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BACKGROUND AND AIM The onset and progression of chronic liver disease (CLD) is a multistage process spanning years or several decades. Some bile acid (BA) features are identified as indicators for CLD progression. However, BAs are highly influenced by various factors and are stage and/or population specific. Emerging evidences demonstrated the association of structure of conjugated BAs and CLD progression. Here, we aimed to investigate the alteration of conjugated BAs and identify new features for CLD progression. METHODS Based on liquid chromatography-mass spectrometry platform, 15 BAs were quantified in 1883 participants including healthy controls and CLD patients (non-alcoholic fatty liver [NAFL], non-alcoholic steatohepatitis [NASH], fibrosis, cirrhosis, and three types of liver cancer). Logistic regression was used to construct diagnostic models. Model performances were evaluated in discovery and test sets by area under the receiver operating characteristic curve, sensitivity, specificity, accuracy, and kappa index. RESULTS Five BA glycine : taurine ratios were calculated, and glycocholic acid/taurocholic acid, glycodeoxycholic acid/taurodeoxycholic acid, and glycochenodeoxycholic acid/taurochenocholic acid were identified as candidates. Three diagnostic models were constructed for the differentiation of healthy control and early CLD (NAFL + NASH), early and advanced CLD (fibrosis + cirrhosis + liver cancer), and NAFL and NASH, respectively. The areas under the receiver operating characteristic curve of the models ranged from 0.91 to 0.97. The addition of age and gender improved model performances further. The alterations of the candidates and the performances of the diagnostic models were successfully validated by independent test sets (n = 291). CONCLUSIONS Our findings revealed stage-specific BA perturbation patterns and provided new biomarkers and tools for the monitoring of liver disease progression.

BACKGROUND: Ghrelin may influence several alcohol-related behaviors in animals and humans by modulating central and/or peripheral biological pathways. The aim of this exploratory analysis was to investigate associations between ghrelin administration and the human circulating metabolome during alcohol exposure in non-treatment seeking, heavy drinking individuals with alcohol use disorder (AUD). METHODS: We used serum samples from a randomized, crossover, double-blind, placebo-controlled human laboratory study with intravenous (IV) ghrelin or placebo infusion in two experiments. A loading dose (3 μg/kg) was followed by continuous infusion (16.9 ng/kg/min) of acyl-ghrelin or placebo during each session. The first experiment included an IV alcohol self-administration (IV-ASA) session, and the second experiment included an IV alcohol clamp (IV-AC) session, both under the counterbalanced infusion of ghrelin or placebo. Serum metabolite profiles were analyzed from repeated blood samples collected during each session. RESULTS: In both experiments, ghrelin infusion was associated with altered serum metabolite profile, specifically with significantly increased levels of cortisol (IV-ASA q-value = 0.0003, and IV-AC q < 0.0001), corticosterone (IV-ASA q = 0.0202, and IV-AC q

International audience; Hepatocellular carcinoma (HCC) development entails changes in liver metabolism. Current knowledge on metabolic perturbations in HCC is derived mostly from case-control designs, with sparse information from prospective cohorts. Our objective was to apply comprehensive metabolite profiling to detect metabolites whose serum concentrations are associated with HCC development, using biological samples from within the prospective European Prospective Investigation into Cancer and Nutrition (EPIC) cohort (>520 000 participants), where we identified 129 HCC cases matched 1:1 to controls. We conducted high-resolution untargeted liquid chromatography-mass spectrometry-based metabolomics on serum samples collected at recruitment prior to cancer diagnosis. Multivariable conditional logistic regression was applied controlling for dietary habits, alcohol consumption, smoking, body size, hepatitis infection and liver dysfunction. Corrections for multiple comparisons were applied. Of 9206 molecular features detected, 220 discriminated HCC cases from controls. Detailed feature annotation revealed 92 metabolites associated with HCC risk, of which 14 were unambiguously identified using pure reference standards. Positive HCC-risk associations were observed for N1-acetylspermidine, isatin, p-hydroxyphenyllactic acid, tyrosine, sphingosine, l,l-cyclo(leucylprolyl), glycochenodeoxycholic acid, glycocholic acid and 7-methylguanine. Inverse risk associations were observed for retinol, dehydroepiandrosterone sulfate, glycerophosphocholine, γ-carboxyethyl hydroxychroman and creatine. Discernible differences for these metabolites were observed between cases and controls up to 10 years prior to diagnosis. Our observations highlight the diversity of metabolic perturbations involved in HCC development and replicate previous observations (metabolism of bile acids, amino acids and phospholipids) made in Asian and Scandinavian populations. These findings emphasize the role of metabolic pathways associated with steroid metabolism and immunity and specific dietary and environmental exposures in HCC development.

