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Biochemical effects of chloroquine therapy in porphyria cutanea tarda
Biochemical Effects of Chl0roquine Therapy in P0rphyria Cutanea Tarda
WILLIAM RALPH VOGLER, M.D. JOHN T. GALAMBOS, M.D. SIDNEY OLANSKY, M.D.
Atlanta, Georgia
From the Department of Medicine. Divisions of Hematology, Digestive Diseases and Dermatology, Emory University School of Medicine. Atlanta, Georgia. This work was supported by U.S. Public Health ServiceResearchGrant FR-39from the Generai Clinical Research Centers Branch. Division of Research Facilities and Resources. and the General Research Fund of Emory University. Requests for reprints should be addressed to Dr. W. R. Vogler, Department of Medicine, Division of Hematology, Emory University, Atlanta, Georgia 30322. Manuscript received October 17, 1969.
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Three patients with porphyria cutanea tarda and evidence of liver disease on biopsy were treated with chloroquine in doses of 0.5 gm daily for eight days. In each instance an initial febrile episode was followed by evidences of hepatocellular injury, which Soon reverted to pretreatment status. Associated with this was an increased excretion of delta-aminolevulinic acid, uroporphyrin, coproporphyrin and Protoporphyrin in the urine and feces. In two of three patients the proportion of type I porphyrin isomers was not influenced by therapy. Porphyrin excretion became normal following therapy, and the Patients experienced prolonged remissions, free of skin manifestations. These findings for the most part support the thesis that the responses are secondary to a reduction in hepatic porphyrins rather than interference of chloroquine in the porphyrin biosynthetic pathway. In One patient transient ascites and a hemolytic episode developed following administration of chloroquine. Porphyria cutanea tarda (PCT) is a type of hepatic porphyria characterized by a bullous dermatosis associated with hirsuties, pigmentary changes, photosensitivity and increased susceptibility to mechanical trauma. In most instances there is evidence of liver disease secondary to alcoholism or history of drug ingestion (i.e., estrogens). Although familial cases have been reported [1,2], it is thought to be an acquired illness. Abdomina complaints, neurologic manifestations and the precipitation of acute attacks by barbiturates do not occur in this illness, clearly separating it from acute intermittent porphyria. The urine and feces usually contain excess uroporphyrin and coproporphyrm as well as 7-carboxyl porphyrin [3]. There is usualty a mild increase in deltaaminolevulinic acid (ALA) excretion [3]. Porphobilinogen (PBG) excretion is normal [3]. Since the report of Linden et al. in 1954 [4] and from subsequent studies [5-8] it has been known that chloroquine administration results in a marked increase in uroporphyrin excretion associated with transient hepatocellular injury. These reports have established that within three days after chloroquine administration there is fever, nausea and malaise followed by excretion of large quantities of uroporphyrin in the urine and feces. Liver enzyme levels (serum glutamic oxalacetic transaminase. SGOT. and serum glutamic pyruvic transaminase. SGPT) become markedly elevated and on biopsy centrolobular necrosis has been documented [8]. There is an associated increase in serum iron [8]. Within a few days, recovery occurs. The liver enzyme levels return to normal and excretion of uroporphyrin falls to normal levels. Felscher and Redeker [8] reported that prolonged clinical rem ssions may be observed after chloroquine therapy. They suggested that this be studied further as a therapeutic modality. Felscher and Redeker [8] reported reduced porphyrin content in liver biopsy specimens obtained after chloroquine therapy, suggesting that administration of chloroqume results in release ot uroporphyrin from hepatic sites. There is no evidence that increased synthesis of urcporphyrin occurs. Once liver stores are reduced subsequent challenge with chloroqume does not result in increased porphyrin excretion. Scholnick and Marver [9] reported that
The AmericanJournalof Medicine
CHLOROQUINE THERAPY OF PORPHYRIA CUTANEA TARDA -- VOGLER ET AL
c h l o r o q u i n e f o r m s a c h l o r o q u i n e - p o r p h y r i n c o m p l e x ident i f i a b l e by s p e c t r o p h o t o m e t r y ; t h e y isolated it by gel filtration. The loss of h e p a t i c p o r p h y r i n was a c c o u n t e d for by release of p o r p h y r i n f r o m m i t o c h o n d r i a a n d l y s o s o m e s , the s u b c e l l u l a r f r a c t i o n s e x h i b i t i n g high c o n c e n t r a t i o n s of c h l o r o q u i n e . Electron m i c r o s c o p y studies s h o w e d e x t e n s i v e mitochondrial damage. This r e p o r t of t h r e e p a t i e n t s with p o r p h y r i a c u t a n e a t a r d a t r e a t e d w i t h c h l o r o q u i n e c o n f i r m s the w o r k of Felscher and R e d e k e r [8]. It gives a d d i t i o n a l i n f o r m a t i o n on t h e effect of c h l o r o q u i n e on t h e e x c r e t i o n of ALA, PBG a n d t h e v a r i o u s p o r p h y r i n isomers. Lastly, t w o c o m p l i c a t i o n s of c h l o r o q u i n e a d m i n i s t r a t i o n are p r e s e n t e d .
