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Jornal de Pediatria
Copyright 2000 by Sociedade Brasileira de Pediatria

REVIEW ARTICLE

Neonatal cholestasis
Mariza Leito Valadares Roquete*

Abstract
Objective: to warn pediatricians about the early recognition of cholestasis in newborns and infants. Methods: a bibliographic research about cholestasis was performed using Medline, and emphasizing the most relevant publications of the last 30 years. Results: the concept of cholestasis and the causes of cholestatic tendency in newborns and infants are described. Several causes of intra and extrahepatic cholestasis are reported as well. In this review, only the diseases with diagnostic, therapeutic or prognostic peculiarities are commented, including extrahepatic biliary atresia, idiopathic neonatal hepatitis, galactosemia, and Alagille s syndrome. Furthermore, several resources are discussed for the diagnosis of cholestasis. Conclusions: the establishment of the diagnosis of cholestasis through the detection of hyperbilirubinemia in newborns who present jaundice after 14 days of life is a goal that could change the prognosis of several diseases responsible for neonatal cholestasis. J Pediatr (Rio J) 2000; 76 (Supl.2): S187-S197: cholestasis, cholestasis intrahepatic, bile duct obstruction extrahepatic.

Introduction Cholestasis results from the reduction in the synthesis of bile salts or from the blockade (intra or extrahepatic) of the excretion of biliary components to the small bowel. The retention of several substances usually excreted in bile does not process with the same intensity; there are situations in which the bile salts are more retained than bilirubin and vice-versa.1 Cholestasis constitutes the main manifestation of hepatobiliary disease,1 with an incidence estimated at 1:2,500 live births.2

* Assistant Professor, Pediatrics, School of Medicine, Universidade Federal de Minas Gerais.

There are numerous studies about the secretion and the metabolism of bile acids carried out with animal models during fetal life and during the first days of life. However, extreme caution is necessary concerning the extrapolation of these results to the human species. In man, the synthesis of bile acids starts in the 12th week of gestation; the biliary secretion is already evident in the 4th month, and bile is present in the intestine in the 5th month of intrauterine life.3 Newborns, especially preterms, are predisposed to cholestasis (physiologic hypercholesteremia) due to the hepatic immaturity related to the metabolism of bile acids: ability to synthesize atypical hepatotoxic bile acids; reduction in the bile salt pool, which results from the decreased synthesis and from the absence of active ileal reabsorption of such salts; decrease in the captivation of bile salts by the S187

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hepatocytes, though in a lower proportion than that of the reduction in biliary secretion, which is also inherent to the newborn.3 In the perinatal period, the immaturity of the canalicular structure and function is evidenced by the irregularity and dilatation of the canaliculi, canalicular hypomotility, and increased paracellular permeability. 3 Because of this immaturity, the newborn usually responds to infectious, toxic, or metabolic aggressions with clinical, biological, and histopathological manifestations of cholestasis. Up to the 4 to 6 months of life, the child presents elevated serum level of bile acids; besides, the activity of the enzymes alkaline phosphatase and gammaglutamyltransferase are also elevated during the 1st semester of life when compared to the one found in adults. The hepatic maturation happens gradually, so that with 1 year of age, the child already presents a pattern of biliary secretion similar to that of adult individuals. Depending on the examiners perspective, cholestasis can be expressed in three forms: Clinical: choluria, hypocholic or acholic stools, jaundice (not obligatory), associated or not with splenomegaly; itching and xanthomas (deposits of cholesterol at the folds and attrition areas of the skin) are related to the duration and cause of cholestasis. Persistent acholic stools are observed in the following conditions: extrahepatic biliary atresia (in 15% of the cases, the discoloration of feces in incomplete or delayed), and intense intrahepatic cholestasis, such as in Alagilles syndrome, cystic fibrosis, alpha 1-antitrypsin deficiency, and sclerosing cholangitis.4 Laboratory: elevation of the level of bile salts and direct bilirubin in blood (>20% of total bilirubin), bilirubinuria, increased activity of aminotransferases and cholestasis enzymes (alkaline phosphatase and gammaglutamyltransferase), and hypercholesteremia; the magnitude of direct hyperbilirubinemia does no allow to define the type or the intensity of cholestasis; 1 in prolonged cholestasis, there is the formation of a covalent ligand complex of direct bilirubin with albumin, delta bilirubin, or biliprotein, which then adopts the half-life of albumin (12 to 14 days, instead of 4 hours), and is manifested as persistent jaundice without evidence of choluria.5 Histopathological: cholestasis is shown by the presence of biliary pigment in the cytoplasm of hepatocytes, in the canalicular light, in ductules, and bile ducts, frequently associated with secondary cellular lesion. Through fetal ultrasound, the diagnostic suspicion of a cholestatic affection can be established in the prenatal period when a subhepatic cystic image is detected, or when the gallbladder is not visible. The low activity of gammaglutamyltransferase in the amniotic liquid in the 18th week, or an elevated gammaglutamyltransferase in fetal blood, or, still, any of the ultrasonographic findings mentioned point out to the diagnosis of extrahepatic biliary atresia until jaundice is present after birth. 4

