Genetic Defects in Bile Acid Conjugation Cause Fat-Soluble Vitamin Deficiency

GASTROENTEROLOGY 2013;144:945–955

Genetic Defects in Bile Acid Conjugation Cause Fat-Soluble Vitamin Deficiency KENNETH D. R. SETCHELL,1 JAMES E. HEUBI,2 SOHELA SHAH,3 JOEL E. LAVINE,4 DAVID SUSKIND,5 MOHAMMED AL–EDREESI,6 CAROL POTTER,7 DAVID W. RUSSELL,8 NANCY C. O’CONNELL,1 BRIAN WOLFE,1 PINKY JHA,1 WUJUAN ZHANG,1 KEVIN E. BOVE,1 ALEX S. KNISELY,9 ALAN F. HOFMANN,10 PHILIP ROSENTHAL,3,11 and LAURA N. BULL3 Department of Pathology and Laboratory Medicine and 2Division of Gastroenterology and Nutrition, Children’s Hospital Medical Center, Cincinnati, Ohio; 3UCSF Liver Center Laboratory and Institute for Human Genetics and 11Departments of Pediatrics and Surgery, University of California, San Francisco Medical Center, San Francisco, California; 4Department of Gastroenterology, Hepatology and Nutrition, Morgan Stanley Children’s Hospital/Columbia University, New York, New York; 5Department of Gastroenterology and Hepatology, Seattle Children’s Hospital and University of Washington Medical School of Medicine, Seattle, Washington; 6Pediatric Specialty Services Division, Dhahran Health Center, Saudi Aramco, Dhahran, Saudi Arabia; 7Department of Gastroenterology and Nutrition, Nationwide Children’s Hospital, The Ohio State University, Columbus, Ohio; 8Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas; 9Institute of Liver Studies, King’s College Hospital, Denmark Hill, London, England; and 10Department of Medicine, University of California San Diego, San Diego, California

This article has an accompanying continuing medical education activity on page e14. Learning Objective: Upon completion of this exercise, successful learners will be able to identify patients with potential defects in bile acid synthesis and be able to differentiate this class of metabolic liver disease from other causes of cholestatic liver disease. Furthermore, the learner will be able to understand the importance of bile acid conjugation for intestinal absorption of lipid-soluble molecules and be able to integrate this knowledge into the clinical practice and management of patients with unexplained fat-soluble vitamin deficiencies and liver disease. See Covering the Cover synopsis on page 859; see editorial on page 870.

BACKGROUND & AIMS: The final step in bile acid synthesis involves conjugation with glycine and taurine, which promotes a high intraluminal micellar concentration to facilitate lipid absorption. We investigated the clinical, biochemical, molecular, and morphologic features of a genetic defect in bile acid conjugation in 10 pediatric patients with fat-soluble vitamin deficiency, some with growth failure or transient neonatal cholestatic hepatitis. METHODS: We identified the genetic defect that causes this disorder using mass spectrometry analysis of urine, bile, and serum samples and sequence analysis of the genes encoding bile acid-CoA:amino acid N-acyltransferase (BAAT) and bile acid-CoA ligase (SLC27A5). RESULTS: Levels of urinary bile acids were increased (432 ⫾ 248 ␮mol/L) and predominantly excreted in unconjugated forms (79.4% ⫾ 3.9%) and as sulfates and glucuronides. Glycine or taurine conjugates were absent in the urine, bile, and serum. Unconjugated bile acids accounted for 95.7% ⫾ 5.8% of the bile acids in duodenal bile, with cholic acid accounting for 82.4% ⫾ 5.5% of the total. Duodenal bile acid concentrations were 12.1 ⫾ 5.9 mmol/L, which is too low for efficient lipid absorption. The biochemical profile was consistent with defective bile acid amidation. Molecular analysis of BAAT confirmed 4 different homozygous mutations in 8 patients tested. CONCLUSIONS: Based on a study of 10 pediatric patients, genetic defects that disrupt bile acid amidation cause fat-soluble vitamin deficiency and growth failure, indicating the importance of bile acid conju-

gation in lipid absorption. Some patients developed liver disease with features of a cholangiopathy. These findings indicate that patients with idiopathic neonatal cholestasis or later onset of unexplained fat-soluble vitamin deficiency should be screened for defects in bile acid conjugation. Keywords: Chronic Liver Disease; Hepatic; Inherited; Nutrient.

H

epatic bile acid conjugation with the amino acids glycine and taurine represents the final step in primary bile acid synthesis in humans.1 The liver has a high capacity for conjugation, and as a result negligible amounts of unconjugated bile acids (⬍2%) typically appear in bile under normal or cholestatic conditions.2 Conjugation significantly alters the physicochemical characteristics of an unconjugated bile acid by increasing the molecular size (Figure 1) and lowering the pKa, thus enhancing aqueous solubility at the pH of the proximal intestine and preventing nonionic passive absorption.3 Conjugation thus promotes a high intraluminal bile acid concentration and therefore efficient solubilization of lipids with low aqueous solubility such as saturated fatty acids and fat-soluble vitamins. Two enzymes catalyze the reactions leading to bile acid amidation. A CoA thioester Abbreviations used in this paper: BAAT, bile acid-CoA:amino acid N-acyltransferase; BACL, bile acid-CoA ligase enzyme; FAB-MS, fast atom bombardment ionization–mass spectrometry; GC-MS, gas chromatography–mass spectrometry; GGT, ␥-glutamyl transpeptidase; PT, prothrombin time. © 2013 by the AGA Institute 0016-5085/$36.00 http://dx.doi.org/10.1053/j.gastro.2013.02.004

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Figure 1. Space-filling models showing the influence of bile acid conjugation with glycine and taurine on the size of the molecule, its physicochemical properties, and its physiological features.

is first formed by the rate-limiting bile acid-CoA ligase enzyme (BACL; encoded by SLC27A5),4,5 and then glycine or taurine is coupled to the carboxyl group of the bile acid in a reaction catalyzed by cytosolic bile acid-CoA:amino acid N-acyltransferase (BAAT; encoded by BAAT).6 In 1997, we first described in a preliminary report a defect in bile acid amidation in a 14-year-old boy with fat malabsorption and fat-soluble vitamin deficiency.7 This child presented in the first 3 months of life with conjugated hyperbilirubinemia, elevated serum transaminase levels, and a normal ␥-glutamyl transpeptidase (GGT) level. Two other patients, a 5-year-old Saudi Arabian boy and his 8-year-old sister, the products of a consanguineous marriage, were later identified with the same bile acid defect. Remarkably, the boy had undergone a portoenterostomy for a diagnosis of “extrahepatic biliary hypoplasia,” whereas his sister was reportedly asymptomatic. We have now identified a bile acid conjugation defect in 10 patients with clinical histories of normal or mildly elevated liver chemistry values but a severe fat-soluble vitamin deficiency, often resulting in coagulopathy and rickets. The main feature, fat-soluble vitamin deficiency, occurs because of reduced biliary secretion of conjugated bile acids and an inability to form mixed micelles because of rapid passive absorption of unconjugated cholic acid in the proximal small intestine. The recognition that genetic defects in bile acid synthesis are associated with unexplained fat-soluble vitamin deficiency warrants a concerted effort to explore this patient population for these disorders. This report describes the clinical, biochemical, and molecular features of defective bile acid conjugation in the largest cohort of patients thus far reported.

Patients and Methods Clinical Descriptions of Patients The demographic information and presentations of 10 patients from 7 families are summarized in the following text and in Table 1, with more detail provided in Supplementary Patients and Methods.

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Evaluation of jaundice and anemia at age 40 days in patient 1, a male infant born at term (2.6 kg) to parents not identified as consanguine, showed ␣-thalassemia. A prolonged prothrombin time (PT) at age 5 months responded to fresh-frozen plasma and vitamin K. Severe anemia, congestive heart failure, pulmonary edema, and rickets with a fracture of the proximal fibula were recorded at 1 year. Marked growth retardation and hepatosplenomegaly at age 14 years prompted reevaluation, with bile acid analysis by mass spectrometry. On evaluation of jaundice with acholic stools at age 28 days in patient 2, a male infant born at term (3.3 kg) to first-cousin parents with 2 nominally well children, sonographic examination showed no gallbladder and cholescintigraphy showed no contrast excretion; an intraoperative cholangiogram was interpreted as consonant with biliary atresia. Jaundice did not resolve with portoenterostomy. At 7 months, rickets was diagnosed, as well as a fracture of the right humerus and low serum vitamin D and E levels. Bile acid analysis of a urine sample was performed at 4 years, with follow-up liver biopsy. Urine and serum samples from patient 3, the 8-year-old full sister of patient 2, were screened for a bile acid synthetic defect by mass spectrometry, although family members considered her healthy. She was later found to have had rickets at age 6 months and fractures at age 3 years. At age 12 years, serum vitamin E level was low without hypocoagulability, other hypovitaminosis, or other abnormal clinical/biochemistry test results. Immunizations at age 4 months in patient 4, a female infant born at 36 weeks’ gestation (2.3 kg) to parents not identified as consanguine and with one well child, immediately produced large ecchymoses. A prolonged PT responded to fresh-frozen plasma and vitamin K. Serum vitamin D and E levels were low, and rickets was seen on imaging. Serum vitamin A level was normal, without other abnormal clinical/biochemistry test results. Liver biopsy was performed at 15 months, with mass spectrometry screening for a bile acid synthetic defect. Hydrocephalus ascribed to aqueductal stenosis, respiratory distress, and hypoglycemia suspect for sepsis led to hospital admission at age 3 days for patient 5, a male neonate born at term (4.58 kg) to consanguine parents. Liver failure developed and was successfully treated with a liver transplant. Evaluation before transplant included liver biopsy and screening for a bile acid synthesis defect. Patient 6, the full sister of patient 5, had never been ill and was clinically well when, at age 9 years, a urine sample was screened by mass spectrometry for abnormal bile acid levels. The only abnormal clinical/laboratory test result was a low total serum tocopherol concentration; hypocoagulability, other hypovitaminoses, or other abnormal clinical/biochemistry test results were not found. Patient 7, the full sister of patients 5 and 6, had severe jaundice as a neonate. Hepatitis was diagnosed. Ursodeoxycholic acid and vitamin A were given for several years. At age 10 years, a urine sample was screened for a bile acid synthesis defect by mass spectroscopy, and serum vitamin A and D and total tocopherol levels were low, without hypocoagulability or other abnormal clinical/biochemistry test results. Growth failure at age 4 months prompted evaluation of patient 8, a female infant born at term (3.63 kg) to parents not identified as consanguine and with one well child. A prolonged PT responded to parenteral vitamin K; serum vitamin A, D, and E levels were low and serum alkaline phosphatase activity was high, without other abnormal clinical/biochemistry test results.

