CLINICAL STUDIES

Erythiopoietic

Protoporphyria

IO Years Experience

VINCENT A. DeLEO, MO. MAUREEN POH-FITZPATRICK,

M.D.

New York, New York MICHELINE MATHEWS-ROTH,

M.D.

Boston, Massachusetts LEONARD C. HARBER, M.D. New York, New York

From the College of Physicians 8 Surgeons, Department of Dermatology, New York, New York, and the Department of Medical Microbiology, Boston City Hospital, Boston, Massachusetts. Requests for reprints should be addressed to Dr. Vincent DeLeo c/o College of Physicians 8 Surgeons of Columbia University, Department of Dermatology, Vanderbilt Clinic-15th Floor, Room 211, 630 West 168th Street, New York, New York 10032. Manuscript accepted June 6, 1975.

8

January 1976

The clinical and laboratory findings in 32 patients with erythropotetic protoporphyria as well as a review of the pertinent literature on this relatively recently described form of porphyria are presented. The disease is thought to be transmitted in an autosomal dominant fashion with variable penetrance and was characterized in these 32 patients by the onset in childhood of burning (97 per cent) and itching (88 per cent) of the skin on exposure to sunlight. This was accompanied by edema (94 per cent) and erythema (89 per cent) of the exposed areas. Vesicles, petechiae and residual scarrkig occurred less frequently. Associated abnormalities included choleltthiasis (12 per cent), anemia (27 per cent) and abnormal ltver function studies (4 per cent). Reports of associated liver disease including nine cases of fatal hepatic failure, are reviewed. Current methods of diagnosis as well as theories of pathophysiology of the disease are presented. Nfneteen of 23 of these patients recently treated with betacarotene responded with significant increase in their tolerance to sun exposure. Erythropoietlc protoporphyria is a heritable error of porphyrin metabolism resulting in increased levels of protoporphyrin in red blood cells, plasma and feces. Unlike several other forms of porphyria, urinary porphyrin levels are not elevated. The disease is characterized clinically by photosensitivity of variable severity developing in early childhood. The exact pathogenesis remains unclear. Unfortunately no experimental animal model is known in which excessive protoporphyrin levels of erythroid origin are naturally present or can be induced to adequately mimic the disease observed in man. The disease was first described in 1953 by Kosenow and Treibs [ 11. It was not until 1961, however, that Magnus et al. [2] clearly defined the clinical and laboratory abnormalities of the disorder and

The American Journal of Medlcine

Volume 60

ERYTHROPOIETIC PROTOPORPHYRIA-D&E0

proposed the name “erythropoietic protoporphyria.” Subsequently, approximately 300 cases have been reported, including cases from the United States, Europe, India, Australia and Africa [3-291. A clinical, laboratory and therapeutic study of erythropoietic protoporphyria was carried out by us in 32 patients observed over a 10 year period. The

Pyridoxal l-l

ET AL.

symptoms and physical findings in these patients are presented and compared with those reported by others. Recent advances in laboratory methods of diagnosis, and current concepts of the nature and origin of abnormally elevated protoporphyrin levels in tissues of patients with erythropoietic protoporphyria are discussed. Histopathologic changes observed in

Phosphate

CH,0P03H,

+ H,N-CH,-COOH

Glycine

PROTOPORPHYRIN III (IX)

CH=N-CH,COOH

H

‘Activated

Ferrochelatase

Glycine”

Fe++

cH *ofw+, T

+

Pr

HEME

R

HOOC-CH,CH,C-COA

ALAsynthetase

PI

PROTOPORPHYRINOGEN

III

COPROPORPHYRlNOGEN

III

Succinyl CoA

-coI 1 0

&Aminolevullnic

H,N-CH,lCH,CH2-COOH

Acid

Porphobilinogen

//4&&

H,N--H$

\

6,

b” ‘4

/ POLYPYRROLE

Pr-(--$.@

UROPORPHYRINOGEN

Ac -

AC

- pr*b$c”,

br I Figure 1.

