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Fatal liver failure in protoporphyria
GASTROENTEROLOGY
1986;90:191-201
Fatal Liver Failure in Protoporphyria Synergism Between Ethanol Excess and the Genetic Defect HERBERT L. BONKOVSKY and ALAN R. SCHNED Departments of Medicine and Pathology, Veterans Administration Medical Center, White River Junction, Vermont, and Dartmouth Medical School, Hanover, New Hampshire
Protoporphyria was diagnosed in a 56-yr-old man based upon a typical clinical and family history, marked increases in erythrocyte and fecal protoporphyrin concentrations, and a marked decrease (21% of normal) in activity of hepatic heme synthase. Routine tests of liver function and histology were normal, except for a slight increase in bromsulphalein retention (9% at 45 min). Liver chemistries remained normal for 8 more years, but deteriorated rapidly when the patient was 63 yr old, with cholestasis precipitated by injury due to excess intake of ethanol. This, in turn, led to a defect in hepatic protoporphyrin excretion and to further worsening of liver injury due to porphyrin deposition. Our patient represents the 21st and oldest patient thus far reported to have died of liver failure complicating protoporphyria. Protoporphyria (also called erythropoietic or erythrohepatic protoporphyria) in humans is a dominantly inherited disorder of heme metabolism in which activity of the final enzyme of heme synthesis, protoheme ferrolyase (commonly called heme synthase or ferrochelatase) is markedly decreased (l-3). The molecular basis for this decrease in activity remains unknown. An analogous disease affects some cattle, but the bovine disease has autosomal recessive inheritance, requiring two abnormal genes for clinical expression (3-5). Both in humans and in cattle, the disease causes a characteristic photosenReceived May 6, 1985. Accepted July 1, 1985. Address requests for reprints to: Herbert L. Bonkovsky, M.D., Director, Clinical Research Facility, Emory University School of Medicine, Atlanta, Georgia 30322. This work was supported by funds from the United States Veterans Administration and by a grant from the National Institutes of Health (HL-28177). The authors thank Dr. J. Bloomer for performing the assay of hepatic protoheme ferrolyase. 0 1986 by the American Gastroenterological 0016-5085i8613.50
Association
sitivity, with life-long solar urticaria. Diagnosis is confirmed by demonstration of increased “free” protoporphyrin concentrations in blood or feces and decreased activity of the ferrolyase enzyme in blood, liver, or other tissues (l-3). Protoporphyria is nearly always a benign disorder and is treated by avoidance of or shielding from sunlight or by ingestion of p-carotene; however, in some patients, severe and fatal liver injury may supervene. Fortunately, this complication is rare. In all previously reported cases a fatal outcome has been attributed to the genetic defect, eventually leading to protoporphyrin deposition in hepatocytes with subsequent development of fibrosis, inflammation, and usually cirrhosis. Typically, such patients become clinically jaundiced late and thereafter deteriorate rapidly (2,3). In this paper we report a man with welldocumented inherited protoporphyria who, at age 56, had a biopsy-documented normal liver with no detectable increase in hepatic protoporphyrin. Examination of his liver and routine liver chemistries remained normal for 8 more years, despite a complicating cardiomyopathy and chronic congestive heart failure. At age 65, however, he developed severe liver injury and marked jaundice due to excessive ethanol intake and to protoporphyria. He improved transiently but died 4 mo later of sepsis and liver failure.
Case Report A 56-yr-old man of Finnish extraction first presented to our Medical Center in March 1974 with a life-long history of sunlight-induced burning, itching, and swelling of the dorsa of the hands, face, neck, and tops of the pinnae of the ears. His father had suffered similar symptoms. The patient claimed to drink little ethanol, although friends reported moderate to heavy (-6 drinks/day) intake. General physical examination, includ-
192
GASTROENTEROLOGY Vol. 90. No. 1
BONKOVSKY AND SCHNED
Table
1. Protoporphyrin
Determinations
PRBC (/.&dIl
Date
Plasma (&dI)
7174
1,350
34
9174
806
6
12174 1175 7180 4182
1,300 1,030 830 970
54 100
4183
3,500
43
12-60
0-Tr
Reference
on Blood, Feces, and Liver of the Patient
(/&
Liver (pgimg protein)
Feces dry wt)
2,210 (111,650 pgL24h) -
-
Hepatic heme synthase activity, 440 pmol hemeimg protein * h
560
-
-
-
670
5.2
Postmortem samples
O-Tr
O-120 10-6.000 ud24 hl
ranee
Comments
TP
PRBC, packed red blood cells. a Tr, trace.
pg/dl and total iron-binding capacity was 315 pg/dl; a bone marrow sample, showed absent iron and slight megaloblastic changes despite normal serum Blz (435 pg/ml; reference range, 150-1000 pg/ml) and increased folate levels (>50 ng/ml; reference range, 4-16 nglml). The erythrocyte mass was 27 ml/kg (reference range, 29 * 4 ml/kg) and total blood volume was 79 ml/kg (reference range, 66 f 7 ml/kg]. Erythrocyte chromium 51 survival was normal. As shown in Table 2, routine tests of liver function were normal; however, retention of bromsulphalein was slightly
ing liver and spleen, was normal, but the skin of the face and dorsa of the hands showed lichenification typical of protoporphyria. Based on these findings, this presumptive diagnosis was made and later confirmed by demonstration of rapidly fading porphyrin fluorescence of some erythrocytes, by a markedly increased concentration of protoporphyrin within erythrocytes, and by increased fecal excretion of protoporphyrin (Table 1). In June 1974, the patient was noted to have a blood hemoglobin concentration of 11.7 g/dl. Serum iron was 40
Table
2. Summary
of Routine
Hematologic
and Clinical
Chemical
Findings
of the Patient
Serum
Date 6174 7174 9174 3175 4175 6178 7180 8180 8181 9181 12181 3182 5182 9182 10182 1183
Admission
Hemoglobin
No.
WJJI
11.7 12.9 13.7 13.8 13.5
3183 416183 4113183 Reference range
76
21 118 18 36 41 36 86 46
61 108 102 73 76 46 177 164 90
15.1
35 66 287 274 300
165 272 315
19
12.9
195
212
4.7
13.8 14.7 10.2
330 490 174
410 234 109
7 9.8 16
14-18
5-40
30-115
Cl.5
13.9 15.9
15.1
2183
10
Total bilirubin (m&W
41 -
15.3 12.1 13.9
9
Alkaline AST phosphatase (IUIL) (NIL)
AST, aspartate transaminase; BSP, bromsulphalein; sis. ’Measured as seconds above control.