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Patient no.

Sex

1 2

M M

3

F

4 5 6 7 8 9 10

Age at diagnosis 14 y 4y

Consanguinity

Origin/ethnicity

Serum AST/ALT

Serum direct bilirubin

Serum fat-soluble vitamin levels

Hepatomegaly Hepatomegaly portoenterostomy —

Normal Elevated

Elevated Elevated

Low Low

Normal

—

Low

Normal Liver failure/orthotopic liver transplantation Normal Normal Normal Hepatomegaly —

Elevated Elevated

Normal Elevated

Low —

Normal Normal Normal Elevated —

Normal Normal Normal Elevated —

Low Low Low Low

Liver

Not known Yes

Laotian Saudi Arabia/Asian

8y

Yes

Saudi Arabia/Asian

F M

1y 3 mo

No Yes

United States/Hispanic United States/Hispanic

F F F M F

11 y 10 y 3 mo 6 mo 6.5 y

Yes Yes Not known No Not known

United United United United United

States/Hispanic States/Hispanic States/Amish States/Hispanic States/white

M, male; F, female; AST, aspartate aminotransferase. weight at birth and 3½ months of age.

aBody

A urine sample was screened by mass spectroscopy for a bile acid synthesis defect. On evaluation at age 5 months of growth retardation, jaundice, and rickets, patient 9, a male infant born at term (2.5 kg), exhibited mild hepatomegaly without splenomegaly. A prolonged PT responded to parenteral vitamin K; serum vitamins D and E levels were low, without hypovitaminosis A. Conjugated and nonconjugated hyperbilirubinemia accompanied elevations in serum transaminase and alkaline phosphatase activities. Liver biopsy was performed, as well as bile acid analysis by mass spectroscopy. Poor weight gain led to evaluation of patient 10, a girl; a urine sample was screened by mass spectroscopy at age 8 years, when duodenal stenosis was surgically palliated, and earlier clinical details are lacking. A urine sample was again screened at age 10 years.

Analytical Techniques The bile acid composition of urine, serum, bile, and feces was examined in detail using a combination of methodologies previously reported, including liquid-solid extraction, lipophilic anion exchange chromatography to isolate bile acids based on conjugate classes, and analysis of these fractions by gas chromatography–mass spectrometry (GC-MS) after derivatization to methyl ester–trimethylsilyl ethers.8 The initial screening procedure for diagnosis of a bile acid synthetic defect was performed by direct analysis of the urine sample using fast atom bombardment ionization–mass spectrometry (FAB-MS) and GC-MS.8,9

Molecular Genetic Analysis of BAAT and SLC27A5 Human genomic DNA was isolated from white blood cells using Puregene DNA Isolation Kits (Qiagen, Valencia, CA). The 3 coding exons of BAAT and the 10 coding exons of SLC27A5 were amplified by polymerase chain reaction. The polymerase chain reaction products were purified and sequenced using standard approaches. Sequences were aligned to a reference gene sequence. Absence of candidate mutations from publically (dbSNP) and locally available control sequence data was confirmed. Predicted functional consequences of missense

changes were evaluated using Polyphen2 (Polymorphism Phenotyping v2; http://genetics.bwh.harvard.edu/pph2/). Control samples. For the mutation in patients 2 and 3, 80 control chromosomes from individuals of Arab ancestry were assayed. For the other mutations, 113 control chromosomes from HapMap families of Northern and Western European ancestry were assayed.10

Histologic Analysis Sections of formalin-fixed paraffin-embedded liver tissue from patients 1, 2, 4, and 5 were stained with H&E, periodic acid–Schiff/diastase, reticulin, and Masson trichrome methods. Patients 1, 2, and 5 had second liver samples obtained at ages 14 years, 4.5 years, and 6 months, respectively. Tissue samples from the second biopsy specimen from patient 2, the only specimen from patient 4, and the first specimen from patient 5 were processed for ultrastructural study (glutaraldehyde fixed, osmium tetroxide postfixed, resin embedded). Ultrathin sections of resin-embedded liver were stained with uranyl oxide/lead citrate and examined using a transmission electron microscope. In patients 2, 4, and 5, expression of BACL and BAAT was assessed immunohistochemically using antibodies against BACL (HPA007292; Sigma, St Louis, MO) and BAAT (ab97455; Abcam, Cambridge, England) with EnVision reaction development (Dako UK, Ely, England) and hematoxylin counterstaining as described elsewhere.11

Results Urinary Bile Acid Analysis The negative-ion FAB-MS spectra of urine from the 10 patients were qualitatively similar to that of the index case (patient 1), as shown in Figure 2. Although the relative intensity of individual ions in the mass spectra varied among patients, remarkable and consistent throughout the spectra was the complete absence of glycine-conjugated (m/z 464/448) and taurine-conjugated (m/z 514/498) bile acids, the usual products of hepatic primary bile acid synthesis, and a dominance of unconju-

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Table 1. Summary of the Demographics, Main Features, and Clinical Findings of 10 Patients With a Confirmed Bile Acid Conjugation Defect and Features of Their Urine and Duodenal Bile Acids

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Table 1. Continued

Body wt (percentile) ⬍5th Normal ⬍50th ⬍3rd 75tha

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50th 10th 25th ⬍3rd ⬍5th

Bone Rickets Rickets with bone fracture Rickets with fractures Rickets — — — — Rickets —

Total urinary Total duodenal Urine FAB-MS analysis: bile acid Unconjugated Cholic bile acid Unconjugated Cholic amidated bile acids concentration bile acids in acid in concentration biliary acid in (present/absent) (␮mol/L) urine (%) urine (%) (␮mol/L) acids (%) bile (%) Absent Absent

217 1111

80.8 64.9

65.5 59.2

5528 35,806

99.7 99.9

95.1 58.7

Absent

—

—

—

—

—

—

Absent Absent

173 153

80.6 78.8

56.5 50.7

76 472

92.0 82.6

76.9 85.8

Absent Absent Absent Absent Absent

135 — 82.5 — 1156

72.4 — 95.7 — 82.7

49.6 — 79.0 — 23.7

3023 24,083 23,509 — 3997

97.1 98.2 99.4 — 96.8

92.0 93.3 94.0 — 63.3

gated and sulfated bile acids (see Supplementary Table 1). A conspicuous feature of all spectra was an intense ion at m/z 407 consistent with the deprotonated molecular ion of an unconjugated trihydroxycholanoic (C24) bile acid, and prominent ions for sulfate conjugates of monohydroxy-cholanoates (m/z 453) and dihydroxy-cholanoates (m/z 471) were observed. Ions of lower abundance were usually present, in particular at m/z 391 for unconjugated dihydroxycholanoic (C24) acids and at m/z 567 and 583 corresponding to glucuronide conjugates of dihydroxycholanoic acids and trihydroxycholanoic acids, respectively. When the urine extracts were fractionated on the lipophilic anion exchanger Lipidex-DEAP to separate bile acids based on mode of conjugation, FAB-MS of the fractions confirmed these structural assignments and further established an absence of any glycine- or taurineconjugated bile acids. GC-MS analysis of the methyl ester–trimethylsilyl ether derivatives of urinary bile acids isolated in these conjugate fractions confirmed the majority of bile acids to be unconjugated, which is in agreement with the findings from FAB-MS analysis. At the time of diagnosis, the mean (⫾SEM) total urinary unconjugated bile acid concentration for the 7 patients for which there was sufficient urine for analysis was 327 ⫾ 195 ␮mol/L (see Supplementary Table 2), representing 79.4% ⫾ 3.9% of the total bile acids excreted. Cholic acid was the predominant urinary bile acid, accounting for 55.8% ⫾ 8.1% of the bile acids in the unconjugated fraction. Low proportions and concentrations of deoxycholic, chenodeoxycholic, and lithocholic acids were found. The mean (⫾SEM) concentration of bile acids excreted in urine as glucuronide and sulfate conjugates was 106 ⫾ 53 ␮mol/L, and cholic acid accounted for 50.0% ⫾ 7.0% of the total bile acids. Qualitatively, the bile acid composition of this conjugate fraction differed from that of the unconjugated fraction (Figure 2) by the presence of a more diverse array of bile acids, notably 1␤-, 2␤-, and 22-hydroxylated metabolites (Figure 2 and Supplementary Table 2). Overall, the mean total urinary

bile acid concentration of these patients was 432 ⫾ 248 ␮mol/L, which was markedly elevated (normal, ⬍20 ␮mol/L), and cholic acid accounted for 54.9% ⫾ 6.9% of all bile acids excreted.

