Neonatal haemochromatosis associated with gastroschisis.

We describe, to our knowledge, the first case of progressive neonatal liver failure due to neonatal haemochromatosis (NH) occurring in an infant with a gastroschisis and review the literature regarding these two conditions. A 1,665 g male infant with antenatally diagnosed gastroschisis was born with a severe coagulopathy, anaemia, thrombocytopenia, hypoglycaemia and jaundice. He developed progressive liver failure, complicated by necrotising enterocolitis. Serum ferritin was elevated at 1,459 microg/L. He died on day 40 and a limited post-mortem examination confirmed significant hepatic siderosis with fibrosis and cholestasis, and siderosis of the pancreas. Although no genetic aetiology for gastroschisis has been identified, an occasional inherited tendency has been observed. There is also evidence to support an autosomal recessive inheritance in NH.

High frequency of missense mutations in glycogen storage disease type VI.

Deficiency of liver glycogen phosphorylase in glycogen storage disease (GSD) type VI results in a reduced ability to mobilize glucose from glycogen. Six mutations of the PYGL gene, which encodes the liver isoform of the enzyme, have been identified in the literature. We have characterized eight patients from seven families with GSD type VI and identified 11 novel PYGL gene defects. The majority of the mutations were missense, resulting in the substitution of highly conserved residues. These could be grouped into those that were predicted to affect substrate binding (p.V456M, p.E673K, p.S675L, p.S675T), pyridoxal phosphate binding (p.R491C, p.K681T), or activation of glycogen phosphorylase (p.Q13P) or that had an unknown effect (p.N632I and p.D634H). Two mutations were predicted to result in null alleles, p.R399X and [c.1964_1969inv6;c.1969+1_+4delGTAC]. Only 7 of the 23 (30%) reported PYGL alleles carry nonsense, splice site or frameshift mutations compared to 68-80% of affected alleles of the highly homologous muscle glycogen phosphorylase gene, PYGM, that underlie McArdle disease. There was heterogeneity in the clinical symptoms observed in affected individuals. These varied from hepatomegaly and subclinical hypoglycaemia, to severe hepatomegaly with recurrent severe hypoglycaemia and postprandial lactic acidosis. We conclude that deficiency of liver glycogen phosphorylase is predominantly the result of missense mutations affecting enzyme activity. There are no common mutations and the severity of clinical symptoms varies significantly.

Copper-induced hepatotoxicosis with hepatic stellate cell activation and severe fibrosis in North Ronaldsay lambs: a model for non-Wilsonian hepatic copper toxicosis of infants.

Copper-sensitive North Ronaldsay sheep represent a possible model for certain hepatic-overload syndromes of infancy and childhood that are clinically, pathologically and genetically distinct from Wilson's disease. The purpose of this study was to simulate in artificially reared lambs the syndrome produced by copper exposure in susceptible human infants. Twenty four North Ronaldsay lambs were assigned to three groups of eight animals, namely, an unsupplemented control group and two trial groups given milk replacer to which copper (CuSO4) had been added at the rate of 5 mg/litre and 10 mg/litre. Four lambs from each group were killed at 40 or 69 days. Livers were fixed in 10% formalin and analysed for copper by mass spectrometry. Paraffin wax-embedded sections were stained with rhodanine for copper and labelled immunohistochemically for alpha smooth muscle actin (ASMA). At 40 days the maximum amounts of copper in the livers of both copper-supplemented groups was 1466-1605 microg/g dry weight (control group 172-201 microg/g Cu dry weight). Histochemically, copper was demonstrated within hepatocytes, together with marked apoptosis. At 69 days there was a florid pericellular fibrosis complemented by strong ASMA immunolabelling, confirming phenotypic modulation of hepatic stellate cells. Such primary copper-induced fibrogenesis confirms the unique status of this animal model in respect of childhood copper toxicosis.

Use of basiliximab in pediatric liver transplantation for Langerhans cell histiocytosis.

