Introduction
Bile acids are synthesized in the liver from cholesterol. They are inherently cytotoxic and can function as detergents to aid digestion and as signaling molecules to regulate gene expression and metabolism. Disruption of bile acid homeostasis that results in the accumulation of hydrophobic bile acids within the hepatocyte or canaliculus can damage hepatocyte membranes because their detergent-like properties,1,2 or by causing a mitochondrial membrane permeability transition that leads to necrosis and apoptosis, and promoting the production of reactive oxygen species and oxidative stress.3–6 This is considered to be the common pathogenesis of cholestatic diseases characterized by jaundice, progressive inflammation, liver fibrosis, cirrhosis, and death.7
More than 20 transporters are involved in the enterohepatic circulation of bile acids.8,9 The bile salt export pump (BSEP) protein, encoded by ABCB11,10 plays a key role in the secretion of bile acids across the canalicular membrane of hepatocytes into bile to provide the osmotic driver for bile flow.11,12 The absence, deficiency, or dysfunction of BSEP, or progressive familial intrahepatic cholestasis, type 2, (PFIC2) is caused primarily by mutations in ABCB11 or, secondarily, by mutations in ATP8B1, which encodes familial intrahepatic cholestasis 1 (FIC1),13,14MYO5B (encoding myosin VB),15,16 or NR1H4 (encoding farnesoid X receptor, FXR),17 or can be due to drug toxicity, leading to the accumulation of bile acids in hepatocytes (cholestasis). Multidrug resistance protein 3 (MDR3), encoded by ABCB4, is expressed on the canalicular membrane of hepatocytes and is responsible for flippase activity in transporting phospholipids from hepatocytes into bile. Mutations in ABCB4 can cause the absence or dysfunction of MDR3 leading to MDR3 deficiency or PFIC3. In PFIC3, the impaired flippase of phosphatidylcholine into bile leads to an overload of bile acids within the canaliculus as biliary bile acids are no longer being sequestered in mixed micelles with phospholipid and cholesterol leading to bile duct injury.18
The toxicity of bile acids depends primarily on their hydrophobicity.19,20 Compensatory modifications of bile acids, such as the formation of hydrophilic taurine-conjugated bile acids, hydroxylated bile acids, sulfated bile acids, and alternative renal excretion routes for bile acids, reduce the toxic detergent effect of hydrophobic bile acids and are considered important compensation mechanisms that affect the disease phenotype.21,22 How dysfunction of MDR3 in human beings affects bile acid metabolism, including the distribution and composition of bile acids, has not been well investigated.
Tetrahydroxylated bile acids (THBAs) are the most hydrophilic and the least cytotoxic bile acids identified in humans to date.23–25 The bile acids found in humans are typically mono-, di-, and tri- hydroxylated. THBAs are often undetectable in healthy humans or are present only in trace amounts. They can be detected in the serum and urine of cholestatic patients and in mouse models of human liver diseases.22 It has been proposed that THBAs are hepatoprotective agents in alleviating cholestatic stress.22 Tetrahydroxylation of bile acids to increase solubility might be an alternative pathway for elimination of bile acids through urine,26 for alleviation of cholestasis.27
In our previous study, the profiling of bile acids revealed increased levels of taurine-conjugated 3α,6α,7α, 12α-THBA (tauro-THBA) in PFIC2 patients, compared with healthy controls.28 In infants with intrahepatic cholestasis of diverse etiologies, a high urine THBA level was associated with better outcomes.29 In a follow-up study of a subset of PFIC2 patients who underwent partial internal biliary diversion, we observed that changes in the level of THBAs were correlated with disease relief and recurrence, which implies a potential use for THBAs as prognostic indicators.30 Our recent study revealed several THBA isoforms that might have potential as prognostic biomarkers in Alagille syndrome patients.25 The correlation of the concentration of tauro-THBA with the prognosis of PFIC2 patients was not evaluated in the previous study owing to lack of sufficient follow-up information. The same was true for PFIC3 patients. We were very interested to know whether the concentration of tauro-THBA may have the potential to be a prognostic biomarker in patients with either a BSEP deficiency or PFIC3. The study goal was to determine the bile acid profile in ABCB4-mutated patients, and to explore the potential use of tauro-THBA as a prognostic indicator in both ABCB4-mutated patients and patients with BSEP deficiency.