UROPORPHYRINOGEN

III

CH3

Pr

COPROPORPHYRINOGEN

Heme biosynthesis.

January 1976

The Amerkan

I

Journal of Medklne

Volume 80

9

EFWlliROPOlETlC

PROTOPORPHYRIA-DeLEO

ET AL.

Skin

Bone Marrow

Erythropoietic

Feces Figure 2. Possible origins of excess protoporphyrin in eryihropoietic protoporphyria. 0 = protoporphyrin IX, JZ = degradedprotoporphyrin IX, 0 = red blood cell, ALA = tieka aminolevulinic acid. Protoporphyrin IX is formed from aminolevulinic acid in either bone marrow or liver and enters the vasculature in red blood cells and plasma. The protoporphyrin then enters the skin and liver. From the liver it is excreted into the gut unchangedor in a degraded form. A portion of the unchanged protoporphyrin recirculates in an enterohepatic fashion. the skin of these patients are reviewed, and effective treatment of these patients with oral beta-carotene is discussed. MECHANISM Porphyrins are a group of ring compounds composed of four pyrrole-type rings linked by methene bridges. The naturally occurring porphyrins vary by substitution of the eight hydrogen atoms in the pyrrole rings with side chains. As seen in Figure 1, the porphyrins are intermediaries or the oxidation products of intermediaries in the synthesis of heme from glycine and succinate. The porphyrins which occur in man are formed by the oxidation of their corresponding porphyrinogen. The porphyrinogens are colorless and do not fluoresce, whereas the porphyrins are colored compounds which fluoresce upon absorption of light in the Soret Band-400 to 410 nm in the long ultraviolet range. Fluorescence is one method of dissipating absorbed light energy: other methods include the formation of free radicals and peroxides. Enzymes and substrates necessary for heme synthesis are present in hepatocytes, the cellular elements of the bone marrow and, less prominently, in many other tissues. Disturbances in porphyrin metabolism may occur in any of these tissues. Porphyrias

10

January 1976

The American Journal of Medicine

have usually been classified as either hepatic or erythropoietic in origin according to the tissue supposedly defective in porphyrin metabolism. In erythropoietic protoporphyria, the metabolic abnormality apparently results in an excessive production of protoporphyrin IX, which can be found in abnormally large amounts in many tissues and tissue fluids including red blood cells, liver, plasma and feces. Because of the elevated levels of red blood cell protoporphyrin in this disease, the defect was originally thought to be erythropoietic [ 21. Various clinical observations and experimental evidence have led many investigators to question this original concept. Cripps and MacEachern [30] found that the level of protoporphyrin in the stool of patients with erythropoietic protoporphyria that they studied was independent of red blood cell levels. They theorized that in these patients the amount of protoporphyrin excreted in the stool per day could not be accounted for by the normal process of red blood cell breakdown with the release number utilizing acid in

of the excessive red cell protoporphyrin. A of studies have been carried out by others isotopically labeled glycine and aminolevulinic patients with erythropoietic protoporphyria

with discordant findings. Gray et al. [31], and later Scholnick et al. [32,33], reported abnormalities in both hepatic and erythropoietic areas. Schwartz et al. [34] believed that the large majority of the excess protoporphyrin was produced erythropoietically, whereas Nicholson et al. [35] believed that the excess was “formed mainly in the liver.” If one accepts a hepatic origin for the defect, it must be postulated that excess protoporphyrin passes from the liver into the plasma and then into the red cells. It is doubtful that this occurs. In mice, elevated red blood cell protoporphyrin levels can be produced by the administration of a number of chemicals, including griseofulvin [36-391. This seems to produce only hepatic protoporphyria. The reported excess red cell protoporphyrin found in these animals is probably limited to that which is ‘absorbed from the plasma onto the red cell membrane [30]. On the other hand, an erythropoietic origin of the excess protoporphyrin leads to the concept that the protoporphyrin leaks from the red blood cells into the plasma and is subsequently cleared by the liver. It is excreted into the bile as degradation products or unchanged protoporphyrin (the protoporphyrin is then eliminated in the feces with a portion of it being recycled in an enterohepatic fashion). Although Redeker et al. 1401 were unable to demonstrate this leak from erythropoietic protoporphyria red cells, Piomelli and his associates [41] recently showed that it does indeed occur. A schematic presentation of these interrelationships is shown in Figure 2.

