Bile acid metabolism in infants and children and its implications for hepatobiliary diseases

Jenner and Howard proposed the hypothesis that idiopathic obstructive cholangiopathies might be caused by the toxic effect of bile acids, especially toxic monohydroxy bile acids. The toxic effects of bile acids on the liver has been recognized in various species of experimental animals. The present author and co-workers have studied the effects of the acids in the hepatobiliary system of rats and rabbits and found a descending order of toxicity for various bile acids: chenodeoxycholic acid, lithocholic acid, ursodeoxycholic acid, deoxycholic acid and cholic acid. We have also studied biles acid metabolism in premature and neonatal infants and demonstrated that the metabolism was particularly primitive. The main bile acids were 3β-hydroxy-5 cholenic and chenodeoxycholic acid which may be relatively toxic in vitro. Also we used the intrinsic bile acid loading test by administering MCT (medium chain triglyceride) milk to differentiate between neonatal hepatitis and congenital biliary atresias.     

Investigation of serum bile acids; seven patients with Alagille syndrome

To clarify whether an abnormal bile acid pattern has a role in the pathogenesis of Alagille syndrome, we compared serum bile acid patterns in seven with Alagille syndrome with those of patients with congenital biliary atresia (CBA), neonatal hepatitis (NH) and normal infants.Of the seven patients with Alagille syndrome, four patients were younger and three were older than 1 year. The mean total serum bile acid level in the infants was higher than in older subjects. There was a dissociation between the levels of serum total bile acid and bilirubin in three of the seven cases. The mean total bile acid levels in serum were in the following decreasing order: CBA, Alagille syndrome, NH and controls.The ratio of cholate to chenodeoxycholate in the younger patients with Alagille syndrome was significantly higher than CBA (P<0.001). However, no specific bile acid pattern was found in Alagille syndrome by high-performance liquid chromatography (HPLC).Abbreviations TBA total bile acids - FBA free bile acids - conj-BA conjugated bile acids - C/CDC ratio of cholate to chenodeoxycholate - G/T ratio of glycine conjugates to taurine conjugates - GPT glutamic pyruvic transaminase - CBA congenital biliary atresia - NH neonatal hepatitis - HPLC high performance liquid chromatography - GCA glycocholate - TCA taurocholate - GCDCA glycochenodeoxycholate - TCDCA taurochenodeoxycholate     

Hepatobiliary effects of cholic and lithocholic acids: experimental study in hamsters

Etiopathogenesis of biliary atresia remains unknown. Among several theories, one proposes that the disorder may be caused by the toxic effect of monohydroxy bile acids on fetal and neonatal hepatobiliary system. In this paper we evaluated toxic effects produced by ingestion of cholic acid, a trihydroxy bile acid, and lithocholic acid, a monohydroxy bile acid in the hepatobiliary system of a hamster during gestational and perinatal periods. A diet composed by 0.5% cholic acid and 0.25% lithocholic acid was administrated to pregnant hamsters. Liver and bile ducts of the adult and newborn animals were analyzed to point out the changes induced by these acids after birth. Because hamsters and humans have a similar bile metabolism, these animals were eligible for the study. The ingestion of 0.5% lithocholic acid, during hamster’s gestation, caused maternal intense ductal/ductular proliferation, inflammatory signs, hepatic cells degeneration and regeneration, hyperplasia of extra hepatic ducts epithelium, and abortion. Both 0.5% cholic acid and 0.25% lithocholic acid ingested by pregnant hamsters, caused ductal/ductular proliferation and hepatobiliary inflammatory damage in a different degree of intensity in adult animals and mild intensity in the young; and also the number of the young was reduced in the litter. We found that the ingestion of these bile acids by hamsters, during gestational period caused different degrees of toxicity on maternal and neonatal hepatobiliary systems. The histopathologic findings observed in biliary atresia patients could not be found in newborn hamsters. New experimental models are needed in the attempt to establish a correlation of these acids with neonatal cholestatic diseases.     

