Theodore Woodward Award. Pathogenesis of biochemical abnormalities in protoporphyria.

In summary, FC gene mutations in patients with protoporphyric liver disease typically cause major structural alterations in the FC protein. However, the gene mutations by themselves do not satisfactorily account for the severe phenotype, as the same mutations are found in asymptomatic family members, and similar mutations are found in patients who do not develop liver disease. Thus there may be unidentified factors in the FC gene locus, or factors outside the locus, which are also important in determining the degree of protoporphyrin accumulation that occurs in an individual patient, hence, the potential for developing significant liver disease. Further studies are needed to clarify this possibility and identify those factors.

Hepatopulmonary syndrome and venous emboli causing intracerebral hemorrhages after liver transplantation: a case report.

Increasing experience has fostered the acceptance of liver transplantation as a treatment for patients with hepatopulmonary syndrome. Morbidity and mortality is most commonly attributed to progressive arterial hypoxemia postoperatively. A cerebral hemorrhage has been reported in one patient with hepatopulmonary syndrome after transplantation. However, a postmortem examination of the brain was not performed and the pathogenesis or type of cerebral hemorrhage was undefined. We report on a patient with severe hepatopulmonary syndrome who developed multiple intracranial hemorrhages after transplantation. The intracerebral hemorrhages were most consistent with an embolic etiology on postmortem examination. We postulate that venous embolization, caused by the manipulation of a Swan Ganz catheter in a thrombosed central vein, resulted in pulmonary emboli that passed through dilated intrapulmonary vessels into the cerebral microcirculation. Special attention to central venous catheters and avoidance of manipulation may be warranted in subjects with severe hepatopulmonary syndrome after liver transplantation.

A novel stop codon mutation (X417L) of the ferrochelatase gene in bovine protoporphyria, a natural animal model of the human disease.

Protoporphyria (PP) is caused by a deficiency of ferrochelatase (FC) activity, which catalyzes the final step in the heme biosynthesis pathway. Bovine are the only species other than man with naturally occurring PP. For expression of the PP phenotype, two copies of the mutated gene are necessary in bovine, whereas one copy is sufficient in humans. We report the first potential disease-causing mutation in the bovine FC gene. The coding region of FC was sequenced from the liver tissue of protoporphyric and normal bovine. A transversion was identified at nucleotide position 1250 which changed the stop codon to leucine (TGA-->TTA) in the protoporphyric FC sequence. As a consequence, the mutant protein is predicted to have an additional 27 amino acids. To screen other bovine for the G-->T transversion, cDNAs from liver tissue of clinically and biochemically normal, and from heterozygous and homozygous affected animals were used for allele-specific polymerase chain reaction. Three normal animals had only the G allele, five affected animals had only the T allele, and three heterozygous animals had both the G and T alleles. These results support our hypothesis that this mutation causes PP in bovine.

Liver metabolism of porphyrins and haem.

The liver is an active site for the biosynthesis of haem and porphyrinogens/porphyrins, which are intermediates of the haem biosynthetic pathway, because haem is required for functional activity of the cytochrome P 450 system and other critical hepatic haemoproteins. The production of hepatic haem is regulated primarily through the activity of aminolaevulinic acid synthase which is the first and normally rate-limiting enzyme of the pathway. This is, in turn, controlled by a putative regulatory haem pool. Hepatic haem can be repleted by the intravenous administration of haem, which is the basis for haem therapy in patients with acute porphyric attacks. The liver catabolizes haem to bilirubin through microsomal haem oxygenase activity and excretes haem into bile along with porphyrins. Biliary excretion of porphyrins increases significantly in patients with some types of porphyria. In protoporphyria this may cause liver damage as a result of protoporphyrin toxicity. The delineation of the pathway for protoporphyrin excretion into bile should facilitate therapy in protoporphyria by identifying ways in which protoporphyrin excretion can be enhanced.

Complications of cirrhosis. Why they occur and what to do about them.

