Disorders of the neonatal liver and Bile Duct - Seminars 2003.pdf

EditorialDisorders of the Neonatal Liver and Bile Ducts Liver and biliary tract disorders in the neonate are relatively rare and often complex. The consequences of delayed diagnosis and inappropriate management may be fatal. As with so much of clinical medicine, awareness of the spectrum of diseases and recognition of the key clinical features of the various disorders is essential to optimizing outcome. It is increasingly hard for today's clinicians to keep up to date when the pace of change is so great and the relevant literature so vast. This issue of Seminars in Neonatology distills the clinical and scientific experience of an international group of experts in an attempt to provide the working neonatologist (and other healthcare workers involved in the care of the newborn) with a ‘state-of-the-art’ review of each topic and a stimulating insight into recent advances. Sue Beath from Birmingham Children's Hospital, UK outlines current concepts of hepatic function and physiology in the newborn, whilst the fresh challenges posed by prenatal diagnosis and molecular genetics are discussed by Mark Davenport and Dino Hadzic from King's College Hospital, London, UK. The myriad of disorders which cause conjugated hyperbilirubinaemia are neatly and suc- cinctly dissected by Eve Roberts from The Hospital for Sick Children in Toronto, Canada. The latter nicely leads on to a more detailed discussion of biliary atresia from a combined Japanese and UK perspective. Stuart Kaufman from The Mount Sinai Hospital in New York, USA brings us up to date with the widespread and potentially hazardous problem of parenteral-nutrition-associated liver disease. Finally, there are important contributions from Paddy McClean and Suzanne Davison in Leeds, UK on the newborn infant with liver failure, and from Dietrich von Schweinitz in Munich on neonatal liver tumours; both are rare but extremely taxing clinical conditions. With the constraints of space, each section is not intended to be encyclopaedic but rather, each expert has provided a readable account combining a useful blend of practical knowledge and analysis of current research. I would like to thank all the contributors. I am also indebted to Seań Duggan, Managing Editor, and his assistant Ann Smiley for their assistance and support. Mark D. Stringer* St James's University Hospital Children's Liver and GI Unit Level 8 Geldhow Wing Leeds LS9 7TF UK *Tel.: +44-1132066689; fax: +44-1132066691 E-mail address: [email protected] (M.D. Stringer). Seminars in NEONATOLOGY www.elsevierhealth.com/journals/siny Seminars in Neonatology (2003) 8, 335 1084-2756/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1084-2756(03)00091-5 Hepatic function and physiology in the newborn S.V. Beath * The Liver Unit, Birmingham Children's Hospital, Steelhouse Lane, Birmingham B4 6NH, UK Summary The liver develops from progenitor cells into a well-differentiated organ in which bile secretion can be observed by 12 weeks' gestation. Full maturity takes up to two years after birth to be achieved, and involves the normal expression of signalling pathways such as that responsible for the JAG1 genes (aberrations occur in Alagille's syndrome), amino acid transport and insulin growth factors. At birth, hepatocytes are already specialized and have two surfaces: the sinusoidal side receives and absorbs a mixture of oxygenated blood and nutrients from the portal vein; the other surface delivers bile and other products of conjugation and metabolism (including drugs) to the canalicular network which joins up to the bile ductules. There is a rapid induction of functions such as transamination, glutamyl transferase, synthesis of coagulation factors, bile production and transport as soon as the umbilical supply is interrupted. Anatomical specialization can be observed across the hepatic acinus which has three distinct zones. Zone 1 borders the portal tracts (also known as periportal hepatocytes) and is noted for hepatocyte regeneration, bile duct proliferation and gluconeogenesis. Zone 3 borders the central vein and is associated with detoxification (e.g. paracetamol), aerobic metabolism, glycolysis and hydrolysis and zone 2 is an area of mixed function between the two zones. Preterm infants are at special risk of hepatic decompensation because their immaturity results in a delay in achieving normal detoxifying and synthetic function. Hypoxia and sepsis are also frequent and serious causes of liver dysfunction in neonates. Stem cell research has produced many answers to the questions about liver development and regeneration, and genetic studies including studies of susceptibility genes may yield further insights. The possibility that fatty liver (increasingly recognized as non-alcoholic steatohepatitis or NASH) may have roots in the neonatal period is a concept which may have important long-term implications. © 2003 Elsevier Ltd. All rights reserved. KEYWORDS Neonatal; Hepatitis; Stem cells; Genetic disorders; Sinusoidal function; Zonal differentiation; Bile acid transport/physiology; Prematurity; Hypoxia; Sepsis; Conjugated; Unconjugated hyperbilirubinaemia Introduction The liver contains a diverse group of cell lines that remain in a state of some plasticity until at least 12 months after birth. The liver differentiates from embryonic liver progenitor cells derived from stem cells, into a mature organ containing hepatocytes, cholangiocytes and immune cells, all existing in a stromal network through which approximately one- quarter of the circulating blood is pumped. 1,2 This process takes place via several important mechanisms which are only just beginning to be understood. These include apoptosis, morphogenesis, proliferation and polarization. 3–5 Aberrations of the normal sequence of embryonic and fetal gene expression can lead to disease, e.g. the notch signalling pathway appears to be important in Alagille's syndrome characterized by cardiac, facial and hepatic abnormalities 6 (Table 1). The liver may play an important role in the maintenance of a healthy feto-placental unit as intra-uterine growth restriction is associated with reduced expression of hepatocyte growth factors. 7 External factors such as the hormonal milieu and hypoxia influence the expression of genes responsible for the transport of amino acids across membranes and the production * Tel.: +44-121-333-8259; fax: +44-121-333-8251 Seminars in NEONATOLOGY www.elsevierhealth.com/journals/siny Seminars in Neonatology (2003) 8, 337–346 1084-2756/03/$ - see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1084-2756(03)00066-6 Table 1 Bile synthesis and transport defects—examples of genetic causes Disease Basis of genetic defect(s) Clinical features Alagille's JAG1, various ligands? Notch signalling pathway Variable—includes paucity of bile ducts, pulmonary artery stenosis, triangular facies Alpha-1 antitrypsin deficiency Mutation of gene coding for protease inhibitor on chromosome 14 Growth restriction in utero, severe cholestasis often with pale stools, 2% present with vitamin-K-sensitive coagulopathy Bile acid synthesis disorders Abnormal expression of the following enzymes: (a) Hydroxy-steroid dehydrogenase (a) Normal GGT, low serum bile acids, no pruritis (b) Oxosteroid reductase (b) Severe jaundice and coagulopathy (c) 25-Dihydroxycholanic cleavage enzymes (c) Normal GGT, elevated ALP and cholesterol, pruritis Cystic fibrosis Mutation in delta 508 or other genes Jaundice, hepatomegaly, meconium ileus, inspissated bile syndrome, pale stools Crigler–Najjar type 1 Various mutations in exon 1–5 of B-UGT1 gene coding for bilirubin glucuronidation Unconjugated hyperbilirubinaemia 200–400 µmol/l, needs phototherapy, phenobarbitone=no effect Crigler–Najjar type 2 Various mutations leading to reduced affinity of the enzyme for substrate Unconjugated hyperbilirubinaemia 100–400 µmol/l, needs phototherapy, phenobarbitone effective Gilbert's disease Mutation in the promoter region of the UDPGT 1 gene Unconjugated hyperbilirubinaemia 100–300 µmol/l is common and usually resolves after brief exposure to phototherapy Dubin–Johnson syndrome Autosomal recessive Conjugated bilirubin (30–400 µmol/l), transaminases normal, centri-lobule pigment granules on liver biopsy PFIC type 1 Byler's disease Mutation of the mixed drug reaction gene 1 (MDR-1) Normal GGT, normal cholesterol, variable jaundice, pruritis (diarrhoea±pancreatitis) PFIC type 2 Mutation on chromosome 2 of bile salt export pump Normal GGT, no intestinal symptoms, giant cell hepatitis common PFIC type 3 Mutation in the P-glycoprotein MDR-3 gene Elevated GGT, troublesome pruritis Rotor syndrome Autosomal recessive Conjugated hyperbilirubinaemia, but no accumulation of bile acids in the liver and normal liver function tests Zellweger's Abnormal expression of peroxisome enzymes involved in side chain modification of bile acids Accumulation of very long-chain fatty acids (C 27 ), large fontanelle, high forehead, profound hypotonia PFIC, progressive familial intrahepatic cholestasis; GGT, gamma glutamyl transferase; ALP, alkaline phosphatase; MDR, multidrug-resistant protein. 3 3 8 S . V . B e a t h of insulin growth factors 8,9 (Table 2). An imbalance in the transport of molecules such as bilirubin and amino acids can lead to cholestasis and a giant cell hepatitis. Fetal development The liver develops from the foregut which folds into the mesoderm. The bile ducts and hepatocytes are derived from stem cells in the endoderm, and Kupffer cells, blood vessels, including the specialized porous endothelium which lines the sinusoids, and fibrous tissue are all derived from mesoderm. The stem cells differentiate into progenitor cells which are then committed to either hepatocyte or cholangiocyte lineage. 10 The hepatocytes develop in long cords into the stroma, initially as plates three to five cells thick. At the time of birth, the architecture of the liver is well established with portal tracts connected to the central veins by plates of hepatocytes in layers which are two cells thick. The hepatocytes have a sinusoidal surface over which blood from the portal vein and hepatic artery flows, and a basolateral canalicular surface from which bile is secreted. The space between the canalicular surface of hepatocytes communicates with biliary cells in the portal tracts via the canals of Hering which also contain oval cells. The oval cells are progenitor cells which proliferate according to normal fetal development or in response to liver injury. 1 Oval cells can develop into hepatocytes or cholangiocytes depending on local environmental factors. 2 Hepatic secretion of bile commences early at around 12 weeks' gestation. The main serum protein of the fetus is alpha- fetoprotein which reaches a peak concentration at the end of the first trimester. Albumin synthesis begins at around 16 weeks' gestation and reaches adult levels at the time of birth. In utero, the liver acts as the main source of red cell production, and even at birth, foci of active haematopoiesis are still present in the parenchyma of the liver. This may explain why disorders of iron metabolism such as neonatal haemochromatosis (which have their origins in the fetus) have such a devastating effect including fulminant liver failure. 11 These areas of haematopoiesis usually disap- pear within six weeks of birth being superseded by the bone marrow. Persistence of extramedullary haemopoiesis is an indication of disease and may be seen in haemolytic anaemia and neonatal hepatitis. 12 Events at birth Two major physiological events at birth affect the liver: the pressure in the lungs drops dramatically Table 2 Environmental and other causes of neonatal jaundice Disease Clinical features Environmental Hypoxia 9,28,29 Sudden rise in AST and ALT up to 20 times baseline, followed by jaundice (conjugated hyperbilirubinaemia) Endocrine (a) Hypothyroidism 8 (a) Jaundice which may be predominantly unconjugated at first, associated with hepatomegaly and elevated AST and ALT (b) Hypopituitarism (b) Hypoglycaemia and jaundice, failure to thrive, poor visual fixing Preterm birth Reduced concentrations of plasma albumin, prolonged coagulation, hypoglycaemia, delay in conjugation of bilirubin, reduced efficiency in transporting bilirubin to canaliculus Interuterine infection, e.g. parvovirus Intra-uterine growth restriction, hepatosplenomegaly, ascites Postnatal infection, e.g. streptococcus 28 Sudden rise in conjugated bilirubin, low platelets, coagulopathy, rise in acute-phase proteins Parenteral nutrition (PN) 32 Early rise in ALP and GGT, jaundice especially during septic episodes, can lead to liver failure Uncertain—susceptibility genes may be interacting with environment Biliary atresia Normal birth weight, 10% have situs inversus, pale stools, high GGT and ALP, bilirubin rises steadily from birth Giant cell hepatitis Growth restriction in utero, ALT and AST may be three to five times normal, haemolysis occasionally present, congenital infection may be detected (especially parvovirus) AST, aspartate transaminase; ALT, alanine transaminase; GGT, gamma glutamyl transferase; ALP, alkaline phosphatase. Hepatic function and physiology in the newborn 339 with the first few breaths; and 50% of the cardiac output previously going to the placenta is rapidly re-distributed as blood flow through the umbilicus ceases. Within minutes, the venous return from vital organs such as the liver and the small bowel increases, and the pulmonary circulation becomes as dynamic as the systemic circulation, producing a steep rise in dissolved oxygen in arterial blood to almost 95% saturation. In addition to these events, a more gradual change in hepatic blood flow occurs. In the fetus, the ductus venosus connects the umbilical vein and the inferior vena cava, pro- viding a functional bypass of the liver for up to 50% of the oxygenated blood from the placenta. Postna- tally, the ductus venosus gradually closes within one to two weeks of birth. Rarely, the ductus fails to close and this has been linked to the subsequent development of encephalopathy and liver tumours in older children. Increase in portal blood flow and bacterial colonization The newborn infant's first feed increases portal blood flow and exposes the intestinal tract to micro-organisms for the first time. Within hours of birth, colonies of bacteria establish themselves in the intestine and a stable microflora develops in the large bowel. Although bacterial colonization may be associated with necrotizing enterocolitis in premature infants, enteric bacteria have a physiological role in that they produce vitamin K. It takes around six weeks for the mass of bacteria to produce sufficient vitamin K, and this is one reason why haemorrhagic disease of the newborn is very rare after this time unless a chronic cholestatic disease is present causing malabsorption of vitamin K (e.g. alpha-1 antitrypsin deficiency 13 ). Induction of enzyme functions (GGT and UDPGT) The rapid assumption of processing functions by the hepatocyte cell mass requires an induction of enzymes such as the cytochrome P450 group and peroxisomal enzymes which were present before birth. 14 Transferase function It is normal to see a rise in gamma glutamyl transferase (GGT) from a low baseline value of around 30 IU/l up to 120–150 IU/l for the first few months of life. A lack of increase in GGT can imply a disorder of the biliary epithelium and canalicular aspect of the hepatocyte cell surface (e.g. progressive familial intrahepatic cholestasis (PFIC) types 1 and 2). In a different type of bile transport disorder, the GGT may be abnormally high as in PFIC type 3, in which one of the genes coding for the bilirubin transporter multidrug-resistant protein 3 (MDR-3) is defective (Table 1). An abnormally high GGT is also a pointer to abnormal biliary function as seen in biliary atresia. Plasma levels of alkaline phosphatase, which is concentrated in the cytoplasm just below the bile canaliculus, are also strikingly elevated in biliary atresia (Table 2). Conjugation Conjugation develops from minimal levels to almost adult levels within two weeks of birth in most cases. Conjugation reactions are an important step in detoxifying by-products of metabolism and drugs, especially relatively insoluble lipid molecules. 15 Bilirubin, which is salvaged from the complex haem molecule when worn-out red blood cells are destroyed, is highly lipophilic and cannot easily cross cytosolic spaces inside the cell. Conjugating the bilirubin molecule with glucuronide is dependent on the function of the enzyme uridine diphosphoglucuronyl transferase (UDPGT), which improves the water solubility of bilirubin. Conjuga- tion also improves the efficiency of transport of bilirubin into the canaliculus and reduces its toxic detergent effect on the bile ducts. A rise in unconjugated bilirubin in the first two weeks of life (up to 50% of babies become visibly jaundiced) may be regarded as almost physiological as it is usually self-limiting once the conjugation capacity of the maturing hepatocytes catches up with the demands of life outside the womb. How- ever, it is important to confirm that plasma bilirubin is reducing after 14 days and also to establish whether the bilirubin is largely unconjugated. An elevated conjugated bilirubin can never be regarded as physiological although it may have a self-limiting cause such as septicaemia. A delay in achieving normal levels of UDPGT is frequently seen in preterm and septic babies who are at risk of developing kernicterus if high levels of unconjugated bilirubin occur. Other causes of an elevation in unconjugated bilirubin are seen in infants experiencing haemolysis, traumatic birth, hypoxia and sepsis (see Table 3). Autosomal-recessive inherited disorders of UDPGT, Crigler–Najjar type 1 and its milder phenotype type 2 are very rare. In contrast, Gilbert's disease affects up to one in 100 of the population. Fortu- nately, the defect in UDPGT expression in Gilbert's disease is mild and only becomes evident under 340 S.V. Beath conditions of stress, such as birth, or, in adult life, in association with prolonged fasting or a systemic illness such as influenza. Babies who are ho- mozygous for the genetic abnormality of Gilbert's disease are more likely to be jaundiced in the first few days of life than unaffected babies. 16 Induction of synthetic functions At birth, the plasma albumin concentration is usually near to adult levels (30–35 g/l), but the concentration of plasma proteins involved in coagulation is usually low. In parallel with the rapid induction of enzymes involved in conjugation, the coagulation proteins increase to adult levels within a few days of birth. Caeruloplasmin is also low at birth and gradually rises during the first three months of life. This can become grossly elevated during sepsis or other stresses because, like alpha-1 antitrypsin, it is an acute-phase protein. 12 Sinusoidal physiology and function It is important to note that a hepatocyte functions as if it had two surfaces: the sinusoidal surface and the canalicular surface (Fig. 1). The sinusoidal surface is exposed to two sources of blood: one is rich in dissolved oxygen (from the hepatic artery), and the other is rich in glucose, amino acids, free fatty acids, larger molecules including intact proteins, insulin, glucagon, free fatty acid binding protein, cholecystokinin, micro-organisms and immune cells (from the portal vein). After a meal, the portal vein delivers approximately twice the volume of blood as the hepatic artery, equivalent to 600 ml/min in an adult. On the other side of a hepatocyte is the canalicular surface. This forms a network of canaliculi bounded by tight junctions, which joins up with bile ducts in the portal tracts via the canals of Hering. Bile is actively transported by specialized transport proteins into the canaliculi and ultimately flows under positive pressure down the biliary tree. 17 Hepatocytes act as a conduit and processor for nutrients, initially from the placenta. Within days of birth, hepatocytes must have the capacity to deal with an enormous range of hormones, food antigens, complex molecules and bacteria derived from the intestinal tract. It is not surprising, therefore, that environmental stress (e.g. intra-uterine infection, respiratory distress, bacterial sepsis) and genetic factors (abnormal transporters of bilirubin) frequently result in cholestasis and disruption of hepatocytes, leading to a giant cell hepatitis (see Tables 1 and 2). Maturing physiology After the initial adaptations to circulatory changes in the newborn have taken place, the liver starts to fulfil its role in maintaining homeostasis. Table 3 Causes of increased plasma unconjugated bilirubin Haemolysis Traumatic birth associated with haemorrhage and tissue contusion Neonatal polycythaemia Congenital hypothyroidism Sepsis Hypoxia Hypoglycaemia Autosomal-inherited disorders Crigler–Najjar types 1 and 2 Gilbert's disease Galactosaemia Fructose intolerance Canaliculas Blood-filled sinusoid Kuppfer cell Specialized endothelium Hepatocyte Tight junction S i n u s o i d Canalicular surface Sinusoidal surface Fig. 1 Arrangement of hepatocytes and the specialized surfaces within the sinusoidal space. Hepatic function and physiology in the newborn 341 Carbohydrate, lipid and protein metabolism Newborn babies do not store much glycogen, and one of the first responses to a feed is a sharp rise in insulin. This has an anabolic effect generally causing glucose to enter cells for energy and for storage as glycogen and triglyceride, especially in the liver. Newborn babies are prone to hypoglycaemia unless they receive frequent feeds, and it is an important physiological adaptation that the cells of the central nervous system can utilize ketones if glucose is not available. The liver plays a key role in the control of free fatty acids which may be converted to ketones during a fast or stored as triglyceride after a feed when glucose and insulin are plentiful. The lack of ketone production during a fast is indicative of abnormal physiology as in disorders of fat oxidation, e.g. long-chain 3-hydroxyacyl dehydrogenase deficiency 18 which is a cause of sudden infant death. 19 The liver is the major organ responsible for the clearance of lactic acid and although lactate may be above 5 mmol/l shortly after birth, it should normalize within 6 h. A persistently elevated lactate should prompt concerns about sepsis, adequacy of the circulation or mitochondrial disorders. 11 Amino acids are transported to the liver along with other nutrients via the portal vein. They are rapidly taken up by the sinusoidal hepatocytes where they are de-aminated, transaminated and enter the urea cycle. Alternatively, amino acids are utilized to make nearly all of the plasma proteins except immunoglobulins. Proteins normally found in high concentrations such as albumin and coagulation factors are reduced in chronic liver disease. Conversely, acute-phase proteins such as alpha-1 antitrypsin and fibrinogen may be raised because the diseased liver fails to clear them. Bile acid formation and the enterohepatic circulation The elaboration of bile requires energy at several points. Bile is a complex fluid derived from cholesterol, phospholipids and haemoglobin. The numerous steps involved in cholesterol metabolism, phospholipid synthesis and the conjugation of bilirubin derived from haem requires enzymes which are dependent on ATPase, as does the transport of these molecules across the canalicular surface of the hepatocyte. 12 Bile is not just a waste product but is crucial in the activation of some of the intestinal lipases and in solubilizing dietary fats. Infants who produce inadequate amounts of bile are at risk of protein energy malnutrition secondary to malabsorption of up to half their dietary fat and also fat-soluble vitamin deficiency (vitamins A, D, E and K). For both these reasons, it is not surprising that there is a physiological mechanism for retrieving bilirubin known as the enterohepatic circulation. It is estimated that a single molecule of bilirubin circulates from hepatocyte to canaliculus to intestinal lumen via the common bile duct to the terminal ileum and back to the liver via the portal vein up to six times a day. 12 Hepatic function by zone The sinusoidal plates are differentiated in two axes: the differentiation between the sinusoidal and canalicular surfaces, and the specialization of function longitudinally from the portal tract down to the central vein. The hepatic acinus can be separated into three zones on the basis of proximity to the portal tracts or the central vein and specialization of function (Fig. 2). Zone 1 represents the area around the portal tracts (also known as the periportal area), zone 3 represents the area around the central vein (also known as the perivenular Zone 1 Zone 1 Zone 1 Zone 1 Zone 1 Zone 2 Zone 3 Zone 3 Zone 2 Central vein (hepatic venule) Portal tract containing artery, portal venule, bile duct Zone 1 Zone 2 Zone 3 Fig. 2 Hepatic acinus showing zonal differentiation of hepatocytes along an axis between the portal tracts and the central vein. Zone 1, periportal; zone 2, mid-acinus; zone 3, perivenular. 342 S.V. Beath area) and zone 2 is an area of mixed functions in the centre of the acinus between zones 1 and 3. The details of zonal function and specialization are still being elucidated but some generalizations are outlined in Table 4. Disorders of bile excretion tend to affect zone 1 where the greatest concentration of bile exists, 20 and where there is the greatest capacity for chylomicron uptake. 21 Regen- eration after hepatocyte injury begins in zone 1, and gluconeogenesis appears to be concentrated in the periportal hepatocytes. 22–24 The fact that zone 3 is rich in detoxification enzymes such as glutathione reductase 25 is important in interpreting the histological features of paracetamol poisoning, in which there is a characteristic pattern of necrosis selectively affecting the hepatocytes around the central veins. Hypoxic events may be evident in zone 3 (haemorrhage around the central vein and apoptosis of neighbouring hepatocytes 26 ), which is furthest away from arterial blood and has the low- est concentration of dissolved oxygen. Glycolysis and hydrolysis have also been associated with the perivenular hepatocytes in zone 3. 23,27 Clinical scenarios Hypoxia The neonatal liver is relatively resistant to the effects of hypoxia, but in conditions of hypoperfusion, such as during circulatory collapse caused by sepsis or blood loss, acute hepatocyte necrosis may be evident, especially around the central vein. 28 Over the next 2–10 days, an increase in plasma transaminases (alanine transaminase, aspartate transaminase) and lactate dehydrogenase are seen which may exceed 500 IU/l, 29 and a coagulopathy may develop. The rise in transaminases is followed by a rise in conjugated bilirubin. Full recovery is possible provided that the cause of hypoperfusion is treated, although the improvement in plasma bilirubin lags behind the improvement in transaminases and may take many weeks to resolve. Septic shock Liver dysfunction commonly develops secondary to severe sepsis in the neonate and can resolve if appropriate antimicrobial treatment is started promptly. However, infection with herpes simplex virus, echo viruses, or Gram-negative organisms is capable of causing fulminant liver failure, 30,31 which may necessitate liver transplantation in ex- ceptional cases. Other causes of fulminant liver failure in the neonate are listed in Table 5 and discussed in detail by McClean and Davison else- where in this issue. Ultrasound scanning of the abdomen in septicaemia may show an ‘echo-bright’ liver which is considered to be due to hyperplasia of the reticuloendothelial system. Biliary sludge and gall stones which may obstruct the biliary tree may also be seen after an episode of sepsis, particularly Table 4 Zonal specialization of hepatocyte functions Zone Functions Marker molecule or enzyme associated with function Zone 1 (periportal) Bile duct proliferation 20 Chylomicron uptake 125 I labelled chylomicron remnant 21 Hepatocyte regeneration Ki-67 antigen detection 22 Gluconeogenesis 23,25 PEPCK 24 Insulin growth factors and binding protein IGF-1 and IGFBP-2 24 Zone 2 Induction of microsomal and peroxisomal enzymes P450 4A and bifunctional enzyme 14 Zone 3 (perivenular) Detoxification Glucuronidation 25 Induction of microsomal and peroxisomal enzymes P450 4A and bifunctional enzyme 14 Aerobic metabolism Relatively more intracellular ionized calcium during aerobic conditions 26 Insulin growth factor binding protein type 1 IGFBP-1 24 Glycolysis Glucose induced release of lactate 23 Hydrolysis of cholesterol esters Cholesterol ester hydrolase 27 Table 5 Causes of fulminant liver failure in babies under six weeks of age Bacterial infection (e.g. Escherichia coli, Meningococcus) Viral infection (e.g. Herpes simplex, adenovirus) Metabolic (e.g. tyrosinaemia, haemachromatosis, galactosaemia) Circulatory collapse (secondary to congenital heart disease or haemorrhage) Hepatic function and physiology in the newborn 343 with necrotizing enterocolitis where enteral starvation exacerbates the problem. Prematurity The great advances in the management of respiratory complications in premature babies have led to an improved survival in younger and more immature infants. This has brought challenges and limitations from other organ systems such as the liver and gastrointestinal tract. Preterm infants are at considerable risk of hypoxia because of a failure to establish a properly dynamic pulmonary circulation. Such infants are also vulnerable to infection via the intestinal tract (e.g. necrotizing enterocolitis) because the surface epithelium of the gut is very permeable and the lamina propria lacks depth. 