Analysis of eight bile acids in urine of gastric cancer patients based on covalent organic framework enrichment coupled with liquid chromatography-tandem mass spectrometry.

Gastric carcinoma is one of the most common and deadly forms of cancer. Early detection is critical for successful treatment of gastric cancer, and examination of BAs in urine may provide a critical diagnostic tool for identifying gastric cancer at stages when it can still be cured. Bile acids (BAs) are a crucial toxic factor correlated with the injury of gastric mucosa and as such, quantifying the amount of BA in patient's urine could provide a new means to quickly and non-invasively identify the presence of gastric cancer in the early stages. Here, a covalent organic framework (COF) material synthesized on the basis of 1,3,5-tris(4-nitrophenyl)benzene (TAPB) and pyromellitic dianhydride (PMDA) was used as stationary phase for SPE column that was coupled to LC-MS/MS for quantitative analysis of eight BAs in human urine, including cholic acid (CA), deoxycholic acid (DCA), glycochenodeoxycholic acid (GCDCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), lithocholic acid (LCA), hyodeoxycholic acid (HDCA), and chenodeoxycholic acid (CDCA). The enrichment effect of synthesized COF material was better than commercial SPE and HLB column. The sensitivity can increase 9.37- to 54.30- fold (calculated by the ratio of peak area between before and after enrichment). The probable mechanism is due to the great porosity and the similar polarity with BAs of the COF material. By compared with previous literatures, our method had the minimum limit of detection, which achieved 46.40, 25.75, 47.40, 47.37, 30.42, and 33.92 pg /mL, respectively, for GCA, GCDCA, CA, CDCA, HDCA and DCA after enrichment. These eight BAs also accomplished excellent linearity from 0.34 to 10,000 ng/mL. This material was successfully applied in the measurements of these six BAs in human urine from 76 gastric cancer patients and 32 healthy people. Compared to healthy people, levels of CA, CDCA, DCA, and HDCA were significantly elevated and levels of GCDCA were depressed, respectively, in gastric cancer patients. Our work suggests that these acids may act as potential biomarkers for gastric cancer and our framework provides a method for "non-invasive" diagnosis of gastric cancer.

Urinary metabolic insights into host-gut microbial interactions in healthy and IBD children.

AIM: To identify metabolic signatures in urine samples from healthy and inflammatory bowel disease (IBD) children. METHODS: We applied liquid chromatography and gas chromatography coupled to targeted mass spectrometry (MS)-based metabolite profiling to identify and quantify bile acids and host-gut microbial metabolites in urine samples collected from 21 pediatric IBD patients monitored three times over one year (baseline, 6 and 12 mo), and 27 age- and gender-matched healthy children. RESULTS: urinary metabolic profiles of IBD children differ significantly from healthy controls. Such metabolic differences encompass central energy metabolism, amino acids, bile acids and gut microbial metabolites. In particular, levels of pyroglutamic acid, glutamic acid, glycine and cysteine, were significantly higher in IBD children in the course of the study. This suggests that glutathione cannot be optimally synthesized and replenished. Whilst alterations of the enterohepatic circulation of bile acids in pediatric IBD patients is known, we show here that non-invasive urinary bile acid profiling can assess those altered hepatic and intestinal barrier dysfunctions. CONCLUSION: The present study shows how non-invasive sampling of urine followed by targeted MS-based metabonomic analysis can elucidate and monitor the metabolic status of children with different GI health/disease status.

NorUrsodeoxycholic acid ameliorates cholemic nephropathy in bile duct ligated mice.

BACKGROUND & AIMS: Severe cholestasis may cause cholemic nephropathy that can be modeled in common bile duct ligated (CBDL) mice. We aimed to explore the therapeutic efficacy and mechanisms of norursodeoxycholic acid (norUDCA) in cholemic nephropathy. METHODS: In 8-week CBDL mice fed with norUDCA (prior or post CBDL) or chow we evaluated serum urea levels, urine cytology and urinary neutrophil gelatinase associated lipocalin (uNGAL), kidney and liver tissue quantification of fibrosis by hydroxyproline content and gene chip expression looking at key genes of inflammation and fibrosis. Moreover, we comprehensively analysed bile acid profiles in liver, kidney, serum and urine samples. RESULTS: NorUDCA-fed CBDL mice had significantly lower serum urea and uNGAL levels and less severe cholemic nephropathy as demonstrated by normal urine cytology, significantly reduced tubulointerstitial nephritis, and renal fibrosis as compared to controls. NorUDCA underwent extensive metabolism to produce even more hydrophilic compounds that were significantly enriched in kidneys. CONCLUSION: NorUDCA ameliorates cholemic nephropathy due to the formation of highly hydrophilic metabolites enriched in kidney. Consequently, norUDCA may represent a medical treatment for cholemic nephropathy. LAY SUMMARY: The term cholemic nephropathy describes renal dysfunction together with characteristic morphological alterations of the kidney in obstructive cholestasis that can be mimicked by ligation of the common bile duct in mice. Feeding the hydrophilic bile acid norUDCA to bile duct ligated mice leads to a significant amelioration of the renal phenotype due to the formation of highly hydrophilic metabolites enriched in the kidney and may therefore represent a medical treatment for cholemic nephropathy.