Cirrhosis is a chronic disease of the liver in which dense bands of fibrosis enclose regenerative hepatocellular nodules. Clinical and radiologic features of advanced liver disease provide presumptive evidence for the presence of cirrhosis. Major complications are related to the increased hepatic resistance, increased sodium and water retention, and hyperdynamic changes of the circulatory system. Patient management should consist of appropriate prophylaxis for the life-threatening complications of variceal bleeding and spontaneous bacterial peritonitis and treatment of other complications as signs and symptoms develop.

Hepatic porphyrias.

The porphyrias are metabolic disorders characterized by abnormal heme biosynthesis with excessive accumulation and excretion of porphyrias or porphyrin precursors. Defects in the enzymes of the heme biosynthetic pathway result in porphyria. Several of the disorders have been classified as hepatic because the major site of the biochemical defect has been localized to the liver. This article describes the enzymes of the heme biosynthetic pathway, the clinical features of the hepatic porphyrias and management of the disorders.

Hepatic protoporphyrin metabolism in patients with advanced protoporphyric liver disease.

Protoporphyria is a genetic disorder in which liver damage is caused by the toxic effect of protoporphyrin accumulation in the liver. In this study protoporphyrin was measured in the resected livers of 7 patients who had liver transplantation and an additional patient from whom liver tissue was obtained post mortem. Comparison of liver, erythrocyte and serum protoporphyrin levels demonstrated a marked gradient between these compartments: erythrocyte, 5781 +/- 655 micrograms/dl; serum, 384 +/- 102 micrograms/dl; liver 377,238 +/- 55,568 micrograms/100 gm wet weight, (mean +/- SE). Protoporphyrin levels in bile of 3 patients were 55,559, and 1,153 micrograms/dl, indicating a gradient between liver and bile as well. Examination of the livers by polarization microscopy and electron microscopy demonstrated protoporphyrin pigment crystals. In one patient who had recurrent liver disease after transplantation, the protoporphyrin concentration in the graft at the time of death was similar to that in the resected liver. These data indicate that liver protoporphyrin levels in patients with advanced protoporphyric liver disease are much higher than levels in blood and bile, in part because protoporphyrin forms crystalline deposits in liver tissue. Thus, progressive hepatic accumulation of protoporphyrin occurs in the face of impaired biliary excretion. An intrinsic defect in hepatic excretion of protoporphyrin is probably not necessary for this condition to develop because liver disease can occur in the graft following transplantation.

Environmental chemical exposures and disturbances of heme synthesis.

Porphyrias are relatively uncommon inherited or acquired disorders in which clinical manifestations are attributable to a disturbance of heme synthesis (porphyrin metabolism), usually in association with endogenous or exogenous stressors. Porphyrias are characterized by elevations of heme precursors in blood, urine, and/or stool. A number of chemicals, particularly metals and halogenated hydrocarbons, induce disturbances of heme synthesis in experimental animals. Certain chemicals have also been linked to porphyria or porphyrinuria in humans, generally involving chronic industrial exposures or environmental exposures much higher than those usually encountered. A noteworthy example is the Turkish epidemic of porphyria cutanea tarda produced by accidental ingestion of wheat treated with the fungicide hexachlorobenzene. Measurements of excreted heme precursors have the potential to serve as biological markers for harmful but preclinical effects of certain chemical exposures; this potential warrants further research and applied field studies. It has been hypothesized that several otherwise unexplained chemical-associated illnesses, such as multiple chemical sensitivity syndrome, may represent mild chronic cases of porphyria or other acquired abnormalities in heme synthesis. This review concludes that, although it is reasonable to consider such hypotheses, there is currently no convincing evidence that these illnesses are mediated by a disturbance of heme synthesis; it is premature or unfounded to base clinical management on such explanations unless laboratory data are diagnostic for porphyria. This review discusses the limitations of laboratory measures of heme synthesis, and diagnostic guidelines are provided to assist in evaluating the symptomatic individual suspected of having a porphyria.

Mortality in patients with acute intermittent porphyria requiring hospitalization: a United States case series.

