Urinary Bile Acids Research Articles

Profiling circulating and urinary bile acids in patients with biliary obstruction before and after biliary stenting.

Bile acids are considered as extremely toxic at the high concentrations reached during bile duct obstruction, but each acid displays variable cytotoxic properties. This study investigates how biliary obstruction and restoration of bile flow interferes with urinary and circulating levels of 17 common bile acids. Bile acids (conjugated and unconjugated) were quantified by liquid chromatography coupled with tandem mass spectrometry in serum and urine samples from 17 patients (8 men and 9 women) with biliary obstruction, before and after biliary stenting. Results were compared with serum concentrations measured in 40 age- and sex-paired control donors (20 men and 20 women). The total circulating bile acid concentration increases from 2.7 µM in control donors to 156.9 µM in untreated patients with biliary stenosis. Serum taurocholic and glycocholic acids exhibit 304- and 241-fold accumulations in patients with biliary obstruction compared to controls. The enrichment in chenodeoxycholic acid species reached a maximum of only 39-fold, while all secondary and 6α-hydroxylated species –except taurolithocholic acids – were either unchanged or significantly reduced. Stenting was efficient in restoring an almost normal circulating profile and in reducing urinary bile acids.ConclusionThese results demonstrate that biliary obstruction affects differentially the circulating and/or urinary levels of the various bile acids. The observation that the most drastically affected acids correspond to the less toxic species supports the activation of self-protecting mechanisms aimed at limiting the inherent toxicity of bile acids in face of biliary obstruction.

Urinary bile acid sulfate levels in patients with primary biliary cirrhosis

Urinary bile acids are mainly conjugated with sulfuric acid. In a previous work, we demonstrated that the levels of urinary sulfated bile acids (USBA) and serum total bile acids (TBA) were correlated very well and also that USBA was considered to be a more useful indicator of hepatic fibrosis than TBA in patients with hepatitis C virus (HCV)-related liver diseases. In the current study we aimed to confirm these finding in patients with primary biliary cirrhosis (PBC), a prototypic cholestatic liver disease. Urinary sulfated bile acids were measured using an automatic assay kit in 50 patients with PBC, of whom 11 were diagnosed as having cirrhosis. We obtained specimens before ursodeoxycholic acid (UDCA) administration in four patients, and during UDCA in 46 patients. The correlations between USBA and various laboratory tests were studied. The median USBA level was 67.9 µmol/g creatinine in PBC; 27.7 without cirrhosis and 210.3 with cirrhosis (P = 0.001). The number of PBC patients with elevated USBA was significantly higher than those with elevated TBA (82% vs. 56%). This significance was remarkable especially in early stages, non-cirrhotic patients (77% vs. 49%). USBA level was well correlated with TBA (r(s) = 0.72), and negatively correlated with platelet (r(s) = -0.34) and albumin (r(s) = -0.31). Urinary sulfated bile acids and TBA are well correlated, and together with the findings that USBA is not affected by meals, USBA is considered to be more beneficial and convenient than TBA for earlier detection of fibrosis in PBC.

Gas chromatography–mass spectrometry-based simultaneous quantitative analytical method for urinary oxysterols and bile acids in rats

A simultaneous quantitative assay method for urinary oxysterols and bile acids using GC–MS was developed to investigate the mechanism of liver toxicity induced by drugs or chemicals. Sample preparations were optimized by exploring various extraction solvents, derivatization reagents, and hydrolysis methods to achieve reliable and maximum sensitivity for these two different compound classes. As a result, satisfactory accuracy, precision, and sensitivity were obtained in the validation. The method was then applied to quantify urinary oxysterols and bile acids produced from liver toxicity induced by atorvastatin (250 mg/kg/day). From the results, increases in bile acid levels and decreases in the concentration ratio between cholic acid and chenodeoxycholic acid, which are the distinguishing phenomena observed in serum or bile for liver toxicity, were also observed in urine. Additionally, the mechanism of liver toxicity was investigated with the urinary concentration ratio of product to precursor in the metabolic pathway from cholesterol to bile acids. The results indicated that enzyme activities related to the production and degradation of bile acids, not oxysterols, were significantly changed from liver toxicity. Thus, it was concluded that urinary levels of oxysterols and bile acids could be useful tools for checking liver toxicity and investigating its mechanism.