Alterations of serum bile acid profile in breast-fed infants with prolonged jaundice

Serum bile acid conjugates in breast-fed infants with prolonged jaundice were analyzed by a newly developed procedure using high-performance liquid chromatography with fluorescence labeling. Major bile acids were cholate and chenodeoxycholate conjugates. Some of the breast-fed jaundiced infants had high levels of serum bile acid conjugates (greater than 25 mumol/L), but the mean levels of individual bile acid conjugates found in jaundiced breastfed infants were not significantly different from those in breast-fed infants without jaundice. The glycine- to taurine-conjugated bile acid ratio in breast-fed jaundiced infants was significantly lower than in breast-fed nonjaundiced infants or bottle-fed nonjaundiced infants. In breast-fed infants, the portion of taurine-conjugated bile acids increased in proportion to serum bilirubin levels. These findings suggest that alteration in conjugated bile acid patterns of breast milk jaundice is related to an increased enterohepatic circulation of bile acids as well as bilirubin in infants fed on breast milk that contains high amounts of taurine.     

Serum Bile Acid Levels in Preoperative and Postoperative Patients with Congenital Biliary Atresia

To elucidate the bile acid metabolism in the preoperative and postoperative stages of congenital biliary atresia (CBA), the unconjugated and conjugated bile acid levels in sera were measured by high performance liquid chromatography (HPLC). The results showed that the mean total serum bile acid (TSBA) level in the preoperative cases of CBA was higher, 122.1±39.0 n?mol/1 (1SD), and was about 12 times higher than the level in the age-matched normal controls (9.9±6.0). Even in the non-icteric patients several years after operation, the mean TSBA level was still much higher (15.2±9.6) than that of normal controls (5.7±3.1). The mean ratio of cholic acid to chenodeoxycholic acid (CA/CDCA) in the preoperative and postoperative cases of CBA was less than 1.0. The mean ratio of glycine-conjugated bile acids to taurine-conjugated bile acids (G/T) in the preoperative cases of CBA was the lowest (1.9±1.1). In the clinically good cases with sufficient bile flow after operation, G/T ratio was the highest (9.3±6.5). In the normal controls and the patients with preoperative and postoperative CBA, the main bile acids were glycocholic acid, taurocholic acid, glycochenodeoxycholic acid and taurochenodeoxycholic acid. These conjugated bile acids comprised more than 85% of the total in amount.     

Abnormal low ratio of cholic acid to chenodeoxycholic acid in a cholestatic infant with severe hypoglycemia

We report a premature infant with severe hypoglycemia (serum glucose: 6 mg/dl) and cholestasis (serum total bile acids: 211.55 mumol/L) caused by hypoplasia of the interlobular bile ducts. This patient had developed intracranial hemorrhage and sepsis while undergoing treatment for hypoglycemia. As a result of endocrine evaluation, we made a diagnosis of idiopathic panhypopituitarism, congenital absence or hypoplasia of the pituitary gland. Moreover, we found abnormal bile acid profiles: The ratio of cholic acid to chenodeoxycholic acid was abnormally low in serum (0.04) and in biliary bile (0.33). However, 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid and bile alcohols were not detected. We therefore suspected that the severe cholestasis and abnormal bile acid profiles in the serum and biliary bile in this patient were related to physiologic immaturity of the enterohepatic circulation of bile acids and immaturity of hepatic 12 alpha-hydroxylation.     

Recent developments in diagnostics and treatment of neonatal cholestasis

Neonatal cholestasis is characterized by conjugated hyperbilirubinemia in the newborn and young infant and is a sign common to over 100 hepatobiliary and/or metabolic disorders. A timely evaluation for its etiology is critical in order to quickly identify treatable causes such as biliary atresia, many of which benefit from early therapy. An expanding group of molecularly defined disorders involving bile formation, canalicular transporters, tight junction proteins and inborn errors of metabolism are being continuously discovered because of advances in genetic testing and bioinformatics. The advent of next generation sequencing has transformed our ability to test for multiple genes and whole exome or whole genome sequencing within days to weeks, enabling rapid and affordable molecular diagnosis for disorders that cannot be directly diagnosed from standard blood tests or liver biopsy. Thus, our diagnostic algorithms for neonatal cholestasis are undergoing transformation, moving genetic sequencing to earlier in the evaluation pathway once biliary atresia, “red flag” disorders and treatable disorders are excluded. Current therapies focus on promoting bile flow, reducing pruritus, ensuring optimal nutrition, and monitoring for complications, without addressing the underlying cause of cholestasis in most instances. Our improved understanding of bile formation and the enterohepatic circulation of bile acids has led to emerging therapies for cholestasis which require appropriate pediatric clinical trials. Despite these advances, the cause and optimal therapy for biliary atresia remain elusive. The goals of this review are to outline the etiologies, diagnostic pathways and current and emerging management strategies for neonatal cholestasis.     

