Heme biosynthesis and the porphyrias.

Porphyrias, is a general term for a group of metabolic diseases that are genetic in nature. In each specific porphyria the activity of specific enzymes in the heme biosynthetic pathway is defective and leads to accumulation of pathway intermediates. Phenotypically, each disease leads to either neurologic and/or photocutaneous symptoms based on the metabolic intermediate that accumulates. In each porphyria the distinct patterns of these substances in plasma, erythrocytes, urine and feces are the basis for diagnostically defining the metabolic defect underlying the clinical observations. Porphyrias may also be classified as either erythropoietic or hepatic, depending on the principal site of accumulation of pathway intermediates. The erythropoietic porphyrias are congenital erythropoietic porphyria (CEP), and erythropoietic protoporphyria (EPP). The acute hepatic porphyrias include ALA dehydratase deficiency porphyria, acute intermittent porphyria (AIP), hereditary coproporphyria (HCP) and variegate porphyria (VP). Porphyria cutanea tarda (PCT) is the only porphyria that has both genetic and/or environmental factors that lead to reduced activity of uroporphyrinogen decarboxylase in the liver. Each of the 8 enzymes in the heme biosynthetic pathway have been associated with a specific porphyria (Table 1). Mutations affecting the erythroid form of ALA synthase (ALAS2) are most commonly associated with X-linked sideroblastic anemia, however, gain-of-function mutations of ALAS2 have also been associated with a variant form of EPP. This overview does not describe the full clinical spectrum of the porphyrias, but is meant to be an overview of the biochemical steps that are required to make heme in both erythroid and non-erythroid cells.

Uroporphyrinogen 3 cosynthetase in bovine erythropoietic porphyria.

The activity of uroporphyrinogen III cosynthetase is much lower in hemolyzates from mature cattle with congenital erythropoietic porphyria than in hemolyzates from mature normal cattle. The porphyric hemolyzates do not inhibit the cosynthetase activity present in normal hemolyzates or in extracts of mouse spleen.

Congenital Erythropoietic Porphyria: A Rare Case of Photosensitivity with Hemolytic Anaemia and Mental Retardation.

Congenital erythropoietic porphyria, also called Gunther's disease, is a very rare genetic autosomal recessive diseaseaffecting less than 1 per 1,000,000 children. Pathogenesis involves genetic mutation encoding uroporphyrinogen-III cosynthase which leads to accumulation of porphyrin in many tissues, leading to extreme skin photosensitivity, red cell lysis, splenomegaly and reduced life expectancy. Herein, we report a 12-year mentally challenged girl with multiple blisters and scars on sun exposed sites since birth. She had hepatomegaly, erythrodontia, severe anaemia with haemolytic blood picture and mildly elevated liver enzymes. Skin biopsy showed deposition of amorphous eosinophilic porphyrins in the dermis, thus confirming a diagnosis of congenital erythropoietic porphyria.

Congenital erythropoietic porphyria: identification and expression of exonic mutations in the uroporphyrinogen III synthase gene.

Congenital erythropoietic porphyria (CEP), an inborn error of heme biosynthesis, results from the deficient activity of uroporphyrinogen III synthase (URO-synthase). This autosomal recessive disorder is heterogeneous; patients with severe disease are often transfusion dependent, while milder patients primarily have cutaneous involvement. To investigate this phenotypic heterogeneity, exonic point mutations in the URO-synthase gene were identified in unrelated CEP patients. Four missense mutations were identified: (a) an A to G transition of nucleotide (nt) 184 that predicted a Thr to Ala substitution at residue 62 (designated T62A); (b) a C to T transition of nt 197 that encoded an Ala to Val replacement at residue 66 (A66V); (c) a T to C transition of nt 217 that predicted a Cys to Arg substitution at residue 73 (C73R); and (d) a C to T transition of nt 683 that resulted in a Thr to Met replacement at residue 228 (T228M). In addition, a G to A transition of nt 27 that did not change the encoded amino acid (A9A) was detected in an African patient. The T62A, C73R, and T228M alleles did not express detectable enzymatic activity, while the A66V allele expressed residual, but unstable activity. The C73R allele was present in eight of 21 unrelated CEP patients (21% of CEP alleles). In three patients, identification of both alleles permitted genotype-phenotype correlations; the A66V/C73R, T228M/C73R, and C73R/C73R genotypes had mild, moderately severe, and severe disease, respectively. These findings provide the first genotype-phenotype correlations and permit molecular heterozygote detection in this inherited porphyria.

