4
Main Types of Human Porphyria
Disorder Defective Enzyme Prevalence a (per million) Neurovisceral Crises Skin Lesions Inheritance
Acute Porphyrias
ADP ALAD − + − AR
AIP HMBS 5.9 + − AD
HCP CPOX 0.9 + + b , c AD
VP PPOX 3.2 + + b , c AD
Nonacute Porphyrias
CEP UROS 0.3 − + c AR
PCT UROD 40 − + c Complex (20% AD)
EPP FECH 9.2 − + d AR
XLEPP ALAS2 0.15 − + d XL
AD, Autosomal dominant; ADP , ALA dehydratase deficiency porphyria; AIP , acute intermittent porphyria; ALA , 5-aminolevulinate; ALAD , 5-aminolevulinic acid dehydratase; ALAS , 5-aminolevulinate synthase; AR, autosomal recessive; CEP , congenital erythropoietic porphyria; EPP , erythropoietic protoporphyria; FECH , ferrochelatase; HCP , hereditary coproporphyria; HMBS , hydroxymethylbilane synthase; PCT , porphyria cutanea tarda; PPOX , protoporphyrinogen oxidase; UROD , uroporphyrinogen decarboxylase; UROS , uroporphyrinogen-III synthase; VP , variegate porphyria; XL , X-linked; XLEPP , X-linked erythropoietic protoporphyria.

a Estimated prevalence of clinically overt disease. , ,

b Skin lesions and neurovisceral crises may occur alone or together.

c Fragile skin, bullae.

d Acute photosensitivity without fragile skin, bullae.

TABLE 41.5
Porphyrias: Patterns of Overproduction of Heme Precursors During Clinically Overt Phase of Disease
Porphyria Urine PBG/ALA Urine Porphyrins Fecal Porphyrins Erythrocyte Porphyrins Plasma Fluorescence Emission Peak
ADP ALA Copro-III Not increased ZPP −
AIP PBG>ALA Mainly uroporphyrin from PBG Normal or increased a
Copro-III/I ratio normal		 Not increased 615–622 nm b
CEP Not increased Uro-I, Copro-I Copro-I ZPP, Proto, Copro-I, Uro-I 615–620 nm
PCT Not increased Uro, Hepta c Isocopro, Hepta, Penta Not increased 615–622 nm
HCP PBG>ALA d Copro-III, uroporphyrin from PBG Copro-III, Copro-III/I ratio increased Not increased 615–622 nm b
VP PBG>ALA d Copro-III, uroporphyrin from PBG Proto IX>Copro-III
X-porphyrin
Copro-III/I ratio increased e 		Not increased 624–628 nm
EPP Not increased Not increased ±Proto f Metal-free proto 626–634 nm g
XLEPP Not increased Not increased ±Proto Metal free proto, ZPP h 626–634 nm g
AIP , Acute intermittent porphyria; ALA , 5-aminolevulinate; ALAD , 5-aminolevulinic acid dehydratase; CEP , congenital erythropoietic porphyria; EPP , erythropoietic protoporphyria; HCP , hereditary coproporphyria; PBG , porphobilinogen; PCT , porphyria cutanea tarda; UROD , uroporphyrinogen decarboxylase; UROS , uroporphyrinogen-III synthase; VP , variegate porphyria; XLEPP , X-linked erythropoietic protoporphyria.

a Slight increase only unless uroporphyrin is present.

b Not always increased.

c Other methylcarboxylate-substituted porphyrins are increased to a smaller extent; uroporphyrin is a mixture of type I and III isomers; heptacarboxylate porphyrin is mainly type III.

d PBG and ALA may be normal when only skin lesions are present.

e Coproporphyrin-III/I ratio increased, but usually less than in overt HCP.

f Not increased in approximately 40% of patients.

g Protoporphyrin (Proto) bound to globin. If hemolysis is present the peak shifts to the left (i.e., at 626–628 nm or is quenched).

h Zn-protoporphyrin (ZPP) 20 to 60% of total protoporphyrin.