CHOLIC ACID AND CHENODEOXYCHOLIC ACID CONCENTRATIONS IN SERUM DURING INFANCY AND CHILDHOOD

Abstract. Heikura, S., Similä, S., Finni, K., Mäentausta, O. and Jänne, O. (Departments of Clinical Chemistry, Biochemistry and Paediatrics, University of Oulu, Oulu, Finland). Cholic acid and chenodeoxycholic acid concentrations in serum during infancy and childhood. Acta Paediatr Scand, 69: 659, 1980.—Concentrations of two primary bile acids (cholic and chenodeoxycholic acids) were determined by radioimmunoassay in the serum of infants and children at ages ranging from 1 hour to 15 years. The same bile acids were also measured in umbilical cord serum. Concentrations of the primary bile acids were significantly higher in the serum of 1-hour old infants than those in the umbilical cord serum or the peripheral vein serum of adults. The levels of cholic and chenodeoxycholic acid remained high until the age of 6 months, being about 5-fold higher than those in the sera of adults. Primary bile acid concentrations reached the adult level by the age of 1–2 years. These results indicate that developmental changes occur in the metabolism and excretion of bile acids in man. The relatively high concentrations of the primary bile acids in serum during the first 6 months of life suggest that up to this age, the mature ability of the liver to excrete the bile salts into the bile and/or to clear them from the circulation has not yet been reached.     

Postnatal development of liver and exocrine pancreas in polycythemic newborn infants.

In 35 newborn infants appropriate for gestational age the influence of neonatal polycythemia (venous hematocrit greater than 60% measured between the second and fourth hour of life) on development of enterohepatic circulation of bile acids, activities of pancreatic enzymes in duodenal juice, and the effects of hemodilution were studied during the second week of life. A significant correlation was found between the initial hematocrit and both the bile acid concentration in serum and lipase and trypsin activity in duodenal juice. Of 35 infants, 10 were not treated with hemodilution due to asymptomatic polycythemia; they had the highest concentration of serum bile acids associated with the lowest lipase and trypsin activity in duodenal juice. However, the 25 infants treated with hemodilution also showed serum bile acid concentrations and lipase and trypsin activity in duodenal juice out of the normal range when compared to normocythemic infants. These data indicate that, during the first days of life, polycythemia results in a delayed postnatal development of enterohepatic circulation of bile acids and exocrine pancreas functions independently from the occurrence of clinical symptoms. Thus, it can be concluded that, on the first day of life, all polycythemic infants should be treated with hemodilution. Moreover, the nutritional management of these infants must also account for the limited functional capacity of the gastrointestinal tract.     

Studies on the conjugating activity of bile acids in children

The unconjugated and conjugated bile acid levels in sera of 98 normal children and nine normal adults were measured by high performance liquid chromatography. The results showed that the mean total bile acid level was high, 11.0 +/- 8.7 mumol/liter (1 SD) during the neonatal period (0-4 wk) and then gradually decreased with age. The ratio of the concentration of conjugated bile acids to total bile acids in serum was as high as 90% or more in infants under 1 yr of age and slowly decreased with age. The mean ratio of cholic acid to chenodeoxycholic acid was high (1.7 +/- 1.1) during the neonatal period but decreased after 3 months to the adult level (0.4 +/- 0.2). The mean ratio of glycine conjugated bile acids to taurine conjugated bile acids was 3.0 +/- 3.1 during the neonatal period and the ratio during the 1st month of life was significantly lower than that after that period with little further change at any age. The mean ratio of the concentration of secondary bile acids to primary bile acids showed significantly lower values in infants less than 1 yr of age. The main bile acid was glycocholic acid in the neonatal period but after 1-3 months glycochenodeoxycholic acid predominated. With age, the serum bile acid pattern which was characteristic in infancy gradually approached that of adults.     