Prognostic roles of tetrahydroxy bile acids in infantile intrahepatic cholestasis.

Tetrahydroxy bile acids (THBAs) are hydrophilic and are present at minimal or undetectable levels in healthy human adults, but are present at high levels in bile salt export pump (abcb11)-knockout mice. The roles of THBAs in human cholestatic diseases are unclear. We aimed to investigate the presence of THBAs in patients with infantile intrahepatic cholestasis and its correlation with outcome. Urinary bile acids (BAs) were analyzed by GC-MS. Data were compared between good (n = 21) (disease-free before 1 year old) and poor prognosis groups (n = 19). Good prognosis patients had a higher urinary THBA proportion than poor prognosis patients [25.89% (3.45-76.73%) vs. 1.93% (0.05-48.90%)]. A urinary THBA proportion >7.23% predicted good prognosis with high sensitivity (95.24%), specificity (84.21%), and area under the curve (0.91) (P < 0.0001). A THBA proportion 7.23% was an independent factor for decreased transplant-free survival (hazard ratio = 7.16, confidence interval: 1.24-41.31, P = 0.028). Patients with a confirmed ABCB11 or tight junction protein 2 gene mutation (n = 7) had a minimally detectable THBA proportion (0.23-2.99% of total BAs). Three patients with an ATP8B1 mutation had an elevated THBA proportion (7.51-37.26%). In conclusion, in addition to disease entity as a major determinant of outcome, a high THBA level was associated with good outcome in the infantile intrahepatic cholestasis patients.

Liver disease in infancy caused by oxysterol 7 α-hydroxylase deficiency: successful treatment with chenodeoxycholic acid.

A child of consanguineous parents of Pakistani origin developed jaundice at 5 weeks and then, at 3 months, irritability, a prolonged prothrombin time, a low albumin, and episodes of hypoglycaemia. Investigation showed an elevated alanine aminotransferase with a normal γ-glutamyl-transpeptidase. Analysis of urine by electrospray ionisation tandem mass spectrometry (ESI-MS/MS) showed that the major peaks were m/z 480 (taurine-conjugated 3ß-hydroxy-5-cholenoic acid) and m/z 453 (sulphated 3ß-hydroxy-5-cholenoic acid). Analysis of plasma by gas chromatography-mass spectrometry (GC-MS) showed increased concentrations of 3ß-hydroxy-5-cholenoic acid, 3ß-hydroxy-5-cholestenoic acid and 27-hydroxycholesterol, indicating oxysterol 7 α-hydroxylase deficiency. The patient was homozygous for a mutation (c.1249C>T) in CYP7B1 that alters a highly conserved residue in oxysterol 7 α-hydroxylase (p.R417C) - previously reported in a family with hereditary spastic paraplegia type 5. On treatment with ursodeoxycholic acid (UDCA), his condition was worsening, but on chenodeoxycholic acid (CDCA), 15 mg/kg/d, he improved rapidly. A biopsy (after 2 weeks on CDCA), showed a giant cell hepatitis, an evolving micronodular cirrhosis, and steatosis. The improvement in liver function on CDCA was associated with a drop in the plasma concentrations and urinary excretions of the 3ß-hydroxy-Δ5 bile acids which are considered hepatotoxic. At age 5 years (on CDCA, 6 mg/kg/d), he was thriving with normal liver function. Neurological development was normal apart from a tendency to trip. Examination revealed pes cavus but no upper motor neuron signs. The findings in this case suggest that CDCA can reduce the activity of cholesterol 27-hydroxylase - the first step in the acidic pathway for bile acid synthesis.

Role of glucuronidation for hepatic detoxification and urinary elimination of toxic bile acids during biliary obstruction.