32 Bacterial translocation from the intestine via the portal vein to the liver can cause direct infections (micro-abscesses and/or large collec- tions). The more common effects of bacterial infection on liver function are indirect, occurring as a result of the toxicity of lipopolysaccharide, e.g. impaired cholesterol and bilirubin metabolism and transport. The detergent properties of bile can be very damaging, causing further disruption to the structure and function of internal plasma membranes, such as the Golgi apparatus, and external membranes, such as the sinusoidal surface of the hepatocyte. Preterm infants are also at risk of hypoglycaemia because of reduced stores of glycogen and adipose tissue. More importantly, gluconeogenesis appears to be ineffective, with energy being diverted to heat rather than glucose generation. Summary Although the blood supply and volume of the liver change dramatically at birth, together with its range of required functions, a healthy baby has sufficient physiological reserve for homeostasis to be well maintained with only a short-lived period of mild unconjugated jaundice being apparent. Babies who are of low birth weight, premature or stressed for other reasons (e.g. infection, hypoxia or congenital heart disease) may present with hypoglycaemia, acidosis and prolonged jaundice. Provided that external factors are corrected, the liver has great powers of repair and regeneration which are located in specific areas of the hepatic acinus and which can be enhanced with good nutrition. The future role of malnutrition on the evolution of non-alcoholic steatohepatitis (NASH) and the place of cyto-protective agents such as vitamin E, ursodeoxycholic acid and N-acetylcysteine are subjects of ongoing research. Practice points • Infants with prolonged jaundice (greater than 14 days) are at risk of developing a vitamin K responsive coagulaopathy and should receive vitamin K supplements. • Mild visible jaundice is common in babies up to 14 days after birth. • Always check that bilirubin levels are reducing from 14 days onwards. • Always measure the type of bilirubin in visibly jaundiced infants after 14 days—is it unconjugated or conjugated? • Sick infants who are jaundiced should have urine saved and frozen, for screening metabolic disorders. • Sepsis is the most common cause of non-physiological jaundice in neonates. • Consider rapid treatment with broad-spectrum antibiotics and acyclovir in collapsed infants. Research agenda • There have been considerable advances in stem cell research, which may ultimately allow human organs to be produced in the laboratory without resorting to cadaveric organ transplantation. Much of this work has been performed in laboratory rats, but more recent experiments with tissues obtained during neonatal surgery and from embryo research may yield fresh insights. • Another key area of research includes collaborative European studies on the genetics of liver disease, and a greater focus on susceptibility genes which may contribute to the pathogenesis of rare diseases, such as biliary atresia and idiopathic reactions to drugs. In future, a better understanding of susceptibility genes may allow high-risk patients to be identified and offered prophylactic or even pre-emptive treatment. New treatments may include the more widespread use of cyto-protective agents such as vitamin E, ursodeoxycholic acid and N-acetylcysteine. 33 • There is increasing appreciation that the disorder NASH occurs in childhood and may 344 S.V. Beath even have its origin prenatally. 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Zonal distribution of chylomicron remnant uptake in rat liver parenchymal cells. Gen Physiol Biophys 1998;17:79–94. 22. Lee VM, Cameron RG, Archer MC. Zonal location of compen- satory hepatocyte proliferation following chemically induced hepatotoxicity in rats and humans. Toxicol Pathol 1998;26:621–7. 23. Ikezawa Y, Yamatani K, Ohnuma H et al. Insulin inhibits glucagons-induced glycogenolysis in perivenular hepatocytes specifically. J Lab Clin Med 2001;138:387–92. 24. Hazel SJ, Nordqvist AC, Hall K et al. Differential expression of IGF-1 and IGF-binding protein-1 and -2 in periportal and perivenous zones of rat liver. J Endocrinol 1998; 157:285–94. 25. Ekberg K, Chandramouli V, Kumaran K et al. Gluconeo- genesis and glucuronidation in liver in vivo and the heterogeneity of hepatocyte function. J Biol Chem 1995; 270:21715–7. 26. Yoneymama K. Validation of confocal laser scanning microscopy for detecting intracellular calcium heterogeneity in liver slices. J Pharmacol Toxicol Methods 2001; 45:187–93. 27. Romero JR, Fresnedo O, Isusi E et al. Hepatic zonation of the formation and hydrolysis of cholesterol esters in periportal and perivenous parenchymal cells. Lipids 1999;34:907–13. 28. Shamir R, Maayan-Metzger A, Bujanover Y et al. Liver enzyme abnormalities in Gram-negative bacteremia of premature infants. Pediatr Infect Dis J 2000;19:495–8. 29. Lackmann GM, Tollner U, Mader R. Serum enzyme activities in full-term asphyxiated and healthy newborns: enzyme kinetics during the first 144 hours of life. Enzyme Protein 1993;47:160–72. 30. Lee WS, Kelly DA, Tanner MS et al. Neonatal liver transplantation for fulminant hepatitis caused by herpes simplex virus type 2. J Pediatr Gastroenterol Nutr 2002;35:220–3. 31. Verboon-Maciolek MA, Swanink CM, Krediet TG et al. Severe neonatal echovirus 20 infection characterized by hepatic failure. Pediatr Infect Dis J 1997;16:524–7. 32. Owings E, Georgeson K. Management of cholestasis in infants with very low birth weight. Semin Pediatr Surg 2000; 9:96–102. 33. Buhimschi IA, Buhimschi CS, Weiner CP. Protective effect of N-acetylcysteine against fetal death and preterm labor induced by maternal inflammation. Am J Obstet Gynecol 2003;188:203–8. 34. Angulo P. Nonalcoholic fatty liver disease. N Engl J Med 2002;346:1221–31. Hepatic function and physiology in the newborn 345 35. Mulhall BP, Ong JP, Younossi ZM. Non-alcoholic fatty liver disease: an overview. J Gastroenterol Hepatol 2002; 17:1136–43. 36. Roberts EA. Steatohepatitis in children. Best Pract Res Clin Gastroenterol 2002;16:749–65. 37. Manton ND, Lipsett J, Moore DJ et al. Non-alcoholic steatohepatitis in children and adolescents. Med J Aust 2000; 173:476–9. 346 S.V. Beath Review article Neonatal hepatitis syndrome Eve A. Roberts * Division of Gastroenterology and Nutrition, Room 8267, Black Wing, The Hospital for Sick Children, and Departments of Paediatrics, Medicine and Pharmacology, University of Toronto, Ontario, Canada Summary Conjugated hyperbilirubinaemia in an infant indicates neonatal liver disease. This neonatal hepatitis syndrome has numerous possible causes, classified as infective, anatomic/structural, metabolic, genetic, neoplastic, vascular, toxic, immune and idiopathic. Any infant who is jaundiced at 2–4 weeks old needs to have the serum conjugated bilirubin measured, even if he/she looks otherwise well. If conjugated hyperbilirubinaemia is present, a methodical and comprehensive diagnostic investigation should be performed. Early diagnosis is critical for the best outcome. In particular, palliative surgery for extrahepatic biliary atresia has the best chance of success if performed before the infant is 8 weeks old. Definitive treatments available for many causes of neonatal hepatitis syndrome should be started as soon as possible. Alternatively, liver transplantation may be life saving. Supportive care, especially with attention to nutritional needs, is important for all infants with neonatal hepatitis syndrome. © 2003 Elsevier Ltd. All rights reserved. KEYWORDS Neonatal hepatitis syndrome; Conjugated hyperbilirubinaemia; Giant-cell hepatitis; Neonatal liver failure; Infant; Metabolic liver disease Jaundice frequently occurs in the first 3 months of life. Most jaundiced infants have unconjugated hyperbilirubinaemia. In contrast, infants with hepatic dysfunction have conjugated hyperbilirubinaemia. This is one of the most important problems in paediatric hepatology. Many different liver disorders can cause neonatal hepatitis syndrome. Possible treatments and outcomes vary greatly among these disorders. It is critically important to find the cause of neonatal hepatitis syndrome as soon as possible, and in the majority of infants, the disease is not idiopathic. Any infant who is still jaundiced at 2–4 weeks of age requires investigation. The first test is to determine whether the hyperbilirubinaemia is conjugated or not. Any infant with conjugated hyperbilirubinaemia has neonatal hepatitis syndrome and requires further investigation. Nomenclature for neonatal liver disease is not straightforward. The simplest term ‘neonatal jaundice’ leads to confusion with physiological jaundice in the newborn. The term ‘neonatal cholestasis’ is imprecise because in the first 3–4 months of life, every infant has some degree of neonatal cholestasis on a physiological basis. This physiological cholestasis is multifactorial. Uptake of bile acids and other organic anions by hepatocytes is inefficient, leading to high concentrations of bile acids in blood; hepatocellular pathways of bile acid conjugation and biliary secretion are also immature and inefficient. The circulating bile acid pool is contracted, and ileal uptake of bile acids is underdeveloped. Hepatic bile canalicular transporters are also regulated developmentally. 1 The term ‘neonatal hepatitis’ is inaccurate because hepatic inflammation is not a feature of every condition. The term ‘neonatal hepatitis syndrome’ is preferred because it emphasizes the uniformity * Division of Gastroenterology and Nutrition, Room 8267, Black Wing, The Hospital for Sick Children, 555 University Avenue, Tronoto, Ontario M5G 1X8, Canada. Tel.: +1-416-813-7733; fax: +1-416-813-4972 E-mail address: [email protected] (E.A. Roberts). Seminars in NEONATOLOGY www.elsevierhealth.com/journals/siny Seminars in Neonatology (2003) 8, 357–374 1084-2756/03/$ - see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1084-2756(03)00093-9 of the clinical presentation as well as the broad spectrum of causative disease processes. A subset of the neonatal hepatitis syndrome is a group of disorders, which is present with liver failure with coagulopathy and metabolic instability (including but not limited to encephalopathy, which may be difficult to evaluate in the newborn) (see paper by McClean and Davison). Liver failure can have an acute-pattern with extremely elevated serum aminotransferases and normal serum albumin, or it can have a chronic-pattern with near-normal serum aminotransferases and low serum albumin, consistent with a prenatal liver injury. Since the conventional definition of acute liver failure does not really apply to the newborn period, it makes sense to use the term ‘neonatal liver failure’ for these disorders. 2,3 As there are so many different causes of neonatal hepatitis syndrome, it is useful to group them: infective, anatomic/structural, metabolic, genetic, neoplastic, vascular, toxic, immune and idiopathic (Table 1). This review will deal mainly with entities in the infectious, metabolic, genetic and immunologic categories, as well as with idiopathic neonatal hepatitis. Liver biopsy is often required to investigate neonatal hepatitis syndrome adequately. 4 The hallmark finding in many neonatal liver disorders is ‘giant-cell hepatitis’ characterized by inflammation and large multi- nucleated hepatocytes in the liver parenchyma. Structural disorders causing obstruction of large bile ducts lead to typical features of duct obstruction in portal tracts and surrounding parenchyma. Typical changes of various metabolic diseases may be evident on liver biopsy in the affected infant. Infection Toxoplasmosis, rubella, cytomegalovirus, herpes simplex (‘TORCH’) infections These congenital infections usually share clinical similarities such as enlargement of the liver and spleen, jaundice, pneumonitis, a petechial or pur- puric rash, and a tendency to prematurity or poor intra-uterine growth. Clinical presentation with neonatal liver failure is possible with any of these agents, but it is most common with Herpes simplex infection. Whenever possible, direct identification of viral infection or measurement of specific IgM antibodies should be sought for rapid diagnosis; relying on conventional TORCH titres is less preferable. Polymerase chain reaction (PCR)-based diagnostic techniques can be extremely useful. Toxoplasmosis Congenital toxoplasmosis is rare and is usually associated with maternal infection in the third trimester. Neonatal hepatitis is prominent. Central nervous system involvement with chorioretinitis (with large pigmented scars), hydrocephaly or microcephaly, and intracranial calcifications usually occurs, leading to convulsions, nystagmus and signs of increased intracranial pressure. In older infants, deafness may occur. Rubella Congenital infection with rubella virus may cause numerous abnormalities including intra-uterine growth retardation, anaemia/thrombocytopenia, congenital heart disease (often patent ductus arteriosus or pulmonary artery stenosis), cataracts, chorioretinitis (‘salt and pepper’ appearance), mental retardation and sensorineural deafness. Conjugated hyperbilirubinaemia with hepatosplenomegaly usually occurs. Liver histology typically shows giant-cell hepatitis. Cytomegalovirus Cytomegalovirus (CMV) is the most common cause of congenital infection, affecting 1–2% of newborns, most of whom are asymptomatic. Clinical findings include a petechial rash, hepatosplenomegaly and jaundice in 60–80%. CMV can cause neonatal liver failure, but this is uncommon. Fetal ascites is not necessarily a poor prognostic sign. CMV infection often affects the central nervous system, producing microcephaly, intracranial calcification, and chorioretinitis; progressive sensorineural deafness may develop later in childhood. Conclusive diagnosis requires CMV to be cultured from the infant within the first 4 weeks of life (usually in urine). In most children, CMV hepatitis is mild and resolves completely. Persisting neurodevelopmental abnormalities become the main problem. A few children develop hepatic fibrosis or non-cirrhotic portal hypertension. Intrahepatic calcification has been reported. Rarely, cirrhosis with chronic cholestasis eventually requires liver transplantation. CMV is an important cause of giant-cell hepatitis. In a study of liver biopsies from infants with neonatal hepatitis or biliary atresia, Chang et al. 5 found evidence of CMV DNA in 23 of 50 infants with neonatal hepatitis by PCR, but in only two of 26 with biliary atresia, and in none of the control specimens. 358 E.A. Roberts Table 1 Neonatal hepatitis syndrome: differential diagnosis Diseases by category Associated with neonatal liver failure? Infection Toxoplasmosis (congenital) Rubella (congenital) CMV (congenital) Herpes simplex (congenital) NLF—acute Syphilis (congenital) HHV-6 NLF—acute (rare) Herpes zoster Hepatitis B (mainly vertical) NLF—acute Hepatitis C (mainly vertical) NLF—acute (rare) HIV (vertical) Parvovirus 19 NLF—chronic Syncytial giant cell hepatitis (?paramyxovirus) Enteric viral sepsis (echoviruses, Coxsackie viruses, adenoviruses) NLF—acute Bacterial infection (extrahepatic or sepsis) NLF—acute Listeriosis Tuberculosis Structural Biliary atresia Choledochal cyst Caroli syndrome Choledocholithiasis Neonatal sclerosing cholangitis Hair-like bile duct syndrome Spontaneous biliary perforation Non-syndromic duct paucity Alagille syndrome Metabolic 1 -Antitrypsin deficiency Cystic fibrosis Galactosaemia NLF—acute or chronic Tyrosinaemia, type 1 NLF—acute or chronic Hereditary fructosaemia NLF—chronic Glycogen storage disease, type IV Niemann–Pick, type A Niemann–Pick, type C NLF—chronic Wolman disease Gaucher disease Progressive familial intrahepatic cholestasis (types 1, 2, 3) North American Indian familial cholestasis Aagenaes syndrome (cholestasis/lymphedema) Primary disorders of bile acid synthesis NLF—chronic Peroxisomal disorders (e.g. Zellweger syndrome) Perinatal haemochromatosis NLF—chronic Citrullinaemia, type II Panhypopituitarism (septo-optic dysplasia) Hypothyroidism X-linked adrenoleukodystrophy NLF—chronic Dubin–Johnson syndrome Genetic Trisomy 18 (biliary atresia) Cat-eye syndrome (biliary atresia) Trisomy 21 (fibrosing hepatitis with transient leukaemia) NLF—chronic Neoplasia Neonatal leukaemia NLF—acute Neuroblastoma NLF—acute Hepatoblastoma Histiocytosis X Erythrophagocytic lymphohistiocytosis NLF—chronic Neonatal hepatitis syndrome 359 Herpes simplex In the newborn, herpes simplex virus (HSV) usually causes a severe multisystem disorder with encepha- litis. Either type 1 or type 2 HSV is capable of causing severe infection, although type 2 virus shed from the infected cervix at birth is more frequent. Neonatal liver failure with an acute-pattern of injury is typical. Liver biopsy shows areas of necrosis with viral inclusions in intact hepatocytes; however, profound coagulopathy may preclude biopsy. Scrapings from vesicular skin lesions typically show the virus, but herpetic skin, mouth or eye lesions may not be present. Antiviral treatment with acyclovir should be administered to avert the otherwise high mortality. In the child with acute- pattern neonatal liver failure, acyclovir should be started immediately, even with results of diagnostic tests still pending. Syphilis Congenital syphilis causes intra-uterine growth retardation and subsequent failure to thrive, severe anaemia and thrombocytopenia, nephrotic syndrome, periostitis, nasal discharge (‘snuffles’), skin rash, diffuse lymphadenopathy and hepatomegaly. Central nervous system involvement occurs in up to 30% of infants. Jaundice may be present within 24 h of birth or develop after treatment, and it may be severe. 6 Diagnosis involves serological testing, including the Venereal Disease Research Laboratory (VDRL) test and confirmatory testing for specific antitreponemal antibodies. Radiographs of long bones may show typical bony abnormalities in the first 24 h of life and permit diagnosis while the other tests are still pending. Treatment with peni- cillin can then be started without delay. Some babies with congenital syphilis never develop jaundice, but present with a typical rash on the palms and soles or only with fever, as well as prominent hepatomegaly. Varicella Varicella may occur in newborn infants if maternal infection occurs within 14 days of delivery. It tends to be more severe in premature infants. Jaundice is a feature of severe disease, which typically involves an extensive rash, pneumonia and multisystem involvement. Hepatotropic viruses: hepatitis A, B, C In general, infection with hepatotropic viruses in neonates does not cause jaundice unless there is acute-pattern neonatal liver failure or severe hepatitis after a typical incubation period. Hepatitis A Hepatitis A is rare in the neonatal period. Congeni- tal infection may occur if the mother is infected 1–2 weeks before delivery. Hepatitis B Vertical (mother-to-infant) hepatitis B infection is generally subclinical in the neonatal period; Table 1 (continued) Diseases by category Associated with neonatal liver failure? Toxic TPN-associated cholestasis Drug-induced (via breast-milk or other) Vascular Budd–Chiari syndrome NLF—acute Severe congestive heart failure Neonatal asphyxia Immune Inspissated bile syndrome Neonatal lupus erythematosus Neonatal hepatitis with auto-immune haemolytic anaemia Idiopathic ‘Le foie vide’ (infantile hepatic non-regenerative disorder) NLF—chronic NLF—acute, ‘acute-pattern’ neonatal liver failure; NLF—chronic, ‘chronic-pattern’ neonatal liver failure. 360 E.A. Roberts prompt administration of both hepatitis B immunoglobulin and hepatitis B immunization provides protection against chronic infection in 93% of infants at risk. Neonatal liver failure may rarely occur with hepatitis B in neonates. Hepatitis C Hepatitis C virus (HCV) has not yet been identified as a cause of neonatal hepatitis syndrome. Vertical transmission of HCV is well documented and occurs at a rate of 4–7% in mothers who are viraemic and not co-infected with the human immunodeficiency virus (HIV) 7 . Women who are co-infected with HCV and HIV are three to four times more likely to transmit HCV to the infant. HIV infection Neonatal conjugated hyperbilirubinaemia is rare in infants with congenital HIV infection, although they may have hepatosplenomegaly. Sometimes, presentation with jaundice and hepatitis occurs later, at approximately 6 months of age. 8 Parvovirus B19 infection Congenital parvovirus B19 infection may cause profound anaemia leading to hydrops and fetal death. It is a cause of chronic-pattern neonatal liver failure, resembling perinatal haemochromatosis. 9 The affected infant may have dermal erythropoiesis (‘blueberry muffin’ rash), anaemia and perinatal distress in addition to conjugated hyperbilirubinaemia, hepatomegaly and severe coagulopathy. Testing by PCR for presence of parvovirus 19 may be positive when all serological tests are negative; placental histology may also suggest prenatal parvovirus infection. Hepatitis has been reported in young children and a 7-month-old infant. Human herpesvirus-6 infection Human herpesvirus-6 (HHV-6) causes exanthem subitum, a common but usually benign febrile illness in infants; other HHV-6 infections are common and self-limiting without a rash. Jaundice and acute-pattern neonatal liver failure has been reported in a 3-month-old infant, based on definite serological evidence for HHV-6 infection and virus isolated from peripheral blood mononuclear cells. Enteric viral sepsis (echovirus, Coxsackie viruses, adenoviruses) Enteroviruses can cause systemic viral infection in the newborn period. Severe hepatitis causing acute-pattern neonatal liver failure may be the prominent feature. Most infants with enteric viral sepsis are less than 5 weeks old at presentation; many are less than 1 week old. The infant is lethargic and jaundiced, with very high serum aminotransferases and severe coagulopathy; meningitis is usually present. Echovirus serotypes 3, 6, 7, 9, 11, 14, 19 and 21 have all been reported in severe infections with hepatitis. 10 Echovirus serotype 11 appears to be most virulent for newborns. The incidence of echovirus infections is greatest between late summer and early autumn. The infant's mother may give a history of abdominal pain just prior to the onset of labour. Vertical infection near the time of birth tends to produce more severe disease in the infant. Coxsackie A and B viruses are capable of causing an identical clinical picture, although myocarditis or heart failure is a clue to Coxsackie virus infection. Adenoviruses have also been reported to cause acute liver failure in the perinatal period. Mortality is high with neonatal liver failure, of the order of 85–90%. Meticulous supportive care is essential. Infants who recover may progress through a recovery phase marked by severe cholestatic jaundice. Subsequent liver function in survivors appears entirely normal. Bacterial infection outside of the liver Conjugated hyperbilirubinaemia may occur in conjunction with sepsis or localized extrahepatic infection. Serum aminotransferases may be slightly elevated. The liver and spleen are not enlarged. In infants, the most common infection is a urinary tract infection, which is typically clinically silent. 11 Gram-negative bacterial septicaemia is usually implicated with conjugated hyperbilirubinaemia, but the mechanism in infants, as in adults, remains unclear. Jaundice in infants may also occur with streptococcal and staphylococcal infections. Listeriosis Congenital infection with Listeria monocytogenes typically involves the liver. Although meningitis is the predominant clinical feature of congenital listeriosis, infants have hepatosplenomegaly and are sometimes jaundiced. Liver biopsy may reveal simply a diffuse hepatitis or, more commonly, diffuse areas of focal necrosis. These micro-abscesses contain numerous Gram-positive bacilli. Pneumo- nia is usually present. A history of maternal illness is common. Neonatal hepatitis syndrome 361 Tuberculosis Congenital tuberculosis is rare. With the world- wide prevalence of tuberculosis rising, tuberculosis in infants may occur somewhat more frequently. Practical criteria for diagnosing congenital tuberculosis are a proven tuberculous infection in the newborn baby and at least one of the following: lesions in the first week of life; tuberculous infection of the placenta or maternal genital organs; primary hepatic complex or caseating granulomas in the liver; exclusion of postnatal infection. 12 Hepatomegaly is usually found in infants with tuberculosis. Jaundice occurs with severe disease. Respiratory distress, poor feeding and fever are frequent. Mortality approaches 30%. Treatment is a quadruple antitubercular antibiotic regimen not including ethambutol. Structural Biliary atresia Confirming or excluding biliary atresia is an important diagnostic issue, because it is frequently responsible for neonatal hepatitis syndrome. Early diagnosis is vital because surgical treatment, the Kasai portoenterostomy, is less likely to be successful the later it is performed. 13 (see paper by Kobayashi and Stringer. Biliary atresia involves a progressive destruction of the extrahepatic bile ducts with scarring, obliteration and concomitant damage to small and medium-sized intrahepatic bile ducts. Biliary atresia is found world-wide in all racial groups, with an incidence of 1:8000–15 000 live births. Cur- rently, biliary atresia is often categorized into two general patterns: embryonal/fetal or ‘early’ and perinatal or ‘late’. The majority of infants have the ‘late’ pattern. They appear to have had a normal biliary system, which has become involved in a fibrosing inflammatory process towards the end of gestation or shortly after birth. By contrast, approximately 10–20% of infants with biliary atresia have additional congenital abnormalities including polysplenia, left atrial isomerism, double-sided left lung, pre-duodenal portal vein, intestinal mal- rotation, and/or congenital heart defects. 14,15 This suggests a different pathogenesis: an early, possibly genetic, developmental abnormality. The ‘late’ pattern of biliary atresia may reflect an acquired inflammatory lesion in originally normal bile ducts. This inflammatory process is not necessarily reversed even if bile drainage is restored after Kasai portoenterostomy. Various infective agents have been implicated in the aetiology of biliary atresia in humans. Early studies suggesting that reovirus-3 infection might be the initiating infection for idiopathic neonatal hepatitis and biliary atresia in humans were not confirmed in later reports. 16 Other studies of liver biopsies and biliary remnants from infants with ‘late’ pattern biliary atresia have not found support for rotavirus infection. 17 CMV infection is found in a proportion of infants with biliary atresia, but CMV does not appear to be an exclusive cause of the condition. In one report of fraternal twins with congenital CMV infection, one had hepatitis only and the other presented with ‘late’ pattern biliary atresia. 18 In one series of infants with biliary atresia, 25% had CMV infection and they tended to be referred later than those without CMV infection. 19 A more complicated mechanistic explanation for ‘late’ pattern biliary atresia is that it occurs when an inflammatory insult sets off an immune-mediated process in a susceptible infant. In a murine model examining bile duct allografts, alloreactive lymphocytes mediated a destructive process histologically similar to that of biliary atresia. 20 CMV remains a candidate virus for causing ‘late’ presentation biliary atresia because CMV can infect bile duct epithelial cells directly and result in increased expression of major histocompatibility complex class II antigens by these cells. Infants with congenital CMV infection and persisting conjugated hyperbilirubinaemia should be evaluated for biliary atresia. Choledochal cyst Choledochal cyst refers to a group of congenital malformations of the biliary system. The most common type is a fusiform, sometimes sausage-shaped, dilatation of the extrahepatic bile ducts (type 1). Choledochal cysts are increasingly identified in the fetus by prenatal sonography and have been found as early as 15–16 weeks' gestation. 21 The diagnosis should be confirmed soon after birth. The majority of these infants have conjugated hyperbilirubinaemia, and surgery should be performed promptly. Spontaneous biliary perforation This condition may present as a severe acute illness resembling acute peritonitis with abdominal pain and distention, jaundice and fever. It can also present as neonatal hepatitis syndrome, often with abdominal distention in addition to jaundice and acholic stools. Biliary ascites is pathognomonic. 362 E.A. Roberts Bacterial superinfection greatly increases morbidity. Surgical repair is usually curative. Neonatal sclerosing cholangitis Neonatal sclerosing cholangitis (NSC) was first reported in 1987 with a few subsequent reports. 22,23 The feature which distinguishes NSC from childhood primary sclerosing cholangitis is that NSC presents in early infancy with conjugated hyperbilirubinaemia which then resolves; although some children present in infancy with primary sclerosing cholangitis, they have not had early cholestatic jaundice. After apparent spontaneous resolution of the neonatal liver disease, NSC progresses to biliary cirrhosis with recurrence of jaundice several years later. Liver transplantation is usually required. NSC may be a metabolic disease 24 or have immunological features without persistent jaundice. In one case, non-specific auto-antibodies were detected. 25 Alagille syndrome Alagille syndrome has an important structural feature: paucity of small (portal) bile ducts. It is a genetic disorder with autosomal-dominant transmission but highly variable expression. The quoted incidence of 1:100 000 probably exaggerates its rarity. Mutations in JAG1 on chromosome 20p have been identified as the genetic basis for Alagille syndrome. Its major clinical features include chronic cholestatic liver disease with decreased numbers of small (portal) intrahepatic bile ducts, structural cardiovascular disease, skeletal abnormalities including ‘butterfly’ vertebrae, posterior embryotoxon of the eye, and typical facies. Minor features include renal abnormalities, small birth size and/or poor growth, delayed puberty or hypogonadism, and an abnormal high-pitched cry/ voice. Associated vascular abnormalities have been noted including decreased intrahepatic portal vein radicals, coarctation of the aorta and other large vessel abnormalities, and moya-moya disease. 26 Neurological abnormalities described in early reports probably were not part of the syndrome itself but instead due to vitamin E deficiency from severe chronic cholestasis. Hypothyroidism and pancreatic insufficiency have also been observed in affected children, and they are more likely to get recurrent otitis media. 27 A spectrum of behavioural problems has been described (mental retardation, learning difficulties or antisocial behaviour), but many children are normal socially and academically. The majority of patients with clinically important Alagille syndrome have conjugated hyperbilirubinaemia in the neonatal period. 28 Cholestasis may be sufficiently severe that the stools are acholic, and hepatobiliary scanning fails to show evidence of biliary excretion. 29 Liver biopsy usually shows reduced numbers of small bile ducts with some giant-cell transformation and cholestasis. The number of portal tracts may also be reduced. In some infants, ongoing damage to bile ducts may be found, or bile ductular proliferation suggestive of extrahepatic bile duct obstruction. Alagille syndrome with a segmental atresia of the common hepatic duct has been found in several infants. The characteristic facies, not always evident in early infancy, has the shape of an inverted triangle and consists of a broad forehead, deep-set eyes, mild hypertelorism, a straight nose and a small pointed chin. The ears may be prominent. The cardiovascular disease is usually relatively benign (peripheral pulmonary artery stenosis), but more severe hypoplasia of the pulmonary artery branches may occur and other congenital heart disease has been found. 