Volume 90

FRYTHROPOIETIC

It has been

shown

that in erythropoietic

PROTOPORPHYRIA

the free base form (not chelated

protopor-

phyria not all red blood cells fluoresce or have elevated protoporphyrin levels [42]. Only the younger cells have appreciably elevated levels 1411. This supports the theory of erythropoietic origin since it seems likely that as cells mature, with a continued leak of protoporphyrin, their protoporphyrin levels would tend to decrease. Regardless of the tissue site of excess protoporphyrin production, two enzymes of the porphyrin synthetic pathway were investigated as to possible defects in erythropoietic protoporphyria. Aminolevulinic acid synthetase (ALAS) is the rate-limiting enzyme of the pathway. An increase in ALAS might result in an overproduction of protoporphyrin. Ferrochelatase (heme synthetase) controls the incorporation of iron into protoporphyrin to form heme. Decreased ferrochelatase activity might result in anemia and a corresponding backup of unutilized protoporphyrin inside the red blood cell. These enzymes were assayed by various methods in both hepatic and erythropoietic tissues. Although assay of these enzymes is difficult, several investigators found increased ALAS activity in patients with erythropoietic protoporphyria [31,34,43,44]. Ferrochelatase activity was found to be decreased in a few cases [34,45], but this was not consistently noted, Neither of these enzyme defects was clearly shown to result in the excess production of protoporphyrin found in the disease. The similarities and differences in two conditions which result in elevated red blood cell protoporphyrin levels but no photosensitivity-lead intoxication and iron deficiency anemia-have also been investigated. Lamola and Yamane [46] recently showed that the protoporphyrin present in red blood cells in erythropoietic protoporphyria differs chemically from that present in red blood cells in lead intoxication and iron deficiency anemia. The protoporphyrin in the latter two disorders (which becomes identical with that in erythropoietic protoporphyria after standard acid-extraction methods used to obtain purified porphyrins for assay) is actually significantly different in that it is chelated with zinc. These investigators postulated that in iron deficiency anemia and lead intoxication, protoporphyrin synthesis is normal whereas heme production from protoporphyrin is decreased-in iron deficiency anemia because of deficiency of iron, and in lead intoxication because of decreased ferrochelatase activity. This may result in globin being formed with abundant vacant heme binding sites, which may then be filled by zinc-protoporphyrin. In erythropoietic protoporphyria, with heme and globin synthesis essentially normal, there are few vacant heme binding sites on the globin molecule. The protoporphyrin is in

January

iblt

0

with any metal

ET

AL

ion)

and it is probably loosely associated to globin at sites other than the heme attachment sites. These sites, unlike the heme binding site, apparently do not or facilitate, complexing with metal ions require,