Exchange Transfusion as a Possible Therapy for Neonatal Hemochromatosis

Exchange Transfusion as a Possible Therapy for Neonatal Hemochromatosis

Developmental pattern of urinary bile acid profile in preterm infants

Bile acid metabolism in preterm infants is yet to be fully characterized. We compared the developmental pattern of urinary bile acid profiles in ten infants born at gestational ages from 25 to 33 weeks with previous data from full-term infants from birth to about 7 months of age. Gas chromatography-mass spectrometry was performed on serial samples. Total urinary bile acid concentrations gradually increased until 1 to 2 months of age. After this peak of excretion (30 to 60 micromol/mmol creatinine), total urinary bile acid concentrations gradually decreased to less than 20 micromol/mmol creatinine. The percentage of usual bile acids (mainly cholic acid) relative to total urinary total bile acids gradually deceased from approximately 30% at birth to less than 15% at 7 months of age. On the other hand, 1beta-hydroxylated bile acids (mainly 1beta,3alpha,7alpha,12alpha-tetrahydroxy-5beta-cholan-24-oic acid) relative to total urinary bile acids were increased gradually from 60% at birth to reach 70% to 80% at 1 month of age. The percentage of 1beta-hydroxylated bile acids relative to total urinary bile acids then remained stable at a high percentage (70% to 90%) until the age of 7 months. Physiological cholestasis in preterm infants persists longer than in full-term infants. Moreover, as large amounts of cholic and 1beta,3alpha,7alpha,12alpha-tetrahydroxy-5beta-cholan-24-oic acids were detected in urine from preterm infants during this study, the 25-hydroxylation pathway may be particularly important for bile acid synthesis in early preterm infants.

Bile acids: the role of peroxisomes

It is well established that peroxisomes play a crucial role in de novo bile acid synthesis. Studies in patients with a peroxisomal disorder have been indispensable for the elucidation of the precise role of peroxisomes. Several peroxisomal disorders are associated with distinct bile acid abnormalities and each disorder has a characteristic pattern of abnormal bile acids that accumulate, which is often used for diagnostic purposes. The patients have also been important for determining the pathophysiological consequences of defects in bile acid biosynthesis. In this review, we will discuss all the peroxisomal steps involved in bile acid synthesis and the bile acid abnormalities in patients with peroxisomal disorders. We will show the results of bile acid measurements in several tissues from patients, including brain, and we will discuss the toxicity and the pathological effects of the abnormal bile acids.

Intestinal Detoxification Limits the Activation of Hepatic Pregnane X Receptor by Lithocholic Acid

The intestinal-derived secondary bile acid (BA) lithocholic acid (LCA) is hepatotoxic and is implicated in the pathogenesis of cholestatic diseases. LCA is an endogenous ligand of the xenobiotic nuclear receptor pregnane X receptor (PXR), but there is currently no consensus on the respective roles of hepatic and intestinal PXR in mediating protection against LCA in vivo. Under the conditions reported here, we show that mice lacking Pxr are resistant to LCA-mediated hepatotoxicity. This unexpected phenotype is found in association with enhanced urinary BA excretion and elevated basal expression of drug metabolism enzymes and the hepatic sulfate donor synthesis enzyme Papss2 in Pxr(-/-) mice. By subsequently comparing molecular responses to dietary and intraperitoneal administration of LCA, we made two other significant observations: 1) LCA feeding induces intestinal, but not hepatic, drug-metabolizing enzymes in a largely Pxr-independent manner; and 2) in contrast to LCA feeding, bypassing first-pass gut transit by intraperitoneal administration of LCA did induce hepatic detoxification machinery and in a Pxr-dependent manner. These data reconcile important discrepancies in the reported molecular responses to this BA and suggest that Pxr plays only a limited role in mediating responses to gut-derived LCA. Furthermore, the route of administration must be considered in the future planning and interpretation of experiments designed to assess hepatic responses to BAs, orally administered pharmaceuticals, and dietary toxins.