0.4 1.0 0.6 1.5
0.8
Albumin W) 5.2 5.2 -
Plasma prothrombin time”
2.5 2.3
Comments BSP retention, 9% Liver biopsy done
0 -
4.0 3.6 4.1
90
-
Globulin (@I
2.4 3.4
0.2
Thoracotomy
0.5
Drinking ethanol
2.9 -
Congestive
4.2
-
4.1
-
3.5-5.0 2.5-3.5
HBsAgIAb, hepatitis B surface antigen/antibody;
2.3 7 9
heart failure
Jaundice noted Weakness, deep jaundice Bleeding gastric ulcer SPEP-@r-bridging HBsAg/Ab negative Fever, weight loss Sepsis
so.5 SPEP, serum protein electrophore-
January 1986
prolonged (9% at 45 min). A needle biopsy of the liver was performed. As shown in Figure lA, the liver was normal histologically. There were no pigment deposits, and fluorescence and polarization microscopy were negative. The patient was treated with oral iron sulfate (325 mg t.i.d.) with increase in his blood hemoglobin to 13.7 g/d1 and decrease in erythrocyte and plasma protoporphyrin concentrations (Table 1). The patient was readmitted in March 1975, having accidently fractured his second cervical vertebra. Although he again denied drinking ethanol, it was believed that he had been drinking; and his serum aspartate transaminase level (AST) rapidly fell from 118 to 18 IU/L after admission (Table 2). Examination of the liver and spleen and other “liver chemistries” were normal. During the ensuing 5 yr, the patient was seen occasionally as an outpatient; examination of the liver remained normal, and serum AST ranged from 36 to 65 IU/L (reference range, 5-40 IUIL) and serum alkaline phosphatase ranged from 73 to 118 IUiL (reference range, 30-115 IUIL). In July 1980, the patient admitted to drinking “two or three beers a day and two or three shots of vodka a week.” In August 1981, admission was required because of increased shortness of breath, edema, weight gain, and low back pain. The patient was disheveled, and relatives reported heavy ethanol ingestion. His pulse rate was 120 and irregular; electrocardiogram showed atria1 fibrillation. The liver span was 12 cm in the right midclavicular line. Chest radiograph showed bilateral pleural effusions and congestive heart failure. Radiographs of the sacroiliac joints showed obliteration, compatible with ankylosing spondylitis. HLA typing was negative for B8 and B27 and positive for A3 and B7. Serum iron was 113 pg/dl and total iron-binding capacity was 268 pgldl. The patient was cardioverted electrically to normal sinus rhythm and was treated with salt restriction and diuretics. He lost 12 lb and his symptoms improved. Congestive cardiomyopathy, due to ethanol, was diagnosed. Admission for recurrent congestive heart failure was required in March 1982. The patient was again noted to have atria1 fibrillation. He had no peripheral stigmata of liver disease, had a normal liver examination, and, as shown in Table 2, had completely normal “liver chemistries.” He was treated with digoxin, diuretics, and salt restriction and was again urged not to drink ethanol. Despite weekly home visits by a nurse or nurse’s aide, the patient resumed heavy drinking of ethanol and, 5 mo later, his serum AST and alkaline phosphatase levels were increased (Table 2). Shortly thereafter he became progressively icteric, prompting readmission in January 1983. The patient was noted to have palmar erythema and ascites, the latter confirmed by abdominal ultrasound. The liver span was 10 cm in the right midclavicular line; no spleen was felt. Ultrasound examination revealed normal intrahepatic and extrahepatic bile ducts and pancreas. A HIDA scan was of poor quality, but radionuclide appeared in the duodenum, ruling out complete obstruction. Tests for hepatitis B surface antigen, hepatitis B surface antibody, hepatitis B core antibody, and hepatitis A virus antibody immunoglobulin M were negative. The patient was pre-
LIVER FAILURE IN PROTOPORPHYRIA 193
sumed to have cholestatic hepatitis due to protoporphyria and alcoholic hepatitis. He bled from a 2-cm-diameter benign antral ulcer and required transfusion of 4 U of packed cells. The patient was also treated with cimetidine and Mylanta-II (Stuart Pharmaceuticals, Wilmington, Del.). During the month of hospitalization, the patient’s serum bilirubin, AST, and alkaline phosphatase levels returned to normal (Table 2). During the same interval, however, serum alanine transaminase increased from 59 to 296 IU/L (reference range, 5-30 IU/L). The patient returned home and, in the ensuing month, the serum bilirubin and AST levels rose to 9.8 m&h and 490 IUIL, respectively. The patient’s final admission, in April 1983, was prompted by a 2-wk history of chills, anorexia, and a 17-lb weight loss. Physical examination showed jaundice and erythematous bullae involving the thick skin over the hands and scalp. These bullae were believed to be due to severe protoporphyrin-mediated actinic injury. As shown in Table 1, erythrocyte protoporphyrin was markedly increased, whereas fecal protoporphyrin was only moderately increased, suggesting limitation of porphyrin excretion by the diseased liver. Five days after admission the patient became hypotensive, acidotic, and hyponatremic due to sepsis. He suffered a respiratory arrest from pneumonia and mucus plugging of bronchi, and died. Autopsy
Findings
The liver at autopsy was slightly enlarged (1500 g). There was moderate splenomegaly and 200 ml of ascitic fluid. The biliary tract was patent, without stones or inflammation. On cut section, the liver was mottled brown-black and firm. On close inspection, there were punctate brown-to-black areas up to 2 mm in diameter separated by a thin reticular network of tan-gray fibrous septa [Figure 1B). Microscopically, the overall liver architecture was distorted by fibrous expansion of portal areas, often with incomplete or complete bridging (Figure 1C). Rare pseudolobules were formed. There was prominent portal and periportal ductular proliferation and a lymphocytic portal infiltrate. In addition to abundant bile, there was a striking accumulation of dark red-brown granular and globular pigment within the cytoplasm of hepatocytes, Kupffer cells, and portal macrophages, as well as within the lumens of bile canaliculi and ductules (Figure 1D). Also noted in the liver were moderate fatty change, mild hemosiderin deposition, central hyaline sclerosis with sinusoidal chicken-wire fibrosis, focal balloon change in hepatocytes, and occasional acidophil bodies (Figure 1F); no Mallory’s hyaline was identified. Fluorescence microscopy of the liver demonstrated red fluorescence within the pigment deposits. By polarization microscopy these deposits were often brightly birefringent, sometimes limited to the periphery of deposits but often with central Maltese crosses (Figure 1E). Electron microscopy showed circumscribed deposits of cytoplasmic crystalline material of several types. Compact masses of blunt rods or bars of variable length formed sunburst patterns or were haphazardly arranged (Figure ZA). Other
Figure
I. Gross and light microscopic appearance of liver biopsy (1974) and of the liver at autopsy (1983). (Parts A-F of this figure have been reduced by 27%.) A. Photomicrograph of a portion of the 1974 needle biopsy specimen of liver showing normal liver. Masson’s trichrome, original magnification x100. B. Close-up photograph of the cut surface of the liver at autopsy, showing punctate black-pigmented areas separated by gray septa. C. Photomicrograph of liver at autopsy, demonstrating distortion of hepatic architecture with bridging fibrosis, portal x100. D. High-power photomicrograph demonstrating abundant granular and ductular proliferation. and abundant deposits of pigment. Masson’s trichrome. original magnification globular pigment within cytoplasm of hepatocytes and Kupffer cells. Hematoxylin and eosin, original magnification x400. E. Same field as .D, by polarization microscopy, showing bright birefringence of pigment and occasional IMaltese crosses. original magnification x400. F. Photomicrograph of liver at autopsy, showing fatty change. balloon degeneration in some x200. scattered inflammatory cells, and chicken-wire sinusoidal fibrosis surrounding central vein (arrow). Masson’s trichrome, original magnification hepatocytes,
Figure
2. Electron micrographs of portions of hepatocytes from liver at autopsy, showing crystalline deposits of protoporphyrin. A. Localized crystalline deposit in starburst pattern comprised of thick, blunt-ended rods or bars. Reduced from ~35,000. B. Deposit of sheavelike spicular crystalline material, haphazardly arranged. Reduced from ~17,500. C. Large, circumscribed crystalline deposit containing an intimate mixture of both spicular and rodlike forms. Reduced from x 14,000.
