Chapter 58: The Porphyrias

The porphyrias are a group of metabolic diseases resulting from derangements, usually of a genetic nature, in the activity of specific enzymes in the heme biosynthetic pathway, leading to overproduction and accumulation of pathway intermediates. Symptoms of these diseases can be neurologic, photocutaneous, or both. The intermediates that accumulate include porphyrins and the porphyrin precursors δ-aminolevulinic acid (ALA) and porphobilinogen (PBG) and their derivatives. Patterns of these substances in plasma, erythrocytes, urine, and feces are characteristic for each porphyria, and are the basis for screening tests and more comprehensive biochemical characterization.

Porphyrias are classified as either erythropoietic or hepatic, depending on the principal site of accumulation of pathway intermediates. The erythropoietic porphyrias are congenital erythropoietic porphyria (CEP), which is very rare, erythropoietic protoporphyria (EPP), the third most common porphyria and the most common in children, and X-linked protoporphyria (XLP), which has the same phenotype as EPP but is less common. Hepatic porphyrias include the acute porphyrias, which cause neurologic symptoms usually in the form of acute attacks, and porphyria cutanea tarda (PCT), which is the most common of the porphyrias, and causes chronic blistering lesions on sun-exposed areas of the skin. The acute porphyrias include ALA dehydratase deficiency porphyria (ADP), acute intermittent porphyria (AIP), hereditary coproporphyria (HCP), and variegate porphyria (VP). VP, and less commonly HCP, can also cause skin manifestations identical to those in PCT.

A type of porphyria is associated with loss-of-function mutations of seven of the eight enzymes in the heme biosynthetic pathway (Table 58–1 and Fig. 58–1) PCT is primarily caused by an acquired deficiency of the fifth pathway enzyme, with heterozygous mutations of that enzyme contributing in some cases. Gain-of-function mutations of ALAS2, the erythroid form of the first pathway enzyme, cause XLP, whereas loss-of-function mutations of this enzyme cause X-linked sideroblastic anemia (Chap. 59). Table 58–2 summarizes the major clinical and laboratory features of the porphyrias.

A case of CEP reported by Schultz in 1874 was the first description of porphyria in the literature. This case was a 33-year-old man with photosensitivity since age 3 months, anemia, splenomegaly, red-wine-colored urine as a result of a pigment resembling hematoporphyrin, and brown-colored bones at autopsy.^1,2 In 1898, T. McCall Anderson described two brothers (ages 23 and 26 years) who most likely had CEP,³ and suffered from hydroa aestivale, with red urine, pruritus and blistering of sun-exposed skin, especially in summer, leading to extensive scarring and mutilation of the ears and nose (see Chap. 58, Fig. 58–5). Using available methods, their urine was also demonstrated to contain a substance related to hematoporphyrin.⁴ In 1889, Stokvis first described a case of acute porphyria in an elderly woman who developed dark-red urine and later died after taking sulphonal, a drug related to the barbiturates.⁵

Hans Günther⁶ published a monograph on porphyrins in 1911 and classified porphyrias into four groups: (1) those that have an acute onset without association with drug ingestion, (2) those that are caused by sulphonal or trional, (3) hematoporphyria congenita, and (4) chronic hematoporphyria. The first two groups correspond to the acute porphyrias, which may present with attacks sometimes related to ingestion of certain drugs, the second group to CEP and hepatoerythropoietic porphyria (HEP), and the fourth to PCT. In 1923, Archibald Garrod proposed the term inborn errors of metabolism for a number of inherited metabolic disorders, including the porphyrias.⁷

Sachs noted an Ehrlich-positive chromogen that was not urobilinogen in urine of patients with acute porphyria in 1931. In the late1930s, Waldenström noted that excretion of this chromogen was an autosomal dominant trait in AIP families, which he identified as PBG in 1939.⁸ The classification of porphyrias as erythropoietic and hepatic was proposed in 1954 by Schmid, Schwartz, and Watson.⁹ An epidemic of hexachlorobenzene-induced PCT in eastern Turkey in 1957^10,11 provided the foundation for the development of animal models of this disorder using halogenated polyaromatic hydrocarbons.^12,13 Strand and coworkers described the enzyme deficiency in AIP for the first time in 1970,¹⁴ and Bonkovsky and coworkers first reported treatment of a porphyria patient with hemin in 1971.¹⁵ In the past several decades, the enzymes of the heme biosynthetic pathway have been defined in terms of their amino acid composition, genomic and complementary DNA (cDNA) sequences, and crystal structures. Erythroid-specific and housekeeping transcripts have been described for at least four enzymes in the pathway, and progress made in understanding the regulation of heme synthesis in specific tissues, especially the marrow and liver. Multiple mutations have been described in each of the human porphyrias, and some specific treatments introduced.

HEME

Heme (iron protoporphyrin IX; Fig. 58–2) is essential for all cells and functions as the prosthetic group of numerous hemoproteins such as hemoglobin, myoglobin, respiratory cytochromes, cytochromes P450 (CYPs), catalase, peroxidase, tryptophan pyrrolase, and nitric oxide synthase. Approximately 85 percent of heme is synthesized in the marrow to meet the requirement for hemoglobin formation; the remainder is synthesized largely in the liver.¹⁶ Most heme synthesized in the liver is required for CYPs, which are located primarily in the endoplasmic reticulum where they turn over rapidly and oxidize a variety of chemicals, including drugs, environmental carcinogens, endogenous steroids, vitamins, fatty acids, and prostaglandins.¹⁷

The term heme may refer more specifically to ferrous protoporphyrin IX, and is readily oxidized in vitro to hemin, that is, ferric protoporphyrin IX. Hemin has one residual positive charge and is usually isolated as a halide, most commonly as the chloride. In alkaline solution the halide is replaced by a hydroxyl ion to form hematin (Fig. 58–3). Heme can form further hexacoordinated complexes with nitrogenous bases to form a hemochrome or hemochromogen; for example, pyridine hemochromogen is useful for identification and quantification of heme and hemoproteins. In medicine, hemin is also a generic term for heme preparations used as intravenous therapies for acute porphyrias, such as lyophilized hematin and heme arginate.

The ferrous iron atom (Fe²⁺) in heme has six electron pairs, of which four are bound to the pyrrolic nitrogens of the porphyrin macrocycle, leaving two unoccupied electron pairs, one above and the other below the plane of the porphyrin ring. In hemoglobin, one of these pairs is coordinated with a histidine residue of the globin chain. The other coordination site in deoxyhemoglobin is protected from oxidation by the nonpolar environment of surrounding amino acid residues, and is available to bind molecular oxygen for transport from the lung to other tissues. To reversibly bind oxygen, the iron in hemoglobin must be in the ferrous state. Methemoglobin (oxidized hemoglobin) that is generated in erythrocytes is continuously reduced to ferrous hemoglobin by the reduced form of nicotinamide adenine dinucleotide–cytochrome b₅ reductase–cytochrome b₅ system (Chap. 50).

Heme Biosynthesis

Figure 58–4 shows the enzymatic steps involved in heme biosynthesis in eukaryotic cells. The first and last three enzymes are mitochondrial and the intermediate four are cytosolic. Erythroid heme synthesis occurs in marrow erythroblasts and reticulocytes, which contain mitochondria. Circulating erythrocytes lack mitochondria and no longer synthesize heme. They contain residual cytosolic enzymes of the heme biosynthetic pathway, zinc protoporphyrin and a small amount of metal-free protoporphyrin. These enzyme activities and protoporphyrin decline during the life span of erythrocytes in the circulation.

δ-Aminolevulinate Synthase (Succinyl Coenzyme A: Glycine C-Succinyl Transferase; Enzyme Commission (EC) 2.3.1.37)

The first enzyme in the heme biosynthetic pathway catalyzes the condensation of glycine and succinyl coenzyme A (CoA) to form ALA (see Fig. 58–4, step 1), and requires pyridoxal 5′-phosphate as a cofactor. δ-Aminolevulinic acid synthase (ALAS) in mammalian cells is localized to the mitochondrial matrix.¹⁸ The enzyme is synthesized as a precursor protein in the cytosol and transported into mitochondria. Two separate ALAS genes encode housekeeping (tissue nonspecific) and erythroid-specific forms of the enzyme (ALAS1 and ALAS2, respectively).¹⁹ The gene locus for human ALAS1 is at 3p.21, and for ALAS2 it is at Xp11.2.¹⁹ The human ALAS2 gene encodes a precursor of 587 amino acids, with an Mr of 64,600 Da. Nucleotide sequences for the ALAS2 and the ALAS1 isoforms are approximately 60 percent similar. No homology is observed between the aminoterminal regions, whereas high homology (approximately 73 percent) is seen after residue 197 of the housekeeping form.²¹ The two human ALAS genes appear to have evolved by duplication of a common ancestral gene that encoded a primitive catalytic site. Subsequently the DNA sequences were modified to encode gene-specific regulatory regions, functioning mostly at the amino termini.²²

The promoter in the human ALAS2 gene contains several putative erythroid-specific cis-acting elements including both a GATA-1 and an NF-E2 binding site.^22,23 Both GATA-1 and NF-E2 are erythroid transcription factors that also bind other DNA sites, such as the promoters of the human β-globin, porphobilinogen deaminase (PBGD), and uroporphyrinogen synthase (UROS) genes.²⁴ Thus, expression of ALAS2 is under the regulatory influence of erythroid transcription factors such as GATA-1 and is coordinated with expression of other genes involved in hemoglobin synthesis. Additionally, ALAS2 mRNA contains an iron-responsive element in its 5′-untranslated region,²³ similar to mRNAs encoding ferritin and the transferrin receptor (Chap. 42).²⁵ Gel retardation analysis shows that the iron-responsive element in ALAS2 mRNA is functional and suggests that translation of the erythroid-specific mRNA is directly linked to the availability of iron, or heme, in erythroid cells.²⁶

In the liver, synthesis of ALAS1 is induced by a variety of chemicals, including drugs and steroids that increase the demand for hepatic CYPs. Upstream enhancer elements in the ALAS1 gene and certain hepatic CYP genes respond to inducing chemicals and interact with the pregnane X receptor (PXR).²⁷ Hemin represses synthesis of ALAS1 in liver,²⁸ accounting for the beneficial effects of intravenous treatment of the acute porphyrias with hemin. At higher concentrations, heme induces heme oxygenase, resulting in its enhanced catabolism.²⁹ Thus, hepatic heme availability is balanced between the rate of synthesis controlled primarily by ALAS1 and the rate of degradation controlled by heme oxygenase, both of which are regulated by heme at different intracellular concentrations. ALAS1 is also upregulated by the peroxisomal proliferator-activated cofactor 1α (PGC-1α),³⁰ a coactivator of nuclear receptors and transcription factors. Transcriptional regulation of ALAS1 by PGC-1α is mediated by interaction of NRF-1 (nuclear regulatory factor 1) and FOXO-1 (a forkhead family member) with the ALAS1 promoter.³¹ When glucose levels are low, transcription of PGC-1α is upregulated,³² in turn increasing ALAS1, which might precipitate an attack of acute porphyria in an individual with the appropriate inherited enzyme deficiency. Thus, upregulation of PGC-1α provides an explanation for the induction of acute attacks of porphyria with fasting, as well as the therapeutic value of glucose loading.

Regulation of heme synthesis in erythroid cells is distinct from the liver. ALAS2 expression in erythroid cells is increased during erythroid differentiation when heme synthesis is increased.^33,34 Experimentally, ALAS2 is often upregulated by heme, whereas in liver ALAS1 is downregulated by heme. The β subunit of human ATP-specific succinyl CoA synthetase (SCS-βA) associates with human ALAS2 but not with ALAS1, and thereby contributes to heme synthesis in the marrow.³⁵

More than 20 ALAS2 mutations are associated with X-linked sideroblastic anemia (Chap. 59); many are in exon 9, which contains the binding site for pyridoxal 5′-phosphate (K391), and these cases are typically responsive to high doses of pyridoxine. At least one mutant enzyme (D190V) in a patient with pyridoxine-refractory X-linked sideroblastic anemia,³⁶ failed to associate with SCS-βA, whereas other ALAS2 mutants did not have this property. The mature D190V mutant protein, but not its precursor protein, underwent abnormal processing; indicating that appropriate association of SCS-βA and ALAS2 is necessary for functioning of ALAS2 in mitochondria.³⁶ Gain-of-function mutations of ALAS2 have been identified in patients with XLP.³⁷

δ-Aminolevulinate Dehydratase (Porphobilinogen Synthase; δ-Aminolevulinate Hydrolase; EC 4.2.1.24)

ALA dehydratase (ALAD) is a cytosolic enzyme that catalyzes the condensation of two molecules of ALA to form the monopyrrole PBG, with removal of two molecules of water (see Fig. 58–4, step 2). The enzyme functions as a homooctamer, and requires intact sulfhydryl groups and zinc for activity. ALAD activity is inhibited by sulfhydryl reagents³⁸ and by lead, which displaces zinc.³⁹ In lead poisoning (Chap. 52), erythrocyte ALAD activity is markedly inhibited, urinary ALA and coproporphyrin excretion increased, erythrocyte zinc protoporphyrin elevated, and neurologic symptoms resemble those seen in acute porphyrias.⁴⁰ 4,6-Dioxoheptanoic acid (succinylacetone) is a substrate analogue and potent inhibitor of ALAD,^41,42 and is a byproduct of the enzyme deficiency in hereditary tyrosinemia type I. This substance is found in urine and blood of patients with this disease, who may also have increased ALA and symptoms resembling acute porphyrias.⁴³

Human ALAD mRNA has an open-reading frame of 990 bp, encoding a protein with an Mr of 36,274.⁴⁴ Sequences known to be essential for enzymatic activity, are those for the active site lysine residues and for the cysteine- and histidine-rich zinc binding sites. The gene for human ALAD is localized to chromosome 9p34.⁴⁵

Studies using [¹⁴C]-ALA have shown that of the two ALA molecules used as substrate, the ALA molecule contributing the propionic acid side is initially bound to the enzyme.³⁸ The tertiary structure of the yeast ALAD has been solved to 2.3-Å resolution, revealing that each subunit adopts a triosephosphate isomerase barrel fold with a 39-residue N-terminal arm. Pairs of monomers then wrap their arms around each other to form compact dimers, and these dimers associate to form a 422 symmetric octamer.⁴⁶ All eight active sites are on the surface of the octamer and possess two lysine residues (210 and 263). The Lys263 residue forms a Schiff base link to the substrate. The two lysine side chains are close to two zinc binding sites. One binding site is formed by three cysteine residues; the other involves Cys234 and His142.