Mouse Organic Solute Transporter α Deficiency Enhances Renal Excretion of Bile Acids and Attenuates Cholestasis

Organic solute transporter alpha-beta (Ostalpha-Ostbeta) is a heteromeric bile acid and sterol transporter that facilitates the enterohepatic and renal-hepatic circulation of bile acids. Hepatic expression of this basolateral membrane protein is increased in cholestasis, presumably to facilitate removal of toxic bile acids from the liver. In this study, we show that the cholestatic phenotype induced by common bile duct ligation (BDL) is reduced in mice genetically deficient in Ostalpha. Although Ostalpha(-/-) mice have a smaller bile acid pool size, which could explain lower serum and hepatic levels of bile acids after BDL, gallbladder bilirubin and urinary bile acid concentrations were significantly greater in Ostalpha(-/-) BDL mice, suggesting additional alternative adaptive responses. Livers of Ostalpha(-/-) mice had higher messenger RNA levels of constitutive androstane receptor (Car) than wild-type BDL mice and increased expression of Phase I enzymes (Cyp7a1, Cyp2b10, Cyp3a11), Phase II enzymes (Sult2a1, Ugt1a1), and Phase III transporters (Mrp2, Mrp3). Following BDL, the bile acid pool size increased in Ostalpha(-/-) mice and protein levels for the hepatic basolateral membrane export transporters, multidrug resistance-associated protein 3 (Mrp3) and Mrp4, and for the apical bilirubin transporter, Mrp2, were all increased. In the kidney of Ostalpha(-/-) mice after BDL, the apical bile acid uptake transporter Asbt is further reduced, whereas the apical export transporters Mrp2 and Mrp4 are increased, resulting in a significant increase in urinary bile acid excretion. These findings indicate that loss of Ostalpha provides protection from liver injury in obstructive cholestasis through adaptive responses in both the kidney and liver that enhance clearance of bile acids into urine and through detoxification pathways most likely mediated by the nuclear receptor Car.

Urinary bile acid sulfate levels in patients with hepatitis C virus‐related chronic liver diseases

Urinary bile acids are mainly conjugated with sulfuric acid, and urinary sulfated bile acid (USBA) levels in hepatobiliary diseases have been reported. However, the relationship between USBA and fasting serum total bile acid (TBA) has not been studied in hepatobiliary diseases. In the present study, we measured USBA levels in patients with hepatitis C virus-related chronic liver diseases, and the relationship between TBA and various laboratory tests was studied. USBA was measured using an automatic assay kit in 66 patients with chronic hepatitis and 28 patients with liver cirrhosis, and its relationship between TBA and various laboratory tests was studied. The median USBA level was 10.7 micromol/g creatinine in patients with chronic hepatitis and 41.1 micromol/g creatinine in liver cirrhosis (P = 0.000). More patients with chronic hepatitis had elevated USBA levels (61%) compared to TBA level (39%) (P = 0.002). USBA level was well correlated with TBA (r(s) = 0.680), and negatively correlated with albumin (r(s) = -0.488), prothrombin time (r(s) = -0.385) and platelet counts (r(s) = -0.394). In patients with liver cirrhosis, USBA was significantly elevated in Child-Pugh class B compared to Child-Pugh class A (P = 0.036). Although the metabolic pathways of USBA and TBA are different, these levels correlated very well, and USBA is considered to be a useful indicator of hepatic function like TBA in patients with chronic hepatitis C.

Diagnostic determination system for high‐risk screening for inborn errors of bile acid metabolism based on an analysis of urinary bile acids using gas chromatography–mass spectrometry: Results for 10 years in Japan

Some patients with cholestasis of unknown cause may have inborn errors of bile acid metabolism (IEBAM) thus causing abnormalities of bile acid biosynthesis. Although seven types of bile acid synthetic defects have thus far been reported for this disorder, no detailed information on its incidence and so on in Japan is yet available. In order to elucidate the current status of IEBAM in Japan, in July 1996 a diagnostic determination system was established for high-risk screening for IEBAM. Urinary bile acids were analyzed on gas chromatography-mass spectrometry (GC-MS) and quantitative analysis was done using selected ion monitoring (SIM). In a total of 576 samples analyzed over the 10 year period prior to June 2005, 159 patients were found with cholestasis of unknown etiology. Of these patients, 10 (6.3%) were found to have IEBAM by this system, while 91 (61.1%) had cholestasis without a definitive diagnosis. This diagnostic determination system with GC-MS of urinary bile acids is therefore considered useful for detecting IEBAM.