Biliary obstruction, a severe cholestatic condition, results in a huge accumulation of toxic bile acids (BA) in the liver. Glucuronidation, a conjugation reaction, is thought to protect the liver by both reducing hepatic BA toxicity and increasing their urinary elimination. The present study evaluates the contribution of each process in the overall BA detoxification by glucuronidation. Glucuronide (G), glycine, taurine conjugates, and unconjugated BAs were quantified in pre- and post-biliary stenting urine samples from 12 patients with biliary obstruction, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The same LC-MS/MS procedure was used to quantify intra- and extracellular BA-G in Hepatoma HepG2 cells. Bile acid-induced toxicity in HepG2 cells was evaluated using MTS reduction, caspase-3 and flow cytometry assays. When compared to post-treatment samples, pre-stenting urines were enriched in glucuronide-, taurine- and glycine-conjugated BAs. Biliary stenting increased the relative BA-G abundance in the urinary BA pool, and reduced the proportion of taurine- and glycine-conjugates. Lithocholic, deoxycholic and chenodeoxycholic acids were the most cytotoxic and pro-apoptotic/necrotic BAs for HepG2 cells. Other species, such as the cholic, hyocholic and hyodeoxycholic acids were nontoxic. All BA-G assayed were less toxic and displayed lower pro-apoptotic/necrotic effects than their unconjugated precursors, even if they were able to penetrate into HepG2 cells. Under severe cholestatic conditions, urinary excretion favors the elimination of amidated BAs, while glucuronidation allows the conversion of cytotoxic BAs into nontoxic derivatives.

Loss of A(1) adenosine receptor attenuates alpha-naphthylisothiocyanate-induced cholestatic liver injury in mice.

Cholestasis has limited therapeutic options and is associated with high morbidity and mortality. The A(1) adenosine receptor (A(1)AR) was postulated to participate in the pathogenesis of hepatic fibrosis induced by experimental extrahepatic cholestasis; however, the contribution of A(1)AR to intrahepatic cholestatic liver injury remains unknown. Here, we found that mice lacking A(1)AR were resistant to alpha-naphthyl isothiocyanate (ANIT)-induced liver injury, as evidenced by lower serum liver enzyme levels and reduced extent of histological necrosis. Bile acid accumulation in liver and serum was markedly diminished in A(1)AR(-/-) mice compared with wild-type (WT) mice. However, biliary and urinary outputs of bile acids were significantly enhanced in A(1)AR(-/-) mice. In the liver, mRNA expression of genes related to bile acid transport (Bsep and Mdr2) and hydroxylation (Cyp3a11) was increased in A(1)AR(-/-) mice. In the kidney, A(1)AR deficiency prevented the decrease of glomerular filtration rate caused by ANIT. Treatment of WT mice with A(1)AR antagonist DPCPX also protected against ANIT hepatotoxicity. Our results indicated that lack of A(1)AR gene protects mice from ANIT-induced cholestasis by enhancing toxic biliary constituents efflux through biliary excretory route and renal elimination system and suggested a potential role of A(1)AR as therapeutic target for the treatment of intrahepatic cholestasis.

Differential diagnosis in patients with suspected bile acid synthesis defects.

AIM: To investigate the clinical presentations associated with bile acid synthesis defects and to describe identification of individual disorders and diagnostic pitfalls. METHODS: Authors describe semiquantitative determination of 16 urinary bile acid metabolites by electrospray ionization-tandem mass spectrometry. Sample preparation was performed by solid-phase extraction. The total analysis time was 2 min per sample. Authors determined bile acid metabolites in 363 patients with suspected defects in bile acid metabolism. RESULTS: Abnormal bile acid metabolites were found in 36 patients. Two patients had bile acid synthesis defects but presented with atypical presentations. In 2 other patients who were later shown to be affected by biliary atresia and cystic fibrosis the profile of bile acid metabolites was initially suggestive of a bile acid synthesis defect. Three adult patients suffered from cerebrotendinous xanthomatosis. Nineteen patients had peroxisomal disorders, and 10 patients had cholestatic hepatopathy of other cause. CONCLUSION: Screening for urinary cholanoids should be done in every infant with cholestatic hepatopathy as well as in children with progressive neurological disease to provide specific therapy.

