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The bile acid (BA) composition in mice is substantially different from that in humans. Chenodeoxycholic acid (CDCA) is an end product in the human liver; however, mouse Cyp2c70 metabolizes CDCA to hydrophilic muricholic acids (MCAs). Moreover, in humans, the gut microbiota converts the primary BAs, cholic acid and CDCA, into deoxycholic acid (DCA) and lithocholic acid (LCA), respectively. In contrast, the mouse Cyp2a12 reverts this action and converts these secondary BAs to primary BAs. Here, we generated Cyp2a12 KO, Cyp2c70 KO, and Cyp2a12/Cyp2c70 double KO (DKO) mice using the CRISPR-Cas9 system to study the regulation of BA metabolism under hydrophobic BA composition. Cyp2a12 KO mice showed the accumulation of DCAs, whereas Cyp2c70 KO mice lacked MCAs and exhibited markedly increased hepatobiliary proportions of CDCA. In DKO mice, not only DCAs or CDCAs but also DCAs, CDCAs, and LCAs were all elevated. In Cyp2c70 KO and DKO mice, chronic liver inflammation was observed depending on the hepatic unconjugated CDCA concentrations. The BA pool was markedly reduced in Cyp2c70 KO and DKO mice, but the FXR was not activated. It was suggested that the cytokine/c-Jun N-terminal kinase signaling pathway and the pregnane X receptor-mediated pathway are the predominant mechanisms, preferred over the FXR/small heterodimer partner and FXR/fibroblast growth factor 15 pathways, for controlling BA synthesis under hydrophobic BA composition. From our results, we hypothesize that these KO mice can be novel and useful models for investigating the roles of hydrophobic BAs in various human diseases.

Oxidative stress and dysregulated cholesterol metabolism are characteristic features of chronic hepatitis C virus infection (CHC). Therefore, we analyzed serum oxysterol profiles in CHC patients and examined the significance of oxysterols in CHC. The concentrations of 7α-hydroxycholesterol, 4β-hydroxycholesterol and 25-hydroxycholesterol as determined by LC-ESI-MS/MS were significantly elevated by +236%, +29% and +44%, respectively, in CHC patients compared with controls. Moreover, the elevated levels were significantly decreased by anti-viral therapy using PEGylated-interferon and ribavirin for 3 months. In contrast, 24S-hydroxycholesterol, 27-hydroxycholesterol and 7α-hydroxy-4-cholesten-3-one concentrations were not affected by CHC or anti-viral treatment. These results suggest that some oxysterols that are elevated in CHC are produced by cholesterol autoxidation due to oxidative stress or inflammation in the liver. Oxysterols may represent novel targets for the inhibition of disease progression and the prevention of hepatocarcinogenesis in CHC patients.

To date, many studies have been conducted using 25-hydroxycholesterol, which is a potent regulator of lipid metabolism. However, the origins of this oxysterol have not been entirely elucidated. Cholesterol 25-hydroxylase is one of the enzymes responsible for the metabolism of 25-hydroxycholesterol, but the expression of this enzyme is very low in humans. This oxysterol is also synthesized by sterol 27-hydroxylase (CYP27A1) and cholesterol 24-hydroxylase(CYP46A1), but it is only a minor product of these enzymes. We now report that CYP3A synthesizes a significant amount of 25-hydroxycholesterol and may participate in the regulation of lipid metabolism. Induction of CYP3A by pregnenolone-16α-carbonitrile caused the accumulation of 25-hydroxycholesterol in a cell line derived from mouse liver. Furthermore, treatment of the cells with troleandomycin, a specific inhibitor of CYP3A, significantly reduced cellular 25-hydroxycholesterol concentrations. In cells that overexpressed human recombinant CYP3A4, the activity of cholesterol 25-hydroxylation was found to be higher than that of cholesterol 4β-hydroxylation, a known marker activity of CYP3A4. In addition, 25-hydroxycholesterol concentrations in normal human sera correlated positively with the levels of 4β-hydroxycholesterol (r = 0.650, P < 0.0001, n = 78), but did not significantly correlate with the levels of 27-hydroxycholesterol or 24S-hydroxycholesterol. These results demonstrate the significance of CYP3A on the production of 25-hydroxycholesterol.