1461. Protoporphyrin is found in the plasma of patients with erythropoietic protoporphyria but not in the plasma of patients with iron deficiency anemia and lead intoxication [ 411. It may be, therefore, that the protoporphyrin in erythropoietic protoporphyria, being less strongly associated with the globin in red blood cells than the zinc protoporphyrin of the other two conditions, is more likely to diffuse from the red cells into the plasma. It also seems probable, that the plasma level of protoporphyrin is the parameter related to the clinical photosensitivity which occurs only in erythropoietic protoporphyria. It has been shown that the protoporphyrin in the plasma is bound to albumin and hemopexin [47,48] ; and’it is interesting that increased catabolism of hemopexin has been noted in patients with erythropoietic protoporphyria [ 491. This relationship of plasma protoporphyrin to photosensitivity is not completely understood. It is known that when a patient with erythropoietic protoporphyria is exposed to light in the 400 to 500 nm range the clinical symptoms of erythema, edema, pruritus and burning can usually be elicited. In contrast, exposure of normal persons to these wavelengths of light has no observable effect [50]. Furthermore, when red blood cells of patients with erythropoietic protoporphyria (or red cells of normal control subjects mixed with protoporphyrin from erythropoietic protoporphyria cells) are suspended in saline solution and exposed to this light, lysis occurs. This photohemolytic phenomenon has not been observed in man in vivo and is inhibited in vitro by numerous substances including albumin, vitamin E and beta-carotene [5 l541. The photohemolysis was shown to occur from the production of defects in the red blood cell membrane accompanied by the formation of lipid and hydrogen peroxides [55]. Other studies have also shown photo-induced lysosomal membrane damage associated with the presence of porphyrins (56,571 (Figure 3). Despite a growing body of data, the pathophysiology of erythropoietic protoporphyria remains unclear. It does seem, however, that an unknown heritable biochemical defect results in excess protoporphyrin production in bone marrow or liver, or both. This protoporphyrin is loosely associated with globin in the red blood cells and from the red cells (or liver) enters the plasma; here it is bound to albumin and hemopexin. This protoporphyrin may absorb light energy

1976

The American

Journal of Medicine

Volume 60

11

ERYTHROPOIETICPROTOPOfWlYRIA-DeLEO

ET AL

eel I membranes,

I

membranes Figure 3. Proposed mechanism of cutaneous photosensitivity in erythropoietic protoporphyria. On absorption of 400 nm light energy the protoporphyrin molecule is raised to an excited state. This energy may be dissipated by the formation of peroxides which cause damage to cell and lysosomal membranes. and become raised to an excited state molecule. A portion of the absorbed energy, as the excited porphyrin returns to the ground state, is dissipated by fluorescence as well as by the formation of free radicals and peroxides. These compounds may attack the integrity of cellular and lysosomal membranes of surrounding cells (probably endothelial cells of dermal capillaries). This damage can result in the exudation of plasma and red cells into the dermis, and possibly the epidermis, resulting in excess protoporphyrin in these tissues. Further excitation of this protoporphyrin may result in damage to membranes of various cells in the area, including mast cells with the release of chemical mediators of inflammation [58]. Activity of these mediators as well as direct light-induced damage to cells of the dermis and epidermis results in the clinical features of photosensitivity. LABORATORY DIAGNOSIS OF ERYTHROPOIETIC PROTOPORPHYRIA The diagnosis of erythropoietic protoporphyria is primarily based on finding increased levels of free protoporphyrin IX in red blood cells. Other biochemical alterations include elevated plasma protoporphyrin IX levels, red blood cell coproporphyrin and elevated fecal copro- and protoporphyrin levels, however, these findings are less consistent. Until recently, a significant difficulty in obtaining laboratory confirmation for clinically suspected erythropoietic protoporphyria was the unavailability of quantitative protoporphyrin determinations in many geographic areas.