Although there are no tissue-specific ALAD isozymes, the ALAD mRNA has two splice variants, a housekeeping (1A) and an erythroid-specific (1B) form.⁴⁵ In both humans and mice, the promoter region upstream of exon 1B contains GATA-1 sites, providing for significant tissue-specific control of these transcripts.⁴⁷

The human enzyme is polymorphic with two common alleles that occur in three combinations (1–1, 1–2, and 2–2).⁴⁴ The allele 2 sequence differs from allele 1 only by a G→C transversion of nucleotide 177 in the coding region, resulting in replacement of lysine by asparagine, a more electronegative amino acid.⁴⁸ ALAD exists primarily as a homooctamer. Mutations associated with ALAD porphyria favor formation of the less active hexamer.^49,50

Porphobilinogen Deaminase (Hydroxymethylbilane Synthase; Porphobilinogen Ammonia-Lyase [Polymerizing], EC 4.3.1.8)

The fourth enzyme in the heme biosynthetic pathway catalyzes the deamination and condensation of four molecules of PBG to yield the linear tetrapyrrole hydroxymethylbilane (HMB; see Fig. 58–4, step 3).⁵¹ PBG deaminase was previously known as uroporphyrinogen I synthase, and the enzyme activity is commonly measured in the laboratory after converting HMB to uroporphyrin I.

This enzyme has a unique cofactor, which is a dipyrromethane that binds the pyrrole intermediates at the catalytic site until six pyrroles (including the dipyrrole cofactor) are assembled in a linear fashion, after which the tetrapyrrole HMB is released.⁵² The apo-deaminase generates the dipyrrole cofactor to form the holo-deaminase, and this occurs more readily from HMB than from PBG.⁵³ High concentrations of PBG may inhibit formation of the holo-deaminase.

The gene encoding human PBG deaminase maps to chromosome 11q23→11qter,⁵⁴ and consists of 15 exons spread over 10 kb of DNA.⁵⁵ Distinct erythroid-specific and housekeeping isoforms are produced through alternative splicing of two distinct primary mRNA transcripts arising from two promoters.⁵⁶ The housekeeping promoter is upstream of exon 1 and is active in all tissues, while the erythroid-specific promoter, which is upstream of exon 2, is active only in erythroid cells. The human housekeeping and erythroid-specific enzymes isoforms contain 361 and 344 amino acid residues, respectively.⁵⁷ Of the additional 17 residues at the N-terminal end of the housekeeping form, 11 are encoded by exon 1, and 6 by a short segment of exon 3 that immediately precedes a methionine codon that initiates translation of the erythroid isoform. Erythroid-specific trans-acting factors, such as GATA-1 and NF-E2, recognize sequences in the erythroid promoter.⁵⁸ A 1320-bp stretch of perfect identity is present between the erythroid and the nonerythroid PBG deaminase, but with a mismatch in the first exon at their 5′ extremities. An additional inframe AUG codon present 51 bp upstream from the initiating codon of the erythroid cDNA accounts for the additional 17 amino acid residues at the N-terminus of the housekeeping isoform. Accordingly, a splice site mutation at the last position of exon 1, or a base transition in intron 1, in certain patients with AIP results in decreased PBG deaminase expression in nonerythroid tissues including the liver, but not in erythroid cells, because transcription of the gene in erythroid cells starts downstream of the site of the genetic lesion.⁵⁹

Uroporphyrinogen III Synthase (Uroporphyrinogen III Cosynthase; EC 4.2.1.75)

UROS, a cytosolic enzyme, catalyzes the formation of uroporphyrinogen III from HMB. The process involves an intramolecular rearrangement that affects only ring D of the porphyrin macrocycle (see Fig. 58–4, step 4).⁵¹ In the absence of this enzyme, HMB spontaneously forms the ring structure uroporphyrinogen I, which, like the III isomer, is a substrate for uroporphyrinogen decarboxylase (UROD). However, because coproporphyrinogen I is not a substrate for coproporphyrinogen oxidase (CPO) the type I porphyrinogen isomers are not further metabolized, and only the type III isomers are precursors of heme.

The UROS cDNA has an open-reading frame of 798 bp, and the predicted protein product consists of 263 amino acid residues, with an Mr of 28,607 Da.²⁰ The amino acid compositions of the hepatic and the purified erythrocyte enzyme are essentially identical, and no tissue-specific isoforms have been described.

The interspecies homology for the UROS proteins is below 10 percent, depending on the number and divergence of the species being compared. However, the crystal structures of uroporphyrinogen III synthase from human and Thermus thermophilus have been solved and are very similar.^60,61 The structure supports a mechanism that includes the formation of a spirolactam intermediate by positioning the A and D rings such that the noncatalytic closure, to form uroporphyrinogen I, is not possible.⁶¹

Uroporphyrinogen Decarboxylase (EC 4.1.1.37)

UROD is a cytosolic enzyme that catalyzes the sequential removal of the four carboxylic groups of the carboxymethyl side chains in uroporphyrinogen to yield coproporphyrinogen (see Fig. 58–4, step 5). The four successive decarboxylation reactions yield 7-, 6-, 5-, and 4-carboxylated porphyrinogens. Increased amounts of these intermediates can be identified as the corresponding oxidized porphyrins in liver, plasma, urine and stool in human PCT and in laboratory animal models in which hepatic UROD is inhibited. An inhibitor of UROD activity, a partially oxidized substrate molecule,⁶² is produced in liver of experimental animals in response to halogenated polycyclic aromatic hydrocarbons such as hexachlorobenzene, dioxin, and polychlorinated biphenyls, as well as other compounds able to activate the Ah receptor.⁶³ This porphomethene compound is believed to explain UROD inhibition in human PCT.⁶² Human UROD is a 42-kDa polypeptide encoded by a single gene containing 10 exons spread over 3 kb and functions as a homodimer.⁶⁴ The gene has been mapped to chromosome 1p34.⁶⁵

Although the UROD gene contains two initiation sites, both sites are used with the same frequencies in all tissues, and the gene is transcribed into a single mRNA.⁶⁶ Recombinant human UROD purified to homogeneity has been crystallized, and its crystal structure was determined at 1.60-Å resolution.⁶⁷ The purified protein is a dimer with a dissociation constant of 0.1 μM.⁶⁷ The 40.8-kDa polypeptide forms a single domain with a distorted (β/α)₈-barrel fold, and a distinctive deep cleft for the enzyme’s active site is formed by loops at the C-terminal ends of the barrel strands. The protein forms a homodimer with one active-site cleft per monomer located adjacent to its neighbor in the dimer. The structure creates a single extended cleft that is large enough to accommodate two substrate molecules in close proximity. Although both uroporphyrinogen I and III are metabolized by UROD, only the coproporphyrinogen III isomer is further metabolized to heme.⁶⁸

Coproporphyrinogen Oxidase (EC 4.1.1.37)

CPO is located on the outer surface of the inner mitochondrial membrane in mammalian cells.⁶⁹ The enzyme catalyzes the removal of the carboxyl group and two hydrogens from the propionic groups of pyrrole rings A and B, forming vinyl groups at these positions (see Fig. 58–4, step 6). The enzyme is isomer specific for coproporphyrinogen III, yielding protoporphyrinogen IX (see Fig. 58–4, step 6). The gene for human CPO has been assigned to chromosome 3q12, spans approximately 14 kb, and consists of seven exons and six introns.⁷⁰ cDNA cloning for this enzyme was first reported in mouse erythroleukemia cells.⁷¹ The predicted mouse protein comprises 354 amino acid residues (Mr = 40,647 Da), with a putative leader sequence of 31 amino acid residues. The result is a mature protein consisting of 323 amino acid residues (Mr = 37,225 Da).⁷¹ Potential regulatory elements exist in the GC-rich promoter region of the gene, such as six Sp1, four GATA-1, one CACCC site, and the CPO gene promoter regulatory element (CPRE).⁷² CPRE binds specifically to a CPRE-binding protein, which has a leucine-zipper-like structure and serves as a DNA sequence-specific transcription factor that regulates gene expression.⁷² Tissue-specific expression of CPO is significant. For example, binding proteins to the Spl-like element, CPRE and GATA-1, cooperatively function in CPO gene expression in erythroid cells. The CPRE-binding protein by itself plays a principal role in basal expression of CPO in nonerythroid cells.⁷³ CPO mRNA increases during erythroid cell differentiation.⁷⁴

Newly synthesized human CPO contains a 110-amino-acid N-terminal signal peptide,⁷⁴ which is removed during transport into the intermembrane space of mitochondria, yielding a mature protein of 354 amino acid residues (Mr = 36,842 Da). A five-base insertional mutation in the middle of this presequence has been described in one patient with HCP.⁷⁵

Protoporphyrinogen Oxidase (EC 1.3.3.4)

The penultimate step in heme biosynthesis is the oxidation of protoporphyrinogen IX to protoporphyrin IX, with removal of six hydrogen atoms. This reaction is mediated by the mitochondrial enzyme protoporphyrinogen oxidase (PPO; see Fig. 58–4, step 7). Human PPO cDNA has been cloned.⁷⁶ The gene is present as a single copy per haploid genome, at chromosome 1q22.⁷⁷ PPO consists of 477 amino acids with an Mr of 50,800 Da. The deduced protein exhibits a high degree of homology over its entire length to the amino acid sequence of PPO encoded by the HEMY gene of Bacillus subtilis. PPO has been crystallized and the structure shows that the enzyme is a homodimer.⁷⁸ Sequences required for import into the mitochondria have been identified.⁷⁹ Expression of PPO is upregulated, approximately fourfold, in the developing erythron from two GATA-1 binding sites located in exon 1.⁸⁰

Ferrochelatase (Protoheme-Ferrolyase; EC 4.99.1.1)

The final step of heme biosynthesis is the insertion of iron into protoporphyrin IX. This reaction is catalyzed by the mitochondrial enzyme ferrochelatase (FECH; see Fig. 58–4, step 8). The enzyme uses protoporphyrin IX, rather than its reduced form, as substrate, but requires the reduced ferrous form of iron.⁸¹ The gene encoding human FECH has been assigned to chromosome 18q.⁸² Two FECH mRNA species, approximately 2.5 kb and approximately 1.6 kb in size, are derived from the utilization of two alternative polyadenylation sites in the mRNA. The human FECH gene contains a total of 11 exons and has a minimum size of approximately 45 kb. A major site of transcription initiation is at an adenine, 89 bp upstream from the translation-initiating ATG. The promoter region contains a potential binding site for several transcription factors, Sp1, NF-E2, and GATA-1, but not a typical TATA or CAAT sequence. The transcripts are identical in all tissues examined.

The crystal structure of B. subtilis FECH has been determined at 1.9-Å resolution.⁸³ Subsequently the structure of human FECH was solved and the location of the substrate binding site determined. The enzyme functions as a homodimer and associates with the inside of the inner mitochondrial membrane.⁸³ The mechanism of catalysis has not been identified nor has a function been assigned to the ²Fe-²S cluster that is present in human FECH. Lead inhibits FECH, and a structure of the protein-lead complex has been solved indicating a critical role for the pi helix in catalysis.⁸⁴ FECH seems to have a structurally conserved core region that is common to the enzyme from bacteria, plants, and mammals.

Control of Heme Synthesis in the Liver and Erythroid Cells

Tissue-specific aspects of heme synthesis have been studied mostly in erythroid cells and hepatocytes, as the marrow and liver have the greatest requirements for heme. The rate of heme synthesis in the liver is largely regulated by ALAS1 activity. The synthesis of ALAS1, in turn, is under feedback control by heme, which regulates ALAS1 at the levels of transcription, translation, and transfer into mitochondria. Many chemicals, hormones, and drugs increase the synthesis of hepatic CYPs, which increases the demand for heme and lead to induction of ALAS1. In addition, the ALAS1 gene contains upstream enhancer elements that are responsive to inducing chemicals and interact with the PXR. Therefore, ALAS1 and CYPs are subject to direct induction by xenobiotics and certain steroids.²⁷ Chemical exposures that induce hepatic heme oxygenase and accelerate the destruction of hepatic heme, or inhibit heme formation, can also induce hepatic ALAS1.

