patients with well-documented hereditary hepatic porphyria. ... intermittent porphyria who had a liver biopsy be- ..... ed into the liver lobules in only 2 cases.
GASTROENTEROLOGY 1983;85:1131-7
LIVER AND BILIARY TRACT
Abnormalities in Liver Function and Morphology and Impaired Aminopyrine Metabolism in Hereditary Hepatic Porphyrias JERZY OSTROWSKI, EW A KOSTRZEWSKA, TOMASZ MICHALAK, BOZENNA ZA WIRSKA, WLODZIMIERZ MJ;:DRZEJEWSKI, and ANITA GREGOR Department of Gastroenterology and Metabolism, Medical Centre of Postgraduate Education; Institute of Haematology; Department of Immunopathology, National Institute of Hygiene, Warsaw; Department of Pathological Anatomy, Wroclaw School of Medicine, Poland
The aminopyrine breath test and measurement of postprandial serum bile acids were performed in 67 patients with well-documented hereditary hepatic porphyria. Elevated postprandial serum bile acids levels were found in 75% and the aminopyrine breath test was abnormal in 42% of the patients studied. These function tests exhibited a statistically significant correlation with the excretion of porphyrin precursors in patients with acute intermittent porphyria. Furthermore, all 12 patients with acute intermittent porphyria who had a liver biopsy because of abnormalities in the aminopyrine breath test or postprandial serum bile acids measurement, or both, were found to have diffuse, moderately severe, but nonspecific ultrastructural changes of hepatocytes, indicative of significant subcellular damage. While a direct relationship between functional and morphologic abnormalities is speculative, it is possible that the defective heme biosynthesis, perhaps related to the reduced aminopyrine demethylation, could form the basis for the ultrastructural hepatic changes in porphyria. This, in turn, could be the cause of the elevated postprandial serum bile acids levels. The three hereditary hepatic porphyrias (HHP), acute intermittent porphyria (AlP), variegate porphyria (VP), and hereditary coproporphyria (Hep) are dominantly inherited metabolic disorders, characterized by excessive production, accumulation, Received May 6, 1982. Accepted April 22, 1983. Address requests for reprints to: Jerzy Ostrowski, M.D., Department of Gastroenterology and Metabolism, ul. Goszczynskiego 1, 02-616 Warsaw, Poland. © 1983 by the American Gastroenterological Association 0016-5085/83/$3.00
and excretion of porphyrins and their precursors. They have one important clinical feature in common: patients with any of these disorders may develop clinically similar acute attacks (1). Although the liver is the main organ in which the overproduction of porphyrin precursors and porphyrins takes place, routine liver function tests in AlP and related disorders have failed to show consistent deviations from normal (1-5). Also, at best only minor histologic changes of hepatic structure have been observed in patients with AlP (6-11). However, prolongation of the antipyrine plasma half-life has been detected in some cases, suggesting impairment of the microsomal cytochrome P450 system (2). The purpose of this study was to evaluate liver function in HHP patients on the basis of the [14C]aminopyrine breath test (ABT) and postprandial serum bile acids (pSBA) levels and to relate the results of these tests to histologic and ultrastructural findings in the liver.
Material and Methods Subjects The study group consisted of 67 patients, aged 2264 yr (43 women and 24 men) with HHP well documented by personal or family history, or both, and by biochemical criteria. The diagnoses of the hereditary hepatic porphyrias were based on quantitative determination of excretion of aminolevulinic acid (ALA), porphobilinogen (PBG) (12), and porphyrins (13), chromatographic separation of urine and fecal porphyrins after their esterification, and assays of PBG deaminase (also called uroporphyrinogen I synthase) activity in erythrocytes (14) and coproporphyrinogen oxidase activity in lymphocytes (15). Fifty-four of 67 HHP patients had AlP. Thirty-eight of
1132
OSTROWSKI ET AL.