Background Bile acids (BAs) are synthesized by the liver and modified by gut bacteria, and may play an intermediary role between the gut microbiome and liver in promoting fibrosis in non-alcoholic fatty liver disease (NAFLD). We investigated the associations between serum and faecal BAs, gut microbiome and fibrosis in patients with and without NAFLD and examined the impact of diet and alcohol consumption on these relationships.Methods Adult patients (n = 122) underwent liver biopsy and BAs characterization by high-performance liquid chromatography/mass spectrometry. Gut microbiome composition was analysed using next-generation 16S rRNA sequencing. Diet and alcohol intake were determined by 3-day food diary.Results Serum and faecal BA concentrations increased progressively among non-NAFLD controls (n = 55), NAFLD patients with no/mild fibrosis (F0-2, n = 58) and NAFLD with advanced fibrosis (F3/4, n = 9). Progressive increases in serum BAs were driven by primary conjugated BAs including glycocholic acid [GCA] and secondary conjugated BAs. In contrast, faecal BA increase was driven by secondary unconjugated BAs (predominately deoxycholic acid [DCA]). Serum GCA levels and faecal DCA levels correlated with the abundance of Bacteroidaceae and Lachnospiraceae, and stool secondary BAs with an unclassifiable family of the order Bacteroidales (Bacteroidales;other). These bacterial taxa were also associated with advanced fibrosis. Modest alcohol consumption was positively correlated with faecal DCA levels and relative abundance of Lachnospiracaea and Bacteroidales;other.Conclusions Higher serum and faecal BA levels are associated with advanced fibrosis in NAFLD. Specific gut bacteria link alterations in BA profiles and advanced fibrosis, and may be influenced by low-level alcohol consumption.

This study assessed the porcine palatal mucosa dissolution, from artificial grooves, by a final rinse of sodium hypochlorite (NaOCl) solely or mixed with Keratobacter ((KB); or agitation) passive ultrasonic irrigation (PUI) or sonic activation (SA). Soft-tissue samples (n = 123) were weighed and placed inside root canal grooves in central maxillary incisors. The specimens were randomly divided into six test groups (n = 20): NaOCl 3% with or without KB and the irrigation dynamics: positive pressure (PP), SA or PUI. An EDTA intermediate rinse was included. Soft-tissue weights were measured subsequently. The NaOCl + KB_PUI group showed the highest mean weight reduction, whilst the NaOCl_PP group recorded the lowest. NaOCl + KB subgroups were associated with hastened reduction compared with NaOCl subgroups. NaOCl_PP presented with significantly lower weight reduction when compared with the remaining experimental groups. The addition of KB to NaOCl or its agitation enhances tissue dissolution ex vivo. The agitation of KB-containing mixtures offers no further benefits. Sin financiación 1.659 JCR (2020) Q3, 78/92 Dentistry, Oral Surgery & Medicine 0.703 SJR (2020) Q1, 34/141 Dentistry (miscellaneous) No data IDR 2020 UEM

Glycocholic acid (GCA) is a newly identified biomarker for hepatocellular carcinoma (HCC) patients. In this study, a method based on macromolecular crowding strategy has been applied for preparation of a molecularly imprinted polymer (MIP), which possesses high adsorption capacity for GCA. Polymethyl methacrylate was used as a macromolecular crowding agent, N-(3-aminopropyl)-methacrylamide hydrochloride as a functional monomer and ethylene dimethacrylate as a cross-linker. The morphology and binding characteristics of MIP were assessed by scanning electron microscopy and absorption experiments. The MIP was used as an adsorbent material to separate GCA, and the molecularly imprinted solid-phase extraction (MISPE) was carefully optimized. The MISPE combined with high-performance liquid chromatographic analysis was successfully used to determine the GCA in plasma and urine samples. When spiked levels ranged from 0.2 to 20 μmol L-1 , the recoveries were between 94.3 and 100.5%. As a proof of principle, this proposed method has been validated on a small subset of HCC patients (n = 10) and healthy volunteers (n = 10). The average GCA concentrations of HCC patients in plasma and urine were about 25 and 2.8 times than that of healthy volunteers. Copyright © 2016 John Wiley & Sons, Ltd.

UNLABELLED Besides aiding digestion, bile salts are important signal molecules exhibiting a regulatory role in metabolic processes. Human ileal bile acid binding protein (I-BABP) is an intracellular carrier of bile salts in the epithelial cells of the distal small intestine and has a key role in the enterohepatic circulation of bile salts. Positive binding cooperativity combined with site selectivity of glycocholate and glycochenodeoxycholate, the two most abundant bile salts in the human body, make human I-BABP a unique member of the family of intracellular lipid binding proteins. Solution NMR structure of the ternary complex of human I-BABP with glycocholate and glycochenodeoxycholate reveals an extensive network of hydrogen bonds and hydrophobic interactions stabilizing the bound bile salts. Conformational changes accompanying bile salt binding affects four major regions in the protein including the C/D, E/F and G/H loops as well as the helical segment. Most of these protein regions coincide with a previously described network of millisecond time scale fluctuations in the apo protein, a motion absent in the bound state. Comparison of the heterotypic doubly ligated complex with the unligated form provides further evidence of a conformation selection mechanism of ligand entry. Structural and dynamic aspects of human I-BABP-bile salt interaction are discussed and compared with characteristics of ligand binding in other members of the intracellular lipid binding protein family. PROTEIN DATA BANK ACCESSION NUMBERS The coordinates of the 10 lowest energy structures of the human I-BABP : GCDA : GCA complex as well as the distance restraints used to calculate the final ensemble have been deposited in the Brookhaven Protein Data Bank with accession number 2MM3.