Methods
Subjects
Study subjects were Chinese children who were admitted to the Children’s Hospital of Fudan University, with consent, under a protocol approved by the Children’s Hospital of Fudan University (2015-178 and 2020-402) and in accord with the ethical guidelines of the 1975 Declaration of Helsinki. Subject screening criteria for ABCB4-mutated patients included: (1) Clinical manifestations of cholestasis. (2) Exclusion of patients with infectious, other metabolic, and obstructive causes of conjugated jaundice by appropriate investigation. (3) Homozygous or compound heterozygous (likely) pathogenic variants in ABCB4 based on a screening panel of 61 cholestasis-related genes or by whole exome sequencing.31 (4) With available fasting serum samples. Sixty-five healthy subjects with fasting samples available and no indications of infectious, endocrinological, or metabolic diseases who were included in a previous study,28,30 were used as controls for comprehensive bile acid profiling. The tauro-THBA concentrations in ABCB4-mutated patients with no jaundice (good prognosis) and those with decompensated cirrhosis or who died/underwent liver transplantation (bad prognosis) during follow-up were compared.
The tauro-THBA concentration in ABCB4-mutated patients were also compared with 17 ABCB11-mutated patients, with primary BSEP deficiency who participated in a previous study.28 The areas under the curve (AUCs) of tauro-THBA in ABCB11-mutated patients with native liver survival (good prognosis) and those who died or underwent liver transplantation (bad prognosis) before 3 years of age were compared by receiver operating characteristic curve (ROC) analysis. The cutoff value derived from the ABCB11-mutated patients (primary BSEP dysfunction) was evaluated in another patient cohort, also previously reported, with secondary BSEP dysfunction, including five patients with ATP8B1, eight with MYO5B deficiency, and 12 with undiagnosed low gamma-glutamyl transferase (GGT) level cholestasis (GGT<100 IU/L)28 to check whether tauro-THBA has the potential to be used as a prognostic biomarker.
Sample preparation and analysis of bile acids
The methods of sample preparation and analysis of bile acids were the same as previous reports.28,30 Briefly, serum was separated from blood by routine centrifugation (1,000 × g for 5 m at 4°C), aliquoted (50 µL), freeze-dried, and stored at −80 °C until bile acid analysis was performed by reverse-phase ultra-high-performance liquid chromatography/multiple-reaction monitoring-mass spectrometry (UPLC/MRM-MS) with negative ion detection at the University of Victoria-Genome British Columbia Proteomics Center.24 The quantitation of bile acids utilized standards of 60 bile acids, and 14 deuterium-labeled analogs of bile acids, as internal standards. The 60 targets, were previously described16,24 and included all the major bile acids and some minor species, e.g., ursodeoxycholic acid (UDCA), hyocholic acid (HCA), hyodeoxycholic acid (HDCA), muricholic acids (MCAs), two THBAs (3α,6α,7α,12α-THBA, and tauro-THBA, which were custom-synthesized and provided by Victor Ling (2British Columbia Cancer Agency, Vancouver, BC, Canada). Linear-regression calibration curves of individual bile acids were constructed with internal calibration (Tables 1 and 2).
Assessment of the hydrophobicity of bile acids
The hydrophobicity of bile acids was calculated by the weighted retention time (RT) of bile acids in LC as:
∑[C(nM)×RT(min)]Ctotal
in which C is the concentration of individual bile acids and Ctotal is the total bile acid concentration.28 A lower hydrophobicity index indicates relatively lower hydrophobicity or higher hydrophilicity.Statistical analysis
The statistical analysis was performed with SPSS 19.0 (IBM Corp., Armonk, NY, USA) to determine the significance of differences between the concentrations of tauro-THBA in the patient groups and healthy controls. Student’s t-test was used when the data had a normal distribution. A nonparametric test, the Mann-Whitney U test, was used when the data did not have a normal distribution. Data were reported as means±standard deviations or medians and interquartile ranges (IQRs). ROC curve analysis was used to calculate AUC. Fisher’s exact test was used in the R × C table. A p-value <0.05 was considered significant.