The porphyrias are characterized clinically by two main features: skin lesions on sun-exposed areas and acute neurovisceral attacks, typically comprising abdominal pain, peripheral neuropathy, and mental disturbance. The skin lesions are caused by porphyrin-catalyzed photodamage, of which singlet oxygen is the main mediator. Acute attacks are associated with increased formation of ALA, and consequently, PBG from induced activity of hepatic ALAS1 and partial hepatic heme deficiency, often in response to induction of hepatic CYPs by drugs and other factors. The relationship of these biochemical changes to the neuronal dysfunction that underlines all clinical features of an acute attack is uncertain. , The observation that correction of the metabolic defect in the liver by transplantation is curative, and that domino transfer of the affected organ to an unaffected recipient causes acute attacks indistinguishable from those suffered by the donor, suggests that their primary cause is release of a hepatic neurotoxin, probably ALA, which may act by interfering with myelin formation and or neuronal gamma aminobutyric acid (GABA) signaling.

In Table 41.4 , the porphyrias are classified as acute, in which acute neurovisceral attacks occur, or as nonacute. Porphyrias may also be classified as hepatic or erythropoietic, according to the main site of overproduction of heme precursors. The main hepatic porphyrias are acute intermittent porphyria (AIP), HCP, VP, and PCT. Erythropoietic porphyrias include CEP, EPP, and XLEPP. Porphyrias may also be classified as cutaneous or acute porphyrias; however, it should be noted that even with these classifications, some porphyrias are difficult to place.

Acute porphyrias

The four acute porphyrias include: aminolevulinate dehydratase deficiency porphyria (ADP), AIP, VP, and HCP. These disorders are autosomal dominant, except for the very rare disorder, ADP, which is autosomal recessive.

Biochemistry and molecular genetics

The inherited defect in each of the autosomal dominant acute porphyrias (see Table 41.4 ) is a mutation in one copy of a gene that encodes an enzyme involved in heme synthesis, resulting in complete or near complete enzyme deficiency from that allele. Enzyme activities are therefore half of normal in all tissues in which they are expressed, reflecting the activity of the normal gene trans to the mutant allele. Heme supply is maintained at normal or near-normal concentration by upregulation of ALAS1, with a consequent increase in the substrate concentration of the defective enzyme. These compensatory changes vary among tissues; they are most prominent in the liver and are undetectable in most other organs. The changes also vary among individuals. Thus in all autosomal dominant acute porphyrias, some individuals show no evidence of overproduction of heme precursors, and others have biochemically manifest disease with or without clinical symptoms.

Low clinical penetrance (the frequency of expression of an allele when it is present in the genotype) is a prominent feature of all the autosomal dominant acute porphyrias. Family studies indicate that many affected individuals are asymptomatic throughout life. Surveys of blood donors suggest that the AIP gene may be present in as many as 1 in 1675 of the population. More recently, two studies interrogated genomic or exomic databases to identify variants in the HMBS gene in Caucasian populations. , The missense variants were then assessed by in silico pathogenicity prediction programs and in vitro enzyme expression studies to determine if these HMBS variants were likely pathogenic or benign. The results showed that the prevalence of deleterious HMBS mutations in the general population was 1:1782 54 and 1:1299. This estimated prevalence is remarkably high and is consistent with the findings of the first study from blood donors. As a consequence, the penetrance of the disease in the general population is estimated at 0.5 to 1%. As AIP is a monogenic disorder, this extremely low penetrance suggests a critical role for modifying factors (environmental and/or genetic) in predisposing heterozygotes to acute attacks. This hypothesis is reinforced by French AIP family studies. Penetrance was estimated at 22.9% in families with AIP contrasting with the penetrance in the general population. Intrafamilial correlation studies and heritability estimation showed that genetic factors (linked or not to HMBS) , as well as shared environmental factors, modulate the risk of having acute attacks in carriers of deleterious HMBS mutations. The high estimate of heritability highlights the importance of performing family studies and offering predictive testing.

For all three disorders, the gene frequency is sufficiently high for rare “homozygous” variants of AIP, HCP, or VP to occur in individuals who are homozygotes or compound heterozygotes for disease-specific mutations, , and for the same person to have two separate types of porphyria. Approximately 25% of patients with overt acute porphyria have no family history of the disease. Such sporadic presentation is a reflection of the high prevalence and low penetrance of mutations in the population; acute porphyria caused by de novo mutation is uncommon.

