The Natural History of Severe Acute Liver Injury.

OBJECTIVES: Acute liver failure (ALF) is classically defined by coagulopathy and hepatic encephalopathy (HE); however, acute liver injury (ALI), i.e., severe acute hepatocyte necrosis without HE, has not been carefully defined nor studied. Our aim is to describe the clinical course of specifically defined ALI, including the risk and clinical predictors of poor outcomes, namely progression to ALF, the need for liver transplantation (LT) and death. METHODS: 386 subjects prospectively enrolled in the Acute Liver Failure Study Group registry between 1 September 2008 through 25 October 2013, met criteria for ALI: International Normalized Ratio (INR)≥2.0 and alanine aminotransferase (ALT)≥10 × elevated (irrespective of bilirubin level) for acetaminophen (N-acetyl-p-aminophenol, APAP) ALI, or INR≥2.0, ALT≥10x elevated, and bilirubin≥3.0 mg/dl for non-APAP ALI, both groups without any discernible HE. Subjects who progressed to poor outcomes (ALF, death, LT) were compared, by univariate analysis, with those who recovered. A model to predict poor outcome was developed using the random forest (RF) procedure. RESULTS: Progression to a poor outcome occurred in 90/386 (23%), primarily in non-APAP (71/179, 40%) vs. only 14/194 (7.2%) in APAP patients comprising 52% of all cases (13 cases did not have an etiology assigned; 5 of whom had a poor outcome). Of 82 variables entered into the RF procedure: etiology, bilirubin, INR, APAP level and duration of jaundice were the most predictive of progression to ALF, LT, or death. CONCLUSIONS: A majority of ALI cases are due to APAP, 93% of whom will improve rapidly and fully recover, while non-APAP patients have a far greater risk of poor outcome and should be targeted for early referral to a liver transplant center.

Testing and management of thrombocytopenia and coagulopathy in the pre- and postliver transplant patient.

Caring for patients with advanced liver disease and acute liver failure requires a thorough understanding of the profound coagulation changes that occur in these conditions. Due to the unique nature of the pathophysiologic changes from hepatic dysfunction, effective interpretation and management of coagulopathy and thrombocytopenia with blood products are important clinical skills, and are likewise required in the post transplant period. Acute superimposed complications such as sepsis and renal dysfunction present additional challenges. The aim of this review was to describe the coagulation changes in liver disease, appropriate coagulation testing, and management strategies in the pre- and post-transplant period.

Hepatocellular copper toxicity and its attenuation by zinc.

We studied the mechanisms by which excess copper exerts, and zinc mitigates, toxic effects on HepG2 cells. Survival and cell growth were reduced in media containing greater than 500 microM copper chloride for 48 h; LD50 was 750 microM. At 1,000 microM copper for 1 h, there was a general reduction of protein synthesis, and no recognizable changes in cellular ultrastructure. Incubation of cells with 200 microM zinc acetate before exposure to copper, raised the LD50 for confluent cells to 1,250 microM copper chloride, improved protein synthesis, and increased synthesis of a 10-kD protein, apparently metallothionein. The mitigation, by zinc, of copper's toxicity may in part be mediated through induction of this protein in the hepatocyte.

ATP7B (WND) protein.

Wilson's disease is a genetic disorder of copper metabolism characterized by the excessive accumulation of this metal in the liver. The gene for Wilson's disease, designated ATP7B, encodes a copper transporting P-type ATPase expressed predominantly in the liver. Over 60 disease specific mutations of ATP7B have now been reported in patients with Wilson's disease. The gene for ATP7B is approximately 80 kb and contains 21 exons that encode an approximately 7.5 kb transcript. Recent studies that focus on the structure and expression of the ATP7B protein support its role as a copper transporter involved in the intracellular trafficking of copper in hepatocytes. The introduction of functional ATP7B protein by recombinant adenovirus mediated gene delivery will be a potential approach for correcting Wilson's disease.

Transplantation for inherited metabolic disorders of the liver.