This report describes a 16-month-old girl with multi-system Langerhans cell histiocytosis (LCH), who developed end-stage liver disease despite intensive chemotherapy. She underwent a liver transplant at 28 months of age while receiving maintenance chemotherapy for bony lesions. In view of previous reports of a high incidence of acute cellular rejection and post-transplant lymphoproliferative disease (PTLD) in children transplanted for LCH, basiliximab was added to the post-transplant immunosuppression regime of tacrolimus and prednisolone. Sixteen months post-transplant, she has had no episodes of acute rejection or PTLD and her LCH has remained in remission. Current literature regarding liver transplantation (LTx) for LCH and the use of basiliximab in pediatric LTx is reviewed.

Contribution of midazolam and its 1-hydroxy metabolite to preoperative sedation in children: a pharmacokinetic-pharmacodynamic analysis.

BACKGROUND: Oral midazolam is widely used for preoperative sedation in children. We have studied the pharmacokinetics (PK) of both midazolam and its active 1-hydroxy metabolite and their contributions to sedative effect in 45 children attending for day surgery. METHODS: Blood samples (two per individual) were collected at the beginning and end of the surgical procedure. Plasma midazolam and 1-hydroxymidazolam (1-OHMDZ) were measured by HPLC. Sedation score (score: 1 = awake, 2 = drowsy/asleep) was recorded at the same time as the first blood sample. The population-PK software P-Pharm was used to analyse the data. Age, weight, sex, concomitant drugs, and the metabolic ratio, 1-OHMDZ/midazolam were investigated as co-variates of the PK of midazolam and 1-OHMDZ. The pharmacokinetic-pharmacodynamic (PK-PD) modelling of the score in relation to plasma midazolam and 1-OHMDZ was performed using logistic regression analysis. RESULTS: A median dose of 0.5 mg kg-1 was given to the children, median age 5 yr (range from 9 months to 12 yr) and weight 21 kg (range 8-75 kg). Average concentrations of midazolam 150 ng ml-1 and 1-OHMDZ 90 ng ml-1 were observed in the first plasma samples. These concentrations resulted in an odds ratio of 4 in favour of score 2 vs 1. The best PK-PD model included both midazolam and 1-OHMDZ as active moieties and predicted correct scores in 86% of cases. CONCLUSION: Studies of midazolam should evaluate the contribution of 1-OHMDZ to the overall PD effect. The metabolite 1-OHMDZ has approximately half the activity of the parent drug and can compensate for at least part of the decreased effect due to increased midazolam metabolism.

Developmental changes in the expression of enterocytic and hepatic cytochromes P4501A in rat.

1. The development of CYP1A enzymes was studied in enterocytic and hepatic microsomes from 1-day-old to adult male and female rats. Microsomes were prepared by calcium precipitation. Enzyme expression was determined by Western blotting using a polyclonal CYP1A1 antibody. 2. The developmental expression of CYP1A in enterocytic and hepatic microsomes was similar for males and females. 3. Enterocytic CYP1A (CYP1A1) showed a sharp increase at weaning, plateauing at adult levels by 60 days. 4. Hepatic CYP1A (mostly CYP1A2) increased sharply just before weaning. However, in contrast to the enterocytic enzyme, there was a 4-fold decrease in enzyme expression down to adult levels by day 60.

Mechanisms of liver injury relevant to pediatric hepatology.

Hepatocyte injury and necrosis from many causes may result in pediatric liver disease. Influenced by other cell types in the liver, by its unique vascular arrangements, by lobular zonation, and by contributory effects of sepsis, reactive oxygen species and disordered hepatic architecture, the hepatocyte is prone to injury from exogenous toxins, from inborn errors of metabolism, from hepatotrophic viruses, and from immune mechanisms. Experimental studies on cultured hepatocytes or animal models must be interpreted with caution. Having discussed general concepts, this review describes immune mechanisms of liver injury, as seen in autoimmune hepatitis, hepatitis B and C infection, the anticonvulsant hypersensitivity syndrome, and autoimmune polyendocrinopathy. Of the monogenic disorders causing significant liver injury in childhood, alpha-1 antitrypsin deficiency and Niemann-Pick C disease demonstrate the effect of endoplasmic or endosomal retention of macromolecules. Tyrosinemia illustrates how understanding the biochemical defect leads to understanding cell injury, extrahepatic porphyric effects, oncogenesis, pharmacological intervention, and possible stem cell therapy. Pathogenesis of cirrhosis in galactosemia remains incompletely understood. In hereditary fructose intolerance, phosphate sequestration causes ATP depletion. Recent information about mitochondrial disease, NASH, disorders of glycosylation, Wilson's disease, and the progressive familial intrahepatic cholestases is discussed.