ALAS2 is not inducible in erythroid cells by drugs that induce ALAS1 in hepatocytes.⁸⁵ The synthesis of ALAS2 is uninfluenced, or often upregulated, by hemin, at both the transcriptional and the translational levels. Hemin treatment of marrow cultures increases erythroid colony-forming units,⁸⁶ whereas hemin treatment of hepatocytes inhibits synthesis of ALAS1 and CYPs. An additional distinct difference in these ALAS isoforms is that SCS-βA associates with ALAS2 but not with ALAS1, suggesting a tissue-specific difference in mitochondrial transport of these isoforms.

There are two major erythropoietic porphyrias in humans. CEP is caused by mutations of the UROS gene. It is one of the least-common porphyrias, but is well known as a result of its long history and the severe photomutilation of exposed areas such as the face and fingers that is a dramatic feature in many cases (Fig. 58–5). EPP, which is caused by FECH mutations, is the third most common porphyria, and the most common in children, but was not well described until 1965. XLP is much less common, has the same phenotype as EPP, but normal FECH activity. In 2008, the discovery of gain-of-function mutations in the last exon of ALAS2 provided an explanation for the increased level of erythrocyte protoporphyrin seen in this type of protoporphyria.³⁷ Characteristics in most patients with erythropoietic porphyrias that are distinct from the hepatic porphyrias include childhood onset, stable symptoms and levels of porphyrins over time, and severity largely determined by genotype rather than factors that affect the heme pathway, primarily in the liver. Substantial increases in erythrocyte zinc protoporphyrin in ADP and homozygous forms of other hepatic porphyrias, such as HEP (the homozygous form of familial PCT), AIP, HCP, and VP, suggest that an erythropoietic component may be important in these conditions.⁸⁷

CONGENITAL ERYTHROPOIETIC PORPHYRIA

Definition and History

CEP is caused by a deficiency of UROS (see Fig. 58–4, step 4), is an autosomal recessive condition, and is also known as Günther disease. It results in accumulation and excretion of isomer I porphyrins, especially uroporphyrin I and coproporphyrin I (see Table 58–1 and Fig. 58–1). Characteristic manifestations of CEP include chronic, severe photosensitivity and hemolytic anemia evident in early childhood. Atypical presentations include milder disease that resembles PCT, and onset during adult life often in association with a myeloproliferative disorder.⁸⁸ Early case descriptions of CEP appeared in 1874 and 1898,³ and approximately 130 cases were reported up to 1997.⁸⁹ However, some of these patients may have had HEP, which has very similar clinical features. Perhaps the most well-known patient was Mathias Petry, who survived until age 34, and beginning in 1915, worked with the porphyrin chemist Hans Fisher, providing samples for early studies of porphyrin chemistry.⁹⁰

Pathophysiology

The uroporphyrinogen III synthase defects in CEP are remarkably heterogeneous at the molecular level, with at least 46 different mutations of the UROS gene, and one GATA-1 mutation reported as of this writing.⁸⁷ The UROS mutations include deletions, insertions, rearrangements, splicing abnormalities, and both missense and nonsense mutations.

The missense mutations are well distributed throughout the gene. Of the 12 single-base substitutions, four (T228M, G225S, A66V, A104V) were hotspot mutations, occurring at CpG dinucleotides.⁹¹ The identification of a mutation that altered the penultimate nucleotide in exon 4, resulting in an E81D mutation, also produced exon skipping on approximately 85 percent of the transcripts from that allele. With the exception of V82F, all CEP missense mutations occurred in amino acid residues that are conserved in both the mouse and the human enzyme.

Genotype–phenotype correlation in CEP was studied by prokaryotic expression of mutant UROS cDNAs. Mean activities of the mutant enzymes expressed in Escherichia coli ranged from 0 to 36 percent of the activity expressed by the normal cDNA. The majority of the mutant cDNAs expressed polypeptides with no enzymatic activity. However, V82F, E81D, A66V, A104V, and V99A showed 36, 30, 15, 8, and 6 percent enzyme activity, respectively, compared with the normal control. A66V and V82F were thermodynamically unstable mutants.⁹¹ Homoallelism for C73R, the most common mutation, found in five patients, was associated clinically with the most severe phenotypes, such as hydrops fetalis and transfusion dependency from birth.

Pathogenesis of the Clinical Findings

Porphyrins in their oxidized state are reddish, fluorescent, and photosensitizing, whereas porphyrin precursors and the reduced porphyrinogens are colorless and nonfluorescent. Most marrow normoblasts in CEP display marked fluorescence as a result of porphyrin accumulation (located principally in the nuclei, probably because of fixation artifact).⁹² Anemia and the excess production and excretion of porphyrins is largely accounted for by ineffective erythropoiesis in the marrow. Porphyrin concentrations are also increased in circulating erythrocytes, and intravascular hemolysis may result from exposure to light in the dermal capillaries, causing erythrocyte damage and lysis or uptake by the spleen. Splenomegaly is very common in CEP and is presumed to be secondary to the hemolytic process. The excess porphyrins that are produced by the marrow or released by hemolysis are transported in plasma to the skin, leading to photosensitivity.

Clinical Features

Severe cutaneous photosensitivity is noted soon after birth in most cases. The disease may be recognized even earlier as a cause of hydrops fetalis. Phototherapy for hyperbilirubinemia may cause severe cutaneous burns and scarring in newborns with unrecognized CEP. Brown staining of the teeth by porphyrins (erythrodontia) is evident when the teeth erupt. Blistering and scarring resemble those found in PCT, but are usually much more severe, reflecting the much higher plasma porphyrin levels observed in CEP. Some cases are relatively mild, and can closely mimic PCT. Late-onset cases are often associated with myeloproliferative disorders, with expansion of a clone of erythroid cells bearing a somatic mutation and displaying UROS deficiency.⁸⁸

Subepidermal bullous lesions are characteristic, and progress to crusted erosions that heal with scarring and areas of hyper- and hypopigmentation. Also common is hypertrichosis, which is sometimes severe, and alopecia. Loss of facial features and digits are common and result from recurrent blisters, infection, and scarring. Fingers may be shortened and tapered as a consequence of scarring and contraction of the skin during childhood growth. Erythrodontia, with brown staining and red fluorescence of the teeth under long-wave ultraviolet light is characteristic, and results from deposition of porphyrins in the developing deciduous and permanent teeth in utero. Porphyrins are also deposited in bone. The skeleton is also affected by expansion of the marrow, leading to pathologic fractures, vertebral compression, short stature, and osteolytic and sclerotic lesions. Vitamin D deficiency resulting from avoiding sunlight might also contribute.

Anemia may be severe and lead to transfusion dependence in the more severe cases. Uncorrected anemia can increase erythropoiesis, which, in turn, is a stimulus to porphyrin production by the abnormal erythropoietic cells in the marrow. Erythrocytes exhibit polychromasia, poikilocytosis, anisocytosis, and basophilic stippling, and reticulocytes and nucleated red blood cells are increased.⁹³

Diagnosis

CEP may be suspected even before birth if a sibling is known to have CEP. However, the family history is often negative. CEP should be suspected as a cause of hydrops fetalis, as the disease can be diagnosed and treated in utero. Aspirated amniotic fluid is dark brown in color and contains large amounts of porphyrins. The diagnosis of CEP is often made after birth when pink to dark-brown staining of the diapers, with red fluorescence under long-wave ultraviolet light, is noted. Cutaneous vesicles and bullae on sun-exposed areas may be severe, with scarring.

Urinary porphyrin excretion is markedly increased, and often in the range of 50 to 100 mg/day (normal: up to ~0.3 mg/day). Uroporphyrin I and coproporphyrin I account for most of the increase, although the III isomers and hepta-, hexa-, pentacarboxylate porphyrins are also increased. Fecal porphyrins are increased, and are predominantly coproporphyrin I. Plasma total porphyrins are markedly increased as well, with a pattern of individual porphyrins similar to that in urine. Markedly increased erythrocyte porphyrins are predominantly uroporphyrin I and coproporphyrin I, although protoporphyrin IX may predominate especially in milder cases.

CEP must be distinguished by biochemical testing from other causes of blistering skin lesions. HEP can present with photosensitivity in early childhood. Mild cases of CEP may be misdiagnosed as PCT.

The diagnosis should be confirmed in all cases by DNA studies, which can identify causative mutations in almost all cases. This is especially important for genetic counseling and for prenatal diagnosis in subsequent pregnancies. Demonstration of a GATA-1 mutation in one case, illustrates that on occasion a genetic defect outside the heme biosynthetic pathway can cause CEP.⁹³

Therapy

Patients should be advised that to avoid severe scarring and loss of facial features and digits it is essential to avoid sunlight, trauma to the skin, and infections. Topical sunscreens that block long-wave ultraviolet light (ultraviolet A light) and oral treatment with β-carotene are somewhat helpful,⁹⁴ but are marginally beneficial in most cases. Erythrocyte transfusions are essential in patients with severe anemia.⁹⁵ Transfusions to maintain the hematocrit above 35 percent, with an iron chelator to avoid iron overload, has been beneficial in some cases.⁹⁶ Hydroxyurea to reduce erythropoiesis and porphyrin production may also be considered.⁹⁷ Splenectomy has provided short-term benefit. Oral charcoal reportedly was quite effective in one patient,⁹⁸ and ascorbic acid and α-tocopherol improved anemia in another.⁸⁹ Unaffected infants born to mothers with CEP may have erythrodontia as a result of exposure to maternal porphyrins before birth.⁹⁹

Hematopoietic stem cell transplantation is the treatment of choice when a suitable donor is available, especially for young patients.¹⁰⁰ When transplantation is successful, there is marked clinical improvement and reduction in porphyrin levels, even if these are not completely normalized. Gene therapy is being explored using retroviral and lentiviral vectors and hematopoietic stem cells from patients with CEP.^101,102

ERYTHROPOIETIC PROTOPORPHYRIA

Definition and History

EPP is caused by a partial deficiency of FECH (see Fig. 58–4, step 8) activity, which results in the accumulation of the substrate protoporphyrin in the marrow. XLP has the same phenotype but is much less common, and is a result of ALAS2 gain-of-function mutations (ALAS; see Fig. 58–1).³⁷ EPP and XLP are characterized by onset of nonblistering cutaneous photosensitivity in early childhood. EPP is the most common porphyria in children and the third most common in adults. Reported prevalence varies between 5 and 15 cases per 1 million individuals.^103,104,105 Protoporphyric hepatopathy is a potentially fatal complication estimated to occur in less than 5 percent of patients.

Pathophysiology

In most families, EPP is best described as an autosomal recessive disease, in which a severe FECH mutation is inherited from one parent and a low-expression (hypomorphic) FECH allele from the other. More than 75 different severe mutations, including nonsense, missense, and splice-site mutations, and deletions, insertions, and rearrangements have been described. Splicing mutations are most common. Recombinant human FECH, when engineered to have individual exon skipping for exons 3 through 11, lacks significant enzyme activity when expressed in E. coli and almost all such variants lacked the [²Fe-²S] cluster.¹⁰⁶

EPP was usually described in the past as an autosomal dominant disorder, with variable penetrance. However, it was noted that EPP patients have only 30 percent or less of normal FECH activity, rather than 50 percent, which would be expected in an autosomal dominant condition. It was then shown that, in addition to a severe FECH mutation, a low-expression (hypomorphic) intronic polymorphism (a –23C→T transition) is found in the other FECH allele of patients with EPP, which is inherited from the other parent.^107,108,109 This transition favors the use of a cryptic acceptor splice site 63 bases upstream of the normal splice site. The aberrantly spliced mRNA contains a premature stop codon and is degraded by a nonsense-mediated decay mechanism.¹⁰⁹ The result is a lower steady-state level of wild-type FECH mRNA. Coinheritance of the hypomorphic allele in trans to a loss-of-function mutant allele was found in 98 percent of French cases with EPP,¹¹⁰ and with a similar frequency in South African patients.¹⁰⁵ The frequency of the IVS3–48C hypomorphic allele is common in the white population, and by itself has no phenotype. Its frequency varies widely in different populations and relates to the observed differences in the prevalence of EPP.^103,104,105

Other underlying genetic mechanisms must be considered in newly identified EPP families. In a few families, a severe FECH mutation, at least one of which must produce some FECH enzyme, is inherited from each parent and the hypomorphic allele is not present. Interestingly, EPP in such families is sometimes associated with seasonal palmar keratoderma, unusual neurologic symptoms, less-than-expected increases in erythrocyte protoporphyrin and absence of liver dysfunction.¹¹¹

XLP was first perceived as a variant form of EPP in which FECH mutations were absent. After family studies suggested sex-linked inheritance, gain-of-function mutations of ALAS2 (the only heme pathway enzyme found on the X chromosome) were discovered.³⁷ This is the only porphyria caused by mutations of ALAS, the first enzyme in the pathway.