Inherited metabolic diseases that affect the liver are a frequent cause of liver failure in children, but other disorders more commonly cause liver failure in adulthood where they may present with chronic liver disease and, less frequently, with acute liver failure. The identification of the underlying genetic defect for many of these inherited disorders has improved our understanding of their pathophysiology and impacted on the indications for and timing of liver transplant, yielding better outcomes. Screening for disease and genetic counseling of family members may help prevent adverse outcomes in relatives of affected individuals. Timely liver transplantation offers correction of the inherited metabolic defect and restores liver function when medical therapy is not possible or when complications of liver disease arise. Some inherited metabolic diseases have their defect based in the liver and lead not to liver disease, but to other end organ damage. Earlier detection of these disorders may prevent pathological injury by treatment of the underlying disease or by pre-emptive liver transplant. In some instances where damage of other organs has already occurred, dual organ transplant with liver and another organ may be needed. Improvement in the technical aspects of performing liver transplantation and posttransplant care has led to better outcomes for those with inherited metabolic disorders of the liver.

Hepatitis B "360".

There is an extremely high burden of liver disease owing to viral hepatitis B (HBV); about 2 billion people are infected and 350 million are chronic carriers of HBV worldwide. More effective medical therapy and liver transplantation are available for those with advancing disease. The interaction between the host immune system and the virus influences the rate of development of advanced liver disease or hepatocellular carcinoma (HCC); treatment that successfully reduces viral replication of HBV also reduces the incidence of development of advanced liver disease and HCC. Liver transplantation for HBV has yielded favorable outcomes since the institution of hepatitis B immune globulin and antiviral therapy. The ability to stabilize and rescue some patients with advanced liver disease owing to HBV has resulted in a changing demographic for patients with HBV undergoing liver transplantation. The main indications for transplant owing to HBV are now acute liver failure (both acute and acute reactivation on the background of chronic HBV) and HCC. Use of donor organs exposed to HBV with positive HBV core antibody is now routinely accepted for its good outcomes, and in selected cases with active HBV, HBV surface antigen-positive donors may be utilized to further expand the donor pool. Another indication for antiviral therapy for HBV is to reduce the risk of reactivation of latent virus in some patients previously exposed to HBV who are being treated with chemotherapy. Health care providers with HBV infection have an obligation to appropriately treat or monitor their disease closely to reduce the risk of transmission of disease from provider to patient. In the future, universal vaccination will reduce the overall burden of HBV liver disease, but until then appropriate utilization of available medical and surgical therapeutic options gives excellent clinical outcomes.

Acute liver failure and liver assist devices.

Survival of patients presenting with acute liver failure (ALF) has improved over the past decades due to earlier disease recognition, advances in supportive measures, intensive care, and liver transplantation. Liver assist devices may have a role in future care of patients with ALF, bridging them to recovery or to transplantation. A multidisciplinary team approach to the care of patients with ALF is critical for achieving good patient outcomes.

Treatment of Wilson's disease: what are the relative roles of penicillamine, trientine, and zinc supplementation?

New options are available for the medical treatment of patients with Wilson's disease. Penicillamine is no longer the treatment of choice, as there is a growing experience with safer and more effective alternatives. Trientine may be the best choice for initial therapy in symptomatic patients requiring chelation therapy, and it may be even more effective when used in combination with zinc, which is recommended for maintenance therapy. Further studies are needed to determine the best therapy for pregnant patients with Wilson's disease, and whether combination therapy using trientine and zinc will be the next treatment of choice for all symptomatic patients with liver or neurologic disease.

Wilson disease: genetic basis of copper toxicity and natural history.

The discovery that the gene for Wilson disease encodes a copper-transporting ATPase has greatly improved our understanding of the pathophysiology of this disorder and of copper metabolism in humans. The abundance of disease-specific mutations and their location at multiple sites across the genome have limited molecular genetic diagnosis to kindred of known patients, and confirm the necessity for de novo screening by well-proven clinical and biochemical means. It is uncertain whether the variety of specific mutations will account for the wide range of presenting clinical signs and symptoms of Wilson disease, and environmental and extragenic factors are likely to be important contributing factors. Chelation therapy with penicillamine and trientine remain effective treatment for most symptomatic hepatic and neurologic Wilson disease. Zinc salts may be used for some asymptomatic patients, and OLT for fulminant hepatitis and patients for whom pharmacotherapy is ineffective. The chelating agent tetrathiomolybdate is under investigation for the treatment of neurologic Wilson disease. Gene therapy is the new horizon for treatment of Wilson disease. However, the ability to treat this disorder effectively by this means awaits further characterization of the gene product and more efficient methods for gene delivery to all hepatocytes in the liver.