Bile acids in serum and bile of infants with cholestatic syndromes

The concentration of individual bile acids in serum was measured in 18 neonates and infants with various cholestatic conditions (extrahepatic biliary atresia, neonatal hepatitis syndrome, chronic intrahepatic cholestasis and posthemolytic cholestasis). The cholate/chenodeoxycholate ratio in serum was smaller than one in all patients with neonatal hepatitis syndrome or extrahepatic biliary atresia, cholestatic conditions which were accompanied by signs of liver cell injury. It was greater than one in the patients with chronic intrahepatic cholestasis. Administration of cholestyramine to patients with patent extrahepatic bile ducts decreased the total concentration bile acids in serum and elevated the cholate/chenodeoxycholate ratio. Thus, cholestyramine administration may be of diagnostic value for evaluation of bile duct patency in cholestasis of infancy. Differences between the bile acid pattern in serum and bile were observed. Thus, the cholate/chenodeoxycholate ratio was always higher in bile than in serum. 3beta-hydroxy-5-cholenoic acid found in serum was not detectable in bile. This finding suggests that impairment of biliary excretion rather than increased hepatic synthesis is responsible for elevation of this monohydroxy bile acid in serum.     

BILE ACID EXCRETION AND MALABSORPTION IN INTRAHEPATIC CHOLESTASIS OF INFANCY ("NEONATAL HEPATITIS")

Bile acid excretion has been studied in four patients with intrahepatic cholestasis of infancy neonatal hepatitis) after intramuscular administration of cholic acid-24-¹⁴.C Bile acid secretion to the intestines was found to be highly impaired and the main route of excretion was via the urine. Practically all of the administered labeled cholic acid was conjugated prior to excretion. The main conjugates were glycocholic and taurocholic acid. At least three additional conjugates of cholic acid were isolated from the urine. Analysis of bile obtained from three of the patients in connection with operative cholangiography showed a very low concentration of bile acids, phospholipids and cholesterol. The bile was of normal colour owing to the presence of bilirubin. Severe steatorrhea and markedly impaired absorption of vitamin A was demonstrated when the patients were jaundiced. The impairment of bile acid excretion to the gut and the degree of steatorrhea were well correlated. In some of the patients steatorrhea persisted after the disappearance of jaundice. In those instances, the impairment of bile acid excretion to the gut was found to persist.     

Bile Acid Metabolism in Children with Cystic Fibrosis

ABSTRACT. Recycling of bile acids through the enterohepatic cycle is very efficacious. Bile acids contribute to bile formation and, by forming micelles, participate in lipid solubilization and absorption. The small fraction which escapes in the feces, is synthesized daily by the liver to compensate for losses. In CF, bile acid malabsorption has been documented; these large losses are accompanied by an interruption in the enterohepatic circulation with concomitant reduction in bile acid pool and disturbances in biliary composition. The various intraluminal factors implicated in bile acid malabsorption include: unhydrolysed triglycerides and phospholipids, precipitation of bile acids in acidic duodenal content, adsorption to residues and modification of colonic microflora. A defect in bile acid ileal uptake has also been advocated. These disturbances in bile acid metabolism associated with CF might lead to aggravation of diarrhea and steatorrhea, cholelithiasis and perhaps liver disease.     

URINARY MONOHYDROXY BILE ACIDS IN YOUNG INFANTS WITH OBSTRUCTIVE JAUNDICE

ABSTRACT. Using an aluminum oxide column, we fractionated and quantitatively determined urinary monohydroxy bile acids in young infants. For comparison purposes, monohydroxy bile acids were also measured in urine from older children and adults with obstructive jaundice. Lithocholic acid was not found in any specimens of the young infants examined, while 3β-hydroxy-5-cholenoic acid was detected in all. In the biliary atresia group, 3β-hydroxy-5-cholenoic acid excreted was 0.45 ± 0.28 μmol per day ( n = 7), and in the neonatal hepatitis group, 0.48 ± 0.44 μmol per day ( n = 9). The mean rate of 3β-hydroxy-5-cholenoic acid to total urinary bile acids in the biliary atresia group was 2.1%, and 1.3% in the neonatal hepatitis group. In the older children and adults with obstructive jaundice ( n =6), 3β-hydroxy-5-cholenoic acid was excreted at a mean rate of 3.9% of total urinary bile acids, ranging from 0.63 to 14.81 mol per day. The excretion rate of 3β-hydroxy-5-cholenoic acid was related to that of chenodeoxycholic acid ( p <0.05) in infants, while it was related to that of both chenodeoxycholic acid ( p <0.01) and cholic acid ( p <0.05) in older children and adults.     