EPP can develop late in life in patients with clonal hematologic disorders and expansion of a clone of hematopoietic cells with mutations of a FECH allele.^112,113 For example, a patient with a myeloproliferative disorder later developed severe EPP because of clonal expansion of a cell of erythropoietic lineage with a FECH deletion and the IVS3–48C/T polymorphism, and died of EPP-induced liver disease.¹¹⁴

Pathogenesis of the Clinical Findings

Marrow reticulocytes are thought to be the primary source of the excess protoporphyrin in EPP.^115,116 Most of the excess erythrocyte protoporphyrin in circulating erythrocytes is found in younger cells as metal-free protoporphyrin (i.e., not complexed with zinc), in contrast to other conditions associated with increased erythrocyte protoporphyrin content. Metal-free protoporphyrin declines much more rapidly with red cell age than it does zinc protoporphyrin.^115,116 Metal-free protoporphyrin, but not zinc protoporphyrin, is released from erythrocytes following solar irradiation, which may explain why lead intoxication and iron deficiency, which are associated with elevated erythrocyte zinc protoporphyrin levels, are not associated with photosensitivity.¹¹⁷ Excess metal-free protoporphyrin enters plasma from reticulocytes, as well as from circulating erythrocytes, and is taken up by hepatocytes, excreted in bile and feces, and may undergo enterohepatic recirculation. Hepatocytes may also provide a limited additional source of excess protoporphyrin in this disease.

Light-excited protoporphyrin in EPP generates free radicals and singlet oxygen,¹¹⁸ which in EPP can lead to peroxidation of lipids¹¹⁹ and crosslinking of membrane proteins. Skin irradiation in EPP patients leads to complement activation and polymorphonuclear chemotaxis, which contributes to the development of skin pathology.¹²⁰ Skin histopathology is not specific but may include thickened capillary walls in the papillary dermis surrounded by amorphous hyaline-like deposits, immunoglobulin, complement, and periodic acid-Schiff–positive mucopolysaccharides.¹²¹ Basement membrane abnormalities are less marked than in other forms of porphyria.¹²²

Protoporphyric hepatopathy is a feared complication that develops in less than 5 percent of patients, and is attributed to the cholestatic effects of excess protoporphyrin presented to the liver. This complication may begin with chronic abnormalities in liver function tests and then progress rapidly as a vicious cycle of increasing protoporphyrin levels in plasma and erythrocytes and worsening liver function and photosensitivity. Hepatopathy is sometimes precipitated by another cause of liver dysfunction such as viral or alcoholic hepatitis. Protoporphyrin is cholestatic, and can form crystalline structures in hepatocytes and impair mitochondrial function, leading to decreased hepatic bile formation and flow.^123,124 Accumulated protoporphyrin may appear as brown pigment in hepatocytes, Kupffer cells, and biliary canaliculi, and these deposits are doubly refractive with a Maltese cross appearance under polarizing microscopy.¹²⁵ DNA microarray studies in explanted livers of patients with hepatopathy revealed significant changes in expression of several genes involved in wound-healing, organic anion transport, and oxidative stress.¹²⁶

Clinical Features

Photosensitivity is present from early childhood in almost all cases. Parents may observe that an affected infant cries and develops skin swelling and erythema when exposed to sunlight. Although EPP is the most common porphyria in children, there is often considerable delay in diagnosis.

Cutaneous photosensitivity in EPP is acute and nonblistering, which is distinctly different from the more chronic, blistering skin manifestations of the other cutaneous porphyrias. Table 58–3 tabulates symptoms in a series of 32 patients with EPP. Skin symptoms are usually worse during spring and summer and affect light-exposed areas, especially of the face and hands. Characteristically, stinging or burning pain develops within 1 hour of sunlight exposure, and if exposure continues is followed by erythema and edema—described as solar urticaria, sometimes with petechiae, and less commonly purpura. Blistering and crusted lesions are uncommon. Artificial lights may contribute to photosensitivity.¹²⁷ Patients typically avoid sunlight and may display no objective cutaneous signs. Repeated light exposure can lead to chronic changes including leathery hyperkeratotic skin especially on the dorsa of the hands and finger joints, mild scarring, and separation of the nail plate (onycholysis).

Mild anemia with microcytosis, hypochromia, reduced iron stores, but usually normal serum iron, and serum transferrin receptor-1, is a common feature of EPP,^{115,128,129,130,131} but there is little evidence for impaired erythropoiesis or abnormal iron metabolism,^129,130 and hemolysis is absent or very mild.^131,153 Iron accumulation in erythroblasts and ring sideroblasts have been noted in marrow in some patients.¹³² Findings in XLP are similar. Also, iron is proposed to have a role in splicing of FECH mRNA, where decreased iron leads to an increase in incorrect splicing of the mRNA.¹³³ Binding of iron-responsive elements binding proteins (IRPs) to the 5′-iron-responsive element (IRE) in ALAS2 mRNA, in low iron conditions, prevents translation of ALAS2 mRNA. When iron is supplemented, the IRPs no longer have high affinity for the ALAS2 5′-IRE, leading to increased translation, import into the mitochondria, and enhanced production of ALA.¹³⁴

Precipitating factors that are important in the hepatic porphyrias do not appear to play an important role in EPP. Although more long-term followup studies are needed, porphyrin levels and symptoms typically do not change over time, unless liver dysfunction develops. Concurrent iron deficiency or other marrow problems might also lead to further increases in porphyrin levels and photosensitivity. Pregnancy is reported to lower erythrocyte protoporphyrin levels somewhat and increase tolerance to sunlight.¹³⁵

Neurovisceral manifestations are absent in uncomplicated EPP. Patients with severe protoporphyric hepatopathy may develop a severe motor neuropathy similar to that seen in the acute porphyrias.¹³⁶ Autosomal recessive EPP associated with palmar keratoderma has also been associated with unexplained neurologic symptoms.¹¹¹

Gallstones containing large amounts of protoporphyrin are common, and may require cholecystectomy at an unusually early age.¹³⁷ Liver function and liver protoporphyrin content are usually normal in EPP. Protoporphyric hepatopathy, which is the most life-threatening complication of EPP, results from the cholestatic effects of protoporphyrin presented in excess amounts to the liver. It can be the major presenting feature of EPP,¹³⁸ and may be chronic or progress rapidly to death from liver failure. Unnecessary surgery for suspected biliary obstruction can be detrimental and should be avoided.¹²⁴ Operating room lights during liver transplantation or other surgery, especially in patients with hepatopathy, can cause marked photosensitivity with extensive burns of the skin and peritoneum and photodamage of circulating erythrocytes.¹³⁹

Diagnosis

Painful, nonblistering photosensitivity suggests the diagnosis. A substantial elevation of erythrocyte protoporphyrin is expected, but is not specific, as erythrocyte zinc protoporphyrin is predominantly increased in conditions such as homozygous porphyrias (other than most cases of CEP), iron deficiency, lead poisoning, anemia of chronic disease,¹⁴⁰ hemolytic conditions,¹⁴¹ and many other erythrocyte disorders. A unique finding in EPP is increased erythrocyte protoporphyrin with a predominance of metal-free rather than zinc protoporphyrin. This occurs because FECH, which can utilize metals in addition to iron, catalyzes the formation of zinc protoporphyrin, and this activity is deficient in EPP. Because FECH is not deficient in XLP, erythrocytes contain increased amounts of both zinc and metal-free protoporphyrin, although the latter still predominates in most cases.

Consequently, the diagnosis of EPP requires demonstration of an increase in metal-free protoporphyrin in red cells. Amounts of metal-free and zinc protoporphyrin in erythrocytes can be measured by ethanol or acetone extraction or high-performance liquid chromatography. There is confusion about terminology used by different laboratories. For example, the term “free erythrocyte protoporphyrin,” refers to protoporphyrin results measured with a hematofluorometer as an indicator of lead exposure, but actually refers to zinc protoporphyrin rather than metal-free or total protoporphyrin. At this writing, we are aware of only two laboratories in the United States (Mayo Clinic Laboratories and the Porphyria Laboratory at the University of Texas Medical Branch), that reliably report the amounts of total, metal-free, and zinc protoporphyrin, as is needed for confirmation of a diagnosis of EPP. The proportions of metal-free and zinc protoporphyrin can also usually distinguish XLP (50 to approximately 85 percent metal-free protoporphyrin) from EPP (>85 percent metal-free protoporphyrin).

The plasma porphyrin concentration is almost always at least mildly increased in EPP, but often less than in other cutaneous porphyrias, and may be normal in mild cases. Plasma porphyrins in EPP are particularly subject to photodegradation during sample processing unless great care is taken to shield the sample from natural or fluorescent light.¹⁴² For these reasons, measurement of erythrocyte rather than plasma porphyrin should be relied upon for diagnosis of EPP and XLP.

Fecal porphyrins are normal or somewhat increased, and consist mostly of protoporphyrin. Urine porphyrins are normal, except after hepatopathy develops, which causes increases in urinary coproporphyrin as is typical for other forms of liver diseases.

Therapy

Avoidance of the sunlight exposure is important, and often requires changes in lifestyle and the working environment. Topical sunscreens that absorb ultraviolet A and sunblocks containing zinc oxide or titanium dioxide may be helpful. Orally administered β-carotene, which probably quenches activated oxygen radicals,^143,144 may afford some protection after 1 to 3 months of therapy, but results are variable. A daily dose of 120 to 180 mg or higher is recommended to achieve a serum β-carotene level of 600 to 800 mcg/dL.¹²⁷ Oral cysteine may also quench excited oxygen species and increase tolerance to sunlight in EPP.¹⁴⁵ Other treatments that aim to either increase skin pigmentation or scavenge activated oxygen species have been reviewed¹⁴⁴ and include dihydroxyacetone/Lawsone, vitamin C and narrow-wave ultraviolet B phototherapy to increase melanin.¹⁴⁶ Afamelanotide, an α-melanocyte–stimulating hormone analogue that increases skin melanin, has shown benefit in clinical trials.¹⁴⁷

It is advisable to monitor liver function tests at least yearly, and avoid severe caloric restriction and drugs or hormone preparations that impair hepatic excretory function.^148,149 Because iron deficiency might be detrimental by further limiting heme synthesis and increasing protoporphyrin accumulation, ferritin levels should be followed in EPP and XLP patients, keeping in mind that ferritin in the lower part of the normal range (especially for women) may indicate depleted iron stores. Iron supplementation has been reported to worsen photosensitivity in some cases (although increases in protoporphyrin levels were not documented) and to correct microcytosis without increasing porphyrin levels in others. Therefore, at present iron supplementation in EPP and XLP is controversial, and systematic studies are needed. Because patients avoid sunlight exposure, vitamin D supplementation is recommended.

Management of protoporphyric liver disease is difficult. The condition may resolve spontaneously especially if another reversible cause of liver dysfunction, such as viral hepatitis or alcohol, is contributing.^122,150 Cholestyramine,^124,151,152 ursodeoxycholic acid,¹⁵³ vitamin E, red blood cell transfusions,¹⁵⁴ plasma exchange, and intravenous hemin may be given in combination to bridge patients until liver transplantation or there is spontaneous improvement.¹⁵⁵ Success of liver transplantation is comparable to that in other liver diseases, even though protoporphyric hepatopathy may recur in the new liver, a result of the continued erythrocyte protoporphyrin release by the marrow.¹⁵⁶ Acute motor neuropathy has developed in some patients with protoporphyric liver disease after transfusion¹⁵⁷ or liver transplantation,^158,159 and is sometimes reversible.¹⁵⁸

Marrow transplantation can achieve remission in human EPP,¹⁶⁰ as well in murine models of protoporphyria.¹⁶¹ Sequential liver and marrow transplantation can correct the overproduction of protoporphyrin by the marrow and prevent recurrence of liver disease.¹⁶² Promising studies in murine models suggest a future role for gene therapy in human EPP.^163,165

The acute porphyrias are comprised of four disorders caused by different enzyme deficiencies, and are distinctive for neurologic symptoms that usually occur as acute exacerbations during adult life. Similar symptoms occur in lead poisoning, hereditary tyrosinemia type I, and in some reported cases of porphyria with dual enzyme deficiencies.

δ-AMINOLEVULINIC ACID DEHYDRATASE PORPHYRIA

Definition and History

ADP is an autosomal recessive disorder resulting from severe deficiency of ALAD activity (see Table 58–1 and Fig. 58–1). This is the rarest of the porphyrias, with only six cases documented at the molecular level.^50,168

Pathophysiology

All reported cases were males.^{164,166,167,168,169} Compound heterozygosity for two distinct ALAD mutations was documented in five cases (see Fig. 58–4, step 2).^167,168 Four (three in Germany and one in the United States) experienced onset of symptoms in their teens, whereas one Swedish case developed severe symptoms in infancy.¹⁶⁶ The sixth patient was a Belgian male who developed ADP at age 63 years and was found to have two inherited base transitions in one allele, and was therefore heterozygous for ALAD deficiency.^164,169 He also developed polycythemia vera and his erythrocyte ALAD activity was less than 1 percent of normal, while lymphocyte ALAD activity was greater than 20 percent of normal. Heterozygous ALAD deficiency was apparently clinically silent in this patient until there was expansion of a clone of erythroid cells that carried the mutant ALAD allele.¹⁶⁹

Thus ADP is highly heterogeneous at the molecular level, with a total of 11 mutant alleles identified in these 6 patients.¹⁶⁸ An additional mutation was found in a healthy Swedish girl with markedly decreased ALAD activity (12% of normal), which was detected by ALAD measurement during neonatal screening for hereditary tyrosinemia.¹⁷⁰ The same mutation was found in a U.S. male patient with acute porphyria who also had a CPO mutation and an unusual pattern of porphyrin precursors and porphyrins reflecting dual enzyme deficiencies.¹⁷¹ Thus, heterozygous ALAD deficiency may rarely be combined with another enzyme deficiency, or may itself cause porphyria if a marrow disorder leads to clonal expansion of the mutant ALAD allele.