Inherited metabolic disease.

Our understanding of the pathophysiology and of new treatments for inherited metabolic diseases that affect the liver continues to grow through the study of gene mutations and their functional effect on the proteins they encode. For genetic hemochromatosis and Wilson's disease, studies focused on the function of their respective gene products provide new insights into metal metabolism. For Crigler-Najjar syndrome, an inherited disorder that results in failure of proper bilirubin glucuronidation, the once futuristic idea of treatment by transplantation of donor hepatocytes has now proven successful in a human recipient. With continued study and experimentation, our diagnostic and therapeutic capabilities will continue to expand for these and other inherited metabolic disorders. Although this increase in new information has sparked numerous reviews of these subjects, the following are highlights from the past year that include information relative to disease diagnosis and treatment, as well as new insights into pathogenesis.

Inherited metabolic disease.

This review focuses on two genetic disorders of metal metabolism, genetic hemochromatosis and Wilson disease, and on the most common lysosomal storage disorder, Gaucher disease, for which recombinant enzyme replacement therapy is available. The discovery of the genes for these disorders has led to an explosion of new information about the function of these gene products and the identification of other proteins involved in their metabolism. These discoveries have altered our current diagnostic and therapeutic approaches to these disorders and have furthered our understanding of disease pathophysiology. New modalities being developed for future use include cell transplant and genetic replacement therapies.

New knowledge of genetic pathogenesis of hemochromatosis and Wilson's disease.

Discovery of the gene for WD has greatly enhanced our understanding of this disorder at the cellular level and has set the stage for future testing of new modes of therapy. Improvements in analytic methods for detecting mutations in genomic DNA will someday enable a rapid and cost-effective method of screening for this disorder. Until then, the time-tested clinical and biochemical evaluation, including measurement of ceruloplasmin oxidase activity, slit-lamp examination for Kayser-Fleischer rings, and measurement of hepatic copper content, will continue to remain the standard for establishing the diagnosis of WD.

Metabolic liver disease.

The discovery of novel metabolic pathways and the genetic basis for diseases of the liver continues to yield new insights into the pathogenesis of inherited metabolic diseases of the liver, whereas the application of new technologies to their treatment continues to advance therapeutic options. This review of selected articles covers a wide range of subjects, from the identification of novel proteins and transport pathways to disease diagnosis and treatment of acute liver failure. Four selected topics, Wilson disease, hemochromatosis and iron overload disorders, alpha-1 antitrypsin disease, and exciting new therapeutic options for lysosomal storage diseases are the focus of this review.

Liver transplantation for Wilson's disease: indications and outcome.

The objective of this study was to determine the indications for and results of liver transplantation in patients with Wilson's disease on the basis of results of a survey with retrospective review of data obtained on 55 transplants performed at centers in the United States and Europe. The study group comprised 32 females and 23 males, aged 8.5 to 51 yr, with features diagnostic of Wilson's disease. Indication for orthotopic liver transplantation included hepatic insufficiency (n = 32), wilsonian fulminant hepatitis (n = 21), intractable neurological Wilson's disease (n = 1) and gastrointestinal hemorrhage (n = 1). Forty-three patients have survived, at this writing, from 3 mo to 20 yr. Mean and median survival after orthotopic liver transplantation were 2.7 and 2.5 yr, respectively. Survival at 1 yr was 79%. Nonfatal complications occurred in five patients. Of the seven patients given transplants for hepatic insufficiency who manifested neurological and/or psychiatric manifestations at the time of orthotopic liver transplantation, four showed improvement of these symptoms. One patient given a transplant for intractable neurological disease improved but died of a vascular complication. Our data demonstrate that liver transplantation is life-saving but not without risk for patients with wilsonian fulminant hepatitis or chronic severe hepatic insufficiency unresponsive to medical therapy. Furthermore, neurological or psychiatric symptoms due to Wilson's disease may improve after liver transplantation; however, the role of this procedure in the management of patients with neurological Wilson's disease in the absence of hepatic insufficiency is still uncertain.

Prognosis of Wilsonian chronic active hepatitis.

