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


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 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 down-regulate 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 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 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.