Human ALAD consists of eight identical oligomers, each with two zinc-binding sites. Lead can bind at least one of these sites and impair enzyme activity. Some mutations found in ADP may affect zinc binding, or favor assembly of a hexameric enzyme with decreased activity. Thus ADP has been described as a conformational disease.⁵⁰

ADP is classified as one of the hepatic porphyrias because it closely resembles the other acute porphyrias. However, the site of overproduction of ALA is not established, and the Swedish infant with severe, early onset disease did not benefit from liver transplantation.¹⁷² Substantial increase in erythrocyte zinc protoporphyrin also suggests an erythroid component. The excess urinary coproporphyrin III in ADP may originate from metabolism of ALA to porphyrinogens in a tissue other than the site of ALA overproduction. Indeed, ALA loading in normal subjects was shown to cause substantial coproporphyrinuria.¹⁷³ The pathogenesis of the neurologic symptoms is poorly understood, as in other acute porphyrias.

Clinical Features

The four adolescent males had intermittent symptoms resembling other acute porphyrias, including abdominal pain, vomiting, extremity pain, and motor neuropathy, although exacerbating factors were less evident.^168,174 Two German cases had initial acute attacks and then remained well during 20 years of followup.¹⁷⁵ The third German case¹⁷⁶ and the U.S. case¹⁶⁸ had further attacks and were maintained on prophylactic hemin infusions. The Swedish infant had more severe neurologic disease, including failure to thrive, and died after liver transplantation.¹⁷⁷ The 63-year-old man in Belgium, developed an acute motor polyneuropathy concurrently with a myeloproliferative disorder.^88,164,178

Diagnosis

A biochemical diagnosis of ADP includes demonstration of markedly deficient erythrocyte ALAD activity, marked elevation in urinary ALA and coproporphyrin III and erythrocyte zinc protoporphyrin, with little or no increase in urinary PBG. Erythrocyte ALAD activity is approximately half-normal in both parents. Lead poisoning is differentiated by increased blood lead and restoration of ALAD activity in vitro by reduced glutathione or dithiothreitol.^167,179 Although biochemical measurements can strongly suggest ADP, the diagnosis must be confirmed by DNA studies.

Patients with hereditary tyrosinemia type I may also have ALAD inhibition and increased excretion of ALA. ⁴³ Dioxoheptanoic acid (succinylacetone), a structural analogue of ALA and a potent ALAD inhibitor, accumulates as a result of an inherited deficiency of fumarylacetoacetate hydrolase in these patients. The presence of this inhibitor can be demonstrated in urine by measuring ALAD activity in normal blood after addition of a patient’s urine. ALAD protein is not reduced in this disease.¹⁸⁰

Therapy

Because few cases have been documented, treatment recommendations are based on limited experience. Hemin was beneficial in the four male patients, but there was little or no response to glucose. A long-term preventive hemin regimen was effective in two of these patients. The Swedish infant did not respond to glucose or hemin, and did not improve greatly after liver transplantation.¹⁷² Whether transplantation would benefit less severe cases is unknown. Hemin produced a biochemical response but no clinical improvement in the late-onset case in Belgium, who had a peripheral neuropathy but no acute attacks.¹⁷⁸

ACUTE INTERMITTENT PORPHYRIA

Definition and History

AIP is an autosomal dominant disorder caused by a partial deficiency of PBG deaminase (see Table 58–1 and Fig. 58–1). Symptoms usually occur as acute attacks and are neurologic in origin. In most countries this is the most common acute porphyria and the second most common porphyria. Most individuals who inherit the enzyme deficiency (probably more than 90 percent) never develop symptoms, but are at some risk to develop symptoms after puberty. The first case of acute porphyria was described in 1889 by Stokvis⁵ who noted a relationship of the symptoms to the drug sulfonal, which is related to the barbiturates.

Prevalence of AIP was estimated to be 1 to 2 per 100,000 population in Europe,¹⁸¹ and 2.4 per 100,000 population in Finland.¹⁸² Up to 300 PBGD mutations have been described in AIP, with many found in only one or a few families.^183,184 The disease occurs in all races, but clusters as a result of founder effects occur in some countries. A founder mutation in northern Sweden is associated with a disease prevalence of 1 per 1500 population.¹⁸⁵ The prevalence of low PBG deaminase activity, which includes latent gene carriers of AIP, is as high as 1 per 500 in the general population of Finland.¹⁸⁶ Based on DNA studies, the minimal prevalence of the AIP-associated genes in France has been calculated to be 1 per 1675 population.¹⁸⁷

Pathophysiology

PBG deaminase is also known as HMB synthase, and formerly as uroporphyrinogen I synthase. PBGD mutations have been classified based in part on the presence or absence of cross-reactive immunologic material (CRIM), which indicates the presence of inactive enzyme protein. Type I mutations are CRIM-negative, with reduction of both enzyme activity and protein to approximately 50 percent of normal in heterozygotes. Type II mutations are associated with reduced PBGD activity only in nonerythroid tissue. These patients with “variant AIP” comprise less than 5 percent of AIP patients, and have normal erythrocyte PBGD activity and decreased hepatic activity because, as explained earlier in the section on hydroxymethylbilane synthase, transcription of the gene to form the erythroid-specific enzyme starts downstream of the site of the mutation. Type III are CRIM-positive mutations that result in decreased activity with structurally abnormal enzyme protein.¹⁸⁸

Pathogenesis of the Clinical Findings

A partial deficiency of PBGD rarely causes clinical expression of AIP, and most individuals who inherit this enzyme deficiency remain healthy with normal porphyrin precursor excretion throughout life. Certain drugs and hormones that exacerbate AIP can directly induce ALAS²⁷ and also increase the demand for heme by inducing CYP enzymes, which turn over rapidly and use most of the heme synthesized in the liver.¹⁸⁹

When heme synthesis is stimulated, the partial enzyme deficiency in AIP apparently impairs heme synthesis sufficiently to compromise negative feedback by the regulatory heme pool, which controls synthesis of the rate-limiting enzyme ALAS1. This leads to marked induction of ALAS1 and overproduction of ALA, PBG and porphyrins in the liver.

It is generally accepted, although not proven, that hepatic PBGD remains constant at approximately 50 percent of normal activity during exacerbations and remissions of AIP, as in erythrocytes. An early report suggested that the enzyme activity is considerably less than half-normal in liver during an acute attack,¹⁴ but additional data is lacking. It has been suggested that once the disease becomes activated, excess PBG may interfere with assembly of the dipyrromethane cofactor for this enzyme.

Clinical improvement and normalization of porphyrin precursor excretion after liver transplantation in patients with severe AIP is a clear indication that the liver plays an essential role in neuropathic processes in the acute porphyrias.¹⁹⁰ Proposed explanations for neurologic dysfunction in the acute porphyrias include the following. (1) Heme pathway intermediates or products derived from them may be neurotoxic. This hypothesis is most favored, although the evidence is not conclusive. (2) PBG deaminase deficiency in the nervous system tissues may limit heme synthesis and formation of important hemoproteins. For example, decreased activity of the hemoprotein nitric oxide synthase might decrease production of nitric oxide and cause vasospasm, which might account for some cerebral manifestations of AIP,^191,192 and possibly compromise intestinal blood flow.¹⁹³ However, regulation of heme and hemoprotein synthesis in nervous tissue and blood vessels is difficult to study, and convincing evidence is lacking. (3) Impaired hepatic heme synthesis during an attack may lead to decreased activity of hepatic tryptophan pyrrolase, which might increase levels of tryptophan in plasma and brain, leading to increased synthesis of the neurotransmitter 5-hydroxytryptamine.

ALA is increased in a number of disorders with similar neurologic manifestations, including all four of the acute porphyrias, lead poisoning, and hereditary tyrosinemia type I, which favors a neuropathic role for this porphyrin precursor or perhaps a derivative. ALA can enter cells readily and be converted to porphyrins, which, in turn, may have toxic potential.¹⁹⁴ ALA is also structurally similar to γ-aminobutyric acid and can interact with γ-aminobutyric acid receptors.^195,196 However, studies of ALA loading have not shown adverse effects.

Impaired motor function with ataxia develops in mice with PBGD deficiency resulting from compound heterozygous or homozygous mutations induced by gene targeting.¹⁹⁷ Induction of hepatic CYPs is impaired in these animals and corrected by heme.¹⁹⁸ But motor neuropathy can develop even with normal or only slightly increased ALA in plasma and urine, suggesting a primary role for heme deficiency in porphyric neuropathy in this murine model.¹⁹⁹

Precipitating Factors

Acute attacks are precipitated in some heterozygotes by various endogenous or exogenous factors that are additive. Additional unknown genetic factors are also likely to contribute. Some individuals remain susceptible to repeated attacks even after avoidance of known precipitants. Many precipitating factors cause induction of hepatic ALAS1, which is closely associated with induction of CYPs and leads to overproduction of ALA and other pathway intermediates.

Drugs and Other Exogenous Chemicals

Most drugs that are harmful in acute porphyrias are known inducers of hepatic CYPs. These drugs increase de novo heme synthesis, thereby derepressing hepatic ALAS1, and also directly induce this rate-limiting enzyme.²⁷ Table 58–4 identifies some drugs that are known to be harmful or safe. Information regarding safety of many drugs in clinical practice is uncertain or lacking. More extensive drug safety databases are available at the websites of the American Porphyria Foundation (www.porphyriafoundation.com) and the European Porphyria Initiative (www.porphyria-europe.com). These drug classifications are often based on limited evidence and may be controversial. Ethanol and other alcohols are inducers of hepatic ALAS1 and some CYPs.²⁰⁰ Smoking is known to increase CYPs in humans, probably from effects of polycyclic aromatic hydrocarbons, and has been associated with more frequent symptoms of acute porphyria.²⁰¹

Endocrine Factors

Rarity of symptoms before puberty and more common clinical expression in women point to hormonal factors as important contributors in AIP. Although estrogens are considered harmful, it is likely that progesterone is mostly responsible for cyclic premenstrual attacks that occur in some women. Progesterone, certain metabolites of testosterone, and synthetic progestins are potent inducers of ALAS1. Thus administration of progestational agents should be avoided. Diabetes mellitus is not known to precipitate attacks of porphyria, and has been observed to decrease the frequency of attacks and lower porphyrin precursor levels, possibly in relation to high circulating glucose levels.²⁰²

Pregnancy

Pregnancy is usually well tolerated.²⁰³ Attacks during pregnancy are sometimes a result of harmful drugs or reduced caloric intake. Metoclopramide, considered at least by some a contraindicated drug, is associated with exacerbation of the disease when used to treat hyperemesis gravidarum.^204,205 But for reasons that are not clear, some women experience attacks during pregnancy even when harmful factors are avoided.

Nutrition

Reduced intake of calories and carbohydrate can exacerbate acute porphyrias. This may occur from efforts to lose weight, bariatric surgery or from metabolic stress from an illness or surgery. Under these conditions, upregulation of PGC-1α can lead to induction of ALAS1, increases in ALA and PBG, and symptoms of acute porphyria, and these effects are reversed by administration of carbohydrate.^30,206 Starvation, may also induce hepatic heme oxygenase,²⁰⁷ which may deplete hepatic heme and contribute to ALAS1 induction.

Stress

Various forms of physical or psychological stress may exacerbate acute porphyrias, although the mechanisms are not well defined. Medical illnesses, fever, infections, alcoholic excess, and surgery may decrease food intake and contribute to induction of hepatic ALAS1 and heme oxygenase. Psychological stress may also lead to decreased food intake and have other metabolic effects.

Clinical Features

Symptoms are almost never seen before puberty, and most commonly develop in women in the third or fourth decade of life. Acute attacks are life-threatening but rarely fatal if promptly recognized and treated. Frequently recurring attacks and chronic symptoms can develop and be disabling. Although the most prominent symptoms are a result of effects on the nervous system, liver and kidney damage may be important in the long-term. In very rare homozygous cases, severe neurologic manifestations are seen early in childhood, and acute attacks are not prominent.^207a,209

Symptoms and signs are nonspecific and highly variable. Abdominal pain is the most common symptom, occurring in 85 to 95 percent of cases.²¹⁰ It is usually severe, steady, and poorly localized, but may be cramping, and is often accompanied by nausea, vomiting, constipation, and abdominal distention because of ileus. Pain in the chest and extremities are also common. Tachycardia is the most common physical sign, occurring in up to 80 percent of acute attacks,²¹¹ and often accompanied by hypertension, sweating, tremors, and other effects of sympathetic overactivity and excess catecholamine production. There is little or no abdominal tenderness, fever, or leukocytosis because inflammation is not prominent. Bowel sounds are usually decreased, but are sometimes increased with diarrhea. The urine is often dark (because of porphobilin, a degradation product of PBG) or reddish (because of porphyrins, including uroporphyrin formed nonenzymatically from PBG). Urinary hesitancy and dysuria may occur as a consequence of bladder dysfunction. Acute mental symptoms may include insomnia, anxiety, restlessness, disorientation, paranoia, and hallucinations.