Biliary Bile Acid Analysis Duodenal bile was available from only 8 patients (patients 1, 2, 4, 5, 6, 7, 8, and 10), and the FAB-MS mass spectra were all similar to that of the index case (Figure 3). Consistent with urine, the striking and significant feature of the mass spectra of the duodenal bile extracts was the absence of ions corresponding to glycine- and taurine-conjugated primary bile acids, typically present when bile acid synthesis is intact. For comparison, the mass spectrum of a patient with liver disease but normal primary bile acid synthesis is shown in Figure 3. The major ion in the spectra of the bile from these patients was at m/z 407, corresponding to unconjugated trihydroxycholanoic acid, and other ions of variable intensity at m/z 391 (unconjugated dihydroxycholanoic), m/z 471 (sulfated dihydroxycholanoic), m/z 567 (dihydroxycholanoic glucuronide), and m/z 583 (trihydroxycholanoic glucuronide) were present. Ions at m/z 499 and 515 represent bile alcohol sulfates. After fractionation of the bile into conjugate classes using Lipidex-DEAP, hydrolysis/solvolysis of the conjugates, and derivatization, GC-MS analysis (Figure 3) established the identity and distribution of the individual bile acids observed in the FAB-MS spectra. No bile acids were found in the glycine and taurine fractions. GC profiles of the unconjugated and glucuronide- and sulfateconjugated bile acid fractions of the bile from the index case confirmed the majority of biliary bile acids to be unconjugated. The major peak in the chromatogram was definitively confirmed from its electron ionization mass spectrum and retention index to be cholic acid. There were traces of other bile acids in this fraction, including

deoxycholic acid, and there was a notable lack of unconjugated chenodeoxycholic acid, which was nevertheless present in low concentrations in the glucuronide and sulfate fractions together with cholic and deoxycholic acids. The biliary bile acid profiles of the 8 patients were qualitatively similar, although quantitatively there was considerable variation in concentrations due to sampling differences during intubation. The total biliary unconjugated bile acid concentration of the bile from the 8 patients was 12.06 ⫾ 5.95 mmol/L, which was significantly greater than the concentration of biliary bile acid glucuronides and sulfates combined (mean, 112 ⫾ 62 ␮mol/L). Unconjugated bile acids in duodenal bile therefore accounted for 95.7% ⫾ 5.8% of the total bile acids, with cholic acid accounting for 82.4% ⫾ 5.5% of all bile acids secreted (Supplementary Table 3).

Figure 2. (A) Typical negativeion FAB-MS spectrum of the urine from a patient with a defect in bile acid amidation. (B) GC-MS total ion current profiles of the methyl ester–trimethylsilyl ether derivatives of urinary bile acids excreted in unconjugated form and as glucuronide and sulfate conjugates combined. No glycine or taurine conjugates were found. Bile acids were identified from their mass spectra and retention indices. Peak numbers correspond to bile acids listed in Supplementary Table 1.

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Serum Bile Acid Analysis Negative-ion FAB-MS analysis of the serum from the index patient (patient 1) yielded a similar mass spectrum to that obtained for the patient’s urine and bile. The major ion and base peak was m/z 407, representing unconjugated trihydroxycholanoic acid. There was an absence of taurine- and glycine-conjugated bile acids. Ions at m/z 453 and 471 were accounted for by sulfate conjugates of monohydroxy-cholanoates and dihydroxy-cholanoates, respectively, while the ions at m/z 567 and 583 were consistent with glucuronides of dihydroxy-cholanoates and trihydroxy-cholanoates, respectively. The mean serum total bile acid concentration of 5 of the patients determined by GC-MS was markedly elevated at 257 ⫾ 157 ␮mol/L (normal, ⬍3.5 ␮mol/L). GC-MS analysis of the

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several stereoisomers of deoxycholic acid, including the 3␤-hydroxy- and 12␤-hydroxy- forms of both the 5␤-H and 5␣-H(allo-)cholanoic acids. Cholic acid was identified, as were several epimers and oxo-derived metabolites of cholic acid. The total bile acid concentration in the feces from this patient was 8.85 mg/g. Notable was the absence of lithocholic acid, normally one of the major bile acids in feces,12 indicating a relatively low level of chenodeoxycholic acid synthesis and consistent with the relative absence of chenodeoxycholic acid in other fluids analyzed.

Molecular Analysis

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Molecular analysis of the 3 coding exons of BAAT in the 8 patients from whom DNA was available resulted in identification of 4 different mutations, each present in homozygous form in one of the families tested (Table 2). In one patient (patient 9), no mutation was identified despite the finding of a urinary profile consistent with defective bile acid conjugation; this patient was also screened for mutation in SLC27A5, and no mutation was identified. Parents of all patients homozygous for a mutation in BAAT were confirmed to be heterozygous carriers of the mutations present in their children; results of genotyping in unaffected siblings are shown (Table 2). None of the 4 mutations detected were found in assayed control chromosomes and these alterations were not present in dbSNP, consistent with these being disease-causing mutations. Furthermore, all 3 missense mutations are predicted to damage protein structure and/or function; the 4th mutation introduces a premature stop codon early in the coding sequence of the gene and is therefore expected to result in lack of functional protein.

Morphologic Findings Figure 3. (A) Typical negative-ion FAB-MS spectra comparing bile from a patient with a defect in bile acid amidation (top spectrum) with bile from a patient with liver disease and intact bile acid synthesis (ions at m/z 448, 464, 498, and 514 represent the glycine- and taurine-conjugated primary bile acids, chenodeoxycholic acid, and cholic acid, respectively). (B) Typical GC-MS profiles of methyl ester–trimethylsilyl ether derivatives of biliary bile acids of a patient with a defect in bile acid amidation. Bile acids were fractionated according to conjugate class on Lipidex-DEAP. No bile acids were found in the glycine or taurine fractions. S1 and S2 represent internal standards (coprostanol and nordeoxycholic acid, respectively), and indicated is the relative volumes (␮L) of bile on-column. (C) Venn diagram showing the mean (n ⫽ 8) relative proportion of the principal bile acids in duodenal bile. Oxo-bile acids refers to all hydroxylated bile acids with a oxo group.

serum revealed cholic acid as the major serum bile acid, accounting for 64.0% ⫾ 6.8% of the total.

Fecal Bile Acid Analysis The GC profile of the methyl ester–trimethylsilyl ethers of bile acids isolated from the feces from patient 1 is shown in Supplementary Figure 1. Mass spectrometry confirmed the major fecal bile acid to be deoxycholic acid, accounting for 47.9% of the total bile acids, and there were

Four of the 10 patients underwent liver biopsy. Biopsies of the livers of 3 patients (patients 1, 2, and 5) were performed in early infancy; biopsies were performed for patients 1 and 5 to investigate unexplained direct hyperbilirubinemia, and biopsy was performed for patient 2 at a hepatic portoenterostomy at age 40 days (Figure 4A). Patient 5 had a small duct cholangiopathy of unusual severity at age 11 weeks (Figure 4B–D) that progressed to cirrhosis, liver failure, and need for transplantation at age 6 months. The explanted liver showed persistent severe small duct injury (Figure 4D), severe intralobular cholestasis, and periportal fibrosis with bridging. In many respects, the findings in the 2 (of 3) early biopsy specimens from patients 2 and 5 resemble those in idiopathic neonatal hepatitis, as do those described in the report of initial findings in patient 1. The prominent, even severe, ductular reaction in D, however, is a point of difference. Samples of liver tissue were obtained beyond infancy in 3 patients. Two of the 3 patients who underwent liver biopsy during infancy had follow-up liver biopsies at ages 4.5 years and 14 years. In patient 1, cholestasis and ductular proliferation had resolved, although during the intervening years he acquired transfusion-related hemosiderosis and mild portal fibrosis. In patient 2, the liver at age

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Table 2. Summary of Molecular Genetic Analyses Identifying Defects in BAAT DNA available and screened

5 6

1 2 3 4 5 6 7 8 9

No Yes Yes Yes Yes Yes Yes Yes Yes

7

10

Family 1 2 3 4

No

Nucleotide change — c1156G¡A c1156G¡A c.206A¡T c.58C¡T c.58C¡T c.58C¡T c.250C¡A No mutation identified —

Predicted effect on protein — p.G386R p.G386R p.D69V p.R20X p.R20X p.R20X p.P84T —

Nature of mutation — Missense Missense Premature stop codon Missense —

—

—

Homozygous

Prediction of functional effecta

— Yesb Yesb Yesb Yesb Yesb Yesb Yesb —

— Probably damaging

—

No. of unaffected siblings genotyped and results — ⫹/⫹ ⫹/⫺ ⫹/⫺ ⫹/⫺

Probably damaging —

1: 1: 1: 1:

Probably damaging —

1: ⫹/⫺ —

—

—

⫹, normal allele; ⫺, mutation present in the family. aUsing Polyphen 2; “probably damaging” is the worst functional category reported by this software. bBoth parents confirmed heterozygous.