Serum and urine metabolite profiling reveals potential biomarkers of human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common malignancy in the world with high morbidity and mortality rate. Identification of novel biomarkers in HCC remains impeded primarily because of the heterogeneity of the disease in clinical presentations as well as the pathophysiological variations derived from underlying conditions such as cirrhosis and steatohepatitis. The aim of this study is to search for potential metabolite biomarkers of human HCC using serum and urine metabolomics approach. Sera and urine samples were collected from patients with HCC (n = 82), benign liver tumor patients (n = 24), and healthy controls (n = 71). Metabolite profiling was performed by gas chromatography time-of-flight mass spectrometry and ultra performance liquid chromatography-quadrupole time of flight mass spectrometry in conjunction with univariate and multivariate statistical analyses. Forty three serum metabolites and 31 urinary metabolites were identified in HCC patients involving several key metabolic pathways such as bile acids, free fatty acids, glycolysis, urea cycle, and methionine metabolism. Differentially expressed metabolites in HCC subjects, such as bile acids, histidine, and inosine are of great statistical significance and high fold changes, which warrant further validation as potential biomarkers for HCC. However, alterations of several bile acids seem to be affected by the condition of liver cirrhosis and hepatitis. Quantitative measurement and comparison of seven bile acids among benign liver tumor patients with liver cirrhosis and hepatitis, HCC patients with liver cirrhosis and hepatitis, HCC patients without liver cirrhosis and hepatitis, and healthy controls revealed that the abnormal levels of glycochenodeoxycholic acid, glycocholic acid, taurocholic acid, and chenodeoxycholic acid are associated with liver cirrhosis and hepatitis. HCC patients with alpha fetoprotein values lower than 20 ng/ml was successfully differentiated from healthy controls with an accuracy of 100% using a panel of metabolite markers. Our work shows that metabolomic profiling approach is a promising screening tool for the diagnosis and stratification of HCC patients.

Clinical study of urinary sulfated bile acids in patients with hepatitis C virus.

BACKGROUND/AIMS: Sulfation is considered to be a detoxifying mechanism for bile acids, which allows the sulfated bile acids to be eliminated in the urine (urinary sulfated bile acids: USBA) by enhancing renal clearance when serum bile acids increase due to hepatobiliary disease. We measured urinary sulfated bile acids in patients positive for hepatitis C virus and studied the relation between USBA and standard liver function tests. METHODOLOGY: Seventy-eight outpatients could be followed for at least one year were enrolled in this study. Blood and urine samples were simultaneously obtained from the patients. The measured urinary sulfated bile acids levels were adjusted for the urinary creatinine concentration and the normal value was defined <8 micromol/g creatinine. RESULTS: Among the 78 patients, the mean USBA level of 26 carriers, 37 patients with chronic hepatitis C, 9 with liver cirrhosis, and 6 with hepatocellular carcinoma were 6.0 +/- 6.1 micromol/g creatinine (mean +/- SD), 27.9 +/- 35.8, 33.1+/- 22.7, and 50.6 +/- 47.3, respectively. The mean urinary sulfated bile acids level was correlated with the clinical progression of these liver diseases. CONCLUSIONS: Urinary sulfated bile acids are considered to be a useful diagnostic indicator of impaired liver function, and can also be used to assess the severity of hepatic disease.

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.

BACKGROUND: 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. METHODS: Urinary bile acids were analyzed on gas chromatography-mass spectrometry (GC-MS) and quantitative analysis was done using selected ion monitoring (SIM). RESULTS AND CONCLUSIONS: 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.

Clinical significance of measuring urinary sulfated bile acids in adult patients with hepatobiliary diseases.

BACKGROUND/AIMS: Measurement of urinary sulfated bile acid (USBA) level is a simple urine test that reflects the degree of cholestasis in newborns. The aim of this study was to clarify the clinical significances of this test for liver diseases in adults. METHODOLOGY: We examined the relationship between USBA level in a urine sample by enzymatic assay and clinical parameters and postoperative complications in 27 patients with hepatobiliary diseases who underwent surgical procedures between 2002 and 2007. RESULTS: Mean USBA in all patients before surgery was 39.8 +/- 64.0 micromol/L (median value was 6.6). USBA level was increased in patients with cholestasis. USBA level was significantly correlated with serum total bile acid, total bilirubin level and serum hyaluronic acid level (r = 0.850, 0.602 and 0.504, respectively) (p < 0.05) and, furthermore, tended to be correlated with liver-uptake ratio (LHL15) by technetium-99m galactosyl human serum albumin (99SmTc-GSA) scintigraphy and alanine aminotransferase level (r = -0.469 and 0.436, respectively but not significant). USBA level tended to be associated with postoperative uncontrolled ascites (p = 0.050, not significant). Postoperative USBA level by day 7 was not changed; however, USBA level in patients with cholestatic diseases was decreased. CONCLUSIONS: USBA is a simple and sensitive noninvasive test for cholestasis and also useful to predict postoperative uncontrolled ascites after hepatic resections.