Aims The aim of this study was to investigate the transport of two kinds of bile acids by organic anion transporting polypeptide 1B3 (OATP1B3) using first-trimester trophoblasts. The mechanisms of damage to fetuses with intrahepatic cholestasis of pregnancy were investigated, providing new potential strategies for targeted therapies aimed at reducing fetal risk. Material and Methods The expression of OATP1B3 was knocked down by lentiviral vector-mediated RNA interference, and silencing efficiency was assessed using real-time polymerase chain reaction and Western blotting. The cytotoxicity of two bile acids (glycocholic acid [GCA] and glycochenodeoxycholic acid [GCDCA]) was assessed using the MTT method. Transport of bile acids was assessed by establishing an in vitro trophoblast monolayer model using polyester Transwell-clear inserts, and the concentration of bile acids in the upper compartment was assessed using high-pressure liquid chromatography. Results GCA and GCDCA (10 and 20 μM) were not cytotoxic to the SWAN cell line (P > 0.05). RNAi treatment decreased the mRNA and protein expressions of OATP1B3 by 94.42% and 49.51%, respectively (P

BACKGROUND & AIMS Metabolomics is comprehensive analysis of low-molecular-weight endogenous metabolites in a biological sample. It could enable mapping of perturbations of early biochemical changes in diseases and hence provide an opportunity to develop predictive biomarkers that could provide valuable insights into the mechanisms of diseases. The aim of this study was to elucidate the changes in endogenous metabolites and to phenotype the metabolic profiling of d-galactosamine (GalN)-inducing acute hepatitis in rats by UPLC-ESI MS. METHODS The systemic biochemical actions of GalN administration (ip, 400 mg/kg) have been investigated in male wistar rats using conventional clinical chemistry, liver histopathology and metabolomic analysis of UPLC- ESI MS of urine. The urine was collected predose (-24 to 0 h) and 0-24, 24-48, 48-72, 72-96 h post-dose. Mass spectrometry of the urine was analysed visually and via conjunction with multivariate data analysis. RESULTS Results demonstrated that there was a time-dependent biochemical effect of GalN dosed on the levels of a range of low-molecular-weight metabolites in urine, which was correlated with developing phase of the GalN-inducing acute hepatitis. Urinary excretion of beta-hydroxybutanoic acid and citric acid was decreased following GalN dosing, whereas that of glycocholic acid, indole-3-acetic acid, sphinganine, n-acetyl-l-phenylalanine, cholic acid and creatinine excretion was increased, which suggests that several key metabolic pathways such as energy metabolism, lipid metabolism and amino acid metabolism were perturbed by GalN. CONCLUSION This metabolomic investigation demonstrates that this robust non-invasive tool offers insight into the metabolic states of diseases.

The presence of a disulfide bridge in liver bile acid binding protein (L-BABP/S-S) allows for site-selective binding of two bile acids, glycochenodeoxycholic (GCDA) and glycocholic acid (GCA), differing only in the presence of a hydroxyl group. The protein form devoid of the disulfide bridge (L-BABP) binds both bile salts without discriminating ability. We investigate the determinants of the molecular recognition process in the formation of the heterotypic L-BABP/S-S complex with GCDA [corrected] and GCA [corrected] located in the superficial and inner protein sites, respectively. The comparison of the NMR spectroscopy structure of heterotypic holo L-BABP/S-S, the first reported for this protein family, with that of the homotypic L-BABP complex demonstrates that the introduction of a S-S link between adjacent strands changes the conformation of three key residues, which function as hot-spot mediators of molecular discrimination. The favoured χ(1) rotameric states (t, g(+) and g(-) for E99, Q100 and E109 residues, respectively) allow the onset of an extended intramolecular hydrogen-bond network and the consequent stabilisation of the side-chain orientation of a buried histidine, which is capable of anchoring a specific ligand.

This study describes the metabonomic characters of the spontaneously hypertensive rats (SHR) and intervention effects of Ping Gan prescription. Ultra performance LC coupled with Q-TOF MS (UPLC/MS-Q-TOF) was employed to develop a metabonomic method of the plasma of SHR. There was a significant difference in metabolic profiling observed between predose group of Wistar Kyoto rats and model group (SHR) by using the principal components analysis (PCA). Some significant changes in metabolites such as LysoPC(22:6), LysoPC(20:4), LysoPC(18:1), cholylglycine, PE(P-16:0e/0:0), sphingosine-1-phosphate, and 2-oxo-4-methylthio butanoic acid were identified. These biochemical changes were associated with the disturbance in sphingolipid metabolism and fat metabolism, which would be helpful to further understand the essence of hypertension and the therapeutic mechanism of Ping Gan prescription. This study suggests that the metabonomic method may be a valuable and feasible tool to explore the therapeutic effect and mechanism of traditional Chinese medicine (TCM).

Aim To explore the effects of matrine (MT) on acute intrahepatic cholestasis induced by 17alpha-ethinyl estradiol (EE) in rats. Methods Acute intrahepatic cholestasis in rats were induced by EE, and the effects of MT on acute intrahepatic cholestasis were explored and compared with ursodeoxycholic acid (UDCA) by serum biochemical determination and bile excretion experiments. Results The serum biochemical and bile biochemical results indicated that MT and UDCA had notable hepatoprotective effects by counteracting cholestasis induced by EE. The bile flow and the bile excretion of glycocholic acid (GC, a substrate of bile salt export pump [Bsep]), ketoprofen glucuronide (KPG) and rhodamine 123 (Rh123, a substrate of multidrug resistance protein 1 [MDR1]) decreased by EE, were significantly improved after administration of MT. Conclusion MT exhibited potential protection against EE-induced acute intrahepatic cholestasis.

