Concentrations of cholestenoic acids in plasma from patients with reduced intestinal reabsorption of bile acids

The concentrations of 3 beta-hydroxy-5-cholestenoic acid, 3 beta,7 alpha-dihydroxy-5-cholestenoic acid, and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid were determined in plasma from patients treated with cholestyramine or subjected to resection of the ileum or colon. The values were compared with those for conjugated and unconjugated C24 bile acids. Patients with an intact ileum but without colon had normal levels of cholestenoic acids. Patients treated with cholestyramine or with ileal resection had elevated levels of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid (median values 189 and 233 ng/ml, respectively, compared to 85 ng/ml in controls). The levels of the other two C27 acids were normal in cholestyramine-treated and low in ileoresected patients and were positively correlated to each other but not to the 3-oxo-delta 4 acid. There were no consistent correlations between the levels of C27 acids and those of conjugated or unconjugated C24 bile acids. The results indicate an increased formation of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid in subjects having a stimulated activity of cholesterol 7 alpha-hydroxylase.     

Occurrence of 3 beta-hydroxy-5-cholestenoic acid, 3 beta,7 alpha-dihydroxy-5-cholestenoic acid, and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid as normal constituents in human blood

Three unconjugated C27 bile acids were found in plasma from healthy humans. They were isolated by liquid-solid extraction and anion-exchange chromatography and were identified by gas-liquid chromatography-mass spectrometry, microchemical reactions, and ultraviolet spectroscopy as 3 beta-hydroxy-5-cholestenoic, 3 beta,7 alpha-dihydroxy-5-cholestenoic, and 7 alpha-hydroxy-3-oxo-4-cholestenoic acids. Their levels often exceeded those of the unconjugated C24 bile acids and the variations between individuals were smaller than for the C24 acids. The concentrations in plasma from 11 healthy subjects were 67.2 +/- 27.9 ng/ml (mean +/- SD) for 3 beta-hydroxy-5-cholestenoic acid, 38.9 +/- 25.6 ng/ml for 3 beta,7 alpha-dihydroxy-5-cholestenoic acid, and 81.7 +/- 27.9 ng/ml for 7 alpha-hydroxy-3-oxo-4-cholestenoic acid. The levels of the individual acids were positively correlated to each other and not to the levels of the C24 acids. The cholestenoic acids were below the detection limit (20-50 ng/ml) in bile and C27 bile acids present in bile were not detected in plasma.     

Occurrence of isomeric dehydrocholesterols in human plasma.

Three isomeric dehydrocholesterols were found in plasma from healthy subjects and patients with abnormal production or metabolism of cholesterol. These chemically labile steroids were isolated by a mild liquid-solid extraction procedure using octadecylsilane-bonded silica as sorbent. Sterol-protein interactions were minimized by diluting plasma with aqueous isopropanol. The dehydrocholesterols were identified by high-performance liquid chromatography-ultraviolet spectroscopy and gas chromatography-mass spectrometry as cholesta-5,7-dien-3 beta-ol (7-dehydrocholesterol), 5 alpha-cholesta-6,8(9)-dien-3 beta-ol (isodehydrocholesterol), and tentatively as cholesta-5,8(9)-dien-3 beta-ol. There was a strong positive correlation between plasma levels of the two former compounds, isodehydrocholesterol levels usually being about 1.4 times higher than those of 7-dehydrocholesterol. The median concentration of 7-dehydrocholesterol in plasma from healthy subjects was 52 ng/ml. Similar concentrations were found in colectomized patients (median concentration 47 ng/ml) and patients with extrahepatic cholestasis and alcoholic liver cirrhosis (median concentrations 79 and 67 ng/ml, respectively). Patients with ileal resection or under treatment with cholestyramine had elevated levels (median concentrations 142 and 160 ng/ml, respectively) whereas patients with primary biliary cirrhosis had subnormal levels (median concentration 26 ng/ml). The results are consistent with a positive correlation between levels of the dehydrocholesterols in plasma and the rate of cholesterol synthesis. The sterols were also analyzed in human skin and bile and the results indicate that the liver may be an important source of isodehydrocholesterol.     