Oral Cholic Acid for Hereditary Defects of Primary Bile Acid Synthesis: A Safe and Effective Long-term Therapy

Oral bile acid replacement has been shown to be an effective therapy in primary bile acid synthesis defects, but to date there have been no reports of the long-term effects of this therapy. The aim of the study was to evaluate the long-term effectiveness and safety of cholic acid (CA) therapy. Fifteen patients with either 3beta-hydroxy-Delta(5)-C(27)-steroid oxidoreductase (3beta-HSD) (n = 13) or Delta(4)-3-oxosteroid 5beta-reductase (Delta(4)-3-oxo-R) (n = 2) deficiency confirmed by mass spectrometry and gene sequencing received oral CA and were followed up prospectively. CA therapy was started at a median age of 3.9 years (range, 0.3-13.1 years). The median follow-up with treatment was 12.4 years (range, 5.6-15 years). The mean daily dose of CA was initially 13 mg/kg and was 6 mg/kg at last evaluation. During CA therapy, physical examination findings, laboratory test results, and findings on sonography normalized. Mass spectrometry analysis of urine showed that excretion of the atypical metabolites was reduced by 500-fold and 30-fold in 3beta-HSD and Delta(4)-3-oxo-R deficiency, respectively, and total urinary bile acid excretion decreased dramatically. Liver biopsies performed in 14 patients after at least 5 years of CA therapy showed marked improvement, especially in patients with the 3beta-HSD deficiency. CA was well tolerated with all children developing normally, including 2 women having 4 normal pregnancies during treatment. Oral CA therapy is a safe and effective long-term treatment of the most common primary bile acid synthesis defects.

Chemical synthesis of 3β-sulfooxy-7β-hydroxy-24-nor-5-cholenoic acid: An internal standard for mass spectrometric analysis of the abnormal Δ 5-bile acids occurring in Niemann-Pick disease

In Niemann-Pick disease, type C1, increased amounts of 3β,7β-dihydroxy-5-cholenoic acid are reported to be present in urinary bile acids. The compound occurs as a tri-conjugate, sulfated at C-3, N-acetylglucosamidated at C-7, and N-acylamidated with taurine or glycine at C-24. For sensitive LC–MS/MS analysis of this bile acid, a suitable internal standard is needed. We report here the synthesis of a satisfactory internal standard, 3β-sulfooxy-7β-hydroxy-24-nor-5-cholenoic acid (as the disodium salt). The key reactions involved were (1) the so-called “second order” Beckmann rearrangement (one-carbon degradation at C-24) of hyodeoxycholic acid (HDCA) 3,6-diformate with sodium nitrite in a mixture of trifluoroacetic anhydride and trifluoroacetic acid, (2) simultaneous inversion at C-3 and elimination at C-6 of the ditosylate derivatives of the resulting 3α,6α-dihydroxy-24-nor-5β-cholanoic acid with potassium acetate in aqueous N, N-dimethylformamide, and (3) regioselective sulfation at C-3 of an intermediary 3β,7β-dihydroxy-24-nor-Δ 5 derivative using sulfur trioxide–trimethylamine complex. Overall yield of the desired compound was 1.8% in 12 steps from HDCA.

Efficacy of urine bile acid as a non-invasive indicator of liver damage in rats

Estimation of liver damage is important in the pathophysiological and toxicological study of liver disease. As a novel, non-invasive marker of liver damage, we studied the efficacy of urine bile acids (UBA) in a rat model of liver disease. Thioacetamide (TAA)-treated rats were used in this study. Single intraperitoneal administration of high-dose TAA induces severe damage to the liver, and thus is used as a model of acute hepatitis. Continuous administration of low-dose TAA yields mild damage to the liver, and induces cirrhosis and hepatic tumors. In this study, it was found that both acute and chronic administration of TAA was associated with a dose-dependent elevation of UBA. The elevation of UBA content correlated with the alteration of blood biochemical indicators, and UBA screening showed a remarkable ability to distinguish liver-damaged rats from healthy rats. In particular, UBA analysis was found to have high sensitivity, specificity, and positive predictive value for the screening of rats with abnormal serum alkaline phosphatase (ALP) activity due to chronic liver damage, which was confirmed to include cholestasis and subsequent cirrhosis by liver histological analysis. In conclusion, we demonstrated that measurement of UBA is a simple, non-invasive and effective method for the screening of cholestasis in TAA-treated rats. We suggest that UBA analysis may have potent applicability for monitoring the progress of liver damage in animal models of chronic liver disease, such as cirrhosis and hepatic encephalopathy.