Twenty of 320 patients with Wilson's disease initially presented with chemical and laboratory features of chronic active hepatitis, confirmed histologically in 17. When first seen, cirrhosis was present in all 20 and was complicated by ascites and/or jaundice in 11. Within 1 week to 8 years of the onset of over liver disease the diagnosis of Wilson's disease was established, and treatment with D-penicillamine was promptly initiated in 19 patients. One man refused treatment and died 4 months later. Treated patients received D-penicillamine or trientine for a total of 264 patient-years (median, 14 patient-years). Abnormal water retention, for which salt restriction and diuretics were added to penicillamine or trientine, disappeared in all but 1 of the patients so affected. Symptomatic improvement and virtually normal levels of serum albumin, bilirubin, aspartate aminotransferase, and alanine aminotransferase followed within 1 year in the majority of subjects. One woman died after 9 months of treatment. Two patients, who became noncompliant with the therapeutic regimen after 9 and 17 years of successful pharmacological treatment, required liver transplants. These results indicate that the prognosis of specifically treated Wilsonian chronic active hepatitis is very good in spite of the presence of cirrhosis.

Caeruloplasmin biosynthesis by the human uterus.

The blue copper protein caeruloplasmin is synthesized mainly by hepatocytes. An alternative transcript for caeruloplasmin produced in certain extrahepatic tissues, CP-1, contains an additional 12 nucleotides encoding 4 amino acids not present in the hepatic transcript, CP-2 [Yang, Friedrichs, Cupples, Bonifacio, S Sanford, Horton & Bowman (1990) J. Biol. Chem. 265, 10780-10785]. We have demonstrated transcription of caeruloplasmin mRNA by a well-differentiated human uterine epithelial adenocarcinoma cell line, Ishikawa, and by human uterine endometrium and purified endometrial glands. Identical CP-2 nucleotide sequences were obtained for partial caeruloplasmin transcripts from human liver and Ishikawa cells, indicating that CP-2 transcripts are produced by uterine epithelial lining cells. The synthesis of caeruloplasmin protein was demonstrated for Ishikawa cells and another uterine adenocarcinoma cell line, ECC1. Peptide-mapping analysis indicated that caeruloplasmin secreted by Ishikawa cells was structurally identical with the protein synthesized by the human hepatoblastoma cell line HepG2. The secretion of a 135,000-M(r) caeruloplasmin by Ishikawa and ECC1 cells, comparable with that of the human hepatoblastoma cell line, HepG2, indicated similar processing of uterine and hepatic caeruloplasmin. Incorporation of 67Cu into caeruloplasmin was demonstrated for Ishikawa and ECC1 cells, suggesting that the human uterus produces a bioactive form of caeruloplasmin and possesses the necessary metal transporters and intracellular machinery for copper incorporation into this protein.

An array of mitochondrial alterations in the hepatocytes of Long-Evans Cinnamon rats.

In an attempt to identify the cellular targets of copper toxicity, we studied the ultrastructure of hepatocytes in the livers of 23 Long-Evans Cinnamon (LEC) rats ranging in age from 10 to 89 weeks. The hepatic copper concentration ranged from 325 to 2,126 (mean, 930) micrograms/g dry weight. Thirteen rats displayed varying degrees of jaundice at the time of killing. Numerous nuclei were indented by cytoplasmic invaginations. Conspicuous abnormalities were displayed by mitochondria. These included marked pleomorphism; increased or diminished matrical density; rearrangement, elongation, dilatation, stacking, or disappearance of cristae; absence of matrical granules; presence of matrical inclusions of flocculent electron-dense deposits; and degenerative changes. The severity of the ultrastructural changes did not correlate with the hepatic copper concentration but did correlate with the degree of the icterus displayed by the rats. Certain mitochondrial changes resembled those encountered in the hepatocytes of patients with Wilson's disease, confirming that these organelles are important targets of copper toxicity.

Pleiotropic effect of LEC mutation: a rodent model of Wilson's disease.

