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.

Activation of the complement system in patients with porphyrias after irradiation in vivo.

Irradiation of the forearms of two patients with erythropoietic protoporphyria and one patient with porphyria cutanea tarda resulted in an in vivo activation of the complement system, as assessed by diminution of the hemolytic titers of the third component of complement by 23-57%, and of the fifth component of complement (C5) by 19-47%. Such treatment also generated chemotactic activity for human polymorphonuclear cells; the chemotactic activity was stable at 56 degrees C and antigenically related to human C5. On Sephadex G-75 chromatography the chemotactic activity eluted with an apparent molecular weight of 15,000. These in vivo results extend our previous in vitro observation of photoactivation of complement in sera from patients with erythropoietic protoporphyria and porphyria cutanea tarda, and suggest that the complement system may participate in the pathogenesis of cutaneous phototoxicity in these patients.

Protoporphyrin hepatopathy. Effects of cholic acid ingestion in murine griseofulvin-induced protoporphyria.

Short-term effects of cholic acid ingestion on hepatic accumulation, fecal excretion, and blood levels of protoporphyrin were studied in vivo in griseofulvin-induced protoporphyric mice. Experimental mice that received feed with 2% griseofulvin and 0.5% cholic acid were compared with control mice that received feed with 2% griseofulvin for 4 wk. Five mice from each group were assessed each week for liver and blood porphyrin levels. Fecal protoporphyrin was compared weekly in the total pooled output of each population. Mean protoporphyrin levels were significantly lower for liver (P less than 0.0001), erythrocytes (P less than 0.05), and plasma (P less than 0.05), and higher for feces (P less than 0.001) for the mice that were fed cholic acid. Microscopic protoporphyrin deposits, inflammation, necrosis, and dysplasia were more severe in livers of control mice. A second experimental design compared four regimens in the feed given to all mice after 1-wk induction with 2% griseofulvin: (a) 0.5% cholic acid, (b) no adulterant, (c) 2% griseofulvin and 0.5% cholic acid, and (d) 2% griseofulvin. No difference in protoporphyrin removal from livers of mice in groups 1 and 2 was observed after 1 and 2 wk of these regimens. The apparent reduction in hepatic protoporphyrin content in mice of group 3 as compared with group 4 at weeks 2 and 3 was not significant at P less than 0.05. These data suggest that in selected circumstances, hepatic protoporphyrin secretion may be enhanced in protoporphyric disease states by bile salt supplementation.

Studies in porphyria: functional evidence for a partial deficiency of ferrochelatase activity in mitogen-stimulated lymphocytes from patients with erythropoietic protoporphyria.

In this paper we show that the ferrochelatase defect in erythropoietic protoporphyria (EPP) can readily be identified in mitogen-stimulated lymphocytes since such cells from patients with EPP accumulate approximately twice as much protoporphyrin IX as cells from normal subjects when incubated with a porphyrin precursor, gamma-aminolevulinic acid (ALA). Treatment of cultures with ALA and with the iron chelator, CaMgEDTA significantly increased the level of protoporphyrin IX in mitogen-stimulated lymphocytes from normal subjects, while the same treatment failed to produce an increase in protoporphyrin IX in cell preparations from EPP patients. In contrast to the results with the chelator treatment, supplementation of the cultures with iron and ALA reduced the level of protoporphyrin IX in normal cells, but not in EPP cells. These findings are compatible with a partial deficiency of ferrochelatase in EPP lymphocytes. The gene defects of acute intermittent porphyria and hereditary coproporphyria have previously been identified using lymphocyte preparations from the gene carriers of these diseases. The present study demonstrates that EPP represents another form of human porphyria in which the gene defect of the disease can now be identified in lymphocyte preparations.

Rates of plasma porphyrin disappearance in fluorescent vs. red incandescent light exposure.

The rates of porphyrin disappearance in plasma specimens were assessed during exposure to standard fluorescent room lighting. Protoporphyrin half-life in specimens from patients with erythropoietic protoporphyria appeared to be less than 30 min under these conditions. Uroporphyrin-coproporphyrin mixtures in plasmas of patients with porphyria cutanea tarda were more photostable, with half-lives measurable in terms of hours. All plasma porphyrins could be protected for several days from similar photodegradation by performing all blood drawing, processing, and assay procedures under ordinary red-incandescent illumination, and by storage in the dark.

