Protoporphyrin IX: the Good, the Bad, and the Ugly

ALA Loading to Bypass ALAS1.

Because the rate-limiting step is bypassed, intermediates after ALA in the heme synthesis pathway accumulate, including PPIX. PPIX is a photosensitizer, and ALA administration can result in enough PPIX accumulation in lesions in the skin and other tissues to be the basis photodynamic therapy (Kennedy et al., 1990). Tissue selectivity is enhanced by direct application of ALA (e.g., to skin lesions) and by lesion-directed laser treatment. An ALA preparation is Food and Drug Administration approved for this purpose. ALA loading in humans also results in increased urinary excretion of coproporphyrin, particularly isomer III (Shimizu et al., 1978).

Gain of Function of ALAS2.

X-linked protoporphyria (XLP) is a recently characterized, rare X-linked disease due to gain-of-function mutations of ALAS2 (Whatley et al., 2008; Holme et al., 2009). These mutations alter the C-terminal portion of the ALAS2 gene (Whatley et al., 2008), which enhances the function of ALAS2 such that amounts of PPIX produced exceed that needed by FECH for heme synthesis. Increased metal-free PPIX in erythrocytes and plasma causes a painful, mostly nonblistering type of photosensitivity. Zinc PPIX, a product of normal FECH activity, is also increased in this condition, but mostly remains in circulating erythrocytes and is less photosensitizing.

Decreased ALAD Activity.

Deficient ALAD activity leads to accumulation of ALA and other intermediates, including PPIX, in large amounts in erythroid cells. This occurs in ALAD deficiency porphyria, the most rare human porphyria, causing elevations in ALA in plasma and urine, coproporphyrin III in urine (as in ALA loading), and zinc PPIX in erythrocytes. The same findings occur in lead poisoning, suggesting that increases in coproporphyrin III and erythrocyte PPIX in lead poisoning result largely from inhibition of ALAD and a resulting oversupply of ALA. In hereditary tyrosinemia type I, succinylacetone accumulates and potently inhibits ALAD, leading to similar biochemical findings and porphyria-like symptoms (Lindblad et al., 1977). Photosensitivity does not occur in these conditions, because the excess PPIX is predominantly zinc PPIX, reflecting adequate FECH activity. ALAD can also be inhibited by styrene in animals and humans (Fujita et al., 1987).

Decreased PPOX Activity.

This penultimate enzyme in the heme biosynthetic pathway catalyzes the oxidation of protoporphyrinogen IX to PPIX with loss of six protons. An inherited partial deficiency of PPOX in variegate porphyria (VP) causes hepatic accumulation of protoporphyinogen IX, which is then oxidized nonenzymatically to PPIX. Chronic, blistering photosensitivity is a common symptom in VP. Phenoxy acid herbicides are potent PPOX inhibitors, and cause PPIX accumulation and phototoxicity in exposed plants (Duke et al., 1990). These chemicals also inhibit PPOX and cause protoporphyrinogen IX and PPIX accumulation in rodent hepatocytes (Sinclair et al., 1994).

Decreased FECH Activity.

Loss-of-function FECH mutations are found in human erythropoietic protoporphyria (EPP) (Bonkowsky et al., 1975; Thapar and Bonkovsky, 2008). The FECH gene is located on the long arm of chromosome 18 on locus 18q21 (Whitcombe et al., 1991). More than 120 different FECH mutations have been identified in EPP (Thapar and Bonkovsky, 2008). Most commonly, a severe FECH mutation is inherited from one parent and a low expression (hypomorphic) FECH alteration that is common in the general population from the other parent (Gouya et al., 1999). FECH deficiency in EPP also impairs zinc PPIX formation, so the excess PPIX that accumulates is mostly metal-free (Nelson et al., 1998; Labbe et al., 1999).

Some xenobiotics and/or their metabolites inhibit FECH and cause PPIX accumulation. Salicylic acid, a nonsteroidal anti-inflammatory drug, binds directly to FECH and inhibits its activity, and can inhibit heme synthesis in cultured cells (Gupta et al., 2013). Certain xenobiotics require P450s to produce metabolites that are FECH inhibitors. 3,5-Diethoxycarbonyl-1,4-dihydrocollidine and the antifungal drug griseofulvin are examples of chemicals that are suicidal substrates of P450s, and their metabolism in mouse liver produces N-methyl- and N-ethyl-protoporphyrin IX, which are potent FECH inhibitors (Cole et al., 1981; Cole and Marks, 1984). These chemicals are also potent inducers of hepatic ALAS1, which further contributes to PPIX accumulation (Cole et al., 1981). Griseofulvin exacerbates acute hepatic porphyrias presumably by its inducing effect on ALAS1 and inhibition of FECH (Felsher and Redeker, 1967).

Decreased Iron Availability.

MFRN1 imports iron into mitochondria (Shaw et al., 2006), and a deficiency of MFRN1 reduces iron supply for FECH, limits heme synthesis, and leads to PPIX accumulation (Troadec et al., 2011). ABCB10 physically interacts with MFRN1 and increases the half-life of MFRN1 (Chen et al., 2009). Deficiency of ABCB10 impairs mitochondrial iron import and causes PPIX accumulation (Yamamoto et al., 2014). At the same time, ABCB10 deficiency in mice results in iron accumulation in cytoplasm and leads to sideroblastic anemia (Yamamoto et al., 2014).