Levels of 7 alpha-hydroxy-4-cholesten-3-one in plasma reflect rates of bile acid synthesis in man

A method for analysis of 7 alpha-hydroxy-4-cholesten-3-one in plasma is described. Following solid-phase extraction/purification the compound is determined by high-performance liquid chromatography using a UV detector. The median concentration in healthy subjects was 12 ng/ml (range 3-40). The levels were lower in diseases associated with a low bile acid production: extrahepatic cholestasis, less than 1.5 ng/ml (range less than 0.9-3); liver cirrhosis less than 1.5 ng/ml (range less than 0.9-38), and higher in diseases associated with a high bile acid production: cholestyramine treatment, 188 ng/ml (range 54-477); ileal resection 397 ng/ml (range 128-750). The levels were essentially normal in patients with colon resection. The results are consistent with a strong positive correlation between the levels of 7 alpha-hydroxy-4-cholesten-3-one in plasma and the rate of bile acid synthesis.     

Bile acids and bile alcohols in a child with hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency: effects of chenodeoxycholic acid treatment

Duodenal bile, urine, plasma, and feces from a child with hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency were analyzed by fast atom bombardment mass spectrometry and gas chromatography-mass spectrometry to investigate the formation and excretion of abnormal bile acids and bile alcohols. The biliary bile salts consisted of glycocholic acid (25%) and of sulfated and glycine conjugated di- and trihydroxycholenoic acids (55%), two C27 bile acids, and eleven sulfated bile alcohols (mainly tetrols, 20%), all having 3 beta,7 alpha-dihydroxy-delta 5 or 3 beta,7 alpha,12 alpha-trihydroxy-delta 5 ring structures. In plasma, sulfated cholenoic acids constituted 65% and unconjugated 3 beta,7 alpha-dihydroxy-5-cholestenoic acid 25% of the total level, 71 micrograms/ml. The urinary excretion of the former was 30.4 mg/day and that of unsaturated bile alcohol sulfates, mainly pentols, 7 mg/day. The predominant bile acid in feces was an unconjugated epimer of 3 beta,7 alpha,12 alpha-trihydroxy-5-cholenoic acid, and small amounts of cholic acid were present. The minimum total excretion was 11.3 mg/day. Treatment with chenodeoxycholic acid resulted in marked clinical improvement and normalized liver function tests. Further studies are needed to define the mechanism of action. Plasma bile acids decreased to 1.6 micrograms/ml and urinary excretion to 3.4 mg/day. Chenodeoxycholic and ursodeoxycholic acids became predominant in all samples. The fecal excretion of unsaturated cholenoic acid sulfates increased to 40 mg/day compared to 89 mg/day of saturated bile acids. The results provide further support for a defective hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency, and indicate that the 3 beta-hydroxy-delta 5 bile acids are formed via 7 alpha-hydroxycholesterol. The formation of glycocholic acid may be due to an incomplete enzyme defect or to transformation of the 3 beta-hydroxy-delta 5 structure by bacterial and hepatic enzymes during an enterohepatic circulation.     

Identification of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid in chronic subdural hematoma.

We detected a novel kind of bile acid in the content of chronic subdural hematoma. This substance was specifically found in chronic subdural hematoma, and not in subdural hygroma, which is pathologically similar except for the lack of capsular membrane. The compound was identified as 7 alpha-hydroxy-3-oxo-4-cholestenoic acid by high performance liquid chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance spectrometry. The structure was confirmed by the comparison with the chemically synthesized compound. The average contents in chronic subdural hematoma were 658.09 +/- 137.53 ng/ml, while those in normal human plasma were 126.27 +/- 17.73 ng/ml. It was not detected in normal cerebrospinal fluid. The higher level in chronic subdural hematoma than human plasma strongly suggests the local, extrahepatic production of this type of C27 bile acids.     

Activation and peroxisomal beta-oxidation of fatty acids and bile acid intermediates in liver from Bombina orientalis and from the rat

1. Bombina orientalis excretes mainly C27 bile acids: trihydroxycoprostanic and varanic acids. More than 90% of the trihydroxycoprostanic acid (THCA) present in the bile, was conjugated with taurine; varanic acid was present in the unconjugated form. 2. Trihydroxycoprostanoyl-CoA (THC-CoA) synthetase activity, required for the formation of the taurine conjugate, was present in the liver of Bombina orientalis. 3. Peroxisomal beta-oxidation, which catalyzes the oxidation of fatty acids as well as the conversion of C27 bile acids into C24 bile acids in rat and human liver, could be detected in liver of Bombina orientalis when palmitoyl-CoA was used as substrate, but not when trihydroxycoprostanoyl-CoA (THC-CoA) was used.     