January 1986
LIVER FAILURE IN PROTOPORPHYRIA 197
deposits were composed of electron-dense crystals in curved or spicular sheaves (Figure ZB). Still other deposits were comprised of both types of crystals (Figure 2C). Fine structural cytoplasmic detail was poorly preserved, but myelin figures were conspicuous in hepatocytes, and there was a suggestion of canalicular dilatation, some loss of cristae in mitochondria, and some loss of microvilli on the sinusoidal cell borders. Additional findings at autopsy included a dilated cardiomyopathy probably related to ethanol, a healing gastric antral ulcer, and extensive necrotizing pneumonia with sepsis. Postmortem blood culture was positive for Streptococcus pneumoniae, and liver, lung, and spleen cultures were positive for Klebsiella pneumoniae. Special
Studies
Liver tissue for electron microscopic Methods. study was retrieved from formalin-fixed stock. Minced tissue was placed overnight in 4% glutaraldehyde. After four changes in 0.1 N cacodylate buffer, the tissue was postfixed for 1 h in cacodylate-buffered 2% (wt/vol) osmium tetroxide and dehydrated in graduated acetones. The tissue was then polymerized and embedded in Epon, sectioned with a DuPont-Sorvall MT-l ultramicrotome (DuPont Co., Wilmington, Del.), stained with uranyl acetate and lead citrate, and examined on a Philips 201 transmission electron microscope (Philips Electronic Instruments, Mahwah, N.J.). Paraffin-embedded sections for light microscopy were stained with hematoxylin and eosin, modified Masson’s trichrome, Perls’ stain for iron, Hall’s bile stain, and periodic acid-Schiff stain, both with and without prior diastase digestion. Erythrocyte and plasma total porphyrins were measured by the method of Piomelli (6). Fluorescent spectra of plasma and erythrocyte porphyrins were performed as previously described (7). Urinary and fecal porphyrins were separated and measured by the methods of Schwartz et al. (8). For measurement of liver porphyrin, liver was homogenized in 99 volumes of 0.1 M NaPO, (pH 7.4). To 50 ~1 of homogenate was added 450 ~1 of a 1 : 1 (vol/vol) mixture of 0.6 M HCIOl/methanol. Precipitated protein was pelleted by centrifugation (1000 g for 5 min) and total porphyrin concentration and porphyrin profile were measured in the supernatant by the method of Grandchamp et al. (9) using a Perkin-Elmer 512 spectrofluorometer (Perkin-Elmer, Norwalk, Conn.) equipped with a R928 photomultiplier tube. An emission fluorescence spectrum (A = 570-700 nm) of the supernatant was obtained with the same instrument (excitation A = 400 nm, slits = 10 nm for both excitation and emission). Activity of hepatic heme synthase was measured by the method of Bonkowsky et al. (10). Urinary Gaminolevulinic acid and porphobilinogen were measured by the method of Mauzerall and Granick (11). Results. As shown in Table 1, concentrations of porphyrins in the patient’s erythrocytes and plasma were persistently and markedly elevated. The fluorescence spectra of erythrocyte and plasma porphyrins were typical for protoporphyria (7). Fecal protoporphyrin in July 1974
was markedly increased at a time when the patient was iron-deficient. The concentration was lower in December 1974, following 6 mo of iron replacement therapy (on hp+h qccasions, fecal coproporphyrin excretion was normal). Urinary excretions of &aminolevulinic acid, porphobilinogen, and porphyrins were normal (data not snown). The two living brothers and four living children of the patient were studied for the possibility that they carried the protoporphyria gene defect. None had symptoms or history of photosensitivity nor any increase in erythrocyte or fecal protoporphyrin concentrations. The needle biopsy specimen of liver obtained from the patient in September 1974 revealed no increase in porphyrin content, despite an activity of heme synthase that was only 21% of normal [patient, 440 pmol heme/mg protein h; normal, 2078 * 298 pmoi heme/mg protein h (mean II: SEM), n = lo], similar to findings in other patients with protoporphyria (2,5). The postmortem liver specimen contained 5.2 pg protoporphyrin/mg protein, or -780 pglg wet wt liver, a concentration within the range previously reported for patients dying with liver failure due to protoporphyria (Table 3). A fluorescence spectrum of the liver porphyrin was typical of protoporphyrin.
Discussion Although protoporphyria is usually a benign and relatively mild disease, it may occasionally be associated with severe, progressive, and fatal liver injury. Table 3 is a summary of fatal cases of liver failure complicating protoporphyria and shows that our patient was the 21st and oldest in whom liver fai!zro occurred. His abnormalities of porphyrin metahnlism and liver function were fairly typical of the group as a whole. Notice that serum AST and bilirubin were increased in every fatal case, whereas serl_m alkaline phosphatase was normal or only slightly increased in most other cases, as well as in our patient just before death. As shown in Table 2, however, our patient’s serum alkaline phosphatase had been increased twofold to fourfold during the 6 mo before his death; the increase was probably due to alcoholic liver injury. As might be expected, near the time of death, our patient and other patients had marked increases in erythrocyte and liver porphyrin concentrations with birefringent pigment deposits present in every insial~c;e in which they were sought. In contrast, fecal porphyrin concentrations or excretions in some patients were normal or only modestly increased, reflecting the effect of cholestasis to reduce hepatic protoporphyrin clearance. In 19 of 21 fatal cases, cirrhosis was observed at autopsy; the two exceptions had moderate to marked fibrosis with bridging. Ultrastructurally, cytoplasmic changes observed in our case are similar to other human cases previously described (12,13), and they also bear some
198
GASTROENTEROLOGY Vol. 90, No. 1
BONKOVSKY AND SCHNED
Table
3. Chronologic Summary of Reported Cases of Protoporphyria With Fatal Liver Failure Protouoruhvrin
Reference
Author lvearl
Age at death (vrl
Sex
PRBC (pg/dU
Plasma (pg/dU
concentrations
Feces (CLslgdry wt)
-
Liver (pg/g wet wtl
23
Barnes et al. (1968)
42
M
24
Hashimoto
et al. (1970)
58
F
4,320
121
25
Donaldson
et al. (1971)
56
M
6,000
-
208
26
Schmidt and Stich (1971)
58 40
M F
3,150
0.4
760
27
Iwanov et al. (1972)
42
F
3,793
-
28
Scott et al. (1973)
43
M
4,000
29
Thompson
31 31 44 33 26
F F F F M
2,000 3,180 4,516
et al. (1973)
4,900
0.7 (?)
79
-
3,200
(2,430 pg/24 h) (7,400 pgi24 h]
30 12 31
Meffert et al. (1974) Bloomer et al. (1975) Ibayashi et al. (1975)
32 13 33
16 29 55
M M M
34
Von Kaltbrunn (1975) Pimstone et al. (1976) Nicholson and Zawirska (1976) Cripps et al. (1977)
11
M
8,700
870
35 36
Singer et al. (1977) Wells et al. (1980)
60 19
M M
3,170 1,404
56
37
Nakanuma et al. (1981)
43
F
38
Romslo et al. (1982)
38
M
36,800
203
65-130
-
Bonkovsky and Schned
65
M
3,500
43
670
780
1120 pg/g dry wt (<600 kg/24 h)
0-Tr
High
790 -
(350 pgi24 h)
1159 to 204 loo-1,500 1,600
16,350 4,700
1,787
High
1,530
1,600 819 57,500
(403 pg/24 h) 368
-
309
-
(1985)
Reference ranges
-
12-60
0-Tr
AP, alkaline phosphatase; AST, aspartate transaminase; Bili, bilirubin; GI, gastrointestinal; Nl, normal; PRBC, packed red blood cells; Tr, used. b NE means not examined. Dashes mean data were not recorded. c Values obtained 8 mo before death when liver biopsy specimens
resemblance to acute changes seen in perfused isolated rat livers (14). The ultrastructural features of the protoporphyrin deposits in our case are also similar to findings previously reported (2,3,12,15). Despite formalin fixation, two types of crystalline material were readily discerned in circumscribed deposits. Many deposits were composed of only a single type of crystal, but others contained an inti-
mate admixture of both types. Although suboptimal preservation precluded a thorough study, we speculate that the blunt, rodlike forms (Figure 2A) may transform or evolve into the curved, spicular sheaves (Figure 2B) over time. We postulate that development of the increase in serum alkaline phosphatase and development of progressive liver injury in our patient was due to
January 1986
LIVER FAILURE IN PROTOPORPHYRIA
Liver pathology
Serum Bili (mgldl)
AST (W/L)
3.4-t 115
14.4-+19.1
150
14 17
(xl&”
>153 91
Pigment deposit
Birefringence
Architecture
+
NEh
Cirrhosis
+
NE
Cirrhosis
1.1
+
NE
Micronodular
cirrhosis
2.3 1.1
+ NE
NE NE
Micronodular Cirrhosis
cirrhosis
1.2
6.4
Increased
5+33
200-+1090
1.5-4.1
+
NE
Mixed microimacronodular active cirrhosis
7.2-+18
580
1.5
+
NE
Micronodular
-
2.0
+ + + + +
NE NE NE + +
Mixed microimacronodular cirrhosis Marked fibrosis Micronodular cirrhosis Micronodular cirrhosis
+ + NE
Cirrhosis Cirrhosis Micronodular
cirrhosis cirrhosis
19
15.6 11+41 7.0 3.2+17.4
17' 190
Nl
cirrhosis
170 100-200 475
Nl
+ + +
282-669
Nl
+
+
Macronodular
+ t
Severe fibrosis Micronodular cirrhosis
11.4 9
204 100
Nl 2
+ +
12.9
198
Nl
t
Nl
NE
NE
2.843.5
199
Moderate fibrosis and inflammation; rare bridging
16
174
Nl
t
t
<1.5
<40
Nl
None
None
NE
Incomplete micronodular cirrhosis and active hepatitis
Comments Terminal upper GI hemorrhage Death from coma and pneumonia Death from variceal hemorrhage Death from GI and other hemorrhage Death from hepatic failure 4 days after cholecystectomy, no common bile duct obstruction found Sideroblastic anemia, terminal course ushered in by crash diet
Severe pneumonia Death from variceal bleeding Death from coma Death from variceal bleeding Death from variceal bleeding, perforated duodenal ulcer 2 wk before Death from liver failure Liver transplant, postop. C. albicans sepsis, died 4 wk after, transplanted liver had 6.4 ~g protoporphyrin/g wet wt Progressive cholestasis, death from GI bleeding, postmortem diagnosis Progressive cholestasis, liver felt cirrhotic, autopsy refused Heavy ethanol, terminal sepsis, pneumonia, cardiomyopathy (alcoholic?)