CASE REPORTS Case 1. This fifty-six year old white woman (C.M.) first admitted to Emory University Hospital on June 26, 1967. She gave a two year history of skin blisters, following minor trauma and exposure to sunlight, and increasing hirsutism. She stated that she had noted a long-standing sensitivity to sunlight expressed as "burning easily." On rare occasions she noted that her urine was dark. Three months before the onset of the skin blisters she had been given Premarin | (conjugated estrogens [equine]) for menopausal symptoms. Skin blisters occurred at sites of trauma, persisted for many months and were unresponsive to local medications. Except for mild chronic bronchitis and arthritis for which she had taken sodium salicylate, the patient has enjoyed good health. She denied the use of alcohol. Upon physical examination her skin was dry, somewhat thickened and glistening over the back and upper portion of her chest. There were scattered ulcerations approximately 1 to 2 cm in diameter with indurated borders on the hands and lower legs. There were numerous 1 to 2 cm scars on the hands and legs. There was growth of fine hair on the face. The liver was not palpable. The remainder of the examination was within normal limits. The hemoglobin was 15.5 gm per cent, hematocrit 46.5 per cent, reticulocytes 2 per cent, platelets 21g,ooO/cu mm, white blood cell count 9,300/cu mm with a normal differential. Liver function studies were within normal limits except for bromsulfalein retention of nine per cent (normal less than 6 per cent). The patient's condition remained essentially unchanged for six months. On December 19, 1967, she was readmitted to the hospital with the same findings. At that time her blood urea nitrogen level was 18 mg per cent, bromsulfalein retention 7 per cent, serum total bilirubin 0.5 mg per cent prothrombin time 11.5 seconds, SGOT 53 (Karmen units), alkaline phosphatase 14.5 King-Armstrong units and serum iron 267/~g per cent. Biopsy of the skin showed atrophy with slight chronic inflammation. Liver biopsy showed periportal parenchymaf necrosis, scattered fatty changes and increased hemosiderin. Most of the iron appeared within the hepatic parenchymal cells. Under ultraviolet light the liver specimen had a red fluorescence. Case 2. This forty-six year old white man (F.F.) was admitted to the hospital on February 26, 1968, complaining of sores on his arms of two years' duration. Over this interval he had had repeated vesicular lesions appearing on his upper extremities, particularly on the exposed areas of his forearms and hands. They were nonpruritic and nontender, except when denuded. The bullae varied in size depending on how long they remained intact. The patient found that by pricking the blisters they did not get as large and healed faster. This condition continued and was made worse by exposure to sunlight. The patient had not noted dark urine. He denied any other constitutional symptoms, had never been anemic and admitted
Volume 49, September 1970
to occasional social drinking. There was no family history of porphyria. He reportedly had a bleeding ulcer in 1955 which was treated medically. Physical examination revealed an obese, white man weighing 120 kg. His blood pressure was 180/110 mm Hg. Examination of the skin revealed numerous scars on the arms and one scab 1 cm long on the scalp. Fundi showed arteriovenous crossing changes with arteriovenous ratio 1:2. No abdominal organs were palpable. The remainder of the examination was within normal limits. The hemoglobin concentration was 18 gm per cent, the hematocrit 51 per cent, white blood cell count 9,600/cu mm with a normal differential. Bromsuifalein test showed 21 per cent retention. The alkaline phosphatase level was 13.2 King-Armstrong units, SGOT 117 Karmen units, lactate dehydrogenase (LDH) 131 Wacker units, serum total bilirubin 1.0 mg per cent, total protein 7.5 gm per cent. Serum iron was 186/~g per cent and prothrombin time 12.3 seconds. A biopsy specimen from the left arm showed hemorrhagic bullae in the subepidermis and necrosis of the upper dermis. These findings were thought to be consistent with porphyria cutanea tarda. Liver biopsy showed moderate steatosis, portal and periportal inflammation with periportal and intralobular necrosis, consistent with alcoholic hepatitis. Fresh frozen sections did not fluoresce for porphyrins. A freshly voided urine specimen did not fluoresce under ultraviolet light.
TEMPERATUREF~
102 I00
]
~,~
~
BILIRUBIN mg~ ALKALINE
PHOSPHATASE
750 500
L
~
SGOT
250
20 IO 0
400
I
,RONUO,
200
A L A U MOLES / 24 HRS.
40 ~ 20
120 80
~ ~
URINE UROPORPHYRIN p MOLES 1 24 HRS,
4O
20 ~ U R I N E ~ O P R O P O R P HYRIN 10 p MOLES124HRS. ,~ : "i ~ ;,' ~ ;,: . i~ : ' ,~ . ~l i I CIHiiIOROQU i , I,N , i J i i . L . _ L . I _ . . L . . ~ . L ~ L . L ~ I ~ L . I _ ~ _ ~ - . ~ ] 2 4 6 8 tO 12 14 ]6 18 47 72 go ED? 157 184 212 218 283
DAYS Fig. I. Effect of chloroquine on temperature, liver function tests, ALA a n d porphyrin excretion in a fifty-six y e a r old w o m a n with porphyria cutanea tarda.
317
CHLOROOUINE
THERAPY
OF P O R P H Y R I A C U T A N E A
I02
T A R D A - - VOGLER ET AL.
TEMPERATURE F ~ TEMPERATURE F~
lOO 98
IOD
1,0
B
BILIRUBIN
mg %
m.. BILIRUBIN
o
ALKALINE
~176 !1 ,,
BSP% SERUM IRON ~g %
2DD t
A L A
2o @
p MOLES i 24 HRS.
NNN
0 20
URINE UROPORPHYRIN p MOLES I 24 HRS. 80
I0
~
D
N
40 URINE COPROPORPHYRIN I~ MOLES I 24 NRS.
2 0 ~
~
~
--
__
L
MOLES I 24 HRS.
~
~'a
URINE COPROPORPHYRIN MOLES / 24 HRS
URINE PROTOPORPHYRIN
2.0
p MOLES I 24 HRS.
F
~
.S
1.0
~
~
--URINE PROTOPORPHYRIN ~ MOLES I 24 HRS. FECAL PORPHYRINS
2 9 . iO ~':;';.':.::;~;;.~,'.k.:'::..',+'..J.:t. :,~....
FECAL PORRHYRINS
p MOLES / 24 HRS.
0
~
p MOLES I 24 HRS.
TO URO
0
1
i
-5
-3
-I
+1
3
5
7
9
iI
13 43
18 I13 141 169 195 233
I
-4
, , ~!~,~i:.~ -2
0 2
4
~;',.~ii, Ii CHLOROQUIN JlJ il illJ_i__L 6 8 I0 12 14 16 18 20
DAYS
DAYS
Fig. 2. Effect of chloroquine on temperature, liver function tests, ALA and urinary and fecal porphyrin excretion in a forty-six year old man with porphyria cutanea tarda.
Fig. 3. Effect of chloroquine on temperature, liver function tests, ALA and urinary fecal porphyrin excretion in a fifty-seven year old man with porphyria cutanea tarda.
Case 3. This fifty-seven year old white man (J.H.) first noted blisters
nitrogen level 18 mg per cent and serum uric acid 8.4 mg per cent. The serum iron was 180 Fg per cent. Skin biopsy showed moderate hyperkeratoses, moderate perivascular infiltrate in the dermis and a nonspecific bulla. Hemosiderosis was present in the bone marrow biopsy. Liver biopsy showed areas of necrosis with polymorphonuclear leukocyte infiltration, mild steatosis and early fibrosis. Liver tissue fluoresced under ultraviolet light.