On the origin of the cholestenoic acids in human circulation

3 Beta-hydroxy-5-cholestenoic acid, 3 beta,7 alpha-dihydroxy-5-cholestenoic acid, and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid are metabolites of cholesterol present at significant concentrations (40-80 ng/ml) in human circulation. The 7 alpha-hydroxylated acids may be formed from cholesterol via two major pathways initiated by oxidations at either the 7 alpha- or 27-positions. In an attempt to clarify the origin and possible precursor-product relationships between these cholestenoic acids, we measured their deuterium enrichment in a unique experiment, after infusion of 10 g of [2H(6)]-cholesterol to a healthy volunteer. The observed extent and time-course of deuterium enrichment of circulating 3 beta-hydroxy-5-cholestenoic and 3 beta,7 alpha-dihydroxy-5-cholestenoic acid were almost identical, while different from that of cholesterol and 7 alpha-hydroxycholesterol. Notably, the deuterium enrichment of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid was similar to that of 7 alpha-hydroxycholesterol (and its metabolite 7 alpha-hydroxy-4-cholesten-3-one), though distinct from the other cholestenoic acids. Finally, the enrichment of unesterified 27-hydroxycholesterol followed a similar, though less pronounced, time curve to the delta(5)-cholestenoic acids. In conclusion, these results suggest that plasma 3 beta-hydroxy-5-cholestenoic acid is formed from a pool of cholesterol distinct from that used for the formation of the bulk of 27-hydroxycholesterol. The results are also in accordance with a formation of 3 beta,7 alpha-dihydroxy-5-cholestenoic acid directly from 3 beta-hydroxy-5-cholestenoic acid, and a formation of most of the circulating 7 alpha-hydroxy-4-cholesten-3-one from 7 alpha-hydroxycholesterol. These results are consistent with a flux of 7 alpha-hydroxycholesterol from the liver into the circulation, and an extrahepatic metabolism of this steroid into 7 alpha-hydroxy-3-oxo-4-cholestenoic acid.     

Rapid and quantitative analysis of unconjugated C(27) bile acids in plasma and blood samples by tandem mass spectrometry

A subgroup of peroxisomal disorders, peroxisome biogenesis defects (PBD), can be differentiated by elevated levels of C(27) bile acids in plasma and bile. Patients with peroxisomal disorders, who lack the ability to chain-shorten the C(27) bile acid intermediates into C(24) bile acids, show elevated levels of C(27) bile acids, notably 3 alpha,7 alpha-dihydroxy-5 beta-cholest-26-oic acid and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid. C(27) bile acids are normally estimated against other bile acid standards, by time-consuming gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry methods, in plasma (minimum of 50 microl). In this article we describe the quantitation of unconjugated di- and trihydroxy C(27) bile acids in 5-microl plasma samples and 3-mm blood spots, using deuterium-labeled internal standards. The synthesis of (2)H(3)-labeled di- and trihydroxycoprostanic acids is described. The sample preparation and analysis by electrospray tandem mass spectrometry (ES-MS/MS) takes less than 1 h and features dimethylaminoethyl ester derivatives. The levels of the di- and trihydroxy bile acids are significantly higher in PBD patients than in age-matched control subjects for both plasma and blood spots collected at birth (some stored for up to 18 years). Excellent correlation is observed between the C(26:0)/C(22:0) very long chain fatty acid (VLCFA) ratio and the levels of trihydroxy C(27) bile acids in plasma from PBD patients.The ES-MS/MS method can be used to rapidly screen for PBD patients in plasma samples with elevated C(26:0)/C(22:0) VLCFA ratios and in archived collections of neonatal blood spots. - Johnson, D. W., H. J. ten Brink, R. C. Schuit, and C. Jakobs. Rapid and quantitative analysis of unconjugated C(27) bile acids in plasma and blood samples by tandem mass spectrometry. J. Lipid Res. 2001. 42: 9;-16.     