Normal
trace; arrow indicates value changed progressively over time. u xULN means “fold” increase above upper limit of normal for the method showed mild chronic hepatitis, microvesicular fatty droplets in hepatocytes, and periportal protoporphyrin deposits.
an interplay between excess ethanol and protoporphyrin. The fact that, despite life-long protoporphyria, the patient’s liver was normal in 1974 (Figure 1A) and apparently remained so at least until March 1982 (Table 2) indicates that protoporphyria per se was not sufficient to cause liver damage. Excess ethanol ingestion, however, led eventually to hepatocytic injury severe enough to cause cho-
lestasis and failure of normal bile formation and, therefore, protoporphyrin excretion. Accumulation of protoporphyrin, which is itself capable of causing cholestasis (16), led to worsening of the process with more liver injury and more accumulation of protoporphyrin. Thus, a vicious positive feedback loop of liver injury was set into motion. Contributions to liver injury from both ethanol and protoporphyrin
200
GASTROENTEROLOGY Vol. 90, No. 1
BONKOVSKYANDSCHNED
were supported by the histopathologic findings at autopsy (Figures lB-1F). The presence of central sclerosis and fat was compatible with ethanolinduced injury while the marked pigment deposition and acidophilic bodies were related to protoporphyrin-induced injury. The portal fibrosis and chronic inflammation were less specific features. In most cases of severe liver damage associated with protoporphyria there has been an inexorable downhill course once jaundice has become apparent (2,3)[Table 3). Our patient, however, showed improvement in January and February of 1983, with a decrease in serum bilirubin from 19 to 4.7 mg/dl (Table 2). At least one other similar patient has been described (17). Thus, despite deep jaundice and evidence of cirrhosis and liver failure in patients with protoporphyria, the inevitability of an immediate fatal outcome should not be assumed. Rather, a search should be made for causes of reversible intercurrent injury (e.g., ethanol, drugs, viruses] and full supportive treatment continued. Efforts to decrease protoporphyrin overproduction by correcting iron deficiency and by a high carbohydrate intake are worthwhile. Other therapies (e.g., hypertransfusion, (18-20)or efforts to increase hematin infusions) protoporphyrin excretion (2,3,21,22) are less wellestablished but deserve consideration and further evaluation.
References 1. DeLeo VA, Poh-Fitzpatrick
M, Mathews-Roth M, Harber LC. Erythropoietic protoporphyria. Ten years experience. Am J Med 1976;60:8-22. and therapy of liver disease in 2. Bloomer JR. Pathogenesis protoporphyria. Yale J Biol Med 1979;52:39-48. and the 3. Bonkowsky HL. Porphyria and heme metabolism porphyrias. In: Zakim D, Boyer TD, eds. Hepatology: a textbook of liver disease. Philadelphia: WB Saunders, 1982: 351-93. 4. Ruth GR,Schwartz S, Stephenson B. Bovine protoporphyria: the first nonhuman model of this hereditary photosensitizing disease. Science 1977;198:199-201. 5. Bloomer JR, Morton KO, Reuter RJ, Ruth GR. Bovine protoporphyria: documentation of autosomal inheritance and comparison with the human disease through measurement of heme synthase activity. Am J Hum Genet 1982;34:322-30. 6. Piomelli S. A micromethod for free erythrocyte porphyrins: the FEP test. J Lab Clin Med 1973;81:932-40. 7. Poh-Fitzpatrick MB. A plasma porphyrin fluorescence marker for variegate porphyria. Arch Dermatol 1980;116:543-7. 8. Schwartz S, Berg MH, Bossenmaier I, Densmore H. Determinations of porphyrins in biological materials. Methods Biochem Anal 1962;8:221-93. 9. Grandchamp B, Deybach JC, Grelier M, de Verneuil H, Nordmann Y. Studies of porphyrin synthesis in fibroblasts of patients with congenital erythropoietic porphyria and one patient with homozygous coproporphyria. Biochim Biophys Acta 1980;629:577-86.