Paresis because of peripheral motor neuropathy usually occurs with prolonged, severe attacks, but is sometimes an early or even initial manifestation.^212,213 Porphyric neuropathy is primarily motor and results from axonal degeneration, which may be followed by demyelinization.²¹⁴ Muscle weakness may not be detected until it is quite advanced because it usually begins in the proximal muscles of the upper extremities. Paresis is usually symmetrical, but may be asymmetrical or focal. Course tremors, clonus and increased reflexes are sometimes prominent. Magnetic resonance imaging may demonstrate cortical densities resembling the posterior reversible encephalopathy syndrome.¹⁹² Sensory loss may develop, especially in the distal extremities. Cranial nerve involvement and cortical blindness have been described.

Motor neuropathy may progress to respiratory and bulbar paralysis and death especially if diagnosis and treatment are delayed and harmful drugs continued. Death may also result from respiratory arrest or cardiac arrhythmia.^214,215 Most attacks treated promptly resolve within days or even hours. Advanced neuropathy from a severe attack is potentially completely reversible, with improvement continuing for up to 1 to 2 years.²¹⁶

Hyponatremia is common during severe attacks and is sometimes a result of hypothalamic involvement and the syndrome of inappropriate antidiuretic hormone secretion. However, hyponatremia may be accompanied by reductions in blood volume,²¹⁷ indicating that increased antidiuretic hormone secretion in this setting is an appropriate physiologic response.²¹⁵ Hyponatremia may sometimes result from gastrointestinal loss, poor intake, and excess renal sodium loss.^215,218 A possible nephrotoxic effect of ALA may explain renal tubular sodium loss and impaired renal function in some patients.²¹⁸ Other electrolyte abnormalities may include hypomagnesemia and hypercalcemia.²¹⁹ Seizures may result from hyponatremia or represent a neurologic effect of acute porphyria.

Chronic mental symptoms, such as depression, are difficult to attribute to AIP. But chronic pain accompanied by depression develops in some patients after frequent exacerbations, and risk for suicide is increased. The disease also predisposes to chronic arterial hypertension and impaired renal function.^203,220,221 The latter may progress and require renal transplantation.^222,223

Mild abnormalities in serum transaminases are common in AIP.²²⁴ More advanced liver disease may develop and the risk of hepatocellular carcinoma is greatly increased (60- to 70-fold) in AIP, and is not related to specific PBGD mutations. Serum α-fetoprotein was not increased and the uninvolved liver was not cirrhotic in most acute porphyria cases with liver cancer reported as of this writing. Increased serum thyroxin levels because of increased thyroxin-binding globulin occurs in some patients with AIP, and occasionally hyperthyroidism and porphyria occur together.²²⁵ Elevated low-density lipoprotein cholesterol is apparently less commonly observed in this disorder than in the past.²²⁶

Diagnosis

A high index of suspicion and knowing when to suspect these diseases contributes to making an initial diagnosis of acute porphyria. Because the disease so often remains latent, there is often no family history of porphyria. Acute porphyria should be considered in patients with unexplained abdominal pain or other characteristic symptoms when initial evaluation does not suggest another more common explanation, and ruled in or out by rapid assessment of urinary PBG, which is both sensitive and specific. A substantial increase in PBG, which can be determined rapidly by a commercial kit,²²⁷ establishes that a patient has either AIP, HCP, or VP. Consensus recommendations are that all major medical centers should retain the capacity for rapid urinary PBG testing on single-void urine specimens, as collection of 24 hour urines and reliance on outside laboratories for screening can greatly delay diagnosis and treatment. The urine specimen should be saved for later quantitative measurement of PBG, ALA, and total porphyrin levels. If PBG is substantially increased, samples of plasma, erythrocytes and feces should also be obtained prior to treatment with hemin. This approach provides for rapid initial diagnosis of AIP, HCP, and VP, subsequent biochemical differentiation of these conditions and diagnosis of ADP. In patients with renal failure, PBG can be measured in serum by a specialized laboratory. Figure 58–6 presents a diagnostic flow chart for use when acute porphyria is suspected.

PBG excretion is generally 50 to 200 mg/day (normal range: 0 to ~4 mg/day) during acute attacks of AIP. Excretion of ALA is usually about half that of PBG (expressed as mg/day). Increases in ALA and PBG can persist for prolonged periods between attacks, especially in AIP. Increases in ALA and PBG are less striking during acute attacks of HCP and VP and often decrease more rapidly.

The diagnosis of an acute attack is largely clinical, and is not based on a specific level of ALA or PBG. Levels of ALA and PBG during an acute attack may be increased compared to baseline levels, which fluctuate considerably and can be difficult to establish between attacks. Intravenous hemin causes dramatic, rapid but often transient decreases in these levels.

Urinary porphyrins are increased in AIP, are predominantly uroporphyrin and account for reddish urine (ALA and PBG are colorless). Uroporphyrin can form nonenzymatically from PBG in urine even prior to excretion. However, there is evidence that porphyrins in this condition are predominantly type III, which may be formed enzymatically,²²⁸ perhaps from ALA transported to tissues other than the liver.¹⁹⁴ Total fecal porphyrins and plasma porphyrins are normal or slightly increased in AIP, and erythrocyte zinc protoporphyrin concentrations may be nonspecifically increased.

Erythrocyte PBGD activity is approximately half-normal in most (70 to 80 percent) patients with AIP. However, this measurement is not definitive for confirming or excluding the diagnosis. As described earlier, some PBGD mutations cause the enzyme to be deficient only in nonerythroid tissues. Moreover, the ranges of activity for normals and AIP are wide and overlapping, and the erythrocyte enzyme is highly age-dependent, such that an increase in the proportion of younger cells in the circulation can raise the activity into the normal range in AIP patients with a concurrent condition such as hemolytic anemia or hepatic disease.^229,230 A decrease in this enzyme also does not distinguish between latent and active disease. For these reasons, and because it does not detect other acute porphyrias, erythrocyte PBGD measurement in not useful for initial diagnosis of ill patients.

Once the diagnosis of AIP is established by biochemical methods, the underlying PBGD mutation should be identified. This confirms the diagnosis and, most importantly, enables reliable and definitive identification of other gene carriers by DNA testing. Erythrocyte PBGD measurement is useful for screening of asymptomatic family members if a known case in the family has low erythrocyte enzyme activity, but is less dependable than DNA testing. PBGD deficiency can be documented in the fetus by measuring the enzyme activity or by identifying the maternal or paternal mutation in amniotic fluid cells. However, prenatal diagnosis is usually not indicated because the great majority of heterozygous carriers of PBGD mutations have a good prognosis.

Therapy

Hospitalization is usually required for treatment of attacks, although well-characterized patients with frequently recurring attacks that respond rapidly to treatment are sometimes managed as outpatients. Hospitalization facilitates treatment of severe symptoms, intravenous therapies and monitoring of respiration, electrolytes and nutritional status. Admission to intensive care is warranted if the vital capacity is impaired. Harmful drugs should be discontinued whenever possible. Pain, nausea, and vomiting are generally severe and require narcotic analgesics, chlorpromazine or another phenothiazine, or ondansetron. Low doses of short-acting benzodiazepines are probably safe for anxiety and insomnia. β-Adrenergic blocking agents may be useful to control tachycardia and hypertension, but may be hazardous in patients with hypovolemia or incipient cardiac failure.²³¹ Seizures are treated by correcting hyponatremia, if present. Almost all anticonvulsant drugs have at least some potential for exacerbating acute porphyrias. Clonazepam may be less harmful than phenytoin, barbiturates, or valproic acid.^232,233 Bromides, gabapentin, and vigabatrin are safe.

Carbohydrate Loading

Glucose and other carbohydrates repress hepatic ALAS1 and reduce porphyrin precursor excretion, but the effects are weak compared to those of hemin. Attacks with mild pain and without severe manifestations such as paresis and hyponatremia may be treated with carbohydrate loading. Oral glucose polymer solutions may be given if tolerated. Intravenous treatment with 300 to 500 g of intravenous glucose, usually administered as a 10 percent solution, is recommended. However, the dilutional effects of a large volume of free water may increase risk of hyponatremia. A more complete parenteral nutrition regimen may be needed if oral or enteral feeding is not possible.

Intravenous Hemin

Hemin is much more potent in reducing levels of ALA and PBG compared to glucose. Although controlled clinical trials are lacking for all current therapies for acute attacks of porphyria, consensus recommendations are that the clinical benefits of hemin are superior to other available therapies.^234,235,243a Hemin is available in the United States as a lyophilized hematin preparation (Panhematin, Recordati Rare Diseases, Northfield, IL), and was the first drug approved under the Orphan Drug Act. Heme arginate (Normosang, Orphan Europe, Paris, France), which is a stable preparation of heme and arginine, is available in Europe and South Africa.^235,236 Hemin, when infused intravenously as hematin or heme arginate, becomes bound to circulating hemopexin and albumin and is then taken up primarily by hepatocytes. It then enters and reconstitutes the regulatory heme pool and represses the synthesis of hepatic ALAS1. This results in a dramatic reduction in porphyrin precursor excretion. The standard regimen for treatment of acute attacks is 3 to 4 mg/kg daily for 4 days. Treatment may be extended if a response is not observed within this time. Hemin has been administered safely during pregnancy.^235,236,243a

Product labeling recommends reconstitution of hematin with sterile water. But it was subsequently discovered that degradation products of hematin begin to form immediately upon reconstitution with water, and these are responsible for phlebitis at the site of infusion, which occurs frequently and can lead to loss of venous access with repeated dosing, and a transient anticoagulant effect.^236a Stabilization of hematin with 25 percent human albumin can prevent these adverse effects,²³⁸ and is currently recommended.²³⁹ Uncommon side effects include fever, aching, malaise, hemolysis, anaphylaxis, and circulatory collapse.^240,241 Excessive dosing caused reversible acute renal tubular damage in one case.²⁴²

Controlled trials comparing initial treatment with either glucose or hemin are lacking, except for one randomized, double-blind, placebo-controlled trial of heme arginate for acute attacks of porphyria, which was underpowered (only 12 patients). Although treatment with hemin was delayed for 2 days, striking decreases in urinary PBG and trends in clinical benefit were noted.²⁴³ In contrast, a larger uncontrolled study enrolled 22 patients who had 51 acute attacks, and heme arginate was initiated within 24 hours of admission in 37 attacks (73 percent); all patients responded and hospitalization was less than 7 days in 90 percent of cases.²³⁵ Therefore, based on this and numerous other uncontrolled clinical studies, it is now recommended that most acute attacks of porphyria be treated promptly with intravenous hemin, without an initial trial of intravenous glucose.^235,243a Response to hemin may be delayed or incomplete when there is advanced neurologic damage. Subacute or chronic symptoms are unlikely to respond.

Liver Transplantation

Liver transplantation has been highly effective in several patients who were disabled by recurrent attacks of AIP.¹⁹⁰ This may be an option for severely affected patients.

Other Therapies

Cimetidine has been recommended for human acute porphyrias based on uncontrolled observations in small numbers of patients.^244,245 This drug inhibits hepatic CYPs, and can prevent experimental forms of porphyria induced by agents such as allylisopropylacetamide that undergo activation by these enzymes.²⁴⁶ However, these mechanisms are not immediately relevant to inherited porphyrias in humans. Therefore, cimetidine cannot be recommended as an alternative to hemin.

Prevention of Acute Attacks

Multiple inciting factors must be avoided especially in patients who continue to have repeated attacks. Consultation with a dietitian may be useful to identify dietary indiscretions, and to help maintain a well-balanced diet somewhat high in carbohydrate (60 to 70 percent of total calories). There is little evidence that additional dietary carbohydrate helps further in preventing attacks. Iron deficiency, if present, should be corrected. Patients who wish to lose excess weight should do so gradually and when they are clinically stable.

Gonadotropin-releasing hormone analogues can prevent repeated attacks that are confined to the luteal phase of the menstrual cycle,^247,248 but are less effective in patients with attacks partially associated with the cycle. If treatment is effective after several months, low-dose estradiol, preferably by the transdermal route, or a bisphosphonate may be added to prevent bone loss and other side effects, or treatment changed to a low-dose oral contraceptive. Hemin administered once or twice weekly can prevent frequent, noncyclic attacks of porphyria in some patients.²⁴⁹

Long-Term Monitoring

Patients with acute porphyrias are at risk for renal damage and hepatocellular carcinoma. Renal function should be monitored, hypertension controlled, and nephrotoxic drugs avoided. Current recommendations are that patients with acute porphyrias who are older than age 50 years, and especially those with continued elevations of ALA and PBG, be screened at least annually by ultrasonogram or an alternative imaging method to detect hepatocellular carcinoma at an early stage.^243a

HEREDITARY COPROPORPHYRIA AND VARIEGATE PORPHYRIA

Definition

These closely related hepatic porphyrias are caused by deficiencies of CPO and PPO, the sixth and seventh enzymes of the heme biosynthetic pathway. They present with neurovisceral symptoms, as in AIP, or with blistering skin lesions identical to those seen in PCT. Cutaneous manifestations are much more common in VP than in HCP. The enzyme deficiency in each is inherited as an autosomal dominant trait with variable penetrance (see Table 58–1 and Fig. 58–1). As in AIP, most individuals who inherit the trait remain asymptomatic. Both disorders are less common and generally less severe than AIP in most countries. The incidence of HCP was estimated to be 2 per 1,000,000 population in Denmark,²⁵⁰ and the incidence of VP in Finland reported at 1.3 per 100,000 population.²⁵¹

Because of a founder effect, VP is especially common among whites of Dutch descent in South Africa, where almost all cases share the same PPO mutation (R59W). The incidence of VP in that country was estimated at 3 per 1000 population.²⁵² Very rare cases of homozygous HCP and VP are manifested by severe neurologic impairment early in life, but not acute attacks, and severe photosensitivity.²⁵³

Pathophysiology

Like other porphyrias, HCP and VP are heterogeneous at the molecular level. At least 43 CPO mutations, mostly missense mutations, have been identified in HCP,²⁵⁴ and 130 PPO mutations in VP (see Table 58–1). Clinical expression is variable and onset of neurologic manifestations is influenced by the same factors that are important in AIP.