Discussion
In this study, we performed an analyses of serum bile acids in genetically-defined ABCB4-mutated patients. To our knowledge, no bile acid profiling in ABCB4-mutated patients has been reported. The principal difference observed was a marked increase in conjugated primary bile acids and hydrophilic bile acids modified by conjugation, sulfation, or ketonization, were significantly elevated. There were also significant increases in atypical hydrophilic trihydroxy, MCAs, and tauro-THBA in ABCB4-mutated patients (Tables 1, 2 and 4). All these modifications resulted in an increase in the hydrophilicity of the bile acid profile, as confirmed by the lower hydrophobicity indices of total serum bile acids in the ABCB4-mutated patient group compared with healthy controls (Fig. 2A). These findings support the hypothesis that abnormal metabolism of bile acids in ABCB4-mutated patients contributed to the activation of multipathway compensatory mechanisms to increase bile acid hydrophilicity, thereby alleviating bile acid toxicity in such patients. The factors that confer or lessen this ability await further definition. Of note, the concentrations and proportions of the main secondary bile acids were not increased compared with the main primary bile acids (Fig. 1 and Table 1), suggesting that the secretion of primary bile acids into the intestine was not increased, as not so many additional secondary bile acids were generated. Quantitation of secondary bile acids in the feces might help to confirm this.
A separate study arm assessed the relationship between tauro-THBA production and the disruption of bile acid homeostasis. We included patients in whom the pathogenesis of cholestasis was clear, i.e. caused by defects of transporters that led to bile acid overload, including the main canalicular bile acid transporter, BSEP, as well as PFIC3, another typical of cholestatic disease. In patients with BSEP deficiency, the secretion of bile acids from hepatocyte into canaliculus is impaired and bile acids are considered to accumulate within the hepatocytes. In patients with PFIC3, impaired flippase of phosphatidylcholine into the bile led to an overload of bile acids within the canaliculus as biliary bile acids were no longer being sequestered in mixed micelles with phospholipids and cholesterol.18 High levels of THBAs have been observed in ABCB11−/− mice.23,33 It has been speculated that the production of THBA was the major compensatory mechanism allowing for reduction of cholestatic stress in the mice. Significant elevation of THBA was not observed in Mdr2−/− mice (ABCB4),18 which had a more severe phenotype of intrahepatic cholestasis. It was speculated that it is only when the bile duct injury eventually spills over onto the hepatocytes that the enzymes for producing THBA were stimulated, as cholestasis and bile acid accumulation eventually become more pronounced because of mechanical blockage of damaged bile ducts. Tauro-THBA concentration was significantly elevated in patients with BSEP deficiency compared with healthy controls and patients with MDR3 deficiency (Fig. 2B). That is in line with the hypothesis that THBA production is triggered by the accumulation of bile acid within hepatocytes, where P450 hydroxylases modify and detoxify bile acids by adding hydroxyl groups. The proportion of trihydroxy and tetrahydroxy bile acids including CAs, MCAs, and THBAs were not significantly elevated in ABCB4-mutated patients, compared with healthy controls (Fig. 1). In addition, differences of the molar ratios of MCA:CDCA, HCA:CDCA, α-MCA:CDCA, β-MCA:CDCA, and ω-MCA:CDCA in ABCB4-mutated patients and healthy controls were not significant (Table 4). The results suggest that, unlike patients with ABCB11-mutations and cholestasis for undiagnosed reasons,28 hydroxylation was not the predominant form of bile acid modification in ABCB4-mutated patients. However, unlike in the mouse model, the concentration of tauro-THBA was significantly elevated in ABCB4-mutated patients, although to relatively low levels compared with BSEP-deficient patients. It is not known whether that was because of the secondary accumulation of bile acids within hepatocytes after bile duct injury or if there are other mechanisms for activating this compensatory mechanism. These observations warrant further follow-up studies.
Study of the Mdr2−/− mouse model has demonstrated that some THBA species stimulate bile flow after either intravenous injection or feeding, suggesting that THBAs have potential as therapeutic agents for cholestatic diseases.34 Recent studies have also indicated that the ability to generate THBA as a compensatory mechanism for the alleviation of cholestasis affects the disease phenotype or prognosis.22 In this limited number of cases, the concentration of tauro-THBA indicated no significant difference between ABCB4-mutated patients with no jaundice (good prognosis) and those suffering with decompensated cirrhosis or who died/underwent liver transplantation (bad prognosis). It would be interesting to know whether, by enhancing the production of THBA or oral administration of THBAs, one might improve the prognosis of ABCB4-mutated patients.