Copper-associated liver disease in North Ronaldsay sheep: a possible animal model for non-Wilsonian hepatic copper toxicosis of infancy and childhood.

Indian childhood cirrhosis (ICC), endemic Tyrolean infantile cirrhosis (ETIC) and idiopathic copper toxicosis (ICT), are clinically and pathologically indistinguishable liver disorders of infants and young children linked with exogenous copper and with increasing evidence for a genetic predisposition. North Ronaldsay sheep are a primitive breed which have adapted to a copper impoverished environment (<5 ppm) and display an abnormal sensitivity to copper poisoning when transferred to a copper replete (11 ppm) habitat. The aetiological parallels prompted a study of copper-associated liver disease in North Ronaldsay sheep (RCT) to see if the pathology could contribute to the understanding of the childhood disorder. A retrospective study was performed in which the livers of 22 mainland-bred North Ronaldsay sheep were compared with three island-bred sheep and categorized for liver copper content and pathomorphology. It was found that all the mainland sheep had accumulated liver copper (>300 microg/g), in contrast to the island sheep, although 10 sheep with increased liver copper (mean 600 SD 270 microg/g) showed no evidence of liver damage. A further 10 sheep with liver copper (mean 1276 SD 508 microg/g) exhibited periportal to panlobular histochemical copper retention, a periportal and/or panlobular pericellular fibrosis, a mixed inflammatory infiltrate and cholangioplasia. Steatosis was absent and regeneration was in abeyance. Finally, two sheep (liver copper >2000 microg/g) had a more active hepatitis with a florid pericellular, panlobular fibrosis and cirrhosis. Electron microscopy identified large numbers of collagen-producing hepatic stellate (Ito) cells in periportal regions. The pathological findings were sufficiently reminiscent of ICC, ETIC and ICT to warrant further exploration of RCT as a putative animal model. The North Ronaldsay sheep liver may be a useful tool for the investigation of copper-induced fibrogenesis.

Enterocytic CYP3A4 in a paediatric population: developmental changes and the effect of coeliac disease and cystic fibrosis.

AIMS: To investigate the effects of age and disease states on the expression and activity of intestinal CYP3A4 in a paediatric population. METHODS: Duodenal biopsies and surgical sections were collected from 104 paediatric patients (age range 2 weeks to 17 years) and from 11 foetuses. An S9 fraction was prepared in each case. CYP3A4 expression was assessed by Western blotting and by immunohistochemistry; activity was measured by the rate of formation of 6beta-hydroxytestosterone from testosterone. Villin expression was used as a marker of enterocyte harvest to normalize CYP3A4 expression and activity data. RESULTS: In the 74 histologically normal paediatric biopsies there were statistically significant increases in CYP3A4 expression (r2 = 0.19, P = 0.001) and activity (r2 = 0.17, P = 0.02) with age. CYP3A4 was practically absent in fetal duodenum and was expressed at relatively low levels in neonates (P < 0.05 between neonates and children > 5 years). Active coeliac disease resulted in significant (P < 0.001) decreases in CYP3A4 expression and activity. CONCLUSIONS: Duodenal CYP3A4 is present at significantly lower levels in neonates and in patients with active coeliac disease. This may have clinical significance with respect to the oral bioavailability of CYP3A4 substrates.

Features of carnitine palmitoyltransferase type I deficiency.

Carnitine palmitoyltransferase type I (CPT I) is unique among long-chain fatty acid oxidation enzymes in that there are two tissue-specific isoforms, 'hepatic' and 'muscle', which are encoded by two separate genes. The 'hepatic' isoform is expressed in liver, kidney and fibroblasts and at low levels in the heart, while the other isoform occurs in skeletal muscle and is the predominant form in heart. Reported patients with CPT I deficiency lack activity of the hepatic isoform and present before 30 months of age with hypoketotic hypoglycaemia, hepatomegaly with raised transaminases, seizures and coma. We discuss four new cases in three families showing, variously, renal tubular acidosis, transient hyperlipidaemia and, paradoxically, myopathy with elevated creatinine kinase or cardiac involvement in the neonatal period as additional features that deserve wider recognition.