CPO catalyzes the two-step decarboxylation of coproporphyrinogen III to yield protoporphyrinogen IX, with intermediate formation of harderoporphyrinogen, a tricarboxyl porphyrinogen. A single active site carries out both decarboxylations, and most of the harderoporphyrinogen formed is not released before being further decarboxylated to protoporphyrinogen IX. However, a variant form of HCP, termed harderoporphyria, is a result of CPO mutations that favor premature release of harderoporphyrinogen from the enzyme.²⁵⁵

Clinical Features

Neurovisceral manifestations are identical to those in other acute porphyrias. Although both HCP and VP are generally less severe than AIP, attacks may be life-threatening. Blistering skin lesions may be seen, and are much more common in VP than in HCP. Factors that contribute to attacks, including drugs, hormones, and dietary factors, are also the same as in AIP. Oral contraceptives may precipitate cutaneous manifestations of VP. Risk of chronic hypertension, renal disease, and hepatocellular carcinoma are increased, as in AIP.

Diagnosis

Urinary PBG is elevated during acute attacks, and usually is the basis for diagnosis of these acute porphyrias. However, increases in PBG may be less than in AIP, and more transient. Levels of coproporphyrin III are markedly increased in urine and feces, whereas in AIP fecal porphyrins are normal or only slightly increased. Fecal porphyrins in HCP are almost entirely coproporphyrin III, whereas in VP both coproporphyrin III and protoporphyrin are approximately equally increased. The fecal coproporphyrin III:I ratio is sensitive for diagnosis of HCP, even in asymptomatic stages of the disease.²⁵⁶ Plasma porphyrin concentration is commonly increased in VP, seldom increased in HCP unless there are cutaneous manifestations, and are normal or only slightly increased in AIP. A characteristic plasma porphyrin fluorescence maximum observed at neutral pH is a very specific marker for VP, and is believed to represent protoporphyrin bound covalently to plasma proteins.²⁵⁷ The fluorescence maximum as at approximately 626 nm in VP, approximately 634 in EPP, and approximately 620 in other porphyrias. This fluorometric method is more effective than examination of fecal porphyrins for detecting asymptomatic VP,²⁵⁷ and is useful for rapidly differentiating VP and PCT. Erythrocyte PBG deaminase activity is normal in HCP and VP, and usually deficient in AIP. Assays for CPO and PPO are not widely available. DNA studies are most reliable for identifying asymptomatic carriers, once the mutation affecting the family is identified.

Harderoporphyria is a variant form of HCP that results from a homozygous defect of a structurally altered CPO, such that harderoporphyrinogen is released prematurely from the enzyme. This variant is identified by finding a predominance of harderoporphyrin in urine and feces. Neonatal hemolytic anemia is a distinctive feature of this condition.²⁵⁸ Increases in porphyrin precursors and porphyrins may be more severe in homozygous HCP and VP, with substantial increases in erythrocyte zinc protoporphyrin.

Therapy

The identification and avoidance of precipitating factors is essential. Treatment of acute attacks is the same as in AIP. Treatment of the phototoxic manifestations is not satisfactory. Although the lesions are identical to the blistering skin lesions seen in PCT, there is no response in HCP and VP to phlebotomies or low-dose chloroquine or hydroxychloroquine. Therefore, avoidance of sunlight and use of protective clothing is most important. Yearly screening for hepatocellular carcinoma by imaging is recommended after age 50 years, especially in individuals with persistent increases in porphyrin precursors or porphyrins.

PORPHYRIA CUTANEA TARDA AND HEPATOERYTHROPOIETIC PORPHYRIA

Definition

PCT is caused by a deficiency of hepatic UROD activity and is manifested by the development of chronic, blistering skin lesions on the dorsal aspects of the hands and other sun-exposed areas of skin in middle or late life. This iron-related disorder is the most common and readily treated form of porphyria (see Table 58–1 and Fig. 58–1). The enzyme deficiency develops specifically in the liver as a result of generation of a UROD inhibitor in the presence of multiple susceptibility factors. The disease has been classified as types 1 to 3, based on the presence or absence of heterozygous UROD mutations and other unknown inherited factors. Patients with familial (type 2) PCT are heterozygous for UROD mutations, which are inherited as an autosomal dominant trait with low penetrance. HEP is the homozygous (or compound heterozygous) form of familial (type 2) PCT, which usually presents in childhood and resembles CEP clinically. Rarely, hepatocellular carcinomas may secrete porphyrins and simulate PCT; however the enzyme defect was not established in such cases.²⁵⁹

PCT must be differentiated from other porphyrias that cause identical blistering skin lesions and from pseudoporphyria (also known as pseudo-PCT). The latter is a poorly understood condition that presents with lesions that closely resemble PCT, but with plasma porphyrins that are not significantly increased. Potentially photosensitizing drugs, such as nonsteroidal antiinflammatory agents, are sometimes implicated.

Pathophysiology

UROD sequentially decarboxylates uroporphyrinogen (which has eight carboxyl side chains) to yield coproporphyrinogen (with four carboxyl groups). When hepatic UROD is profoundly inhibited, the substrate and the intermediate and final products of the reaction accumulate as the oxidized porphyrins in the liver (mostly uroporphyrin and heptacarboxylporphyrin), and then appear in plasma and urine. Photosensitivity results from activation of porphyrins in the skin by long-wave ultraviolet light and generation of reactive oxygen species.

Hepatic UROD activity is inhibited to less than approximately 20 percent of normal in all patients with PCT. Types 1, 2, and 3 are not fundamentally different or clinically distinguished from each other. Patients with type 1 or “sporadic” PCT have no UROD mutations and no family history of PCT. Approximately 80 percent of patients fall into this category. Type 2 or “familial” PCT comprises approximately 20 percent of cases who are heterozygous for UROD mutations; but because the penetrance of this trait is low there are usually no other documented cases in the family. In families with type 3, which is rare, more than one member has PCT, but there is no UROD mutation; these familial cases presumably share other inherited or environmental susceptibility factors.

Although hepatic UROD activity must be reduced to approximately 20 percent of normal for PCT to be manifest, the amount of enzyme protein, when measured immunochemically in liver, remains at its genetically determined level, which in type 2 cases is approximately 50 percent of normal.²⁶⁰ Mice heterozygous for mutant UROD alleles are much more sensitive to porphyrinogenic stimuli than wild-type animals.²⁶¹ In heterozygous mice that display porphyric phenotypes, hepatic UROD protein is half normal, but the catalytic activity of the enzyme is reduced to approximately 20 percent, suggesting the existence of an inhibitor of hepatic UROD.²⁶¹

Although iron does not directly inhibit UROD, there is considerable evidence that PCT is an iron-related disease, with hepatic siderosis seen in many cases. This explains why HFE (hemochromatosis gene) mutations that lead to increased intestinal iron absorption predispose to development of PCT (Chap. 42). Individuals who inherit a UROD mutation have approximately 50 percent of normal enzyme activity from birth, such that a UROD inhibitor can more readily reduce enzyme activity to less than approximately 20 percent of normal. How iron and other known or suspected susceptibility factors such as alcohol, smoking, estrogens, hepatitis C, HIV, hepatic steatosis, and other suspected factors contribute to the development of PCT is less-well understood, but these may act in part by increasing oxidative stress in hepatocytes. A deficiency of ascorbic acid,^261a and perhaps other antioxidants,²⁶³ may play a role in some patients. Smoking may also act by inducing hepatic CYPs, including CYP1A2, which is essential for causing uroporphyria in rodent models,^264,265 and may produce a UROD inhibitor, which has been characterized as a uroporphomethene. This substance is a product of partial oxidation of uroporphyrinogen, and is found in the liver of mice that are heterozygous for a UROD mutation, homozygous for an HFE mutation (C282Y), and develop uroporphyria spontaneously.⁶² Other potential mechanisms for lowering of hepatic UROD activity in PCT, such as oxidative damage to UROD active site residues, are less favored but have not been excluded.²⁶⁶

At least 70 different UROD mutations have been identified in type 2 PCT and HEP (see Table 58–1). These reduce UROD activity and immunoreactivity to approximately 50 percent of normal in all tissues from birth. Most are missense mutations, with each occurring in one or a few families. Homozygosity for a null UROD mutation is lethal in early neonatal life. Therefore, in HEP at least one of the mutant UROD alleles must preserve at least some catalytic activity. Knowledge of the crystal structure of UROD allows mapping of specific mutations and prediction of their impact on enzyme structure and function. Expression studies in eukaryotic cells suggest that some mutations may destabilize the enzyme protein in a tissue-specific manner.²⁶⁷

Pathogenesis of the Clinical Findings

A distinctive feature of PCT is massive accumulation of porphyrins in the liver. As a result, fresh hepatic tissue shows strong red fluorescence on exposure to long-wave ultraviolet light. Microscopic, birefringent, needle-like inclusions are found in lysosomes, and paracrystalline inclusions in mitochondria. Increased stainable iron is very common. Other nonspecific hepatic findings are probably partly a result of the disease itself, although the effects of associated factors such as alcohol and hepatitis C are difficult to differentiate. Liver histopathology includes hepatocyte necrosis, inflammation, increased iron, and increased fat. Mild abnormalities in liver function tests, especially serum transaminases and γ-glutamyltranspeptidase, are present in almost all cases, but cirrhosis is unusual. The risk of hepatocellular carcinoma is increased, especially in those with more prolonged disease, cirrhosis, or other risk factors such as hepatitis C or alcoholic liver disease.^268,269,270

Excess porphyrins are transported in plasma from the liver. Skin histopathology includes subepidermal blistering and deposition of periodic acid-Schiff–positive material around blood vessels and fine fibrillar material in the upper dermis and at the dermoepithelial junction. Immunoglobulin G, other immunoglobulins, and complement are deposited around dermal blood vessels and at the dermoepithelial junction. Splits in the lamina lucida of the basement membrane lead to formation of fluid-containing bullae.²⁷¹ These histologic changes are found in other cutaneous porphyrias, as well as pseudoporphyria, and are not diagnostic for PCT. Activation of the complement system after irradiation has been demonstrated in PCT patients both in vivo and in vitro in sera,²⁷² and is thought to result from generation of reactive oxygen species.

Susceptibility Factors

PCT is a highly heterogeneous disease, with multiple susceptibility factors expected in the individual patient.²⁷³ Multiple factors are important in familial as well as sporadic PCT, because heterozygosity for a UROD mutation is a susceptibility factor that does not of itself reduce hepatic enzyme activity sufficiently to cause the disease. The environmental, infectious, and inherited factors discussed below, none of which is invariably present, are known or suspected to play an important role. Their prevalence may show considerable geographic variation in PCT patients as well as healthy subjects.

Alcohol

PCT has long been associated with excess alcohol use. Alcohol and its metabolites may induce ALAS1 and CYP2E1, generate active oxygen species that contribute to oxidative damage, cause mitochondrial injury, deplete reduced glutathione and other antioxidant defenses, increase production of endotoxin, activate Kupffer cells, decrease the iron regulatory hormone hepcidin²⁷⁴ and increase iron absorption.

Smoking and Cytochrome P450 Enzymes

Smoking is less extensively studied as a risk factor but is commonly associated with alcohol use in PCT.²⁷³ Smoking may increase oxidative stress in hepatocytes, and induces CYP1A2, which is essential to the development of uroporphyria in rodent models. CYP levels have been found to be increased in liver in human PCT, but it is not clear which CYP might be important in pathogenesis of the human disease. A study of caffeine metabolism did not find evidence for increased CYP1A2 activity in vivo in PCT, even when smokers and nonsmokers were analyzed separately.²⁷⁵ However, a more inducible polymorphism of CYP1A2 has been found to be more common in PCT than in normal subjects.²⁷⁶

Estrogens

Estrogen use is very common in women with PCT.^273,277,278 In the past, some men developed the disease after treatment of prostate cancer with estrogens.²⁷⁷ Female rats or males treated with estrogens are more susceptible to development of chemically induced uroporphyria than untreated males.²⁷⁹ The mechanism is not established, although estrogens can generate reactive oxygen species in some experimental systems.²⁶⁶

Hepatitis C

Reported prevalence of hepatitis C in PCT has ranged from 21 to 92 percent in various countries, and greatly exceeds the prevalence of this viral infection in healthy subjects, which shows considerable geographic variation. Hepatitis C is associated with excess fat, some iron accumulation, mitochondrial dysfunction, and oxidative stress in hepatocytes, which may contribute to the development of PCT. Dysregulation of hepcidin may contribute to iron accumulation in hepatitis C.²⁸⁰

Human Immunodeficiency Virus

PCT is less commonly associated with HIV infection than with hepatitis C.²⁸¹ Occasionally PCT is the initial manifestation of this infection. The mechanism is unknown.