Mortality and morbidity resulting from several hepatobiliary diseases motivated the performance, in the United Kingdom, of a screening, similarly to what is done for the neonatal diagnosis of phenylketonuria and congenital hypothyroidism. A pilot population tracking was performed with the dosage of direct bilirubin in liquid blood samples, collected in a opaque recipient, also used for the conventional screening in newborns 4 to 28 days old.6 This initiative aimed at improving the prognosis of several hepatobiliary affections through their early diagnosis and treatment. The cholestatic syndrome in infants constitutes one of the most important diagnostic challenges of pediatric hepatology. This denomination refers to several clinical affections, as well as to a small group of diseases that require surgical treatment. Approximately 70 to 80% of the cholestases in infants are caused by idiopathic neonatal hepatitis and by extrahepatic biliary atresia, in similar proportions, but with a predominance of the former. As these two affections have not had their etiopathogenesis defined yet, only 15 to 20% of the cases have their cause determined. Even in situations in which there are highly sophisticated lacid biliaryseratorial and imaging resources, it is frequently not possible to define the genesis of the cholestatic process. On the other hand, depending on the studied population, there will be significant frequency differences of cholestatic nosologies: while in the United Kingdom the deficiency of alpha 1-antitrypsin was responsible for 17% of the cholestasis cases in infants,7 in South Africa, congenital syphilis contributed with almost 22% for the causes of cholestasis.8 Besides, the scenery of causes of cholestasis varies according to the place where the patient receives care: surgical diseases predominate in children hospitalized at reference centers, while clinical causes are assessed at an outpatient unit; so, cholestases caused by congenital infections are rarely referred to an hepatology outpatient service. The diagnostic challenge in the approach of cholestatic newborns and infants is due to the variety of extra and mainly intrahepatic affections that manifest clinically in an indistinct way, with jaundice due to direct hyperbilirubinemia, usually with no indices that point out to a specific diagnosis. To this initial difficulty, we may add limitations that are inherent to the public health care system in our country and that concern the deepening of investigation, which is indispensable for the diagnosis of metabolic diseases. Besides, there are surgical conditions extrahepatic biliary atresia and choledochal cyst - that require correction before 2 months of age in order to achieve a better prognosis. Concerning clinical affections, the early diagnosis is of paramount importance in those entities that already have a specific treatment, such as some metabolic (fructosemia, galactosemia) and infectious diseases (infection of the urinary tract, sepsis, tuberculosis, syphilis, toxoplasmosis, etc.). The adequate handling of a cholestasis case starts when the general pediatrician is asked to evaluate the jaundice of

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a newborn or infant during the first 6 months of life. Without disregarding recent advances in complementary investigation, the general pediatrician is acknowledged as the most important medical professional in the correct identification of cholestasis cases. Since he/she has the opportunity to have the first contact with the jaundiced child through the medical appointment, the pediatrician should be aware of his/her responsibility, and have a minimal knowledge about cholestasis for the initial evaluation and the fast reference of the infant in this situation. Aiming at dynamizing the investigation and allowing an early diagnosis of treatable cholestatic diseases, some recommendations are indispensable to health professionals that give primary care to the population, especially pediatricians. Although most part of jaundice cases happening in the neonatal period are due to indirect hyperbilirubinemia, the pediatrician should be alert to the possibility of diagnosing a case of cholestasis in every newborn whose jaundice persists for over 14 days of life. In this situation, the dosage of fractionated bilirubins, and the observation of urine color for the identification of choluria, which may either be evident due to the patients diuresis dependence or not, are necessary. This practice is indicated according to the observation that physiological jaundice (indirect hyperbilirubinemia) rarely remains longer than the 2 first weeks of life, except for preterms and newborns with jaundice caused by breast milk. As in the physiological jaundice, the latter two situations are causes of jaundice due to increased indirect bilirubin, without any other evidence of choluria, hypocholic or acholic stools. If there are evident clinical signs of cholestasis, or if only the dosage or serum bilirubins reveals that direct bilirubin levels equal or inferior to 20% of total bilirubin, the pediatrician can conclude that it is a case of direct hyperbilirubinemia - an event that is always pathological. When facing a newborn or infant with direct hyperbilirubinemia, the investigation of an hepatobiliary disease becomes indispensable. This situation requires urgent submission to a reference center, which has adequate investigation conditions and experienced clinical and surgical teams, aiming at recognizing early the affections that can be clinically or surgically treated, so that the prognosis improves and the cure is achieved according to the case. Although some hereditary diseases do not have a specific treatment, the definitive diagnosis is greatly important to the genetic counseling to the patients relatives. There is no specific treatment for Alagilles syndrome, but this condition has to be quickly diagnosed in order to prevent an unnecessary surgery due to the presence of persistent acholic stools, which are similarly found in extrahepatic biliary atresia. The administration of vitamin K to the cholestatic newborn is also necessary, in order to prevent intracranial hemorrhage resulting from the malabsorption of vitamin K in this situation.9

Causes of cholestasis In Table 1, the main extra and intrahepatic causes of cholestasis in the infant are displayed. The highlighted affections are those that will be commented detailedly, due to their diagnostic, therapeutic, and prognostic particularities. Extrahepatic biliary atresia Extrahepatic biliary atresia is an affection that results from the obliterate and fibrosing process of intra and extrahepatic bile ducts due to a perinatal inflammatory process of unknown etiology, which presents progressive evolution to biliary cirrhosis, despite the successful surgical