4.5 years showed mild persistent ductular reaction and focal periportal fibrosis. Signs of obstructive cholangiopathy and lobular cholestasis were absent. Light microscopy of a single liver biopsy specimen obtained from patient 4 at age 15 months showed mild steatosis and rare necrotic

hepatocytes but no changes in bile ducts or ductules and no fibrosis. Liver ultrastructure at age 10 weeks in patient 5 was of note for extremely prominent autophagy, diffuse disorganization of mitochondrial cristae, and a severe but non-

Figure 4. (A) Patient 2. Open liver biopsy performed at hepatic portoenterostomy (“Kasai”): mild portal mononuclear cell infiltration, absent bile plugs in interlobular bile ducts, lobular cholestasis with spotty zone 1 hepatocyte necrosis, and prominent zone 3 giant cell transformation. H&E stain; original magnification 10⫻. (B) Patient 2. Mild proliferation of small bile ducts and ductular reaction at the limiting plate are highlighted. H&E stain; original magnification 25⫻. (C) Patient 5. Open liver biopsy performed at age 10 weeks: severe periportal fibrosis with bridging and lobular cholestasis with prominent giant cell transformation in zones 2 and 3. Giant cells have slightly foamy cytoplasm. Periportal fibrosis accompanies florid ductular and mild small duct proliferation. Lumina of ductules and ducts contain wispy bile residue and degenerate cholangiocytes but no bile plugs. Focally, a brisk pericholangitis is associated. H&E stain; original magnification 10⫻. (D) Patient 5. At age 6 months, the explanted liver showed a severe cholangiopathy with florid ductular proliferation and focally extreme dilatation without bile plugs. Periportal fibrosis had progressed to cirrhosis since the previous biopsy. H&E stain; original magnification 10⫻.

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Figure 5. Electron microscopy of biopsy 1 for patient 5. (A) Canaliculus exhibits unusual tortuous folding of microvilli (original magnification 5000⫻). (B) Patient 2 at age 4.5 years. A dilated canaliculus is surrounded by a prominent circumferential band of thin filaments, a sign of chronic injury (original magnification 5000⫻). (A and B) Osmium tetroxide postfixation, uranyl acetate, and lead citrate stain. (C) Patient 4. All hepatocytes exhibit uniform cytoplasmic background stain without the strong punctate granular reaction product present in the cytoplasm of normal hepatocytes (positive control, inset). Anti-BAAT antibody, hematoxylin counterstain (original magnification 200⫻). (D) Patient 4. Diffuse cytoplasmic reaction of variable intensity is observed in hepatocytes of both the patient and a normal control (inset). Anti-BACL antibody, hematoxylin counterstain (original magnification 200⫻).

specific pattern of injury to cholangiocytes of small ducts and ductules with substantial accumulation of bulky residual bodies in cholangiocyte cytoplasm. In addition, architectural distortion of canaliculi was unexpectedly severe and unusual, similar to that reported in another bile acid synthesis defect, 5-␤ reductase deficiency13 (Figure 5A). The ultrastructure of canaliculi and cholangiocytes at age 15 months in patient 4 was minimally altered. However, prominently dilated endoplasmic reticulum was universally present, as was mild mitochondrial pleomorphism with occasional matrix crystalloids. Canaliculi at age 4.5 years in patient 2 were normal or were dilated with accumulation of pericanalicular filaments (Figure 5B). Immunostaining for BAAT showed strong punctate diffuse cytoplasmic localization in normal hepatocytes that was uniformly depleted in liver biopsy tissue from patients 2, 4, and 5 (Figure 5C). Immunostaining for BACL, also involved in amidation, was normal in these 3 patients (Figure 5D), with nonuniform intensity ascribed to lobular unrest.

Discussion We describe the clinical, biochemical, and molecular characterization of 10 patients with a defect in bile acid conjugation. These cases illustrate the essential role that bile acids play in facilitating the absorption of fat-

soluble vitamins and dietary fatty acids, while conversely highlighting serum fat-soluble vitamin status as a sensitive marker for disturbances in hepatic bile acid synthesis and intraluminal bile acid composition. Our findings indicate that bile acid conjugation is essential for the normal enterohepatic circulation of bile acids and suggest that patients with unexplained fat-soluble vitamin deficiency should be investigated for the possibility of defects in bile acid conjugation. Bile acids are synthesized in the liver from cholesterol by a complex series of chemical reactions catalyzed by 17 different hepatic enzymes located in different subcellular fractions. The enzymes and their genes are well characterized and complementary DNAs described.14 There are multiple pathways in bile acid synthesis,15 but irrespective of the pathway by which unconjugated cholic and chenodeoxycholic acids are formed, the final step leads to the formation of the glycine and taurine conjugates,1 and these account for ⬎95% of the bile acids secreted in bile and are responsible for driving bile flow. Although inborn errors in bile acid synthesis involving impaired synthesis of cholic and chenodeoxycholic acids usually present as well-defined progressive familial cholestatic liver disease,9 by contrast, cholestasis is generally not the primary manifestation of a bile acid conjugation defect. The variable degree of cholestasis is difficult to explain. We speculate that in some patients

high levels of unconjugated cholic acid maintain bile flow and do not accumulate to toxic levels in hepatocytes. Alternatively, unconjugated bile acids are not well transported by canalicular transporters and in some patients may accumulate in hepatocytes, causing direct injury and/or recruitment of inflammatory factors. There was evidence of an interface inflammation in the liver biopsy specimens we were able to obtain, which would support the latter. The phenotype of defective bile acid conjugation is quite variable, with patients having little or mild to severe liver disease, presumably because cholic acid is synthesized at a normal rate and its efficient intestinal absorption leads to a recycling pool of bile acids that can generate bile flow. In one patient (patient 5), severe cholestasis and liver failure required liver transplantation; however, all the patients we describe shared the common feature of severe fat-soluble vitamin deficiency with subnormal levels of retinol, vitamin E, 25-hydroxyvitamin D, and prolonged PT. Chronically, these deficiencies led to rickets in 4 of the 10 patients described and fractures in 2 of the 10 patients. Poor growth is variable and largely limited to infants and young children. While a low serum GGT level is a characteristic feature of patients with PFIC1 and PFIC2,16 this is also the case for most patients with bile acid synthetic defects,9 including the 4 patients with this amidation defect in which serum GGT level was measured at baseline. Differential diagnosis of PFIC1 and PFIC2 from bile acid synthetic defects can be established from the presence in the case of PFIC or absence in the case of bile acid synthetic defects of primary bile acids. The clinical presentation and biochemical features of defective amidation closely parallel the predicted features hypothesized by Hofmann and Strandvik some 6 years before this first discovery.17 Their hypothesis was based on studies of C23-nor bile acids, bile acids that are poorly conjugated with glycine or taurine, enter the smooth endoplasmic reticulum, and undergo glucuronidation or sulfation followed by secretion into bile and/or urine but do not undergo an enterohepatic circulation.18 In our patients, newly synthesized chenodeoxycholic and deoxycholic acids (formed by bacterial 7-dehydroxylation of cholic acid) should, in the absence of amidation, undergo such glucuronidation (and possibly some sulfation) and be rapidly eliminated from the body, explaining the low proportions in bile. Definitive diagnosis of a defect in bile acid amidation in all 10 patients was accomplished by mass spectrometry using FAB-MS analysis of the urine,8,9 which is the same approach used to identify other bile acid synthetic defects. ESI-MS can also be used to make this diagnosis,19 as was recently reported for a patient with defective amidation due to a bile acid-CoA ligase deficiency.20 The striking feature of the mass spectra of the urine, bile, and serum of patients with defective amidation is the complete absence of ions corresponding to glycine- and taurine-conjugated bile acids and the presence of a dominant ion at m/z 407 representing unconjugated cholic acid; this conclusion

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was confirmed by GC-MS analysis. Although these patients conjugate bile acids with glucuronic and sulfuric acids, these conjugates collectively accounted for on average only ⬍5% of the bile acids secreted in bile and ⬍0.2% in 3 patients and are apparently of little help in promoting intestinal lipid absorption. Unconjugated bile acids in duodenal bile accounted for 95.7% ⫾ 5.8% of the bile acids. Quantitatively, duodenal bile obtained after induced gallbladder concentration by cholecystokinin administration had relatively high concentrations of unconjugated bile acids (mean ⫾ SEM, 12.06 ⫾ 5.95 mmol/L), of which cholic acid accounted for 82.4% ⫾ 5.5% of the bile acids secreted. Cholic acid was likewise quantitatively the major bile acid in serum and urine, and concentrations were markedly elevated. The duodenal bile acid concentrations were on average close to the critical micellar concentration for unconjugated cholic acid, which is approximately 11 mmol/L,3 meaning that the concentration of bile acids in micelles is quite low. It is likely that the postprandial intraluminal bile acid concentrations would be even lower after a meal, as has been reported previously.21 Conjugation of cholic acid with glycine and taurine has only a small effect on critical micellar concentration. The reduced fat-soluble vitamin concentrations and prolonged PT in these patients is explained by the rapid nonionic passive diffusion of unconjugated cholic acid from the proximal intestine, which reduces its intraluminal effectiveness for absorption of lipophilic compounds. Amidation of bile acids is an important final step in bile acid synthesis because this modification serves to lower the pKa of the unconjugated bile acid and promotes ionization at intestinal pH, thus preventing absorption from the proximal small bowel. The secondary bile acid, deoxycholic acid, was quantitatively the second most abundant bile acid in duodenal bile, albeit in low concentrations, and interestingly chenodeoxycholic acid was only found in traces in all biological fluids. The marked reduction in chenodeoxycholic acid was supported by the finding of negligible amounts of its secondary bile acid metabolite, lithocholic acid, in the feces of the index case, the only patient whose feces were available for analysis. It is probable that the reduced synthesis of chenodeoxycholic acid is caused by the excessive production of unconjugated cholic acid because cholic acid down-regulates chenodeoxycholic acid synthesis. Diarrhea, previously hypothesized as a possible feature of an amidation defect,17 was not seen in any patient. This is perhaps explained by a rapid recycling of unconjugated bile acids in the proximal small bowel, thus preventing excessive loss into the colon where they would be cathartic. Furthermore, it could be speculated that release of FGF19 might downregulate bile acid synthesis or that liver disease in some patients resulted in a failure of a compensatory increase in bile acid synthesis. Discerning whether an amidation defect resides in the bile acid CoA ligase (encoded by SLC27A5) or in the bile acid-CoA:amino acid N-acyltransferase (encoded by BAAT) requires the use of molecular techniques to sequence