Foetal ‘flat’ bile acids reappear during human liver regeneration after surgery

Changes in bile acid (BA) pool, such as the reappearance of typically foetal-type molecular species with a 'flat' structure at the steroid ring, occur during hepatocarcinogenesis, both in humans and rodents. Moreover flat-BAs also appear during rat liver regeneration. These changes can be detected in urine. The aim of the present study was to investigate whether flat-BAs also reappear during human liver regeneration, and whether this change correlates with the magnitude of liver resection. Patients undergoing partial hepatectomy were divided in two groups: major hepatectomy group (> 50% of hepatic tissue resection, n = 17) and minor hepatectomy group (< 50%, n = 13). BAs were extracted from serum and urine (collected over 24 h) and analysed by gas chromatography-mass spectrometry. Samples were obtained before surgery (day 0) and on the third and seventh days after hepatectomy. In serum, total BAs significantly increased on day seven after hepatectomy, but only a moderate increase in flat-BA concentrations was observed. By contrast, urinary excretion of total as well as flat-BAs significantly increased at day three and day seven after hepatectomy. Moreover, the amount of flat-BAs excreted in urine during the first week after partial hepatectomy correlated with the magnitude of the resection. Urinary BA output increases and flat-BAs reappear in urine during human liver regeneration. These results suggest that determination of BAs in urine may be an interesting parameter obtained by non-invasive techniques whose actual clinical value during human liver regeneration warrants further evaluation.

Immobilized enzymatic fluorescence capillary biosensor for determination of sulfated bile acid in urine

The authors have proposed an immobilized enzymatic fluorescence capillary biosensor (SBAs-IE-FCBS) for the determination of sulfated bile acids (SBAs). The reaction principle of the biosensor is that under the catalysis of the bile acid sulfate sulfatase (BSS) and β-hydroxysteroid dehydrogenase (β-HSD) immobilized on inner surface of a medical capillary, SBAs desulfates to 3β-hydroxyl bile acids, then the latter reacts with nicotinamide adenine dinucleotide (NAD +), and is converted into 3-ketosteroid; meanwhile, NAD + is converted to reduced nicotinamide adenine dinucleotide (NADH). NADH continuously reacts with 1-methoxy-5-methylphenazinium methyl sulfate (1-MPMS) and is converted into NAD + circularly and 1-MPMSH 2. Finally resazurin is reduced into resorufin by 1-MPMSH 2, the formed resorufin ( λ ex/ λ em: 540 nm/580 nm) is used for quantifying the concentration of SBAs. Optimized conditions being suitable with the biosensor are as follows: the concentrations of BSS and β-HSD used for the immobilization all are 5 kU L −1; the concentrations of 1-MPMS and resazurin all are 25 μmol L −1; the concentrations of Tris–HCl buffer and NAD + are 100 and 400 μmol L −1, respectively; total volume of the enzyme, reagent and sample is only 18 μL per time for determining; the reaction temperature is 37 °C; the reaction time is 15 min. The concentration of SBAs is directly proportional to the fluorescence intensity of the biosensor measured from 0.5 to 5.0 μmol L −1. The relative standard deviation is less than 3.4%, and the detection limit was 0.16 μmol L −1. The recoveries are in the range 95.5–106%. This SBA-IE-FCBS can be used for quantifying SBAs in urine to diagnose and judge hepatobiliary diseases, etc.

Current concept about postoperative cholangitis in biliary atresia

Postoperative cholangitis characterized by fever and acholic stool and positive blood culture is a common and serious complication following Kasai's operation for biliary atresia. The aim of this review was to describe the pathogenesis, clinical manifestations, medical treatment and outcome of postoperative cholangitis. Articles on biliary atresia retrieved from Pubmed and MEDLINE in the recent 10 years were reviewed. The pathogenesis of postoperative cholangitis is still controversial. Recent methods for the diagnosis of postoperative cholangitis include urinary sulfated bile acids (USBA) and magnetic resonance cholangio-pancreaticography (MRCP). High-dose steroids and oral antibiotics have been used to reduce the incidence of postoperative cholangitis, and recurrent cholangitis leads to a lower survival rate. Cholangitis is one of the most important determinants of long-term survival after the Kasai's procedure. The knowledge on postoperative cholangitis has been increasing in the past 10 years, showing a lower incidence of the disease and better therapeutic results.