BACKGROUND: Bile salt deconjugation by Lactobacillus strains is often closely linked to bile tolerance and survival of the strains in the gut and lowering of cholesterol in the host. The present study investigated the deconjugation of bile salts and removal of cholesterol by 12 Lactobacillus strains in vitro. The 12 strains were previously isolated from the gastrointestineal tract of chickens. RESULTS: The 12 Lactobacillus strains could deconjugate sodium glycocholate (GCA, 16.87-100%) and sodium taurocholate (TCA, 1.69-57.43%) bile salts to varying degrees, with all strains except L. salivarius I 24 having a higher affinity for GCA. The 12 Lactobacillus strains also showed significant (P < 0.05) differences in their ability to remove cholesterol from the growth medium (26.74-85.41%). Significant (P < 0.05) correlations were observed between cholesterol removal and deconjugation of TCA (r = 0.83) among the L. reuteri strains (C1, C10 and C16) and between cholesterol removal and deconjugation of TCA (r = 0.38) and GCA (r = 0.70) among the L. brevis strains (I 12, I 23, I 25, I 211 and I 218). In contrast, although L. gallinarum I 16 and I 26 and L. panis C 17 showed high deconjugating activity, there was no correlation between cholesterol removal and deconjugation of bile salts in these strains. CONCLUSION: The results showed that the 12 Lactobacillus strains were able to deconjugate bile salts and remove cholesterol in vitro, but not all strains with high deconjugating activity removed cholesterol effectively. © 2009 Society of Chemical Industry.

The possible influence of a bile salt on production of conjugated linoleic acid (CLA) by Lactobacillus reuteri ATCC 55739 was evaluated. Cells of the lactobacilli grown in MRS broth with and without linoleic acid (LA, 0.2%) were harvested and washed. The washed cells were added to buffer containing 0.2% LA and incubated 18 h at 37 degrees C. The cells, which had been grown without LA, transformed LA into CLA (mainly c9t11-C18:2) better than did those cells grown with it. When sodium glycocholate (0.3%) was added to the washed cell suspensions, about the same level of CLA was formed as in its absence regardless of whether or not the cells had been grown in broth supplemented with free LA. Thus, glycocholate that occurs in humans did not influence production of CLA by resting cells of the lactobacilli.

Background Until now, hydrophilic and lipophilic vitamin preparations had to be administered separately during total parenteral nutrition. By addition of glycocholic acid, a vitamin supplement (Cernevit, Baxter, Heidelberg, Germany) was developed that combines all vitamins into one vial. However, little information exists about possible consequences of bile acid administration such as glycocholic acid, especially if liver disease is pre-existing. Aim To evaluate the effects of total parenteral nutrition with a vitamin preparation containing high doses of glycocholic acid in patients with and without liver disease. Methods In a prospective, randomized-controlled trial, 74 patients, 36 of them with hepatobiliary disease, received total parenteral nutrition for 16 +/- 11 days, either with Cernevit or control vitamin supplements. Patients were closely monitored for clinical and biochemical parameters including serum bile acid profiles measured by high-performance liquid chromatography. Results Serum glycocholic acid increased in patients with liver disease treated with Cernevit, whereas total bile acids did not significantly change. Other liver function tests remained stable during treatment. No adverse events during Cernevit administration were noted except for a reversible slight increase of transaminases in one patient. Conclusions Cernevit was well tolerated after repeated dosing, even in patients with severe liver disease. Apart from standard controls of liver biochemistry, no specific surveillance is necessary during treatment with Cernevit.

The unconjugated bile acids cholic acid, deoxycholic acid, and chenodeoxycholic acid; their glycine and taurine conjugates glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, and taurochenodeoxycholic acid; and a taurine conjugated ursodeoxycholic acid, tauroursodeoxycholic acid, were characterized through 1H and 13C NMR in aqueous media under the physiological pH region (7.4 +/- 0.1). Assignments of 1H and 13C signals of all the bile acids were made using a combination of several one- and two-dimensional, homonuclear (1H-1H) and heteronuclear (1H-13C) correlations as well as spectral editing NMR methods. Stereochemical assignment of the five-membered ring of the bile acids is reported here for the first time. The complete characterization of various bile acids in aqueous media presented here may have implications in the study of the pathophysiology of biliary diseases through human biliary fluids using NMR spectroscopy.

The regulation of drug metabolic activity of cultured hepatocytes can be applied to the evaluation of pharmacokinetics, analysis of drug delivery and the bioartificial liver system. It is very difficult to maintain the drug metabolic activity mediated by cytochrome P-450 (CYP) 3A. Recently we found that the CYP3A aminopyrine N-demethylase (AMND) activity of hepatocytes cultured on collagen surface oscillated with culture time. This phenomenon was related to the concentration of bile acid in the culture medium. CYP3A, multidrug resistant gene 2 (MDR2) and heat shock protein 84 (HSP84) mRNA appeared in a manner corresponding to this oscillation. When a large quantity of bile acid was taken up into hepatocytes from the medium, low AMND activity was observed, and these proteins did not appear. When bile acid was secreted and the bile acid concentration inside the hepatocytes was low, high AMND activity was obtained, and these proteins appeared. In order to clarify the mechanism of oscillation between AMND activity and bile acid, 8 μM glycocholic acid was added to the culture medium 15 h before the measurement. No oscillation in AMND activity was observed in the presence of 8 μM glycocholic acid. Bile acid controls the AMND activity in the transcription of hepatocytes. Copyright © 2001 John Wiley & Sons, Ltd.