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these 2 genes for mutations, or immunostaining of a liver tissue to detect absence of one enzyme, because both defects yield seemingly indistinguishable negative-ion mass spectra of the urine. Screening of SLC27A5 and BAAT for mutations can be performed in suspected cases of defects in bile acid conjugation. DNA was obtained from 8 of the 10 patients with a biochemically confirmed diagnosis, and homozygous mutations (Table 2) were identified in all but one patient. Because we did not detect a mutation in BAAT in patient 9, we sequenced the coding exons of SLC27A5 in his DNA; however, we also found no mutations in this gene. In each family in which a BAAT mutation was detected, the affected children were found to be homozygous for the familial mutation, and other unaffected family members were heterozygous or did not carry the mutation. These results indicate that this amidation defect behaves as an autosomal recessive trait. Of interest is that the BAAT mutation in patient 8, who is Amish, is different from the BAAT mutation previously reported in individuals with Lancaster County Old Order Amish ancestry,22 consistent with the finding of genetic heterogeneity for some other rare genetic disorders among the Amish. Liver biopsy findings in 4 of 10 patients suggest that transient and potentially severe cholestatic liver disease may be associated with BAAT deficiency only during infancy. On the other hand, the findings in the late liver biopsy specimens from patients 1 and 2 and clinical evidence in the other 8 patients indicate that BAAT deficiency does not regularly produce cholestasis in infancy or serious chronic liver disease. Most unusual in symptomatic infants was excessive proliferation of bile ductules that exceeds what is usual for idiopathic neonatal hepatitis or in other genetic defects in bile acid synthesis. This overlaps with findings in both biliary atresia and severe cholestasis related to parenteral alimentation. Also of interest is that periportal and pericellular fibrosis was already established in patient 5 at age 10 weeks, a feature generally considered a hallmark of an underlying metabolic disease. These findings permit postulation that transient hepatocyte injury with small duct cholangiopathy occurs in BAAT deficiency; it may have a biochemical basis and, when severe, may produce direct hyperbilirubinemia with potential to progress to liver failure in infants. The common lesion in those infants who underwent liver biopsy suggests biliary obstruction (as seen with biliary atresia). Of importance is that no obstruction of large bile ducts was shown, although a cholangiogram reportedly was abnormal in patient 2. The cause of the ductular injury pattern is not apparent. That nonamidated bile acids or salts themselves are not strongly irritant to mature hepatocytes or cholangiocytes can be inferred from the absence of clinical hepatobiliary disease in most patients with BAAT deficiency. Defective bile acid conjugation associated with mutations in BAAT has been described in a number of patients from an Amish kindred; hypercholanemia in Amish patients carrying a homozygous mutation in TJP2 and

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heterozygous mutation in BAAT occurred more often than expected by chance, suggesting that heterozygosity for the BAAT mutation may increase penetrance of disease associated with a TJP2 mutation.22 Recently, the first confirmed defect associated with a mutation in SLC27A5 was reported.20 The patient, of Pakistani origin and born to consanguineous parents, presented with cholestasis, elevated serum bilirubin and transaminase levels, normal serum GGT level, and low serum fat-soluble vitamin levels, which is a similar presentation to that of the patients with BAAT deficiency described here. A liver biopsy specimen from this child showed extensive fibrosis. The patient was homozygous for a missense mutation C.1012C⬎T in SLC27A5. No mutations were found in BAAT, but interestingly a second mutation was found in ABCB11, encoding the bile salt export pump (BSEP). Treatment with ursodeoxycholic acid was reportedly beneficial in a single patient with defective bile acid amidation caused by a mutation in SLC27A5.20 However, oral administration of primary conjugated bile acids should provide a better and rational therapeutic approach to correcting the fat-soluble vitamin malabsorption in patients with amidation defects.23 A trial of oral glycocholic acid is currently ongoing in 5 of the patients described here.

Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at www.gastrojournal.org, and at http:// dx.doi.org/10.1053/j.gastro.2013.02.004. References 1. Sjövall J. Dietary glycine and taurine conjugation in man. Proc Soc Exp Biol Med 1959;100:676 – 678. 2. Matoba N, Une M, Hoshita T. Identification of unconjugated bile acids in human bile. J Lipid Res 1986;27:1154 –1162. 3. Hofmann AF, Roda A. Physicochemical properties of bile acids and their relationship to biological properties: an overview of the problem. J Lipid Res 1984;25:1477–1489. 4. Vessey DA, Zakim D. Characterization of microsomal choloyl-coenzyme A synthetase. Biochem J 1977;163:357–362. 5. Killenberg PG. Measurement and subcellular distribution of choloyl-CoA synthetase and bile acid-CoA:amino acid N-acyltransferase activities in rat liver. J Lipid Res 1978;19:24 –31. 6. Johnson MR, Barnes S, Kwakye JB, et al. Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from human liver. J Biol Chem 1991;266:10227–10233. 7. Setchell KDR, Heubi JE, O’Connell NC, et al. Identification of a unique inborn error in bile acid conjugation involving a deficiency in amidation. In: Paumgartner G, Stiehl A, Gerok W, eds. Bile acids in hepatobiliary diseases: basic research and clinical application. Dordrecht: Kluwer, 1997:43– 47. 8. Sjövall J, Griffiths WJ, Setchell KDR, et al. Analysis of bile acids. In: Makin HLJ, Gower DB, eds. Steroid analysis. Dordrecht: Springer, 2010:837–966. 9. Setchell KDR, Heubi JE. Defects in bile acid biosynthesis— diagnosis and treatment. J Pediatr Gastroenterol Nutr 2006; 43(Suppl 1):S17–S22. 10. Consortium TIH. A second generation human haplotype map of over 3.1 million SNP’s. Nat Genet 2007;449:851– 861. 11. Hadzic N, Bull LN, Clayton PT, et al. Diagnosis in bile acid-CoA: amino acid N-acyltransferase deficiency. World J Gastroenterol 2012;18:3322–3326.

12. Setchell KDR, Street JM, Sjövall J. Fecal bile acids. In: Setchell KDR, Kritchevsky D, Nair PP, eds. The bile acids: methods and applications. New York: Plenum, 1988:441–570. 13. Daugherty CC, Setchell KDR, Heubi JE, et al. Resolution of liver biopsy alterations in three siblings with bile acid treatment of an inborn error of bile acid metabolism (delta 4-3-oxosteroid 5 betareductase deficiency). Hepatology 1993;18:1096 –1101. 14. Russell DW. The enzymes, regulation, and genetics of bile acid synthesis. Annu Rev Biochem 2003;72:137–174. 15. Axelson M, Sjövall J. Potential bile acid precursors in plasma— possible indicators of biosynthetic pathways to cholic and chenodeoxycholic acids in man. J Steroid Biochem 1990;36:631– 640. 16. Bezerra JA, Balistreri WF. Intrahepatic cholestasis: order out of chaos. Gastroenterology 1999;117:1496 –1498. 17. Hofmann AF, Strandvik B. Defective bile acid amidation: predicted features of a new inborn error of metabolism. Lancet 1988;2: 311–313. 18. Yoon YB, Hagey LR, Hofmann AF, et al. Effect of side-chain shortening on the physiologic properties of bile acids: hepatic transport and effect on biliary secretion of 23-nor-ursodeoxycholate in rodents. Gastroenterology 1986;90:837– 852. 19. Haas D, Gan-Schreier H, Langhans CD, et al. Differential diagnosis in patients with suspected bile acid synthesis defects. World J Gastroenterol 2012;18:1067–1076. 20. Chong CP, Mills PB, McClean P, et al. Bile acid-CoA ligase deficiency—a new inborn error of bile acid metabolism. J Inherit Metab Dis 2011;35:521–530. 21. Porter HP, Saunders DR. Isolation of the aqueous phase of human intestinal contents during the digestion of a fatty meal. Gastroenterology 1971;60:997–1007. 22. Carlton VEH, Harris BZ, Puffenberger EG, et al. Complex inheritance of familial hypercholanemia with associated mutations in TJP2 and BAAT. Nat Genet 2003;34:91–96.

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23. Heubi JE, Setchell KDR, Rosenthal P, et al. Oral glycocholic acid treatment of patients with bile acid amidation defects improves growth and fat soluble vitamin absorption (abstr). Hepatology 2009;50:895A.

Received November 20, 2012. Accepted February 6, 2013. Reprint requests Address requests for reprints to: Kenneth D. R. Setchell, PhD, Department of Pathology and Laboratory Medicine, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229. e-mail: [email protected]; fax: (513) 6367853. Acknowledgments Preliminary details of the index case were presented at the Falk Symposium No. 93 (XIV International Bile Acid Meeting), Freiberg, Germany, October 22–24, 1996. Sohela Shah’s current affiliation is: Clinical Genetics Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York. The authors thank U. Sanford (University of California San Francisco) for technical support with the molecular genetic studies. Conflicts of interest The authors disclose no conflicts. Funding Supported by National Institutes of Health grants 8 UL1 TR000077-04 (to J.E.H.), U01 DK62497 (to K.D.R.S., J.E.H., P.R., K.E.B., and L.N.B.), and R01 DK58214 (to L.N.B.).