10. Bonkowsky
HL, Bloomer JR, Ebert PS, Mahoney MJ. Heme synthetase deficiency in human protoporphyria. Demonstration of the defect in liver and cultured skin fibroblasts. J Clin Invest 1975;56:1139-48. 11. Mauzerall D, Granick S. The occurrence and determination of Gaminolevulinic acid and porphobilinogen in urine. J Biol Chem 1956;219:435-46. 12. Bloomer JR, Phillips MJ, Davidson DL, Klatskin G. Hepatic disease in erythropoietic protoporphyria. Am J Med 1975; 58:869-82. 13. Pimstone NR, Webber BL, Blekkenhorst GH, Eales L. The hepatic lesion in protoporphyria (PP): preliminary studies of haem metabolism, liver structure and ultrastructure. Ann Clin Res 1976;8(Suppl 17):122-32. 14. Lee RG, Avner DL, Berenson MM. Structure-function relationships of protoporphyrin-induced liver injury. Arch Path01 Lab Med 1984;108:744-6. 15. MacDonald DM, Germain D, Perrot H. The histopathology and ultrastructure of liver disease in erythropoietic protoporphyria. Br J Dermatol 1981;104:7-17. 16. Avner DL, Lee RG, Berenson MM. Protoporphyrin-induced cholestasis in the isolated in situ perfused rat liver. J Clin Invest 1981;67:385-94. 17. Conley CL, Chisholm JJ Jr. Recovery from hepatic decompensation in protoporphyria. Johns Hopkins Med J 1979; 145:237-g. 18. Bechtel MA, Bertolone SJ, Hodge SJ. Transfusion therapy in a patient with erythropoietic protoporphyria. Arch Dermatol 1981;117:99-101. 19. Lamon JM, Poh-Fitzpatrick MB, Lamola A. Hepatic protoporphyrin production in human protoporphyria. Effects of intravenous hematin and analysis of erythrocyte protoporphyrin distribution. Gastroenterology 1980;79:115-25. 20. Bloomer JR, Pierach CA. Effect of hematin administration to patients with protoporphyria and liver disease. Hepatology 1982;2:817-21. 21. Avner DL, Berenson MM. Effect of choleretics on canalicular transport of protoporphyrin in the rat liver. Am J Physiol 1982;242:347-53. 2.2.Poh-Fitzpatrick MB, Sklar JA, Goldsman C, Lefkowitch JH. Protoporphyrin hepatopathy. Effects of cholic acid ingestion in murine griseofulvin-induced protoporphyria. J Clin Invest 1983;72:1449-58. 23. Barnes HD, Hurworth E, Millar JHD. Erythropoietic porphyrin hepatitis. J Clin Path01 1968;21:157-9. 24. Hashimoto K, Kanie T, Mibukura Y, Okumura H, Sakai Y, Kajita A. Protoporphyria erythropoietica associated with abnormalities of blood cells: an autopsied case. Rinsho Ketsueki 1970;11:49-57 (in Japanese). 25. Donaldson EM, McCall AJ, Magnus IA, Simpson JR, Caldwell RA, Hargreaves T. Erythropoietic protoporphyria: two deaths from hepatic cirrhosis. Br J Dermatol 1971;84:14-24. Protoporphyrie mit 26. Schmidt D, Stich W. Erythropoietische Porphyrinurie. Blut 1971;22:202-10. 27. Iwanov E, Taschev T, Kraster L, Braikov N. Leberschaedigung bei Protoporphyria erythropoietica. Derm Monatsschr 1972; 158:806-16. 28. Scott AJ, Ansford AJ, Webster BH, Stringer HCW. Erythropoietic protoporphyria with features of a sideroblastic anemia terminating in liver failure. Am J Med 1973;54:251-9. 29. Thompson RPH, Molland EA, Nicholson CD, Gray CH. Erythropoietic protoporphyria and cirrhosis in sisters. Gut 1973;14:934-8. H, Schmidt B, Sonnichsen N. 30. Meffert H, Barthelmes Leberbeteiligung bei erythropoetischer Protoporphyrie. Derm Monatsschr 1974;160:748-57. H, Honmatsu T, Sakamoto S, Matsusaka T, 31. Ibayashi
January 1986
Sumiyoshi A. Erythropoietic protoporphyria with atypical liver cirrhosis. Nihon Rhynsho 1975;33(Suppl 1):1486-g (in Japanese). 32. Von Kaltbrunn PF. Erythropoetische Protoporphyrie mit leberbeteiligun und haemolytischer Anaemie. M. D. thesis, 1975. Faculty of Medicine, University of Zurich, Switzerland. 33. Nicholson DC, Zawirska B. Porphyrin production in terminal erythropoietic protoporphyria. In: Doss M, ed. Porphyrins in human diseases. Basel: Karger, 1976:137-40. 34. Cripps DJ, Gilbert LA, Goldfarb S. Erythropoietic protoporphyria: juvenile protoporphyrin hepatopathy, cirrhosis and death. J Pediatr 1977;91:744-8.
LIVER FAILURE IN PROTOPORPHYRIA
201
35. Singer JA, Plaut AG, Kaplan MM. Hepatic failure and death from erythropoietic protoporphyria. Gastroenterology 1977; 74:588-91. 36. Wells MM, Golitz LE, Bender BJ. Erythropoietic protoporphyria with hepatic cirrhosis. Arch Dermatol 1980;116:429-32. 37. Nakanuma Y, Wada M, Kono N, Miyamura H, Ohta G. An autopsy case of erythropoietic protoporphyria with cholestatic jaundice and hepatic failure, and a review of the literature. Virchows Arch [A] 1981;393:123-32. 38. Romslo I, Gadeholt HG, Hovding G. Erythropoietic protoporphyria terminating in liver failure. Arch Dermatol 1982; 118:668-71.
1986;90:191-201
Fatal Liver Failure in Protoporphyria Synergism Between Ethanol Excess and the Genetic Defect HERBERT L. BONKOVSKY and ALAN R. SCHNED Departments of Medicine and Pathology, Veterans Administration Medical Center, White River Junction, Vermont, and Dartmouth Medical School, Hanover, New Hampshire
Protoporphyria was diagnosed in a 56-yr-old man based upon a typical clinical and family history, marked increases in erythrocyte and fecal protoporphyrin concentrations, and a marked decrease (21% of normal) in activity of hepatic heme synthase. Routine tests of liver function and histology were normal, except for a slight increase in bromsulphalein retention (9% at 45 min). Liver chemistries remained normal for 8 more years, but deteriorated rapidly when the patient was 63 yr old, with cholestasis precipitated by injury due to excess intake of ethanol. This, in turn, led to a defect in hepatic protoporphyrin excretion and to further worsening of liver injury due to porphyrin deposition. Our patient represents the 21st and oldest patient thus far reported to have died of liver failure complicating protoporphyria. Protoporphyria (also called erythropoietic or erythrohepatic protoporphyria) in humans is a dominantly inherited disorder of heme metabolism in which activity of the final enzyme of heme synthesis, protoheme ferrolyase (commonly called heme synthase or ferrochelatase) is markedly decreased (l-3). The molecular basis for this decrease in activity remains unknown. An analogous disease affects some cattle, but the bovine disease has autosomal recessive inheritance, requiring two abnormal genes for clinical expression (3-5). Both in humans and in cattle, the disease causes a characteristic photosenReceived May 6, 1985. Accepted July 1, 1985. Address requests for reprints to: Herbert L. Bonkovsky, M.D., Director, Clinical Research Facility, Emory University School of Medicine, Atlanta, Georgia 30322. This work was supported by funds from the United States Veterans Administration and by a grant from the National Institutes of Health (HL-28177). The authors thank Dr. J. Bloomer for performing the assay of hepatic protoheme ferrolyase. 0 1986 by the American Gastroenterological 0016-5085i8613.50
Association
sitivity, with life-long solar urticaria. Diagnosis is confirmed by demonstration of increased “free” protoporphyrin concentrations in blood or feces and decreased activity of the ferrolyase enzyme in blood, liver, or other tissues (l-3). Protoporphyria is nearly always a benign disorder and is treated by avoidance of or shielding from sunlight or by ingestion of p-carotene; however, in some patients, severe and fatal liver injury may supervene. Fortunately, this complication is rare. In all previously reported cases a fatal outcome has been attributed to the genetic defect, eventually leading to protoporphyrin deposition in hepatocytes with subsequent development of fibrosis, inflammation, and usually cirrhosis. Typically, such patients become clinically jaundiced late and thereafter deteriorate rapidly (2,3). In this paper we report a man with welldocumented inherited protoporphyria who, at age 56, had a biopsy-documented normal liver with no detectable increase in hepatic protoporphyrin. Examination of his liver and routine liver chemistries remained normal for 8 more years, despite a complicating cardiomyopathy and chronic congestive heart failure. At age 65, however, he developed severe liver injury and marked jaundice due to excessive ethanol intake and to protoporphyria. He improved transiently but died 4 mo later of sepsis and liver failure.