on the dorsum of the left hand while playing golf in 1959. The lesions failed to heal. Shortly after this he noticed that his urine was.red and shortly thereafter a diagnosis of porphyria cutanea tarda was made. He was given a dose of chloroquine and became violently ill, but the skin lesions subsided for one to two years. SubsequenUy he has had repeated vesicles on his hands and face which worsen in the spring and fall. There has been a gradual splenic enlargement from 1964 to 1968. Liver function tests in 1966 showed a bromsulfalein retention of 10 per cent. The patient gave a history of past ingestion of alcohol9 He had a peptic ulcer in 1963 and chest roentgenograms have shown interstitial fibrosis. There was no family history of porphyria. On physical examination his skin showed areas of scarring, depigmentation and hyperpigmentation on exposed portions of arms, face, scalp and shoulders. The liver was palpable 2 cm below the costal margin. The spleen was soft, the tip was felt 8 cm below the left costal margin. Laboratory data revealed a hemoglobin of 14.8 gm per cent, hematocrit 43 per cent, white blood cell count 5,700/cu mm, platelet count 117,000/cu ram, reticulocytes 2.7 per cent. Serum total bilirubin was 0.6 mg per cent, SGOT 60 Karmen units, alkaline phosphatase 15 King-Armstrong units, bromsulfalein retention 15 per cent, prothrombin time 12.7 seconds, blood urea
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J
SGOT
~
!1
PHOSPHATASE
40U
B S P %
|
mq %
METHODS All patients were hospitalized on the Clinical Research Facility and fed a regular diet. Preliminary studies included blood counts, bone marrow examination, liver biopsy, blood urea nitrogen, uric acid, bilirubin, prothrombin time, serum glutamic oxalacetic transaminase, alkaline phosphatase, sodium sulfobromophthalein excretion, serum iron and iron-binding capacity. Twenty-four hour urine collections for total volume and creatinine were made. ALA and PBG were measured by the method of Marver et al. [10]. The urinary porphyrins were measured according to Schlenker et al. [11] with spectrophotometric corrections of Mingioli [12]. Three to four day aliquots of feces were pooled, a 100 gm aliquot was mixed with 200 ml of 5N hydrochloric acid. This was allowed to stand for two hours, filtered and the precipitate was washed with 100 ml 5N hydrochloric acid. The filtrate was then brought to 500 ml volume with water, and
The American Journal of Medicine
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CHLOROOUINE THERAPY OF PORPHYRIA CUTANEA TARDA -- VOGLER ET AL.
aliquots were frozen for porphyrin determinations9The aliquots were thawed, 100 ml of saturated sodium acetate was added, and uroporphyrin was extracted by the method of Dresel and Falk [13]. Coproporphyrin and protoporphyrin were isolated by the method of Wranne [14]. Porphyrins were assayed by measurements of absorbance in the Soret region using a Beckman DU Spectrophotometer, applying the correction factors of Mingioli [12]. Separation of Porphyrin Isomers. Selected samples of isolated urine porphyrins were chromatographed by the method of Falk and Benson [15] to determine changes in porphyrin isomers. After conversion to methyl esters, chromatography revealed two major spots with RF values similar to those reported by Falk and Benson [15] for isomers I and III. The spots were eluted with ethanolic chloroform and absorbance at 402 m# measured9 After four to five days in the hospital, chloroquine in a dose of 0.5 gm daily was given for eight days. After treatment the patients were continued under observation for ten to fourteen days in the hospital and then followed at monthly intervals as outpatients. RESULTS
The results of the clinical and biochemical changes are summarized in Figures 1, 2 and 3. Clinical Response. Beginning approximately thirty-six hours after the first dose of chloroquine, all three patients had malaise, myalgias and fever which persisted over a seventy-two hour period and gradually returned to normal 9 One patient (Case 2), who received the smallest dose of chloroquine on a weight basis (4 m g / k g ) , experienced the least discomfort. The first patient (Case 1) noted slight nausea the first day. One patient (Case 3) had anorexia for six days,
TABLE I
nausea for five days and v o m i t i n g for two days beginning the day after initiation of t r e a t m e n t . After the fever subsided the patients had no further complaints with the exception of one patient (Case 3) in w h o m ascites developed. Liver Function Tests, Abnormalities and liver function tests suggested hepatocellular injury manifest by a p r o m p t increase in SGOT, increased bromsulfalein retention and a subsequent increase in serum alkaline phosphatase. One patient (Case 1) had no change in serum bilirubin but the other two (Cases 2 and 3) had a definite increase in direct-reacting bilirubin. In all instances the serum total bilirubin remained less than 3 mg per cent, and clinical jaundice was not evident. S e r u m Iron. In two patients (Cases 1 and 3) there was a very striking increase in serum iron highest at approximately the time that the SGOT level was most elevated. One patient (Case 2) failed to show an increase in serum iron, indeed there was a reduction in serum iron following therapy, from 192 to 98 #g per cent by the fourth day of treatment. Urinary ALA, PBG and P o r p h y r i n Excretion. In all three patients ALA excretion increased, as shown in the figures and Table I. Porphobilinogen excretion was somewhat variable and did not exceed normal values (Table I). One patient (Case 1) showed no essential change; one (Case 2) had a transient rise on the third and fourth days and one (Case 3) a reduction of porphobilinogen excretion after therapy. Uroporphyrin was the major porphyrin excreted in all three patients, rising briskly during the second day of t r e a t m e n t
Effect of Chloroquine on Daily Excretion of ALA and PBG ALA (/zM/24hr)"
Day of Study -
5 4 3 2 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
PBG (#M/24 hot
Chloroquine (gm/day)
Case 1
Case 2
Case 3
Case 1
Case 2
Case 3
0 0 0 0 0 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0 O 0 0 0 0 0 0 0
22.44 24.69 28.44 28.50 27.54 28.17 40.90 46.54 45.29 39.51 49.54 37.26 30.62 27.63 27.62 24.26 23.91 23.25 25.38 ... ... ...
15.02
9 .. 16.14
5.23 4.77 4.68 4.96
49 ... 3.56 ...
. .. 7.70 . .. 7.30
5.18
3.61
...
Normal values 6.33 to 19.78/LM/24 hours.
Volume 49, September 1970
15.45
...
16.95 15.48 17.45 16.65 22.18 24.47 22.16 21.30 25.91 23.42 18.97 16.82 19.61
9
19.07 24.63 33.00 24.47 29.04 28.04 33.38
21.53 19.17 ... 17.96 9. . 17.43 9 14.41 8.73
5.14 5.87 5.22 3.56 3.55 4.57 5.19 5.81 6.37 6.62 4.40 4.85 4.17 5.00 . . . .
.
.
.
.
4.41 7.10 4.36 8.36 4.93 4.85 5.39 5.10 4.36 6.50 4.72 6.72 4.12 9.34 3.63 4.19 3.24 3.91 . . . . . . 3.60 3.95 . . . . . . 3.79 3.09 . . . . . . . 5.34
.
.
.
39
t Normal values 0.73 to 6.38/~M/24 hours [11].