Successful treatment of refractory ascites in a child with transjugular intrahepatic portosystemic shunt.

UNLABELLED: A 16-y-old boy who had undergone bone marrow transplantation for relapsed acute lymphoblastic leukaemia developed liver cirrhosis and refractory ascites, which did not respond to salt restriction, diuretics and abdominal paracentesis. Liver transplantation was not feasible because of poor nutritional status, pre-existing renal dysfunction and uncertainty about the prognosis of his leukaemia. The patient underwent a successful transjugular intrahepatic portosystemic shunt (TIPS), with immediate resolution of ascites, enabling cessation of diuretics and improvement in nutritional status. At 24 mo following TIPS there has been no re-accumulation of ascites. CONCLUSION: TIPS may have a role in the management of refractory ascites secondary to liver cirrhosis in selected children.

A comparison of the ontogeny of enterocytic and hepatic cytochromes P450 3A in the rat.

Enzymes of the cytochrome P450 3A (CYP3A) sub-family are abundant in adult liver and gut and contribute significantly to the first-pass metabolism of many orally administered drugs. The development of CYP3A enzymes with regard to their expression and activity in enterocytic and hepatic microsomes from 1-day-old through to adult male and female rats has been studied. Microsomes were prepared by calcium precipitation. Enzyme expression was assessed semi-quantitatively by Western blotting using rat polyclonal CYP3A2 and 2C11 antibodies and peptide antibodies specific to rat CYPs 3A1, 3A2, 2C12, and 2C13. The formation of 6beta-hydroxytestosterone (6OHT), determined by HPLC, was used as a measure of enzyme activity. Formation of 6OHT by enterocytic microsomes was similar for males and females and showed a sharp increase at weaning. This pattern was mirrored by levels of immunoquantifiable CYP3A2 (CYP3A9), but CYP3A1 followed a more gradual development. CYPs 2C11, 2C12, or 2C13 were not detected in gut microsomes. In contrast, CYPs 3A1, 3A2, 2C11, 2C12, and 2C13 were all expressed in hepatic microsomes. There was no surge in hepatic enzyme expression or hepatic 6OHT formation at weaning, and a marked sex difference in 6OHT formation was apparent from day 25. The surge in gut activity at weaning may be a protective mechanism against ingested toxins.

Copper toxicity affects proliferation and viability of human hepatoma cells (HepG2 line).

In Wilson's disease and Indian childhood cirrhosis (ICC) copper accumulates in the liver resulting in poor hepatocyte regeneration and fibrosis. An inhibition of hepatocyte proliferation and an increase in cell death could account for these outcomes. To establish how the toxicity of this metal ion impacts upon the proliferation and viability of the HepG2 cells they were cultured in 4-32 microM copper(II) sulphate (CuSO4)). These levels were comparable to the circulatory and tissue concentrations of copper recorded for these two diseases. Specific uptake comparable to levels of copper recorded in the livers of patients with Wilson's disease and ICC was measured in the HepG2 cells. After 48 h acid vesicle function increased from 4 to 32 microM Cu2+ but significantly declined at 64 microM compared to the controls. Lysosomal acid phosphatase showed a concentration dependent decline in activity at 72 h. Cellls exposed to 64 microM Cu2+ had a potential doubling time (Tpot) 21 h longer than the control cells due to a prolonged DNA synthesis phase. At 64 microM Cu2+, increases of necrosis up to 18% were seen whereas comparable levels of apoptotic and necrotic cells (<5%) were seen below this concentration. Chronic exposure over 8 weeks impaired colony-forming efficiency at concentrations of 16 microM Cu2+ and above. This study suggests that when liver cells sequester large amounts of copper, the toxic effects include delayed cell-cycle progression, a gradual loss of replicative capacity, and an increased incidence of cell death.

Assessment of stool colour in community management of prolonged jaundice in infancy.