Table 1 -

Causes of cholestasis in the infant

EXTRAHEPATIC CAUSES Extrahepatic biliary atresia Choledochal cyst Stenosis at the bile duct Mucous tampon INTRAHEPATIC CAUSES Idiopathic neonatal hepatitis Hypoplasia of interlobular ducts Syndromic (Alagilles syndrome or arteriohepatic dysplasia) Nonsyndromic Familial progressive intrahepatic cholestasis (Bylers disease)10 Zellweger syndrome (cerebrohepatorenal syndrome, peroxisomal disease) Trihydroxycoprostanic acidemia Metabolic diseases Tyrosinemia Galactosemia Hereditary intolerance to fructose Alpha 1-antitrypsin deficiency Cystic fibrosis11 Idiopathic hypopituitarism12 Hypothyroidism Lipidoses (Gauchers disease, Niemann-Picks disease, Wolmans disease) * Infectious diseases (cytomegalovirus, herpes, mumps, Coxsackie, echovirus, HIV, 13 hepatitis B, toxoplasmosis, syphilis, tuberculosis,14 listeriosis) Toxic Parenteral nutrition Sepsis Urinary tract infection Genetic/chromosomal (trisomy E, Downs syndrome) Miscellaneous (shock, intestinal obstruction, histiocytosis)

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correction. Extrahepatic biliary atresia is believed to be the phenotypic expression of several underlying diseases,15 being responsible for 90% of cases of obstructive cholestasis in children.1 Its rare occurrence among stillborn and preterm children points to the possibility of its onset in the perinatal period. A higher frequency of antigen HLA B12, and of the haplotypes A9-B5 and A28-B35 was observed among patients affected by extrahepatic biliary atresia.16 The incidence ranges from 1:8,000 to 1:23,000 live births.17,18 It is classified in three types: atresia restrict to the common bile duct (type I), atresia of the common hepatic duct (type II), and atresia of the right and left hepatic ducts (type III), which is responsible for 70% to 90% of the cases. Most children with extrahepatic biliary atresia are term newborns, with normal weight. Extrahepatic biliary atresia may be associated with polysplenia syndrome in 5% to 21% of patients. This syndrome is characterized by intestinal malrotation, polysplenia, preduodenal portal vein, absence of inferior vena cava, bilobate symmetric liver, situs inversus, anomalous supply to the hepatic artery, bilobate right lung, dextrocardia, and other cardiac defects. The palliative correction of extrahepatic biliary atresia is performed through the Kasai procedure (portoenterostomy), which consists of the excision of the hepatic hilum fibrous cone, with exposure of the area that contains the residual microscopic bile ducts, followed by anastomosis of this area with the jejunal loop in Y-Roux. The success of the surgery is associated with the childs age at the occasion of the procedure; this means that it should be performed before 60 days of life (or before 45 days of life, as it is recommended by the French), considering that the liver bile ducts at the region close to the hepatic hilum are patent in the first weeks of life, but are progressively destroyed.19 So, the agile and adequate reference of infants with cholestasis constitutes the most important prognostic determinant for the postoperative biliary flow and for the survival of individuals affected by extrahepatic biliary atresia. In the Frenchs experience, the following independent factors were observed to improve the global survival prognosis: previous Kasai procedure, age at surgery (<45 days), anatomical type of atresia (more favorable in type I), absence of polysplenia, and teams experience (expressed by the annual number of Kasai procedures).20 About 15 to 20% of children with extrahepatic biliary atresia submitted to Kasai procedure will not need liver transplantation; on the other hand, in the other children, Kasai procedure will postpone the need for a transplantation to a more favorable occasion, allowing adequate growth and the development of immunity to viral infections, especially to Epstein-Barr virus, which is responsible for the lymphoproliferative disease.21 Choledochal cyst The incidence of choledochal cyst ranges from 1:13,000 to 1:2,000,000 live births, and it is more frequent among Oriental people, with a predominance in females (more than

80%). It represents the second most common cause of extrahepatic cholestasis (the first is extrahepatic biliary atresia). It is classified in five different types 22: type I consists of the saccular or fusiform dilatation of the common bile duct, which is responsible for 90% of the cases; type II has the form of a diverticulum; type III is represented by choledochocele (dilatation of the intraduodenal portion of the common bile duct); type IV presents multiple dilatations of the intra and extrahepatic bile ducts; type IVb, with multiple extrahepatic cysts, rarely occurs; type V, also denominated Caroli disease, consists of an isolated or multiple dilatation of large intrahepatic bile ducts (segmental) - actually, type V is a cystic disease of the bile duct, not a type of choledochal cyst. The etiology of choledochal cyst in infancy is not clarified yet, and it occurs in infants younger than 6 months. Its clinical expression is indistinguishable from the extrahepatic biliary atresia. Contrarily to the latter, its diagnosis is easily established by abdominal ultrasound, even inside the uterus, allowing diagnosis, early surgical correction, and, consequently, improvement in the prognosis. Surgery consists of the performance of hepaticojejunostomy in Y-Roux, associated with resection of the cyst, aiming at reducing biliary stasis and the subsequent risk for cholangitis and for becoming malignant (cholangiocarcinoma). Idiopathic neonatal hepatitis This definition includes most unknown causes of intrahepatic cholestasis. It is obvious that with the advances in investigation methods and with the growing experience regarding cholestatic syndrome, mainly in developed countries, there is a tendency towards the reduction of cases of idiopathic neonatal hepatitis, and an increase in the diagnosis of other clinical entities. The predominance of the diagnosis of idiopathic neonatal hepatitis is the unequivocal result of the still incomplete domain of the knowledge about the causes of cholestasis and of the current diagnostic limitations. Idiopathic neonatal hepatitis is an affection that dos not have treatment indication, and it is limited to clinical and laboratory follow-up to the patient. The prognosis is different for sporadic and familial cases. Among the sporadic cases, 60% recover, 10% have persistent inflammation or fibrosis (2% with cirrhosis), and 30% die. On the other hand, among the familial cases, only 30% recover, while 60% die, and 10% develop chronic hepatic disease or cirrhosis. 23 Hipoplasia of interlobular bile ducts The hypoplasia of interlobular bile ducts may present in two forms: syndromic (Alagilles syndrome or arteriohepatic dysplasia), and nonsyndromic. Both forms are distinguished mainly through the best prognosis of the former in relation to the latter, and through the evidence of clinical findings that are typical of Alagilles syndrome. From the histopathologic point of view, hypoplasia of interlobular