them previously had manifested neurologic-visceral symptoms of acute attacks. Latent gene carriers of AlP comprised 16 family members of AlP patients. The diagnosis of AlP was based on low red cell PBG deaminase activity «25 nmol porphyrin/ml RBC . h). Four of 6 patients with HCP had clinically manifest porphyria and 2 were family members of HCP patients. The chemical characteristic in these patients was the increased excretion of coproporphyrin mainly in feces (653-4944 fLg/g dry wt) and reduced coproporphyrinogen oxidase activity in lymphocytes. * Among the 3 patients with VP. 1 previously had an acute attack with neurologic as well as cutaneous manifestations and 2 had a positive family history and the typical chemical findings, i.e., excretion offecal protoporphyrin in amounts of 95-173 fLg/g dry wt. Four patients had atypical hepatic porphyria. They had had one or more acute attacks characteristic of porphyria but the pattern of porphyrin excretion did not fit neatly into any of the categories of hepatic porphyria recognized. Three of these patients had increased excretion of porphyrin precursors and porphyrins characteristic of AlP but the activity of red cell PBG deaminase, assayed repeatedly, was normal. One of them had acute attacks with neurologic and cutaneous manifestations associated with increased amounts of fecal protoporphyrin, but the activity of his PBG deaminase was reduced. Among the investigated cases, there were 47 patients with a clinical history and chemical findings of hepatic porphyria (clinically manifest porphyria) and 20 patients who were adult family members of HHP patients but who had never had acute attacks (clinically latent porphyria). In the latter group, the diagnosis of porphyria was based on family history and generally accepted biochemical criteria, as previously described. None of the patients with clinically manifest porphyria had any clinical symptoms at the time of this study. None of the studied patients was an alcoholic. None had physical signs of chronic liver disease or any evidence of biliary tract disease. Only 1 patient had a history of acute viral hepatitis, type B. All patients were on a normal diet for at least 2 mo before the study. At least 6 mo had elapsed since the last administration of intravenous glucose to abort a porphyric attack. Thirty-seven percent of patients with porphyria smoked cigarettes. The control group comprised 34 healthy volunteers, aged 24-48 yr (22 women and 12 men), who had no evidence of past or present liver disease. Their daily urinary excretion of porphyrins and porphyrin precursors was normal. Forty-four percent of this group smoked cigarettes. None of patients observed had received any drugs for 2 wk prior to the study. In all 67 patients with HHP and in controls, liver function was evaluated on the basis of ABT and pSBA levels. Both tests were made in the morning, after an overnight fast. Written informed consent was obtained from each subject.
* The assays of coprooxidase were performed by Professor Yves Nordmann, Department of Biochemistry, Faculty of Medicine, Colombes, France.
GASTROENTEROLOGY Vol. 85, No. 5
The routine liver function tests also were done in all 67 patients with HHP. Thymol test was elevated in 8 patients, total bilirubin in 6, alkaline phosphatase in 8, serum glutamic pyruvic transaminase in 5, and serum glutamic oxaloacetic transaminase in 3. Prothrombin time was normal in all patients.
Aminopyrine Breath Test In the study, dimethylamine-[ 14 C]aminopyrine, obtained from the Radiochemical Centre, Amersham, United Kingdom, was used (chemical purity ~98%; sp act 12.2 mCi/mmol). The labeled compound was dissolved in 0.9% NaCl to obtain 1 fLCi/ml solution. The patients orally received 3 fLCi aminopyrine dissolved in 50 ml of water. Two hours after aminopyrine administration, radioactive CO 2 was collected in counting vials containing 2 ml of 1 M solution of hyamine hydroxide in methanol and ethanol (1:1, vollvol), with 0.6% phenolphthalein as indicator. After adding 10 ml of scintillation mixture, radioactivity was assessed using a Packard liquid scintillation spectrometer (Packard Instrument Co., Downers Grove, Ill.). The aminopyrine standard was prepared as described by Galizzi et al. (16). The results were calculated as disintegrations per minute per millimole of CO 2 and expressed as percentage of the dose administered, assuming that the endogenous CO 2 production level was 9 mmollkg' h (17).