Jaundice persisting beyond the first 2 wk of life is often regarded as an indication for investigation to exclude cholestatic liver disease. Most babies with prolonged jaundice have breast milk-related jaundice, which is a benign condition. Cholestatic liver disease is usually accompanied by pale stools and yellow or orange urine. A community programme was established to ascertain the incidence of prolonged jaundice and determine whether abnormal stool and urine colour could be used to assist primary care staff in referral decisions. Data were collected on normal stool and urine colour and used to devise a colour chart and information sheet for parents. Babies with prolonged jaundice were identified and referred for investigation. In all, 3661 babies were recruited into the study, of which 127 were jaundiced at 28 d of age. Of these, 125 were breastfed. The incidence of jaundice in breastfed babies at 28 d was 9.2% (95% CI 7.8%-11.0%) Abnormal liver function tests (LFTs) were common, but no baby had abnormal stool or urine colour and none was found to have liver disease. Jaundiced breastfed babies who are well are unlikely to have serious disease. Elevated LFTs are compatible with a diagnosis of breast milk-related jaundice. Prolonged jaundice in bottle-fed babies, and persistent pallor of stools or yellow/orange urine, are rare and merit immediate referral. Parents and professionals can be advised to report pale stools without generating a large number of unnecessary referrals. Further work is needed to determine whether a colour chart reduces the mean age of referral and treatment of infants with cholestatic liver disease.

Recommended approaches for the laboratory measurement of homocysteine in the diagnosis and monitoring of patients with hyperhomocysteinaemia.

Several recent studies have indicated that an increased concentration of plasma homocysteine is an independent risk factor for the premature development of vascular disease. These important findings emphasize the need for careful selection of an appropriate analytical approach to diagnose and treat individuals who may be at risk. We compared the results obtained from the measurement of plasma total homocysteine (free + protein-bound fractions) by high-performance liquid chromatography (HPLC) with the measurement of plasma free homocystine (free fraction) by conventional ion-exchange chromatography in 10 patients with inherited defects of homocysteine metabolism and 13 obligate heterozygote individuals. This study can be used to formulate recommendations on the appropriate use of these assays in different clinical circumstances. Our results show that the concentration of total plasma homocysteine must exceed 60 mumol/L before plasma free homocystine becomes detectable by conventional ion-exchange chromatography. Similarly, assessment of the urinary excretion of homocysteine in these patients indicates that it may not become consistently detectable by conventional ion-exchange chromatography or HPLC until plasma total homocysteine exceeds 150 mumol/L. On this basis, while most patients with classical homocystinuria would be detected by analysis of plasma using conventional ion-exchange chromatography or by measurement of of the urinary homocysteine excretion, occasional patients would be missed. When monitoring patients receiving treatment for classical homocystinuria, in whom metabolic control is good, and when investigating individuals with a suspected inherited defect of cobalamin or folate metabolism, a method which measures plasma total homocysteine should be used. The identification of moderate hyperhomocysteinaemia of undefined cause investigated in relation to a history of early vacsular disease can only be identified by this approach.

The allopurinol load test lacks specificity for primary urea cycle defects but may indicate unrecognized mitochondrial disease.

Thirty-three children ranging from 2 weeks to 12 years of age were selected for allopurinol loading, 16 on the basis of an increased urinary ourotate excretion detected by routine organic acid analysis (group A), and 17 for clinical reasons suggesting a urea cycle defect (group B). The allopurinol load test proved positive in 13 of 16 patients from group A, mean peak orotate 64.0 mumol/mmol creatinine (upper limit of reference range, 13.2) and 11 of 17 patients from group B, mean peak orotate 41.0 mumol/mmol creatinine (upper limit of reference range, 13.2). Thorough investigation of these patients including urinary and plasma amino acid analysis and, in 17 cases, liver biopsy for histology and measurement of ornithine carbamyltransferase (OCT) and carbamyl-phosphate synthetase (CPS) activity failed to identify any evidence of a urea cycle disorder. However, muscle biopsies performed in 11 patients showed some evidence of mitochondrial disease in four cases, two defined on the basis of reduced respiratory chain enzyme activity and two on the basis of mtDNA abnormalities. These findings indicate that an increased excretion of orotate in sick children may not be uncommon and that a positive allopurinol load test result may not indicate a specific inherited urea cycle defect. In addition, these results raise the interesting possibility that defective ureagenesis may be a feature of mitochondrial disease in some individuals.