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bile ducts is characterized by the number of interlobular bile ducts/0.5 inferior portal spaces ratio; however, about 20% of the cases have hypoplasia defined only after 6 to 12 months of life. Alagilles syndrome24 is a rare entity (1:40,000 to 1:100,000 live births) of genetic transmission, with dominant autosomal character, reduced penetrance, and variable expressivity, related to microdeletions of the short arm of chromosome 20; most patients do not present familial history, which reinforces the characteristic of incomplete penetrance, and the possibility of new genetic mutation.1 It is characterized by five major signs: chronic cholestasis (temporary, persistent, or intermittent jaundice, itching starting at 6 months; xanthomas at skin attrition areas), dimorphic facies (protuberant forehead, hollow eyes, with discrete hypertelorism, pointed chin, flat nose, with a bulbshape point), cardiovascular anomalies (stenosis of the peripheral branch of the pulmonary artery, which is present in 90% of the cases, without clinical repercussion), defects of the vertebral arches (butterfly vertebras), and posterior embryotoxon (this sign is present in 10% of the general population, and consists of the thickening of the line formed by Descemet membrane and the angle of the anterior eye chamber, which is evident with the use of slit lamp or through gonioscopy). Facial dimorphism and the evidence of butterfly vertebras may be better characterized as the patients age advances. The syndrome is considered complete when at least four signs present together, while the presence of only three of them characterizes the incomplete form. Other clinical findings are less frequent in Alagilles syndrome, such as delayed growth, hypogonadism, shortening of distal phalanges, deforming polyarticular arthritis,1 renal compromising (mesangiolipidosis, renal tubular acidosis, nephrocalcinosis, etc.), and alteration in the voice volume and tone. The enzymes alkaline phosphatase and gammaglutamyltransferase are significantly elevated in most cases. Cholesterol may reach up to 3,000 mg/dL in 10% of the patients with Alagilles syndrome.1 The course of cholestasis is variable, and the cofrequency is exacerbated during a viral infection. Itching, xanthomas (cholesterol deposits), and neuromuscular symptoms resulting from vitamin E deficiency are responsible for the morbidity. Tyrosinemia Tyrosinemia type 1 is an inborn error of metabolism, and results from the deficiency of the enzyme fumarylacetoacetate hydrolase, of recessive autosomal transmission. It is a serious and rare disease, but its frequency is especially elevated in the north of Quebec, in Canada. It may be acute or chronic. The acute form has an early onset, with high mortality in the first 6 months of life, except for infants that have access to liver transplantation. It evolves with cirrhosis, vitamin D-resistant rickets, hemorrhagic disorders, sepsis, and porphyria crises. The porphyria crises manifest with vomiting, intense abdominal pain, fever, hypotonia,

tachycardia, hypertension, pain in the extremities, peripheral polyneuropathy, difficulty for deambulation, flaccid paralysis, respiratory paralysis, syndrome of inappropriate secretion of antidiuretic hormone, and hyponatremia. The chronic form presents hepatomegaly associated or not with hypophosphatemic rickets resulting from proximal tubulopathy; porphyria attacks are usually caused by prolonged fasting, infection, or transgression of the diet. The hepatocellular carcinoma is the most feared complication of the chronic form, and requires semestral monitoring of the alpha-fetoprotein level, and the performance of abdominal ultrasound or tomography in every patient presenting tyrosinemia. 25 The levels of serum phenylalanine, tyrosine, and methionine are high in tyrosinemia; the alpha-fetoprotein exhibits extremely elevated levels. In urine, succinyl acetone and delta-aminolevulenic acid are elevated, and there is the identification of glycosuria, generalized aminoaciduria, phosphaturia, and hypercalciuria.25 Treatment consists of dietary restriction of phenylalanine and tyrosine, with a favorable result only with proximal renal tubulopathy. The hepatic disease requires liver transplantation. The 2-(2-nitro-4-trifluoromethylbenzoyl)1,3-cyclohexanedione (NTBC), is still in experimental phase, but has been considered a promising drug for the treatment of hepatic compromising and tyrosinemia.25 Galactosemia Galactosemia results from deficiency of galactose-1phosphate uridyltransferase, of recessive autosomal genetic character. Its clinical expression varies from the mild form, with diarrhea and vomiting coinciding with the intake of milk, up to severe acute hepatic failure. The classical form of galactosemia has the following characteristics: growth deficit, digestive symptoms (vomiting and diarrhea), occasional hypoglycemia, hepatic alterations (hepatomegaly, coagulation disorders, jaundice, portal hypertension, ascites, etc.), ocular alterations (cataracts, retinal detachment, and bilateral intraocular hemorrhage), mental retardation, renal tubulopathy, and sepsis caused by Escherichia coli. The clinical suspicion of galactosemia indicates the immediate need for a metabolic screening through the assay of reducing substances in the urine, which are not reactive to glucose-oxidase, performed in children using milk and presenting no evidence of vomiting during the examination. The pediatrician should be careful in the interpretation of the exam, since there are other reducing substances, such as lactose, maltose, fructose, uric acid, vitamin C, and cephalosporins. Moreover, galactosuria (identified through urinary chromatography) is nonspecific for the diagnosis of galactosemia, and may be observed in normal term newborns during the first 5 days of life, and in preterm, up to 2 weeks of life. On the other hand, about 50% of the children with chronic hepatic diseases present galactosuria, although they do not have enzyme deficiency. Through Beutler & Baludas spot test, it is possible to