12

January 1976

The American Journal of Medlcine

Easily performed screening studies yield qualitative estimates of increased levels of erythrocyte protoporphyrin. Two such methods utilizing whole blood were described by Rimington and Cripps [ 591. The patient’s blood may be diluted with saline solution and examined with the fluorescence microscope with appropriate filter systems. The presence of more than an occasional fluorescent erythrocyte (fluorocyte) is considered abnormal. In a modification of their second screening test 5 to 10 drops of the patient’s whole blood are added to a test tube containing 2 ml of a 4:l (volume/volume) ethyl acetate:acetic acid mixture and stirred well. Two milliliters of 1.5N hydrochloric acid is then added. After thorough mixing, layers will reform. The tube is then exposed to Wood’s lamp light in a dark room. Red fluorescence in the lower layer constitutes a positive test. Any color change in the upper layer should be disregarded. These screening tests may be positive in Gunther’s disease (erythropoietic porphyria), iron deficiency anemia, lead intoxication and other dyserythropoietic states, as well as in erythropoietic protoporphyria. A positive or questionable screening test calls for quantitative, as well as more discerning qualitative, porphyrin measurement. Quantitative determinations of protoporphyrin for confirmation of diagnosis until recently consisted of tedious, prolonged, volumetric, analytical technics such as the Wranne method [60]. Poh-Fitzpatrick et al. [61] showed that a rapid quantitative microfluorometric assay for free erythrocyte porphyrins

Volume 60

ERYTHROPOIETIC PROTOPORPHYRIA-

(FEP), developed

originally by Piomelli 162,631,

could

be used accurately and reproducibly in the diagnosis of erythropoietic protoporphyria. This method can be used in determinations on fresh whole blood specimens as well as on packed cells and plasma. With minor modifications, this method can also accurately assay porphyrins in specimens of whole blood collected by finger puncture on special filter paper (903: Schleicher and Schuell) and mailed to any laboratory equipped to perform the determination [ 6 11. Both the standard methods and the newer microfluorometric methods of quantitative and qualitative assay require a number of extractions of the porphyrins from tissue into various solvents. Poh-Fitzpatrick and Lamola [64] recently described a direct method in which the specimen (whole blood, packed cells, plasma) is diluted with phosphate buffered saline solution (pH 7.4) and examined directly (spectrofluorometrically) allowing for a more rapid diagnosis. Protoporphyrin is photodegradable and all specimens should be protected from light exposure. Red blood cell protoporphyrin levels remain relatively stable for several weeks when the blood is stored at 4’C in the dark. Plasma levels are less stable and decrease rapidly if exposed to light even for short periods of time [65]. For this reason plasma determination is best done immediately. If this is impossible then the specimen should be drawn and stored in the dark until the determination can be performed. MATERIALS

AND METHODS

The study group is composed 10 year period, January Protoporphyrin

of 32 patients

levels were determined

red blood cells obtained

we used the spectrophotometric assay

used [61].

ber of these ometric

patients

method.

inogen [67]

method of Wranne

for free erythrocyte

of

[60];

porphyrins

has been of a num-

were

determined

and spectrophotometric

by a similar fluortest for porphobil-

determination

of total

porphyrins 1671 were performed on urine specimens from all patients. Stool specimens were obtained from a number of patients Holti [ 681. Most patients sugar,

and

nitrogen, total

determinations

before

and

carotene

CLINICAL

of porphyrins

had complete

blood urea

transaminase These

for assay

during

by the method

of

blood counts, fasting blood serum

serum

glutamic

bilirubin

were

performed

treatment

with

Erythropoietic

Case NO.

Age (vr) and Sex

1

35, F 19, F

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

9. 24, 51, 26, 25. 14, 12, 10, 12. 14, 19, 11, 39, ll,M 10, 30, 32, 16, 10, 12, 22, 14,

M M F F M M M F PA M M F M

25 26 27 28 29 30 31

9. M 4. F 11.M 21, M 35, F 41, F 48, M

F M F M F M M F

Data on 32 Patients

Age at Onset Family History* (vr) 13 4 2 2 12 13 1 3

1 1 2 4 6 2 4 4 4 4 8 2 2 2 8 2 1 2 6 8 4 1 4

wrth

Protoporphyria

Associated Diseases Cholelrrhiasrs

+ + t + + + -1. + _ + c + + _ _ i + i_~ + + + + + -

Anemia

Anemia

Cholelithiasis

Anemia Anemia

Anemia Anemia

Cholelithiasis Anemia; abnormal liver function studies 40. F 4 Cholelithiasis 32 ______-* c indicates additional family members with photosensitivity, - indicates no family members similarly affected.

from four to 5 1 years with an average

microfluo-

levels in the plasma

The Watson-Schwartz

Demographic

Until June 1973,

of a rapid quantitative

Protoporphyrin

1974.

on specimens

from all patients.

since then. a modification rometric

seen over a

1965 through December

TABLE I

DeLEO ET AL

oxaloacetic

determinations.

several

times both

beta-carotene.