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CHLOROQUINETHERAPYOF PORPHYRIACUTANEATARDA-- VOGLERET AL.
and falling to below pretreatment levels by the second week following chloroquine therapy. There was a three to fourfold increase in coproporphyrin excretion in the urine in all patients. Protoporphyrin excretion slightly increased in two patients during treatment; however, the amount excreted was small and remained within normal limits. Fecal Porphyrins. Fecal porphyrin excretions were measured in two patients (Cases 2 and 3). As shown in the figures, there was an increase in total porphyrin during treatment. In both instances fecal uroporphyrin decreased and coproporphyrin and protoporphyrin increased somewhat. Porphyrin Isomers. The results of chromatography of uroporphyrins during the pretreatment period, at the height of excretion and during follow-up are shown in Table I1. In
TABLE II
Effect of Chloroquine on Uroporphyrin Isomers Measured Prior to, at Peak of Urinary Uroporphyrin Excretion and Five to Seven Days After Stopping Therapy % UroporphyrinI Data
Pretreatment Peak excretion Follow-up
Case 1
Case 2
Case 3
63 79 76
70 65 65
18 63 36
two patients (Cases 1 and 2) the major uroporphyrin excreted throughout the study was the type I isomer. One patient (Case 3) had an apparent marked increase in percentage of type I during the peak porphyrin excretion period. Complications. In addition to the myalgias and the evidence of hepatocellular injury (with recovery) already described, one patient (Case 3) had an hemolytic episode during treatment; the plasma hemoglobin on the eleventh day after initiation of treatment was 20.8 mg per cent, falling to 1.4 mg per cent by the fourteenth day. His hematocrit fell :from 44 to 40 per cent and reticulocytes increased from 3.8 to 5.2 per cent. This patient's course was also complicated by the development of ascites which responded to salt restriction and diuretic therapy. When seen a month later for follow-up examination, at which time he had relaxed his strict salt curtailment, ascites had recurred although results of his liver function studies had returned to pretreatmerit values. Two months after treatment he had no further ascites, no blisters and skin pigmentation was reduced. The patients have been followed for twelve, ten and three
months, respectively, and have shown impressive clinical improvement with disappearance of all manifestations of cutaneous porphyria. The excretion of ALA and porphyrins remained within normal limits during this period of study. COMMENTS
The results of these studies, as well as those reported by others [8], indicate that chloroquine has a specific effect on the hepatic cell of the patient with porphyria cutanea tarda, resulting in hepatic necrosis and release of preformed porphyrins from liver and apparently from skin. Recent evidence has suggested that the binding of the porphyrin with chloroquine most likely takes place within the mitochondria and lysosomes, as these subcellular fractions contain the highest concentration of chloroquine [9]. Electron microscopy studies have shown extensive mitochondrial damage. The increase in ALA excretion here recorded has not been reported previously. It is likely that this increased excretion results from injury to the mitochondria resulting in release of ALA, since the enzyme necessary for its synthesis is localized in mitochondria [16,17]. If the increase in ALA excretion had resulted from inhibition of the enzyme ALA dehydrase by chloroquine one would expect porphobilinogen excretion to decrease during chloroquine administration. This was not the case in our patients. It is not known whether chloroquine stimulates the synthesis of ALA in porphyria cutanea tarda by increasing activity of ALA synthetase, as has been reported following barbiturate administration in acute intermittent porphyria. To our knowledge ALA synthetase activity has not been measured after chloroquine therapy. The role of iron in this disease is uncertain. Some recent evidence has indicated that the reduction in iron stores results in increased activity of heme synthetase in a patient with pyridoxine responsive anemia, suggesting that iron does inhibit this enzyme [18]. If heme synthetase activity can be increased by phlebotomies it is conceivable that accumulating porphyrinogens would be converted more rapidly to heme and the metabolic effects of increased porphyrins eliminated. The response of patients with porphyria cutanea tarda to phlebotomies is of interest in this connection [19]. Further investigation is needed to elucidate the role of iron in porphyria. ACKNOWLEDGMENT
We are indebted to Mrs. Elizabeth Mingioli for the measurements of ALA, PBG and porphyrins.
REFERENCES
1. Barnes HD, Overton J, Sweet RD: Familial cutaneous porphyria. Brit J Derm 77: 130, 1965. 2. Waldenstr6m J, Haeger-AronsenB: Different patterns of human porphyria. Brit Med J 2: 272, 1963. 3. Taddeini L, Watson FJ: The clinical porphyrias. Seminars Hemat 5: 335, 1968. 4. Linden IH, Steffen CG, NewcomerVD, Chapman M: Development
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of porphyria during chloroquine therapy for chronic discoid lupus erythematosus. Calif Med 81: 235, 1954. 5. Marsden CW: Porphyria during chloroquine therapy. Brit J Derm 71: 219, 1959. 6. Cripps DJ, Curtis AC: Toxic effect of chloroquine on porphyria hepatica. Arch Derm 86: 575, 1962. 7. SweeneyGD, SaundersSJ, Dowdle EB, Eales L: Effects of chloro-
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C H L O R O Q U I N E T H E R A P Y OF PORPHYRIA C U T A N E A TARDA -- VOGLER ET AL.
8.
9.
10.
11.
12.
quine on patients with cutaneous porphyria of the "symptomatic" type. Brit Med J l(supp): 1281, 1965. Felscher BF, Redeker AC: Effect of chloroquine on hepatic uroporphyrin metabolism in patients with porphyria cutanea tarda. Medicine 45: 575, 1966. Scholnick P, Marver H: The molecular basis of chloroquine (C) responsiveness in porphyria cutanea tarda (PCT). Clin Res i6: 258, I968. Marver HS, Tschudy DP, Perlroth MG, Collins A: The determination of aminoketones in biological fluids. Anal Biochem 14: 53, 1966. Schlenker FS, Davis CL, Kitchell CL: Urinary total, aqueous and ether-soluble porphyrins. Techn Bull Regist Med Techn 33: 57, 1963. Minl]ioli ES: New correction factors for spectrophotometric assay of erythrocyte porphyrins. Anal Biochem 22: 47, ]968.
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13. Dresel EIB, Falk JE: Studies on the biosynthesis of blood pigments. Biochem J 63: 72, ]956. 14. Wranne L: Free erythrocyte copro- and protoporphyrins. Acta Paediat 49 (supp 124): 449, 1960. 15. Falk JE, Benson A: Separation of uroporphyrin esters I and III by paper chromatography. Biochem J 55: 101, 1953. 16. Laver WG, Newberger A, Udenfriend S: Initial stages in the biosynthesis of Porpflyrins. Biochem J 70: 4, 1958. 17. Urata G, Granick S: Biosynthesis of ~-aminoketones and the metabolism of aminoacetone. J Biol Chem 238: 811, 1963. 18. Vogler WR, Mingioli ES: Porphyrin synthesis and heine synthetase activity in pyridoxine-responsive anemia. Blood 32: 979, 1968. 19. Epstein JH, Redeker AG: Porphyria cutanea tarda: a study of the effect of phlebotomy. New Eng J Med 279: ]301, 1968.