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determine qualitatively the presence of absence of galactose1-phosphate uridyltransferase. The exam should be postponed for 3 to 4 months in children that have received blood transfusion. The proof exam consists of the determination of activity of the enzyme galactose-1phosphate uridyltransferase in red blood cells or in cultivated fibroblasts.26 The institution of a diet free from galactose (without milk and dairy products) may revert the status if it is started early. Every mother that already has a child with diagnosis of galactosemia should be prescribed a low-galactose diet in the subsequent pregnancies. However, dietary restriction of galactose during pregnancy does not totally prevent the installation of galactosemia in the child with deficiency of galactose-1-phosphate uridyltransferase, considering the endogenous synthesis of galactose in the mother.26 Hereditary intolerance to fructose The hereditary intolerance to fructose results from deficiency in the activity of aldolase B, transmitted through recessive autosomal inheritance. Clinical manifestations depend on the introduction time and on the amount of fructose, saccharose, and sorbitol in the diet; the younger the child is and the larger the quantity of sugar ingested is, the more severe the disease will be. In the acute form of the disease, vomits predominate (they are related to the introduction of foods containing fructose in the diet), as well as symptomatic hypoglycemia. The chronic form is characterized by delayed growth, vomiting, diarrhea, aversion to foods containing fructose after 2 years of age, hepatic alterations (cholestasis, hepatomegaly, prolonged prothrombin time, hepatic insufficiency, ascites, steatosis, and cirrhosis), renal tubulopathy, anemia, plateletopenia, hypoglycemia, hypophosphatemia, hypermagnesemia, hyperuricemia, lactic acidemia, and hyperacidoaminemia. The diagnostic screening is done through the assay of reducing sugars in the urine of patients that are known to use foods with fructose. The diagnostic confirmation is performed by the determination of the activity of fructose1-phosphate aldolase in a fragment of hepatic tissue or of small bowel. The treatment consists of the withdrawal of all foods and medications containing fructose, saccharose, and sorbitol, and, from 2 to 3 years of age on, the introduction of fructose in small quantities is allowed. Deficiency of alpha 1-antitrypsin The phenotype PiZZ is the responsible for the deficiency of alpha 1-antitrypsin (in a level 40% lower than the reference value) and for hepatic alterations that result from this deficiency. Up to the present moment, the pathogenesis of hepatic disease in alpha 1-antitrypsin deficiency remains unknown. In the experience of Kings College Hospital,

London, the deficiency of alpha 1-antitrypsin was responsible for 17% of the cases of cholestasis.7 In the study carried out by Sveger, in Sweden, among the newborns with phenotype PiZZ (alpha 1-antitrypsin from 20 to 160 mg/dL), 12% presented cholestasis in the beginning of life, with remission of jaundice between the 2nd and the 4th month of life.27 Of the 127 patients with phenotype PiZZ at 18 years of age, 85% remained with normal aminotransferases, without any clinical or biochemical evidence of hepatic dysfunction. 28 Pulmonary emphysema appears later in individuals with phenotypes PiZZ or PiNul. The histopathologic diagnosis is carried out through the identification of PAS-positive globules resistant to diastase in the periportal hepatocytes after 12 weeks of life. Through the immunohistochemical assay, the diagnosis is confirmed by the presence of alpha 1-antitrypsin in the PAS-positive globules. There is no specific treatment for the hepatic disease in alpha 1-antitrypsin deficiency yet. The only therapeutic resource is still the liver transplantation. Parenteral nutrition Cholestasis resulting from parenteral nutrition has a pathogenetic multifactorial explanation. It is more frequent among low-weight newborns that use parenteral nutrition for more than 2 weeks. Cholestasis associated with total parenteral nutrition occurs in up to 33% of the infants that receive it for less than 8 weeks; it may affect 80% of those that receive it for more than 2 months.29 The hepatic alteration advances slowly, since laboratory alterations (elevation of serum bile acids, increase in bilirubins, with predominance of the conjugated or direct fraction in aminotransferases and in alkaline phosphatase), jaundice, and hepatomegaly, going through steatosis, hepatic histopathology, comparable to that of extrahepatic biliary atresia, peaking with the biliary cirrhosis in cases of extremely prolonged duration of the parenteral nutrition, besides the distension of the gallbladder and the formation of biliary sludge (precursor of the biliary lithiasis), which is visible through abdominal ultrasound. Several factors are implicated in the genesis of cholestasis resulting from prolonged total parenteral nutrition: immaturity of the enterohepatic circulation, perinatal aggressions (fasting, hypoxia, hypovolemia, hepatotoxic drugs, gastrointestinal diseases, sepsis, exposure to hepatotropic viruses),30,31 nutrient deficiency (taurine, essential fatty acids, carnitine, trace metals, vitamin E, selenium, etc.), and toxicity of the nutrients (amino acids, lipids, metals, etc.). The best strategy is the prevention through the early institution of oral nutrition or through continuous infusion with the use of probe. The limit of protein should also be established at less than 3 g/kg/day. The prognosis of cholestasis secondary to parenteral nutrition is relatively good, with a low remission of the alterations within 4 to 6