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.

Efficacy of urinary sulfated bile acids for diagnosis of bacterial cholangitis in biliary atresia.

This study evaluated the efficacy of measuring urinary sulfated bile acids (USBA) for diagnosis of bacterial cholangitis in patients with biliary atresia. Eight infants with biliary atresia were recruited. The USBA level was measured when they were admitted to hospital with a fever of unknown origin. Clinical manifestations and laboratory data were reviewed. The standard USBA value for each patient (S-USBA) was defined as the level measured when they had no fever, and USBA ratio (R-USBA) was calculated as the USBA level during the febrile episode divided by the S-USBA. Then R-USBA values were compared between febrile episodes with and without cholangitis to assess the diagnostic ability of USBA. Twenty-three febrile episodes occurred in eight patients during a 15-month period. Nine episodes were diagnosed as being due to cholangitis, five were due to non-cholangitis, and nine were of undetermined origin. The R-USBA value ranged from 1.5 to 15.4 during cholangitis episodes and from 0.4 to 1.2 during non-cholangitis febrile episodes. When fever was of undetermined origin, R-USBA was found to be increased during some episodes and not in others. USBA increased immediately in patients with cholangitis. The measurement of USBA is a useful non-invasive test for cholangitis in patients with biliary atresia who had undergone Kasai's operation.

Are bile acids involved in the renal dysfunction of obstructive jaundice? An experimental study in bile duct ligated rats.

BACKGROUND: Surgery on patients with obstructive jaundice is associated with a significant risk of postoperative renal failure. Bile acids are implicated as nephrotoxins because they accumulate in the plasma and the kidney becomes their only excretory route in cholestasis. The experimental evidence favoring this proposal is inadequate and unconvincing. Therefore, we designed an animal experiment involving bile duct ligated (BDL) rats in which we could correlate variations in serum and urine bile acids with indices of nephrotoxicity and renal function. HYPOTHESIS: Bile acids are putative nephrotoxins. MATERIALS AND METHODS: Total serum and urine bile acid concentrations and profiles were determined using liquid chromatography/gas chromatography/mass spectrometry selected ion monitoring. Nephrotoxicity was assessed by renal histopathology and by determination of the urinary activities of the following enzymes: muramidase, glutamate dehydrogenase, alkaline phosphatase, N-acetyl-beta-D-glucosaminidase, and lactate dehydrogenase. Renal function was assessed by measuring urine osmolality, daily osmolar excretion, sodium excretion (U(Na)V), potassium excretion (U(K)V), and total protein and albumin excretion. RESULTS: Maximum plasma concentrations and renal clearance of bile acids occurred between the third or fourth postoperative day following BDL. This peak coincided with maximal disruption of proximal convoluted tubule architecture and postoperative changes in renal function-increased urine flow rate and decreases in urine osmolality and sodium excretion. Thereafter, 1) plasma levels of bile acids returned toward normal levels, 2) urinary bile acid clearance declined, 3) normal renal histology was restored, and 4) normal renal function was reestablished. Throughout this period, fluctuations in enzymuria were evident. However, these shifts did not coincide with plasma and urine bile acid concentrations and histological and functional changes. DISCUSSION AND CONCLUSIONS: Transient functional impairment of renal cation and water transport and nonspecific morphological changes in the proximal convoluted tubule occur 3 to 4 days following bile duct ligation in rats. These functional and morphological changes occurred when plasma total and urinary bile acids were at their peaks. Although it is tempting to equate association with causality, we cannot implicate bile acids as being responsible for the aberrations in renal function and structure following BDL. Accordingly, we have concluded that elevated plasma concentrations of bile acids are renal exacerbates acting in concert with other factors, be they prerenal or renal in origin to precipitate a cascade of events leading to postoperative renal failure in cholestasis.

Multidrug resistance-associated protein 4 is up-regulated in liver but down-regulated in kidney in obstructive cholestasis in the rat.