Measurement of Postprandial Serum Bile Acids Bile acids were determined in sera by the enzymatic spectrofluorometric method according to Fausa (18), using Sterognost 30: produced by Nyegaard, Oslo, Norway. Blood samples were taken 2 h after the administration of a standard meal (15% protein, 30% fat, and 55% carbohydrate).
Statistics The results of the ABT and pSBA measurement were statistically evaluated by the nonparametric test (Mann-Whitney) and Spearman correlation rank analysis.
Histologic Studies Histologic studies were performed on liver biopsy specimens obtained by Menghini needle from 12 women with AlP, aged 26-62 yr, who had abnormally low values of the ABT or elevated pSBA levels, or both. All the patients who had a liver biopsy were seronegative for hepatitis B surface antigen by enzyme-linked immunoabsorbent assay (ELISA) (Hepanostica, Organon Teknika) and 2 patients were seropositive for hepatitis B core antibody (anti-HBc) by indirect immunofluorescence test (19). All liver biopsy specimens were free of hepatitis B antigens as determined by direct immunofluorescent staining of the cryostat sections with anti-HBs and antiHBc conjugated with fluorescein isothiocyanate (FITC). In all of these patients serum iron level was normal.
November 1983
LIVER ABNORMALITIES IN HEPATIC PORPHYRIAS
The tissue for histologiC investigation was fixed in 4% formalin buffered with a phosphate buffer, pH 7.4, and, after routine processing, embedded in paraffin. The following stains were used: hematoxylin and eosin, Masson's trichrome, Gomori's , Van Gieson's, Mallory's, PerIs', periodic acid-Schiff (PAS) after diastase digestion, and Oil Red 0 method.
Electron Microscopic Studies For ultrastructural examinations, fragments of the liver tissue were fixed in 2% solution of glutaraldehyde buffered with 0.2 M cacodyl buffer, pH 7.4, routinely processed, and embedded in Epon 1312. Ultrathin sections were double-stained with uranyl acetate and lead citrate. All observations were carried out using a JEM lODe electron microscope , equipped with a gonometriC stage, at . magnifications of x 5300-x 100,000.
Reslllts Aminopyrine Metabolism and Serum Bile Acids In 34 healthy volunteers, the lowest ABT result was 5.41% and this value was taken as the lower normal limit. In 31 controls , the highest pSBA level, subsequently taken as the higher normal limit, was 6.56 f1moliL. As shown in Figure 1, abnormal results of pSBA
measurement were obtained in 75% and of ABT in 42% of 67 patients with HHP. The abnormal results were found mainly in clinically manifest porphyries, whose ABT results were significantly lower (p < 0.001) and pSBA levels significantly higher (p < 0.001) than those obtained in patients with clinieally latent porphyria. Furthermore, there was a highly significant correlation between the function tests when compared for fill 67 patients in the series (rs = ~0.6115, P < 0.001) and also for the 47 clinically manifest porphyries (rs = -0.5315 , P < 0.001) . The abnormal results of ABT and pSBA measurement were not dependent either on time elapsed since the last attack, severity of clinical course, or number of previous acute attacks. Cigarette smoking also did not affect the aminopyrine demethylation in porphyries, as no significant difference of ABT values was observed among smoking and nonsmoking HHP patients. Daily urinary excretion of porphyrin precursors, determined at the time of this study, in patients with clinically manifest AlP was significantly higher than in those with latent AlP (p < 0.001). In patients with manifest AlP, ALA excretion was 4.24-155.0 mg/24 h (median, 23 .55) and PBG excretion was 1.23-191.6 mg/~4 h (median, 67.1). In patients with latent AlP, ALA excretion was 2.5-28.0 mg/2 4 h (median, 5.13) and PBG excretion was 0.95-61.7 mg/24 h (median, 4.25). A highly significant correlation was found postprandial serum bile acids
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Figure 1. The results of 2-h aminopyrine breath tests, expressed as a percentage of dose, and of measurements of postprandial serum bile acids in 47 patients with clinically manifest HHP (closed circles) and in 20 patients with clinically latent HHP (O J, compared with controls (open circles). The horizontal lines indicate the normal limits .
1134 OSTROWSKI ET AL.
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