Iron deficiency impairs heme formation because ferrous iron is a substrate for FECH. However, zinc can substitute for iron, so with iron deficiency FECH catalyzes an increase in formation of zinc PPIX (Braun, 1999), which accumulates in reticulocytes and is increased in circulating erythrocytes. An increase in erythrocyte zinc PPIX is an early indicator of iron deficiency and precedes the development of anemia (Labbe et al., 1999). Iron chelators can bind to ferrous iron in the cytoplasm of cells, such as hepatocytes, and prevent iron from entering mitochondria to reduce FECH function and cause PPIX accumulation (Amo et al., 2009; Blake and Curnow, 2010; Juzeniene et al., 2013). In rodent and chick embryo livers and isolated hepatocytes, iron chelation can limit heme synthesis sufficiently to potentiate induction of ALAS1 by drugs and steroids (Anderson et al., 1982). In lead and other heavy metal poisoning, disturbed iron hemostasis may account in part for zinc PPIX accumulation (Schauder et al., 2010). Impaired iron delivery to reticulocytes accounts in part for anemia of chronic disease, which is also associated with an increase in erythrocyte zinc PPIX (Braun, 1999).

PPIX Accumulation with Normal PPOX and FECH Activity and Iron Supply.

Amounts of zinc PPIX under normal and abnormal conditions are higher in younger than older erythrocytes, so increased erythropoiesis and shortened erythrocyte life span will increase the average concentration of PPIX. Therefore, an increase in zinc PPIX in erythrocytes is expected in any erythrocytic disorder (e.g., hemolytic anemias), even if heme synthetic enzymes and iron availability are not impaired (Anderson et al., 1977).

PPIX Transporters in PPIX Accumulation.

Excess PPIX synthesized in mitochondria needs to be pumped out to the cytoplasm and then to the extracellular space (Fig. 2). Transporters are required because the two carboxylate side chains of PPIX make the movement of PPIX across a lipid bilayer energetically unfavorable. PBzR locates in the outer membrane of mitochondria and its expression is increased during erythroid differentiation, suggesting that PBzR might be responsible for transporting PPIX (Pastorino et al., 1994; Taketani et al., 1994). Overexpression of recombinant PBzR in Escherichia coli supported the role of PBzR in PPIX movement (Wendler et al., 2003). Apart from PBzR, ABCG2 also contributes to PPIX movement (Jonker et al., 2002). ABCG2 is primarily localized on extracellular membranes and responsible for PPIX efflux (Solazzo et al., 2009). ABCG2 also locates in mitochondrial membranes and contributes to PPIX efflux from mitochondria (Kobuchi et al., 2012). ABCG2-deficient mice have a higher amount of PPIX in red blood cells, liver, and the Harderian gland than wild-type mice (Jonker et al., 2002, 2007).

In summary, PPIX homeostasis is maintained by close coordination of multiple factors, including ALAS, FECH, iron supply, and transporters of iron and PPIX (Fig. 2). These are the targets for genetic and environmentally induced disturbances in PPIX homeostasis. PPIX homeostasis can also be disturbed by alterations in enzymes, cofactors, and transporters downstream of ALAS and upstream of FECH (Fig. 2). For example, severe, homozygous, or compound heterozygous deficiency of any enzyme in the heme biosynthetic pathway after ALAS leads to an increase in erythrocyte PPIX, which is zinc chelated if FECH activity is preserved. Heme synthesis is increased in the marrow in an effort to compensate for the severe hemolytic anemia seen in some of these disorders.

PPIX-Mediated Skin Damage.

Metal-free PPIX is an endogenously produced photosensitizer. An acute, painful, and mostly nonblistering type of photosensitivity is the most common and significant clinical manifestation of increased metal-free PPIX in the protoporphyrias (EPP and XLP) (Thapar and Bonkovsky, 2008). To prevent symptoms, patients are forced to avoid light, which interferes with many professional and other everyday activities and impairs quality of life (Thapar and Bonkovsky, 2008). PPIX either in blood in the dermal vessels or after uptake into skin cells absorbs light energy and transfers this energy to oxygen, generating reactive oxygen species (ROS), which can lead to cytotoxicity through proteins, DNA, and lipid damage (Lim, 1989). These ROS can also cause local vasodilatation and edema by complement activation and mast cell degranulation (Baart de la Faille et al., 1991). Patients who remove themselves from exposure as soon as symptoms begin will recover quickly. Longer exposure can lead to more marked reactions with severe pain, redness, swelling, and even blistering, as well as systemic reactions, and may require several days or more for recovery (Dubrey et al., 2015). Repeated reactions to sunlight can have residual effects, such as waxy thickening of the skin, especially over the knuckles, hands, nose, and cheeks, and mild scarring (Horner et al., 2013). Because sunlight exposure is avoided, EPP and XLP patients are often vitamin D deficient (Wahlin et al., 2011a). Mild microcytic anemia with evidence of iron deficiency is common in EPP and XLP and is poorly understood (Holme et al., 2007), because elemental iron absorption is not impaired (Bossi et al., 2015).