Case Report A 56-yr-old man of Finnish extraction first presented to our Medical Center in March 1974 with a life-long history of sunlight-induced burning, itching, and swelling of the dorsa of the hands, face, neck, and tops of the pinnae of the ears. His father had suffered similar symptoms. The patient claimed to drink little ethanol, although friends reported moderate to heavy (-6 drinks/day) intake. General physical examination, includ-
192
GASTROENTEROLOGY Vol. 90. No. 1
BONKOVSKY AND SCHNED
Table
1. Protoporphyrin
Determinations
PRBC (/.&dIl
Date
Plasma (&dI)
7174
1,350
34
9174
806
6
12174 1175 7180 4182
1,300 1,030 830 970
54 100
4183
3,500
43
12-60
0-Tr
Reference
on Blood, Feces, and Liver of the Patient
(/&
Liver (pgimg protein)
Feces dry wt)
2,210 (111,650 pgL24h) -
-
Hepatic heme synthase activity, 440 pmol hemeimg protein * h
560
-
-
-
670
5.2
Postmortem samples
O-Tr
O-120 10-6.000 ud24 hl
ranee
Comments
TP
PRBC, packed red blood cells. a Tr, trace.
pg/dl and total iron-binding capacity was 315 pg/dl; a bone marrow sample, showed absent iron and slight megaloblastic changes despite normal serum Blz (435 pg/ml; reference range, 150-1000 pg/ml) and increased folate levels (>50 ng/ml; reference range, 4-16 nglml). The erythrocyte mass was 27 ml/kg (reference range, 29 * 4 ml/kg) and total blood volume was 79 ml/kg (reference range, 66 f 7 ml/kg]. Erythrocyte chromium 51 survival was normal. As shown in Table 2, routine tests of liver function were normal; however, retention of bromsulphalein was slightly
ing liver and spleen, was normal, but the skin of the face and dorsa of the hands showed lichenification typical of protoporphyria. Based on these findings, this presumptive diagnosis was made and later confirmed by demonstration of rapidly fading porphyrin fluorescence of some erythrocytes, by a markedly increased concentration of protoporphyrin within erythrocytes, and by increased fecal excretion of protoporphyrin (Table 1). In June 1974, the patient was noted to have a blood hemoglobin concentration of 11.7 g/dl. Serum iron was 40
Table
2. Summary
of Routine
Hematologic
and Clinical
Chemical
Findings
of the Patient
Serum
Date 6174 7174 9174 3175 4175 6178 7180 8180 8181 9181 12181 3182 5182 9182 10182 1183
Admission
Hemoglobin
No.
WJJI
11.7 12.9 13.7 13.8 13.5
3183 416183 4113183 Reference range
76
21 118 18 36 41 36 86 46
61 108 102 73 76 46 177 164 90
15.1
35 66 287 274 300
165 272 315
19
12.9
195
212
4.7
13.8 14.7 10.2
330 490 174
410 234 109
7 9.8 16
14-18
5-40
30-115
Cl.5
13.9 15.9
15.1
2183
10
Total bilirubin (m&W
41 -
15.3 12.1 13.9
9
Alkaline AST phosphatase (IUIL) (NIL)
AST, aspartate transaminase; BSP, bromsulphalein; sis. ’Measured as seconds above control.
0.4 1.0 0.6 1.5
0.8
Albumin W) 5.2 5.2 -
Plasma prothrombin time”
2.5 2.3
Comments BSP retention, 9% Liver biopsy done
0 -
4.0 3.6 4.1
90
-
Globulin (@I
2.4 3.4
0.2
Thoracotomy
0.5
Drinking ethanol
2.9 -
Congestive
4.2
-
4.1
-
3.5-5.0 2.5-3.5
HBsAgIAb, hepatitis B surface antigen/antibody;
2.3 7 9
heart failure
Jaundice noted Weakness, deep jaundice Bleeding gastric ulcer SPEP-@r-bridging HBsAg/Ab negative Fever, weight loss Sepsis
so.5 SPEP, serum protein electrophore-
January 1986
prolonged (9% at 45 min). A needle biopsy of the liver was performed. As shown in Figure lA, the liver was normal histologically. There were no pigment deposits, and fluorescence and polarization microscopy were negative. The patient was treated with oral iron sulfate (325 mg t.i.d.) with increase in his blood hemoglobin to 13.7 g/d1 and decrease in erythrocyte and plasma protoporphyrin concentrations (Table 1). The patient was readmitted in March 1975, having accidently fractured his second cervical vertebra. Although he again denied drinking ethanol, it was believed that he had been drinking; and his serum aspartate transaminase level (AST) rapidly fell from 118 to 18 IU/L after admission (Table 2). Examination of the liver and spleen and other “liver chemistries” were normal. During the ensuing 5 yr, the patient was seen occasionally as an outpatient; examination of the liver remained normal, and serum AST ranged from 36 to 65 IU/L (reference range, 5-40 IUIL) and serum alkaline phosphatase ranged from 73 to 118 IUiL (reference range, 30-115 IUIL). In July 1980, the patient admitted to drinking “two or three beers a day and two or three shots of vodka a week.” In August 1981, admission was required because of increased shortness of breath, edema, weight gain, and low back pain. The patient was disheveled, and relatives reported heavy ethanol ingestion. His pulse rate was 120 and irregular; electrocardiogram showed atria1 fibrillation. The liver span was 12 cm in the right midclavicular line. Chest radiograph showed bilateral pleural effusions and congestive heart failure. Radiographs of the sacroiliac joints showed obliteration, compatible with ankylosing spondylitis. HLA typing was negative for B8 and B27 and positive for A3 and B7. Serum iron was 113 pg/dl and total iron-binding capacity was 268 pgldl. The patient was cardioverted electrically to normal sinus rhythm and was treated with salt restriction and diuretics. He lost 12 lb and his symptoms improved. Congestive cardiomyopathy, due to ethanol, was diagnosed. Admission for recurrent congestive heart failure was required in March 1982. The patient was again noted to have atria1 fibrillation. He had no peripheral stigmata of liver disease, had a normal liver examination, and, as shown in Table 2, had completely normal “liver chemistries.” He was treated with digoxin, diuretics, and salt restriction and was again urged not to drink ethanol. Despite weekly home visits by a nurse or nurse’s aide, the patient resumed heavy drinking of ethanol and, 5 mo later, his serum AST and alkaline phosphatase levels were increased (Table 2). Shortly thereafter he became progressively icteric, prompting readmission in January 1983. The patient was noted to have palmar erythema and ascites, the latter confirmed by abdominal ultrasound. The liver span was 10 cm in the right midclavicular line; no spleen was felt. Ultrasound examination revealed normal intrahepatic and extrahepatic bile ducts and pancreas. A HIDA scan was of poor quality, but radionuclide appeared in the duodenum, ruling out complete obstruction. Tests for hepatitis B surface antigen, hepatitis B surface antibody, hepatitis B core antibody, and hepatitis A virus antibody immunoglobulin M were negative. The patient was pre-
LIVER FAILURE IN PROTOPORPHYRIA 193
sumed to have cholestatic hepatitis due to protoporphyria and alcoholic hepatitis. He bled from a 2-cm-diameter benign antral ulcer and required transfusion of 4 U of packed cells. The patient was also treated with cimetidine and Mylanta-II (Stuart Pharmaceuticals, Wilmington, Del.). During the month of hospitalization, the patient’s serum bilirubin, AST, and alkaline phosphatase levels returned to normal (Table 2). During the same interval, however, serum alanine transaminase increased from 59 to 296 IU/L (reference range, 5-30 IU/L). The patient returned home and, in the ensuing month, the serum bilirubin and AST levels rose to 9.8 m&h and 490 IUIL, respectively. The patient’s final admission, in April 1983, was prompted by a 2-wk history of chills, anorexia, and a 17-lb weight loss. Physical examination showed jaundice and erythematous bullae involving the thick skin over the hands and scalp. These bullae were believed to be due to severe protoporphyrin-mediated actinic injury. As shown in Table 1, erythrocyte protoporphyrin was markedly increased, whereas fecal protoporphyrin was only moderately increased, suggesting limitation of porphyrin excretion by the diseased liver. Five days after admission the patient became hypotensive, acidotic, and hyponatremic due to sepsis. He suffered a respiratory arrest from pneumonia and mucus plugging of bronchi, and died. Autopsy