Iron and HFE Mutations

Mild to moderate iron overload is found in most patients with PCT, and iron deficiency is protective. The importance of iron has been confirmed in animal models, such as rodents treated with hexachlorobenzene and other halogenated polyaromatic hydrocarbons.²⁶⁶ Mice with disruption of one UROD allele (UROD[+/–]) and two disrupted HFE alleles (HFE[–/–]) develop uroporphyria without administration of exogenous chemicals.²⁶¹ Prevalence of the C282Y mutation of the HFE gene, which is the major cause of hemochromatosis in whites, is increased in both sporadic and familial PCT, and 10 to 20 percent of patients may be C282Y homozygotes (Chap. 43).²⁸² In southern Europe, where the C282Y is less prevalent, the H63D mutation is more commonly associated with PCT.²⁸³ Excess iron may contribute to UROD inhibition by providing an oxidative environment that is apparently needed for generation of a UROD inhibitor. Hepatic hepcidin expression is reduced in hemochromatosis, and is also reduced in PCT patients without hemochromatosis genotypes when compared to patients without PCT and comparable iron overload, suggesting that reduced expression of this peptide is important in causing hepatic siderosis in PCT.²⁷⁴

Antioxidants

Substantial reductions in plasma levels of ascorbate and carotenoids have been noted in some patients with PCT.²⁶³ Ascorbate deficiency in rodents enhances susceptibility to development or uroporphyria, and ascorbate decreases uroporphyrin accumulation except in animals treated with large amounts of iron.²⁶²

A large outbreak of PCT occurred during a period of food shortage in a population in eastern Turkey in the 1950s from consumption of seed wheat treated with the fungicide hexachlorobenzene.¹¹ Smaller outbreaks and individual cases have occurred after exposures to other chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin).²⁸⁴ These chemicals were subsequently shown to cause hepatic UROD deficiency and biochemical features resembling PCT in laboratory animals, and many studies that followed have greatly increased our understanding of this acquired enzyme deficiency.^266,285 But such exposures are rarely identified in PCT patients in current clinical practice.

Clinical Features

Disease onset is usually in the fourth or fifth decade of life, and is more common in men. Onset may occur earlier in familial (type 2) disease or in cases with the C282Y/C282Y HFE genotype.²⁸⁶ Fluid-filled vesicles develop most commonly on the backs of the hands (Fig. 58–7A). Skin friability is increased and blisters often follow minor trauma. These also occur on the forearms, face, ears, neck, legs, and feet. The blisters often rupture, crust over, and are prone to infection before healing slowly. Milia may precede or follow vesicle formation. Facial hypertrichosis and hyperpigmentation are particularly noticed by female patients (Fig. 58–7B). Severe thickening of affected areas of skin is termed pseudoscleroderma and resembles systemic scleroderma.

Blistering skin lesions in VP and HCP are identical to those in PCT. Those in CEP and HEP resemble PCT but are usually much more severe and mutilating. Mild or moderate erythrocytosis is common in PCT, and is not well explained. Chronic lung disease from smoking may contribute.

Drugs that exacerbate the acute porphyrias are only occasionally reported to play a role in PCT.²⁸⁷ PCT may occur with other conditions predisposing to iron overload such as myelofibrosis^288,289 and end-stage renal disease,²⁹⁰ and with diabetes mellitus²⁷⁰ and cutaneous and systemic lupus erythematosus. PCT associated with end-stage renal disease is usually more severe, sometimes with severe mutilation. Lack of urinary porphyrin excretion in these patients leads to much higher concentrations of porphyrins in plasma, and the excess porphyrins are poorly dialyzable.²⁹⁰ The disease occasionally develops during pregnancy, perhaps related to effects of estrogen.

The clinical manifestations of HEP usually resemble CEP, with onset of blistering skin lesions, hypertrichosis, scarring, and red urine in infancy or childhood. Sclerodermoid skin changes are sometimes prominent. Excess porphyrins originate mostly from the liver in this condition. Unusually mild cases have been described.²⁹¹

Diagnosis

A diagnosis of PCT is established by finding a substantial elevation of porphyrins in urine or plasma, with a predominance of highly carboxylated porphyrins (uroporphyrin and hepta-, hexa-, and pentacarboxylporphyrins); coproporphyrins are also increased. Levels of PBG are normal, and urinary ALA is normal or slightly increased. The pattern of porphyrins in feces is complex, and includes heptacarboxylporphyrins and isocoproporphyrins. The latter are overproduced in the presence of UROD deficiency because pentacarboxylporphyrinogen is a substrate for CPO, leading to formation of dehydroisocoproporphyrinogen, which is excreted in bile and undergoes oxidation by intestinal bacteria to isocoproporphyrins.²⁹²

Measurement of plasma porphyrins and determination of the fluorescence emission peak at neutral pH is especially useful for screening patients with blistering skin lesions. A substantial increase with a peak at approximately 620 nm is most commonly caused by PCT, and excludes VP and pseudoporphyria, which are the most common conditions that mimic PCT clinically.^292a Plasma porphyrin measurements are essential for diagnosis of PCT in patients with advanced renal disease; the reference range is higher in patients with renal failure than in normals.²⁹³

Erythrocyte porphyrins are substantially increased in CEP and HEP, but are normal or only modestly increased in PCT. Rare cases of HCP with blistering lesions are identified by a predominance of coproporphyrin III in urine and especially feces. Familial (type 2) cases are identified by half-normal erythrocyte UROD activity or preferably by DNA studies to identify a UROD mutation. Erythrocyte UROD activity is 5 to 30 percent of normal in HEP, and DNA studies reveal that a UROD mutation is inherited from each parent.

Biochemical findings in HEP resemble PCT, with predominant accumulation and excretion of highly carboxylated porphyrins and isocoproporphyrins. However, in contrast to PCT, erythrocyte zinc protoporphyrin is substantially increased. At least one genotype may be associated with predominant excretion of pentacarboxylporphyrin.²⁹¹

Therapy

Treatment is highly effective but specific in both sporadic and familial PCT, and therefore should be initiated only after the diagnosis is well established. It is sometimes reasonable to start treatment after PCT is validated with a plasma porphyrin screen and excludes VP and pseudoporphyria (see “Diagnosis” above). Patients should be questioned and tested for all known susceptibility factors, including use of alcohol, tobacco, and estrogens, hepatitis C, HIV, HFE mutations, and inherited UROD deficiency (erythrocyte UROD activity or, preferably, UROD mutations), because their presence influences management. Serum ferritin should be measured before starting treatment. Patients are advised to stop drinking and smoking and to discontinue estrogens. A nutritionally adequate intake of ascorbic acid and other nutrients should be assured, but this vitamin should not be administered as primary therapy.

Improvement may occur after removing one or more susceptibility factor, but without phlebotomy or low-dose hydroxychloroquine recovery is unpredictable or slow.²⁹⁴ Repeated phlebotomy is the preferred treatment at most centers. The original rationale proposed by Ippen in 1961 was to decrease the commonly associated mild or moderately increased hemoglobin, stimulate erythropoiesis, and perhaps channel excess heme pathway intermediates to hemoglobin synthesis.²⁹⁵ However, the oxidized porphyrins that accumulate in PCT cannot reenter the heme biosynthetic pathway and be converted to heme, and it is now understood that phlebotomy is effective by reducing body iron stores and liver iron content. Treatment with an iron chelator such as desferrioxamine is less efficient than phlebotomies in reducing iron, but may be tried when the latter are contraindicated.²⁹⁶

Approximately 450 mL of blood is removed, usually at 2-week intervals. In one series an average of 5.4 phlebotomies was required for remission, but many more are needed in some patients with coexisting hemochromatosis and marked increases in serum ferritin levels. Hemoglobin or hematocrit levels are followed as safety (not therapeutic) targets, to prevent symptomatic anemia. Usually, the hemoglobin should not fall below 10 to 11 g/dL, but the baseline value and the age and clinical condition of the patient are also considered. The therapeutic target is a serum ferritin near 15 ng/mL, which is close to the lower limit of normal and associated with tissue iron depletion but usually not anemia. Additional iron depletion is not beneficial, and causes anemia. Treatment is also guided by plasma (or serum) porphyrin levels, which are more convenient to measure repeatedly than urine porphyrins, and fall more slowly than the serum ferritin. Plasma porphyrins usually decline from initial levels of 10 to 25 mcg/dL during treatment, to below the upper limit of normal (~1 mcg/dL) within weeks after phlebotomies are completed.²⁹⁷ New skin lesions are generally decreased at the end of treatment, but some may occur for a few weeks after plasma porphyrin levels become normal. Severe sclerodermatous changes and liver function abnormalities can also improve.

After a remission, continued phlebotomies are generally not needed. However, relapses may occur, especially in patients who resume use of alcohol, and are treated by another course of phlebotomies. For patients with the C282Y/C282Y or C282Y/H63D HFE genotypes, management guidelines for hemochromatosis should be followed. Continued phlebotomies as needed to maintain a serum ferritin below approximately 50 ng/mL may also be beneficial in patients who have experienced recurrences of PCT, although published experience is limited. It is also advisable to follow porphyrin levels and reinstitute phlebotomies promptly if porphyrin levels begin to rise. Liver imaging and a serum α-fetoprotein determination should be repeated as screening for hepatocellular carcinoma. After remission, transdermal estrogen can be resumed in women, if needed, with little risk for recurrence of PCT.²⁷⁵

A low-dose regimen of hydroxychloroquine or chloroquine is also effective,^{266,298,299,300,301,302,303} and most appropriate when phlebotomy is contraindicated or poorly tolerated, if iron overload is not severe. However, this treatment is preferred at some centers because it is more convenient and much less expensive. These 4-aminoquinoline antimalarials do not appear to deplete hepatic iron, and the mechanism for their effect in PCT is not fully understood. Full therapeutic doses of these drugs exacerbate photosensitivity in PCT, induce fever, malaise, and nausea, markedly increase urinary and plasma porphyrins, and increase serum transaminases, other liver function tests, and ferritin. This reaction can even unmask previously unrecognized PCT.³⁰⁴ Although these adverse effects, which are unique to PCT, are followed by complete remission,³⁰⁵ they should be avoided by a low-dose treatment regimen (hydroxychloroquine 100 mg or chloroquine 125 mg—one-half of a standard tablet—twice weekly) at least until plasma or urine porphyrins are normalized.^298,301,302 However, some patients may respond poorly and require later treatment with larger doses, or phlebotomy.³⁰⁵ There is a small risk of retinopathy,³⁰⁶ which may be lower with hydroxychloroquine. A recent prospective study found that time to biochemical remission with low-dose hydroxychloroquine was comparable to that with phlebotomy.³⁰⁷ In a retrospective study, low-dose chloroquine was ineffective in patients homozygous for the C282Y mutation of the HFE gene,³⁰⁸ which suggests that the degree of excess hepatic iron may influence response to this treatment.

These 4-aminoquinolines are not effective in other porphyrias, and do not mobilize all types of porphyrins from liver and other tissues.³⁰⁹ Chloroquine may form complexes with a variety of porphyrins, which might promote their mobilization from liver,^310,311 but this does not appear to explain the effects in PCT. It was suggested that mobilization of hepatic iron may be important,^302,312,313 but serum ferritin concentrations do not change significantly during treatment. Most likely, these drugs colocalize with excess porphyrins in lysosomes and other intracellular organelles and promote their release by a process that involves transient cell damage.

PCT may improve after treatment of coexisting hepatitis C. However, for several reasons PCT should be treated first and hepatitis C later in most cases. First, PCT is usually more symptomatic and can be treated more quickly and effectively. Second, there is some evidence that treatment of hepatitis C may be more effective after iron reduction. Third, interferon and ribavirin commonly cause anemia, which usually precludes phlebotomy for PCT. Hydroxychloroquine may be an option during treatment with interferon and ribavirin, but initial worsening of liver function tests, even with a low-dose regimen, may cause concern. It is reasonable to consider continuing low-dose hydroxychloroquine after PCT is in remission to avoid a reoccurrence of PCT during treatment of hepatitis C, but there is little experience with this approach. Reports that PCT patients are often resistant to treatment of hepatitis C,^314,315 contrast with reports of successful treatment, and prospective studies are needed. Studies are needed with newer and more effective agents for hepatitis C as these become available.

Treatment of PCT associated with end-stage renal disease is more difficult, as phlebotomy is often contraindicated by anemia. Erythropoietin administration can correct anemia, mobilize iron, and support phlebotomy in many cases.^290,316,317 High-flux hemodialysis may remove porphyrins from plasma and provide some benefit.³¹⁸ PCT is not a contraindication to renal transplantation, which is likely to lead to remission³¹⁹ partly because of resumption of endogenous erythropoietin production. The level of plasma porphyrins are often especially high in these patients, and should be assessed prior to surgery, because there may be some risk of skin and peritoneal burns from exposure to operating room lights.

Management of HEP emphasizes avoiding sunlight, as in CEP. Oral charcoal was helpful in a severe case associated with dyserythropoiesis.⁹⁸ Phlebotomy has shown little or no benefit. Retrovirus-mediated gene transfer can correct porphyria in cell lines from patients with this disease, which suggests that gene therapy may be applicable in the future.³²⁰