Acute intermittent porphyria (AIP) is a genetic disorder in which patients may have life threatening attacks of neurologic dysfunction. This study examined the prognosis during the past 50 years of patients in the United States who required hospitalization for porphyric attacks. The cumulative survival was determined for 136 patients with AIP who were hospitalized for porphyric attacks between 1940 and 1988. Diagnosis was established on the basis of clinical symptoms, in combination with increased urinary excretion of porphobilinogen. The patient group had an average age of 32 years (range 9 to 75) at diagnosis and consisted of 43 males and 93 females. At follow-up, 19 males (44%) and 31 females (33%) were decreased. The standardized mortality ratio for the 136 patients, compared to an age-matched hypothetical population experiencing USA 1970 Census Death Rates was 3.2, with a 95% confidence interval of 2.4-4.0. Most deaths occurred during the initial porphyric attack (20% of deaths) or a subsequent attack (38% of deaths). Suicide was also common (five deaths). Comparison was made between 50 patients who were diagnosed before 1971, the year in which hematin therapy became available, and 86 patients who were diagnosed afterward. There was improved survival in the latter group, particularly after 10 years from the time of diagnosis, but this did not reach statistical significance. In conclusion, the proportionate increase in mortality due to symptomatic AIP was three-fold compared to the general population during the past 50 years. The major cause of the increased mortality was the porphyric attack itself.

Follow-up after liver transplantation for protoporphyric liver disease.

Protoporphyria is a genetic disorder in which patients may develop severe protoporphyrin-induced liver damage and require transplantation. Because unique problems occur in the perioperative period and because excess production of protoporphyrin by the bone marrow continues after liver transplantation, the efficacy of this procedure for protoporphyric liver disease is uncertain. We present follow-up of nine patients who underwent liver transplantation. Two patients died within 2 months of transplantation, one from complications of abdominal bleeding and the other from sepsis after bowel perforations. The remaining seven patients had follow-up at 14 months to 8 years after transplantation (mean, 3.8 years). Two of the seven had suffered skin burns from exposure to operating room lights, which healed without scarring. Three had axonal neuropathies in the postoperative period requiring prolonged mechanical ventilation, and motor defects persisted in two. Five patients had normal liver chemistries at follow-up (mean, 3.5 years), with liver biopsy results normal or showing mild portal triad abnormalities, but erythrocyte protoporphyrin levels remained significantly elevated (1,765 +/- 365 mcg/dL; normal, < 65). The other two patients, both of whom had rejection, cytomegalovirus infection, and biliary tract obstruction requiring endoscopic therapy, had a recurrence of protoporphyric liver disease as indicated by liver biopsy features. One died 5 years after transplantation from complications of the liver disease. The other was stable 3.3 years after transplantation and was being monitored for possible retransplantation. Thus, liver transplantation can be performed successfully in patients with protoporphyric liver disease, with intermediate survival rates comparable to the general transplant population. However, disease may recur in the graft, particularly if there are complications that cause cholestasis.

Review of support systems used in the management of fulminant hepatic failure.

Fulminant hepatic failure has an exceedingly high mortality. Liver transplantation is the treatment option of choice. Unfortunately, one-third of patients with fulminant hepatic failure die awaiting a donor liver. For over 35 years attempts to remove or dilute putative toxins in the blood have been unsuccessful in improving survival rates. The use of biocompatible interfaces with blood or plasma and current hepatocyte culture techniques have led to the development of new support systems. This generation of bioartificial livers will hopefully provide the necessary hepatic functions and prevent many of the complications associated with fulminant hepatic failure. This paper will review the support systems tried and currently under investigation, with an emphasis on bioartificial livers.

Gel-entrapment bioartificial liver therapy in galactosamine hepatitis.