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WILLIAM RALPH VOGLER, M.D. JOHN T. GALAMBOS, M.D. SIDNEY OLANSKY, M.D.
Atlanta, Georgia
From the Department of Medicine. Divisions of Hematology, Digestive Diseases and Dermatology, Emory University School of Medicine. Atlanta, Georgia. This work was supported by U.S. Public Health ServiceResearchGrant FR-39from the Generai Clinical Research Centers Branch. Division of Research Facilities and Resources. and the General Research Fund of Emory University. Requests for reprints should be addressed to Dr. W. R. Vogler, Department of Medicine, Division of Hematology, Emory University, Atlanta, Georgia 30322. Manuscript received October 17, 1969.
3:16
Three patients with porphyria cutanea tarda and evidence of liver disease on biopsy were treated with chloroquine in doses of 0.5 gm daily for eight days. In each instance an initial febrile episode was followed by evidences of hepatocellular injury, which Soon reverted to pretreatment status. Associated with this was an increased excretion of delta-aminolevulinic acid, uroporphyrin, coproporphyrin and Protoporphyrin in the urine and feces. In two of three patients the proportion of type I porphyrin isomers was not influenced by therapy. Porphyrin excretion became normal following therapy, and the Patients experienced prolonged remissions, free of skin manifestations. These findings for the most part support the thesis that the responses are secondary to a reduction in hepatic porphyrins rather than interference of chloroquine in the porphyrin biosynthetic pathway. In One patient transient ascites and a hemolytic episode developed following administration of chloroquine. Porphyria cutanea tarda (PCT) is a type of hepatic porphyria characterized by a bullous dermatosis associated with hirsuties, pigmentary changes, photosensitivity and increased susceptibility to mechanical trauma. In most instances there is evidence of liver disease secondary to alcoholism or history of drug ingestion (i.e., estrogens). Although familial cases have been reported [1,2], it is thought to be an acquired illness. Abdomina complaints, neurologic manifestations and the precipitation of acute attacks by barbiturates do not occur in this illness, clearly separating it from acute intermittent porphyria. The urine and feces usually contain excess uroporphyrin and coproporphyrm as well as 7-carboxyl porphyrin [3]. There is usualty a mild increase in deltaaminolevulinic acid (ALA) excretion [3]. Porphobilinogen (PBG) excretion is normal [3]. Since the report of Linden et al. in 1954 [4] and from subsequent studies [5-8] it has been known that chloroquine administration results in a marked increase in uroporphyrin excretion associated with transient hepatocellular injury. These reports have established that within three days after chloroquine administration there is fever, nausea and malaise followed by excretion of large quantities of uroporphyrin in the urine and feces. Liver enzyme levels (serum glutamic oxalacetic transaminase. SGOT. and serum glutamic pyruvic transaminase. SGPT) become markedly elevated and on biopsy centrolobular necrosis has been documented [8]. There is an associated increase in serum iron [8]. Within a few days, recovery occurs. The liver enzyme levels return to normal and excretion of uroporphyrin falls to normal levels. Felscher and Redeker [8] reported that prolonged clinical rem ssions may be observed after chloroquine therapy. They suggested that this be studied further as a therapeutic modality. Felscher and Redeker [8] reported reduced porphyrin content in liver biopsy specimens obtained after chloroquine therapy, suggesting that administration of chloroqume results in release ot uroporphyrin from hepatic sites. There is no evidence that increased synthesis of urcporphyrin occurs. Once liver stores are reduced subsequent challenge with chloroqume does not result in increased porphyrin excretion. Scholnick and Marver [9] reported that
The AmericanJournalof Medicine
CHLOROQUINE THERAPY OF PORPHYRIA CUTANEA TARDA -- VOGLER ET AL
c h l o r o q u i n e f o r m s a c h l o r o q u i n e - p o r p h y r i n c o m p l e x ident i f i a b l e by s p e c t r o p h o t o m e t r y ; t h e y isolated it by gel filtration. The loss of h e p a t i c p o r p h y r i n was a c c o u n t e d for by release of p o r p h y r i n f r o m m i t o c h o n d r i a a n d l y s o s o m e s , the s u b c e l l u l a r f r a c t i o n s e x h i b i t i n g high c o n c e n t r a t i o n s of c h l o r o q u i n e . Electron m i c r o s c o p y studies s h o w e d e x t e n s i v e mitochondrial damage. This r e p o r t of t h r e e p a t i e n t s with p o r p h y r i a c u t a n e a t a r d a t r e a t e d w i t h c h l o r o q u i n e c o n f i r m s the w o r k of Felscher and R e d e k e r [8]. It gives a d d i t i o n a l i n f o r m a t i o n on t h e effect of c h l o r o q u i n e on t h e e x c r e t i o n of ALA, PBG a n d t h e v a r i o u s p o r p h y r i n isomers. Lastly, t w o c o m p l i c a t i o n s of c h l o r o q u i n e a d m i n i s t r a t i o n are p r e s e n t e d .