Erythropoietic protoporphyria and lead intoxication: the molecular basis for difference in cutaneous photosensitivity. I. Different rates of disappearance of protoporphyrin from the erythrocytes, both in vivo and in vitro.

In lead intoxication photosensitivity is usually absent, despite concentrations of protoporphyrin in the erythrocytes equal to or greater than in erythropoietic protoporphyria. Profound differences in the distribution of protoporphyrin in aging erythrocytes were demonstrated by age-dependent fractionation of cells on discontinuous density gradients. In erythropoietic protoporphyria the concentration of protoporphyrin declined extremely rapidly with erythrocyte age; the bulk of the protoporphyrin was lost in less than 3 days and the concentration of fluorescent erythrocytes in the gradient paralleled the decline of protoporphyrin. In lead intoxication the protoporphyrin concentration declined only slightly with cell aging and erythrocytes of all ages fluoresced. In the bone marrow from a patient with erythropoietic protoporphyria all reticulocytes, but only occasional late normoblasts, fluoresced, suggesting a single population. Sterile incubation in plasma (pH 7.5) demonstrated rapid diffusion of protoporphyrin from the erythrocytes in erythropoietic protoporphyria, but not in lead intoxication. Plasma protoporphyrin was elevated in erythropoietic protoporphyria, but not in lead intoxication. Estimates of the daily loss of protoporphyrin from erythropoietic tissue in erythropoietic proporphyria suggested an order of magnitude similar to the total blood protoporphyrin. Therefore, it is not necessary to postulate a preponderant extraerythropoietic source to explain the amount of fecal excretion. A significant amount of the diffused protoporphyrin probably reaches the skin with resulting photosensitivity. In contrast, in lead intoxication protoporphyrin remains within the erythrocyte throughout its life span ; there is no diffusion into the plasma and hence no photosensitivity.

Comparative study of protoporphyrins in erythropoietic protoporphyria and griseofulvin-induced murine protoporphyria. Binding affinities, distribution, and fluorescence spectra in various blood fractions.

Excess erythrocyte protoporphyrins of human congenital erythropoietic protoporphyria and of griseofulvin-induced murine hepatic protoporphyria were found to be associated with hemoglobin and stroma fractions in similar relationships. More than 99.5% of total erythrocyte protoporphyrin was bound to hemoglobin in each case. However, profound differences were found when protoporphyrin concentration was measured in erythrocytes that had been segregated into populations of progressive age on discontinuous density gradients. In erythropoietic protoporphyria, porphyrin content diminished rapidly with age; in murine protoporphyria, the aging erythrocyte populations became progressively more porphyrin rich. In vitro diffusion of protoporphyrin from plasma across the intact erythrocyte membrane was demonstrated. The equimolar binding affinity of protoporphyrin to hemoglobin was shown to be 40 times that of protoporphyrin to serum albumin. This strong affinity provides the driving force for the observed transmembrane diffusion, and explains the high erythrocyte/plasma porphyrin ratio in murine hepatic protoporphyria. The opposite rapid efflux of intra-erythrocytic protoporphyrin into plasma previously shown in uncomplicated erythropoietic protoporphyria occurs despite this strong hemoglobin affinity, implying continuous efficient clearance of protoporphyrin from plasma by the liver. Furthermore, these and other data suggest that a hepatic synthetic source for any significant fraction of the blood protoporphyrin in erythropoietic protoporphyria is highly improbable.

Erythropoietic protoporphyria and lead intoxication: the molecular basis for difference in cutaneous photosensitivity. II. Different binding of erythrocyte protoporphyrin to hemoglobin.