Low water solubility and development of resistance are important drawbacks in the use of cisplatin as a cytostatic agent. A novel bile acid-cisplatin complex, Bamet-R2 [cis-diamminechlorocholylglycinateplatinum (II)], with liver vectoriality, has been synthesized. Our aim was to investigate the usefulness of this compound to overcome cisplatin resistance and to determine whether its encapsulation into liposomes increases its water solubility, uptake by liver tumor cells and cytostatic activity. Highly efficient incorporation of Bamet-R2 into liposomes permitted an increase in the concentration of the drug compared with that in the initial free solution by more than 6 x 10(6)-fold, which is 10(3)-fold higher than the encapsulation obtained for cisplatin. A partially cisplatin-resistant (87-fold) monoclonal cell line (Hepa 1-6/10R) was obtained by 2 subcloning steps of a population of mouse hepatoma Hepa 1-6 cells grown in step-wise increasing cisplatin concentrations up to 10 microM. Decreased sensitivity to cisplatin was accompanied by a 3.2-fold lower drug accumulation compared to wild-type cells. Uptake was markedly increased by the binding of cisplatin to glycocholic acid in both Hepa 1-6 and Hepa 1-6/10R cells. This probably accounts for the partial overcoming (-82%) of resistance when used on Hepa 1-6/10R cells. Inclusion of Bamet-R2 into liposomes further increased the amount of the drug accumulated in both cell types and, hence, enhanced its cytostatic activity. Since both plain liposomes and Bamet-R2 have little toxicity, the formulation of this compound in liposomes may offer a substantial advantage over cisplatin in the treatment of tumors resistant to this anti-neoplastic agent.

The use of collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) has been shown to produce fragmentation that is useful for the structural analysis of bile acids and their conjugates. Low-energy CID using a triple quadrupole has been used to help characterise bile acid identity but the majority of work has been conducted using high-energy CID on specialised instrumentation. This paper describes the use of low-energy CID as a rapid method for identification of urinary bile acids and presents some examples of its use in the diagnosis of liver disease in infants. These include the differential diagnosis of peroxisomal disorders, identification of compounds (e.g. 3 beta,7 alpha-dihydroxy-5-cholenoic acid 3-sulphate) indicative of 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase/isomerase deficiency and the confirmation of the identity of an unusual bile acid series consisting of different conjugates of lithocholic acid. The use of lithium cationisation and derivatisation with aminosulfonic acids for the analysis of unconjugated and glycine-conjugated bile acids has also been evaluated.

The single pass albumin dialysis (SPAD) was reported to be an alternative to the Molecular Adsorbent Recirculating System (MARS) for the effective removal of protein bound substances in liver failure. Three SPAD experiments using different albumin concentrations and dialysate flow rates were performed. In each experiment, 1000 ml human donor plasma, spiked with 250 mg unconjugated bilirubin, 200 mg sulfobromophthalein (BSP) and 115 mg glycocholic acid (N-[3alpha,7alpha,12alpha-trihydroxy-24-oxycholan-24-yl]glycine) - a conjugated bile acid (BA), circulated in a closed loop with 150 ml/min and was dialysed against albumin solution. These substances are bound to the different binding sites of albumin and have different association constants. For the comparison, the standard MARS experiment was performed using the same plasma flow rate of 150 ml/min. Moreover, the clearances of bilirubin for MARS and SPAD during clinical treatments were calculated using own data and those reported by Seige, Kreymann, Jeschke, et al. in Transplant Proc 1999; 31: 1371-5. The concentrations of bilirubin, BSP and BA were measured in plasma and dialysate and for these substances clearances (Cl) were calculated. It is known that the elimination rate of bilirubin is not very high during albumin dialysis in comparison to other substances, like bile acids, due to the high association constant. An increase of albumin concentration or the flow rate improved the efficacy but also raised the costs substantially. In this study, we have shown that MARS is the more effective kind of albumin dialysis for the important substances like bile acids. By SPAD an improvement of efficacy can be reached only by dramatic increase of the costs. Also, the earlier experiments showed that MARS is safer because of the removal of the stabilizers, which are normally included in the commercial albumin solutions.

Iron and/or calcium were added to psyllium, guar gum, locust bean gum, citrus rag, high methoxy pectin, and low methoxy pectin. This mixture was then incubated with either cholic acid, deoxycholic acid, glycocholic acid, or taurocholic acid to evaluate the effect of minerals on fiber-bile acid binding. The addition of iron with or without calcium increased the binding of deoxycholic acid to all fibers (p≤0.01 and p≤0.025) except low methoxy pectin. Iron and calcium caused increased binding of high methoxy pectin to all bile acids (p≤0.01 and p≤0.025). Dietary fiber's ability to bind certain bile acids may be maximized through mineral supplementation.