Closely woven clothing, hats, and gloves are manufactured for patients with protoporphyria and other photosensitizing diseases. Pharmacological approaches have included pharmaceutical-grade β-carotene, narrow wave UVB phototherapy, cysteine, afamelanotide, antihistamines, and vitamin C (Table 1) (Sawyer et al., 1980; Krook and Haeger-Aronsen, 1982; Mathews-Roth et al., 1994; Collins and Ferguson, 1995; Warren and George, 1998; Wahlin et al., 2011a; Fabrikant et al., 2013; Tintle et al., 2014; Biolcati et al., 2015; Langendonk et al., 2015; Lengweiler et al., 2015; Minder and Schneider-Yin, 2015). Minder et al. (2009) reviewed the treatment options for dermal photosensitivity in EPP in 2009 and concluded that the data were insufficient to prove efficacy of any treatments studied in EPP. Since then, controlled trials have demonstrated improved light tolerance in EPP and XLP with afamelanotide, an α-melanocyte–stimulating hormone analog that increases skin melanogenesis (Harms et al., 2009; Fabrikant et al., 2013; Biolcati et al., 2015; Langendonk et al., 2015).

PPIX-Mediated Hepatobiliary Disease.

Because PPIX concentrations in bile are high in EPP and XLP, PPIX-containing gallstones are common and may require cholecystectomy at an early age (Bloomer and Enriquez, 1982; Doss and Frank, 1989; Todd, 1991). PPIX-mediated liver damage, which occurs in less than 5% of patients with protoporphyria, is the most serious clinical manifestation and can be life threatening and require liver transplantation. The marrow is the major source of PPIX in EPP and XLP. Hepatic de novo synthesis is at best a minor contributor (Fig. 3). After uptake from plasma, hepatocytes transport PPIX into bile canuliculi. Bile is stored and concentrated in the gallbladder and delivered to the small intestine during meals, and some may be reabsorbed in the small intestine and delivered back to the liver by an enterohepatic circulation. This is the only route for excretion of PPIX because it is a large, hydrophobic molecule that cannot be excreted by the kidneys (Jonker et al., 2002, 2007).

Large amounts of PPIX are hepatotoxic, damaging both hepatocytes and cholangiocytes. PPIX impairs bile flow when administered in large amounts to bile fistula rodents (Lee et al., 1984). Once liver damage ensues, PPIX elimination by the liver is further compromised and levels of PPIX in plasma and erythrocytes can increase progressively, and bile composition becomes altered (Szechcinski et al., 1986; Meerman et al., 1999b). Increased delivery of PPIX to the liver can lead to further inflammation and accelerated damage to hepatocytes and cholangiocytes. Liver damage can progress slowly, as indicated by mild and often unexplained abnormal liver function tests, or advance rapidly to hepatic failure with evidence of both acute and chronic liver disease (Bruguera and Herrero, 2005; Anstey and Hift, 2007; Ma et al., 2010). Treatment with a combination of plasmapheresis to remove PPIX from plasma, hemin infusions to reduce marrow production of PPIX (Bloomer and Pierach, 1982; Gordeuk et al., 1986), cholestyramine (or activated charcoal) to interrupt the enterohepatic circulation of PPIX (Gorchein and Foster, 1999), bile acids such as ursodeoxycholic acid to enhance bile formation and flow (Van Hattum et al., 1986; Gross et al., 1998), and vitamin E to reduce oxidative stress (Komatsu et al., 2000; Singal et al., 2014) may achieve remission or slow the progression of liver disease and bridge the patient to hepatic transplantation. Such approaches are depicted in Fig. 3. These measures are not always effective and have not been applied in sufficient numbers of patients with hepatopathy to allow rigorous evaluation of efficiency (Anstey and Hift, 2007; Casanova-Gonzalez et al., 2010). New approaches to treatment and prevention of this severe complication of protoporphyria are needed.

Survival after liver transplantation for protoporphyric hepatopathy is comparable to that after other liver diseases (McGuire et al., 2005). However, because overproduction of PPIX by marrow continues, protoporphyric hepatopathy may recur (Samuel et al., 1988; de Torres et al., 1996; Meerman et al., 1999a; Dellon et al., 2002; McGuire et al., 2005). Marrow stem cell transplantation after a temporary remission of hepatopathy or liver transplantation can prevent recurrent liver damage (Rand et al., 2006; Wahlin and Harper, 2010; Dowman et al., 2011; Wahlin et al., 2011b; Singal et al., 2014). At present, identification in advance of patients at risk for development of hepatopathy is impossible. Motor neuropathy, which is characteristic of the acute porphyrias but not EPP and XLP, can develop in patients with severe protoporphyric hepatopathy, suggesting that PPIX or a product of PPIX may be neurotoxic under some conditions (Rank et al., 1993). Gene therapy targeting FECH, which is undergoing preclinical development, will, like bone marrow transplantation, restore FECH activity in the marrow (Poh-Fitzpatrick et al., 2002; Oustric et al., 2014).