Acidic solvents extract the same porphyrin-protoporphyrin-from the erythrocytes of patients with either erythropoietic protoporphyria or lead intoxication. However, extractable protoporphyrin disappears rapidly, both in vivo and in vitro, from erythrocytes in erythropoietic protoporphyria but slowly, if at all, in lead intoxication. Consistent with these observations, fluorescence spectroscopy revealed that the intracellular state of the erythrocyte protoporphyrin is different in the two diseases. Spectrofluorometric measurements coupled with fractionations and biochemical syntheses showed that in erythropoietic protoporphyria the protoporphyrin is bound as the free base to hemoglobin molecules at sites other than the heme binding sites. In lead intoxication the fluorescent porphyrin is also bound to hemoglobin but is present as zinc protoporphyrin. The data suggest that the zinc protoporphyrin is bound at heme binding sites. Acidic extraction solvents remove the chelated zinc, but zinc protoporphyrin may be extracted intact from erythrocytes with acetone, ethanol, or the detergent Ammonyx-LO.

Erythropoietic porphyria of the fox squirrel Sciurus niger.

Uroporphyrin I is found in high concentration in the bones, teeth, blood, soft tissues, and urine of the fox squirrel, Sciurus niger. The concentration of uroporphyrin in fox squirrel spleen is much higher than in liver, kidney or bone marrow, probably because of accumulation from phagocytosed red cells. Bleeding causes a marked increase in the uroporphyrin concentration of red cells and spleen, and a 3-8-fold increase in uroporphyrin excretion. Urinary excretion of delta-aminolevulinic acid and porphobilinogen is not greater in fox squirrels than in nonporphyric gray squirrels. Sciurus carolinensis, used as controls. In all these characteristics, as well as in the previously demonstrated deficiency of the enzyme uroporphyrinogen III cosynthetase in red cells, the physiological porphyria of fox squirrels resembles congenital erythropoietic porphyria, a hereditary disease of man and cattle. For squirrels differ in showing no evidence of cutaneous photosensitivity or hemolytic anemia. Uroporphyrinogen III cosynthetase activity is present in fox squirrel bone marrow at 1/10 its concentration in gray squirrel marrow. The fox squirrel enzyme is much more unstable than the gray squirrel enzyme, which provides a possible explanation for its low activity and for the overproduction of uroporphyrin I. It is unlikely that the deficiency of cosynthetase is due to its inactivation by excessive amounts of uroporphyrinogen I synthetase, because activity of the latter enzyme is the same in blood from fox and gray squirrels.Fox squirrel porphyria provides a convenient model for studies of pathogenesis of human congenital erythropoietic porphyria.

Toxicologic aspects of photosensitization in livestock.

Two types of plant-caused photosensitizations are recognized in livestock: 1) primary, wherein the phototoxic agent in the plant is ingested and reaches the skin chemically unchanged; and 2) secondary, wherein the phototoxic agent in the porphyrin phylloerythrin produced by chlorophyll degradation in ruminant stomachs. Phylloerythrin is normally excreted in bile but is allowed to reach the skin when hepatic damage interferes with the phylloerythrin-excreting mechanism. Primary photosensitizing plant toxins are few, whereas secondary photosensitizations can be caused by damage to the liver by a variety of plant and other toxins. Plants causing each type of photosensitization are discussed and clinical manifestations of the disease in livestock are summarized. Tetradymia species are one of the most economically important causes of phototoxicity in livestock. The etiology of this phototoxic syndrome in sheep and the importance of sagebrush species as preconditioning agents for phototoxicity are discussed.

Chemical and biochemical aspects of photosensitization in livestock and poultry.

Certain synthetic and naturally occurring chemicals, particularly those found in some range plants, may interact with livestock and poultry in the presence of activating light to produce photosensitization. Such photosensitization may have serious implications for livestock producers as a result of causing reduced performance of and even death of affected animals. The mechanisms producing photosensitization in livestock and poultry are discussed in context with the chemical nature of major livestock photosensitizers. The possibility that photosensitizing agents for livestock may have toxicologic significance in humans consuming photoactive residues in meat or animal byproducts is considered.