CASE REPORTS Case 1. This fifty-six year old white woman (C.M.) first admitted to Emory University Hospital on June 26, 1967. She gave a two year history of skin blisters, following minor trauma and exposure to sunlight, and increasing hirsutism. She stated that she had noted a long-standing sensitivity to sunlight expressed as "burning easily." On rare occasions she noted that her urine was dark. Three months before the onset of the skin blisters she had been given Premarin | (conjugated estrogens [equine]) for menopausal symptoms. Skin blisters occurred at sites of trauma, persisted for many months and were unresponsive to local medications. Except for mild chronic bronchitis and arthritis for which she had taken sodium salicylate, the patient has enjoyed good health. She denied the use of alcohol. Upon physical examination her skin was dry, somewhat thickened and glistening over the back and upper portion of her chest. There were scattered ulcerations approximately 1 to 2 cm in diameter with indurated borders on the hands and lower legs. There were numerous 1 to 2 cm scars on the hands and legs. There was growth of fine hair on the face. The liver was not palpable. The remainder of the examination was within normal limits. The hemoglobin was 15.5 gm per cent, hematocrit 46.5 per cent, reticulocytes 2 per cent, platelets 21g,ooO/cu mm, white blood cell count 9,300/cu mm with a normal differential. Liver function studies were within normal limits except for bromsulfalein retention of nine per cent (normal less than 6 per cent). The patient's condition remained essentially unchanged for six months. On December 19, 1967, she was readmitted to the hospital with the same findings. At that time her blood urea nitrogen level was 18 mg per cent, bromsulfalein retention 7 per cent, serum total bilirubin 0.5 mg per cent prothrombin time 11.5 seconds, SGOT 53 (Karmen units), alkaline phosphatase 14.5 King-Armstrong units and serum iron 267/~g per cent. Biopsy of the skin showed atrophy with slight chronic inflammation. Liver biopsy showed periportal parenchymaf necrosis, scattered fatty changes and increased hemosiderin. Most of the iron appeared within the hepatic parenchymal cells. Under ultraviolet light the liver specimen had a red fluorescence. Case 2. This forty-six year old white man (F.F.) was admitted to the hospital on February 26, 1968, complaining of sores on his arms of two years' duration. Over this interval he had had repeated vesicular lesions appearing on his upper extremities, particularly on the exposed areas of his forearms and hands. They were nonpruritic and nontender, except when denuded. The bullae varied in size depending on how long they remained intact. The patient found that by pricking the blisters they did not get as large and healed faster. This condition continued and was made worse by exposure to sunlight. The patient had not noted dark urine. He denied any other constitutional symptoms, had never been anemic and admitted
Volume 49, September 1970
to occasional social drinking. There was no family history of porphyria. He reportedly had a bleeding ulcer in 1955 which was treated medically. Physical examination revealed an obese, white man weighing 120 kg. His blood pressure was 180/110 mm Hg. Examination of the skin revealed numerous scars on the arms and one scab 1 cm long on the scalp. Fundi showed arteriovenous crossing changes with arteriovenous ratio 1:2. No abdominal organs were palpable. The remainder of the examination was within normal limits. The hemoglobin concentration was 18 gm per cent, the hematocrit 51 per cent, white blood cell count 9,600/cu mm with a normal differential. Bromsuifalein test showed 21 per cent retention. The alkaline phosphatase level was 13.2 King-Armstrong units, SGOT 117 Karmen units, lactate dehydrogenase (LDH) 131 Wacker units, serum total bilirubin 1.0 mg per cent, total protein 7.5 gm per cent. Serum iron was 186/~g per cent and prothrombin time 12.3 seconds. A biopsy specimen from the left arm showed hemorrhagic bullae in the subepidermis and necrosis of the upper dermis. These findings were thought to be consistent with porphyria cutanea tarda. Liver biopsy showed moderate steatosis, portal and periportal inflammation with periportal and intralobular necrosis, consistent with alcoholic hepatitis. Fresh frozen sections did not fluoresce for porphyrins. A freshly voided urine specimen did not fluoresce under ultraviolet light.
TEMPERATUREF~
102 I00
]
~,~
~
BILIRUBIN mg~ ALKALINE
PHOSPHATASE
750 500
L
~
SGOT
250
20 IO 0
400
I
,RONUO,
200
A L A U MOLES / 24 HRS.
40 ~ 20
120 80
~ ~
URINE UROPORPHYRIN p MOLES 1 24 HRS,
4O
20 ~ U R I N E ~ O P R O P O R P HYRIN 10 p MOLES124HRS. ,~ : "i ~ ;,' ~ ;,: . i~ : ' ,~ . ~l i I CIHiiIOROQU i , I,N , i J i i . L . _ L . I _ . . L . . ~ . L ~ L . L ~ I ~ L . I _ ~ _ ~ - . ~ ] 2 4 6 8 tO 12 14 ]6 18 47 72 go ED? 157 184 212 218 283
DAYS Fig. I. Effect of chloroquine on temperature, liver function tests, ALA a n d porphyrin excretion in a fifty-six y e a r old w o m a n with porphyria cutanea tarda.
317
CHLOROOUINE
THERAPY
OF P O R P H Y R I A C U T A N E A
I02
T A R D A - - VOGLER ET AL.
TEMPERATURE F ~ TEMPERATURE F~
lOO 98
IOD
1,0
B
BILIRUBIN
mg %
m.. BILIRUBIN
o
ALKALINE
~176 !1 ,,
BSP% SERUM IRON ~g %
2DD t
A L A
2o @
p MOLES i 24 HRS.
NNN
0 20
URINE UROPORPHYRIN p MOLES I 24 HRS. 80
I0
~
D
N
40 URINE COPROPORPHYRIN I~ MOLES I 24 NRS.
2 0 ~
~
~
--
__
L
MOLES I 24 HRS.
~
~'a
URINE COPROPORPHYRIN MOLES / 24 HRS
URINE PROTOPORPHYRIN
2.0
p MOLES I 24 HRS.
F
~
.S
1.0
~
~
--URINE PROTOPORPHYRIN ~ MOLES I 24 HRS. FECAL PORPHYRINS
2 9 . iO ~':;';.':.::;~;;.~,'.k.:'::..',+'..J.:t. :,~....
FECAL PORRHYRINS
p MOLES / 24 HRS.
0
~
p MOLES I 24 HRS.
TO URO
0
1
i
-5
-3
-I
+1
3
5
7
9
iI
13 43
18 I13 141 169 195 233
I
-4
, , ~!~,~i:.~ -2
0 2
4
~;',.~ii, Ii CHLOROQUIN JlJ il illJ_i__L 6 8 I0 12 14 16 18 20
DAYS
DAYS
Fig. 2. Effect of chloroquine on temperature, liver function tests, ALA and urinary and fecal porphyrin excretion in a forty-six year old man with porphyria cutanea tarda.
Fig. 3. Effect of chloroquine on temperature, liver function tests, ALA and urinary fecal porphyrin excretion in a fifty-seven year old man with porphyria cutanea tarda.
Case 3. This fifty-seven year old white man (J.H.) first noted blisters
nitrogen level 18 mg per cent and serum uric acid 8.4 mg per cent. The serum iron was 180 Fg per cent. Skin biopsy showed moderate hyperkeratoses, moderate perivascular infiltrate in the dermis and a nonspecific bulla. Hemosiderosis was present in the bone marrow biopsy. Liver biopsy showed areas of necrosis with polymorphonuclear leukocyte infiltration, mild steatosis and early fibrosis. Liver tissue fluoresced under ultraviolet light.