A study was carried out on the negative ion thermospray liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry of a group of bile acids and their glycine conjugates. The liquid chromatographic/tandem mass spectrometric experiments were performed using low-energy collisions on a triple-quadrupole mass spectrometer. It was found that relatively high collision energies and collision gas pressures were required to produce fragmentation and that some unusual fragments were produced.

A breath test using [1-(13)C]glycocholic acid as a substrate is a potential diagnostic method for small intestinal bacterial overgrowth syndrome. [1-(13)C]Glycocholic acid has been thus synthesized in an excellent yield from ethyl [1-(13)C]glycinate hydrochloride in a one-pot reaction. This method is suitable for the preparation of the labeled compound on a laboratory scale, which helps to perform extensive clinical studies of the breath test.

Abstrat Inour country, congenital biliary atresia (CBA) has occurred in approximately 1 of 10,000 live births, but its prognosis has been extremely poor. In the mass screening of this disease, glycocholic acid (GCA) or chenodeoxycholic acid (CDCA) level in dried blood spots on filter paper in5-day-old neonates was determined by radioimmunoassay (RIA). To determine GCA in dried blood spots on filter paper, the procedures in this experiment were modified using commercial kit for serum. The mean GCA level of controls (n=391) was 5.88±4.28 nM/ml and that of CBA (n=22) was 14.1 ±3.1 nM/ml. On the other hand the mean CDCA level of controls (n=86) was 5.86 ±3.07 pM/l disc that of CBA (n=22) was 30.0 ±35.9 pM/l disc. When the criterion is assumed to be more than 16 nM/ml of GCA level or 10 pM/l disc of CDCA level, false negative diagnoses of CBA are 31.8% and 18.2% respectively. On the other hand false positive diagnoses of CBA are 6.9% and 3.5% respectively. It was thought that false negative diagnosis on mass screening with GCA level would decrease using fresh dried blood spot within 2 weeks after taking blood. In 22 CBA cases, the GCA levels in dried blood spots on filter paper were not always parallel with the CDCA levels.

The bile acid composition and metabolism of rainbow troutSalmo gairdneri has been investigated by thin layer chromatography, gas liquid chromatography, and radio gas liquid chromatography methods. For this purpose gallbladder bile was collected from fed fish at 6 and 13 months and from starved fish at 12 months of age. Cholic acid was found to be the main component and constituted over 85% of total. Chenodeoxycholic acid accounted for 14% or less and the 3α, 12α-7-keto- and 7α, 12α-3-keto-5β-cholanoates for 1% or less of total. The bile acids were conjugated mainly with taurine, only small amounts of glycocholic acid being detected. Ca. 5% of the taurocholate was sulfated, as were trace amounts of cholic and glycocholic acids. The size of the bile acid pool was found to increase in the older fish and to decrease in starved fish. Unlike mammalian livers, the livers of the trout converted radioactive chenodeoxycholic acid into cholic acid.

The bile acids, taurocholic, taurodeoxycholic and glycocholic acid, were reacted with bis(tri-n-butyltin) oxide (TBTO) to form 1:1 derivatives. The water of reaction was removed with 2, 2-dimethoxypropane or by azeotropic distillation with benzene. The compounds were characterized by 13C and 119Sn NMR, IR, elemental analysis, molecular weight determination, DTA, TGA, and conductance measurements. The data indicated that tri-n-butyltin glycocholate forms a covalent ester, tin being tetracoordinated. The taurocholic and taurodeoxycholic acid derivatives contain one molecule of coordinated water. They partially dissociate in polar solvents. The taurocholic and glycocholic acid derivatives were tested in vitro as anticancer agents and exhibited ED50 in the 0.2–0.4 ppm (mg kg−1) range against KB epidermoid tumor and P-388 leukemia using NCI protocols.

Eighteen normal women, 20 with active urinary schistosomiasis, and 25 with past histories of viral hepatitis were given a contraceptive pill containing 0.05 mg ethinyl estradiol and 0.5 mg levonorgestrel for six consecutive cycles. Serum bile acid levels were measured by enzyme immunoassay method before and after 3 and 6 months of use. Simultaneously, conventional liver function tests (serum bilirubin, transaminases, alkaline phosphatase and albumin) were done. Serum bile acid concentration was not significantly changed by the contraceptive use in any group. The concentration of cholylglycine (the main bile acid measured) did not correlate with the values of any of the other tests. Pretreatment values of serum cholylglycine were significantly lower in the past-hepatitis group. The difference was maintained during treatment.18 normal women, 20 with active schistosomiasis, and 25 with past histories of viral hepatitis were given a contraceptive pill containing 0.05 mg ethinyl estradiol and 0.5 mg levonorgestrel for 6 consecutive cycles. Serum bile acids were measured by enzyme immunoassay method before and after 3 and 6 months of use. Simultaneously, conventional liver function tests (serum bilirubin, transaminases, alkaline phosphatase, and albumin) were done. Serum bile acid concentration was not significantly changed by contraceptive use in any group. The concentration of cholylglycine (the main bile acid measured) did not correlate with the values of any of the other tests. Pretreatment values of serum cholylglycine were significantly lower in the past-hepatitis group. The difference was maintained during treatment.