All the autosomal dominant acute porphyrias show extensive allelic heterogeneity. More than 490 disease-specific mutations have been identified in the HMBS gene in AIP, approximately 75 in the CPOX gene in HCP, and more than 180 in the PPOX gene in VP. Approximately 5% of families with AIP have HMBS mutations that only impair expression of the ubiquitous isoform and therefore do not decrease activity in erythroid cells. All other mutations in the autosomal dominant acute porphyrias affect all tissues. Most are restricted to one or a few families, but founder mutations are present in some populations and explain the high frequency of VP in South Africans of Dutch descent and of AIP in Sweden. ,

Clinical features

The life-threatening, acute neurovisceral attacks that occur in AIP, VP, and HCP are clinically identical. , , Acute attacks are more common in women, usually occurring first between the ages of 15 and 40 years, and are rare before puberty. The main clinical features are summarized in Table 41.6 . The clinical features of ADP, which has been reported in only six patients, are similar but may start in childhood.

TABLE 41.6
Clinical Features of an Acute Neurovisceral Attack of Porphyria
Data from Harper P, Sardh E. Management of acute intermittent porphyria. Exp Opin Orphan Drugs 2014;2:349–368.
Symptom/Sign Percent of Acute Attacks
Abdominal pain 85–90
Nonabdominal pain 25–70
Vomiting and nausea 30–90
Constipation/diarrhea 50–80
Psychologic symptoms (insomnia, anxiety, depression, confusion, hallucinations) 20–85
Acute encephalopathy (headache, somnolence, seizures, altered consciousness) 2–20
Motor neuropathy (muscle weakness, pain, low/absent tendon reflexes) 10–90
Hemi/tetraparesis 30–40
Respiratory paralysis 10–55
Sensory neuropathy 10–40
Hypertension 40–75
Tachycardia (>80/min) 30–85
Hyponatremia (<135 nmol/L) 30–60

Acute attacks almost always start with abdominal pain that rapidly becomes very severe but is not accompanied by other signs of an acute surgical condition. , Pain may also be present in the back and thighs and may occasionally be most severe in these regions. Signs of autonomic neuropathy, such as vomiting, constipation, tachycardia, and hypertension, are frequent. When convulsions occur, they may be a consequence of hyponatremia or secondary to central nervous system involvement and may be associated with posterior reversible encephalopathy syndrome, a serious complication of longstanding attacks. , Pain may dissipate within a few days, but in severe cases, a predominant motor neuropathy develops that may progress to flaccid quadriparesis. Persistent pain and vomiting may lead to weight loss and malnutrition. The acute phase may be accompanied by mental confusion with abrupt changes in mood, hallucinations, and other psychotic features. However, these mental disturbances disappear with remission. Persistent psychiatric illness is not a feature of the acute porphyrias, although mild anxiety or depression may be present in some patients. Abdominal pain usually resolves within 2 weeks, but recovery from neuropathy may take many months, and is not always complete. Most patients have one or a few attacks followed by complete recovery and prolonged remission. Approximately 5% have repeated acute attacks, which in women may be premenstrual and this subgroup of patients reports a high disease burden and diminished quality of life. Precipitating factors have been identified in approximately two-thirds of patients who present with acute attacks. The most important are drugs, alcohol (especially binge drinking), the menstrual cycle, calorie restriction, infection, and stress. Acute attacks may complicate a small proportion of pregnancies in affected patients. Drugs are frequent precipitants of acute attacks in VP, and hormonal factors appear to be more important in AIP. Drugs known to provoke acute attacks include barbiturates, sulfonamides, progestogens, and some anticonvulsants, but many others have been implicated in the precipitation of acute attacks.

Acute attacks are always accompanied by increased excretion of PBG and ALA in the urine, as a consequence of the primary deficiency of HMBS activity in AIP, and secondary to allosteric inhibition of HMBS activity in HCP and VP. The diagnosis of an acute attack is therefore based on demonstrating increased PBG excretion in a random urine sample. The patterns of porphyrin accumulation specific to each acute porphyria are described in detail in the section Differentiation Among the Acute Porphyrias and in Table 41.5 .

Where porphyrins also accumulate, as is the case in HCP and VP, skin lesions similar to those of PCT and other bullous porphyrias may occur. These are present in approximately 80% of patients with clinically manifest VP (see Table 41.4 ). Approximately 60% of patients with this condition present with skin lesions alone. The skin is less commonly affected in HCP; skin lesions without an acute attack are uncommon and are usually provoked by intercurrent cholestasis.