30 Butterfly vertebrae, due to failure of the anterior arches of the vertebral body to fuse, are most commonly found in the thoracic spine. Eye signs may be very diverse; 31 posterior embryotoxon is most frequent. Alagille syndrome seems to be rather benign in many children. The clinical features of severe cholestasis—jaundice, pruritus, hypercholestero- laemia with or without xanthomas, elevated serum bile acids, alkaline phosphatase and -glutamyl transpeptidase (GGT)—resolve or improve during the first year of life. The hepatic lesion does not progress inexorably to cirrhosis. However, young children with protracted jaundice usually have a poorer prognosis, with progressive liver disease. Conservative estimates put overall mortality at 20– 25%, due to cardiac disease, intercurrent infection or progressive liver disease. Liver transplantation for severe hepatic disease is warranted and catch-up growth may occur afterwards. 32,33 JAG1 is the human homologue of the rat gene Jagged1. It encodes a ligand of Notch 1, which is involved in determining cell fate during differentiation, especially in tissues where epithelial– mesenchymal interactions are important. These include many of the organs that are potentially abnormal in Alagille syndrome. Haploinsufficiency of JAG1 causes Alagille syndrome; mutations result in truncated and thus inactive proteins and residual gene expression cannot compensate. 34 Many mutations are sporadic. No clear relationship between genotype and phenotype has been found, although Neonatal hepatitis syndrome 363 the Delta/Serrate/Lag-2 (DSL) domain in the JAG1 protein may influence the severity of liver disease. 35–37 Non-syndromic bile duct paucity In a full-term neonate in whom Alagille syndrome has been excluded, various other disorders may cause portal ductopenia (small duct paucity). This ‘non-syndromic duct paucity’ may be idiopathic or associated with other specific conditions (Table 2). Among congenital infections, CMV is most important and, in such cases, CMV inclusions may be found in bile duct epithelial cells. When idiopathic neonatal hepatitis is clinically severe, duct paucity may be present. Metabolic 1 -Antitrypsin deficiency This is the most common inherited cause of neonatal hepatitis syndrome. The protease inhibitor, 1 -antitrypsin, a member of the serpin superfamily, is produced mainly in the liver. 1 -Antitrypsin binds and inactivates leukocyte elastase. More than 90 variants have been reported. The deficiency status is caused by mutations in the 1 -antitrypsin genes on chromosome 14. Deficiency occurs in 1:1600– 2000 live births in North American and European populations, but it is much less common with other ethnic backgrounds. Only a small proportion of individuals with 1 -antitrypsin deficiency ever develops liver disease, but 85–90% of the children with 1 -antitrypsin deficiency who develop liver disease present with neonatal hepatitis syndrome. In most of these infants, liver disease eventually resolves. Cholestasis may be severe with totally acholic stools and a non-draining hepatobiliary scan. Small duct paucity may be present and portends a poor prognosis. The rare infant has been reported with both 1 -antitrypsin deficiency and biliary atresia. The 10–15% who do not have neonatal hepatitis syndrome present later with non- jaundiced hepatomegaly. Some newborn infants present with potentially serious haemorrhagic complications associated with severe coagulopathy. Clinical diagnosis rests upon finding low serum concentrations of 1 -antitrypsin and identifying an allelic variant of 1 -antitrypsin. The most common deficiency variant is ‘Z’, a slow-moving protein on electrophoresis, with a point mutation resulting in a single amino acid substitution (lysine replacing glutamic acid at position 342). Some variants such as M Malton and M Duarte show only subtle electro- phoretic differences from the normal ‘M’ and may be difficult to recognize. Since 1 -antitrypsin is an acute-phase reactant, diagnostic low serum concentrations may not be found due to hepatic inflammation. Determining the phenotype (‘PI’ type) by iso-electric focusing or identifying a specific gene defect by molecular methods such as PCR is essential to the diagnosis. In individuals with the Z- or M-variant allele, liver biopsy shows globu- lar inclusions, which are abnormal 1 -antitrypsin protein retained in the endoplasmic reticulum. These globules stain pink with periodic acid–Schiff- diastase (PAS-D) stain. They are not reliably found in liver biopsies from infants less than 3 months old. Most infants with 1 -antitrypsin deficiency and neonatal hepatitis syndrome have PI type ZZ (although PI Z/null cannot be excluded without family studies). Liver disease may occur with PI SZ at a relatively young age, and with PI FZ and PI MZ later in adulthood. 38 The long-term outlook for infants with jaundice and 1 -antitrypsin deficiency is often very good. Approximately half do well; of these infants, half are entirely normal and the other half have mildly abnormal serum aminotransferases, no jaundice and no enlargement of liver or spleen. The rest go on to chronic liver disease with cirrhosis or die in the first year of life. Early prognostication of individual infants with 1 -antitrypsin deficiency is difficult. In one study of children with neonatal hepatitis, persisting elevation of serum aminotransferases and serum GGT through 6–12 months of Table 2 Causes of non-syndromic paucity of bile ducts (ductopenia) in infants Prematurity Infection CMV Rubella Syphilis Hepatitis B Metabolic 1 -Antitrypsin deficiency Cystic fibrosis Zellweger syndrome Byler syndrome Ivemark syndrome Prune belly syndrome Hypopituitarism Genetic: chromosomal disorders Trisomy 18, 21 Partial trisomy 11 Monosomy X Immune-related: graft–host disease Severe idiopathic neonatal hepatitis Isolated/idiopathic 364 E.A. Roberts age, or the presence of bile ductular proliferation, bridging fibrosis or cirrhosis on the initial liver biopsy presaged rapidly progressive liver disease. 39 Although the severity of jaundice at presentation may not be predictive of outcome, its duration appears to be critical. Infants in whom jaundice resolves within a few months, usually by 6 months old, are likely to have a good outcome, but those with prolonged jaundice pursue a downhill course. Liver transplantation is generally tolerated well, although attention to potential kidney disease associated with 1 -antitrypsin deficiency is required through the early postoperative period. 40 The PI type of the donor effectively replaces the abnormal phenotype. Cystic fibrosis Abnormalities of liver function tests or on liver biopsy are found in as many as one-third of infants with cystic fibrosis. However, even in infants, hepatic pathology is highly variable. The spectrum of hepatic pathology includes giant-cell hepatitis, extrahepatic bile duct obstruction by inspissated bile, massive hepatic steatosis usually without conjugated hyperbilirubinaemia, and paucity of small (portal tract) bile ducts. Neonatal hepatitis is very uncommon. 41 Many infants who have severe liver disease also have meconium ileus. Galactosaemia The incidence of galactosaemia is approximately 1:50 000. Clinical features are extremely variable in the neonatal period and include vomiting, diarrhoea, jaundice, poor weight gain and malnutrition. Eye manifestations include cataracts, intraocular haemorrhage and retinal detachment. Although mental retardation may occur, many children have normal intelligence. Some infants present with septicaemia. Galactosaemia can present as an acute or chronic type of neonatal liver failure. A few infants never have any symptoms and are diagnosed later in childhood. The definitive diagnostic test is measurement of erythrocyte galactose-1-phosphate uridyltransferase (GALT), which must be performed before the infant has had any blood transfusions. Testing the urine for reducing substances can be misleading, as reducing substances may be present in other severe neonatal liver disease. Galactosuria may be present in normal newborns for the first few days of life, and well into the second week in premature babies. Conversely, galactosuria may not be present in an affected infant who is too unwell to take lactose-containing formula. Cataracts found on physical examination require definitive assessment by an ophthalmologist. ‘Oil-drop’ cataracts are highly typical of galactosaemia and may resolve with treatment if the disease is diagnosed early. Treatment consists of elimination of galactose from the diet. Liver disease usually improves. Later complications, mainly neurodevelopmental problems, may develop later despite good dietary control. Hereditary tyrosinaemia, type 1 Hereditary tyrosinaemia type 1 is an autosomal- recessive disease of tyrosine metabolism due to lack of fumaryl acetoacetate hydrolase (FAH), expressed mainly in the liver and kidneys. 42 The classic clinical presentation is liver disease with rickets and aminoaciduria. However, babies with this disorder may present with neonatal liver failure in the perinatal period, with classic neonatal hepatitis syndrome, or at a later age (between 4 and 24 months) with hepatomegaly, ascites and coagulopathy but no jaundice. Untreated hereditary tyrosinaemia type 1 carries a high mortality in infancy; the proportion of patients presenting in later childhood is comparatively small. Children with hereditary tyrosinaemia type 1 who survive to mid-childhood have a very high incidence of hepatocellular carcinoma, with a prevalence approaching 40% in mid-childhood. The disease is found world-wide, but it is common in the Saguenay–Lac St. Jean region of Canada (1:500), Pakistan and northern Europe. Although presentation of hereditary tyrosinaemia type 1 as neonatal hepatitis syndrome may be less common than other presentations, hereditary tyrosinaemia type 1 has to be considered in any infant with clinically or histologically severe neonatal hepatitis (Table 3) or neonatal liver failure. Coagulopathy may be prominent, attributed in part to dysfibrinogenaemia. Hypoglycaemia may occur. The liver biopsy shows parenchymal changes with inflammation of unusual severity. Typically, the -fetoprotein level is disproportionately high Table 3 Diseases causing histologically severe neonatal hepatitis 1 -Antitrypsin deficiency Hereditary tyrosinaemia, type 1 Niemann–Pick disease, type C Syncytial giant cell hepatitis Primary disorders of bile acid synthesis (mainly ∆ 4 -3-oxosteroid 5-reductase deficiency) Idiopathic neonatal hepatitis Neonatal hepatitis syndrome 365 for an infant, often 40 000–70 000 µg/l. Rickets may be present at an early age. Laboratory findings include an abnormal plasma amino acid profile with elevations of tyrosine, phenylalanine and methionine. Succinylacetone can be detected in the urine in virtually all patients. Treatment with 2-(2-nitro- 4-trifluoromethyl-benzoyl)-1,3-cyclohexanedione (NTBC: an inhibitor of 4-hydroxyphenylpyruvate- dioxygenase), in addition to a low-tyrosine– phenylalanine diet, has revolutionized the management of this disease and extended survival dramatically, 43 although a long-term risk of hepatocellular carcinoma persists. Hereditary fructosemia Hereditary fructose intolerance is an autosomal- recessive disorder and is due to deficiency of aldolase B (fructose biphosphate aldolase) in the liver, kidney and intestine. The genetic basis of this disorder is mutations in the aldolase B gene, leading to a spectrum of functional changes in the enzyme. 44,45 Age of presentation depends on dietary exposure to fructose, not frequent in early infancy. Sucrose-containing medications or unduly early introduction of fruit juice or bananas must be considered. The usual presentation is with vomiting and hepatomegaly, but jaundice may be present in nearly half of affected children. Elevated serum aminotransferases, mild coagulopathy, proteinuria and aminoaciduria are common. Liver biopsy reveals macrovesicular fat with fibrosis and pathognomonic changes in hepatocyte cytoplasm (so- called ‘fructose holes’) on electron microscopy. Aldolase B activity can be measured in the liver biopsy specimen. Treatment is to remove all fructose (and sucrose) from the diet. Niemann–Pick disease, type A or type C There are two types of Niemann–Pick disease associated with neonatal liver disease. Type A Although hepatosplenomegaly is frequently found with type A (acute neuronopathic) Niemann– Pick disease, due to lysosomal sphingomyelinase deficiency, jaundice is rare. Type C This is a disorder of cholesterol esterification, with progressive neurological deterioration during childhood in most, but not all, cases. The gene product of NPC1 appears to mediate trafficking of sterols and various other substrates out of lysosomes to other subcellular compartments. 46 In addition to abnormal cholesterol homeostasis, peroxisomal function may be impaired. 47 In approximately 30–60% of cases, Niemann–Pick disease type C presents with conjugated hyperbilirubinaemia, often with prominent splenomegaly. The infants appear neurologically normal, although subsequent motor and speech development may lag. 48 Fetal ascites may occur. Liver biopsy shows a histologically severe neonatal hepatitis, with pericellular fibrosis and pseudo- acinar formation; features suggesting that extrahepatic biliary obstruction may be found. 48 Storage cells typical of Niemann–Pick disease are often not found in the liver this early in life. Rectal biopsy may show foamy macrophages in the lamina propria, or typical ultrastructural changes (lamellar cytoplasmic inclusions) in rectal ganglion cells. Studies of cholesterol esterification in the patient's cultured fibroblasts are definitive. Progressive familial intrahepatic cholestasis Currently, three types of ‘progressive familial intrahepatic cholestasis’ (PFIC) are recognized, which are due to defects in different transporters in the bile canalicular membrane of the hepatocyte. The term ‘Byler disease’ has been replaced by ‘PFIC-1’. PFIC-1 Clinically, PFIC-1 presents with conjugated hyperbilirubinaemia in the first 3 months of life or often a little later, around 4–6 months old. The degree of jaundice may vary. Fat-soluble vitamin deficiencies, and associated rickets, may be severe. Pruritus is severe and does not respond well to treatment. Growth retardation may not be evident initially. Liver biopsy shows little inflammation but has distinctive canalicular bile plugs. Small duct paucity may be found. The serum GGT is normal, as is serum cholesterol. The total serum bile acid concentration is elevated but the biliary chenodeoxycholic acid concentration is extremely low. 49 Children with PFIC-1 have persistent diarrhoea with fat malabsorption and protein loss, recurrent pancreatitis, and poor growth leading to short stature. Sensorineural hearing loss may occur. Cirrhosis usually develops in early childhood and liver transplantation is required. After liver transplant, pancreatitis may still occur, and the diarrhoea may get worse. Patients with PFIC-1 have a mutation in the gene FIC1 on chromosome 18q21–22. 50 FIC1 encodes a 366 E.A. Roberts P-type ATPase (ATP8B1) involved in aminophos- pholipid transport between membrane leaflets. FIC1 is expressed in numerous tissues including the gastrointestinal tract, pancreas and lung. Mutations in FIC1 are also responsible for Greenland Eskimo cholestasis 51 and for benign recurrent intrahepatic cholestasis, a disease mainly of adults but sometimes symptomatic in childhood. 52,53 PFIC-2 Children with PFIC-2 have mutations in the human bile salt export pump (BSEP, ABCB11), an ATP- binding cassette transporter formerly known as sister of P-glycoprotein (SPGP) 54,55 on chromosome 2q24. They have cholestasis and normal serum GGT, and more severe hepatic abnormalities than in PFIC-1, with inflammation, giant-cell transformation of hepatocytes, fibrosis and ductular proliferation on liver biopsy. Clinical presentation is like PFIC-1 except that there is no extrahepatic involvement. PFIC-3 Affected children have progressive intrahepatic cholestasis but, in contrast to PFIC-1 and PFIC-2, children with PFIC-3 have elevated serum GGT. 56 PFIC-3 frequently presents in infancy, or else later in childhood. Jaundice may be less striking than pruritus; despite the clinical appearance of biliary tract obstruction, imaging reveals a normal biliary tree. Portal fibrosis with or without bile ductular proliferation is found on liver biopsy. Mutations in the P-glycoprotein MDR-3 gene (ABCB4) have been identified, and mutations resulting in a truncated protein appear to be associated with more severe disease than mis-sense mutations. 57 The affected protein is the bile canalicular membrane transloca- tor of phospholipids. PFIC-3 patients have bile phospholipid concentrations which are extremely low, <15% of normal. Most children with severe disease eventually require liver transplantation. North American Indian familial cholestasis (North American Indian childhood cirrhosis) Chronic cholestatic liver disease was described in 14 North American Indians living in North-west Quebec, Canada; familial clustering was prominent, and consanguinity was a possible factor. Nine of the 14 presented with neonatal conjugated hyperbilirubinaemia, and in these infants, jaundice disappeared during the first year of life. Chronic cholestatic disease was similar in all 14; hepatosplenomegaly, pruritus, facial telangiectasia and, eventually, cirrhosis and portal hypertension. Serum GGT was elevated. 58,59 The genetic basis of this disorder was recently determined; it is due to mutations in FLJ14728, conventionally called cirhin, on chromosome 16q22, and it encodes a protein of unknown function which localizes to mitochondria. 60 Aagenaes syndrome Aagenaes syndrome is a very rare disorder with cholestasis and lower limb oedema. It was initially reported in a Norwegian kindred but has also been reported in children of Norwegian descent and in other ethnic groups. The principal features are neonatal hepatitis syndrome evolving to a chronic cholestatic condition and a lymphatic disorder. This may be localized lower limb lymphedema, a more subtle disorder with generalized oedema despite normal serum albumin, or haemangioma(s) and/or lymphangioma(s). The lymphatic abnormalities may present clinically somewhat later than the jaundice. The neonatal hepatitis evolves into a predominantly cholestatic problem with pruritus and fat-soluble vitamin deficiencies requiring supplementation. The genetic basis of this familial cholestatic disorder is not known, but its genetic locus has been mapped to chromosome 15q. 61 Primary disorders of bile acid synthesis Inherited defects in some of the enzymes in the complex process of bile acid synthesis may cause neonatal hepatitis syndrome or chronic cholestasis later in childhood. While these diseases are extremely rare, they can be treated successfully by supplementation of critical bile acids, if the diagnosis is made relatively early in the course of disease. 62 3-Hydroxy-∆ 5 -C 27 -steroid dehydrogenase/isomerase deficiency This microsomal enzyme is the second in the bile acid synthetic pathway. Infants lacking it present with jaundice and acholic stools in the first few days of life; neonatal hepatitis may be histologically severe or the cholestatic disease may be somewhat more indolent, resembling progressive intrahepatic cholestasis and presenting later in childhood. Typically, infants and children with 3- hydroxy-∆ 5 -C 27 -steroid dehydrogenase/isomerase deficiency have normal serum GGT and low serum total bile acid concentrations, but no pruritus. They produce excessive amounts of C 24 -bile acids with a 3-hydroxy-∆ 5 structure. The currently preferred Neonatal hepatitis syndrome 367 treatment strategy is cholic acid with or without ursodeoxycholic acid. ∆ 4 -3-Oxosteroid 5-reductase deficiency ∆ 4 -3-Oxosteroid 5-reductase is an important cytosolic enzyme in the bile acid synthetic pathway. The original description of this disorder included two infants with early severe cholestasis and coagulopathy. Subsequent reports have included infants with a clinical presentation resembling perinatal haemochromatosis. Serum GGT is usually, but not invariably, normal. Liver biopsy may reveal abnormal bile canaliculi in a focal, ‘mosaic’ pattern. In this disorder, excess, potentially toxic, ∆ 4 -3-oxo bile acids are produced. Treatment with cholic acid (with or without ursodeoxycholic acid) appears to be beneficial in patients without iron overload. The hereditary disorder has to be distinguished from acquired deficiency of the enzyme due to severe liver disease of any cause. 24,25-Dihydroxycholanoic cleavage enzyme deficiency Infants have been described with a defect in the 25-hydroxylase pathway. 63 Jaundice and hepatomegaly were noted in the first week of life; serum GGT was normal but alkaline phosphatase and cholesterol were elevated, hepatobiliary scanning showed no drainage, and pruritus developed later. Treatment with chenodeoxycholic plus cholic acid appeared beneficial. Other bile acid synthesis disorders This is an evolving field. Two other inborn errors of bile acid metabolism have recently been described in single patients presenting with neonatal liver disease. 64,65 Neonatal hepatitis syndrome with a defect in bile acid conjugation (ligase deficiency) has also been observed. 62 Zellweger syndrome Zellweger syndrome is the prototype of the peroxisomal biogenesis disorders, characterized by multiple abnormalities of peroxisome function. The molecular and cell biology of these disorders is complex, involving multiple PEX genes which encode peroxins, proteins required for peroxisome assembly. Zellweger syndrome is most often associated with mutations in PEX1 and PEX6. 66–68 Zellweger syndrome is rare, occurring in 1:100 000, and affects both genders equally. Multiple systems besides the liver are affected; features include profound hypotonia, facial dysmorphism with a high forehead and large fontanelles, developmental delay, seizures, bony abnormalities such as epi- physeal calcifications, and cystic malformations in the brain and kidneys. In the first 3 months of life, hepatic involvement may not be prominent, although some babies have persistent conjugated hyperbilirubinaemia. Others are not jaundiced but have hepatosplenomegaly with evidence of poor hepatic synthetic function. Hepatic fibrosis is typical, and paucity of the small (portal) bile ducts may be found. Electron microscopy reveals the absence of peroxisomes in hepatocytes. Mitochondria may also appear abnormal. These infants may develop cirrhosis, although extrahepatic features of the syndrome almost always overshadow the hepatic disease. Perinatal haemochromatosis This disorder is also called neonatal haemochromatosis or neonatal iron storage disease. It is a severe liver disease with extensive iron overload in the newborn, suggesting fetal liver injury. It is thought to be extremely rare, but approximately 120 cases have been reported. Its pathogenesis remains uncertain. The dispute as to whether perinatal haemochromatosis is a single liver disease or a common clinical presentation for multiple disease processes is justified. Some cases have a definable aetiology but pathogenesis remains unclear in a significant proportion of patients. These appear to have a hereditary or at least familial pattern. Most babies present shortly after birth, although a few have been diagnosed at 2–3 months of age. 69–71 The affected infant has neonatal liver failure with a classic chronic-pattern. The biochemical features are those of end-stage cirrhosis: near-normal serum aminotransferases, low serum concentrations of proteins produced in the liver (e.g. albumin), and variable jaundice with conjugated hyperbilirubinaemia. Jaundice may be somewhat more prominent in infants presenting at a few weeks old. Ascites, including fetal ascites, may be present. Serum iron and transferrin are normal, but serum ferritin is usually increased to the 2000– 3000 µg/l range. The liver and certain other organs (pancreas, kidneys, adrenal glands and heart—not the reticulo-endothelial system) show iron accumulation. 72 Finding iron deposition in salivary glands on buccal biopsy or evidence of iron overload on magnetic resonance imaging supports the diagnosis. 368 E.A. Roberts Treatment is supportive in a well-equipped neonatal intensive care setting; liver transplantation may be required for survival. Recently, multiple drugs aimed at reducing oxidative stress have been used with some success, if treatment is commenced very early. 73 This ‘anti-oxidant cocktail’ includes anti-oxidants (N-acetylcysteine, selenium and -tocopheryl polyethylene glycol succinate), a hepatocytoprotective agent (prostaglandin E 1 , omitted if a patent ductus arteriosus is present) and a chelator (desferrioxamine, used until the serum ferritin is <500 µg/l). Not all infants respond to this regimen, which has never been subjected to a controlled clinical trial, and there is a risk of septicaemia complicating desferrioxamine therapy. Infants surviving the early liver disease appear to stabilize clinically and may end up with inactive fibrosis or even no residual liver disease. An incidental hepatocellular carcinoma has been reported in three infants. Certain clinical patterns associated with perinatal haemochromatosis are unusually severe: a renal tubular disorder 74 or ∆ 4 -3- oxosteroid-5-reductase deficiency. Citrullinaemia, type II Although jaundice is rare with the classic form of citrullinaemia (type I, argininosuccinate synthetase deficiency), infants with neonatal hepatitis syndrome were recently described who had type II citrullinaemia, confirmed by genetic analysis. 75–77 A distinguishing feature was the presence of steatosis and iron deposition histologically. Liver disease was severe enough in one infant to require liver transplantation. Type II citrullinaemia is due to a deficiency in citrin, a carrier protein of unknown function associated with the urea cycle, encoded by the gene SLC25A13. Disorders of bilirubin conjugation Dubin–Johnson syndrome is due to mutations in the human gene MRP2, which encodes the bile canalicular membrane transporter for anion conjugates. 78,79 Numerous mutations have been described, most of which cause functional deficits through defects in protein maturation and localiz- ation. 80,81 Neonatal hepatitis syndrome has been reported rarely in Dubin–Johnson syndrome. 82 Diagnosis is hampered by the difficulty in recogniz- ing the typical melanin-containing pigment in the liver during infancy, as little accumulates until later in childhood. Treatment of severely affected neonates with ursodeoxycholic acid may be beneficial. Genetic Trisomy 18 Trisomy 18 is associated with growth retardation, skeletal abnormalities and complex congenital heart disease. In a series of 10 infants with cyto- genetically confirmed trisomy 18, giant-cell hepatitis was found in three and biliary atresia in two. In one infant with trisomy 18, serial liver biopsies suggested late evolution of neonatal hepatitis to biliary atresia. Other cytogenetic abnormalities, including trisomy 13, deletion of the short arm of chromosome 18 and 49, and XXXXY, 83 have been reported rarely in association with biliary atresia. Trisomy 21 An association between trisomy 21 and biliary atresia is not well substantiated. Severe liver disease has been reported with Down's syndrome. Some patients had severe hepatic fibrosis associated with a transient myeloproliferative disorder, raising the possibility of hepatic fibrogenesis due to high concentrations of growth factors derived from megakaryocytes. 84 Neonatal liver failure may occur. Treatment with low-dose cytosine arabinoside may be curative. 85 Immune Inspissated bile syndrome The ‘inspissated bile syndrome’ is the term traditionally used for conjugated hyperbilirubinaemia complicating severe jaundice associated with haemolysis, usually due to Rhesus factor or ABO incompatibility or erythrocyte abnormalities. Intra- hepatic cholestasis is found on liver biopsy, and cholestasis may be due to direct hepatocellular toxicity of unconjugated bilirubin. A multifactorial cause cannot be entirely excluded as these infants are often premature and present complex medical problems. The outlook is generally good, although early reports showed cirrhosis in some infants. Neonatal lupus erythematosus Neonatal lupus erythematosus is due to passage of maternal anti-Ro and anti-La antibodies across the placenta, leading to damage to fetal tissues, which express Ro and La antigens. The heart, skin and liver are most likely to be involved, rarely with thrombocytopenia and leukopenia. 86 Congenital heart block is the most dramatic cardiac manifestation; a discoid lupus erythematosus rash may be Neonatal hepatitis syndrome 369 present in the newborn period or develop some weeks later. Hepatic involvement, evident in approximately 10%, is often limited to elevated serum aminotransferases, but neonatal hepatitis syndrome is found. Occasionally, this is severe enough to mimic extrahepatic biliary tract obstruction, with acholic stools and a non-draining hepatobiliary scan. One infant with severe liver disease resembled perinatal haemochromatosis. Transient unexplained isolated conjugated hyperbilirubinaemia in the perinatal period and later presentation at 2–3 months old with transient elevations of serum aminotransferases are other possible clinical presentations. 87 In most infants, the liver disease resolves completely between 6 and 12 months of age, as the maternal antibodies are degraded. Mild fibrosis was found in one child on repeat liver biopsy. The diagnosis of neonatal lupus erythematosus is difficult in the child who does not have congenital heart block or a typical skin rash. Some infants have only transient jaundice and myocarditis with an abnormal electrocardiogram. If the mother is known to have systemic lupus erythematosus or Sjo¨gren's syndrome, the diagnosis should be suspected. Frequently, however, the mother is asymptomatic and has no obvious rheumatological disease. Routine methods may fail to detect anti-Ro and anti-La in the infant, and in any case, these studies need to be performed at as young an age as possible. Very high titres of ANA in the infant may be due to neonatal lupus erythematosus. Deposits of associated antibodies (anti-Ro and/or anti-La) may be found in affected liver tissue by immuno- fluorescence. 