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Supplementary Patients and Methods Ten patients from 7 different families are presented, and the demographics and main clinical presentations are summarized in Table 1. A more detailed description of these patients follows. Patient 1 was a male infant born weighing 2.6 kg after an uncomplicated term pregnancy in an 18-year-old primigravida of Laotian descent. Specific details on his parents are not available. He presented at age 40 days with jaundice and anemia (serum total bilirubin level of 10 mg/dL, direct bilirubin level of 5 mg/dL, hemoglobin level of 8.5 g/dL, and reticulocyte count of 11%). Hemoglobin electrophoresis found Hemoglobin H with Constant Spring, a form of ␣-thalassemia with an ␣-hemoglobin chain variant.1 At age 90 days, laboratory values were as follows: total bilirubin, 21 mg/dL; alanine aminotransferase (ALT), 216 IU/L; aspartate aminotransferase (AST), 294 IU/L; lactate dehydrogenase, 765 IU/L; and alkaline phosphatase, 466 IU/L. Findings on hepatobiliary iminodiacetic acid scanning and on ultrasound evaluations of the liver and gallbladder were normal. At age 5 months, he presented with failure to thrive, voluminous stools, epistaxis, and hematochezia. Physical examination revealed weight and height below the 5th percentile and marked hepatosplenomegaly but no ascites. His skin bore petechiae and ecchymoses. PT was 83 seconds (control, 10 seconds) and partial thromboplastin time (PTT) was 200 seconds (control, 30 seconds); these values normalized after administration of fresh-frozen plasma and vitamin K. Light microscopy of a percutaneous liver biopsy specimen found marked bile duct proliferation with bile plugs, hepatocellular pseudo-acini with intrahepatocytic cholestasis, sinusoidal disarray, expanded portal tracts with infiltration of neutrophils and lymphocytes, and mild bridging fibrosis, without extramedullary hematopoiesis. He remained on oral vitamin K (2.5 mg/day) for 4 years. Voluminous stools were treated with a change in formula to one with significant amounts of medium-chain triglycerides and hydrolyzed cow milk protein. At 1 year of age, the patient was admitted with anemia, congestive heart failure, and pulmonary edema. He had radiographic evidence of rickets with a fracture of the proximal fibula. Laboratory evaluation revealed the following: serum calcium, 6.5 mg/dL; phosphorus, 2.1 mg/dL; magnesium, 2.2 mg/dL; alkaline phosphatase, 1090 IU/L; ALT, 32 IU/L; AST, 26 IU/L; direct bilirubin, 1.4 mg/dL; albumin, 3.2 g/dL; and total protein, 6.5 g/dL. He responded to treatment with ergocalciferol and calcium supplements and remained on ergocalciferol for 4 years. The patient required multiple blood transfusions due to his hemoglobinopathy and treatment with the iron chelator deferoxamine throughout his childhood. No further evaluation of his liver or digestive problems was performed until age 14 years, when physical examination

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revealed marked growth retardation and hepatosplenomegaly. Laboratory evaluations revealed markedly decreased serum concentrations of 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, carotene, and vitamin K. Serum ALT and total and direct bilirubin levels were normal (Table 1). Hepatobiliary ultrasound studies showed hepatomegaly with homogeneity and no gallstones. At liver biopsy, severe hemosiderosis within hepatocytes and Kupffer cells, a well-preserved liver lobular architecture, minimal periportal fibrosis, and mild focal portal inflammation were seen; cholestasis and ductular proliferation had resolved. A urine sample was then screened for a bile acid synthetic defect using mass spectrometry. Patient 2 was a male infant, the third baby born to Saudi Arabian parents who were first cousins. He had a normal delivery at term and weighed 3.3 kg. A 7-year-old brother and an 8-year-old sister were reportedly both healthy. There was no history of liver disease in either parent’s family. He was admitted at age 28 days because of worsening jaundice since birth, weighing 3.8 kg (75th percentile) with acholic stools. Physical findings included a slightly enlarged liver without splenomegaly. Laboratory tests revealed the following: total bilirubin, 18 mg/ dL; direct bilirubin, 13 mg/dL; AST, 185 IU/L; ALT, 228 IU/L; and alkaline phosphatase, 863 IU/L. Ultrasonography failed to identify the gallbladder or common bile duct, although the liver was reportedly of normal size with slightly nonhomogeneous echogenicity. Hepatobiliary iminodiacetic acid scanning showed no excretion of isotope into the intestine. At age 40 days, an intraoperative cholangiogram revealed opacified intrahepatic ducts and small hypoplastic cystic and common bile ducts with contrast seen in the duodenum. Extrahepatic biliary hypoplasia was diagnosed, and a portoenterostomy was performed at the surgeon’s discretion. At age 3 months, the patient was asymptomatic and gaining weight. Serum total bilirubin level was 7.4 mg/dL, and direct bilirubin level was 6.7 mg/dL. The liver was firm. At age 7 months, a fracture of the right humerus and rickets were identified. The patient was noncompliant to oral fat-soluble vitamins. A low serum vitamin E level (1.7 mg/dL) was measured at age 9 months. Regular administration of Aquasol E (Columbus Labs, Ottawa, Ontario, Canada) did not result in any significant change in the vitamin E level after 1 month. A vitamin A level was normal, as were PT and PTT values. Pruritus developed at age 1 year, and he was started on ursodeoxycholic acid 4 months later (10 mg/kg body wt per day). Growth and development continued normally, and pruritus persisted. Serum vitamin E levels were persistently low. At age 4 years, urine and serum samples were screened for a bile acid synthetic defect. A follow-up liver biopsy was also performed at that time. He was then lost to follow-up until age 15.5 years, when he presented with severe bilateral knee valgus deformity and low serum vitamin D and E levels. His weight was 32.7 kg (⬍3rd percentile) and

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body mass index was 15.7 kg/m2; he reported no pruritus. On examination, he was nonicteric with a 2-cm firm liver, no splenomegaly, and no evidence of portal hypertension. Liver disease was substantial, as evidenced by elevated serum transaminase (2⫻ the upper limit of normal), alkaline phosphatase (20⫻ the upper limit of normal), and serum total bile acid levels (99 ␮mol/L). His serum GGT and bilirubin levels were normal. Ultrasonography showed mild coarsening of liver echotexture but no evidence of portal hypertension. Patient 3 was the 8-year-old sister of patient 2. Clinical information about this patient was not available at the time of diagnosis because the father claimed the child was asymptomatic and refused medical examination other than screening of urine and serum samples by mass spectrometry for a bile acid synthetic defect. The early clinical history is scant; a chest x-ray showed cupping of anterior ribs consistent with rickets at age 6 months, and she had fractures of the right 3rd and 4th fingers with high serum alkaline phosphatase and phosphorus levels at age 3 years. At age 12 years, serum vitamin E level was low (3.8 mg/mL), with a normal vitamin A level and normal serum transaminase level, GGT level, PT, and PTT. At age 15 years, her weight was 53 kg (50th percentile). Patient 4 was a 2.3-kg female infant born to a secundigravida by cesarean section at 36 weeks’ gestation because of preeclampsia. There was no parental consanguinity or family history of liver disease, growth failure, or diarrhea. A 4-year-old brother was well. On routine 4-month immunizations, the patient developed large ecchymoses on both legs immediately after injections and a complete blood cell count revealed a hemoglobin level of 4.6 g/dL with 182,000/mm3 platelets and a leukocyte count of 3900/mm3. PT was ⬎110 seconds, and PTT was 100 seconds. Plasma fibrinogen concentrations were normal. The patient received packed red blood cells, vitamin K, and fresh-frozen plasma, and her coagulopathy resolved. She weighed 5.2 kg, and her height was 57.5 cm (both ⬍3rd percentile for age). There was no hepatosplenomegaly, ascites, or edema. Serum AST, ALT, and bilirubin levels were normal. Ultrasonography of the abdomen revealed no abnormalities. Serum levels of vitamin A were normal (34 ␮g/L; expected, 20 – 43 ␮g/L) with low serum levels of 25-hydroxyvitamin D (9 ng/mL; expected, ⬎20 ng/mL) and vitamin E (⬍ 1.5 mg/dL; expected, 3–15 mg/dL). Skeletal radiography revealed moderate to severe rickets. The patient was begun on water-soluble vitamins A, D, E, and K (ADEK; Axcan Scandipharm, Inc, Birmingham, AL) and elemental formula. Over the next month, the patient’s fat-soluble vitamin deficiency resolved (serum vitamin A, 47.7 ␮g/L; vitamin E, 7.7 mg/dL) but her weight for age and height worsened, falling further below the 3rd percentile, and serum transaminase values rose mildly (AST, 46 IU/L; ALT, 66 IU/L) with normal serum

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GGT and bilirubin levels. Percutaneous liver biopsy was performed at age 15 months to investigate mild transaminasemia. A nasogastric tube was placed, and the patient was given a minimum of 4 cans of Peptamen Junior (medium-chain triglyceride– containing formula; Nestle Nutrition, Vevey, Switzerland) per day with improvement in growth. Urine and serum samples from the child were screened for a bile acid synthetic defect. Patient 5 was a 5½-month-old male infant with a history of developmental delay; he was the 5th of 6 siblings born to parents who shared a pair of great-greatgrandparents. He was born at term weighing 4.58 kg following an uneventful pregnancy and was admitted to the hospital at age 2 days for hydrocephalus ascribed to aqueductal stenosis and for jaundice, respiratory distress, hypoglycemia, and possible sepsis. He was moderately icteric (total bilirubin, 13.8 mg/dL; direct bilirubin, 2.3 mg/dL) with petechiae over the right eyebrow. The abdomen was soft and without hepatosplenomegaly. Computed tomography of the brain showed dilated lateral ventricles and large third ventricles. Phototherapy was given, and a ventricular shunt was placed. He developed progressive liver failure with prolonged international normalized ratio (INR) and underwent evaluation, including liver biopsy, for liver transplantation. A urine sample was screened for inborn errors of bile acid synthesis. The patient subsequently received a liver allograft from a deceased donor; his course thereafter was benign. Patient 6 was the sister of patients 5 and 7. She was in excellent health with a history of only a single urinary tract infection, for which she received intravenous antibiotics. At age 9 years, her height was 134.9 cm (50th percentile), weight 32.7 kg (50th percentile), and body mass index 18 kg/m2 (80th percentile), without abnormality on physical examination, including absence of jaundice and hepatosplenomegaly. PT was 12.6 seconds with an INR of 1.0. Serum alkaline phosphatase level was 384 IU/L, AST level was 30 IU/L, ALT level was 17 IU/L, and total bilirubin level was 0.4 mg/dL. Serum 25-OH vitamin D level was 25 ng/mL (expected, ⬎20 ng/mL), vitamin A level was 24 ␮g/L (expected, 26 –72 ␮g/L), and total tocopherol levels were low at 3 mg/dL (expected, 3–15 mg/dL). A urine sample was screened for a bile acid synthetic defect. Patient 7 was the sister of patients 5 and 6. Severe jaundice as a neonate prompted extensive evaluation (including liver biopsy; findings unavailable). All results of genetic studies were normal. Her parents were told that she had hepatitis but were given no further information. She was subsequently treated with ursodeoxycholic acid until approximately 3 years of age and was given vitamin A until 5 years of age. No further treatments or diagnostic studies were performed. At age 4 years, severe anemia was treated with iron. When screened for an inborn error of bile acid amidation at age 10 years, her height was 134.6 cm (10th percentile), weight was 26.3 kg (10th