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months after its interruption. Less than 10% of the cases evolve to hepatic cirrhosis. A pilot study showed the efficacy of the ursodeoxycholic acid (30 mg/kg/day) in the treatment of cholestasis associated with total parenteral nutrition.32 There was also a successful try with the use of cholecystokinin in the prophylaxis of cholestasis in risk newborns submitted to total parenteral nutrition.33

injected drugs, homosexuality, promiscuous sexual life, hemodyalisis treatment, blood or blood derivative transfusion, health professional), prenatal history of infection, use of medications, and hepatic disease in relatives. Some characteristics of the clinical manifestations and of the clinical examination of infants presenting cholestasis may point to determined diagnoses, according to what is presented in Table 3.

Clinical diagnosis The clinical status of several cholestatic diseases is usually similar - jaundice, choluria, hypocholic or acholic stools, hepatomegaly with or without splenomegaly -, in spite of the cause of cholestasis. However, in 288 infants younger than 3 months of age, Alagille used as clinical criteria: observation of color in feces during 10 days, birthweight, average age at the beginning of acholic stools, and the clinical characteristics of hepatomegaly. These characteristics allowed to distinguish between the cholestatic diseases of extrahepatic origin and those of intrahepatic nature, with an confidence level of 82%34 (Table 2). For a correct assessment of the color of the feces, it is indispensable that the child is not ingesting colorful foods or medications during the observation period; it is recommended that the pediatrician does at least one rectal touch on the child, in order to have the opportunity to examine the feces recently emitted, and check if they are homogeneously colored or acholic. The evidence of feces persistently acholic associated with persistent hepatomegaly indicate the probable diagnosis of extrahepatic biliary atresia.4 Itching is part of the clinical expression only after 4 or 6 months of life; xanthomas are manifested later, in intrahepatic affections that go along with important hypercholesterolemia (example: Alagilles syndrome). In the anamnesis of an infant with suspicion of cholestatic disease, some inquiries about the relatives and particularly about the mother are indispensable: parents consanguinity, father and/or mother pertaining to a risk group (use of

Complementary examinations There are two main goals to be achieved through a fast and effective investigation: to establish if the cholestatic disease requires surgical treatment (extrahepatic cause) or clinical treatment (intrahepatic cause), and, simultaneously, to verify if the cholestasis results from an intrahepatic disease that requires specific treatment.

Table 3 -

Important clinical signs in the diagnosis of cholestasis Diagnostic suspicion Congenital infection, Alagilles syndrome Congenital infection, sepsis, galactosemia, tyrosinemia Sepsis, galactosemia, fructosemia, tyrosinemia, urinary tract infection Galactosemia, congenital mumps Galactosemia, fructosemia, idiopathic hypopituitarism Alagilles syndrome

Clinical sign Low birthweight

General status compromised

Vomiting

Cataracts

Hypoglycemia

Facies (protuberant forehead, hollow eyes, bulbous nose, and pointed chin) Table 2 Differential diagnosis between extra and intrahepatic cholestases (Alagille, 1979) Intrahepatic diseases Birthweight (average) Onset of acholic stools (days) Frequency of colored feces (10-day observation) Liver with large volume and consistency 2,680 g 30 79% 53% Extrahepatic biliary atresia Myocarditis 3,230 g 16 26% 87% Polysplenia syndrome Butterfly vertebra Posterior embryotoxon Micropenis Rickets Congenital cardiopathy

Alagilles syndrome, extrahepatic biliary atresia, congenital mumps Coxsackie Extrahepatic biliary atresia Alagilles syndrome Alagilles syndrome Idiopathic hypopituitarism Tyrosinemia