Findings
The liver at autopsy was slightly enlarged (1500 g). There was moderate splenomegaly and 200 ml of ascitic fluid. The biliary tract was patent, without stones or inflammation. On cut section, the liver was mottled brown-black and firm. On close inspection, there were punctate brown-to-black areas up to 2 mm in diameter separated by a thin reticular network of tan-gray fibrous septa [Figure 1B). Microscopically, the overall liver architecture was distorted by fibrous expansion of portal areas, often with incomplete or complete bridging (Figure 1C). Rare pseudolobules were formed. There was prominent portal and periportal ductular proliferation and a lymphocytic portal infiltrate. In addition to abundant bile, there was a striking accumulation of dark red-brown granular and globular pigment within the cytoplasm of hepatocytes, Kupffer cells, and portal macrophages, as well as within the lumens of bile canaliculi and ductules (Figure 1D). Also noted in the liver were moderate fatty change, mild hemosiderin deposition, central hyaline sclerosis with sinusoidal chicken-wire fibrosis, focal balloon change in hepatocytes, and occasional acidophil bodies (Figure 1F); no Mallory’s hyaline was identified. Fluorescence microscopy of the liver demonstrated red fluorescence within the pigment deposits. By polarization microscopy these deposits were often brightly birefringent, sometimes limited to the periphery of deposits but often with central Maltese crosses (Figure 1E). Electron microscopy showed circumscribed deposits of cytoplasmic crystalline material of several types. Compact masses of blunt rods or bars of variable length formed sunburst patterns or were haphazardly arranged (Figure ZA). Other
Figure
I. Gross and light microscopic appearance of liver biopsy (1974) and of the liver at autopsy (1983). (Parts A-F of this figure have been reduced by 27%.) A. Photomicrograph of a portion of the 1974 needle biopsy specimen of liver showing normal liver. Masson’s trichrome, original magnification x100. B. Close-up photograph of the cut surface of the liver at autopsy, showing punctate black-pigmented areas separated by gray septa. C. Photomicrograph of liver at autopsy, demonstrating distortion of hepatic architecture with bridging fibrosis, portal x100. D. High-power photomicrograph demonstrating abundant granular and ductular proliferation. and abundant deposits of pigment. Masson’s trichrome. original magnification globular pigment within cytoplasm of hepatocytes and Kupffer cells. Hematoxylin and eosin, original magnification x400. E. Same field as .D, by polarization microscopy, showing bright birefringence of pigment and occasional IMaltese crosses. original magnification x400. F. Photomicrograph of liver at autopsy, showing fatty change. balloon degeneration in some x200. scattered inflammatory cells, and chicken-wire sinusoidal fibrosis surrounding central vein (arrow). Masson’s trichrome, original magnification hepatocytes,
Figure
2. Electron micrographs of portions of hepatocytes from liver at autopsy, showing crystalline deposits of protoporphyrin. A. Localized crystalline deposit in starburst pattern comprised of thick, blunt-ended rods or bars. Reduced from ~35,000. B. Deposit of sheavelike spicular crystalline material, haphazardly arranged. Reduced from ~17,500. C. Large, circumscribed crystalline deposit containing an intimate mixture of both spicular and rodlike forms. Reduced from x 14,000.
January 1986
LIVER FAILURE IN PROTOPORPHYRIA 197
deposits were composed of electron-dense crystals in curved or spicular sheaves (Figure ZB). Still other deposits were comprised of both types of crystals (Figure 2C). Fine structural cytoplasmic detail was poorly preserved, but myelin figures were conspicuous in hepatocytes, and there was a suggestion of canalicular dilatation, some loss of cristae in mitochondria, and some loss of microvilli on the sinusoidal cell borders. Additional findings at autopsy included a dilated cardiomyopathy probably related to ethanol, a healing gastric antral ulcer, and extensive necrotizing pneumonia with sepsis. Postmortem blood culture was positive for Streptococcus pneumoniae, and liver, lung, and spleen cultures were positive for Klebsiella pneumoniae. Special
Studies
Liver tissue for electron microscopic Methods. study was retrieved from formalin-fixed stock. Minced tissue was placed overnight in 4% glutaraldehyde. After four changes in 0.1 N cacodylate buffer, the tissue was postfixed for 1 h in cacodylate-buffered 2% (wt/vol) osmium tetroxide and dehydrated in graduated acetones. The tissue was then polymerized and embedded in Epon, sectioned with a DuPont-Sorvall MT-l ultramicrotome (DuPont Co., Wilmington, Del.), stained with uranyl acetate and lead citrate, and examined on a Philips 201 transmission electron microscope (Philips Electronic Instruments, Mahwah, N.J.). Paraffin-embedded sections for light microscopy were stained with hematoxylin and eosin, modified Masson’s trichrome, Perls’ stain for iron, Hall’s bile stain, and periodic acid-Schiff stain, both with and without prior diastase digestion. Erythrocyte and plasma total porphyrins were measured by the method of Piomelli (6). Fluorescent spectra of plasma and erythrocyte porphyrins were performed as previously described (7). Urinary and fecal porphyrins were separated and measured by the methods of Schwartz et al. (8). For measurement of liver porphyrin, liver was homogenized in 99 volumes of 0.1 M NaPO, (pH 7.4). To 50 ~1 of homogenate was added 450 ~1 of a 1 : 1 (vol/vol) mixture of 0.6 M HCIOl/methanol. Precipitated protein was pelleted by centrifugation (1000 g for 5 min) and total porphyrin concentration and porphyrin profile were measured in the supernatant by the method of Grandchamp et al. (9) using a Perkin-Elmer 512 spectrofluorometer (Perkin-Elmer, Norwalk, Conn.) equipped with a R928 photomultiplier tube. An emission fluorescence spectrum (A = 570-700 nm) of the supernatant was obtained with the same instrument (excitation A = 400 nm, slits = 10 nm for both excitation and emission). Activity of hepatic heme synthase was measured by the method of Bonkowsky et al. (10). Urinary Gaminolevulinic acid and porphobilinogen were measured by the method of Mauzerall and Granick (11). Results. As shown in Table 1, concentrations of porphyrins in the patient’s erythrocytes and plasma were persistently and markedly elevated. The fluorescence spectra of erythrocyte and plasma porphyrins were typical for protoporphyria (7). Fecal protoporphyrin in July 1974
was markedly increased at a time when the patient was iron-deficient. The concentration was lower in December 1974, following 6 mo of iron replacement therapy (on hp+h qccasions, fecal coproporphyrin excretion was normal). Urinary excretions of &aminolevulinic acid, porphobilinogen, and porphyrins were normal (data not snown). The two living brothers and four living children of the patient were studied for the possibility that they carried the protoporphyria gene defect. None had symptoms or history of photosensitivity nor any increase in erythrocyte or fecal protoporphyrin concentrations. The needle biopsy specimen of liver obtained from the patient in September 1974 revealed no increase in porphyrin content, despite an activity of heme synthase that was only 21% of normal [patient, 440 pmol heme/mg protein h; normal, 2078 * 298 pmoi heme/mg protein h (mean II: SEM), n = lo], similar to findings in other patients with protoporphyria (2,5). The postmortem liver specimen contained 5.2 pg protoporphyrin/mg protein, or -780 pglg wet wt liver, a concentration within the range previously reported for patients dying with liver failure due to protoporphyria (Table 3). A fluorescence spectrum of the liver porphyrin was typical of protoporphyrin.