Studies of porphyrin synthesis in fibroblasts of patients with congenital erythropoietic porphyria and one patient with homozygous coproporphyria.

Partial deficiencies in enzymes activity of the heme biosynthesis pathway have been demonstrated in cultured skin fibroblasts and other tissues from patients suffering from congenital erythropoietic porphyria and hereditary coproporphyria. Using a new fluorimetric method, we have assessed quantitatively porphyrin biosynthesis from added delta-aminolevulinic acid in cultured fibroblasts of two congenital erythropoietic porphyria patients and one homozygous case of hereditary corproporphyria. The results were compared with those of the patients' parents and those of normal controls. All the porphyrins synthesized remained within the cells of normal subjects and of patients with congenital erythropoietic porphyria; these porphyrins were mostly (95%) protoporphyrin. The fibroblasts of the patient with homozygous hereditary coproporphyria synthesized the same amount of porphyrins, but only 25% were found within the cells, whereas 75% were found in the medium. The porphyrins found within the cells were coproporphyrin (25%) and protoporphyrin (75%); in the medium, only coproporphyrin was identified.

The osteodystrophy of congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) is a rare disorder of heme biosynthesis that results in the production of large quantities of photoactive porphyrins. The clinical syndrome is dominated by extreme photosensitivity with mutilation of light exposed extremities and hemolytic anemia. Bone disease has been occasionally noted, but is not well characterised. We describe a man with CEP who developed bone pain and spinal crush fractures at the age of 22. Skeletal radiographs revealed features typical of other severe hemolytic anemias, but in addition there was loss of the terminal phalanges of the hand as a result of photomutilation. Spinal bone density (assessed by DPA) was reduced and at the hip bone density was at the lower limit of normal. The metacarpal cortical bone density was 2.9 standard deviations below normal. Biochemical and histological studies accelerated bone turnover. Although the serum 250H vitamin D concentration was very low (because of light avoidance) there was no evidence that the bone disease was a consequence of this. Treatment for one year with clodronate and a high transfusion regime was associated with small reductions in serum alkaline phosphatase and urine hydroxyproline excretion, but there was no improvement in bone mineral density. We conclude that CEP has a distinctive osteodystrophy comprising osteolysis of light-exposed extremities and a high turnover type of osteoporosis. Privational vitamin D deficiency may also occur. The effect upon bone of the new therapies for CEP should be considered.

Beta-carotene therapy for erythropoietic protoporphyria and other photosensitivity diseases.

This paper describes the development of the use of carotenoid pigments in the treatment of light-sensitive skin diseases. It also discusses the animal and human studies involved in determining whether carotenoids have any anti-cancer activity. The possible mechanisms of carotenoid photoprotective and anti-cancer actions are briefly discussed.

Brown amiotic fluid in congenital erythropoietic porphyria.

Brown amniotic fluid due to congenital erythropoietic porphyria has been observed at the 15th week of pregnancy in a fetus proved after abortion to have the disease. The color is produced by abnormal porphyrins resulting from a deficiency of uroporphyrin cosynthetase. The mother, a carrier of the autosomal recessive gene, has since delivered a normal infant whose amniotic fluid was unremarkable.

Congenital erythropoietic porphyria. A mild variant with low uroporphyrin I levels due to a missense mutation (A66V) encoding residual uroporphyrinogen III synthase activity.

BACKGROUND AND DESIGN: Congenital erythropoietic porphyria, an inborn error of heme biosynthesis, results from the deficient activity of the enzyme uroporphyrinogen III synthase. The clinical manifestations in unrelated patients with this autosomal recessive disorder are remarkedly variable, ranging from mild cutaneous involvement to severe transfusion-dependent hemolytic anemia. Biochemical and molecular studies were undertaken to investigate the nature of the unusually mild phenotype in a 15-year-old boy with only cutaneous manifestations. RESULTS: The proband's levels of total porphyrins, urinary uroporphyrin I, and erythrocyte coproporphyrin I were elevated, but not as dramatically as in other patients with this porphyria. Interestingly, the erythrocyte uroporphyrinogen III synthase activity in the proband was about 21% of the normal mean, indicating the presence of significant residual activity. In cultured lymphoblasts from the proband, his father, and mother, the enzymatic activities were 10%, 70%, and 50% of the normal mean, respectively. Molecular analyses revealed that the proband was heteroallelic for two uroporphyrinogen III synthase missense mutations: the C73R allele inherited from his mother and the A66V allele transmitted by his father. The A66V allele encoded residual enzymatic activity in vitro while the C73R allele did not. CONCLUSIONS: The A66V allele accounted for the proband's low levels of porphyrin accumulation and mild clinical manifestations. Such genotype-phenotype correlations should provide understanding of the remarkable clinical variability in other patients with this inherited porphyria.