88 The risk of neonatal lupus erythematosus in subsequent pregnancies appears variable, estimated at 10–50%. Idiopathic neonatal hepatitis In a large proportion of infants presenting with conjugated hyperbilirubinaemia before 3 months old, no aetiology is found. Liver biopsy shows an extensive giant-cell transformation of hepatocytes with inflammation, but bile ducts appear generally normal. A few infants with histologically severe inflammation also have small duct paucity. All these infants are classified as having idiopathic neonatal hepatitis, a condition of unknown and not necessarily unitary aetiology. In one series, babies with idiopathic neonatal hepatitis accounted for approximately one-quarter of all infants who underwent liver biopsy in the first year of life. This figure probably underestimates the incidence of idiopathic neonatal hepatitis. An important subset of idiopathic neonatal hepatitis includes instances where more than one child in a single family is affected, accounting for 5–15% of cases in most series. Cholestasis in idiopathic neonatal hepatitis may be sufficiently severe clinically that supportive measures, including high-calorie formula feeds containing medium-chain triglycerides and supplementation of fat-soluble vitamins, are required at least temporarily. Differentiation from biliary atresia and other severe cholestatic conditions is the critically important issue. In general, there are no easy discriminators between severe idiopathic neonatal hepatitis and biliary atresia, and thorough methodical investigation is essential. An operative cholangiogram may be required, and there is no evidence that diagnostic laparotomy for assessment of the extrahepatic biliary tree is detrimental to infants with idiopathic neonatal hepatitis. Although idiopathic neonatal hepatitis can occur in preterm babies, some will have cholestasis due to immaturity of the biliary tree. These infants are prone to early hypoglycaemia and also have a func- tionally immature gastrointestinal tract resulting in difficulties with feeding. Premature babies can also have congenital infection or biliary atresia. Some disorders, notably perinatal haemochromatosis, seem to predispose to premature birth. The prognosis for idiopathic neonatal hepatitis is generally good. Mortality runs at 13–25%. 89 Predictors of poor prognosis include prolonged (>6 months) or severe jaundice, acholic stools, familial occurrence, persistent hepatomegaly and severe inflammation on biopsy. Peak bilirubin level is not necessarily predictive of outcome, and the prognostic importance of ductopenia has not been rigorously investigated. Sepsis may shift an infant from a relatively good prognosis to a poor outlook. The long-term outlook for infants whose liver disease resolves in the first year of life is very good, without residual liver disorder. Management of neonatal hepatitis syndrome Management should be supportive and, if possible, definitive. Generally, treatments involve dietary manipulation to remove toxins or surgical interven- tion to relieve obstruction. For some metabolic disorders, such as hereditary tyrosinemia type 1 and bile acid synthesis disorders, unloading the metabolic pathway is effective. Orthotopic liver transplant is often the only definitive treatment for severe infantile liver disease and can be performed 370 E.A. Roberts safely in the first year of life, especially if nutrition is maintained (see paper by McClean and Davison). It is advisable to place an infant with conjugated hyperbilirubinaemia on a lactose-free formula until the results of testing for galactosaemia are known. However, if the infant is breast-feeding well and is clinically stable, and if GALT results can be obtained with little delay, the child may be left breast-feeding. Brief use of a more restrictive diet is sometimes justifiable; an infant with severe neonatal hepatitis syndrome might be placed on a lactose-free/low-protein formula (to minimize aromatic amino acid intake) until the results of tests for both galactosaemia and hereditary tyrosinaemia type 1 are available. All infants with severe cholestatic jaundice require special formulas to ensure that caloric intake is adequate. A nearly elemental formula containing medium-chain triglycerides, which can be absorbed regardless of luminal concentrations of bile acids, is preferable. Caloric density can be increased further by concentrating the formula or adding starch powder. These formulas are relatively expensive and not particularly palatable, although many infants take them satisfactorily. An alternative strategy is to modify a standard infant formula by adding medium-chain triglyceride liquid and additional starch. This is often the best approach for an infant presenting later in the neonatal period with jaundice. A more difficult problem is how to manage the infant who is satisfactorily breast-feeding; if breast-feeding is continued, weight gain must be monitored closely. Should growth falter, supplementation with a highly digestible high-caloric density formula should be added, or else the baby should be weaned and the special formula substituted for breast- feeding at that point. A breast-feeding device to provide supplementary formula at the nipple may be useful. In biliary atresia, resting energy expenditure runs approximately 30% higher than in normal infants of the same age and sex; 90 an aggressive approach to feeding is required, including naso- gastric supplementation if oral feeding cannot meet caloric needs. In idiopathic neonatal hepatitis, this type of special formula, along with fat-soluble vitamin supplementation, may be required until the jaundice abates, at which point the baby can be placed on an appropriate regular diet. Special diets are used life-long for children with inborn errors of carbohydrate and amino acid metabolism. Infants with chronic cholestasis, whether jaundiced or not, require supplementation of fat- soluble vitamins. Vitamins A and D are potentially toxic in high dose. Administration of the more polar 25-hydroxyvitamin D may be more effective than plain vitamin D because the hydroxylated form of vitamin D is better absorbed. However, residual hepatic 25-hydroxylation activity is usually adequate. Vitamin E transferred via the placenta to the fetus may keep the infant replete until the age of 3 months, but the sufficiency of maternal stores varies greatly. Most babies require supplementation after 2 months of age or earlier if the baby was born preterm. Vitamin E attached to polyethylene glycol 1000 through a succinate linkage (-tocopheryl polyethylene glycol succinate or ‘TPGS’) has been shown to have the best bioavail- ability in severe cholestasis, since its absorption depends on simple passive absorption of the polyethylene glycol. Since vitamin E absorption is exquisitely dependent on luminal bile acids, any jaundiced infant with deficiency of another fat- soluble vitamin should be assumed to require extra vitamin E. Coagulation should be monitored closely in all infants with cholestasis. Infants with a coagulopathy (measured by prothrombin time or INR) should receive oral vitamin K daily. An alternate approach is to use a combination of fat-soluble vitamin preparation that includes vitamin K daily; parenteral vitamin K may be needed periodically. Infants receiving rifampicin for pruritus should receive extra vitamin K. Pruritus due to severe cholestasis interferes with the infant's sleep and compromises social inter- action and play during waking hours. It is often difficult to treat. Local measures such as non- perfumed skin creams and colloidal oatmeal bath preparations may help. If there is some bile flow, as in Alagille syndrome, cholestyramine or ursodeoxycholic acid may be effective. Cholestyramine can cause intestinal obstruction or hypernatraemia in small infants and therefore must be used carefully and given with adequate fluids. If bile flow is totally obstructed, therapeutic choices are more limited. Phenobarbital is relatively ineffective, causes sedation and may exacerbate rickets. Rifampicin (5–10 mg/kg/day given by mouth in two equally divided doses) relieves pruritus in at least 50%, although experience in very young infants is limited. 91 Side effects include hepatotoxicity in 5–10% and thrombocytopenia, and the urine turns to orange–red colour. Surgical biliary diversion may be effective in some conditions, including Alagille and PFIC syndromes. 92 Specific attention to the infant's developmental needs is often highly beneficial. Physiotherapy may improve gross motor development; lower limb weakness seems to be especially common in older Neonatal hepatitis syndrome 371 infants with biliary atresia. Stimulation programs enhance mental development of infants who require frequent hospitalization or for those with syndromes associated with central nervous system involvement, such as congenital CMV infection. Practice points • Any infant jaundiced at 2–4 weeks of age must be evaluated for conjugated hyperbilirubinaemia. • Conjugated hyperbilirubinaemia in the first 24 h of life strongly suggests congenital infection. • Structural abnormalities of the biliary tree found on prenatal ultrasound require assessment as soon as possible after birth. • 1 -Antitrypsin deficiency is the most frequent metabolic disease causing neonatal hepatitis syndrome in Caucasian infants. • Conjugated hyperbilirubinaemia with cholestasis but a normal GGT suggests PFIC (types 1 or 2) or a primary disorder of bile acid synthesis. • Chronic-pattern neonatal liver failure presents with conjugated hyperbilirubinaemia, unremarkable AST and ALT concentrations, a low serum albumin, and a marked coagulopathy. Research directions • The role of congenital infection in the pathogenesis of biliary atresia. • The mechanism for duct paucity in Alagille syndrome. • The existence of other types of progressive familial intrahepatic cholestasis. • The mechanism of hepatic fibrosis in trisomy 21 with transient myeloproliferative syndrome/leukaemia. • The pathogenesis of perinatal haemochromatosis. References 1. Tomer G, Ananthanarayanan M, Weymann A et al. Differ- ential developmental regulation of rat liver canalicular membrane transporters bsep and mrp2. Pediatr Res 2003; 53:288–94. 2. Shneider BL. Neonatal liver failure. Curr Opin Pediatr 1996; 8:495–501. 3. Jackson R, Roberts EA. Identification of neonatal liver failure and perinatal hemochromatosis in Canada. Paediatr Child Health 2001;6:248–50. 4. Lichtman S, Guzman C, Moore DL et al. Morbidity after percutaneous liver biopsy. Arch Dis Child 1987;62:901–4. 5. Chang MH, Huang HH, Huang ES et al. Polymerase chain reaction to detect human cytomegalovirus in livers of infants with neonatal hepatitis. Gastroenterology 1992; 103:1022–5. 6. Wolf MJ, Beunen G, Casaer P et al. Extreme hyperbilirubinaemia in Zimbabwean neonates: neurodevelopmental outcome at 4 months. Eur J Pediatr 1997;156: 803–7. 7. Roberts EA, Yeung L. Maternal–infant transmission of hepatitis C virus infection. Hepatology 2002;36:S106–13. 8. Persaud D, Bangaru B, Greco MA et al. Cholestatic hepatitis in children infected with the human immunodeficiency virus. Pediatr Infect Dis J 1993;12:492–8. 9. Silver MM, Hellmann J, Zielenska M et al. Anemia, blueberry-muffin rash, and hepatomegaly in a newborn infant. J Pediatr 1996;128:579–86. 10. Gillam GL, Stokes KB, McLellan J et al. Fulminant hepatic failure with intractable ascites due to an echovirus 11 infection successfully managed with a peritoneo-venous (LeVeen) shunt. J Pediatr Gastroenterol Nutr 1986;5: 476–80. 11. Garcia FJ, Nager AL. Jaundice as an early diagnostic sign of urinary tract infection in infancy. Pediatrics 2002; 109:846–51. 12. Cantwell MF, Shehab ZM, Costello AM et al. Brief report: congenital tuberculosis. N Engl J Med 1994;330:1051–4. 13. Chardot C, Carton M, Spire-Bendelac N et al. Prognosis of biliary atresia in the era of liver transplantation: French national study from 1986 to 1996. Hepatology 1999;30: 606–11. 14. Karrer FM, Hall RJ, Lilly JR. Biliary atresia and the polysplenia syndrome. J Pediatr Surg 1991;26:524–7. 15. Vasquez J, Lopez Gutierrez JC, Gamez M et al. Biliary atresia and the polysplenia syndrome: its impact on final outcome. J Pediatr Surg 1995;30:485–7. 16. Steele MI, Marshall CM, Lloyd RE et al. Reovirus 3 not detected by reverse transcriptase-mediated polymerase chain reaction analysis of preserved tissue from infants with cholestatic liver disease. Hepatology 1995;21:697–702. 17. Bobo L, Ojeh C, Chiu D et al. Lack of evidence for rotavirus by polymerase chain reaction/enzyme immunoassay of hepatobiliary samples from children with biliary atresia. Pediatr Res 1997;41:229–34. 18. Hart MH, Kaufman SS, Vanderhoof JA et al. Neonatal hepatitis and extrahepatic biliary atresia associated with cytomegalovirus infection in twins. Am J Dis Child 1991; 145:302–5. 19. Tarr PI, Haas JE, Christie DL. Biliary atresia, cytomegalovirus, and age at referral. Pediatrics 1996;97:828–31. 20. Schreiber RA, Kleinman RE, Barksdale EM et al. Rejection of murine congenic bile ducts: a model for immune mediated bile duct disease. Gastroenterology 1992;102:924–30. 21. Stringer MD, Dhawan A, Davenport M et al. Choledochal cysts: lessons from a 20 year experience. Arch Dis Child 1995;73:528–31. 22. Amedee-Manesme O, Bernard O, Brunelle F et al. Sclerosing cholangitis with neonatal onset. J Pediatr 1987;111:225–9. 23. Mulberg AE, Arora S, Grand RJ et al. Expanding the spectrum of neonatal cholestatic liver disease. 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Yuan ZR, Okaniwa M, Nagata I et al. The DSL domain in mutant JAG1 ligand is essential for the severity of the liver defect in Alagille syndrome. Clin Genet 2001; 59:330–7. 38. Primhak RA, Tanner MS. Alpha-1 antitrypsin deficiency. Arch Dis Child 2001;85:2–5. 39. Francavilla R, Castellaneta SP, Hadzic N et al. Prognosis of alpha-1-antitrypsin deficiency-related liver disease in the era of paediatric liver transplantation. J Hepatol 2000; 32:986–92. 40. Prachalias AA, Kalife M, Francavilla R et al. Liver transplantation for alpha-1-antitrypsin deficiency in children. Transpl Int 2000;13:207–10. 41. Lykavieris P, Bernard O, Hadchouel M. Neonatal cholestasis as the presenting feature in cystic fibrosis. Arch Dis Child 1996;75:67–70. 42. St-Louis M, Tanguay RM. Mutations in the fumarylaceto- acetate hydrolase gene causing hereditary tyrosinemia type I: overview. Hum Mutat 1997;9:291–9. 43. Holme E, Lindstedt S. Tyrosinaemia type I and NTBC (2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione). J Inherit Metab Dis 1998;21:507–17. 44. Tolan DR. Molecular basis of hereditary fructose intolerance: mutations and polymorphisms in the human aldolase B gene. Hum Mutat 1995;6:210–8. 45. Esposito G, Vitagliano L, Santamaria R et al. Structural and functional analysis of aldolase B mutants related to hereditary fructose intolerance. FEBS Lett 2002;531:152–6. 46. Neufeld EB, Wastney M, Patel S et al. The Niemann–Pick C1 protein resides in a vesicular compartment linked to retrograde transport of multiple lysosomal cargo. J Biol Chem 1999;274:9627–35. 47. Sequeira JS, Vellodi A, Vanier MT et al. Niemann–Pick disease type C and defective peroxisomal beta-oxidation of branched-chain substrates. J Inherit Metab Dis 1998;21: 149–54. 48. Kelly DA, Portmann B, Mowat AP et al. Niemann–Pick disease type C: diagnosis and outcome in children, with particular reference to liver disease. J Pediatr 1993;123: 242–7. 49. Jacquemin E, Dumont M, Bernard O et al. Evidence for defective primary bile acid secretion in children with progressive familial intrahepatic cholestasis (Byler disease). Eur J Pediatr 1994;153:424–8. 50. Bull LN, van Eijk MJ, Pawlikowska L et al. A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis. Nat Genet 1998;18:219–24. 51. Klomp LW, Bull LN, Knisely AS et al. A missense mutation in FIC1 is associated with Greenland familial cholestasis. Hepatology 2000;32:1337–41. 52. Bull LNCV, Stricker NL, Baharloo S et al. Genetic and mor- phological findings in progressive familial intrahepatic cholestasis (Byler disease [PFIC-1] and Byler syndrome): evidence for heterogeneity. Hepatology 1997;26:155–64. 53. van Ooteghem NA, Klomp LW, van Berge-Henegouwen GP et al. Benign recurrent intrahepatic cholestasis progressing to progressive familial intrahepatic cholestasis: low GGT cholestasis is a clinical continuum. J Hepatol 2002;36: 439–43. 54. Jansen PL, Strautnieks SS, Jacquemin E et al. Hepato- canalicular bile salt export pump deficiency in patients with progressive familial intrahepatic cholestasis. Gastroenterology 1999;117:1370–9. 55. Wang L, Soroka CJ, Boyer JL. The role of bile salt export pump mutations in progressive familial intrahepatic cholestasis type II. J Clin Invest 2002;110:965–72. 56. Jacquemin E, de Vree JML, Sturm E et al. Mutations in the MDR3 gene are responsible for a subtype of progressive familial intrahepatic cholestasis (PFIC) [abstr]. Hepatology 1997;26:248A. 57. Jacquemin E, De Vree JM, Cresteil D et al. The wide spectrum of multidrug resistance 3 deficiency: from neonatal cholestasis to cirrhosis of adulthood. Gastroenterology 2001;120:1448–58. 58. Weber AM, Tuchweber B, Yousef I et al. Severe familial cholestasis in North American Indian children: a clinical model of microfilament dysfunction? Gastroenterology 1981;81:653–62. 59. Drouin E, Russo P, Tuchweber B et al. North American Indian cirrhosis in children: a review of 30 cases. J Pediatr Gastro- enterol Nutr 2000;31:395–404. 60. Chagnon P, Michaud J, Mitchell G et al. A missense mutation (R565W) in cirhin (FLJ14728) in North American Indian childhood cirrhosis. Am J Hum Genet 2002;71:1443–9. 61. Bull LN, Roche E, Song EJ et al. Mapping of the locus for cholestasis-lymphedema syndrome (Aagenaes syndrome) to a 6.6-cM interval on chromosome 15q. Am J Hum Genet 2000;67:994–9. 62. Bove KE. Liver disease caused by disorders of bile acid synthesis. Clin Liver Dis 2000;4:831–48. Neonatal hepatitis syndrome 373 63. Clayton PT, Casteels M, Mieli-Vergani G et al. Familial giant cell hepatitis with low bile acid concentration and increased urinary excretion of specific bile alcohols: a new inborn error of bile acid synthesis? Pediatr Res 1995;37:424–31. 64. Setchell KD, Schwarz M, O'Connell NC et al. Identification of a new inborn error in bile acid synthesis: mutation of the oxysterol 7alpha-hydroxylase gene causes severe neonatal liver disease. J Clin Invest 1998;102:1690–703. 65. Setchell KD, Heubi JE, Bove KE et al. Liver disease caused by failure to racemize trihydroxycholestanoic acid: gene mutation and effect of bile acid therapy. Gastroenterology 2003; 124:217–32. 66. Moser HW. Genotype–phenotype correlations in disorders of peroxisome biogenesis. Mol Genet Metab 1999;68:316–27. 67. Gould SJ, Valle D. Peroxisome biogenesis disorders: genetics and cell biology. Trends Genet 2000;16:340–5. 68. Preuss N, Brosius U, Biermanns M et al. PEX1 mutations in complementation group 1 of Zellweger spectrum patients correlate with severity of disease. Pediatr Res 2002;51: 706–14. 69. Knisely AS, Magid MS, Dische MR et al. Neonatal hemochromatosis. Birth Defects 1987;22:75–102. 70. Vohra P, Haller C, Emre S et al. Neonatal hemochromatosis: the importance of early recognition of liver failure. J Pediatr 2000;136:537–41. 71. Kelly AL, Lunt PW, Rodrigues F et al. Classification and genetic features of neonatal haemochromatosis: a study of 27 affected pedigrees and molecular analysis of genes implicated in iron metabolism. J Med Genet 2001;38: 599–610. 72. Silver MM, Valberg LS, Lines LD et al. Tissue iron and copper quantitation in perinatal hemochromatosis and other perinatal liver diseases. Comparison with a large perinatal control population, including cases with chronic liver disease. Am J Pathol 1993;143:1312–25. 73. Flynn DM, Mohan N, McKiernan P et al. Progress in treatment and outcome for children with neonatal haemochromatosis. Arch Dis Child Fetal Neonatal Ed 2003;88:F124–7. 74. Bale PM, Kan AE, Dorney SF. Renal proximal tubular dysgen- esis associated with severe neonatal hemosiderotic liver disease. Pediatr Pathol 1994;14:479–89. 75. Ben-Shalom E, Kobayashi K, Shaag A et al. Infantile citrullinemia caused by citrin deficiency with increased dibasic amino acids. Mol Genet Metab 2002;77:202–8. 76. Saheki T, Kobayashi K. Mitochondrial aspartate glutamate carrier (citrin) deficiency as the cause of adult-onset type II citrullinemia (CTLN2) and idiopathic neonatal hepatitis (NICCD). J Hum Genet 2002;47:333–41. 77. Tamamori A, Okano Y, Ozaki H et al. Neonatal intrahepatic cholestasis caused by citrin deficiency: severe hepatic dysfunction in an infant requiring liver transplantation. Eur J Pediatr 2002;161:609–13. 78. Kartenbeck J, Leuschner U, Mayer R et al. Absence of the canalicular isoform of the MRP gene-encoded conjugate export pump from the hepatocytes in Dubin–Johnson syndrome. Hepatology 1996;23:1061–6. 79. Paulusma CC, Kool M, Bosma PJ et al. A mutation in the human canalicular multispecific organic anion transporter gene causes the Dubin–Johnson syndrome. Hepatology 1997;25:1539–42. 80. Hashimoto K, Uchiumi T, Konno T et al. Trafficking and functional defects by mutations of the ATP-binding domains in MRP2 in patients with Dubin–Johnson syndrome. Hepatology 2002;36:1236–45. 81. Keitel V, Nies AT, Brom M et al. A common Dubin–Johnson syndrome mutation impairs protein maturation and transport activity of MRP2 (ABCC2). Am J Physiol Gastrointest Liver Physiol 2003;284:G165–74. 82. Regev RH, Stolar O, Raz A et al. Treatment of severe cholestasis in neonatal Dubin–Johnson syndrome with ursodeoxycholic acid. J Perinat Med 2002;30:185–7. 83. Silveira TR, Salzano FM, Howard ER et al. Congenital structural abnormalities in biliary atresia: evidence for etio- pathogenic heterogeneity and therapeutic implications. Acta Paediatr Scand 1991;80:1192–9. 84. Schwab M, Niemeyer C, Schwarzer U. Down syndrome, transient myeloproliferative disorder, and infantile liver fibrosis. Med Pediatr Oncol 1998;31:159–65. 85. Al-Kasim F, Doyle JJ, Massey GV et al. Incidence and treatment of potentially lethal diseases in transient leukemia of Down syndrome: Pediatric Oncology Group Study. J Pediatr Hematol Oncol 2002;24:9–13. 86. Silverman ED, Laxer RM. Neonatal lupus erythematosus. Rheum Dis Clin North Am 1997;23:599–618. 87. Lee LA, Sokol RJ, Buyon JP. Hepatobiliary disease in neonatal lupus: prevalence and clinical characteristics in cases enrolled in a national registry. Pediatrics 2002;109:E11. 88. Selander B, Cedergren S, Domanski H. A case of severe neonatal lupus erythematosus without cardiac or cutaneous involvement. Acta Paediatr 1998;87:105–7. 89. Suita S, Arima T, Ishii K et al. Fate of infants with neonatal hepatitis: pediatric surgeons' dilemma. J Pediatr Surg 1992; 27:696–9. 90. Pierro A, Koletzko B, Carnielli V et al. Resting energy expenditure in infants and children with extrahepatic biliary atresia. J Pediatr Surg 1989;24:534–8. 91. Gregorio GV, Ball CS, Mowat AP et al. Effect of rifampin in the treatment of pruritus in hepatic cholestasis. Arch Dis Child 1993;69:141–3. 92. Emerick KM, Whitington PF. Partial external biliary diversion for intractable pruritus and xanthomas in Alagille syndrome. Hepatology 2002;35:1501–6. 374 E.A. Roberts Review article Neonatal liver failure Patricia McClean * , Suzanne M. Davison Children's Liver and GI Unit, St James's, University Hospital, Beckett Street, Leeds LS9 7TF, UK Summary Liver failure in the neonatal period is challenging to diagnose and manage, and still carries a high mortality. With ongoing developments in the field of metabolic disorders and antiviral therapy, and the ability to offer liver transplantation to small babies, an overall survival of 40% has been achieved. Early recognition of liver failure, good supportive care and prompt referral to a paediatric liver transplant centre are essential elements in improving the outcome for these babies. Decisions about contra-indications to and timing of transplantation are complex as many of the disease processes are still evolving in the neonatal period, and extrahepatic disease, which cannot be corrected by a transplant, may appear later. © 2003 Elsevier Ltd. All rights reserved. KEYWORDS Neonatal liver failure; Viral hepatitis; Neonatal haemochromatosis; Mitochondrial hepatopathy Neonatal liver failure (NLF) is rare and may be difficult to recognize initially, as jaundice can be a late feature. Coagulopathy, unresponsive to intravenous vitamin K, is always present, although this is not uncommon in ill neonates (Table 1). Some conditions, such as neonatal haemochromatosis (NH), are due to chronic in-utero liver disease and present as decompensated cirrhosis with low levels of albumin and normal transaminases, whilst others have the typical features of acute liver failure of perinatal onset with high transaminases. 1 Hypoglycaemia, hyperammonaemia and encephalopathy (often difficult to define in a neonate) are common, although these are seen in many sick infants. The diagnosis of liver failure must be considered in any neonate with coagulopathy. 2 If other features of liver dysfunction are absent or non- specific, measurement of the individual clotting factors will show low levels of II, V, VII, IX and X, and normal or elevated levels of factor VIII in infants where the coagulopathy is due to liver disease. Fibrinogen and factors XI and XII are frequently normal but may be decreased. Aetiology Table 2 lists most of the recognized causes of NLF. Published series from paediatric liver centres will not include cases that resolve spontaneously (e.g. hypoxic/ischaemic), respond quickly to treatment (e.g. bacterial infection), or die early. 2,3 Infection and metabolic disorders are the two main causes of NLF. Many of these diseases may also present as a less fulminant neonatal hepatitis syndrome (see * Corresponding author. Tel.: +44-113-2066689; fax: +44-113- 2088891 E-mail address: [email protected] (P. McClean). Table 1 Differential diagnosis of a prolonged prothrombin time in the neonate Congenital bleeding disorder Afibrinogenaemia Dysfibrinogenaemia Deficiencies of individual clotting factors Acquired bleeding disorder Disseminated intravascular coagulation Vitamin K deficiency Antibodies/inhibitors affecting coagulation NLF Drug induced For normal laboratory ranges of clotting parameters in the newborn see the work by Williams et al. 53 Seminars in NEONATOLOGY www.elsevierhealth.com/journals/siny Seminars in Neonatology (2003) 8, 393–401 1084-2756/03/$ - see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1084-2756(03)00095-2 paper by Roberts). This paper will only describe diseases presenting primarily as NLF. Clinicopathological features Presentation at birth implies an intra-uterine insult such as congenital infection, NH or mitochondrial disorders. A later presentation may be related to infection or a metabolic condition unveiled by the introduction of feeding. A detailed obstetric history, including information on consanguinity, previous miscarriages and neonatal deaths, is important. There are various patterns of presentation which are not mutually exclusive and may progress to fibrosis or cirrhosis. Hepatocyte necrosis is the characteristic pathological feature of infants with acute viral infections, toxic or ischaemic injury and some metabolic diseases. This results in initially high serum transaminases, but as the degree of necrosis progresses, the transaminase levels fall, the liver shrinks in size and coagulopathy and hyperbilirubinaemia worsens. Recovery is heralded by a sustained fall in prothrombin time in association with falling transaminases and bilirubin levels. In contrast, in some metabolic conditions, such as fatty acid oxidation defects and mitochondrial respiratory chain defects, there is little cell necrosis and the liver failure is at a subcellular level. Histologically, there may be diffuse hepatic steatosis and/or swelling of hepatocytes, and clinically there is hepatomegaly with moderate elevation of transaminases and minimal to moderate jaundice. Often, with prompt and appropriate medical treatment, the liver recovers completely, but if the liver disease is chronic and has progressed to cirrhosis, as occurs in many mitochondrial disorders, recovery will not occur. Infiltrative and storage disorders result in hepatosplenomegaly with raised transaminases and bilirubin. Finally, NH is a typical example of decompensated cirrhosis, presenting at birth with a small liver, normal transaminases, mildly raised bilirubin, markedly reduced serum albumin and, possibly, features of portal hypertension. Investigations The initial investigations necessary to establish the cause of NLF are shown in Table 3. It is important to recognize medically treatable causes early, such as galactosaemia or tyrosinaemia, before pro- ceeding to more invasive procedures. Specific investigations are discussed under the relevant disease headings. Performing a liver biopsy is hazardous due to coagulopathy. The potential benefits must be balanced against this risk. Some units advocate an open surgical biopsy in these circumstances following vigorous correction of coagulopathy. Management There are no randomized trials of the following interventions in neonates. Recommendations are extrapolated from experience in paediatric practice. Intravenous dextrose to maintain normoglycaemia may require concentrations of 20–50% via central venous access. Hyponatraemia usually reflects hyperaldosteronism and/or fluid retention: fluids should be restricted and excessive sodium Table 2 Aetiology of NLF Durand et al. 2 (<1 year) n=80 Aw et al. 3 (<4 weeks) n=33 Infection Hepatitis B 6 0 HSV 1 and 2 2 5 HHV6 4 0 Enterovirus 0 0 Bacterial 0 1 Metabolic Tyrosinaemia 1 12 1 Mitochondrial 17 0 Urea cycle 2 1 Galactosaemia 2 3 Fatty acid oxidation 0 0 HFI 1 0 IE bile salts 0 0 CDGS 0 0 NH 13 16 Infiltrative/storage HLH 3 4 Leukaemia 1 0 Tumours 0 0 Niemann–Pick C 0 0 Other Drugs 1 1 AIH 3 0 Hypocortisolism 0 1 Hypoxic/ischaemic 0 0 Unknown 13 0 Most described causes of NLF are listed in the first column. Columns 2 and 3 give the number of cases in the series by Durand et al., who reported all infants under 1 year old with liver failure and Aw et al. who documented neonatal cases only. HSV, herpes simplex virus; HHV6, human herpes virus 6; HFI, hereditary fructose intolerance; IE bile salts, inborn error of bile salt synthesis; CDGS, congenital disorder of glycosylation syndrome; HLH, haemophagocytic lymphohistiocytosis; AIH, autoimmune hepatitis. 