Although it has been speculated that chlorpromazine may alter the transhepatic movement of bile acids from plasma to bile, the effect of chlorpromazine on various determinants of bile acid transport in isolated rat hepatocytes remains incompletely defined. In particular, there is little information about the effect of chlorpromazine on the release of bile acids from freshly isolated hepatocytes. Therefore, we examined the effect of chlorpromazine, administered in vivo and in vitro, on the efflux rate of radiolabeled bile acids in freshly isolated rat hepatocytes. In an isolated haptocyte system, it is not possible to distinguish the sinusoidal plasma membrane function of efflux (back diffusion) from the canalicular plasma membrane function of excretion. Therefore, efflux, as used in this manuscript, reflects both back diffusion and excretion. In vitro, chlorpromazine produced a rapid dose dependent significant (P

Mixed micelles (MM) formed from glycocholic acid and lecithin are suited to solubilize lipophilic drugs for intravenous use. To test for possible drug-drug interactions, the protein binding of a series of agents known to bind to different sites on albumin (diazepam, warfarin, ketoprofen, frusemide, probenecid) and additionally (prazosin, quinidine, propranolol) or exclusively (disopyramide) to alpha 1-acid glycoprotein or to transcortin (prednisolone) was determined in the presence and absence of MM. Concentrations of MM, corresponding to the maximum possible plasma concentration achieved by injecting the highest clinical doses of MM into the systemic circulation, had little or no effect on the unbound fractions of drugs known to bind exclusively to albumin. Only at five times higher MM concentrations were the free fractions substantially increased (by up to 45%). Unbound fractions of drugs bound with high affinity but low capacity to alpha 1-acid glycoprotein were increased between 50-85% even at 'therapeutic' doses of MM. The present study suggests that drugs solubilized by MM should be given by slow injection or infusion to patients already receiving drugs which are highly bound to alpha 1-acid glycoprotein.

The stereochemical course of the hydrolysis of synthetic sn-glycerol-1-palmitate-2-oleate-3-linoleate, sn-glycerol-1,2-dipalmitate-3-oleate and their antipodes by pancreatic and milk lipoprotein lipase was investigated by thin layer and gas liquid chromatographies of the diacylglycerol intermediates. The enzymic hydrolyses were made with bile salts or lysolecithin in a 1∶1 molar ratio to the substrate as emulsifiers and were limited to short time intervals which minimized isomerization and the reversal of lipolysis. In all instances, the products of hydrolysis by lipoprotein lipase contained a marked preponderance of the 2,3-diacylglycerols, while the composition of the diacylglycerol intermediates in the products of pancreatic lipase varied with the nature of the fatty acid in the 1 and 3 positions of the triacylglycerol molecule. Pancreatic lipase, but not lipoprotein lipase, gave a preferential release of unsaturated fatty acids. The above results are similar to those obtained with radioactive trioleoylglycerol and conventional stereospecific analyses and suggest that lipoprotein lipase may favor attack on the sn-1 position. It is hypothesized that the small amounts of the 1,2-diacylglycerols present may have arisen from a reversal of lipolysis also catalyzed by this enzyme.

Hypercholesterolemic rats, fed 1% cholesterol and 0.5% glycocholate, were treated with arylsulfonates in various ways to observe the pattern of cholesterol elimination. Dietary linoleyl p-toluene-sulfonate (LTS) hastened return to normocholesterolemia and lowered hepatic cholesterol either with or without continued cholesterol feeding. LTS administered via the portal vein significantly lowered plasma cholesterol in 48 hr; ethyl linoleate and monoolein produced no lowering. LTS administered via the portal vein to glycocholate-infused rats increased the biliary excretions of label from [4-14C]cholesterol administered intracardially and also increased total bile acid excretion 21% without increased bile volume when compared to similar injection of ethyl linoleate. No change in biliary excretion of cholesterol was seen. Bile acid kinetics were studied by using isotopic dilution techniques. Cholate turnover was enhanced by feeding oleyl p-toluenesulfonate (OTS) and oleylp-(n-decyl)-benzenesulfonate (ODS) as suggested by a 16–35% decrease in half-life in both normal and hypercholesterolemic rats. Rats consuming a grain-based colony diet had a 54% increase in cholate synthesis when OTS was included in the diet. The composition of bile was changed when either OTS or ODS was fed; an increase in chenodeoxycholate was noted. This change was gradual with OTS but rapid with ODS and paralleled enhanced decay of chenodeoxycholate specific radioactivity in response to treatment. ODS and OTS also increased14CO2 expiration from oral [26-14C] cholesterol in hypercholesterolemic rats. Dietary OTS and ODS elevated hepatic free cholesterol in hypercholesterolemic rats; ODS also elevated plasma free cholesterol and increased cholesteryl ester hydrolase activity in the liver. The data suggest that arylsulfonates stimulate cholesterol catabolism, in addition to the reported inhibition of cholesterol absorption [Lipids 12:819 (1977)].

The content of the rabbit colon has been examined and found to contain the secondary bile acid deoxycholate. Infusions of sodium deoxycholate stimulated colonic motor activity, but the infusion of the primary bile acid sodium glycocholate did not have this effect. Infusions of control amounts of distilled deionized water and isotonic saline were not followed by a significant change in motor activity. Histological damage was caused by 15 mmol.l-1 solutions but was not severe till above 24 mmol.l-1. The possibility that mucosal damage produced motor activity was discounted by the fact that this damage was also present in rabbits treated by sodium glycocholate with no observed motor response. It is postulated that the secondary bile acid, sodium deoxycholate, could have a role in the production of colonic motor activity in the rabbit.