Discussion Although protoporphyria is usually a benign and relatively mild disease, it may occasionally be associated with severe, progressive, and fatal liver injury. Table 3 is a summary of fatal cases of liver failure complicating protoporphyria and shows that our patient was the 21st and oldest in whom liver fai!zro occurred. His abnormalities of porphyrin metahnlism and liver function were fairly typical of the group as a whole. Notice that serum AST and bilirubin were increased in every fatal case, whereas serl_m alkaline phosphatase was normal or only slightly increased in most other cases, as well as in our patient just before death. As shown in Table 2, however, our patient’s serum alkaline phosphatase had been increased twofold to fourfold during the 6 mo before his death; the increase was probably due to alcoholic liver injury. As might be expected, near the time of death, our patient and other patients had marked increases in erythrocyte and liver porphyrin concentrations with birefringent pigment deposits present in every insial~c;e in which they were sought. In contrast, fecal porphyrin concentrations or excretions in some patients were normal or only modestly increased, reflecting the effect of cholestasis to reduce hepatic protoporphyrin clearance. In 19 of 21 fatal cases, cirrhosis was observed at autopsy; the two exceptions had moderate to marked fibrosis with bridging. Ultrastructurally, cytoplasmic changes observed in our case are similar to other human cases previously described (12,13), and they also bear some
198
GASTROENTEROLOGY Vol. 90, No. 1
BONKOVSKY AND SCHNED
Table
3. Chronologic Summary of Reported Cases of Protoporphyria With Fatal Liver Failure Protouoruhvrin
Reference
Author lvearl
Age at death (vrl
Sex
PRBC (pg/dU
Plasma (pg/dU
concentrations
Feces (CLslgdry wt)
-
Liver (pg/g wet wtl
23
Barnes et al. (1968)
42
M
24
Hashimoto
et al. (1970)
58
F
4,320
121
25
Donaldson
et al. (1971)
56
M
6,000
-
208
26
Schmidt and Stich (1971)
58 40
M F
3,150
0.4
760
27
Iwanov et al. (1972)
42
F
3,793
-
28
Scott et al. (1973)
43
M
4,000
29
Thompson
31 31 44 33 26
F F F F M
2,000 3,180 4,516
et al. (1973)
4,900
0.7 (?)
79
-
3,200
(2,430 pg/24 h) (7,400 pgi24 h]
30 12 31
Meffert et al. (1974) Bloomer et al. (1975) Ibayashi et al. (1975)
32 13 33
16 29 55
M M M
34
Von Kaltbrunn (1975) Pimstone et al. (1976) Nicholson and Zawirska (1976) Cripps et al. (1977)
11
M
8,700
870
35 36
Singer et al. (1977) Wells et al. (1980)
60 19
M M
3,170 1,404
56
37
Nakanuma et al. (1981)
43
F
38
Romslo et al. (1982)
38
M
36,800
203
65-130
-
Bonkovsky and Schned
65
M
3,500
43
670
780
1120 pg/g dry wt (<600 kg/24 h)
0-Tr
High
790 -
(350 pgi24 h)
1159 to 204 loo-1,500 1,600
16,350 4,700
1,787
High
1,530
1,600 819 57,500
(403 pg/24 h) 368
-
309
-
(1985)
Reference ranges
-
12-60
0-Tr
AP, alkaline phosphatase; AST, aspartate transaminase; Bili, bilirubin; GI, gastrointestinal; Nl, normal; PRBC, packed red blood cells; Tr, used. b NE means not examined. Dashes mean data were not recorded. c Values obtained 8 mo before death when liver biopsy specimens
resemblance to acute changes seen in perfused isolated rat livers (14). The ultrastructural features of the protoporphyrin deposits in our case are also similar to findings previously reported (2,3,12,15). Despite formalin fixation, two types of crystalline material were readily discerned in circumscribed deposits. Many deposits were composed of only a single type of crystal, but others contained an inti-
mate admixture of both types. Although suboptimal preservation precluded a thorough study, we speculate that the blunt, rodlike forms (Figure 2A) may transform or evolve into the curved, spicular sheaves (Figure 2B) over time. We postulate that development of the increase in serum alkaline phosphatase and development of progressive liver injury in our patient was due to
January 1986
LIVER FAILURE IN PROTOPORPHYRIA
Liver pathology
Serum Bili (mgldl)
AST (W/L)
3.4-t 115
14.4-+19.1
150
14 17
(xl&”
>153 91
Pigment deposit
Birefringence
Architecture
+
NEh
Cirrhosis
+
NE
Cirrhosis
1.1
+
NE
Micronodular
cirrhosis
2.3 1.1
+ NE
NE NE
Micronodular Cirrhosis
cirrhosis
1.2
6.4
Increased
5+33
200-+1090
1.5-4.1
+
NE
Mixed microimacronodular active cirrhosis
7.2-+18
580
1.5
+
NE
Micronodular
-
2.0
+ + + + +
NE NE NE + +
Mixed microimacronodular cirrhosis Marked fibrosis Micronodular cirrhosis Micronodular cirrhosis
+ + NE
Cirrhosis Cirrhosis Micronodular
cirrhosis cirrhosis
19
15.6 11+41 7.0 3.2+17.4
17' 190
Nl
cirrhosis
170 100-200 475
Nl
+ + +
282-669
Nl
+
+
Macronodular
+ t
Severe fibrosis Micronodular cirrhosis
11.4 9
204 100
Nl 2
+ +
12.9
198
Nl
t
Nl
NE
NE
2.843.5
199
Moderate fibrosis and inflammation; rare bridging
16
174
Nl
t
t
<1.5
<40
Nl
None
None
NE
Incomplete micronodular cirrhosis and active hepatitis
Comments Terminal upper GI hemorrhage Death from coma and pneumonia Death from variceal hemorrhage Death from GI and other hemorrhage Death from hepatic failure 4 days after cholecystectomy, no common bile duct obstruction found Sideroblastic anemia, terminal course ushered in by crash diet
Severe pneumonia Death from variceal bleeding Death from coma Death from variceal bleeding Death from variceal bleeding, perforated duodenal ulcer 2 wk before Death from liver failure Liver transplant, postop. C. albicans sepsis, died 4 wk after, transplanted liver had 6.4 ~g protoporphyrin/g wet wt Progressive cholestasis, death from GI bleeding, postmortem diagnosis Progressive cholestasis, liver felt cirrhotic, autopsy refused Heavy ethanol, terminal sepsis, pneumonia, cardiomyopathy (alcoholic?)
Normal
trace; arrow indicates value changed progressively over time. u xULN means “fold” increase above upper limit of normal for the method showed mild chronic hepatitis, microvesicular fatty droplets in hepatocytes, and periportal protoporphyrin deposits.
an interplay between excess ethanol and protoporphyrin. The fact that, despite life-long protoporphyria, the patient’s liver was normal in 1974 (Figure 1A) and apparently remained so at least until March 1982 (Table 2) indicates that protoporphyria per se was not sufficient to cause liver damage. Excess ethanol ingestion, however, led eventually to hepatocytic injury severe enough to cause cho-
lestasis and failure of normal bile formation and, therefore, protoporphyrin excretion. Accumulation of protoporphyrin, which is itself capable of causing cholestasis (16), led to worsening of the process with more liver injury and more accumulation of protoporphyrin. Thus, a vicious positive feedback loop of liver injury was set into motion. Contributions to liver injury from both ethanol and protoporphyrin
200
GASTROENTEROLOGY Vol. 90, No. 1
BONKOVSKYANDSCHNED
were supported by the histopathologic findings at autopsy (Figures lB-1F). The presence of central sclerosis and fat was compatible with ethanolinduced injury while the marked pigment deposition and acidophilic bodies were related to protoporphyrin-induced injury. The portal fibrosis and chronic inflammation were less specific features. In most cases of severe liver damage associated with protoporphyria there has been an inexorable downhill course once jaundice has become apparent (2,3)[Table 3). Our patient, however, showed improvement in January and February of 1983, with a decrease in serum bilirubin from 19 to 4.7 mg/dl (Table 2). At least one other similar patient has been described (17). Thus, despite deep jaundice and evidence of cirrhosis and liver failure in patients with protoporphyria, the inevitability of an immediate fatal outcome should not be assumed. Rather, a search should be made for causes of reversible intercurrent injury (e.g., ethanol, drugs, viruses] and full supportive treatment continued. Efforts to decrease protoporphyrin overproduction by correcting iron deficiency and by a high carbohydrate intake are worthwhile. Other therapies (e.g., hypertransfusion, (18-20)or efforts to increase hematin infusions) protoporphyrin excretion (2,3,21,22) are less wellestablished but deserve consideration and further evaluation.
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