Erythropoietic protoporphyria: four novel frameshift mutations in the ferrochelatase gene.

Human erythropoietic protoporphyria is an inherited disorder of the heme metabolic pathway caused by defects in the gene for ferrochelatase, the terminal enzyme of the pathway that catalyzes chelation of ferrous iron into protoporphyrin IX to form heme. Mutation analysis was performed for families with erythropoietic protoporphyria and four novel frameshift mutations were identified. Two of the mutations, 205insA and 215insT in exon 3 of the ferrochelatase gene, are single bp insertions. The other two, 400delA in exon 4 and 678delG in exon 6, are single bp deletions. All of the mutations result in premature termination codons downstream shortly after the mutation sites, and in one case the premature termination codon caused by 400delA was also shown to reduce mRNA level via nonsense-mediated mRNA decay.

Haplotype analysis of families with erythropoietic protoporphyria and novel mutations of the ferrochelatase gene.

Ferrochelatase, the enzyme that catalyzes the terminal step in the heme biosynthetic pathway, is the site of the defect in the human inherited disease erythropoietic protoporphyria. Molecular genetic studies have shown that the majority of erythropoietic protoporphyria cases are transmitted in dominant fashion and that mutations underlying erythropoietic protoporphyria are heterogeneous. We performed haplotype analysis of American families that shared recurrent ferrochelatase gene mutations yet had forbearers from several European countries. This was to gain insight into whether these mutations represent mutational hotspots at the ferrochelatase gene, or propagation of ancestral alleles bearing the mutations. Two recurrent mutations were found to occur on distinctive chromosome 18 haplotypes, consistent with being hotspot mutations. On the other hand, we found three sets of two unrelated families that shared the same haplotypes bearing these mutations, which could reflect geographic dispersion of ancestral mutant alleles. In addition, we report novel mutations associated with erythropoietic protoporphyria: g(+ 1)-->t transversion of the exon 4 donor site, g(+ 1)-->a transition of the exon 6 donor site, and t(+ 2)-->a substitution at the exon 9 donor site; these mutations are predicted to cause splicing defects of the associated exons. We also identified a g(+ 5)-->a transition of the exon 1 donor site in four unrelated families with erythropoietic protoporphyria, and a G(- 1)-->A substitution at the exon 9 donor site in an additional family. The probability that these sequence changes are normal polymorphisms was virtually excluded (p < 0.0001) by their absence in 120 ferrochelatase alleles from 30 normal subjects and 30 individuals with manifested erythropoietic protoporphyria with or without a known mutation.

Changes in protoporphyrin distribution dynamics during liver failure and recovery in a patient with protoporphyria and Epstein-Barr viral hepatitis.

Acute liver failure with cholestasis, histologic and serologic evidence of Epstein-Barr viral infection, and associated autoimmune hemolytic anemia occurred in a patient with lifelong protoporphyria. Changes in previously established baseline protoporphyrin distribution dynamics in erythrocyte, plasma, and fecal excretion compartments were observed during the period of severe hepatic dysfunction and recovery. These changes were consistent with predictions of a previously described conceptual model for human protoporphyria.

Porphyria cutanea tarda. Clinical features and laboratory findings in 40 patients.

PIP: Porphyria cutanea tarda is the most common disorder of porphyrin metabolism in the United States and Europe. This report presents the clinical, laboratory and pathologic features of 40 patients with porphyria cutanea tarda. Each patient was followed up for variable times during 1960-76 at the Clinical Research Center and the Dermatology Service of the Columbia-Presbyterian Medical Center; at the New York University Medical Center; or at the Rockefeller University Hospital. Earlier age at onset; diminution of alcohol ingestion as the major etiologic factor; and, an increased incidence in females indicate new environmental influences. The most frequently associated etiologic factor, aside from alcohol intake, was use of estrogens for contraception; postmenopausal syndrome; or treatment of prostatic carcinoma. Cutaneous findings in the patients included bullae (85%); increased skin fragility (75%); facial hypertrichosis (63%); hyperpigmentation (55%); sclerodermoid changes (18%); and, dystrophic calcification with ulceration (8%). Diabetes mellitus was found in 15%; systemic lupus erythematosus in 5%; elevated serum iron level in 62%; and, abnormal liver function test results in 60%. Histologic abnormalities were seen in liver biopsies of 34 patients. Phlebotomy is the treatment of choice. In 32 patients so treated, clinical remissions averaged 30.9 months. 31% (10 patients) had a relapse but additional phlebotomies resulted in 2nd remissions. Chloroquine and plasmaphoresis treatments were also briefly discussed.^ieng

A plasma porphyrin fluorescence marker for variegate porphyria.