Blood

and vitamin A levels were also determined.

FEATURES

Pertinent demographic data of 32 patients with erythropoietic protoporphyria are presented in Table I. These patients (18 male and 14 female) range in age

of 2 1.4 years.

Two-thirds of these patients reported that other members of their families were similarly photosensitive: these 32 cases occurred in 20 families, Most patients reported their first episode of photosensitivity early in life at an average age of 4.3 years. Three patients, however, thought that they had been free of symptoms until early adolescence, the latest reported age of onset being 13 years. Similar findings were reported in other large series [ 7,8]. The clinical features of the 32 patients are presented in Table II. The most frequently reported symptom was a burning sensation in the skin after exposure to sunlight (97 per cent). This was followed in frequency by pruritus (88 per cent) and pain (67 per cent). Most patients reported visible skin changes usually consisting of edema (94 per cent) and/or erythema (69 per cent). In contrast to other reports [7,22], only one of our patients reported vesicle formation, and only a small number (19 per cent) pre-

January 1976

The American Journal of Medicine

Volume 60

13

ERYTHROPOIETIC PROTOPORPHYRIA-DeLEO

ET AL

Clinical Features of 32 Patients with Erythrbpoietic Protoporphyria

sented with residual scarring. These scars were shallow depressions 2 to 4 mm in diameter, scattered over the nose, cheeks and dorsa of the hands (Figures 4 and 5). The scar formation usually followed episodes of sun exposure which had resulted in severe edema, erythema and necrosis of the skin resulting in crusting. Most patients had minimal thickening and lichenification of skin over the dorsa of the hands without evidence of true scarring. Signs and symptoms were confined to sun exposed areas of

patients’ skin. No hypertrichosis [ 14,161 or nail changes [6,7], as described by other investigators, were observed in any of these patients. A petechial rash was observed in one patient 24 hours after prolonged sun exposure (Case 3 1). One patient (Case 15) reported only subjective symptoms (burning and pruritus) without any objective cutaneous lesions on exposure to sunlight. Similar cases have been reported in which, the same symptoms in the absence of concomitant cutaneous lesions, resulted in the incorrect diagnosis of “psychogenic pruritus” [ 69] _ Some patients reported changes occurring almost immediately after sun exposure: others were able to tolerate as much as 4 hours of sunlight before the onset of symptoms. Some patients reported cutaneous photosensitivity not only to natural sunlight but also to fluorescent lighting. Most patients reported abatement of symptoms in fall and winter. Although some investigators report a decrease in symptoms with an increase in the age of patients, this is not always found [7,9] and was not the case in our patients.

Case 27. Photograph of patient showing scarFigure 4. ring of face and thickening of the skin of the dorsa of hands.

Figure 5. Case 27. Photograph ring of the nose.

TABLE

II

Patients No.

Feature

31 28 5 30 22 6 1 1

Burning Pruritus Pain Edema Erythema Scarring Papulo-vesicle Petechiae

14

January

1976

The American

% 97 88 16 94

___-__

69 19 3 3

Journal of Medicine

Volume

60

of patient showing

scar-

ERYTHROPOIETIC PROTOPORPHYRIP---nel.EO

Decrease

in photosensitivity

during pregnancy

has

recently been reported [ 71 and was noted in one patient (Case 19). Protoporphyrin levels are shown in Table III. Red blood cell levels ranged from 131 to 1,617 pg/lOO cc (normal,