Effect of milk feeding on intestinal bilirubin absorption in the rat

The hypothesis that the etiologic mechanism of the late-onset, prolonged, unconjugated hyperbilirubinemia of the breast-fed infant, known as the breast milk jaundice syndrome, results from exaggeration of intestinal bilirubin absorption has been investigated in an adult rat model, which permits quantitative measurement of the enterohepatic circulation of bilirubin. After instillation of unconjugated bilirubin in buffer into the duodenum, 25% of the dose was absorbed and appeared in bile. Administration of bilirubin in human milk or cow milk formula resulted in a marked reduction in absorption to 2%. Administration of bilirubin in milk from mothers of infants with breast milk jaundice syndrome not only failed entirely to prevent the absorption of bilirubin, but enhanced late absorption, to produce a total absorption of 60% of the bilirubin dose. Thus, although normal milk significantly retarded intestinal bilirubin absorption and diminished the bilirubin load to the liver, milk from mothers of infants with breast milk jaundice syndrome appeared to enhance the enterohepatic circulation of bilirubin and to increase the total hepatic bilirubin load. This exaggeration of the enterohepatic circulation of bilirubin may be related to the increased concentrations in these milks of long-chain nonesterified fatty acids.     

Alpha1-fetoprotein in neonatal hepatobiliary disease.

It has been suggested that the quantitative estimation of serum alpha-1-fetoprotein may help in distinguishing the neonatal hepatitis syndrome from biliary atresia. We measured the serum AFP concentration in 52 neonates and infants with various hepatobiliary disorders, including neonatal hepatitis syndrome (group I), biliary atresia (group II), and other hepatopathies such as choledochal cyst (group III). The mean serum AFP concentration in patients with neonatal hepatitis was significantly greater than the mean concentration in the other two groups. There was no significant difference between the mean serum AFP concentrations in patients with biliary atresia and in group III patients. Patient age was noted to be an important factor: Serum AFP levels greater than 35 microgram/ml in infants one to four months of age suggpst the diagnosis of neonatal hepatitis syndrome. Serum AFP levels below 10 microgram/ml in infants less than four months of age suggest the diagnosis of biliary atresia or hepatopathies other than neonatal hepatitis. However, the variable and significant overlapping of serum AFP values between 10 and 35 microgram/ml limit the diagnostic value of this test.     

EXCRETION OF BILE ACIDS IN ERYTHROBLASTOSIS FOETALIS

The excretion pattern of intramuscularly injected cholic acid-24–¹⁴C was studied for 4 days after the injection in 10 cases of erythro-blastosis (EB). Seven patients with EB and raised serum conjugated bilirubin excreted 3643% of the injected isotope in the urine, whereas the amounts of isotope in the faeces varied greatly. In 3 cases without raised serum conjugated bilirubin less isotope was recovered in the urine and always more than 10% of injected isotope was recovered in the faeces. Cholic acid-24–¹⁴C was excreted essentially unchanged in all cases but in conjugated form. In all cases of EB the urine was found to contain bile acids, chiefly cholic acid. The infants with EB associated with cholestasis excreted 4.8–132.3 μmol of these acids per day; the corresponding values in the absence of cholestasis being 0.4–0.9 μmol per day. In the infants with physiological jaundice the excretion ranged from less than 0.01 to 0.7 μmol per day; the correspondign values in the 2 patients with hyperbilirubinaemia were about 0.2 μmol per day. The infants with EB associataed with cholestasis were found to excrete as large amounts of bile acids in the urine as the infants with intrahepatic cholestasis. These findings strongly suggest that increased serum conjugated bilirubin, irrespective of the patho-genesis of the liver damage, is associated with an impaired bile acid excretion to the intestine. EB without increased serum conjugated bilirubin did not seem to alter the bile acid metabolism, since the urinary excretion of cholic acid and chenodeoxycholic acid in these cases was practically the same as in jaundiced newborn infants.     