Metabolic studies with 67Cu were undertaken to identify the site of the cellular defect in copper metabolism in the Long-Evans Cinnamon (LEC) rat. The apparent rate of copper uptake by LEC primary hepatocytes was increased [maximal velocity (Vmax) = 259 pmol.min-1.mg protein-1] compared with controls (Vmax = 161 pmol.min-1.mg protein-1); however, Michaelis-Menten constant (Km) values were comparable (11.8 and 12.7 microM, LEC and control, respectively). Efflux of copper from LEC and control hepatocytes was similar from 0 to 15 min, but was reduced from 15 to 60 min in the former. Although hepatic copper contents were markedly elevated in LEC rats compared with controls (658 +/- 199 vs. 21.5 +/- 6.6 micrograms/g dry wt), biliary copper concentration was reduced in LEC rats compared with controls (0.187 vs. 1.39 +/- 0.66 microgram/ml). Subcellular fractionation of LEC liver homogenates revealed approximately 75% of copper to be present in cytosol, with gradients of copper concentration from cytosol to either lysosome or microsomal subcellular fractions. LEC rat bile and hepatic microsome and lysosome fractions contained smaller fractions of 67Cu administered intravenously as cupric acetate compared with control rats. However, recovery of 67Cu in bile and in lysosomal subcellular fractions were similar for LEC and controls following administration of 67Cu-labeled asialoceruloplasmin, which is targeted to lysosomes. This discordance suggests a possible defect in the entry of copper into lysosomes but normal delivery of lysosomal copper to bile. Based on these findings, we conclude that the mutation in LEC rats alters copper transport at more than one cellular site.

Biliary copper excretion capacity in intact animals: correlation between ATP7B function, hepatic mass, and biliary copper excretion.

Copper toxicosis can occur in the absence of biliary copper excretion. To demonstrate whether biliary copper excretion capacity is correlated with hepatic mass and ATP7B function, we undertook studies in intact animals. Copper-histidine was injected intrasplenically after baseline bile collection, followed by measurement of copper excretion in Long-Evans Cinnamon (LEC) rats lacking atp7b function and in normal, syngeneic Long-Evans Agouti (LEA) rats. The basal biliary copper excretion was very low in LEC rats compared with LEA rats, 8+/-4 and 37+/-18 ng copper/min, respectively; p<0.05. After addition of copper, copper excretion increased significantly (by two- to five-fold) in LEA rats during the 30 minute study period, whereas LEC rats showed only a slight and transient increase in copper excretion. After one-third and two-thirds partial hepatectomy immediately before copper loading, copper excretion decreased progressively. The studies indicate that biliary copper excretion depends on hepatocyte mass and ATP7B gene function. Analysis of copper excretion with our non-radioactive method will facilitate testing of novel therapies and pathophysiological mechanisms in copper toxicity.

Spontaneous cholangiofibrosis in Long-Evans Cinnamon rats: a rodent model for Wilson's disease.

The Long-Evans Cinnamon (LEC) rat is a rodent model of Wilson's disease characterized by ceruloplasmin deficiency, hepatic copper accumulation, and hepatocellular injury. So far, the LEC rat appears to be the only strain in which cholangiofibrosis develops spontaneously. The aim of the study reported here was to characterize the time course of development and investigate the structural and ultrastructural features of cholangiofibrosis and their possible relationship to hepatic copper and iron content. The livers of 54 rats (22 males), ages 5 to 113 weeks, were examined by light microscopy and graded for statistical analysis, with respect to extent of replacement of liver tissue by cholangiofibrosis. The study was complemented by electron microscopy, and by measurements of copper and iron contents by atomic absorption spectroscopy. Cholangiofibrosis was present in LEC rats by 20 weeks of age. The hyperplastic biliary epithelial cells varied markedly in size and shape, ranging from flat to cuboidal or elongated. Epithelial cells did not exhibit characteristics of intestinal cells. Some basement membranes had splits, duplications, or multiplications. Cytoplasmic organelles within hyperplastic biliary cells appeared unremarkable in contrast to the characteristic mitochondrial abnormalities present in neighboring hepatocytes. There was a positive correlation between histologic grades of cholangiofibrosis and ages of the animals (r = 0.68, P < 0.001), but no significant correlation between histologic grade and hepatic copper or iron content. We conclude that cholangiofibrosis is the predominant pathologic response to chronic liver injury induced by excess copper in LEC rats. The pathogenic role of copper in the development of cholangiofibrosis requires clearer definition.