BACKGROUND/AIMS: Multidrug resistance-associated protein 4 (Mrp4, ABCC4) transports cyclic nucleotides, anti-retroviral compounds, and sulfated bile acids. Mrp4 expression is increased in farnesyl/bile acid receptor knockout mice. Our aim was to investigate Mrp4 expression and function in rat liver and kidney in obstructive cholestasis. METHODS: Male Sprague-Dawley rats were subjected to bile duct ligation (BDL) or sham-surgery. Animals were sacrificed after 3, 7, and 14 days and tissues were harvested for Western blot analysis, real-time reverse transcriptase-mediated polymerase chain reaction (RT-PCR), and immunohistochemistry. RESULTS: Western blot analysis revealed a progressive, more than seven-fold increase (P < 0.05) of Mrp4 expression in cholestatic livers, 14 days after BDL. In contrast, Mrp4 in 14-day BDL kidneys decreased to 26+/-4% of controls (P < 0.005). Immunohistochemistry localized Mrp4 to the basolateral hepatocyte membrane and corroborated its hepatic up-regulation after BDL. Real-time RT-PCR demonstrated no major changes of Mrp4 mRNA levels in liver and kidney after BDL. Cyclic adenosine monophosphate, an MRP4 substrate, was increased in plasma and urine, consistent with these findings. CONCLUSIONS: Obstructive cholestasis in rats results in progressive up-regulation of Mrp4 protein in liver but down-regulation in kidney. The absence of corresponding changes in Mrp4 mRNA suggests posttranscriptional mechanisms as predominant regulators of Mrp4 expression in BDL rats.

Urinary bile acid profile in children with inborn errors of bile acid metabolism and chronic cholestasis; screening technique using electrospray tandem mass-spectrometry (ES/MS/MS).

BACKGROUND: It is well known that urine becomes the major route for bile acid excretion in liver diseases and thus we examined bile acid profile in urine obtained from normal children and children having chronic liver diseases using electrospray tandem mass spectrometry (ES/MS/MS). MATERIAL/METHODS: Bile acid were extracted from 5 ml of urine obtained from five healthy children or from twenty patients with various liver diseases including patients with unknown chronic liver diseases, Zellweger syndrome, peroxisomal bifunctional protein deficiency disease, tyrosinema type 1, biliary atresia, and patients with progressive familial intrahepatic cholestasis (PFIC) of undetermined type. Identification and quantification of bile acids were achieved in 5 minutes using electrospray tandem mass spectrometry (ES/MS/MS). RESULTS: Urinary bile acid excretion increased in liver diseases an average of 100 times as compared to control values. There was a specific profile for different liver disease which confirms the pathology of the disease and could be used for its diagnosis. The results also show that the ions used for the diagnosis of oxo-steroid reductase deficiency disease were present in other chronic liver diseases suggesting that these atypical bile acids may not be a result of an inborn error of bile acid metabolism. CONCLUSIONS: The urinary bile acid profile obtained in this study by ES/MS/MS can be of use for the diagnosis of certain chronic liver diseases.

Liver disease caused by failure to racemize trihydroxycholestanoic acid: gene mutation and effect of bile acid therapy.

BACKGROUND & AIMS: Inborn errors of bile acid metabolism may present as neonatal cholestasis and fat-soluble vitamin malabsorption or as late onset chronic liver disease. Our aim was to fully characterize a defect in bile acid synthesis in a 2-week-old African-American girl presenting with coagulopathy, vitamin D and E deficiencies, and mild cholestasis and in her sibling, whose liver had been used for orthotopic liver transplantation (OLT). METHODS: Bile acids were measured by mass spectrometry in urine, bile, serum, and feces of the patient and in urine from the unrelated recipient. RESULTS: Liver biopsy specimens showed neonatal hepatitis with giant cell transformation and hepatocyte necrosis; peroxisomes were reduced in number. High concentrations of (25R)3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid in the urine, bile, and serum established a pattern similar to that of Zellweger syndrome and identical to the Alligator mississippiensis. Serum phytanic acid was normal, whereas pristanic acid was markedly elevated. Biochemical, MRI, and neurologic findings were inconsistent with a generalized defect of peroxisomal function and were unique. Analysis of the urine from the recipient of the deceased sibling's liver confirmed the same bile acid synthetic defect. A deficiency in 2-methylacyl-CoA racemase, which is essential for conversion of (25R)THCA to its 25S-isomer, the substrate to initiate peroxisomal beta-oxidation to primary bile acids, was confirmed by DNA analysis revealing a missense mutation (S52P) in the gene encoding this enzyme. Long-term treatment with cholic acid normalized liver enzymes and prevented progression of symptoms. CONCLUSIONS: This genetic defect further highlights bile acid synthetic defects as a cause of neonatal cholestasis.