Long-term complications of acute porphyria include chronic renal failure, hypertension, and primary hepatocellular carcinoma. ,

Treatment

As soon as an attack of acute porphyria is suspected as the cause of illness, drugs and other potential provoking agents should be withdrawn, and supportive treatment should begin using drugs that are known to be safe. , Opiates are usually required to control pain and an antiemetic such as ondansetron may be required to control nausea and vomiting. Patients with acute porphyria are prone to severe hyponatremia and are particularly susceptible to cerebral edema. Therefore careful administration of any intravenous fluids, with avoidance of hypotonic solutions, is essential. If hyponatremia develops, it should be corrected slowly to avoid osmotic demyelination (for more details on hyponatremia and its treatment, see Chapter 50 ). Adequate caloric intake must be maintained, preferably by giving carbohydrate-rich supplements orally or if necessary via a nasogastric tube (see below). When vomiting prevents enteral administration, dextrose given intravenously as a 10% solution in saline should suffice initially. In prolonged acute attacks, total parenteral nutrition should be considered.

Unless the attack is mild and is clearly resolving, specific treatment with intravenous human hemin should be started as soon as the diagnosis has been established. , This treatment increases the concentration of heme in the liver, thus decreasing the activity of ALAS1 and the formation of ALA and PBG. Treatment does not require biochemical monitoring as clinical improvement is the required endpoint. However, heme administration will not reverse an established neuropathy. If heme preparations are not available, carbohydrate loading can ameliorate an acute attack, probably by decreasing ALA synthase activity, , but this treatment is less effective than intravenous heme.

Repeated attacks are difficult to control. Cyclic premenstrual attacks in women may be prevented by suppression of ovulation with gonadotropin-releasing hormone (gonadorelin) analogs, but many patients require repeated courses of intravenous heme or regular intermittent heme infusions to reduce the frequency and severity of attacks. , , Orthotopic liver transplantation leads to immediate and prolonged remission with restoration of PBG excretion to normal.

Givosiran (ALN-AS1) is a recently approved treatment that prevents acute attacks by targeting hepatic ALAS1 activity. It consists of a N -acetylgalactosamine-conjugated liver-targeted small interfering RNA that binds to and silences ALAS1 mRNA, reducing translation and expression of the ALAS1 protein. When compared to placebo, givosiran reduces ALA and PBG urinary excretion to near normal concentrations and reduces the frequency of acute attacks. Adverse events reported from the clinical trial on recurrent acute porphyria patients treated with givosiran included increased liver enzymes and chronic kidney disease.

Management of families

Diagnosis of autosomal dominant acute porphyria should be followed by investigation of the patient’s family to identify affected, often asymptomatic, relatives, so that they can be advised to avoid drugs and other factors known to provoke potentially fatal acute attacks and recent data indicate that a predictive diagnosis may reduce the risk of symptomatic disease. Furthermore, a presymptomatic diagnosis also has the benefit that specific treatment can be started promptly if an attack does develop without a delay while a diagnosis is sought. Although attacks are rare before puberty, children should be tested at as young an age as is practicable to ensure that their status is known by the time they reach puberty and to enable the very low risk for affected children to be further reduced. Counseling to reduce the risk of an acute attack should include comprehensive information about the disease, including specific advice to guide selection of safe drugs, as well as provision of jewelry, an identity card, or some other means to identify the individual as having a predisposition for acute porphyria. Where available, patients should be made aware of the relevant national patient support group. A list of online resources for patients and professionals can be found in Box 41.1 .

BOX 41.1
Online Resources

  • American Porphyria Foundation: http://www.porphyria foundation.com . Provides information for patients and professionals, including a searchable drug database for acute porphyrias.

  • European Porphyria Network: http://www.porphyria.eu . A multilingual website providing information for patients and professionals. Provides contact details for porphyria specialist centers. Specific guidance for certain clinical areas or topics are covered.

  • The Drug Database for Acute Porphyria: http://www.drugs -porphyria.org . The database is searchable and provides evidence-based monographs for each drug that has been assessed with regard to safety in acute porphyrias.

  • South African Porphyria Centre: http://www.porphyria.uct .ac.za . Provides information for patients and professionals. Section on prescribing in acute porphyrias includes advice on specific disorders, including malaria, tuberculosis, and HIV.

POINTS TO REMEMBER

Acute porphyrias

  • Present clinically after puberty.

  • Low clinical penetrance means that there is often no family history.

  • Clinical effects are due to autonomic, central, and motor neuropathy.

  • Common precipitants are drugs, hormonal fluctuations, excess alcohol consumption, starvation, infection, and stress.

  • Acute attacks are diagnosed by demonstrating increased porphobilinogen excretion in a random urine sample.

  • Mild hyponatremia is common.

  • Specific treatment is with intravenous human hemin, or givosiran for severe recurrent attacks.

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