Ten patients with variegate porphyria were uniformly found to have distinctive plasma porphyrin fluorescence wavelength maxima in saline-diluted plasma specimens. The porphyrin complex in each of these plasma samples had a fluorescence emission maximum at 626 +/- 1 nm. Twelve patients with porphyria cutanea tarda, eight patients with erythropoietic protoporphyria, one patient with congenital erythropoietic porphyria, two patients with acute intermittent porphyria, and four patients with hereditary coproporphyria, whose plasma specimens were similarly examined, had plasma fluorescence characteristics that were different from those of the patients with variegate porphyria. Plasma fluorescence emission that is maximal at 626 +/- 1 nm is a diagnostic marker for variegate porphyria.

Porphyrialike bullous dermatosis after chronic intense tanning bed and/or sunlight exposure.

Five fair-skinned patients had porphyrialike blisters and mechanical fragility of skin chronically exposed to intense radiation from tanning devices and/or the sun, but they had normal red blood cell, plasma, urine, and stool porphyrin levels. Use of several weak photosensitizing drugs during the year before or while developing lesions was identified or suspected in four patients.

Methotrexate and ultraviolet radiation.

Reactivation of a sunburn has been reported after the administration of methotrexate for cancer chemotherapy. A similar reaction is described in a patient with psoriasis who was receiving both coal tar with ultraviolet radiation for cutaneous lesions and methotrexate for arthritis. Knowledge of this poorly understood side effect of methotrexate is particularly important for physicians administering phototherapy and, perhaps, photochemotherapy.

Porphyria cutanea tarda associated with chronic renal disease and hemodialysis.

A fourth case of symptomatic porphyria associated with hemodialysis for chronic renal failure is reported. Subepidermal bullous dermatoses of patients who have undergone hemodialysis have not usually been associated with elevated porphyrin levels. However, this patient and three previously reported cases have been found to have abnormal porphyrin study results in association with skin lesions typical for porphyria cutanea tarda, occurring after hemodialysis. Hemodialysis does not effectively decrease circulating plasma uroporphyrin levels, although some dialysis of uroporphyrin into the dialysate could be measured in this case. Evaluation of bullous or porphyrialike dermatoses in patients treated with hemodialysis should include adequate testing for increased porphyrin levels.

Correlation of serum and urinary porphyrin levels in porphyria cutanea tarda.

A significant linear correlation was found between serum total porphyrin concentration and 24-hour total urinary porphyrin excretion in 18 patients with porphyria cutanea tarda sampled at diagnosis and during and after treatment on 73 occasions. This confirms that the serum porphyrin level parallels urinary porphyrin excretion and is an appropriate indicator of disease activity useful for monitoring patients in clinical practice.

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.

Cutaneous porphyrialike photosensitivity after liver transplantation.

BACKGROUND: Liver disease and cholestasis are often associated with abnormal coproporphyrin levels, but photosensitivity eruptions do not usually occur with these conditions. We present a case of a cutaneous porphyrialike photosensitivity in a liver transplant patient with only mildly elevated coproporphyrin levels. OBSERVATIONS: A 5-year-old girl developed a cutaneous porphyria cutanea tarda-like photosensitivity after liver transplantation for congenital biliary atresia. Her cutaneous eruption consisted of fluid-filled vesicles and crusted erosions involving her face and arms. These lesions eventually healed with atrophic scarring and milia. The patient was in a state of chronic hepatic rejection, but her serum and 24-hour urine specimens contained only mildly elevated levels of coproporphyrin. Results of histopathologic examination and direct immunofluorescence of a skin biopsy specimen resembled those of porphyria. CONCLUSIONS: Although clinically porphyria cutanea tarda was suspected, her porphyrin profile was not consistent with that diagnosis. Liver transplantation has become relatively common, but porphyria cutanea tarda-like clinical and histopathologic findings associated with coproporphyrinemia in a liver transplant patient have not been reported to date.