394 P. McClean, S.M. Davison administration should be avoided. Fluid restriction is also indicated for deteriorating oliguric renal impairment, which may ultimately require dialysis support and for control of ascites. Ascites may respond to optimizing serum albumin (infusing 5 ml/kg of 20% human albumin solution), and di- uretic therapy [oral spironolactone or intravenous potassium canrenoate (1 gϭ0.7 g spironolactone)]. If causing respiratory compromise, percutaneous drainage may be required. Ventilatory support should be considered early for neurological as well as respiratory deterioration. Inotropic support may be required, and as peripheral vasodilation may accompany liver failure, vasoconstrictors such as noradrenaline should be considered. Acidosis, reflecting hepatic and/or renal dysfunction, sepsis or a metabolic disorder may require bicarbonate correction. Hepatic encephalopathy manifests as irritability, poor sucking or excessive somnolence. Manage- ment includes restriction of protein intake to 2 g/ kg/24 h, and enteral lactulose. Cerebral oedema may accompany encephalopathy; fluid overload should be strictly avoided. Convulsions or deteriorating conscious level should prompt imaging to exclude intracerebral haemorrhage. Feeds should be withheld until galactosaemia, tyrosinaemia and urea cycle defects have been excluded. Withdrawal of lactose or fructose from infants with galactosaemia or hereditary fructose intolerance, respectively, leads to a dramatic improvement in clinical symptoms. Enteral feeding should be recommenced as soon as possible, although this may be compromised by fluid restriction or poor tolerance, necessitating temporary parenteral nutrition. Thrombocytopenia, coagulopathy and disseminated intravascular coagulation contribute to the risk of bleeding. Ranitidine should be prescribed for gastric protection. Intravenous vitamin K (300 µg/kg/24 h) is used to ensure adequate substrate for coagulation factors. Active bleeding requires correction of thrombocytopenia and coagulopathy. However, as the trend in the prothrombin time or international normalized ratio (INR) provides the best indicator of hepatic function and recovery, coagulopathy is not routinely corrected, unless severe. Antibiotics effective against Gram-negative organisms, streptococci and listeria should be administered even in the absence of overt sepsis, together with antifungal prophylaxis. Intravenous acyclovir (30 mg/kg/24 h) should be continued until herpes simplex virus (HSV) infection has been excluded. Intravenous n-acetylcysteine may improve survival in liver failure by enhancing tissue oxygen- ation. 4 Although firm evidence is lacking, its use in supportive therapy is becoming widespread. In preterm infants, the pharmacokinetics and excretion Table 3 General investigation of NLF Blood Haematology Full blood count and film, Blood group and Coombs test Prothrombin time, partial thromboplastin time, fibrinogen D dimers/fibrin degradation products Biochemistry Urea and electrolytes, creatine kinase, amylase Bilirubin (unconjugated/conjugated), transaminases, GT, alkaline phosphatase, albumin Acid–base balance Glucose, lactate, ammonia Cholesterol, triglycerides, free fatty acids, hydroxy butyrate Ferritin and transferrin saturation Plasma amino acids Galactose-1-phosphate uridyl transferase Carnitine/acyl carnitines Cortisol (9am) Serum bile salts Transferrin iso-electric focusing Alpha fetoprotein Toxicology including paracetamol Microbiology Bacterial/viral culture and PCR detection Viral serology: mother and infant (IgM in infant may be negative for several weeks) Other Storage for DNA Urine Biochemistry Amino acids, organic acids including succinyl acetone, orotic acid pH, ketones, reducing substances Toxicology Urinary bile salts Microbiology Bacterial/viral culture and PCR detection Other samples for viral culture/PCR viral detection Stool/rectal swab Guthrie card blood spots (HSV diagnosis) 28,29 Nasopharyngeal secretions Vesicle fluid Cerebrospinal fluid Eye swab Ascitic fluid Radiology Chest X-ray Echocardiography Doppler ultrasound scan of abdomen PCR, polymerase chain reaction. Neonatal liver failure 395 of intravenous n-acetylcysteine depend on weight and gestational age. 5 The role of plasmapheresis has diminished as techniques in transplantation have advanced. In one study of 49 children, including neonates, the major benefit from plasmapheresis was improved coagulation, with no effect on neurological complications. 6 There is increasing experience of extra- corporeal support systems such as the molecular adsorbent recirculation system (MARS) 7 in children, but none in the neonatal period. Liver transplantation The role of orthotopic liver transplantation (OLT) in the management of acute liver failure in adults and children is well established. However, there are only a few small series describing the particular medical and surgical challenges of OLT in neonates or small infants. 8,9 Infants with multi-organ failure, uncontrolled sepsis, generalized mitochondrial disorders or haemophagocytic lymphohistiocytosis should not be considered for liver transplantation because they will succumb to their underlying disease. Infants with NH or viral-induced liver failure are currently the most common groups undergoing OLT. 8,9 In spite of the introduction of techniques which use part of larger livers (reduced, split or monosegment grafts), there are still difficulties obtaining suitable donor organs, and the death rate on the waiting list remains high; 31% in one series. 3 The mortality after transplant is 40–50% compared with 30–40% in older children or adults trans- planted for acute liver failure. Complications include vascular thrombosis, sepsis and haemorrhage, but the incidence of acute rejection in this age group is very low. 8,9 Infectious causes of neonatal liver failure Enterovirus Enteroviruses are small, single-stranded RNA viruses, comprising polio-, Coxsackie A and B and Echoviruses. Severe infection may occur in neonates with multi-organ involvement, including hepatic necrosis. Echovirus, particularly serotype 11, is the most frequently identified virus. It is postulated that Echovirus causes hepatic failure by vascular rather than direct hepatocyte damage. 10 Symptoms typically develop between day 4 and 7, with fever, lethargy, poor feeding and abdominal distension due to hepatosplenomegaly and ascites. Convulsions may reflect meningoencephalitic involvement (in one-third of cases). The outcome ranges from spontaneous recovery to a rapidly fatal fulminant course. Mortality was estimated to be as high as 80% in 1986. 11 The role of the oral antiviral agent pleconaril in neonatal infection is being evaluated in a multicentre study. 12 Of three infants with life-threatening enteroviral hepatitis treated in an open study, two recovered completely. 13 Herpes simplex virus infection Herpes simplex virus (HSV) 1 and 2 are usually acquired due to exposure to infected maternal genital secretions or lesions at delivery, although intra-uterine and postnatal infection may occur. Risk of transmission is highest in seronegative mothers with primary infection at delivery. Ma- ternal viral shedding, however, is frequently asymptomatic. Delivery by Caesarean section significantly reduces the risk of neonatal infection. 14 Of 186 neonates with HSV infection, 60% presented after day 5. 15 HSV infection may involve the skin, eyes, mucous membranes, brain, lung and liver. Liver failure may occur with disseminated disease or as the only manifestation; onset may be sudden with lethargy, circulatory collapse and mild jaundice. Absence of typical skin lesions is common. 15 Standard treatment is intravenous acyclovir 30 mg/kg/24 h in three divided doses for 10–21 days. However, there is evidence that 60 mg/kg/ 24 h is associated with improved survival. 16 Acyclo- vir resistance occurs in 0.3% of immunocompetent and 4–7% of immunocompromised patients, 17 and may emerge during therapy. 18,19 Cross-resistance occurs to penciclovir and famciclovir. Alternative antiviral agents include foscarnet and cidofovir, but have increased toxicity. A novel group of antiviral agents has been described that inhibit viral helicase–primase enzymes; potential advantages observed in animal models include superior efficacy and reduced resistance. 20 Despite acyclovir therapy, the clinical course is often rapid deterioration, with death from multi- organ failure within days. Of 59 infants with disseminated disease treated with acyclovir between 1981 and 1997, survival was 47%. OLT should be considered. Hepatitis B virus infection Liver failure due to fulminant HBV infection is now rare due to screening and immunization 396 P. McClean, S.M. Davison policies. 2,21 In the World Health Organization European Region, 41 of 51 countries have implemented universal immunization. 22 In the UK, where this has not been implemented, universal HBV screening of all women during pregnancy since April 2000 has facilitated identification of at-risk infants. Fulminant HBV infection is particularly associated with transmission from a ‘low risk’ surface- antigen-positive mother who is e-antigen negative and e-antibody positive. The diagnosis should be considered irrespective of immunization history. Diagnosis is made by the detection of HBV DNA in peripheral blood. Spontaneous recovery may occur, but progressive deterioration may necessitate consideration for OLT. Experience of antiviral strategies for fulminant hepatitis B in infancy and childhood is limited. In adults, a potential role for lamivudine in fulminant hepatitis B has been suggested. 23 Pharmacokinetics and safety of lamivudine in neonates has been established. 24 Human herpes virus 6 Human herpes virus 6 (HHV6) as a cause of neonatal liver failure (NLF) has been reported occasionally. Of two infants presenting at day 3 and day 5 of life, 25 symptoms included fever, hypotonia, lethargy and shock. Both had raised hepatic transaminases and thrombocytopenia. One recovered spontaneously and the other died at day 15. Four infants with HHV6 in Durand et al's 2 series underwent OLT. Ganciclovir is effective treatment, 26 although resistance after prolonged exposure may occur. 27 Other viruses Adenovirus and parvovirus have also been associated with NLF. It is likely that other viruses may also be responsible. In one series, three of nine infants had ‘nonA–nonB’ liver failure, 8 and in another series, aetiology was undetermined in 13 of 80 (16%) infants. 2 Metabolic causes of NLF Neonatal haemochromatosis NH or neonatal iron storage disease is a rare disorder of abnormal iron storage presenting as liver failure within the first weeks of life. Iron accumulates in the fetal liver, pancreas, heart, thyroid and salivary glands but spares the reticuloendothelial system. The aetiology of this condition is unclear, and it most likely represents a clinicopathological endpoint of different in-utero insults to the fetal liver, including viral infection, immunological mechanisms and an inherited predisposition. 30 There is no evidence that these babies have the HFE gene for hereditary haemochromatosis seen in adults. Family studies have indicated both an autosomal recessive and a maternal mode of inheritance. In some families, once a child is born with NH, all subsequent children have been affected, and this has occurred irrespective of having different fathers. 30 Recurrent re-activation of maternal viral infection or immunological factors have also been postulated for maternal transmission. Early results suggest that repeated immunoglobulin administration during subsequent pregnancies in these mothers can significantly ameliorate the disease in the offspring. 31 Infants with NH are often premature and/or small for dates. The obstetric history may reveal previous miscarriages or stillborn infants. Oligo- hydramnios or polyhydramnios can complicate the pregnancy. The usual presentation is of acute decompensation of endstage liver disease in the first 24 h of life with hypoglycaemia, coagulopathy, hypoalbuminaemia and ascites. Jaundice does develop subsequently, but the serum transaminases are often within the normal range. Occasionally, infants present with a more protracted course and a milder coagulopathy. Abnormalities of serum bile acids suggestive of delta 4-3-oxosteroid 5-beta- reductase deficiency have been associated with a particularly severe presentation of NH with a bad prognosis. It has been suggested that this may reflect severe hepatocellular failure per se, and the infants do not respond to treatment with bile salts. 32 The diagnosis is reached by excluding other recognized causes of NLF and demonstrating evidence of iron overload. Much weight has been placed on finding high serum ferritin levels in this condition, but this is a frequent finding in neonatal liver disease of any aetiology. 33 A 95–100% saturation of transferrin is probably more specific for NH. 34,35 Extrahepatic iron deposition can be demonstrated in the salivary glands of the lip, although ensuring an adequate sample is difficult. 36 Decreased signal intensity on T2-weighted images on magnetic resonance imaging identifies iron in the liver and other tissues, and demonstrates a lack of siderosis in the spleen. 37 Liver histology shows varying degrees of hepatocyte loss, stromal collapse and fibrosis with Neonatal liver failure 397 regenerative nodules. Grade 3–4 siderosis with sparing of the Kupffer cells is a diagnostic feature. 31 Overall, the prognosis is poor with a mortality rate of 75% in a recent review. 30 General supportive management is instigated as soon as the diagnosis of NLF is recognized. There have been some reports of spontaneous recovery. Three infants responded to an anti-oxidant cocktail (Table 4) described in 1993. 38 Success with this treatment may be more likely in a milder subgroup if commenced early. 39 Liver transplantation has been successful in NH but with a high mortality on the waiting list (25–64%) and post-transplant (40–60%). 30,35,39 Mitochondrial respiratory chain disorders The mitochondrial respiratory chain consists of five protein complexes, plus ubiquinone and cytochrome c, located on the inner mitochondrial membrane. Other oxidative reactions within the mitochondria (the tricarboxylic acid cycle and fatty acid oxidation) generate reduced cofactors, such as flavin adenine dinucleotide (FADH 2 ) and nicotina- mide adenine dinucleotide (NADH), which pass electrons down the respiratory chain resulting in the formation of ATP. This process is called oxidative phosphorylation. NLF has been recognized in deficiencies of complex I, III and IV, multiple complex deficiencies and in mitochondrial DNA (mtDNA) depletion syndrome. Mitochondrial disorders of the electron transport proteins in the liver can present as NLF or as gradually progressive liver disease which can suddenly decompensate. 40 Most infants have extrahepatic features, although a few patients with respiratory chain defects isolated to the liver have been described. 41 Typically, severe liver failure develops in the first few weeks of life. Some cases have evidence of prenatal liver disease. Non-specific symptoms are common with lethargy, hypotonia, poor feeding and vomiting. Some of these features may be early signs of neurological involvement. More specific features of liver disease are conjugated hyperbilirubinaemia, coagulopathy, ascites and moderately raised transaminases (between two and 12 times normal). Hypoglycaemia is common and may be due to secondary inhibition of oxidation of fatty acids. Involvement of other organs result in neurological symptoms, myopathy, proximal renal tubular dysfunction, hypertrophic cardio- myopathy, and haematological and gastrointestinal disorders. 42 Elevated blood lactate should raise suspicion of a respiratory chain defect, particularly if it rises further either postprandially or following an intravenous glucose load. In the presence of renal tubular dysfunction, plasma lactate may be lower, but urinary lactate is raised. Ketone bodies are usually raised, with the plasma 3-OH- butyrate:acetoacetate ratio often greater than 2. Intermediates of the tricarboxylic acid cycle plus 3-methyl-glutaconic and 3-methylglutaric acid may be detected in urine. 42 Evidence of involvement of other organs should be sought by evaluating renal tubular function, echocardiography, and visual and auditory evoked responses. Neurological disease may be implied by a raised cerebrospinal fluid (CSF):plasma lactate ratio, an elevated CSF protein or abnormalities on magnetic resonance imaging. Liver histology almost invariably shows steatosis and fibrosis, which may have progressed to micronodular cirrhosis. Cholestasis, hepatocyte necrosis and increased iron staining may be present. Electron microscopy reveals increased numbers of abnormal mitochondria. Muscle may be histologically normal or show steatosis. Ragged red fibres are rare in infancy, but if present, these are very suggestive of a respiratory chain defect. The definitive diagnosis is made by measuring the enzymatic activity of respiratory chain complexes in affected tissues. Muscle is traditionally used as it is safer to obtain, and demonstration of extrahepatic disease in a patient with NLF is a contra-indication to OLT. 43 However, due to the variability of abnormal mtDNA within tissues (heteroplasmy), and the wide range of normal values, the results can be difficult to interpret. 40 Liver biopsies are potentially hazardous, and abnormalities may be artifactual due to the severity of the liver damage. mt DNA depletion syndrome is diagnosed by demonstrating a low ratio of mtDNA to nuclear DNA using Southern blotting techniques. Table 4 Anti-oxidant cocktail for NH N-acetylcysteine 140 mg/kg orally, then 70 mg/kg 4 hourly for 19 doses Selenium 2–3 µg/kg/day intravenously over 24 h Alpha tocopheryl polyethylene glycol succinate 20–30 IU/kg/24 h orally Prostaglandin E1 0.4–0.6 µg/kg/h intravenously for 2–4 weeks Desferrioxamine 30 mg/kg/24 h intravenously over 8 h until ferritin <500 µg/l 398 P. McClean, S.M. Davison Supportive treatment is usually the only therapeutic option. The use of ubiquinone, riboflavin and chloroacetate have not been shown to affect prognosis in patients presenting with NLF. Liver transplantation has been successful in a few patients with isolated liver disease and a less fulminant presentation 41,44 , but many patients succumb to neurological disease after transplant. Therefore, Thompson et al. 43 recommend that any evidence of extrahepatic disease is a contra-indication to OLT. Genetic counselling is difficult as many of these defects are sporadic. However, mtDNA originates from the ovum and maternal inheritance has been reported. 45 mtDNA depletion syndrome is probably due to a defect of a nuclear gene that codes for replication of the mitochondrial genome. Consan- guinity is common and the mode of inheritance appears to be autosomal-recessive. 46 Tyrosinaemia type 1 Hereditary tyrosinaemia type 1 (HT1), a recessive condition, is caused by a deficiency of fumaryl- acetoacetate hydoxylase (FAH), resulting in accumulation of toxic metabolites, fumaryl- acetoacetate and maleylacetoacetate, and their reduced derivatives succinylacetoacetate and succinylacetone. These are thought to be responsible for liver and proximal renal tubular dysfunction, and porphyria-like crises. The clinical presentation is variable but, in infants less than 6 months old, HT1 causes acute liver failure. Coagu- lopathy is a dominant feature and has been described in the absence of other signs of liver failure. 47 The serum transaminases are only mildly raised and some infants do not develop jaundice. Plasma tyrosine, phenylalanine and methionine are raised, but these can be elevated in liver disease. The presence of succinyl acetone in the urine is diagnostic. Very high levels of alpha fetoprotein (AFP) are typical. The diagnosis can be confirmed by measuring FAH activity in skin fibroblasts or liver cells. 2-(2-Nitro-4-trifluoromethylbenzoyl)-1-3-cyclohexanedione (NTBC), a bleaching herbicide, has been used to block the formation of toxic metabolites in patients with HT1 since 1992. Plasma tyrosine and phenylalanine remain raised unless the patient's dietary intake is restricted. In 80 patients commenced on NTBC before 6 months of age, the response rate was 90%, but there are still concerns that the future risk of developing hepatocellular carcinoma, a well-recognized complication of HT1, is not removed. 48 Ongoing monitoring of plasma amino acids, urinary succinylacetone, serum AFP, ophthalmological examination and hepatic imaging are important. Infants who do not respond to NTBC, or in whom hepatocellular carcinoma is suspected, are considered for OLT. This removes the risk of hepatocellular carcinoma in the future, and the children can return to a normal diet, but the renal tubular defect may not be corrected. 49 Haemophagocytic lymphohistiocytosis Haemophagocytic lymphohistiocytosis (HLH) is a rare disorder involving inappropriate activation of macrophages. It is divided into a primary, familial form and a secondary form usually triggered by infection in an immunocompromised host. The primary form can present as NLF with hepatosplenomegaly, markedly abnormal liver function tests and a high serum ferritin, which may cause confusion with NH. 50 Other diagnostic clues are fever, raised triglycerides, hypofibrinogenaemia and cytopenia. The diagnosis is usually confirmed by evidence of haemophagocytosis in a bone marrow aspirate. Initial management includes chemotherapy, usually dexamethasone and etoposide, but long-term survival requires a bone marrow transplant. 51 In one series, 5-year survival was 21%. 52 OLT is contra-indicated due to recurrence in the graft. 50 Conclusions NLF is an uncommon but challenging condition. It must be considered early in the differential diagnosis of coagulopathy in the newborn. Infection and metabolic disorders are the most common aetiologies. The overall mortality rate in the two series described in this paper was 60%. Sepsis, haemorrhage and multi-organ failure were the main causes of death. In each series, 24% of patients survived with their native liver following intensive supportive management and appropriate specific medical therapy. 2,3 In one series, five of 16 infants listed for a liver transplant died before an organ became available, 3 but in both series, the survival after transplant was 50%. Children with mitochondrial respiratory chain defects may succumb to previously unrecognized neurological disease post-transplant. The recognition and management of these infants require intensive co-operation between neonatal, hepatology and transplant teams. Neonatal liver failure 399 Practice points • NLF should be considered in any neonate with a coagulopathy which does not respond to intravenous vitamin K. • Jaundice and raised transaminases are not always present in infants with in-utero onset of cirrhosis. • Infection and metabolic diseases are the most frequent causes of NLF. • Feeds should be withheld until the results of galactose-1-phosphate uridyl transferase and urine organic and amino acids are available. • Monitor and maintain normoglycaemia. Research directions • Genetics of mitochondrial cytopathies. Can the ratio of mutant to normal mtDNA be influenced? • Pathogenesis of NH. References 1. Shneider BL. Neonatal liver failure. Curr Opin Pediatr 1996; 8:495–501. 2. Durand P, Debray D, Mandel R et al. Acute liver failure in infancy: a 14-year experience of a pediatric liver transplantation center. J Pediatr 2001;130:871–6. 3. Aw MM, Rela M, Heaton ND et al. Neonatal liver failure—a ten year experience [abst]. J Pediatr Gastroenterol Nutr 2001;32:381A. 4. Harrison PM, Wendon JA, Gimson AE et al. Improvement by acetylcysteine of hemodynamics and oxygen transport in fulminant hepatic failure. N Engl J Med 1991;324:1852–7. 5. Ahola T, Fellman V, Laaksonen R et al. Pharmacokinetics of intravenous N-acetylcysteine in pre-term newborn infants. Eur J Clin Pharmacol 1999;55:645–50. 6. Singer AL, Olthoff KM, Kim H et al. Role of plasmapheresis in the management of acute hepatic failure in children. Ann Surg 2001;234:418–24. 7. Stange J, Mitzner S, Risler T et al. Molecular adsorbent recycling system (MARS). Clinical results of a new membrane based blood purification system for bioartificial liver support. Artif Organs 1999;23:319–30. 8. Bonatti H, Muiesan P, Connelly S et al. Hepatic transplantation in children under 3 months of age: a single centre's experience. J Pediatr Surg 1997;32:486–8. 9. Noujaim HM, Mayer DA, Buckels JA et al. Techniques for and outcome of liver transplantation in neonates and infants weighing up to 5 kilograms. J Pediatr Surg 2002;37:159–64. 10. Wang J, Atchison RW, Walpusk J et al. Echovirus hepatic failure in infancy: report of four cases with speculation on the pathogenesis. Pediatr Dev Pathol 2001;4:454–60. 11. Modlin JF. Perinatal echovirus infection: insight from a literature review of 61 cases of serious infection and 16 outbreaks in nurseries. Rev Infect Dis 1986;8:918–26. 12. Sawyer MH. Enterovirus infections: diagnosis and treatment. Pediatr Infect Dis J 1999;18:1033–40. 13. Aradottir E, Alonso EM, Shulman ST. Severe neonatal enteroviral hepatitis treated with pleconaril. Pediatr Infect Dis J 2001;20:457–9. 14. Brown Z, Wald A, Morrow R et al. Effect of serologic status and Caesarean delivery on transmission rates of herpes simplex virus from mother to infant. JAMA 2003;289:203–9. 15. Kimberlin DW, Lin CY, Jacobs RF et al. Natural history of neonatal herpes simplex virus infections in the acyclovir era. Pediatrics 2001;108:223–9. 16. Kimberlin DW, Lin CY, Jacobs RF et al. Safety and efficacy of high-dose intravenous acyclovir in the management of neonatal herpes simplex virus infections. Pediatrics 2001; 108:230–8. 17. Bacon TH, Levin MJ, Leary JJ et al. Herpes simplex virus resistance to acyclovir and penciclovir after two decades of antiviral therapy. Clin Microbiol Rev 2003;16:114–28. 18. Oram RJ, Marcellino D, Strauss D et al. Characterization of an acyclovir-resistant herpes simplex virus type 2 strain isolated from a premature neonate. J Infect Dis 2000; 181:1458–61. 19. Levin MJ, Weinberg A, Leary JJ et al. Development of acyclovir resistant herpes simplex virus early during the treatment of herpes neonatorum. Pediatr Infect Dis J 2001; 20:1094–7. 20. Kleymann G, Fischer R, Betz UA et al. New helicase–primase inhibitors as drug candidates for the treatment of herpes simplex disease. Nat Med 2002;8:392–8. 21. Kao JH, Hsu HM, Shau WY et al. Universal hepatitis B vaccination and the decreased mortality from fulminant hepatitis in infants in Taiwan. J Paediatr 2001;139:349–52. 22. Van Damme P, Vorsters A. Hepatitis B control in Europe by universal vaccination programmes: the situation in 2001. J Med Virol 2002;67:433–9. 23. Santantonio T, Mazzola M, Pastore G. Lamivudine is safe and effective in fulminant hepatitis B. J Hepatol 1999;30:551. 24. Moodley D, Pillay K, Naidoo K et al. Pharmacokinetics of zidovudine and lamivudine in neonates following coadmin- istration of oral doses every 12 hours. J Clin Pharmacol 2001;41:732–41. 25. Mendel I, de Matteis M, Bertin C et al. Fulminant hepatitis in neonates with human herpesvirus 6 infection. Pediatr Infect Dis J 1995;14:993–7. 26. Tokimasa S, Hara J, Osugi Y et al. Ganciclovir is effective for prophylaxis and treatment of human herpesvirus-6 in allo- geneic stem cell transplantation. Bone Marrow Transplant 2002;29:595–8. 27. Manichanh C, Olivier-Aubron C, Lagarde JP et al. Selection of the same mutation in the U69 protein kinase gene of human herpesvirus-6 after prolonged exposure to ganciclovir in vitro and in vivo. J Gen Virol 2001;82:2767–76. 28. Ilona LF, Pia O, Marianne F et al. Detection of herpes simplex virus DNA in dried blood spots making a retrospec- tive diagnosis possible. J Clin Virol 2003;26:39–48. 29. Barbi M, Binda S, Primache V et al. Use of Guthrie cards for the early diagnosis of neonatal herpes simplex virus disease. Pediatr Infect Dis J 1998;17:251–2. 30. Kelly AL, Lunt PW, Rodrigues F et al. Classification and genetic features of neonatal haemochromatosis: a study of 27 affected pedigrees and molecular analysis of genes implicated in iron metabolism. J Med Genet 2001;38:599–610. 31. De Boissien D, Knisely AS. Neonatal hemochromatosis. Suchy FJ, Sokol RJ, Balistreri WF, editors. Liver disease in children. Philadelphia: Lippincott, Williams and Wilkins, 2001;641–7. 400 P. McClean, S.M. Davison 32. Siafakas CG, Jonas MM, Peraz-Atayde AR. Abnormal bile acid metabolism and neonatal hemochromatosis: a subset with poor prognosis. J Pediatr Gastroenterol Nutr 1997; 25:321–6. 33. Lee WS, McKiernan PJ, Kelly DA. Serum ferritin level in neonatal fulminant liver failure [letter]. Arch Dis Child Fetal Neonatal Ed 2001;85:F226. 34. Vohra P, Haller C, Emre S et al. Neonatal hemochromatosis: the importance of early recognition of liver failure. J Pediatr 2000;136:537–41. 35. Sigurdsson L, Reyes J, Kocoshis SA et al. Neonatal hemochromatosis: outcomes of pharmacologic and surgical therapies. J Pediatr Gastroenterol Nutr 1998;26:85–9. 36. Knisely AS, O'Shea PA, Stocks JF et al. Oropharngeal and upper respiratory tract mucosal-gland siderosis in neonatal hemochromatosis: an approach to biopsy diagnosis. J Pediatr 1988;113:871–4. 37. Hayes AM, Jaramillo D, Levy HL et al. Neonatal hemochromatosis: diagnosis with MR imaging. Am J Roentgenol 1992; 159:623–5. 38. Shamieh I, Kibort PK, Suchy FJ et al. Antioxidant therapy for neonatal iron storage disease [abst]. Pediatr Res 1993; 33:109A. 39. Flynn DM, Mohan N, McKiernan P et al. Progress in treatment and outcome for children with neonatal haemochromatosis. Arch Dis Child Fetal Neonatal Ed 2003;88:F124–7. 40. Cormier-Daire V, Chretien D, Rustin P et al. Neonatal and delayed-onset liver involvement in disorders of oxidative phosphorylation. J Pediatr 1997;130:817–22. 41. Goncalves I, Hermans D, Chretien D et al. Mitochondrial respiratory chain defect: a new etiology for neonatal cholestasis and early liver insufficiency. J Hepatol 1995; 23:290–4. 42. Munnich A, Rotig A, Chretien D et al. Clinical presentations and laboratory investigations in respiratory chain deficiency. Eur J Pediatr 1996;155:262–74. 43. Thompson M, McKiernan P, Buckels J et al. Generalised mitochondrial cytopathy is an absolute contraindication to orthotopic liver transplant in childhood. J Pediatr Gastroen- terol Nutr 1998;26:478–81. 44. Dubern B, Broue P, Dubuisson C et al. Orthotopic liver transplantation for mitochondrial respiratory chain disorders: a study of 5 children. Transplantation 2001;71:633–7. 45. Rotig A, Bessis J-L, Romero N et al. Maternally inherited duplication of the mitochondrial genome in a syndrome of proximal tubulopathy, diabetes mellitus and cerebellar ataxia. Am J Hum Genet 1992;50:364–70. 46. Bakker HD, Scholte HR, Dingemans KP, et al. Depletion of mitochondrial deoxyribonucleic acid in a family with fatal neonatal liver disease. J Pediatr 1996;128:683–7. 47. Croffie JM, Gupta SK, Chung SKF et al. Tyrosinemia type 1 should be suspected in infants with severe coagulopathy even in the absence of other signs of liver failure. Pediatrics 1999;103:675–8. 48. Holme E, Lindstedt S. Nontransplant treatment of tyrosinemia. Clin Liver Dis 2000;4:805–14. 49. Mohan N, McKiernan P, Preece MA et al. Indications and outcome of liver transplantation in tyrosinaemia type 1. Eur J Pediatr 1999;158(Suppl 2):S49–54. 