Summary. Bacterial overgrowth in the small bowel has been recognised in 11 patients with the aid of the breath-test with [glyco-1-14C] cholic acid and by the determination of the amount of bacteria in the jejunal juice. In all but one patient hypoalbuminaemia, from 21.9 to 42.9 g/1, was observed. This low level of serum albumin can be due to an excessive intestinal protein-loss or to a decreased rate of synthesis of albumin as the result of an insufficient delivery of amino acids to the liver. In 11 patients with bacterial overgrowth the rate of synthesis of albumin has been determined by the 14C-carbonate method. Compared to a control group the rate of albumin synthesis and the serum level of valine, leucine, lysine and tryptophan were significantly decreased in the patients with bacterial overgrowth. A positive correlation was found between the rate of albumin synthesis and the serum levels of valine, isoleucine, leucine and tryptophan. When the serum levels of these 4 essential amino acids were low, the rate of albumin synthesis was depressed.

To elucidate the bile acid metabolism in the preoperative and postoperative stages of congenital biliary atresia (CBA), the unconjugated and conjugated bile acid levels in sera were measured by high performance liquid chromatography (HPLC). The results showed that the mean total serum bile acid (TSBA) level in the preoperative cases of CBA was higher, 122.1±39.0 nmol/1 (1SD), and was about 12 times higher than the level in the age-matched normal controls (9.9±6.0). Even in the non-icteric patients several years after operation, the mean TSBA level was still much higher (15.2±9.6) than that of normal controls (5.7±3.1). The mean ratio of cholic acid to chenodeoxycholic acid (CA/CDCA) in the preoperative and postoperative cases of CBA was less than 1.0. The mean ratio of glycine-conjugated bile acids to taurine-conjugated bile acids (G/T) in the preoperative cases of CBA was the lowest (1.9±1.1). In the clinically good cases with sufficient bile flow after operation, G/T ratio was the highest (9.3±6.5). In the normal controls and the patients with preoperative and postoperative CBA, the main bile acids were glycocholic acid, taurocholic acid, glycochenodeoxycholic acid and taurochenodeoxycholic acid. These conjugated bile acids comprised more than 85% of the total in amount.

Fasting and post-prandial conjugated bile salt concentrations and intravenous glycocholate tests were normal in seven patients with Gilbert's syndrome. These findings indicate that the hepatic handling of conjugated bile salts in Gilbert's syndrome is normal.

Measurement of serum bile acids has been claimed to be a sensitive and specific biochemical test of hepatic function. We have prospectively measured post-prandial serum glycocholate (cholylglycine) concentrations in 31 patients with alcoholic liver disease and compared these measurements with those of bromsulphalein (BSP) retention, prothrombin time, and serum albumin. In the patients with early (non-cirrhotic) alcoholic liver disease (N = 14) BSP retention was abnormal significantly more frequently than was serum cholylglycine concentration (100% vs 29%, p less than 0.001). In contrast, amongst patients with late (cirrhotic) alcoholic liver disease, BSP retention and serum cholylglycine were abnormal with equal frequency (94%). In both groups of patients BSP retention and serum cholylglycine were abnormal significantly more often than were prothrombin time and serum albumin concentrations. We conclude that moderately severe hepatocellular dysfunction is required before serum cholylglycine can become a reliable biochemical indicator of liver disease.

The presence of sulfated bile acid in serum of healthy persons and patients with hepatobiliary diseases was recognized by using an Amberlite XAD-2 column for extraction of bile acid and utilizing a Sephadex LH-20 column for separation of sulfated bile acid (sulfate of either taurine or glycine conjugate), and nonsulfated bile acid (taurine and glycine conjugate. The values of nonsulfate determined in this study were almost similar to the level of total serum bile acid reported by previous workers. A small amount of sulfated bile acid was found in normal serum, and the percentage of sulfated to total bile acid was ca. 10%. In patients with hepatobiliary diseases, both levels of sulfated and nonsulfated bile acid in serum rose, but sulfate did not increase in parallel to the level of nonsulfate. A remarkable increase of serum-sulfated bile acid could be observed in patients with cholestasis, while a slight elevation was noted in patients with chronic hepatocellar insufficiency. The percentage of sulfated to total bile acid was from 1.8 to 21.2% in patients with hepatobiliary diseases. Sulfated bile acid in serum consisted of only dihydroxycholanoic acid, deoxycholic and chenodeoxycholic acid. As no solvolysis was not carried out in previous works, bile acid sulfate in serum as described in this study was not determined at all.

A new technique using T-cannulated bile ducts has been developed for studying bile acid turnover in the rat by isotope dilution. Applicability of this technique to study the effect of diet on cholate and chenodeoxycholate metabolism is suggested. In tests with the method, the rate of cholate synthesis for rats fed colony diet was twice that for rats fed semisynthetic diet. When sodium glycocholate was fed in the latter diet the half-life of cholate decreased; the secretion rate of deoxycholate and cholate increased without affecting chenodeoxycholate. Although curves for cholate log specific activity vs. time were linear, those obtained simultaneously for chenodeoxycholate were nonlinear. A double exponential curve was obtained for chenodeoxycholate of colony-fed rats. Separation of free deoxycholate and chenodeoxycholate was improved through continuous development thin layer chromatography.