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The investigational approach should use diagnostic methods that allow a minimal biochemical evaluation (hepatic profile, glycemia, renal function), hemogram with coagulation assay, biochemical and serologic studies for the diagnosis of certain clinical entities, and mainly the use of diagnostic resources that evidence the existence or not of biliary flow. Table 4 displays all the examinations available that are indispensable to a broad evaluation of infants with cholestasis. It is necessary to clarify that not all the exams are available in the public health care system. Some diagnostic methods will receive a special analysis, according to their relevance. Direct bilirubin, in a level equal or higher than 20% of the total bilirubin - direct hyperbilirubinemia -, or when it goes over 2 mg/dL, constitutes one of the main laboratory expressions of cholestasis. However, using the conventional dosage methods, there is disagreement between laboratories concerning bilirubin fractions, with an overestimate of direct bilirubin when the total bilirubin is inferior to 5 mg/ dL; this mistake makes the distinction between hepatobiliary and hemolytic diseases difficult sometimes.35 In the extrahepatic biliary atresia, there is a moderate and fluctuating increase in the serum level of bilirubin: so, a reduction of bilirubin in two successive dosages does not allow to discard the diagnosis of this affection.4 Several comparative studies tried to distinguish, through serum dosages of bilirubin and through the evaluation of the activity of aminotransferases and gammaglutamyltransferase, between extrahepatic cholestases and intrahepatic diseases. However, they found a low discriminatory power and an elevated rate of superposing levels of those dosages in both intrahepatic and extrahepatic affections. Abdominal ultrasound The abdominal ultrasound constitutes, together with biochemical and hematological evaluations, the initial investigation in any patient with cholestasis. First, the hepatic biopsy through punction should be carried out, aiming at detecting images indicative of contraindication for the procedure, such as hemangiomas, intrahepatic cystic lesions, and voluminous ascites. The main objective of abdominal ultrasound in the investigation of cholestasis is the fast and secure identification - even inside the uterus - of choledochal cysts, whose prognosis is related to early surgical correction. Besides, it is possible to assess if there is dilatation of the bile ducts, which is a finding related to extrahepatic obstructions, excluding the extrahepatic biliary atresia. As a resource for the differential diagnosis between extrahepatic biliary atresia and intrahepatic conditions, ultrasound has some limitations. So, it should not be used as an isolated diagnostic method, but should be analyzed within a clinical context, with the help of the subsidiary exams available in each case. Through this imaging method,

Table 4 -

Complementary examinations for the diagnosis of cholestasis in infants

1 - Hemogram 2 - Coagulogram - Platelets - Prothrombin time - Activated partial thromboplastin time 3 - Biochemical (blood) - Bilirubins - Aminotransferases, alkaline phosphatase, gammaglutamyltransferase - Albumin - Glycemia - Urea and creatinine - Triglycerides and cholesterol - Calcium and phosphor - Protein electrophoresis 4 - Urine - Routine urine, assay for reducing substances in the urine (Clinitest*) and urine culture 5 - Screening or inborn errors of metabolism in blood and urine 6 - Alpha 1-antitrypsin: dosage and phenotype (isoelectric electrofocalization) 7 - Serology - VDRL - HBsAg and total anti-HBc - Toxoplasmosis: immunofluorescence and ELISA IgM - Cytomegalovirus: ELISA IgM - Mumps: ELISA IgM - HIV: ELISA IgM, western blot, and polymerase chain reaction 8 - Imaging - Simple radiographs of thorax, cranium, column, and long bones - Biliaresdominal acid ultrasound - Hepatobiliary scintigraphy with 99m-Tc DISIDA - Preoperative (transhepatobiliary or endoscopic) or operative cholangiography 9 - Hepatic biopsy 10 - Others - Duodenal tubage - Sodium and chlorate in sweat - Alpha-fetoprotein - Dosage of succinyl acetone in urine - Galactosemia: urinary chromatography of carbon hydrates Bleuter-Baluda test Activity of galactose-1-phosphate uridyltransferase in red blood cells or in cultivated fibroblasts - T3, T4, thyrotropin, cortisol, growth hormone - Hemoculture - Myelogram - Ophthalmologic exam

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it is possible to verify the presence of polysplenia, or of preduodenal portal vein, which constitute anomalies associated with extrahepatic biliary atresia. Abdominal ultrasound should be preceded by 4 to 6 hours of fasting, so that the gallbladder remains full of bile. The normal gallbladder presents length equal or superior to 1.5 cm, and is not visible in 60% of the patients with extrahepatic biliary atresia. The nonidentification of the gallbladder imposes the repetition of the exam within 24 hours. Through the motor proof (diet intake), it is possible to show the temporary increase of the gallbladder during the diet, followed by a contraction in individuals that have totally pervious bile ducts. The verification of contraction of the gallbladder is an indirect proof that there is permeability between the cystic duct and the duodenum, but this does not implicate patency of the proximal ducts. A new ultrasonographic finding - the sign of triangular cord -, evidenced at the cranial portion of the portal vein bifurcation, defined by some Korean authors, has sensitivity of 85%, specificity of 100%, and accuracy of 95% for the diagnosis of extrahepatic biliary atresia. 36 The reproductibility of this ultrasonographic finding will allow a faster investigation, considering that all the patients with this finding had the diagnosis of extrahepatic biliary atresia confirmed, while none of the cases of intrahepatic cholestasis presented the triangular cord. An ultrasonographist of the Radiology Service, at Hospital das Clnicas da Universidade Federal de Minas Gerais, assessing 27 cases of cholestasis, observed a sensitivity of 62.5% and a specificity of 100% for the triangular cord sign.37 Duodenal tubage Duodenal tubage consists of the introduction of a radiopaque probe at the second or third portion of the duodenum, with the collection of duodenal juice for 24