50. Parizhskaya M, Reyes J, Jaffe R. Hemophagocytic syndrome presenting as acute hepatic failure in two infants: clinical overlap with neonatal hemochromatosis. Pediatr Develop Pathol 1999;2:360–6. 51. Sung L, King SM, Carcao M et al. Adverse outcomes in primary hemophagocytic lymphohistiocytosis. J Pediatr He- matol Oncol 2002;24:550–4. 52. Arico M, Janka G, Fischer A et al. Hemophagocytic lymphohistiocytosis. Report of 122 children from the International Registry. Leukaemia 1996;10:197–203. 53. Williams MD, Chalmers EA, Gibson BES. The investigation and management of neonatal haemostasis and thrombosis. Br J Haematol 2002;119:295–309. Neonatal liver failure 401 Review article Parenteral nutrition associated liver disease Stuart S. Kaufman a* , Gabriel E. Gondolesi b , Thomas M. Fishbein c a Department of Gastroenterology and Nutrition, Children’s National Medical Center, 111 Michigan Avenue, N.W. Washington, DC 20010, USA b Recanti/Miller Transplantation Institute, The Mount Sinai Hospital and School of Medicine, One Gustave L. Levy Place, Box 1104, New York, NY 10029, USA c Department of Surgery, Georgetown University Hospital, 4 PHC 3800 Reservoir Road, N.W. Washington, DC 20007, USA Summary Liver disease is relatively common during parenteral nutrition (PN). Cholestasis predominates in infants, and ranges in severity from mild increases in plasma conjugated bilirubin to progressive liver failure that results in death of the patient. Severity of liver disease depends primarily on the magnitude of the underlying intestinal problem that indicated PN. Transient ileus resulting from a non- intestinal disorder usually results in trivial, self-limited liver injury. Removal of a large segment of the intestinal tract because of necrotizing enterocolitis or a congenital malformation predicts a more prolonged course with a guarded prognosis, particularly when initially complicated by sepsis. Pathogenesis of PN-associated liver disease is not completely understood. There is no proven treatment short of ending PN through adaptation of remnant intestine or intestinal transplantation, with or without a concurrent liver graft. Effective interventions that are less radical than transplantation are needed. Research that includes prospective trials of novel therapies in PN-associated liver disease is the key to improving outcome. © 2003 Elsevier Ltd. All rights reserved. KEYWORDS Intestine; Liver disease; Intestinal failure; Short bowel syndrome; Parenteral nutrition; Transplant Introduction Parenteral nutrition (PN) therapy has been available for infants with inadequate gastrointestinal tract function for more than 30 years. Hepatobiliary complications of PN were recognized early in the experience. PN-associated liver disease (PNALD) remains the leading cause of neonatal cholestasis 1 and the primary indication for combined liver and intestinal transplantation in children. 2,3 These facts emphasize that understanding of the aetiology of PNALD remains incomplete, and that no preventative measure or treatment has proven unequivocally to be effective. The early assumption that a specific shortcoming of PN, e.g. a toxic excess of one or more nutrients or a critical deficiency of others, causes liver injury is simplis- tic. Rather, liver injury during PN therapy results from the entire clinical setting that prompted utilization of PN, and for that reason, the term ‘PN-induced liver disease‘ has largely been dis- carded in favour of less-biased terms such as PNALD. Uncomplicated PNALD When ileus owing to extreme prematurity and severe respiratory tract disease is the indication for PN, incidence of conjugated hyperbilirubinaemia ranges from around 10 to 25%. 4 Premature infants receiving PN may be more likely to develop cholestasis than full-term infants. 5 Conjugated * Corresponding author. Tel.: +1-202-884-3058; fax: +1-202-884-4156 E-mail address: [email protected] (S.S. Kaufman). Seminars in NEONATOLOGY www.elsevierhealth.com/journals/siny Seminars in Neonatology (2003) 8, 375–381 1084-2756/03/$ - see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1084-2756(03)00094-0 bilirubin and gamma-glutamyl transferase (GGT) rise within 1–4 weeks of initiating therapy. 4,6 When adequate gastrointestinal tract function returns within 4–6 weeks, biochemical indications of liver injury resolve quickly. 4,5 As biochemical abnormalities are not only mild but also transient, liver biopsy is now performed rarely. Hepatocellular and canalicular cholestasis, bile duct proliferation, fibrous expansion of portal tracts (stage 1 fibrosis), and extramedullary haematopoiesis have been described after 2 weeks of PN. 7 These lesions are predictably reversible. Complicated PNALD When a serious gastrointestinal disorder indicates PN in the neonate, both incidence and potential severity of PNALD increase substantially. 8–10 Incidence of PNALD in surgical patients approxi- mates 50–60%, although improvements in PN may have reduced the incidence recently. 8 Full- term infants may be as vulnerable as premature infants. 9,11 Major intestinal surgery in the newborn period places affected patients at risk of developing what is now termed ‘intestinal failure’, 12 which may be defined operationally in infancy when PN has been required for a minimum of 3 months. Intestinal failure in neonates is most often the result of anatomic short bowel syndrome from severe necrotizing enterocolitis (NEC), and congenital malformations such as gastroschisis and intestinal atresia. Much less commonly, intestinal failure results from severe functional disturbances of the gastrointestinal tract, including the intestinal pseudo-obstruction syndromes 13 and congenital enterocyte transport disorders such as microvillus inclusion disease and tufting enteropathy. 14,15 Hirschsprung disease is also likely to result in intestinal failure if the aganglionic segment includes the entire colon and a portion of small bowel, in which case both loss of gut length and dysmotility of remnant bowel probably contribute to PN dependence. 16 In infants with intestinal failure, PNALD frequently becomes apparent with hyperbilirubinaemia that begins during an initial bout of sepsis and continues to rise over the next 12–18 months as PN is continued. 9 Subsequent episodes of bacterae- mia and fungaemia often worsens hyperbilirubinaemia, which may not return to baseline despite successful treatment. 17 As PN maintains appropriate nutritional status, patients look surprisingly well except for persistent jaundice. Physical examination of the abdomen betrays the overall well appearance of these infants, as it demonstrates enlargement of the liver and features of portal hypertension that include progressive splenomegaly and visible abdominal wall collateral circulation. Eventually, the liver and spleen may occupy almost the entire abdominal cavity. Typical features of portal hypertension that are observed with normal gastrointestinal anatomy, including clinically notable ascites and oesophagogastric varices, are uncommon. 2 In patients with intestinal failure, severe gastrointestinal bleeding usually emanates from gastrostomy orifices and the surfaces of enterostomies, if present, although dilated veins are almost never visible. Laboratory tests demonstrate conjugated hyperbilirubinaemia and functional hypersplenism (thrombocytopenia and, later, neutropenia), which are of prognostic value. A platelet count of 100 000/µl is associated with a 1-year survival of only about 30%, 18 while a total plasma bilirubin of about 10 mg/dl predicts death within 6 months. 19 Elevations of other blood tests related to liver injury and cholestasis, viz. alanine aminotransferase and GGT, do not correlate with disease severity, and routine laboratory indications of impaired synthetic function, e.g. prolongation of prothrombin time and hypoglycaemia during brief fasting, occur very late in the evolution of PNALD. Death most commonly results from multi-organ failure precipitated by bacterial or fungal sepsis. While irritability and lethargy consistent with hepatic encephalopathy and fetor hepaticus are often observed late in the course of progressive PNALD, death directly attributable to cerebral oedema and brain-stem herniation is unusual. Histopathology of complicated PNALD Histological changes in progressive PNALD are those of increasing intracellular and canalicular cholestasis, portal and lobular inflammation, macrophage hyperplasia, interlobular bile duct proliferation, and fibrosis. The appearance may simulate mech- anical biliary tract obstruction, e.g. biliary atresia. 20 Fibrosis is initially concentrated in portal areas (stage 1), and later extends as septae into the hepatic lobule (stage 2). More extensive fibrosis that connects adjacent portal zones define bridging fibrosis (stage 3). In the most advanced stage of fibrosis, i.e. cirrhosis (stage 4), regenerative nodules are superimposed on extensive bridging. Cirrhosis has been confirmed after just 4 months of PN, 7 development of which yields an actuarial 1-year survival of about 25–30%. 18 Prospective 376 S.S. Kaufman et al. studies delineating a relationship between deteriorating clinical and laboratory markers of liver function and portal hypertension vs. liver histology are not available. Response of the liver to cessation of PN, made possible either by adaptation of remnant bowel to full enteral nutrition or by intestinal transplantation, is variable. Cholestasis associated with stage 1 or 2 fibrosis is reversible. 21 Cholestasis often diminishes before complete discontinuation of PN, although the quantity of enteral calories that must be delivered or assimilated has not been established. 9 Despite clinical resolution of cholestasis and portal hypertension, fibrosis may not regress completely; the long-term significance of which is unclear. 20,22–24 Infants with stage 3 or 4 fibrosis need a combined liver and intestinal transplant, because this degree of fibrosis is probably not reversible. PNALD and indications for transplantation Reported mortality of paediatric patients with neonatal intestinal failure who are anticipated to require PN indefinitely varies markedly, between 0 and 90%, depending largely upon how indefinite PN is defined. 25–29 Death usually results from complications of liver failure. When there is a significant risk of liver failure with no reasonable prospect of ending PN, intestinal transplantation should be considered. 2 Severity of PNALD determines the type of transplant to be performed. Rapidly progressive and advanced disease requires combined liver and intestinal transplantation. Due to the severe shortage of suitable donor organs, around 50% of infants waiting for a combined liver and intestinal transplant die before transplant. 18,21,30 This fact, combined with the common uncertainty about the rate of progression of PNALD in individual patients, requires early referral and listing for transplantation, even before it is clear that PNALD shall progress to end-stage. A plasma bilirubin that is continuously elevated to more than 3–4 mg/dl by age 6 months despite tolerance of some enteral feeding justifies referral to an intestinal transplant centre. 2 If cholestasis appears to have resolved before suitable donor organs become available, which may take more than a year, transplantation is deferred or cancelled. In other infants, PNALD may progress slowly, albeit relentlessly, and permit isolated intestinal transplantation. Patients with PNALD who are likely to tolerate an isolated intestinal transplant generally have few or no visible abdominal veins, less marked hepatosplenomegaly, a platelet count greater than 150 000/µl, and total plasma bilirubin less than 6–7 mg/dl. 31 There are no data to show that repeated liver biopsy is superior to ongoing clinical assessment in establishing the need for liver replacement during intestinal transplantation. Pathogenesis and prevention of PNALD The risk of death from PNALD in infants and young children is not directly related to the duration of PN. 9,32 The median age of death on PN in three recent studies, mainly from liver failure, was only 19 months, 25,26,29 which is similar to the duration of PN in paediatric patients able to end therapy. 27,29,33 However, the risk of death and, hence, the need for referral for transplantation, can be predicted based on the probability of successfully ending PN. This apparent contradiction is reconciled by the observation that advanced liver disease rarely develops in infants who, based on favourable remnant bowel anatomy and related factors, can be predicted to end PN eventually, even if the duration of PN is especially long. 24 Factors predicting progressive PNALD include the following. Extreme short bowel syndrome The liver is particularly vulnerable to injury during PN therapy when loss of small bowel is severe. 34 Most paediatric patients with neonatal-onset intestinal failure succumbing to liver failure have no more than 50 cm of small bowel, often much less. 9,29 Termination of small bowel as a stoma, i.e. absence of enterocolonic continuity, may place patients at further risk. 27 Therapeutic implications Surgery designed to lengthen remnant small bowel may become technically feasible beyond the neonatal period. A bowel-lengthening operation is most likely to reduce PN requirements when sub- stantial enteral calories are already tolerated, and PNALD is absent or mild and non-progressive. As the length of bowel added is modest, this type of operation is not likely either to reduce PN requirements markedly or forestall liver failure in infants with less than 30–50 cm of remnant small bowel; the population at greatest risk of progressive PNALD. 35 These infants should be referred for isolated intestinal transplantation before PNALD progresses to end-stage. 31 Parenteral-nutrition-associated liver disease 377 Local and systemic sepsis Infants who develop progressive and ultimately fatal PNALD are more likely to have experienced early sepsis in association with bowel resection, on average within 30 days of birth. 9 They may also have had an increased frequency of recurrent sepsis. 17,27 Animal studies indicate that following massive intestinal resection, intestinal bacteria and their byproducts, including endotoxins and peptidoglycans, translocate to the liver via the portal venous and lymphatic systems. 36 Bacterial byproducts inhibit hepatocellular bile acid transport and activate hepatic macrophages via locally produced cytokines such as tumour necrosis factor- . 37,38 Hepatocellular cholestasis and necrosis, inflammation and fibrosis then supervene, mediated by factors that include increased levels of reactive oxygen species. 39 PN may directly contribute to the production of pro-cholestatic cytokines when other systemic stresses are present. 40 Furthermore, enteral feeding, that is essential to intestinal growth (and ending PN) after major resection, may have the undesirable effect of promoting overgrowth of potentially toxic gut flora. The greater the number of potentially toxic bacteria in the gut lumen, the more deleterious the potential impact of translocation on the liver. 41 Chronic stasis of intestinal content secondary either to partial obstruction or dysmotility of remnant small bowel exacerbates bacterial overgrowth and PNALD. 8 Therapeutic implications In experimental models, suppression of intestinal bacteria, particularly of strict anaerobes using agents such as metronidazole, may be beneficial to the liver. However, clinical studies have only rarely indicated benefit. 42 Current animal research suggests that elevation of hepatic glutathione, by means of supplementation with dietary precursors including glutamine, may minimize hepatic injury associated with PN and, possibly, massive intestinal resection and intra-abdominal sepsis. 39,43 Similarly, any manoeuvre that reduces the prevalence of venous-catheter-associated sepsis may also spare the livers of PN-dependent patients, 17 although not all clinical experience supports this assertion. 25 Lack of early enteral feeding Tolerance of little, if any, enteral nutrition after major intestinal resection is another risk factor for fatal PNALD. 9 Enteral feeding may protect the liver by promoting enterohepatic recirculation of bile acids, particularly if initiated early and if some ileum remains after intestinal resection. Enteral feeding may also promote bile flow by stimulating gallbladder contraction and by lessening postresection increases in intestinal permeability. Lack of enteral nutrition and consequent gallbladder stasis are risk factors for gallbladder disease which includes acalculous cholecystitis, gallbladder sludge and gallstones. 44 It is unclear whether failure to tolerate enteral feeding represents an independent risk for progressive PNALD, or if prolonged fasting and PNALD are simply co- dependent consequences of the original surgical disorder. 17 Therapeutic implications Early enteral feeding to the extent permitted by remnant bowel anatomy and function may be important to promote bile flow and prevent or retard PNALD. However, overfeeding may exacerbate bacterial overgrowth that is potentially hepatotoxic. Although suppression of bacterial overgrowth may improve digestive function, 29 beneficial effects on the liver remain uncertain. Development of gallstones in a jaundiced patient with PNALD despite some enteral feeding is not prima facie evidence of biliary tract obstruction, because advanced liver disease is an independent risk factor for cholelithiasis. In our experience, cholestasis is only likely to be improved by cholecystectomy, a relatively high-risk operation with advanced liver disease, when the clinical presentation suggests obstruction, i.e. biliary colic, pancreatitis or inflammatory disease. These symptoms are uncommon in infants with PNALD. Cholecystokinin (CCK), by means of increasing intrahepatic bile flow, gallbladder contraction or both, has been used to prevent and treat PN- associated hyperbilirubinaemia at a dose of about 0.02 µg/kg two or three times daily. 44,45 Whether CCK therapy reduces fibrosis or affects long-term outcome of potentially progressive PNALD remains to be demonstrated. There is no convincing evidence that enterally administered ursodeoxycholic acid is useful in PNALD. 46 The possibility that poor absorption inhibits therapeutic efficacy awaits the availability and testing of a suitable intravenous preparation. PN composition and delivery There is an association between parenteral calorie intake greater than around 70% of the calculated energy requirement and an increased propensity 378 S.S. Kaufman et al. for PNALD. A high percentage of PN may not be an independent risk factor for PNALD but, rather, simply reflect the severity of co-existing gut loss. 34 However, parenteral calorie intake consistently greater than metabolic expenditures may also be deleterious to the liver. 37 There is no evidence that intravenous glucose is inherently hepatotoxic, but intravenous emulsified lipid intake greater than 1 g/kg/day is associated with fatal liver disease. 47 The effect may reflect direct hepatotoxicity of some components in lipid emulsion. 48 Additionally, lipid may also provide substrates to fuel the systemic inflammatory response and its deleterious effect on the liver. 49 Copper and manganese, which are routinely included in PN, are potentially hepatotoxic. 23,50 As excretion of copper and manganese is predominantly biliary, retention of these elements by a cholestatic liver may produce additional hepatic injury and justify the monitoring of blood concentrations. Taurine may be conditionally essential in young infants for hepatocellular bile salt conjugation and secretion. Although use of taurine- containing amino acid solutions, e.g. TrophAmine ® (Kendall McGaw, Inc., Irvine, CA, USA), in infants with intestinal failure is logical and without recognized risk, these solutions do not definitively alter the incidence and severity of PNALD. 51 Carnitine, which is required for efficient oxidation of long- chain fatty acids, may also be conditionally essential in patients receiving long-term PN. Inclusion of carnitine in PN based on plasma concentrations is also logical, but there is no evidence that carnitine constitutes effective prophylaxis against PNALD. 52 Choline deficiency in long-term PN may, like carnitine, contribute to PNALD, given the essential nature of this substance and its requirement for efficient hepatic lipid metabolism. 53 Whether routine choline supplementation of PN solutions in intestinal failure patients reduces the frequency and severity of potentially severe PNALD is the subject of current clinical investigation. Inter- mittent infusion of PN may protect the liver by promoting efficient energy utilization, particularly in the presence of hepatotoxic pro-inflammatory stresses. 54 Benefit is most likely to be obtained when total bilirubin is less than 10 mg/dl. Optimal duration of the parenteral fast has not been established. 55 Conclusion PNALD remains a relatively common complication of PN therapy. Prognosis relates primarily to the type and severity of the underlying intestinal disorder that prompted use of PN. Mild and transient gastrointestinal dysfunction causes trivial and self- limited liver injury. Major intestinal resection, particularly in conjunction with abdominal sepsis, predicts a more prolonged and complicated course with a guarded prognosis. Prevention of PNALD remains difficult because the key to successful prevention requires a better understanding of its pathophysiology. Although combined liver and intestinal transplantation may permit survival of some patients, less drastic and more effective interventions are needed. Research that includes prospective trials of novel therapies in PNALD is the key to improving outcome. Practice points • Occurrence and prognosis of PNALD is a function of the severity and chronicity of the gastrointestinal dysfunction that originally prompted PN. • Risk of chronic hepatic failure secondary to PNALD is highest in infants with intestinal failure. • Apart from jaundice, infants with severe PNALD and portal hypertension look well until terminal hepatic decompensation. • Infants requiring indefinite PN with liver disease that has not improved by age 6 months should be evaluated for intestinal transplantation. • There remains no proven, effective therapy for PNALD, but early enteral feeding, minimization of sepsis, and avoidance of excess calorie intake may be beneficial. Research directions • Clarifying the effect of protracted or recurring abdominal and systemic inflammation on course of liver disease. • Clarifying inflammatory pathways involved in producing cholestasis, hepatic necrosis and fibrosis. • Improving identification of early clinical risk factors for progressive PNALD to guide transplant referral. • Investigation of novel anti-inflammatory therapies for treatment of PNALD. Parenteral-nutrition-associated liver disease 379 References 1. Stormon MO, Dorney SFA, Kamath KR et al. The changing pattern of diagnosis of neonatal cholestasis. J Paediatr Child Health 2001;37:47–50. 2. Kaufman SS, Atkinson JB, Bianchi A et al. Indications for pediatric intestinal transplantation: a position paper of the American Society of Transplantation. Pediatr Transplant 2001;5:80–7. 3. Smith R. 24 Aug 2002. The intestinal transplant registry. Available from: ^http://www.intestinaltransplant.org&. 4. Beale EF, Nelson RM, Bucciarelli RL et al. 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Trophic enteral nutrition increases hepatic glutathione and protects against peroxidative damage after exposure to endotoxin. J Pediatr Surg 2003;38:844–7. 44. Angelico M, Della Guardia M. Review article: hepatobiliary complications associated with total parenteral nutrition. Aliment Pharmacol Ther 2000;14:54–7. 45. Teitelbaum DH, Han-Markey T, Drongowski RA et al. Use of cholecystokinin to prevent the development of parenteral nutrition-associated cholestasis. J Parenter Enteral Nutr 1997;21:100–3. 46. Heubi JE, Wiechmann DA, Creutzinger V et al. Taurourso- deoxycholic acid (TIDCA) in the prevention of total parenteral nutrition-associated liver disease. J Pediatr 2002;141:237–42. 47. Cavicchi M, Beau P, Crenn P et al. Prevalence of liver disease and contributing factors in patients receiving home parenteral nutrition for permanent intestinal failure. Ann Intern Med 2000;132:525–32. 48. Iyer KR, Spitz L, Clayton P. New insight into mechanisms of parenteral nutrition-associated cholestasis; role of plant sterols. J Pediatr Surg 1998;33:1–6. 49. McCowen K, Burke PA, Bistrian BR. Liver disease and home parenteral nutrition [Letter]. Ann Intern Med 2000; 133:1009–10. 50. Fuhrman MP, Herrmann V, Masidonski P et al. Pancyto- penia after removal of copper from total parenteral nutrition. J Parenter Enteral Nutr 2000;24:361–6. 51. Hata S, Kubota A, Okada A. A pediatric amino acid solution for total parenteral nutrition does not affect liver function test results in neonates. Surg Today 2002;32:800–3. 52. Moukarzel AA, Dahlstrom KA, Buchman AL et al. Carnitine status of children receiving long-term total parenteral nutrition: a longitudinal prospective study. J Pediatr 1992; 120:759–62. 53. Buchman AL, Ament ME, Sohel M et al. Choline deficiency causes reversible hepatic abnormalities in patients receiving parenteral nutrition: proof of a human choline requirement: a placebo-controlled trial. J Parenter Enteral Nutr 2001;25:260–8. 54. Morikawa N, Suematsu M, Kyokane T et al. Discontinuous total parenteral nutrition prevents postischemic mitochondrial dysfunction in rat liver. Hepatology 1998;28:1289–99. 55. Hwang T-L, Lue M-C, Chen L-L. Early use of cyclic TPN prevents further deterioration of liver functions for the TPN patients with impaired liver function. Hepatogastro- enterology 2000;47:1347–50. Parenteral-nutrition-associated liver disease 381 Biliary atresia Hiroyuki Kobayashi a , Mark D. Stringer b* a Department of Pediatric Surgery, Juntendo University, School of Medicine, Bunkyo-ku, Tokyo, Japan b Children's Liver & GI Unit, St James's University Hospital, Leeds LS9 7TF, UK Summary Biliary atresia (BA) is a congenital obliterative cholangiopathy of unknown aetiology, affecting both the intra- and extrahepatic bile ducts. Although relatively rare, BA must be excluded in any infant with conjugated hyperbilirubinaemia since the prognosis is improved by early diagnosis and prompt surgery. At least two phenotypes of BA are currently recognized; the syndromic variety is associated with other congenital anomalies and a poorer outcome. The results of treatment have steadily improved and, with a combination of timely expert surgery (Kasai portoenterostomy) and liver transplantation in specialist centres, good quality long-term survival is now possible in more than 90% of affected patients. A better understanding of the aetiology of BA and the pathogenesis of hepatic fibrosis is needed in order to develop new therapeutic strategies. © 2003 Elsevier Ltd. All rights reserved. KEYWORDS Biliary atresia; Neonatal jaundice; Portoenterostomy The aims of this article are to focus on important clinical aspects of biliary atresia (BA) relevant to routine clinical practice, and to highlight some of the more recent scientific and clinical advances in our understanding of this condition. BA is a congenital obliterative cholangiopathy of unknown aetiology. It is a major cause of obstruc- tive jaundice in neonates. Untreated infants succumb to liver failure within a year or two. In the late 1950s, Morio Kasai, a Japanese surgeon, reported the presence of patent microscopic biliary channels at the porta hepatis in young infants with BA. Exposure of these channels by radical excision of atretic extrahepatic biliary remnants could result in effective drainage of bile, especially if the operation was performed within eight weeks of birth. The Kasai portoenterostomy operation is now accepted as the standard operation for the condition. However, despite this procedure, BA remains the foremost indication for liver transplantation in infants and children. Incidence BA is rare. Reliable incidence figures are available from France (one in 19 500 live births), the UK and Eire (one in 16 700 live births), Georgia in the USA and Sweden (one in 14 000 live births). 1–4 The highest recorded incidence is in French Polynesia. 1 No significant seasonal variation or clustering was found in the French study. In most large series, there is a slight female preponderance. Aetiology and pathogenesis Despite intensive interest and investigation, the cause of BA remains unknown. Two different forms are described. 5 In syndromic BA (also known as the embryonic type), there are associated congenital anomalies such as an interrupted inferior vena cava, preduodenal portal vein, intestinal mal- rotation, situs inversus, cardiac defects and polysplenia. In this variety, which accounts for about 10–20% of all cases, BA is likely to be due to a developmental insult occurring during differentiation of the hepatic diverticulum from the foregut of the embryo. A possible relationship between syndromic BA and maternal diabetes has been reported. 6 Non-syndromic BA (also known as the * Corresponding author. Tel.: +44-113-206-6689; fax: +44-113-206-6691 E-mail address: [email protected] (M.D. Stringer). Seminars in NEONATOLOGY www.elsevierhealth.com/journals/siny Seminars in Neonatology (2003) 8, 383–391 1084-2756/03/$ - see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1084-2756(03)00065-4 perinatal type) may have its origins later in gestation and run a different clinical course. There is no ideal animal model for BA, and this has slowed our understanding of its pathogenesis. Various aetiologic mechanisms have been postulated including intra-uterine or perinatal viral infection, genetic mutation, abnormal ductal plate remodelling, a vascular or metabolic insult to the developing biliary tree, pancreaticobiliary ductal malunion and immunologically mediated inflammation. Recent observations would suggest that BA is not a single disease entity. Viral agents Reovirus type 3 infection, rotavirus, cytomegalovirus, papillomavirus and Epstein–Barr virus have all been proposed as possible aetiologic agents but conclusive evidence is lacking. Reovirus type 3 can cause an inflammatory cholangiopathy in weaning mice but the condition is not progressive and the animals recover. Genetic factors Generally, BA is not considered to be an inherited disorder. However, genetic mutations that result in defective morphogenesis may be important in syndromic BA. Transgenic mice with a recessive deletion of the inversin gene have situs inversus and an interrupted extrahepatic biliary tree. 5,7 Muta- tions of the CFC1 gene, which is involved in left– right axis determination in humans, have recently been identified in a few patients with syndromic BA. 8 Abnormal ductal plate remodelling Intrahepatic bile ducts are derived from primitive hepatocytes which form a sleeve (the ductal plate) around intrahepatic portal vein branches and associated mesenchyme in early gestation. Remodel- ling of the ductal plate in fetal life results in the formation of the intrahepatic biliary system. Tan et al. (1994) emphasized the similarities in cytokeratin immunostaining between the biliary ductules in BA and normal first-trimester fetal bile ducts. 