In a previous study, the urinary excretion of bile acids was investigated by gas-liquid chromatography-mass spectrometry (GC-MS) in seven infants with α1-antitrypsin deficiency. It was found that the proportion of THBAs increased but that the patients who developed cirrhosis during the observation period had a lower concentration of THBAs than those with a more favorable course.27 A follow-up study by Lee et al.29 that studied the proportion of THBAs in the urine of 40 infants with intrahepatic cholestasis by GC-MS found that a higher THBA proportion was associated with good outcomes and that urinary tauro-THBA proportion predicted good prognosis with high sensitivity (95.24%) and specificity (84.21%, p<0.0001). However, the case numbers in these studies were small, and most of the cases in Lee et al.29 had an undefined etiology. In addition, the sensitivity of the GC-MS method was lower than that of the LC-MS method, and THBA is weakly detectable in serum samples using the GC-MS method,22,24,27,29 detects unconjugated forms of bile acids. A deconjugation step is needed to convert conjugated bile acids, such as glycine-conjugated, taurine-conjugated, glucuronidated, and sulfated forms. Therefore, what was measured in those studies was the total unconjugated and variously modified forms of the bile acids for each compound.27,29 Only the proportion of urinary THBA was evaluated in those studies. The THBA concentration was not quantitated or was only partially quantitated by the GC-MS method. Therefore, it is not known whether any of the conjugated bile acids, including tauro-3α,6α,7α,12α-THBA, could be a potential prognostic biomarker for the disease, because GC-MS is not suitable for direct analysis of any of the very polar and conjugated bile acids. In our study, LC-MRM/MS was used24 with custom-synthesized taurine-3α,6α,7α,12α-THBA as standard substance.28 Compared with GC-MS, it provides higher sensitivity, allows direct analysis of tauro-3α, 6α, 7α, and 12α-THBA, and does not need time-consuming sample preparation before LC-MS runs. Therefore, the results for tauro-3α,6α, 7α,12α-THBA in the serum of patients with confirmed hereditary cholestasis, as seen in this study, is new and complements previously published results using urine.
Multiple THBA isomers have been identified in patients with liver diseases.27,29 The prognostic value of taurine-3α,6α,7α,12α-THBA is explored in the current study, as it is one of the main forms of THBAs in patients with PFIC228 and with Alagille syndrome.25 However, a study limitation as that only taurine-3α,6α,7α,12α-THBA was measured, as we only had access to custom-synthesized taurine-3α,6α,7α,12α-THBA when we performed the study. No authentic compounds of other tauro-THBAs are commercially available. In addition, tauro-3α,6α,7α,12α-THBA has been reported as a major THBA in animal models of PFIC2. That is why we hypothesized it might be a biomarker in patients of PFIC2. It will be very interesting to know whether there are other THBA isomers that have even higher specificity and sensitivity for prognostic purposes, and that warrants further study.
The distribution of bile acids in both serum and urine reflected the metabolism of bile acids during disease. The detection of urine samples is noninvasive, but serum samples were much easier to collect for liver function tests in children. Collection of urine samples was usually not convenient as the majority of our patients were infants who were being followed at outpatient clinics. For this study, only serum samples collected from the patients were available in our biobank and not enough urine samples were available for analysis. This study focused on the analysis of bile acids in serum only. In addition, serum bile acids are derived from two main sources, the intestine and the liver, which directly reflect the bile acid metabolism. Our study of serum samples complements previous reports in urine samples. Whether the concentration of taurine-3α,6α,7α,12α-THBA in urine is also associated with the severity of disease and has the potential to work as a prognostic biomarker warrants further study.
In the current study, we asked whether the THBA concentration was associated with the severity of disease and if it has the potential to work as a prognostic biomarker. We assessed the relationship between levels of tauro-THBA and prognosis in ABCB11-mutated patients and found that serum tauro-THBA concentrations of >60 nM were a sensitive biomarker for predicting clinical outcome in patients with hereditary cholestasis caused by both BSEP dysfunction and cases of undiagnosed low GGT cholestasis (Fig. 2C–D and Table 5).