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

Kenneth D. R. Setchell; James E. Heubi; Sohela Shah; Joel E. Lavine; David Suskind; Mohammed Al–Edreesi; Carol Potter; David W. Russell; Nancy C. O'Connell; Brian Wolfe; Pinky Jha; Wujuan Zhang; Kevin E. Bove; Alex S. Knisely; Alan F. Hofmann; Philip Rosenthal; Laura N. Bull


Gastroenterology. 2013;144(5):945-955. 

In This Article


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 unconjugated 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 taurine-conjugated bile acids.

Figure 2.

(A) Typical negative-ion 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.

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 μmol/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 μmol/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 1β-, 2β-, and 22-hydroxylated metabolites (Figure 2 and Supplementary Table 2 ). Overall, the mean total urinary bile acid concentration of these patients was 432 ± 248 μmol/L, which was markedly elevated (normal, <20 μmol/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.

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 (μL) 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.

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 sulfate-conjugated 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 μmol/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).

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 μmol/L (normal, <3.5 μmol/L). GC-MS analysis of the 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 several stereoisomers of deoxycholic acid, including the 3β-hydroxy- and 12β-hydroxy- forms of both the 5β-H and 5α-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

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

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 4BD) 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.

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×.

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 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 nonspecific 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-β reductase deficiency[13] (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).

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×).

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.