9 They suggested that non-syndromic BA might be caused by a failure of bile duct remodelling at the hepatic hilum, with persistence of fetal bile ducts poorly supported by mesenchyme. Immune-mediated mechanisms Biliary obstruction in non-syndromic BA is progressive. A few such infants even have a history of pigmented stools at birth. Pathogenesis may involve an immune-mediated inflammatory destruction of intra- and extrahepatic bile ducts. Several studies have investigated whether bile duct epithelial cells are susceptible to immune/ inflammatory attack because of abnormal expression of human leukocyte antigens (HLA) or intercellular adhesion molecules on their surface. 10,11 Silveira et al. (1993) showed a greater than three-fold increase in HLA-B12 antigen in BA patients compared with controls, particularly in those with no associated malformations; haplo- types A9–B5 and A28–B35 were also increased. 12 Aberrant expression of class II HLA-DR antigens on biliary epithelial cells and damaged hepatocytes in BA patients may render these tissues more susceptible to immune-mediated damage by cytotoxic T cells or locally released cytokines. 13 Dillon et al. (1994) reported increased expression of intercellular adhesion molecule-1 (ICAM-1) on bile duct epithelium in patients with BA, and suggested that this might have a role in immune-mediated damage. 11 Kobayashi et al. (2001) found a strong expression of ICAM-1 on proliferating bile ductules, endothelial cells and hepatocytes in BA. 14 There was a direct relationship between the degree of ductular expression of ICAM-1 and disease severity. They postulated that ICAM-1 might be important in the development of cirrhosis. More recently, interest has focused on co- stimulatory molecules. Two processes are involved in the activation of T lymphocytes by antigen presenting cells (APC). One relates to the expression of major histocompatibility complex class II molecules which interact directly with T-cell receptors. The other depends on the expression of B7 antigens on APC, and provides the second (co-stimulatory) signal to T lymphocytes through CD28. 15 In postoperative BA patients with good liver function, co-stimulatory antigens (B7-1, B7-2 and CD40) are expressed only on bile duct epithelial cells, whilst in patients with failing livers, these markers are found on the surface of Kupffer cells, dendritic cells, sinusoidal endothelial cells (SEC) and in the cytoplasm of hepatocytes. 16 This suggests that the biliary epithelium and hepatocytes in BA are susceptible to immune recognition and destruction. Agents that block or prevent co-stimulatory pathways might offer a new therapeutic approach to limiting liver damage. None of these mechanisms are mutually exclusive. In addition, for some theories, it is not known whether the observations are primary or secondary events. One current hypothesis is that in the aetiology of non-syndromic BA, a viral or other toxic 384 H. Kobayashi, M.D. Stringer insult to the bile duct epithelium induces the expression of new antigens on the biliary epithelial cell surface. 17 Coupled with a genetically predeter- mined susceptibility mediated via histocompatibility antigens, these neoantigens are recognized by circulating T lymphocytes, resulting in a cell- mediated, immune, fibrosclerosing bile duct injury. Pathology BA is a cholangiopathic disease affecting both the intra- and extrahepatic bile ducts. The lumen of the extrahepatic biliary tree is obliterated by inflammatory tissue to a variable extent. There are three main types of obliteration, of which type 3 (occlusion at the level of the porta hepatis) is the most common, occurring in almost 90% of cases. Atresia of the common bile duct (type 1) or common hepatic duct (type 2) with patent proximal ducts is much less common but carries a better prognosis. In type 3, the gallbladder is typically small and contains clear mucus only. Occasionally, a cyst is found proximal or distal to the atretic bile duct but this should not be confused with a true choledochal cyst. In its early stages, histopathologic examination of a liver biopsy by light microscopy typically shows portal tract oedema, cholestasis, bile plugging, ductular proliferation and a lymphocytic inflammatory infiltrate (predominantly CD4+); under electron microscopy de- generate biliary ductular cells containing bile pigment are also seen. 18 Disease progression results in hepatic fibrosis and cirrhosis with concomitant portal hypertension and liver failure. Clinical features and diagnosis Although BA may occur in premature infants, birth weight and gestation are usually normal. Presenting features include conjugated hyperbilirubinaemia, dark urine and pale stools. Malabsorption of fat- soluble vitamin K may lead to coagulopathy and bleeding (which can be intracranial). On examination, the liver is usually enlarged and there may be splenomegaly. Early diagnosis and prompt surgery improve the outcome of infants with BA. Neonatal jaundice that persists beyond two weeks of age demands clinical assessment and prompt investigation (see paper by Roberts in this issue). The cardinal biochemical feature of BA is conjugated hyperbilirubinaemia, but there is considerable overlap between the clinical, biochemical, radiologic and histologic features of BA and other causes of the neonatal hepatitis syndrome. No single pre-operative investigation can diagnose BA with certainty. However, by using a combination of tests, it is possible to be reason- ably certain about the diagnosis in most cases. The following investigations are particularly helpful. • Investigations to exclude metabolic and infective causes of conjugated hyperbilirubinaemia. • Hepatobiliary ultrasound will exclude other surgical causes of jaundice such as choledochal cyst and inspissated bile. In BA, the intrahepatic ducts are not dilated on ultrasonography because they are affected by the inflammatory process. Various sonographic features have been targeted in attempts to distinguish BA from other causes of conjugated hyperbilirubinaemia in infants 19–23 (Table 1). Irrespec- tive of interobserver variation, failure to visualize the common bile duct is not diagnostic of BA because a patent distal common bile duct can be found in up to 20% of affected infants. An absent gallbladder or one with an irregular outline is suggestive of BA. 23 In some cases, a Table 1 Diagnostic accuracy of ultrasound scanning in biliary atresia Sonographic feature n BA Sensitivity (%) Specificity (%) Absent common bile duct Azuma et al. (2001) 19 30 23 83 71 Triangular cord sign Park et al. (1999) 20 79 25 84 98 Kotb et al. (2001) 21 65 25 100 100 Tan Kendrick et al. (2003) 22 217 31 84 100 GB absent/irregular shape and wall thickness Farrant et al. (2000) 23 346 71 90 92 GB triad (length <19 mm, indistinct wall and irregular contour) Tan Kendrick et al. (2003) 22 217 31 97 100 GB, gallbladder. Biliary atresia 385 well-defined triangular area of high reflectivity is seen at the porta hepatis corresponding with fibrotic ductal remnants (the ‘triangular cord’ sign). 20,21 • Radionuclide hepatobiliary imaging using technetium-99m iminodiacetic acid (IDA) derivatives fails to demonstrate bile excretion into the bowel in BA. Pretreatment with phenobarbitone 5 mg/kg/day and a 24 h scan enhance the accuracy of the test. • Percutaneous needle liver biopsy. • Magnetic resonance cholangiography may be used to visualize the bile ducts and gallbladders of infants with cholestatic jaundice without BA. Han et al. (2002) reported a diagnostic accuracy of 98%, sensitivity of 100% and specificity of 96% in the diagnosis of 23 infants with BA. 24 Some centres have found duodenal intubation and aspiration looking for bile to be an accurate test, but false-positive and false-negative results are found in a small proportion of patients. 25 If the diagnosis remains in doubt, consideration should be given to endoscopic retrograde cholangiography which is technically demanding but nevertheless possible in up to 90% of infants, and gives few false positives. 26 Alternatively, laparoscopy or mini- laparotomy and cholangiography may be required to exclude the diagnosis. Screening tests Only 0.04–0.2% of newborns have conjugated hyperbilirubinaemia due to cholestatic hepatobiliary disease. In an attempt to achieve earlier diagnosis of BA, various screening tests have been explored but none has satisfied criteria for widespread use. The use of tandem mass spectrometry to measure conjugated bile acids in dried blood spots from newborn infants has proved disappointing. 27 Gamma-glutamyl transpeptidase (GGT) is a hepatic enzyme which may be found in amniotic fluid from the second trimester onwards consequent on fetal bile production and defaecation. In a large study of amniotic fluid samples, MacGillivray and Adzick (1994) showed that infants born with BA had minimal levels of GGT in amniotic fluid dating back to 18 weeks' gestation. 28 A small number of cases of BA complicated by cyst formation have been detected prenatally on maternal ultrasound scans (see paper by Davenport in this issue). Other potential community screening tests, including those based on stool colour analysis, are under evaluation, but yellow skin pigmentation alone is too broad a criterion to be practical. 29 Surgery The diagnosis of BA is confirmed at laparotomy (Fig. 1). A cholangiogram is performed if there is doubt about ductal patency. The obliterated extrahepatic biliary tract is separated from the underlying portal vein and adjacent hepatic artery, and then transected high in the porta hepatis where patent microscopic biliary ductules may be present. 30 Biliary continuity is restored using a Roux loop of jejunum, which is anastomosed to the transected tissue (portoenterostomy). Infants tolerate the procedure well and early postoperative complications are uncommon. Peri-operative antibiotic prophylaxis is given to try and prevent cholangitis. Some units use choler- etics such as phenobarbitone and ursodeoxycholic acid to try and promote bile flow. There is some evidence from uncontrolled studies that corticosteroids might facilitate bile flow postoperatively; 31 this is currently being evaluated by a randomized controlled trial in the UK. Fat-soluble vitamins (A, D, E, and K) and formula feeds enriched with medium-chain triglycerides are also prescribed. Results of portoenterostomy The results of surgery have steadily improved during the last 30 years. The age at which surgery is carried out is the single most widely quoted prognostic variable. Resolution of jaundice is more likely if surgery is performed at less than eight weeks of age, but this age correlation is not linear. Results are considerably worse if the infant is older than 100 days at the time of portoenterostomy 32,33 because the obliterative cholangiopathy and hepatic fibrosis are more advanced. However, the Kasai procedure is still worthwhile provided that the patient does not have cirrhosis with impaired Figure 1 Operative view of the porta hepatis in a 10-week-old infant with BA. Note the liver fibrosis and atretic biliary remnants. A stay suture has been placed in the tiny gallbladder. 386 H. Kobayashi, M.D. Stringer synthetic liver function or ascites. Types 1 and 2 BA generally have a good prognosis if treated early. In the more typical type 3 BA, the presence of larger bile ductules at the porta hepatis (>150 µm in diam- eter) is associated with a better prognosis. The subgroup of infants with syndromic BA has a worse outcome both in terms of clearance of jaundice and overall mortality. 1,6 The latter is related to associated malformations, particularly congenital heart disease, a predisposition to developing the hepatopulmonary syndrome and possibly immune compromise from functional hyposplenism. There is anecdotal evidence to suggest that infants with concomitant cytomegalovirus infection fare less well after the Kasai procedure. In the UK, a survey of all infants with BA con- ducted between 1993 and 1995 demonstrated that outcome was also related to centre experience. 2 Consequently, in 1999, the management of BA in England and Wales was centralized to three supra- regional paediatric liver units. Approximately 60% of all infants undergoing portoenterostomy for BA in these centres now achieve clearance of jaundice (plasma bilirubin <20 µmol/l). Another measure of success after Kasai portoenterostomy is survival with the native liver; this is currently about 50% at five years in the UK. In common with other series from specialist units around the world, this figure is likely to decrease to around 30–40% at 10 years. Infants who fail to clear their jaundice after portoenterostomy and those who develop complicated or end-stage chronic liver disease despite an initially successful Kasai procedure require liver transplantation. BA is the most common indication for liver transplantation in children, and the majority of affected children will eventually come to transplant. Most of these cases require a transplant in the first few years of life. The timing of transplant is dictated by liver function, nutritional status, symptoms and the presence of complications. A high hepatic artery resistance index measured on Doppler ultrasound is an ominous sign and an indication for relatively urgent transplantation. 34 For some patients, hepatic decompensation may be precipitated by ado- lescence or pregnancy. However, as many as 20% of patients undergoing portoenterostomy will reach maturity with good native liver function. Five-year survival after liver transplantation for BA is currently 80–90%, and techniques such as split-liver grafting and living-related liver transplantation have minimized the risk of dying on the waiting list. The combination of Kasai portoenterostomy and liver transplantation has transformed a disease that was almost invariably fatal in the 1960s into one with an overall five-year survival of about 90%. Furthermore, long-term studies have shown a relatively good quality of life in BA survivors after portoenterostomy alone 35 and after liver transplantation. 36 Long-term complications In addition to the risk of progressive liver disease, numerous other complications may occur after portoenterostomy for BA. These include: • bacterial cholangitis; • portal hypertension; • metabolic and nutritional sequelae; • intrahepatic cysts; • hepatopulmonary syndrome; • hepatic malignancy. Bacterial cholangitis This is most likely to occur in the first year following surgery and is due to infection complicating impaired bile flow. Approximately 40% of infants are affected. Cholangitis typically manifests as worsen- ing jaundice, fever and pale stools, but more subtle effects such as poor feeding and irritability may be dominant. Blood culture (and sometimes liver biopsy culture) may yield a Gram-negative organ- ism but it is important to treat suspected cases early and empirically with broad-spectrum intravenous antibiotics. The occurrence of postoperative cholangitis in patients with established bile flow is associated with a poorer overall outcome and a higher likelihood of eventual liver transplantation. Portal hypertension Hepatic fibrosis is present at the time of portoenterostomy, and about two-thirds of children will have endoscopically visible oesophageal varices by two to three years of age, although only half of these will ever bleed. 37,38 Endoscopic sclero- therapy or variceal banding is an effective method of controlling variceal bleeding, but children with poor underlying liver function are best treated by liver transplant. Metabolic and nutritional sequelae Persistent cholestasis may cause malabsorption of fat and fat-soluble vitamins. Vitamin K-dependent coagulopathy, rickets and fractures from vitamin D deficiency and calcium malabsorption 39 and neurological disturbances consequent on vitamin E deficiency are potential complications. Oral, and sometimes parenteral, vitamin supplements are Biliary atresia 387 needed. Malabsorption of long-chain triglycerides and fatty acids may impair weight gain and induce steatorrhoea. Formula feeds enriched with medium-chain triglycerides are generally beneficial but an adequate intake of essential fatty acids must be maintained. Calorie supplementation is often required to maintain nutrition and growth in the presence of chronic liver disease. Abnormali- ties of copper and zinc metabolism have also been reported in children after portoenterostomy. Intrahepatic cysts Biliary cysts or ‘lakes’ may develop within the livers of long-term survivors and cause recurrent attacks of cholangitis 40 (Fig. 2). Prolonged antibiotic treatment and ursodeoxycholic acid may be helpful in preventing further cholangitis, but unremitting infection is an indication for liver transplantation. Hepatopulmonary syndrome Diffuse intrapulmonary shunting may occur as a complication of chronic liver disease in children with BA, probably as a result of vasoactive com- pounds from the mesenteric circulation bypassing sinusoidal inactivation. The syndrome is characterized by cyanosis, dyspnoea on exertion, hypoxia and finger clubbing. It is more prevalent in children with syndromic BA. The diagnosis is confirmed using a combination of arterial blood gas estimations with and without inspired oxygen, radionuclide lung scans using macro-aggregated albumin to quantify the degree of shunting and contrast bubble echocardiography. This complication is progressive but can usually be reversed by liver transplantation. Pulmonary hypertension is a rarer complication but may also develop in long-term survivors after portoenterostomy. Hepatic malignancy Rarely, malignant changes (hepatocellular carcinoma or cholangiocarcinoma) may complicate long- standing biliary cirrhosis after portoenterostomy. Prognostic markers From the preceding account, it is apparent that the Kasai procedure revolutionized the treatment of BA, but numerous complications may still jeopard- ize long-term outcome. Can long-term prognosis after portoenterostomy be determined at an early stage? Rapid resolution of jaundice and complete normalization of biochemical liver function are associated with a good long-term outcome but this scenario is uncommon. Conventional biochemical liver function tests are neither specific nor particularly sensitive markers of hepatic fibrosis and functional liver mass. Liver histology is the only certain way of assessing the severity of fibrosis, but even this is not a reliable guide to the timing of liver transplantation. Recent research has focused on the search for early noninvasive markers of hepatic fibrosis in postoperative BA patients. Potential candidate markers, all of which have shown some correlation with clinical status, include the following. Collagen type IV and N-terminal procollagen-III peptide (PIIIP) Hepatic fibrosis is associated with an increased production of various extracellular matrix components including types III and IV collagen. Raised levels of serum type IV collagen and PIIIP have been observed clinically and experimentally in fibrotic liver disease, and shown to be markers of ongoing fibrosis. 41 Hyaluronic acid (HA) In the liver, HA is synthesized primarily by fat- storing Ito cells and its metabolism has recently been elucidated. Normally, more than 90% of the HA in circulation is taken up by HA receptors on hepatic SEC and degraded. Provided that increased tissue production of HA can be excluded, elevation of serum HA is due to impaired uptake and metabolism of HA by hepatic SEC. Therefore, plasma HA levels might reflect hepatic dysfunction and fibrosis. Kobayashi et al. (1999) have shown a positive Figure 2 MRI image (T2) showing multiple bile lakes (arrows) in the liver of a patient with BA after portoenterostomy. 388 H. Kobayashi, M.D. Stringer correlation between serum HA and serum bilirubin in patients with BA, supporting this hypothesis. 42 Endothelin Endothelin from endothelial cells is capable of in- ducing sustained vasoconstriction of portal veins. High levels of serum endothelin have been observed in patients with BA, particularly in those with portal hypertension. 43 In contrast, no significant differ- ence in serum nitric oxide levels between BA patients with or without portal hypertension has been identified. 44 -Glutathione-s-transferase (GST) The -GST, a hepatocyte-derived enzyme found in high concentrations within the hepatic cytosol, is a more sensitive and specific marker for hepatocellular damage than serum aminotransferases. In one study, serum -GST levels were significantly higher in stable patients with BA complicated by intrahepatic bile cysts. 45 Transforming growth factor-beta 1 (TGF-1) TGF-1 is an important mediator of liver cell proliferation and replication. Hepatic stellate cells (HSC) are activated by TGF-1 and are the main precursor cells involved in fibrogenesis. Several studies have concluded that HSC are responsible for increased collagen production in patients with BA and play a key role in fibrogenesis. Kobayashi et al. (2001) found that activated HSC displayed a strongly positive immunoreactivity to TGF-1 in liver biopsy specimens from patients with BA. 46 TGF-1 was strongly expressed in apparently clinically stable patients, indicating that hepatic fibrosis may be progressing silently in such patients. Interferon-inducible protein-10 (IP-10) One current hypothesis for the mechanism of chronic liver damage implicates resident and invasive hepatic macrophages as playing a central role in fibrosis through the release of inflammatory cytokines which perpetuate cellular injury and promote net collagen synthesis. The recruitment and maintenance of macrophages are dependent on cell adhesion molecules as well as chemo-attractants. IP-10 is a chemokine originally isolated in response to interferon- stimulation of monocytes, fibroblasts and endothelial cells. IP-10 is expressed in chronic hepatitis and its production is related to the severity of liver dysfunction and histologic damage. 47 More recently, it has been shown that serum IP-10 levels increase in parallel with the number of infiltrating mononuclear cells present in liver fibrosis. They correlate well with aminotransferase activity, suggesting that this chemokine is released during active inflammation and reflects ongoing hepatic tissue damage. 48 Hepatic duplex sonography A decreased maximum portal flow velocity and an increased hepatic artery resistance index correlate with measures of hepatic fibrosis and a poorer prognosis after portoenterostomy. 49 However, in common with the prognostic markers cited above, it has yet to be determined whether these parameters offer a better measure of long-term prognosis compared with standard biochemical liver function tests after portoenterostomy. Conclusions BA is a rare, complex disorder and demands expert multidisciplinary management. Despite progressive improvement in the results of treatment, the condition is extremely distressing for parents and families who not only face the anxieties of newborn surgery with an uncertain outcome, but also the possibility of eventual liver transplantation and its attendant risks. Parental education and support is essential, and charities such as the Chil- dren's Liver Disease Foundation (http:// www.childliverdisease.org) provide invaluable assistance to the families of affected children. Practice points • The prognosis of BA is improved by early detection. • BA must be excluded in any infant who has conjugated hyperbilirubinaemia after 14 days of age. • Infants with suspected BA should be referred to a specialist unit for further investigation and management. • A combination of timely expert surgery (Kasai portoenterostomy) and liver transplantation enables good long-term survival in more than 90% of affected patients. • At least two phenotypes of BA are recognized; the syndromic variety is associated with other congenital anomalies and a poorer outcome. Biliary atresia 389 • The aetiology of BA remains unknown, but immunologic mechanisms appear to be important in pathogenesis. Research agenda • An improved understanding of the pathogenesis and key mediators of hepatic fibrosis might enable the development of new therapeutic strategies. • The role of adjunctive therapies after portoenterostomy, particularly the use of corticosteroids and other choleretic agents, should be established by prospective randomized controlled trials. References 1. Chardot C, Carton M, Spire-Bendelac N et al. Prognosis of biliary atresia in the era of liver transplantation: French national study from 1986 to 1996. Hepatology 1999; 30:606–11. 2. McKiernan PJ, Baker AJ, Kelly DA. The frequency and outcome of biliary atresia in the UK and Ireland. Lancet 2000; 355:25–9. 3. Yoon PW, Bresee JS, Olney RS et al. Epidemiology of biliary atresia: a population-based study. Pediatrics 1997; 99:376–82. 4. Fischler B, Haglund B, Hjern A. A population-based study on the incidence and possible pre- and perinatal etiologic risk factors of biliary atresia. J Pediatr 2002;141:217–22. 5. Perlmutter DH, Shepherd RW. Extrahepatic biliary atresia: a disease or a phenotype? Hepatology 2002;35:1297–304. 6. Davenport M, Savage M, Mowat AP et al. The biliary atresia splenic malformation syndrome. Surgery 1993;113:662–8. 7. Mazziotti MV, Willis LK, Heuckeroth RO et al. Anomalous development of the hepatobiliary system in the Inv mouse. Hepatology 1999;30:372–8. 8. Jacquemin E, Cresteil D, Raynaud N et al. CFC1 gene mutation and biliary atresia with polysplenia syndrome. J Pediatr Gastroenterol Nutr 2002;34:326–7. 9. Tan CEL, Driver M, Howard ER et al. Extrahepatic biliary atresia: a first-trimester event? Clues from light microscopy and immunohistochemistry. J Pediatr Surg 1994;29:808–14. 10. Seidman SL, Duquesnoy RJ, Zeevi A et al. Recognition of major histocompatibility complex antigens on cultured human biliary epithelial cells by alloreactive lymphocytes. Hepatology 1991;13:239–46. 11. Dillon P, Belchis D, Tracy T et al. Increased expression of intercellular adhesion molecules in biliary atresia. Am J Pathol 1994;145:263–7. 12. Silveira TR, Salzano FM, Donaldson PT et al. Association between HLA and extrahepatic biliary atresia. J Pediatr Gastroenterol Nutr 1993;16:114–7. 13. Kobayashi H, Puri P, O'Briain DS et al. Hepatic overexpres- sion of MHC class II antigens macrophage-associated antigen (CD68) in patients with biliary atresia of poor prognosis. J Pediatr Surg 1997;32:590–3. 14. Kobayashi H, Horikoshi K, Li L et al. Serum concentration of adhesion molecules in postoperative biliary atresia patients: relationship to disease activity and cirrhosis. J Pediatr Surg 2001;36:1297–301. 15. Allison JP. CD28-B7 interactions in T-cell activation. Curr Opin Immunol 1994;6:414–9. 16. Kobayashi H, Li Z, Yamataka A, et al. Role of immunologic co-stimulatory factors in the pathogenesis of biliary atresia. J Pediatr Surg 2003;38: 892–6. 17. Sokol RJ, Mack C. Etiopathogenesis of biliary atresia. Semin Liver Dis 2001;21:517–24. 18. Davenport M, Gonde C, Redkar R et al. Immunohistochemis- try of the liver and biliary tree in extrahepatic biliary atresia. J Pediatr Surg 2001;36:1017–25. 19. Azuma T, Nakamura T, Moriuchi T, et al. Preoperative ultrasonographic diagnosis of biliary atresia—with reference to the presence or absence of the extrahepatic bile duct. Presented at the 38th Annual Congress of the Japanese Society of Pediatric Surgeons, Tokyo, Japan, June 2001. 20. Park WH, Choi SO, Lee HJ. The ultrasonographic ‘triangular cord’ coupled with gallbladder images in the diagnostic prediction of biliary atresia from infantile intrahepatic cholestasis. J Pediatr Surg 1999;34:1706–10. 21. Kotb MA, Kotb A, Sheba MF et al. Evaluation of the triangular cord sign in the diagnosis of biliary atresia. Pediatrics 2001; 108:416–20. 22. Tan Kendrick AP, Ooi BC, Tan CE. Biliary atresia: making the diagnosis by the gallbladder ghost triad. Pediatr Radiol 2003;33:311–5. 23. Farrant P, Meire HB, Mieli-Vergani G. Ultrasound features of the gall bladder in infants presenting with conjugated hyperbilirubinaemia. Br J Radiol 2000;73:1154–8. 24. Han SJ, Kim MJ, Han A et al. Magnetic resonance cholangiography for the diagnosis of biliary atresia. J Pediatr Surg 2002;37:599–604. 25. Larrosa-Haro A, Caro-Lopez AM, Coello-Ramirez P et al. Duodenal tube test in the diagnosis of biliary atresia. J Pediatr Gastroenterol Nutr 2001;32:311–5. 26. Iinuma Y, Narisawa R, Iwafuchi M et al. The role of endoscopic retrograde cholangiopancreatography in infants with cholestasis. J Pediatr Surg 2000;35:545–9. 27. Mushtaq I, Logan S, Morris M et al. Screening of newborn infants for cholestatic hepatobiliary disease with tandem mass spectrometry. Br Med J 1999;319:471–7. 28. MacGillivray TE, Adzick NS. Biliary atresia begins before birth. Pediatr Surg Int 1994;9:116–7. 29. Kelly DA, Stanton A. Jaundice in babies: implications for community screening for biliary atresia. Br Med J 1995; 310:1172–3. 30. Howard ER. Biliary atresia: etiology management and complications. Howard ER, Stringer MD, Colombani PM, editors. Surgery of the liver, bile-ducts and pancreas in children. London: Arnold, 2002;103–32. 31. Meyers RL, Book LS, O'Gorman MA et al. High-dose steroids ursodeoxycholic acid and chronic intravenous antibiotics improve bile flow after Kasai procedure in infants with biliary atresia. J Pediatr Surg 2003;38:406–11. 32. Davenport M, Kerkar N, Mieli-Vergani G et al. Biliary atresia: the King's College Hospital experience. J Pediatr Surg 1997; 32:479–85. 33. Chardot C, Carton M, Spire-Bendelac N et al. Is the Kasai operation still indicated in children older than 3 months diagnosed with biliary atresia. J Pediatr 2001;138:224–8. 34. Broide E, Farrant P, Reid F et al. Hepatic artery resistance index can predict early death in children with biliary atresia. Liver Transpl Surg 1997;3:604–10. 35. Howard ER, MacClean G, Nio M et al. Biliary atresia: survival patterns after portoenterostomy, and comparison of a 390 H. Kobayashi, M.D. Stringer Japanese with a UK cohort of long-term survivors. J Pediatr Surg 2001;36:892–7. 36. Bucuvalas JC, Britto M, Krug S et al. Health-related quality of life in pediatric liver transplant recipients: a single- center study. Liver Transpl 2003;9:62–71. 37. Stringer MD, Howard ER, Mowat AP. Endoscopic sclero- therapy in the management of esophageal varices in 61 children with biliary atresia. J Pediatr Surg 1989; 24:438–42. 38. Kang N, Davenport M, Driver M et al. Hepatic histology and the development of oesophageal varices in biliary atresia. J Pediatr Surg 1993;28:63–6. 