Ontogenic development-associated changes in the expression of genes involved in rat bile acid homeostasis

Ontogenic changes in the rat bile acid (BA) pool, measured enzymatically and by GC-MS, and expression of enzymes (5alpha-reductase, 5beta-reductase, and cytochrome P450 enzymes Cyp7a1, Cyp8b1, Cyp27 and Cyp3a11), transporters [bile salt export pump, sodium taurocholate-cotransporting polypeptide, apical sodium-dependent bile acid transporter, and organic solute transporter alpha/beta (Ostalpha/Ostbeta)], and nuclear receptors [fetoprotein transcription factor (Ftf), farnesoid X receptor (Fxr), small heterodimer partner (Shp), and hepatic nuclear factor 4alpha (HNF-4alpha)], determined by quantitative PCR, were investigated. The absolute size of the BA pool increased progressively up to adulthood, whereas the complexity of its composition was high in fetuses, decreased after birth, increased again progressively up to adulthood, and decreased in aged animals. Allo-cholic acid only appeared early in development, in spite of low 5alpha-reductase expression. The relative size of the BA pool, corrected by liver weight, was maintained from 1 week after birth, except at weaning, when a transient peak accompanied by Shp downregulation and Cyp7a1 upregulation was observed. An imposed weaning delay of 1 week had no effect on the time course of the BA pool size but decreased the proportion of chenodeoxycholic and alpha-muricholic acids, whereas the proportion of cholic acid was increased, probably as a result of Cyp8b1 upregulation. In conclusion, changes in the expression of genes involved in BA homeostasis may play a role in physiological adaptations to digestive functions during the rat life span.

Intrahepatic cholestasis of pregnancy: detection with urinary bile acid assays

To determine sensitivity and specificity of urine bile acid sulfate (UBAS) and non-sulfated urine bile acid (UBA) assays for detection of intrahepatic cholestasis of pregnancy (ICP). Urine was collected prospectively from healthy and pruritic patients evaluated for ICP. Results were expressed as a ratio to urinary creatinine. Of 20 patients evaluated for ICP, 14 had confirmed ICP by serum testing and six had pruritus only. UBAS results were higher with ICP (P<0.001) and increased with gestational age in healthy controls. Adjusted for gestational age, the multiples of the median (MoM) were still higher (2.64+/-1.11, 1.05+/-0.54, P<0.001). The sensitivity was 100% and specificity 83% at 1.5 MoM in pruritic patients. UBA results were higher with ICP (23.0+/-9.8, 12.8+/-7.4, P<0.001), with sensitivity of 100% and specificity of 50% at 10.2 micromol/g cretinine. Urinary bile acids can detect or exclude ICP with serum abnormalities. Urine bile acid sulfates have higher specificity than non-sulfated urine bile acids at equivalent sensitivities, but co-detection of progestin sulfates is suspected.

Random urinary bile acids as a diagnostic method for intrahepatic cholestasis of pregnancy (ICP)

Random urinary bile acids as a diagnostic method for intrahepatic cholestasis of pregnancy (ICP)

Defects in Bile Acid Biosynthesis‐Diagnosis and Treatment

Bile acid synthetic defects represent a specific category of metabolic liver disease. This article highlights the history and summarizes our analytical approach to the diagnosis and treatment of genetic defects in bile acid synthesis. By the application of mass spectrometry as a screening tool, it is possible to perform rapid diagnosis of potential inborn errors in bile acid synthesis from urinary bile acid analysis. Molecular techniques then afford the identification of specific mutations in genes encoding the enzymes responsible for bile acid synthesis. Using this approach, 6 of the 7 known genetic defects that are causes of progressive cholestatic liver disease, syndromes of fat-soluble vitamin malabsorption, or neurological disease, have been characterized. Bile-acid therapy using oral cholic acid has proven effective in most of these bile acid synthetic defects making early diagnosis crucial to optimum clinical prognosis.