Intestinal absorption of calcium and magnesium in hepatobiliary disease in infancy

The intestinal absorption of calcium and magnesium was measured by metabolic balance studies in 6 normal infants, 13 infants with biliary atresia, 5 infants with successfully repaired biliary atresia, 7 infants with neonatal hepatitis, and 2 infants with choledochal cyst. The absorption of both elements was impaired in these disorders. The malabsorption of these elements was most marked in biliary atresia. In successfully repaired biliary atresia the absorption was increased to the normal levels. In neonatal hepatitis the degree of the malabsorption was variable in individual cases. In choledochal cyst the reduction of the absorption was less marked than in biliary atresia and neonatal hepatitis. In biliary atresia parenteral vitamin D increased moderately the absorption of both elements, though oral vitamin D had little effect. In infants with biliary atresia receiving a milk containing medium-chain triglycerides the absorption was moderately raised. There was a clear relation between the absorption of calcium and that of magnesium: the per cent. absorption of magnesium was almost the same as that of calcium in most cases. The serum calcium level determined during the studies was within the normal ranges in hepatobiliary diseases. The serum magnesium level was, however, found to be generally reduced in these conditions. It was greatly reduced in the patients with biliary atresia.     

Malabsorption of liposoluble vitamins in a child with bile acid deficiency

A male born to first cousins presented at 12 months with hypocalcemic convulsions, rickets, epistaxis due to vitamin K deficiency, and extremely low serum levels of beta-carotene and vitamin A. Liver function was altered moderately (glutamic-oxaloacetic transaminase, 55 U/L; glutamic-pyruvic transaminase, 37 U/L; lactate dehydrogenase, 255 U/L; alkaline phosphatase, 437 U/L). To correct the deficiencies, 8,000 IU vitamin D/day, 10,000 IU vitamin A/day, and intramuscular administration of vitamin K1 were required. At 9 years, he presented signs of neuromuscular affection, and the serum vitamin E level (measured for the first time) was extremely low. Classic lipid malabsorption syndromes (abetalipoproteinemia, chronic cholestasis, mucoviscidosis, coeliac disease, Whipple's disease) were excluded by appropriate examinations. Composition of duodenal bile acids was characterized by undetectable levels of cholic acid metabolites, and only chenodeoxycholic acid metabolites were present. Serum total bile acid concentration was normal, with an atypical low cholic acid/chenodeoxycholic acid ratio and abnormal presence of 3 beta-OH-delta 5-cholenic acid and 6-OH-bile acids. Urinary bile acid composition was also characterized by elevated 6-OH-bile acids. Known enzymopathies of the bile acid synthetic pathway were excluded (cerebrotendinous xanthomatosis, cerebro-hepato-renal syndrome of Zellweger, coprostanic acidemia). Bile acid pool sizes were determined by using stable isotopes: cholic acid pool size [2.90 (N, 32 +/- 16) microM/kg] and chenodeoxycholic acid pool size [10.8 (N, 32.6 +/- 9.9) microM/kg] were extremely low; fractional turnover rates of both bile acids were in a normal range.(ABSTRACT TRUNCATED AT 250 WORDS)     

SERUM BILE ACIDS IN NEWBORNS: EVIDENCE FOR AN HEPATIC DYSFUNCTION IN LOW-BIRTH-WEIGHT INFANTS

ABSTRACT. The post-prandial pattern of total serum bile acids was studied in 47 newborns: 12 prematures (less than 36 weeks), 17 term low-birth-weight infants (less than the 3rd percentile), 18 term normals. The study was made at the end of the first month. Blood was collected in a peripheral vein using a microcatheter. Samples were taken at fasting time and 30, 60, 120, 180 min after a test meal intake (40 ml/kg of "humanized" milk based formula). Bile acids were assayed using an original enzymatic micromethod which needed only 50 μl of serum and showed a sensitivity of 0.3 pmol in 200 μl of reaction medium. The response of serum bile acids after the test meal was very similar in normal term newborns and in adults. Prematures exhibited bile acid levels slightly higher than normals, but this difference was significant only at 0 and 180 min. Low-birth-weight infants showed very high values of serum bile acids at all times during the test, compared to normal and premature infants. Serum levels of total bilirubin and alkaline phosphatase were similar in all 3 groups. These results are not consistent with cholestasis but rather indicate a specific dysfunction in bile acid metabolism in low-birth-weight infants.