Corneal and conjunctival changes in congenital erythropoietic porphyria.

Penetrating keratoplasty and conjunctival resection were performed on a 53-year-old woman with confirmed congenital erythropoietic porphyria. Corneal and conjunctival tissue samples, studied by light and electron microscopy, revealed inflammatory cells and a reduced keratocyte population. Vessels within the conjunctival and corneal stroma displayed a thickened basement membrane. Microfibrillar material was seen in the extracellular spaces of the conjunctival stroma. These changes are similar to those reported for ultraviolet light-exposed skin of porphyria patients. The Descemet's membrane lacked the normal fetal and postnatal banding. It presented as a homogeneous layer, consisting of uniform, densely packed collagen fibers, which suggests endothelial damage in utero. The corneal endothelium was severely damaged.

The porphyrias.

The porphyrias are a group of disorders of heme metabolism that result from partial defects in the several enzymes that control heme biosynthesis. Accumulation of porphyrins or porphyrin precursors in several different patterns results from these defects and biochemically characterizes each specific syndrome. Patterns of cutaneous photosensitivity and associated systemic symptoms among the several porphyrias result from the types of porphyrins or precursors accumulated in each.

Rapid improvement in the chemical pathology of congenital erythropoietic porphyria with treatment with superactivated charcoal.

Non-absorbable sorbents that bind porphyrins in the gastrointestinal tract may be useful in the treatment of porphyrias whose manifestations result from porphyrin excess. To test this, we assessed the effect of oral charcoal on porphyrin economy in a patient with a probable congenital erythropoietic porphyria. Treatment with a superactivated charcoal (Super Char), 25 g three times daily, was associated with a precipitous drop in erythrocyte porphyrin (from 21.4 +/- 2.9 [SD] to 7.4 +/- 0.4 nmole/ml; p less than 0.025) and plasma porphyrin (from 1.56 +/- 0.24 to 0.70 +/- 0.08 nmole/ml; p less than 0.01). Urinary porphyrin excretion appeared to rise, from 103 +/- 45 to 160 +/- 30 mumole/d, but the change was not statistically significant. Constipation appeared to limit compliance with the charcoal regimen by the end of the study period. Nonetheless, superactivated charcoal may be a useful therapy in this disfiguring porphyria.

Corneoscleral ulceration in congenital erythropoietic porphyria (a case report).

A case of congenital erythropoietic porphyria, with corneoscleral ulceration, a rare association in this disorder, was reported. Anterior segment ischemia is believed to play an important role in the etiopathogenesis of corneoscleral ulceration. Remarkable improvement occurred with use of topical heparin drops (500 units/ml).

[Acute intermittent porphyria]. / Porfiria acuta intermittente.

Acute intermittent porphyria (AIP) is a congenital disease which as its name suggests, runs intermittently. Biochemically it is characterised by over-production of hepatic ALA synthetase (ALA-s), inducible mitochondrial enzyme and an increase in prophyrinic precursors (PBG, ac S-ALA). Clinically it is characterised by an abdominal nervous symptomatology. The primary metabolic error has been identified as a deficiency in enzyme activity which partially blocks haem biosynthesis. During the appearance of clinical manifestations, certain factors are present which have the capacity of inducing hepatic ALA-s production in vitro. Apart from some preventive measures treatment is mainly of symptomatology and complications. More recently the use of ALA-s inhibitors has been introduced.