Mitofusin 2 Protects Hepatocyte Mitochondrial Function from Damage Induced by GCDCA

Mitochondrial impairment is hypothesized to contribute to the pathogenesis of chronic cholestatic liver diseases. Mitofusin 2 (Mfn2) regulates mitochondrial morphology and signaling and is involved in the development of numerous mitochondrial-related diseases; however, a functional role for Mfn2 in chronic liver cholestasis which is characterized by increased levels of toxic bile acids remain unknown. Therefore, the aims of this study were to evaluate the expression levels of Mfn2 in liver samples from patients with extrahepatic cholestasis and to investigate the role Mfn2 during bile acid induced injury in vitro. Endogenous Mfn2 expression decreased in patients with extrahepatic cholestasis. Glycochenodeoxycholic acid (GCDCA) is the main toxic component of bile acid in patients with extrahepatic cholestasis. In human normal hepatocyte cells (L02), Mfn2 plays an important role in GCDCA-induced mitochondrial damage and changes in mitochondrial morphology. In line with the mitochondrial dysfunction, the expression of Mfn2 decreased significantly under GCDCA treatment conditions. Moreover, the overexpression of Mfn2 effectively attenuated mitochondrial fragmentation and reversed the mitochondrial damage observed in GCDCA-treated L02 cells. Notably, a truncated Mfn2 mutant that lacked the normal C-terminal domain lost the capacity to induce mitochondrial fusion. Increasing the expression of truncated Mfn2 also had a protective effect against the hepatotoxicity of GCDCA. Taken together, these findings indicate that the loss of Mfn2 may play a crucial role the pathogenesis of the liver damage that is observed in patients with extrahepatic cholestasis. The findings also indicate that Mfn2 may directly regulate mitochondrial metabolism independently of its primary fusion function. Therapeutic approaches that target Mfn2 may have protective effects against hepatotoxic of bile acids during cholestasis.     

Potential bile acid precursors in plasma--possible indicators of biosynthetic pathways to cholic and chenodeoxycholic acids in man

The plasma concentrations of 3 beta-hydroxy-5-cholestenoic acid, 3 beta,7 alpha-dihydroxy-5-cholestenoic acid and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid have been compared with that of 7 alpha-hydroxy-4-cholesten-3-one in healthy subjects and in patients with an expected decrease or increase of the bile acid production. In controls and patients with liver disease, the level of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid was positively correlated to that of 3 beta,7 alpha-dihydroxy-5-cholestenoic acid and not to that of 7 alpha-hydroxy-4-cholesten-3-one. In patients with stimulated bile acid formation the levels of the acids were not correlated to each other but there was a significant positive correlation between the levels of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid and 7 alpha-hydroxy-4-cholesten-3-one. These findings indicate that the precursor of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid differs depending on the activity of cholesterol 7 alpha-hydroxylase. Since the activity of this enzyme is reflected by the level of 7 alpha-hydroxy-4-cholesten-3-one in plasma the findings are compatible with a formation of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid from 3 beta,7 alpha-dihydroxy-5-cholestenoic acid when the rate of bile acid formation is normal or reduced and from 7 alpha-hydroxy-4-cholesten-3-one under conditions of increased bile acid synthesis. In support of this interpretation, 7 alpha,26-dihydroxy-4-cholesten-3-one was identified at elevated levels in plasma from patients with ileal resection or treated with cholestyramine. The levels of 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one were also higher than normal in these patients. Based on these findings and previous knowledge, a model is proposed for the biosynthesis of bile acids in man. Under normal conditions, two major pathways, one "neutral" and one "acidic" or "26-oxygenated", lead to the formation of cholic acid and chenodeoxycholic acid, respectively. These pathways are separately regulated. When the activity of cholesterol 7 alpha-hydroxylase is high, the "neutral" pathway is most important whereas the reverse is true when cholesterol 7 alpha-hydroxylase activity is low. In cases with enhanced activity of cholesterol 7 alpha-hydroxylase, the "neutral" pathway is connected to the "acidic" pathway via 7 alpha,26-dihydroxy-4-cholesten-3-one, whereas a flow from the acidic pathway to cholic acid appears to be of minor importance.     

Identification of unconjugated bile acids in human bile

Unconjugated bile acids in the bile of healthy and diseased humans were determined qualitatively and quantitatively by means of gas-liquid chromatography and gas-liquid chromatography-mass spectrometry, after their isolation by ion-exchange chromatography. In a healthy person and three patients with cholelithiasis, unconjugated bile acids comprised 0.1-0.4% of total biliary bile acids. The bile acid composition of the unconjugated fraction was quite different from that of the glycine- or taurine-conjugate fraction, in that it contained a relatively large proportion of unusual bile acids including C23 and C27 bile acids. In two patients with cerebrotendinous xanthomatosis, C22 and C23 bile acids were the major constituents of the biliary unconjugated bile acids, and comprised about 0.8% of total bile acids; no detectable amounts of C27 bile acids were found in their bile. The analysis of biliary unconjugated bile acids may be useful for the diagnosis of metabolic diseases concerning bile acids, particularly the accumulation or disappearance of unusual bile acids.     