on the dorsum of the left hand while playing golf in 1959. The lesions failed to heal. Shortly after this he noticed that his urine was.red and shortly thereafter a diagnosis of porphyria cutanea tarda was made. He was given a dose of chloroquine and became violently ill, but the skin lesions subsided for one to two years. SubsequenUy he has had repeated vesicles on his hands and face which worsen in the spring and fall. There has been a gradual splenic enlargement from 1964 to 1968. Liver function tests in 1966 showed a bromsulfalein retention of 10 per cent. The patient gave a history of past ingestion of alcohol9 He had a peptic ulcer in 1963 and chest roentgenograms have shown interstitial fibrosis. There was no family history of porphyria. On physical examination his skin showed areas of scarring, depigmentation and hyperpigmentation on exposed portions of arms, face, scalp and shoulders. The liver was palpable 2 cm below the costal margin. The spleen was soft, the tip was felt 8 cm below the left costal margin. Laboratory data revealed a hemoglobin of 14.8 gm per cent, hematocrit 43 per cent, white blood cell count 5,700/cu mm, platelet count 117,000/cu ram, reticulocytes 2.7 per cent. Serum total bilirubin was 0.6 mg per cent, SGOT 60 Karmen units, alkaline phosphatase 15 King-Armstrong units, bromsulfalein retention 15 per cent, prothrombin time 12.7 seconds, blood urea
318
J
SGOT
~
!1
PHOSPHATASE
40U
B S P %
|
mq %
METHODS All patients were hospitalized on the Clinical Research Facility and fed a regular diet. Preliminary studies included blood counts, bone marrow examination, liver biopsy, blood urea nitrogen, uric acid, bilirubin, prothrombin time, serum glutamic oxalacetic transaminase, alkaline phosphatase, sodium sulfobromophthalein excretion, serum iron and iron-binding capacity. Twenty-four hour urine collections for total volume and creatinine were made. ALA and PBG were measured by the method of Marver et al. [10]. The urinary porphyrins were measured according to Schlenker et al. [11] with spectrophotometric corrections of Mingioli [12]. Three to four day aliquots of feces were pooled, a 100 gm aliquot was mixed with 200 ml of 5N hydrochloric acid. This was allowed to stand for two hours, filtered and the precipitate was washed with 100 ml 5N hydrochloric acid. The filtrate was then brought to 500 ml volume with water, and
The American Journal of Medicine
!
CHLOROOUINE THERAPY OF PORPHYRIA CUTANEA TARDA -- VOGLER ET AL.
aliquots were frozen for porphyrin determinations9The aliquots were thawed, 100 ml of saturated sodium acetate was added, and uroporphyrin was extracted by the method of Dresel and Falk [13]. Coproporphyrin and protoporphyrin were isolated by the method of Wranne [14]. Porphyrins were assayed by measurements of absorbance in the Soret region using a Beckman DU Spectrophotometer, applying the correction factors of Mingioli [12]. Separation of Porphyrin Isomers. Selected samples of isolated urine porphyrins were chromatographed by the method of Falk and Benson [15] to determine changes in porphyrin isomers. After conversion to methyl esters, chromatography revealed two major spots with RF values similar to those reported by Falk and Benson [15] for isomers I and III. The spots were eluted with ethanolic chloroform and absorbance at 402 m# measured9 After four to five days in the hospital, chloroquine in a dose of 0.5 gm daily was given for eight days. After treatment the patients were continued under observation for ten to fourteen days in the hospital and then followed at monthly intervals as outpatients. RESULTS
The results of the clinical and biochemical changes are summarized in Figures 1, 2 and 3. Clinical Response. Beginning approximately thirty-six hours after the first dose of chloroquine, all three patients had malaise, myalgias and fever which persisted over a seventy-two hour period and gradually returned to normal 9 One patient (Case 2), who received the smallest dose of chloroquine on a weight basis (4 m g / k g ) , experienced the least discomfort. The first patient (Case 1) noted slight nausea the first day. One patient (Case 3) had anorexia for six days,
TABLE I
nausea for five days and v o m i t i n g for two days beginning the day after initiation of t r e a t m e n t . After the fever subsided the patients had no further complaints with the exception of one patient (Case 3) in w h o m ascites developed. Liver Function Tests, Abnormalities and liver function tests suggested hepatocellular injury manifest by a p r o m p t increase in SGOT, increased bromsulfalein retention and a subsequent increase in serum alkaline phosphatase. One patient (Case 1) had no change in serum bilirubin but the other two (Cases 2 and 3) had a definite increase in direct-reacting bilirubin. In all instances the serum total bilirubin remained less than 3 mg per cent, and clinical jaundice was not evident. S e r u m Iron. In two patients (Cases 1 and 3) there was a very striking increase in serum iron highest at approximately the time that the SGOT level was most elevated. One patient (Case 2) failed to show an increase in serum iron, indeed there was a reduction in serum iron following therapy, from 192 to 98 #g per cent by the fourth day of treatment. Urinary ALA, PBG and P o r p h y r i n Excretion. In all three patients ALA excretion increased, as shown in the figures and Table I. Porphobilinogen excretion was somewhat variable and did not exceed normal values (Table I). One patient (Case 1) showed no essential change; one (Case 2) had a transient rise on the third and fourth days and one (Case 3) a reduction of porphobilinogen excretion after therapy. Uroporphyrin was the major porphyrin excreted in all three patients, rising briskly during the second day of t r e a t m e n t
Effect of Chloroquine on Daily Excretion of ALA and PBG ALA (/zM/24hr)"
Day of Study -
5 4 3 2 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
PBG (#M/24 hot
Chloroquine (gm/day)
Case 1
Case 2
Case 3
Case 1
Case 2
Case 3
0 0 0 0 0 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0 O 0 0 0 0 0 0 0
22.44 24.69 28.44 28.50 27.54 28.17 40.90 46.54 45.29 39.51 49.54 37.26 30.62 27.63 27.62 24.26 23.91 23.25 25.38 ... ... ...
15.02
9 .. 16.14
5.23 4.77 4.68 4.96
49 ... 3.56 ...
. .. 7.70 . .. 7.30
5.18
3.61
...
Normal values 6.33 to 19.78/LM/24 hours.
Volume 49, September 1970
15.45
...
16.95 15.48 17.45 16.65 22.18 24.47 22.16 21.30 25.91 23.42 18.97 16.82 19.61
9
19.07 24.63 33.00 24.47 29.04 28.04 33.38
21.53 19.17 ... 17.96 9. . 17.43 9 14.41 8.73
5.14 5.87 5.22 3.56 3.55 4.57 5.19 5.81 6.37 6.62 4.40 4.85 4.17 5.00 . . . .
.
.
.
.
4.41 7.10 4.36 8.36 4.93 4.85 5.39 5.10 4.36 6.50 4.72 6.72 4.12 9.34 3.63 4.19 3.24 3.91 . . . . . . 3.60 3.95 . . . . . . 3.79 3.09 . . . . . . . 5.34
.
.
.
39
t Normal values 0.73 to 6.38/~M/24 hours [11].