Quinidine photosensitivity.

A 55-year-old woman developed a dermatitis confined to light-exposed areas while taking quinidine gluconate, warfarin sodium, furosemide, spironolactone, and digoxin after cardiac surgery. Phototesting indicated a normal erythematous response to 290- to 320-nm ultraviolet radiation, but she developed erythema from 6 joules/sq cm of 320- to 400-nm radiation (ultraviolet A [UV-A]), a much lower dose than needed to produce a reaction in normal individuals. Two days after she discontinued quinidine and warfarin, phototesting showed no reaction to as much as 20 joules/sq cm of UV-A. One week after resuming quinidine (but not warfarin), she again reacted to 8 joules/sq cm of UV-A. No reactivity was elicited when the preparation was applied to the skin or injected into the dermis either with or without subsequent UV-A irradiation.

Hydroa vacciniforme.

Two patients with hydroa vacciniforme, a rare photodermatosis of unknown etiology, demonstrated distinctive scarring and vesiculobullous skin lesions on light-exposed body areas. Results of blood and urine porphyrin studies were normal, and no systemic abnormalities were noted. A small bullous lesion was produced in normal skin in case 1 with 15 times the minimal erythema dose of ultraviolet energy. The conditions of both patients improved while they were taking beta carotene orally.

A tightly bound protein-porphyrin complex isolated from the plasma of a patient with variegate porphyria.

Free acid porphyrins were isolated from plasma of a patient with variegate porphyria. Part of the total porphyrin content--which included protoporphyrin IX, harderoporphyrin and uroporphyrin in a molar ratio of 1.2:1:0.5 and traces of pentacarboxylic porphyrin--was extractable with ethyl acetate/acetic acid as described previously [1]. Unextractable porphyrins remained in the precipitate formed after mixing the lower liquid layer and precipitate from the extraction procedure (Fig. 1, [1]) with excess ethyl acetate/acetic acid. A portion of this precipitate was hydrolyzed in 8 mol/l HCl; its porphyrins were extracted with N-butanol and analyzed by high pressure liquid chromatography; another portion was chromatographed on Sephadex G-150 with 1 mol/l MgCl2, and the major porphyrin-protein pool was hydrolyzed in 8 mol/l HCl, reacted separately with AgNO3 and Ag2SO4, and subjected to cellulose acetate and polyacrylamide-gel electrophoresis. The results support the hypothesis that a dicarboxylic porphyrin, a major portion of which was unextractable by standard procedures [1] and which appeared to be covalently bound to a protein of approximately 68 000 mol. wt. that moved with human serum albumin during cellulose acetate electrophoresis, is the preponderant porphyrin in this plasma.

The genetic basis of "Scarsdale Gourmet Diet" variegate porphyria: a missense mutation in the protoporphyrinogen oxidase gene.

The porphyrias are disorders of porphyrin or porphyrin-precursor metabolism that result from inherited or acquired aberrations in the control of the porphyrin-heme biosynthetic pathway. Variegate porphyria (VP), one of the acute hepatic porphyrias, is characterized by a partial reduction in the activity of protoporphyrinogen oxidase (PPO), and recently, mutations in the PPO gene on chromosome 1q22-23 have been described. Our purpose was to identify the underlying genetic lesion in a severely affected patient with VP and to detect the silent mutation carriers in her family. The disease in this patient was precipitated by carbohydrate restriction as outlined in the "Scarsdale Gourmet Diet". Our mutation detection and confirmation strategy included PCR, automated sequencing, and restriction enzyme digestion. We identified a missense mutation in the patient and five family members. The mutation consisted of a previously unreported C-to-T transition in exon 5 of the PPO gene, resulting in the substitution of arginine by cysteine, designated R152C. This arginine residue is evolutionarily highly conserved in humans, mice, bacteria, yeast, and plants, indicating the importance of this residue in PPO. Our study established that a missense mutation in the PPO gene was the underlying mutation in this patient with VP and explained the occurrence of the phenotype in this family.