Cholic acid synthesis from 26-hydroxycholesterol and 3-hydroxy-5-cholestenoic acid in the rabbit

Intravenous administration of 26-hydroxycholesterol to the rabbit with a bile fistula yielded cholic acid in proportions (84 and 86%) not significantly different from that derived from cholesterol. By contrast, the naturally occurring C27 bile acid 3 beta-hydroxy-5-cholestenoic acid yielded not more than 8% cholic acid. Thus initial 26-hydroxylation of cholesterol followed by 7-alpha-hydroxylation can provide sufficient amounts of cholic acid to be considered a quantitatively significant pathway for bile acid synthesis, and in addition it is the only pathway that can be the source of the circulating levels of C24 and C27 monohydroxy bile acids.     

Plasma amino acid imbalance in alcoholic liver cirrhosis

Plasma amino acid concentrations and plasma glucagon and serum insulin levels were studied in male patients with compensated alcoholic and nonalcoholic liver cirrhosis. Age, nutritional status, and liver function tests were similar in both groups; none of the patients presented hepatic encephalopathy. Plasma valine and leucine concentrations were lower, and tyrosine, higher in alcoholic than nonalcoholic liver cirrhosis. As a result, the molar ratios of branched-chain amino acids (BCAA) to aromatic amino acids (AAA) were reduced markedly in this group. Although correlation coefficients comparing BCAA/AAA ratios and KICG in alcoholic and nonalcoholic liver cirrhosis were similar, a steeper regression line was observed in alcoholics. Plasma glucagon and proline levels were significantly higher in alcoholic than nonalcoholic liver cirrhosis, the former correlated with AAA concentrations only in alcoholic liver cirrhosis, but not with BCAA levels. These results indicated that alcoholic liver cirrhosis presented a more deranged plasma amino acid pattern than nonalcoholic, and the amino acid imbalances, except for depressed BCAA and elevated proline, were derived, in part, from the hyperglucagonemia.     

Hepatocyte nuclear factor 4alpha is a central regulator of bile acid conjugation

Hepatocyte nuclear factor 4alpha (HNF4alpha) has an important role in regulating the expression of liver-specific genes. Because bile acids are produced from cholesterol in liver and many enzymes involved in their biosynthesis are preferentially expressed in liver, the role of HNF4alpha in the regulation of bile acid production was examined. In mice, unconjugated bile acids are conjugated with taurine by the liver-specific enzymes, bile acid-CoA ligase and bile acid-CoA:amino acid N-acyltransferase (BAT). Mice lacking hepatic HNF4alpha expression exhibited markedly decreased expression of the very long chain acyl-CoA synthase-related gene (VLACSR), a mouse candidate for bile acid-CoA ligase, and BAT. This was associated with markedly elevated levels of unconjugated and glycine-conjugated bile acids in gallbladder. HNF4alpha was found to bind directly to the mouse VLACSR and BAT gene promoters, and the promoter activities were dependent on HNF4alpha-binding sites and HNF4alpha expression. In conclusion, HNF4alpha plays a central role in bile acid conjugation by direct regulation of VLACSR and BAT in vivo.     

Itch in Liver Disease: Facts and Speculations

Pruritus in hepatobiliary disease is commonly believed to be caused by retention of bile acids with their sequestration in the skin. HOwever, we have recently demonstrated that skin levels of bile acids in patients with cholestasis correlate poorly with pruritus. In this report, we present additional data concerning the relationship of pruritus to bile acid retention: (1) the urinary excretion of sulfated and nonsulfated bile acids was not significantly different in patients with cholestasis who itched compared to those who did not; (2) one patient with itch associated with a liver abscess had normal levels of bile acids in serum, skin, and urine; (3) patients with primary biliary cirrhosis who itched had lower serum bile acid levels than patients with mechanical biliary obstruction who did not itch.These studies support our premise that pruritus in hepatobiliary diseases is not directly related to bile acid retention. They suggest that the type of cholestatic disorder, and not simply the magnitude of the cholestasis, as estimated by the elevation of serum bile acids, is important. We propose that the agent responsible for pruritus is produced in response to cholestasis, possibly through activation of the alternate pathway of bile acid synthesis. Properties of the hypothetical pruritogen are discussed.     