A need exists for an effective, safe bioartificial liver to support patients in fulminant hepatic failure (FHF). The purpose of this study was to determine the treatment efficacy of the novel gel-entrapment porcine hepatocyte bioartificial liver (BAL) in a fatal model of canine hepatic failure. FHF was produced in 27- to 30-kg halothane-anesthetized dogs by bolus infusion of the hepatotoxin D-galactosamine (D-Gal). Three groups were studied during the 48-hr experiment: Group D-Gal (n = 5) received galactosamine, 1.0 g/kg, iv at Time O, Group HepBAL (n = 5) received D-Gal followed by continuous hemoperfusion with the BAL device loaded with approximately 6 billion viable pig hepatocytes starting at Time 24 hr, and three dogs served as healthy controls (Group Control) and received no galactosamine. The primary endpoints were survival and coma development. Group D-Gal demonstrated 100% mortality from liver failure by 42 hr, characterized by a progressive rise in liver enzymes, total bilirubin, ammonia, and lactate and associated with coagulopathy, hypoglycemia, coma, and brain death. BAL therapy significantly delayed the onset of coma and improved survival (median 47 hr vs D-Gal median 36 hr). A significant delay in the rise of lactate and ammonia was also noted. BAL therapy prolonged survival and improved both laboratory and clinical markers of fatal liver failure. These data indicate that this BAL may have clinical utility in supporting human liver failure.

An anesthetized model of lethal canine galactosamine fulminant hepatic failure.

A reproducible large animal model of fulminant hepatic failure was developed in the anesthetized dog by the administration of the amino sugar D-galactosamine. Galactosamine in 5% dextrose in water (D5W), was given as an intravenous bolus to 10 young male dogs weighing 27 to 30 kg. Three dogs that received an equal volume of D5W alone served as controls. Galactosamine at 0.5 g/kg (n = 5) produced significant biochemical evidence of liver injury with 100% survival at 48 hours. Galactosamine 1.0 g/kg (n = 5) yielded in 100% 48-hour mortality resulting from fulminant liver failure characterized by a progressive increase in liver enzymes, total bilirubin, ammonia, and lactate and associated coagulopathy, hypoglycemia, coma, and increased intracranial pressure. Necropsy showed liver pallor, ascites, and brain swelling. Liver histology showed significant hepatocellular necrosis. This clinically relevant large animal model will enable the quantitative evaluation of new technologies, such as the bioartificial liver, for the support of hepatic failure in humans.

Hepatic levels of cyclosporine and metabolites in patients after liver transplantation.

Despite the critical role of the liver in the metabolism of cyclosporine, only a few studies have measured hepatic levels (CSAH) in patients receiving the drug, and none has directly assayed hepatic levels of the metabolites. In this study we measured CSAH and its principal metabolites (mono-OH and di-OH CSA) by HPLC/mass spectroscopy in 19 liver biopsy specimens collected from 14 patients who had undergone liver transplantation, in order to determine how they correlated with blood levels (CSAB). The hepatic concentrations were also compared with biochemical and histological parameters of cholestasis. A positive correlation was observed between CSAH and CSAB (r = 0.47), irrespective of the length of time the patients had received the drug (7 to 1662 days) as defined by the relationship: CSAH(ng/g wet weight) = 6.7 x CSAB(ng/ml)+338. Hepatic levels of metabolites exceeded those of the parent compound in 11 biopsy specimens. No correlation was found for CSAH and the metabolites and serum bilirubin or the degree of cholestasis in the liver biopsy specimens. These findings indicate that: (1) CSA is concentrated in liver tissue several-fold over blood; (2) The hepatic concentration can be estimated from the blood concentration even in the presence of cholestasis; (3) Significant levels of CSA metabolites are found in liver tissue, frequently exceeding the concentration of the parent compound.

Hemolytic anemia in protoporphyria: possible precipitating role of liver failure and photic stress.

Hemolytic anemia is not a common clinical feature in protoporphyria. In this report, we describe two patients in whom we have encountered severe hemolytic anemia. Both individuals had advanced hepatic disease as a complication of their porphyria and were undergoing orthoptic liver transplantation. The onset of hemolysis appeared to be related to the development of liver disease, and in both cases, the operative procedure acutely exacerbated the red cell destructive process. We suggest that liver involvement in protoporphyria may unmask hemolytic anemia, and that red cells in these individuals are hypersensitive to photooxidative stress, such as might occur during a prolonged operative procedure.