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percentile), and body mass index was 16.2 kg/m2 (25th percentile). Physical examination showed neither jaundice nor hepatosplenomegaly. Laboratory results included a PTT of 14.9 seconds with an INR of 1.2, AST level of 50 IU/L, ALT level of 33 IU/L, and total bilirubin level of 0.5 mg/dL. Serum 25-hydroxyvitamin D level was low at 12 ng/mL, and serum vitamin A level was marginally low at 21 ␮g/L with total tocopherol levels reduced at 0.3 mg/dL. Patient 8 was a female infant, born at term weighing 3.63 kg to an Amish secundigravida, who presented at age 4 months with growth failure and unexplained fatsoluble vitamin deficiency. There was no family history of liver disease, malabsorption, or growth failure. Her sibling was well. Physical examination was unremarkable; she weighed 5.64 kg (25th percentile) with a height of 63.2 cm (25th percentile). Laboratory values were as follows: total serum bilirubin, 0.4 mg/L with 0 mg/dL direct; ALT, 19 IU/L; AST, 54 IU/L; alkaline phosphatase, 2108 IU/L; and GGT, 10 IU/L. Serum ␣-tocopherol level was 1.8 mg/dL (expected, 3–15 mg/dL), retinol level was 0.36 ␮mol/L (expected, 0.63–1.75 ␮mol/L), serum 25hydroxyvitamin D level was ⬍5 ng/mL, and PTT was ⬎120 seconds, which normalized with parenteral vitamin K. A urine sample was screened for a bile acid synthetic defect using mass spectrometry. Patient 9 was a male infant weighing 2.5 kg at birth after a 34-week pregnancy, complicated by glucose intolerance, in a 37-year-old Hispanic woman. It was her 6th pregnancy; no information is available on the patient’s 5 siblings. There was no family history of liver disease, growth failure, or rickets. He was evaluated at age 5 months for jaundice, growth retardation, rickets, and

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hypocalcemia. Physical examination revealed jaundice and mild hepatomegaly with no splenomegaly or ascites. PT was 24.5 seconds with an INR of 2.2. Laboratory studies showed the following: total bilirubin, 10.1 mg/dL (direct, 7.8 mg/dL); albumin, 4.3 g/dL; alkaline phosphatase, 1716 IU/L; total calcium, 5.9 mg/dL; AST, 756 IU/L; ALT, 37 IU/L; hemoglobin, 12 g/dL; and platelet count, 450,000/mm3. PT values normalized with parenteral vitamin K. Serum 25-hydroxyvitamin D level was 14.5 ng/mL (expected, ⬎20 ng/mL), and vitamin E level was ⬍1.5 mg/dL (expected, 3–15 mg/L). Light microscopy of a liver biopsy specimen showed giant cell transformation of hepatocytes. He was given intravenous calcium, fatsoluble vitamins, ursodeoxycholic acid, and Alimentum (a medium-chain triglyceride– based formula; Abbot Nutrition, Columbus, OH). A urine sample was screened for a bile acid synthetic defect using mass spectrometry. Patient 10 was a 10-year-old girl evaluated for poor weight gain. Weight was 20.9 kg (⬍5th percentile); findings on physical examination were otherwise unremarkable. She received intensive nutritional therapy by nasogastric tube but had only modest weight gain. A urine sample was screened for a bile acid synthetic defect in 2006, when the child 8 years of age, and again at 10 years of age. She subsequently was hospitalized for operative repair of duodenal stenosis and has been lost to follow-up. Supplementary Reference 1. Hofmann AF, Strandvik B. Defective bile acid amidation: predicted features of a new inborn error of metabolism. Lancet 1988;2:311– 313.

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BILE ACID CONJUGATION VITAMIN DEFICIENCY

955.e4

Supplementary Table 1. Relative Intensity of Principal Ions Present in the Negative-Ionization FAB-MS Spectra of the Urine of 10 Patients With a Defect in Bile Acid Conjugation Involving Impaired Bile Acid Conjugation Patient no. Iona Unconjugated bile acids m/z 407 (trihydroxycholanoic) m/z 391 (dihydroxycholanoic) Sulfates m/z 471 (dihydroxycholanoic) m/z 453 (monohydroxycholanoic) Glucuronides m/z 583 (trihydroxycholanoic) m/z 567 (dihydroxycholanoic)

1

2

3

4

5

6

7b

8

9

10

100 7

20 4

72 30

100 16

58 8

57 33

20 (25) 18 (23)

100 4

100 10

72 34

57 46

100 8

86 20

11 4

100 32

95 35

80 (100) 10 (13)

8 2

58 35

6 4

16 6

31 9

100 92

15 3

9 12

100 78

37 (46) 60 (75)

19 4

22 15

69 3

NOTE. The base peak in the spectrum is represented by 100% relative intensity, and the intensity of other ions in the spectra is expressed relative to the base peak. All values are expressed as percentages. aIons at m/z 448 and 464 corresponding to glycine conjugates of dihydroxy-cholanoates and trihydroxy-cholanoates, respectively, and m/z 498 and 514 corresponding to taurine conjugates of dihydroxy-cholanoates and trihydroxy-cholanoates, respectively, were absent in the negative-ion mass spectra of the urine from all patients. bBase peak was at m/z 417. The numbers in parentheses are relative intensities normalized to the major bile acid species.

2 6 7 8 9 10 12 13 14 15 16 17 19 20 22 23 24 25

1

Unconjugated bile acids 3␣,7␣,12␣-trihydroxy-5␤-cholan-23-oic (nor-cholic) 3␣,12␣-dihydroxy-5␤-cholanoic (deoxycholic) 3␤,7␣,12␣-trihydroxy-5␤-cholanoic (iso-cholic) 3␣,7␣,12␣-trihydroxy-5␣-cholanoic (allo-cholate) 3␣,7␣-dihydroxy-5␤-cholanoic (chenodeoxycholic) 3␣,7␣,12␣-trihydroxy-5␤-cholanoic (cholic acid) 3␣,7␤,dihydroxy-5␤-cholanoic (ursodeoxycholic) Trihydroxy-cholanoic isomer Trihydroxy-cholanoic isomer 3␣,7␤,12␣-trihydroxy-5␣-cholanoic 3␣,7␣,12␣-trihydroxy-5␤-cholan-25-oic 1␤,3␣,12␣-trihydroxy-5␤-cholanoic acid 1␤,3␣,7␣,12␣-tetrahydroxy-cholanoic 3,7,12,22-tetrahydroxy-cholanoic isomer 3-oxo-7␣,12␣-dihydroxy-4-cholenoic 7-oxo-3␣,12␣-dihydroxy-5␤-cholanoic 3,7,12,22-tetrahydroxy-isomer 2␤,3␣,7␣,12␣-trihydroxy-5␤-cholanoic Total unconjugated bile acids Sulfates and glucuronides combined 3␣,7␣-trihydroxy-5␤-cholan-23-oic (nor-chenodeoxycholic) 3␣,7␣,12␣-trihydroxy-5␤-cholan-23-oic (nor-cholic) 3␣-hydroxy-5␤-cholanoic (lithocholic) 3␣,12␣-dihydroxy-5␣-cholanoic (allo-deoxycholic) 3␣,12␤-dihydroxy-5␣-cholanoic 3␣,12␣-dihydroxy-5␤-cholanoic (deoxycholic acid) 3␣,7␣,12␣-trihydroxy-5␣-cholanoic (allo-cholate) 3␣,7␣-dihydroxy-5␤-cholanoic acid (chenodeoxycholic) 3␣,7␣,12␣-trihydroxy-5␤-cholanoic (cholic) trihydroxy-cholanoic isomer 3␣,7␤,dihydroxy-5␤-cholanoic (ursodeoxycholic) 3␣,7␣,12␣-trihydroxy-5␤-cholan-25-oic 12-oxo-3␣-hydroxy-5␤-cholanoic 3,7,12,22-tetrahydroxy-cholanoic isomer trihydroxy-cholanoic isomer Total conjugated bile acids

Summary Total urinary bile acid concentration Unconjugated bile acids as proportion of total bile acids (%) Cholic acid as proportion of total bile acids (%) NOTE. Values are expressed as ␮mol/L unless otherwise indicated.