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.     

Differentiated quantification of human bile acids in serum by high-performance liquid chromatography-tandem mass spectrometry

Determination of quantitative changes in the pattern of serum bile acids is important for the monitoring of diseases affecting bile acid metabolism. A sensitive and specific high-performance liquid chromatography (HPLC)-MS/MS method was developed for the differentiated quantification of unconjugated as well as glycine- and taurine-conjugated cholic, chenodeoxycholic (CDCA), deoxycholic (DCA), ursodeoxycholic (UDCA) and lithocholic acid (LCA) in serum samples. After solid-phase extraction and reversed-phase HPLC separation, detection of the conjugated bile acids was performed using electrospray ionization (ESI)-MS/MS and selected reaction monitoring mode, whereas unconjugated bile acids were determined by ESI-MS and selected ion monitoring mode. The within-day and between-day coefficients of variation were below 7% for all bile acids and the recovery rates of the extraction procedure were between 84.9 and 105%. The developed method was applied to a group of 21 healthy volunteers and preliminary reference intervals in serum were established. In patients with drug-induced cholestasis, an elevation of primary bile acids has been shown.     

Circulating transforming growth factor beta(1) as an indicator of hepatic function impairment in liver cirrhosis

In the liver, transforming growth factor (TGF) -beta(1)is primarily responsible for activation of fat-storing cells, which are the main source of extracellular matrix proteins. Their deposition play a key role in the development of liver cirrhosis. The aim of this study was to evaluate plasma TGF-beta(1)in patients with different stages of liver cirrhosis and its possible use as an indicator of liver function impairment. TGF-beta(1)was measured in the plasma of 40 patients with liver cirrhosis. To estimate possible effect of liver insufficiency on plasma TGF-beta(1), patients were divided into three groups: A, B and C, univocal with Child-Pugh classes. Normal values were collected from 13 healthy volunteers. Liver cirrhosis resulted in a significant increase of plasma concentration of TGF-beta(1)(39.3+/-3.8 ng/ml), which doubled normal values (18.3+/-1.6 ng/ml). The highest concentrations were observed in alcoholic patients (44.4+/-4.7 ng/ml). TGF-beta(1)level increased depending on the degree of liver insufficiency, demonstrated by a significant positive correlation with Child-Pugh score (r=0.591). Values in group A were similar to normal, but were significantly elevated in groups B and C. These findings suggest possible use of plasma TGF-beta(1)measurement as an indicator of liver function impairment and possible marker of hepatic fibrosis progression in cirrhotic patients.     

Differential regulation of cytosolic and peroxisomal bile acid amidation by PPAR alpha activation favors the formation of unconjugated bile acids

In human liver, unconjugated bile acids can be formed by the action of bile acid-CoA thioesterases (BACTEs), whereas bile acid conjugation with taurine or glycine (amidation) is catalyzed by bile acid-CoA:amino acid N-acyltransferases (BACATs). Both pathways exist in peroxisomes and cytosol. Bile acid amidation facilitates biliary excretion, whereas the accumulation of unconjugated bile acids may become hepatotoxic. We hypothesized that the formation of unconjugated and conjugated bile acids from their common substrate bile acid-CoA thioesters by BACTE and BACAT is regulated via the peroxisome proliferator-activated receptor alpha (PPARalpha). Livers from wild-type and PPARalpha-null mice either untreated or treated with the PPARalpha activator WY-14,643 were analyzed for BACTE and BACAT expression. The total liver capacity of taurochenodeoxycholate and taurocholate formation was decreased in WY-14,643-treated wild-type mice by 60% and 40%, respectively, but not in PPARalpha-null mice. Suppression of the peroxisomal BACAT activity was responsible for the decrease in liver capacity, whereas cytosolic BACAT activity was essentially unchanged by the treatment. In both cytosol and peroxisomes, the BACTE activities and protein levels were upregulated 5- to 10-fold by the treatment. These effects caused by WY-14,643 treatment were abolished in PPARalpha-null mice. The results from this study suggest that an increased formation of unconjugated bile acids occurs during PPARalpha activation.