hours. So, the presence of bile is verified through the observation of the liquid drained or aspirated. It is a simple, little invasive, and inexpensive method, but it requires patience and perseverance from who is performing the procedure. The same care measures recommended for the observation of the color of feces (suspension of colorful foods or medications) are also valid for the tubage. Hepatic biopsy In several reports of the medical literature, the hepatic histopathology has revealed an excellent diagnostic resource to distinguish between cholestases resulting from extrahepatic obstruction and those of intrahepatic origin, mainly allowing the differentiation between extrahepatic biliary atresia and idiopathic neonatal hepatitis. According to several studies, the results show variations in the precision of the histopathology: sensitivity (83.3-95.6%), specificity (82.5-100%), accuracy (86.5-96.8%). Some histopathological characteristics allow to differentiate idiopathic neonatal hepatitis from extrahepatic biliary obstructions, according to what is shown in Table 5. In a study developed in Porto Alegre, Rio Grande do Sul, Brazil, the following histopathological variables were observed, in a decrescent order, for the diagnosis of extrahepatic cholestasis: periportal ductal proliferation, portal ductal proliferation, portal expansion, cholestasis in neoductules, myeloid metaplasia, and porto-portal shunts.38 Hepatobiliary scintigraphy The radiopharmaceutical Tc-99m DISIDA is captured by the liver and excreted by the bile ducts to the duodenum in normal individuals. Scintigraphy with this radiopharmaceutical is destined to establish the differential diagnosis between partial and complete cholestasis in young infants. The intravenous administration of Tc-99m DISIDA should be preceded by the use of phenobarbital (5 mg/kg/day,

Table 5 -

Histopathologic differences between idiopathic neonatal hepatitis and extrahepatic biliary obstructions* Extrahepatic biliary obstruction Intracytoplasmic and canalicular cholestasis Preserved architecture Rare giant cells Discrete inflammatory infiltrate Portal and periportal fibrosis Ductal proliferation Biliary thrombus in the interlobular ducts

Idiopathic neonatal hepatitis Intracytoplasmic and canalicular cholestasis Disarranged architecture Gigantocellularis transformation Inflammatory infiltrate in the portal space Little fibrosis Extramedullary hematopoiesis

* The characteristics highlighted refer to the most important histopathological findings for the diagnosis of each of the affections.

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twice a day, orally) during 3 to 5 days, aiming at reducing the number of undetermined and false positive exams through the increased conjugation and excretion of the radiotracer, and increased bile acid independent bile flow. At first, this method was considered a promising diagnostic resource. However, in several experiences, it revealed to be a high-sensitivity exam, but of low specificity for the diagnosis of surgical cholestases, with accuracy ranging from 52.5 to 87%. Due to the limitations that were inherent to the exam, mainly in cases of intense intrahepatic cholestasis and in infants with extrahepatic biliary atresia referred late, the Pediatric Clinics and Surgery Service, at Hospital das Clnicas da Universidade Federal de Minas Gerais, abandoned the hepatobiliary scintigraphy, since the intense majority of results, independently on the cause of the cholestasis, showed absence of radioisotope flow to the duodenum. Cholangiography Preoperative cholangiography through retrograde laparoscopic or endoscopic via, which are unavailable in most urban centers, have their use quite limited. The experience with this method has been restrict to small groups of patients. Operative cholangiography, performed after a minilaparotomy, constitutes the last diagnostic method in cases of probable extrahepatic biliary atresia or in those patients with persistent acholic stools in which is was impossible to discard an extrahepatic biliary obstruction. In infants with extrahepatic biliary atresia, surgical cholangiography is possible to be performed in only 17 to 25% of the cases, due to the impossibility of injecting contrast agents through the gallbladder in atresia. The exclusion of diagnosis of extrahepatic biliary atresia can be guaranteed only when the permeability of bile ducts from the interior of the liver up to the duodenum is evidenced unequivocally. Considering the difficulty in the execution of cholangiography, the experienced surgeon establishes the diagnosis of extrahepatic biliary atresia through the macroscopic evaluation of hepatobiliary structures.

to define the diagnosis of diseases that require a specific treatment, such as galactosemia, hereditary intolerance to fructose, tyrosinemia, sepsis, etc. Currently, abdominal ultrasound and hepatic histopathology constitute the most accurate diagnostic methods for the distinction between intra and extrahepatic cholestases; the duodenal tubage and the hepatobiliary scintigraphy were left aside, since they do not allow good specificity and diagnostic accuracy, and also because of the morosity that is inherent to the former method. The patients referred to the surgeon are mainly those affected by extrahepatic biliary atresia or by choledochal cyst; so, the surgery team should work together with the clinical team, in order to perform the surgical correction quickly. The success of the surgery will depend also on the surgeons experience and ability, and he/she will still have to face the postoperative complications. The team responsible for the clinical follow-up of patients submitted to Kasai procedure frequently faces consequences of an unsuccessful surgery: protein-energy insufficiency, hypovitaminoses, itching, infections, complications resulting from portal hypertension (upper gastrointestinal bleeding, ascites, spontaneous bacterial peritonitis). Moreover, when the patient develops severe hepatic insufficiency, the medical team has to skillfully administrate the patients financial difficulties, the familial unstructure, and the perspective of a magical solution with the liver transplantation.

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Correspondence: Profa. Mariza Leito Valadares Roquete Rua Prof. Norton Kaiserman, 82/102 Bairro Anchieta CEP 30310-570 Belo Horizonte, MG, Brazil E-mail: roquetemlv@uol.com.br