1

2

4

5

6

8

10

Concentration (mean ⫾ SEM)

Mean composition (%)

4.7 2.5 1.5 1.1 1.4 117.9 0.0 0.9 2.4 2.8 1.3 11.8 7.4 5.0 5.3 1.9 4.4 3.1 175.3

17.0 1.9 4.3 2.4 1.4 394.3 4.1 2.6 2.5 1.8 4.2 22.9 72.9 24.6 119.0 15.8 6.9 22.6 721.0

0.8 0.3 0.3 1.6 3.3 99.9 0.2 0.0 1.5 0.0 0.0 1.2 1.8 10.3 1.7 4.8 10.1 1.5 139.2

11.2 0.0 0.6 0.8 2.2 55.2 1.7 0.8 1.1 0.7 0.9 9.1 12.8 3.0 13.5 1.7 0.8 4.6 120.8

6.0 0.3 3.2 0.4 1.1 50.4 0.3 1.0 1.5 1.0 1.6 6.5 9.1 2.3 7.4 1.8 1.0 2.8 97.8

0.4 0.2 0.0 0.4 0.0 62.9 2.0 0.3 0.0 0.5 0.0 0.0 4.0 0.0 3.5 4.1 0.4 0.3 79.0

5.3 1.3 2.2 4.9 8.4 193.5 26.0 0.0 20.5 46.0 1.6 6.3 20.0 5.1 25.9 294.9 281.7 13.0 956.7

6.5 ⫾ 2.4 0.9 ⫾ 0.4 1.7 ⫾ 0.7 1.6 ⫾ 0.7 2.5 ⫾ 1.1 139.2 ⫾ 50.2 4.9 ⫾ 3.8 0.8 ⫾ 0.4 4.2 ⫾ 2.9 7.5 ⫾ 6.9 1.4 ⫾ 0.6 8.3 ⫾ 3.1 18.3 ⫾ 10.1 7.2 ⫾ 3.4 25.2 ⫾ 17.2 46.4 ⫾ 44.8 43.6 ⫾ 42.9 6.8 ⫾ 3.3 327 ⫾ 195

3.2 0.4 0.8 0.6 1.0 55.8 1.1 0.4 1.1 1.3 0.6 3.7 6.1 2.7 6.7 6.4 6.0 2.1

0.0

0.7

0.0

0.0

0.0

0.0

0.3

0.1 ⫾ 0.1

0.1

0.0 0.0 1.5 0.4 9.8 0.0 3.2 24.2 0.0 0.5 1.2 0.7 0.0 0.0 41.6

1.0 1.8 3.7 3.1 48.8 3.5 9.8 254.3 10.0 24.6 4.0 17.9 3.0 4.1 390.3

0.0 0.0 0.0 0.0 2.9 0.0 19.6 7.4 0.7 0.7 0.0 0.5 0.2 1.6 33.5

0.0 0.9 0.3 0.9 2.2 0.0 5.4 18.3 1.0 0.0 0.0 2.2 0.0 1.4 32.6

0.0 0.0 3.1 0.0 12.4 0.0 5.8 14.1 0.0 0.4 0.5 1.1 0.0 0.0 37.4

0.0 0.0 0.0 0.0 0.0 0.0 0.4 2.5 0.0 0.0 0.0 0.3 0.0 0.3 3.5

0.4 0.1 0.3 0.7 1.0 0.0 22.9 81.0 0.1 60.2 0.7 17.7 0.3 13.7 199.5

0.2 ⫾ 0.1 0.4 ⫾ 0.2 1.3 ⫾ 0.5 0.7 ⫾ 0.4 11.0 ⫾ 5.6 0.5 ⫾ 0.5 9.6 ⫾ 2.7 57.4 ⫾ 29.5 1.7 ⫾ 1.2 12.3 ⫾ 7.2 0.9 ⫾ 0.6 5.8 ⫾ 2.5 0.7 ⫾ 0.4 3.0 ⫾ 1.5 105.5 ⫾ 53.0

0.1 0.5 2.0 0.7 12.1 0.1 17.8 50.0 1.1 5.9 0.8 5.1 0.2 3.6

216.9 80.8 65.5

1111.3 64.9 58.4

172.7 80.6 62.1

153.4 78.8 47.9

135.2 72.4 47.8

82.5 95.7 79.3

1156.1 82.7 23.7

432 ⫾ 196 79.4 ⫾ 3.9 54.9 ⫾ 7.2

GASTROENTEROLOGY Vol. 144, No. 5

2 3 4 5 6 8 9 10 11 12 16 18 20 21

Bile acid

SETCHELL ET AL

Patient Peak no.

955.e5

Supplementary Table 2. Urinary Bile Acid Concentrations and Relative Composition of the Individual Bile Acids Identified in the Unconjugated and Combined Sulfate and Glucuronide Conjugate Fractions From 7 Patients With a Confirmed Genetic Defect in Bile Acid Conjugation

May 2013

Supplementary Table 3. Bile Acid Concentrations and Relative Composition of Individual Bile Acids Identified in the Unconjugated and Combined Sulfate and Glucuronide Conjugate Fractions of Duodenal Bile From 7 Patients With a Confirmed Genetic Defect in Bile Acid Conjugation Patient Peak no.

Bile acid

NOTE. Values are expressed as ␮mol/L unless otherwise indicated.

2

4

5

6

7

8

10

Mean ⫾ SEM

Mean composition (%)

129 ⫾ 118 24 ⫾ 15 43 ⫾ 20 53 ⫾ 21 71 ⫾ 35 9531 ⫾ 3889 23 ⫾ 20 35 ⫾ 28 74 ⫾ 35 214 ⫾ 157 571 ⫾ 500 165 ⫾ 164 754 ⫾ 745 194 ⫾ 85 67 ⫾ 52 11,949 ⫾ 5885

2.1 0.1 0.4 0.4 2.0 82.4 0.1 0.3 0.6 1.1 2.7 1.1 3.0 2.2 0.4 — 2.2 1.1 7.8 0.5 17.1 53.3 2.0 2.2

0 0 47 42 0 5235 0 0 120 12 7 5 5 31 4 5509

901 84 127 118 128 21,035 153 205 250 1195 3831 1240 5629 503 400 35,798

8 0 0 0 4 51 0 0 0 0 3 3 0 0 0 69

0 0 0 0 0 366 0 0 0 0 0 0 25 0 0 390

14 0 13 28 47 2765 0 0 0 0 24 0 31 17 0 2938

57 94 0 153 90 22,328 0 0 154 0 377 0 0 323 73 23,649

54 0 114 60 29 22,035 0 0 0 344 197 42 339 153 0 23,397

0 10 39 26 268 2435 28 78 70 164 128 31 9 526 59 3871

0 0 0 0 0 20 0 0 20

3 4 2 0 0 0 0 0 9

0 0 0 0 0 7 0 0 7

17 0 15 0 10 40 0 0 82

4 4 35 0 20 16 0 7 85

17 4 9 17 204 146 27 9 433

2 0 0 0 56 69 15 0 142

0 0 0 0 19 97 0 9 125

5⫾3 2⫾1 8⫾5 2⫾2 39 ⫾ 26 49 ⫾ 19 5⫾4 3⫾2 112 ⫾ 62

3997 96.8 63.3

12,061 ⫾ 5947 95.7 ⫾ 5.8 82.4 ⫾ 5.5

5528 35,806 99.7 99.9 95.1 58.7

76 92.0 76.9

472 3023 24,083 23,509 82.6 97.1 98.2 99.4 85.8 92.0 93.3 94.0

BILE ACID CONJUGATION VITAMIN DEFICIENCY

Unconjugated bile acids 3␣,7␣,12␣-trihydroxy-5␤-cholan-23-oic (nor-cholic) 3␣,12␣-dihydroxy-5␤-cholanoic (deoxycholic) 3␤,7␣,12␣-trihydroxy-5␤-cholanoic (iso-cholic) 3␣,7␣,12␣-trihydroxy-5␣-cholanoic (allo-cholate) 3␣,7␣-dihydroxy-5␤-cholanoic (chenodeoxycholic) 3␣,7␣,12␣-trihydroxy-5␤-cholanoic (cholic acid) trihydroxy-bile acid isomer 3␣,7␤,dihydroxy-5␤-cholanoic (ursodeoxycholic) 3␣,7␣,12␣-trihydroxy-5␤-cholan-25-oic (homocholic) 1␤,3␣,12␣-trihydroxy-5␤-cholanoic acid 1␤,3␣,7␣,12␣-tetrahydroxy-cholanoic 3,7,12,22-tetrahydroxy-cholanoic isomer 3-oxo-7␣,12␣-dihydroxy-4-cholenoic 7-oxo-3␣,12␣-dihydroxy-5␤-cholanoic 3,7,12,22-tetrahydroxy-isomer Total unconjugated bile acids Sulfates and glucuronides combined 2 3␣,7␣,12␣-trihydroxy-5␤-cholan-23-oic (nor-cholic) 5 3␣,12␤-dihydroxy-5␣-cholanoic 6 3␣,12␣-dihydroxy-5␤-cholanoic (deoxycholic acid) 8 3␣,7␣,12␣-trihydroxy-5␣-cholanoic (allo-cholate) 9 3␣,7␣-dihydroxy-5␤-cholanoic acid (chenodeoxycholic) 10 3␣,7␣,12␣-trihydroxy-5␤-cholanoic (cholic) 12 3␣,7␤,dihydroxy-5␤-cholanoic (ursodeoxycholic) 18 12-oxo-3␣-hydroxy-5␤-cholanoic Total conjugated bile acids Summary Total biliary bile acid concentration Unconjugated bile acids as proportion of total bile acids (%) Cholic acid as proportion of total bile acids (%)

2 6 7 8 9 10 11 12 16 17 19 20 22 23 24

1

955.e6