39. Chongsrisawat V, Ruttanamongkol P, Chaiwatanarat T et al. Bone density and 25-hydroxyvitamin D level in extrahepatic biliary atresia. Pediatr Surg Int 2001;17:604–8. 40. Bu LN, Chen HL, Ni YH et al. Multiple intrahepatic biliary cysts in children with biliary atresia. J Pediatr Surg 2002; 37:1183–7. 41. Kobayashi H, Miyano T, Horikoshi K et al. Prognostic value of serum procollagen III peptide and type IV collagen in patients with biliary atresia. J Pediatr Surg 1998;33:112–4. 42. Kobayashi H, Horikoshi K, Yamataka A et al. Hyaluronic acid: a specific prognostic indicator of hepatic damage in biliary atresia. J Pediatr Surg 1999;34:1791–4. 43. Kobayashi H, Miyano T, Horikoshi K et al. Clinical significance of plasma endothelin levels in patients with biliary atresia. Pediatr Surg Int 1998;13:491–3. 44. Kobayashi H, Ohara N, Watanabe S et al. Serum nitric oxide in patients with biliary atresia. Asian J Surg 2000;23:294–6. 45. Kobayashi H, Horikoshi K, Yamataka A et al. -Glutathione-s transferase as a new sensitive marker of hepatocellular damage in biliary atresia. Pediatr Surg Int 2000;16:302–5. 46. Kobayashi H, Horikoshi K, Yamataka A et al. Are stable postoperative biliary atresia patients really stable? Pediatr Surg Int 2001;17:104–7. 47. Narumi S, Tominaga Y, Tamaru M et al. Expression of IFN- inducible protein-10 in chronic hepatitis. J Immunol 1997; 158:5536–44. 48. Kobayashi H, Narumi S, Tamatani T et al. Serum IFN- inducible protein-10: a new clinical prognostic predictor of hepatocyte death in biliary atresia. J Pediatr Surg 1999; 34:308–11. 49. Kardorff R, Klotz M, Melter M et al. Prediction of survival in extrahepatic biliary atresia by hepatic duplex sonography. J Pediatr Gastroenterol Nutr 1999;28:411–7. Further reading Howard, ER, Stringer, MD, Colombani, PM. Surgery of the liver, bile ducts, and pancreas in children, 2nd ed. London: Arnold, 2002. Biliary atresia 391 Review article Neonatal liver tumours Dietrich von Schweinitz * Paediatric Surgery, Dr. von Hauner'sches Kinderspital, Lindwurmstr. 4, D-80337 Munich, Germany Summary Primary liver tumours are very rare during the neonatal period, but increasing numbers of them are now diagnosed prenatally by routine ultrasound scan. A precise diagnosis is sometimes problematic because of non-specific clinical symptoms, misleading imaging and difficulties with histological interpretation. Benign infantile haemangioendothelioma usually undergoes spontaneous regression, but may be life-threatening due to congestive heart failure and/or consumptive coagulopathy when treatment with resection, embolization or arterial ligation is necessary. Malignant hepatoblastoma may occur in the newborn, and often has to be treated with chemotherapy to achieve resectability. Symptoms are less specific and the prognosis is worse than in older children. Mesenchymal hamartoma is a benign cystic lesion that should be resected whenever possible. Rarely, germ cell tumours occur in the neonatal liver. Benign teratomas have to be resected, while malignant choriocarcinomas may respond to chemotherapy and can be cured in some cases. © 2003 Elsevier Ltd. All rights reserved. KEYWORDS Neonatal liver tumour; Infantile haemangioendothelioma; Hepatoblastoma; Mesenchymal hamartoma; Teratoma; Choriocarcinoma General considerations Perhaps the first description of a neonatal liver tumour was published in 1854 by Noeggerath of the Obstetrical Clinic in Bonn, Germany. He reported a huge ‘liver carcinoma’ which caused obstructed labour. 1 According to his microscopic findings, this tumour would now be classified as a hepatoblastoma. Since Noeggerath's findings, it has taken more than 100 years to develop a pathological classification of childhood liver tumours; this is now used uniformly. 2 Liver tumours are very rare and account for only 5% of all neoplasms in the fetus and newborn. 3 They include a variety of benign and malignant neoplasms with a different distribution than in older children (Table 1). The most frequent are infantile haemangioendotheliomas, cavernous haemangiomas, mesenchymal hamartomas, the highly malignant hepatoblastomas and, rarely, benign or malignant germ cell tumours. All other entities mentioned in Table 1 have only been reported in single cases. Thus, in the National German Co-operative Paediatric Liver Tumour Studies from 1989 to 2002, out of 302 liver tumours, 26 were in infants under 6 weeks of age. Of these, 17 were infantile haemangioendotheliomas or * Tel.: +49-89-5160-3101; fax: +49-89-5160-4726 E-mail address: [email protected]. uni-muenchen.de (D. von Schweinitz). Table 1 Primary liver tumours and tumour-like lesions in the newborn Malignant Benign Hepatoblastoma Infantile haemangioendothelioma/ cavernous Hepatocellular carcinoma a Mesenchymal hamartoma Rhabdoid tumour a Teratoma Yolk sac tumour a Adenoma a Choriocarcinoma a Focal nodular hyperplasia a Undifferentiated sarcoma a Hepatic cysts Rhabdomyosarcoma Liver abscess Inflammatory pseudotumour a Only single cases reported in the literature. Seminars in NEONATOLOGY www.elsevierhealth.com/journals/siny Seminars in Neonatology (2003) 8, 403–410 1084-2756/03/$ - see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1084-2756(03)00092-7 cavernous haemangiomas, one was a mesenchymal hamartoma, six were hepatoblastomas and two were germ cell tumours. All neonatal tumours grow from immature liver tissue. In the fourth gestational week, the hepatic diverticulum of endoderm develops from the foregut. It expands into the mesoderm of the septum transversum, and differentiates into the liver cell cords and the epithelial lining of the bile ducts. Mesenchymal cells from the septum transversum form fibrous tissue, haematopoietic elements and Kupffer cells. 3 There is increasing evidence that undifferentiated cells persist in the liver until adulthood. It is as yet unclear from precisely which cellular components and at what time the various neonatal liver tumours arise. 4 With increasing use of prenatal ultrasound screening, many tumours are detected before birth; therefore, perinatal emergency situations can be avoided. After birth, ultrasound is the most convenient imaging procedure and can often give information on the type of neoplasm encountered. For more precise anatomical classification and staging, however, a computed tomography (CT) scan or, preferably, a magnetic resonance imaging (MRI) scan is necessary. Other radiological techniques such as scintigraphy and vascular contrast imaging are sometimes indicated for tumour diagnosis or planning therapy. 5 Laboratory diagnostics should include a differential blood count, liver parameters, serology for hepatotropic viruses, and the tumour markers alpha-fetoprotein (AFP), beta-human chorionic gonadotrophin (beta-HCG), lactate dehydrogenase and markers for neuroblastoma (catecholamine metabolites, neuron- specific enolase). It should be noted that clinical symptoms and laboratory tests are often non- specific in newborns with a liver tumour, and that imaging results can be misleading. 6,7 Furthermore, histological differentiation can be difficult, causing confusion between fetal hepatoblastoma and infantile haemangioendothelioma for example, and needle biopsies may not yield enough material for a reliable diagnosis. 2,6 Therefore, in most cases, an early laparotomy with an open tumour biopsy is necessary for correct diagnosis and rapid initiation of treatment. Infantile haemangioendothelioma and cavernous haemangioma The majority of neonatal liver tumours are vascular neoplasms. Many of these are diagnosed during the first few weeks of life. Infantile haemangioendothelioma, also termed capillary haemangioma by some authors, 3 seems to become symptomatic much more frequently than the cavernous type of haemangioma in this age group. Many hepatic vascular lesions are incidental findings during pre- or postnatal ultrasound investigations. However, infantile haemangioendothelioma, in particular, can cause severe symptoms with abdominal distension and hepatomegaly, severe arteriovenous shunting with congestive heart failure, haemo- dynamic anaemia, thrombocytopenia and consumptive coagulopathy (Kasabach–Merrit syndrome), rupture with intraperitoneal haemorrhage and respiratory distress (Fig. 1a). Rarely, biliary obstruction and jaundice can occur. 8,9 In cases with rapid growth or in patients with multiple lesions, a life-threatening status can develop shortly after birth or sometimes even during the fetal period with hydrops fetalis and intra-uterine heart failure. In about 50% of affected newborns, there are multiple haemangiomas on the skin and in other organs, i.e. a haemangiomatosis syndrome. 2 Associ- ations with omphalocele and other congenital malformations have also been reported. 3 In gross appearance, infantile haemangioendothelioma and cavernous haemangioma are like soft sponges filled with blood. Lesions may also contain homogeneous grey/white areas of fibrosis, calcification or focal haemorrhage. CT and MRI scans show these lesions as areas of reduced density. After administration of intravenous contrast, an enhancement from the periphery towards the centre of the lesion is typical. Large areas of haemorrhage, however, may imitate areas of necrosis as in a malignant tumour (Fig. 1b). With sonography, infantile haemangioendothelioma and cavernous haemangioma are seen as highly vascu- larized single or multiple tumours with a more-or- less homogeneous pattern. The technique of colour Doppler sonography is particularly valuable in these cases, since the often extremely high blood flow through the coeliac trunk and hepatic artery into the lesion can be visualized and measured (Fig. 1c). Accordingly, the flow through the corresponding liver veins is also enhanced. In cases with severe arteriovenous shunting, an abrupt decrease of the width of the abdominal aorta directly caudal to the branching of the coeliac trunk can be seen. Usually, these findings are typical for infantile haemangioendothelioma and may function as a basis for this diagnosis. 5 The various types of radionuclide scans cannot deliver additional information, but angiography may be necessary for planning treatment such as arterial ligation or embolization. The clinical symptoms and imaging findings typical for infantile haemangioendothelioma may 404 D. von Schweinitz also occur in the very rare congenital choriocarcinoma of the liver, as we have encountered in one case. 7 Therefore, caution is necessary and tumour markers, such as AFP and beta-HCG, must be measured in the serum. If the clinical diagnosis is not clear, a biopsy should be taken at laparotomy, which in an appropriate situation can be combined with a ligation of the involved hepatic arteries. 9 An open biopsy usually establishes the diagnosis of infantile haemangioendothelioma. However, even here we have encountered difficulties since the differentiation between an infantile haemangioendothelioma with only slightly enlarged cavernous spaces and a fetal hepatoblastoma or a benign hyperplastic regenerative node of the liver can be extremely difficult. 6 Therefore, before starting specific treatment, histology should be reviewed by an experienced paediatric pathologist. Usually, histology shows normal, still immature cords of hepatic cells divided by more-or-less vascular spaces lined by a single layer of plump, regular endothelial cells. 2 The dimensions of these vascular spaces determine whether they are categorized as capillary haemangioendothelioma or cavernous haemangioma. 3 Increased mitotic activity is usually not apparent in these tumours. In addition to this type 1 lesion described by Dehner and Ishak, 10 a type 2 haemangioendothelioma with more pleomorphic endothelial cells forming papil- lary structures occurs rarely. These lesions, which usually occur in older children, have a tendency to transform into malignant angiosarcoma. 3,10 The pathogenesis of these vascular lesions is currently unclear. 3 One would presume some enhanced angiogenic potential in the fetal liver in these cases, but this has not been shown to be true, and as yet there is no solid data or measurements of angiogenic factors in these lesions or in the patients' blood. 11 The management of infantile haemangioendotheliomas and haemangiomas is controversial. Most asymptomatic lesions can be managed expectantly, using serial ultrasound to visualize the anticipated spontaneous regression. 8,9 In cases with a gradual onset of controllable symptoms, medical treatment alone may occasionally be sufficient. This includes Figure 1 (a) Two-week-old infant with a large infantile haemangioendothelioma resulting in cardiac failure and Kasabach–Merrit syndrome. (b) Magnetic resonance scan of an infantile haemangioendothelioma in a newborn. (c) Duplex sonography of the right hepatic artery with hugely increased blood flow, surrounding a portal vein branch into an infantile haemangioendothelioma. Neonatal liver tumours 405 the use of digitalis and diuretics for congestive heart failure, and the administration of blood products to correct anaemia and coagulopathy. This can be accompanied by steroid therapy (prednisolone 2–5 mg/kg/day) in an attempt to suppress continued growth of the lesion or even encourage regression. Results are, however, often disappointing. 12 Treatment with alpha-2A-interferon (1–3 µg/m 2 / day) seems to be more effective, but this is associated with potentially severe side effects. 13 In infants with a rapid onset of severe symptoms, more invasive measures can be life saving. Relatively small solitary tumours are best treated by complete resection. However, most infantile haemangioendotheliomas are very large and extend diffusely throughout the liver; hepatic arterial ligation or embolization are then necessary. When technically feasible, the latter may be favoured as the less-invasive procedure whilst the former gives the opportunity for an open biopsy. These measures often stabilize the situation rapidly, but not infrequently, new collateral feeding vessels develop within days with the risk of recurrent problems. Despite this fact and the danger of other complications, at least 80% of such cases have a successful outcome after these procedures. 9 There have been several reports of orthotopic liver transplantation for massive, uncontrollable infantile haemangioendothelioma. Apart from the young age of the patients and the urgency of the situation, there are major logistical problems, and the results in the few reported cases have not been good enough to propose orthotopic liver transplantation as a standard procedure in the treatment of large infantile haemangioendotheliomas. 14 There is considerable optimism about the development of potent anti-angiogenic drugs. The angiogenesis inhibitor AGM-1470 was able to inhibit growth of haemangioendothelioma in a mouse model. 15 There are, however, no controlled clinical studies on such substances, and a better understanding of pathogenesis may be necessary before more specific medical treatments can be developed. Mesenchymal hamartoma Mesenchymal hamartomas are typically diagnosed during the first 2 years of life. Some cases are symptomatic in the neonate, and a few cases are detected by prenatal ultrasound scan. Many authors suggest that mesenchymal hamartoma is not a true neoplasm, but a developmental lesion, which originates from the connective tissue of the portal (ductal) plates. 3 The pathogenesis is not clear, but abnormal blood supply to a liver lobule 3 and/or abnormal expression of fibroblast growth factors may be relevant. 16 Mesenchymal hamartoma usually presents as a palpable mass, most often in the right liver, in an otherwise asymptomatic child. Typically, the lesion contains multiple large and small cysts containing some debris and divided by septae of variable thickness (Fig. 2a). Occasionally, the cysts are so small that the mass appears to be completely solid. The morphologic pattern on CT, MRI and ultrasound imaging is usually predominantly cystic. Some- times, a high portal blood flow into the lesion can be seen on colour Doppler sonography. Mesenchy- mal hamartomas do not normally produce proteins which could serve as specific markers, although some patients have a moderately elevated serum AFP. 17 Histologically, the multilocular cysts are lined by endothelium or by bile duct epithelium, and are surrounded by fibrous or myxoid tissue containing bile ducts and multiple vessels, particularly portal Fig. 2 (a) Gross section of a cystic mesenchymal hamartoma. (b) Histological appearance of a mesenchymal hamartoma with fibrous tissue, multiple cysts and large portal vessels. 406 D. von Schweinitz vein branches. The lesion is often surrounded by a thick fibrous capsule, but can also grow into adjacent compressed or fibrous hepatic parenchyma (Fig. 2b). 2,3 There is some uncertainty about the natural history of mesenchymal hamartomas. Meyers and Scarife 8 stated that ‘the tumour tends to increase in size during the first several months of life and subsequently may either stabilize, continue to grow or undergo regression’. While complete spontaneous regression of mesenchymal hamartomas has been reported, 18 instances of massive local recurrence and later transformation to undifferentiated sarcoma are well documented. 17,19,20 There- fore, small lesions should be resected completely. Non-resectable mesenchymal hamartomas should be biopsied. If the mass stabilizes during the first few months of life, it may be followed by regular ultrasound investigation with a chance of spontaneous regression or at least a relative reduc- tion of size during growth of the patient. If the lesion does not vanish completely during the first 4–5 years of life, we undertake resection because of the risk of malignant transformation at a later stage. 16 The resection is performed with the established techniques of liver surgery, and a radical excision should be attempted. Usually, the complication rate is low. However, if there is a very high portal blood flow into the lesion, its resection may lead to venous congestion in the intestinal tract, ischaemia, toxaemia and shock, as we encountered in one case. 21 Hepatoblastoma Although malignant hepatoblastoma is the most common liver tumour of early childhood, less than 10% of cases occur during the neonatal period. In the German national studies, six out of 194 hepatoblastomas presented during the first 6 weeks of life. Hepatoblastomas are significantly associated with genetic anomalies and malformation syndromes, the most important of which are Beckwith–Wiedemann syndrome, trisomy 18, familial adenomatous polyposis coli and fetal alcohol syndrome. Extremely premature children have a significantly increased risk of developing a hepatoblastoma. 22 Hepatoblastoma is an embryonal neoplasm com- posed of malignant epithelial tissue with variable differentiation, most often with embryonal or fetal components (Fig. 3a). Some hepatoblastomas also contain malignant mesenchymal tissue with immature fibrous areas, spindle cells and cartilage-like osteoid, and are then called mixed hepatoblastoma. 2,3,22 In neonates, the relatively differentiated, pure fetal histology seems to predominate compared with older children. 23 The clinical picture of the often extensive hepatoblastoma tumour in neonates (Fig. 3b) may also differ in other ways to hepatoblastomas in the typical age group of 6 months to 3 years. According to some reports, metastases occur earlier and are often systemic, sometimes bypassing the lungs because of differences in the fetal circulation. 23 Neonatal hepatoblastomas do not seem to produce such excessive amounts of AFP as those in older children, whilst the natural blood concentrations are still high in this period of life. 6 Hepatoblasto- mas can be detected prenatally by abdominal ultrasound and may cause polyhydramnios and still- birth. 23 During labor and birth, tumour rupture with massive haemorrhage can occur. 1,24 Due to diagnostic uncertainty, all neonatal tumours suspected to be a hepatoblastoma should Fig. 3 (a) Histological appearance of a hepatoblastoma with embryonal (left) and fetal (right) differentiation. (b) Large hepatoblastoma in a young infant involving the right liver and segment IV of the left liver. Neonatal liver tumours 407 be investigated histologically. 6,25 If a resection is not possible, a biopsy should be taken. Chemo- therapy containing the effective cytotoxic agents, cisplatin and doxorubicin, 26–28 should be administered, carefully considering the dose limitations in young infants. With this regimen, some extensive hepatoblastomas may be reduced to an operable size, and metastases can regress. Thus, cure can become possible even in neonates. However, due to their different biological behaviour, as well as the greater risks of surgery and chemotherapy, neonatal hepatoblastomas are associated with a worse overall prognosis. 3,23 In contrast to previous reports, all six neonates registered in the German Co-operative Trials survived tumour-free; four after a primary tumour resection and two after inductive chemotherapy and delayed resection. None of these children had distant metastases. Germ cell tumours Primary teratomas of the liver, although very rare, occur most often in the newborn period. 3 They are typically cystic and contain mature or immature elements. These tumours usually lack malignant components. Serum AFP may be markedly elevated in these patients. Mesenchymal hamartoma and hepatoblastoma are the main differential diagnoses. Diagnosis is complicated by the fact that some hepatoblastomas have teratoid features on histology. 2 The same is true of the malignant yolk sac tumour, which has only been described in combination with hepatoblastoma, 3 but this might also be defined as a hepatoblastoma with a malignant germ cell tumour differentiation. 2 In these cases, an adequate biopsy is important to define the different malignant and benign tumour components. In cases of benign teratoma, surgical excision results in cure. How- ever, as in teratomas at other sites, it is essential that the resection is radical, since recurrent tumour may be highly malignant and associated with a poor outcome. 8 The liver seems to be a favourite organ for growth of the very rare choriocarcinoma in neonates. It is not clear whether this tumour originates in the fetus or is a metastasis from a choriocarcinoma in the placenta of the mother. In some, but not all, of the observed cases, a primary tumour was found in the placenta. 29 Histologically, neonatal choriocarcinomas resemble those of older patients. The clinical symptoms of choriocarcinoma in the neonatal liver can be very similar to those of infantile haemangioendothelioma. We encountered one case with congestive heart failure and typical Kasabach–Merrit syndrome. With colour Doppler sonography, the appearance of the tumour was like that of an infantile haemangioendothelioma with arteriovenous shunts through cavernous vascular spaces. However, both serum AFP and beta-HCG were markedly elevated, and a biopsy proved the diagnosis. 7 Recent experience with single cases shows that these tumours may respond to chemotherapy according to the different co-operative germ cell tumour protocols, i.e. regimens containing cisplatin, etoposide and ifosfamide. 7,29 If regression occurs, the tumour may become resectable. With such a strategy, the previously very dismal prognosis has improved, and tumour-free survivors, even from metastasizing neonatal choriocarcari- noma, have now been reported. 7,29 Conclusions Liver tumours occur very rarely in neonates. The most common neoplasms are benign infantile haemangioendothelioma and, less frequently, malignant hepatoblastoma. Other benign and malignant tumours, such as mesenchymal hamartoma and germ cell tumours, can occur. Differen- tial diagnosis of these tumours can be difficult because of non-specific clinical symptoms, incon- sistent expression of tumour markers, and sometimes confusingly similar histological appearances. Treatment strategies, ranging from pure observation to chemotherapy combined with extensive surgery, have to be tailored to the specific neoplasm. With the development of better therapeutic regimens, prognosis has improved during recent years. Intensive research is, however, essential to gain insight into the pathogenetic mechanisms of these different neoplasms. Practice points • Clinical symptoms and imaging are often non-specific in neonatal liver tumours. • Histological diagnosis is best confirmed by a reference pathologist. • The most common entity is the benign infantile haemangioendothelioma. • Infantile haemangioendothelioma type 1 usually regresses spontaneously. • Differential diagnosis comprises mainly hepatoblastoma and choriocarcinoma. • Medical treatment is often disappointing. • Resection, embolization or arterial ligation are therapeutic options in severe cases. 408 D. von Schweinitz • Diagnosis of mesenchymal hamartoma should be confirmed histologically. • Primary or delayed complete resection is the treatment of choice. • Differential diagnosis of neonatal hepatoblastoma largely concerns benign infantile haemangioendothelioma. • Treatment of hepatoblastoma consists of primary resection of small tumours, or initial chemotherapy with cisplatin and doxorubicin followed by surgery for large lesions. • Neonatal teratoma should be resected completely. • Choriocarcinoma may respond to chemotherapy and thus become curable after resection. Research directions • The role of genetic modifications and angiogenic factors in the pathogenesis of infantile haemangioendothelioma. • The efficacy of systemic or local anti-angiogenic substances on tumour growth suppression. • The possible role of differentiating and growth factors in the aetiology of mesenchymal hamartomas. • Mechanisms of malignant transformation in mesenchymal hamartomas. • Evaluation of the clinical and molecular genetic conditions leading to intra-uterine growth of hepatoblastoma. • Factors leading to the growth of choriocarcinoma in the placenta and/or in the fetus. References 1. Noeggerath E. Geburtshindernis in Folge eines Leberkarzinoms. Deutsche Klinik 1854;6:496–7. 2. Weinberg AG, Finegold MJ. Primary hepatic tumors of childhood. Hum Pathol 1983;14:512–37. 3. Isaacs H. Tumors of the fetus and newbornPhiladelphia: W.B. Saunders, 1997. 4. Ruck P, Xiao JC, Pietsch T et al. Hepatic stem-like cells in hepatoblastoma: expression of cytokeratin 7, albumin and oval cell associated antigens detected by OV-1 and OV-6. Histopathology 1997;31:324–9. 5. Von Schweinitz D. Treatment of liver tumors in children. Clavien P-A, editor. Malignant liver tumors. Current and emerging therapies. Malden, MA: Blackwell Science, 1999; 323–40. 6. Von Schweinitz D, Gluër S, Mildenberger H. Liver tumors in neonates and very young infants: diagnostic pitfalls and therapeutic problems. Eur J Pediatr Surg 1995;5:72–6. 7. Szavay PhO, Wermes C, Fuchs J et al. Effective treatment of infantile chriocarcinoma in the liver with chemotherapy and surgical resection: a case report. J Pediatr Surg 2000;35: 1134–5. 8. Meyers RL, Scaife ER. Benign liver and biliary tract masses in infants and toddlers. Semin Pediatr Surg 2000;9:146–55. 9. Davenport M. Hemangiomas and other vascular anormalies. Howard ER, Stringer MD, Colombani PM, editors. Surgery of the liver, bile ducts and pancreas in children. London: Arnold, 2002;219–38. 10. Dehner LP, Ishak KG. Vascular tumors of the liver in infants and children. Arch Pathol 1971;92:101–11. 11. Prokurat A, Kluge P, Chrupek M et al. Hemangioma of the liver in children: proliferating vascular tumor or congenital vascular malformation? Med Pediatr Oncol 2002;39:524–9. 12. Prokurat A, Chrupek M, Kosciesza A et al. Hemangioma of the liver in children—conservative versus operative treatment. Surg Child Intern 2000;3:202–7. 13. Deb G, Donfrancesco A, Ilari I et al. Hemangioendothelioma: successful therapy with interferon-alpha. A study in association with the Italian Pediatric Haematology/Oncology Society (AIEOP). Med Pediatr Oncol 2002;38:118–9. 14. Daller JA, Bueno J, Gutienez J et al. Hepatic hemangioendothelioma: clinical experience and management strategy. J Pediatr Surg 1999;34:98–106. 15. O'Reilly MS, Brem H, Folkman J. Treatment of murine hemangioendotheliomas with the angiogenesis inhibitor AGM-1470. J Pediatr Surg 1995;30:325–392. 16. Von Schweinitz D, Gomez Dammeier B, Gluër S. Mesen- chymal hamartoma of the liver—new insight into histogenesis. J Pediatr Surg 1999;34:1269–71. 17. Stringer MD. Benign tumors. Howard ER, Stringer MD, Co- lombani PM, editors. Surgery of the liver, bile ducts and pancreas in children. London: Arnold, 2002;247–60. 18. Barnhart DC, Hirschl RB, Garver KA et al. Conservative management of mesenchymal hamartoma of the liver. J Pediatr Surg 1997;32:1495–8. 19. Ramanujam TM, Ramesh JC, Goh DW et al. Malignant transformation of mesenchymal hamartoma of the liver: case report and review of the literature. J Pediatr Surg 1999; 34:1684–6. 20. Murray JD, Ricketts RR. Mesenchymal hamartoma of the liver. Am Surg 1998;64:1097–103. 21. Fuchs J, von Schweinitz D, Feickert HJ. Acute respiratory distress syndrome following resection of a mesenchymal hamartoma of the liver. Med Pediatr Oncol 1999;32:151–3. 22. Tomlinson GE, Finegold MJ. Tumors of the liver. Pizzo PA, Poplack DG, editors. Principles and practice of pediatric oncology. Philadelphia: Lippincott Williams and Wilkins, 2002;847–64. 23. Ammann RA, Plaschkes J, Leibundgut K. Congenital hepatoblastoma: a distinct entity. Med Pediatr Oncol 1999; 32:466–8. 24. Sinta L, Freud N, Dulitzki F. Hemoperitoneum as the presenting sign of hepatoblastoma in a newborn. Pediatr Surg Int 1992;7:131–3. 25. Von Schweinitz D, Bu¨rger D, Mildenberger H. Is laparotomy the first step in treatment of childhood liver tumors? The experience from the German Cooperative Pediatric Liver Tumor Study HB89. Eur J Pediatr Surg 1994;4:82–6. 26. Ortega JA, Douglass E, Feusner J et al. Randomized comparison of cisplatin/vincristine/fluorouracil and cisplatin/ continuous infusion doxorubicin for treatment of pediatric hepatoblastoma: a report from the Children's Cancer Group and the Pediatric Oncology Group. J Clin Oncol 2000;18: 2665–2675. Neonatal liver tumours 409 27. Pritchard J, Brown J, Shafford E et al. Cisplatin, doxorubicin and delayed surgery for childhood hepatoblastoma: a successful approach—results of the first prospective study of the International Society of Pediatric Oncology. J Clin Oncol 2000;18:3819–28. 28. Von Schweinitz D, Byrd DJ, Hecker H et al. Efficiency and toxicity of ifosfamide, cisplatin and doxorubicin in the treatment of childhood hepatoblastoma. Eur J Cancer 1997; 33:1243–9. 29. Blohm MEG, Calaminus G, Gnekow AK et al. Disseminated choriocarcinoma in infancy is curable by chemotherapy and delayed tumour resection. Eur J Cancer 2001;37:72–8. 410 D. von Schweinitz

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