The functional size of ferrochelatase determined in situ by radiation inactivation.

Ferrochelatase (EC 4.99.1.1) catalyzes the final step of heme biosynthesis, the insertion of iron(II) into protoporphyrin. It is an integral protein of the inner mitochondrial membrane. The functional size of bovine hepatic ferrochelatase has been studied in situ using radiation inactivation analysis. The functional unit required for enzymic activity in intact mitochondria was found to have a mass of 82 +/- 13 kDa. In contrast, the structural unit (evaluated in immunoblots following sodium dodecyl sulfate-polyacrylamide gel electrophoresis) has a mass of 40 +/- 10 kDa. Similar results were obtained when irradiation was performed on sodium cholate-solubilized mitochondria. The presence or absence of dithiothreitol during irradiation had no effect on target sizes obtained from either intact or solubilized mitochondria. Pairwise comparison of the functional and structural target sizes from each set of irradiated samples yielded a ratio of 2.0 +/- 0.4. Previous studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography have shown that a Mr 40,000 peptide is associated with ferrochelatase activity. This study shows that the functional size of bovine ferrochelatase is approximately 80 kDa; the data are most consistent with a model for active ferrochelatase composed of two structural subunits of about 40 kDa each.

Early liver transplantation is indicated for tyrosinemia type I.

Liver transplantation is now accepted as the treatment of choice for tyrosinemia type I (hereditary tyrosinemia). In an effort to determine whether any factors in these patients would aid in predicting optimal timing of the transplant procedure, we evaluated several clinical, biochemical, and radiographic parameters in five successive patients undergoing liver transplant for tyrosinemia type I at the University of Minnesota. All five patients evidenced prolonged periods of clinical and metabolic stability with dietary therapy and four of five remained stable at the time of evaluation for transplantation. Nevertheless, all five suffered significant and unexpected complications of tyrosinemia prior to the time of liver transplant. Four developed renal stones, two were in liver failure, and one developed a neurologic crisis that left him completely paralyzed. Hepatocellular carcinoma was found in one of the five at transplant. We could identify no clinical, biochemical, or radiographic study that was predictive of the likelihood of significant complications of the disorder. Survival from the transplant procedure itself was 100%. The inability to predict or prevent significant complications of tyrosinemia and the favorable outcome from transplantation lead us to recommend liver transplant for all patients with tyrosinemia type I by 12 months of age.

Bile porphyrin analysis in the evaluation of variegate porphyria.

BACKGROUND: Variegate porphyria is a genetic disorder of porphyrin metabolism in which patients may have both neurologic dysfunction and photocutaneous lesions. Biochemical confirmation of the diagnosis can be difficult, particularly in patients without neurologic dysfunction at the time of testing. The demonstration of increased fecal excretion of porphyrin is frequently used for this purpose, but levels may be normal. Since elevated fecal porphyrin levels in variegate porphyria are presumably a consequence of increased biliary excretion, we evaluated whether analysis of porphyrins in bile distinguishes better between patients with variegate porphyria and controls. METHODS: Bile samples were collected by duodenal aspiration from 10 patients with proved variegate porphyria who had no neurologic symptoms when they were studied and 17 control subjects. Bile and fecal porphyrin levels were measured fluorometrically. RESULTS: The mean total porphyrin concentration in bile in the patients with variegate porphyria was significantly higher than that in the controls (67.8 vs. 0.71 mumol per liter; P less than 0.00002). There was more than a ninefold difference between the highest level in any control subject and the lowest level in any patient with variegate porphyria. The mean fecal porphyrin level in the patients with variegate porphyria also differed significantly from that in the controls (0.79 vs. 0.14 mumol per gram of dry weight; P less than 0.007), but four patients had levels within the control range. CONCLUSIONS: The concentration of porphyrin in bile is higher in patients with variegate porphyria than in controls, and the difference is greater than that for fecal porphyrin. Bile porphyrin measurements may be helpful in the evaluation of asymptomatic patients suspected of having variegate porphyria.