319
CHLOROQUINETHERAPYOF PORPHYRIACUTANEATARDA-- VOGLERET AL.
and falling to below pretreatment levels by the second week following chloroquine therapy. There was a three to fourfold increase in coproporphyrin excretion in the urine in all patients. Protoporphyrin excretion slightly increased in two patients during treatment; however, the amount excreted was small and remained within normal limits. Fecal Porphyrins. Fecal porphyrin excretions were measured in two patients (Cases 2 and 3). As shown in the figures, there was an increase in total porphyrin during treatment. In both instances fecal uroporphyrin decreased and coproporphyrin and protoporphyrin increased somewhat. Porphyrin Isomers. The results of chromatography of uroporphyrins during the pretreatment period, at the height of excretion and during follow-up are shown in Table I1. In
TABLE II
Effect of Chloroquine on Uroporphyrin Isomers Measured Prior to, at Peak of Urinary Uroporphyrin Excretion and Five to Seven Days After Stopping Therapy % UroporphyrinI Data
Pretreatment Peak excretion Follow-up
Case 1
Case 2
Case 3
63 79 76
70 65 65
18 63 36
two patients (Cases 1 and 2) the major uroporphyrin excreted throughout the study was the type I isomer. One patient (Case 3) had an apparent marked increase in percentage of type I during the peak porphyrin excretion period. Complications. In addition to the myalgias and the evidence of hepatocellular injury (with recovery) already described, one patient (Case 3) had an hemolytic episode during treatment; the plasma hemoglobin on the eleventh day after initiation of treatment was 20.8 mg per cent, falling to 1.4 mg per cent by the fourteenth day. His hematocrit fell :from 44 to 40 per cent and reticulocytes increased from 3.8 to 5.2 per cent. This patient's course was also complicated by the development of ascites which responded to salt restriction and diuretic therapy. When seen a month later for follow-up examination, at which time he had relaxed his strict salt curtailment, ascites had recurred although results of his liver function studies had returned to pretreatmerit values. Two months after treatment he had no further ascites, no blisters and skin pigmentation was reduced. The patients have been followed for twelve, ten and three
months, respectively, and have shown impressive clinical improvement with disappearance of all manifestations of cutaneous porphyria. The excretion of ALA and porphyrins remained within normal limits during this period of study. COMMENTS
The results of these studies, as well as those reported by others [8], indicate that chloroquine has a specific effect on the hepatic cell of the patient with porphyria cutanea tarda, resulting in hepatic necrosis and release of preformed porphyrins from liver and apparently from skin. Recent evidence has suggested that the binding of the porphyrin with chloroquine most likely takes place within the mitochondria and lysosomes, as these subcellular fractions contain the highest concentration of chloroquine [9]. Electron microscopy studies have shown extensive mitochondrial damage. The increase in ALA excretion here recorded has not been reported previously. It is likely that this increased excretion results from injury to the mitochondria resulting in release of ALA, since the enzyme necessary for its synthesis is localized in mitochondria [16,17]. If the increase in ALA excretion had resulted from inhibition of the enzyme ALA dehydrase by chloroquine one would expect porphobilinogen excretion to decrease during chloroquine administration. This was not the case in our patients. It is not known whether chloroquine stimulates the synthesis of ALA in porphyria cutanea tarda by increasing activity of ALA synthetase, as has been reported following barbiturate administration in acute intermittent porphyria. To our knowledge ALA synthetase activity has not been measured after chloroquine therapy. The role of iron in this disease is uncertain. Some recent evidence has indicated that the reduction in iron stores results in increased activity of heme synthetase in a patient with pyridoxine responsive anemia, suggesting that iron does inhibit this enzyme [18]. If heme synthetase activity can be increased by phlebotomies it is conceivable that accumulating porphyrinogens would be converted more rapidly to heme and the metabolic effects of increased porphyrins eliminated. The response of patients with porphyria cutanea tarda to phlebotomies is of interest in this connection [19]. Further investigation is needed to elucidate the role of iron in porphyria. ACKNOWLEDGMENT
We are indebted to Mrs. Elizabeth Mingioli for the measurements of ALA, PBG and porphyrins.
REFERENCES
1. Barnes HD, Overton J, Sweet RD: Familial cutaneous porphyria. Brit J Derm 77: 130, 1965. 2. Waldenstr6m J, Haeger-AronsenB: Different patterns of human porphyria. Brit Med J 2: 272, 1963. 3. Taddeini L, Watson FJ: The clinical porphyrias. Seminars Hemat 5: 335, 1968. 4. Linden IH, Steffen CG, NewcomerVD, Chapman M: Development
320
of porphyria during chloroquine therapy for chronic discoid lupus erythematosus. Calif Med 81: 235, 1954. 5. Marsden CW: Porphyria during chloroquine therapy. Brit J Derm 71: 219, 1959. 6. Cripps DJ, Curtis AC: Toxic effect of chloroquine on porphyria hepatica. Arch Derm 86: 575, 1962. 7. SweeneyGD, SaundersSJ, Dowdle EB, Eales L: Effects of chloro-
The American Journal of Medicine
C H L O R O Q U I N E T H E R A P Y OF PORPHYRIA C U T A N E A TARDA -- VOGLER ET AL.
8.
9.
10.
11.
12.
quine on patients with cutaneous porphyria of the "symptomatic" type. Brit Med J l(supp): 1281, 1965. Felscher BF, Redeker AC: Effect of chloroquine on hepatic uroporphyrin metabolism in patients with porphyria cutanea tarda. Medicine 45: 575, 1966. Scholnick P, Marver H: The molecular basis of chloroquine (C) responsiveness in porphyria cutanea tarda (PCT). Clin Res i6: 258, I968. Marver HS, Tschudy DP, Perlroth MG, Collins A: The determination of aminoketones in biological fluids. Anal Biochem 14: 53, 1966. Schlenker FS, Davis CL, Kitchell CL: Urinary total, aqueous and ether-soluble porphyrins. Techn Bull Regist Med Techn 33: 57, 1963. Minl]ioli ES: New correction factors for spectrophotometric assay of erythrocyte porphyrins. Anal Biochem 22: 47, ]968.
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13. Dresel EIB, Falk JE: Studies on the biosynthesis of blood pigments. Biochem J 63: 72, ]956. 14. Wranne L: Free erythrocyte copro- and protoporphyrins. Acta Paediat 49 (supp 124): 449, 1960. 15. Falk JE, Benson A: Separation of uroporphyrin esters I and III by paper chromatography. Biochem J 55: 101, 1953. 16. Laver WG, Newberger A, Udenfriend S: Initial stages in the biosynthesis of Porpflyrins. Biochem J 70: 4, 1958. 17. Urata G, Granick S: Biosynthesis of ~-aminoketones and the metabolism of aminoacetone. J Biol Chem 238: 811, 1963. 18. Vogler WR, Mingioli ES: Porphyrin synthesis and heine synthetase activity in pyridoxine-responsive anemia. Blood 32: 979, 1968. 19. Epstein JH, Redeker AG: Porphyria cutanea tarda: a study of the effect of phlebotomy. New Eng J Med 279: ]301, 1968.
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