We describe a new sensitive and specific method for the quantification of serum malonate (malonic acid, MA), which could be a new biomarker for de novo lipogenesis (fatty acid synthesis). This method is based upon a stable isotope-dilution technique using LC-MS/MS. MA from 50 microl of serum was derivatized into di-(1-methyl-3-piperidinyl)malonate (DMP-MA) and quantified by LC-MS/MS using the positive electrospray ionization mode. The detection limit of the DMP-MA was approximately 4.8 fmol (500 fg) (signal-to-noise ratio = 10), which was more than 100 times more sensitive compared with that of MA by LC-MS/MS using the negative electrospray ionization mode. The relative standard deviations between sample preparations and measurements made using the present method were 4.4% and 3.2%, respectively, by one-way ANOVA. Recovery experiments were performed using 50 microl aliquots of normal human serum spiked with 9.6 pmol (1 ng) to 28.8 pmol (3 ng) of MA and were validated by orthogonal regression analysis. The results showed that the estimated amount within a 95% confidence limit was 14.1 +/- 1.1 pmol, which was in complete agreement with the observed X(0) = 15.0 +/- 0.6 pmol, with a mean recovery of 96.0%. This method provides reliable and reproducible results for the quantification of MA in human serum.

We have developed a highly sensitive and specific method for the analysis of serum sterol profiles. Sterols in 1 mul of dried serum were derivatized into picolinyl esters (3beta-picolinate) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the electrospray ionization (ESI) mode. In addition to cholesterol, 19 cholesterol precursors, cholestanol, campesterol, sitosterol, and sitostanol were identified simultaneously. Quantitative analyses for the picolinyl esters of 11 available sterols were performed, and detection limits were found to be less than 1 pg on-column. Reproducibilities and recoveries of 8 noncholesterol sterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.6% to 8.2% and 2.5% to 16.5%, respectively. The recovery experiments were performed using 1 mul aliquots of normal human serum spiked with 1 ng to 6 ng of sterols, and recoveries of the sterols ranged from 88.1% to 102.5% with a mean recovery of 98.1%. The present method provides reliable and reproducible results for the identification and quantification of neutral sterols, especially in small volumes of blood samples, which is useful for serological diagnosis of inherited disorders in cholesterol metabolism and for noninvasive evaluation of cholesterol biosynthesis and absorption in humans.

We have developed a new sensitive and specific nonradioisotope assay method to measure the activity of HMG-CoA reductase, the rate-controlling enzyme in the cholesterol biosynthetic pathway. This method was based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry using electrospray ionization in positive mode. Mevalonic acid, the product of HMG-CoA reductase, was converted to mevalonolactone (MVL) in an incubation mixture, extracted by a salting-out procedure, derivatized into the mevalonyl-(2-pyrrolidin-1-yl-ethyl)-amide, and then purified using a disposable silica cartridge. The resulting mevalonylamide was quantified by selected reaction monitoring using the positive electrospray ionization mode. The detection limit of this mevalonylamide was found to be 240 amol (signal-to-noise ratio=3), approximately 833 times more sensitive than that of MVL measured by a conventional radioisotope (RI) method (200 fmol). The variances between sample preparations and between measurements by this method were analyzed by one-way layout and calculated to be 3.2% and 1.8%, respectively. The recovery experiments were performed using incubation mixtures spiked with 0.77-2.31 nmol MVL/mg protein and were validated by a polynomial equation. These results showed that the estimated concentration within a 95% confidence limit was 0.47+/-0.07 nmol/mg protein, which coincided completely with the observed X0 nmol/mg protein with a mean recovery of 94.6%. This method made it possible to measure HMG-CoA reductase activity with a high degree of reproducibility and reliability, and especially with sensitivity superior to that of the conventional RI method.

Background/Aims The aim of the study was to examine the effects of taurine on hepatic fibrogenesis and in isolated hepatic stellate cells (HSC). Methods The rats of the hepatic damage (HD) group were administered carbon tetracholoride (CCl 4 ) for 5 weeks and a subgroup received, in addition, a 2% taurine containing diet for 6 weeks (HDT). The HSC were isolated from normal rats and cultured for 4 days. Results The hepatic taurine concentration was decreased in the HD group. This loss and the hepatic histological damage and fibrosis (particularly in the pericentral region), were reduced following taurine treatment. Furthermore, the hepatic alpha-SMA, lipid hydroperoxide and 8-OHdG levels in serum and liver, as well as hepatic TGF-β 1 mRNA and hydroxyproline levels were significantly increased in the HD group, and most of these parameters were significantly reduced following taurine treatment. In contrast to the MAP-kinase and Akt expressions, which remained unchanged, the lipid hydroperoxide and hydroxyproline concentrations, as well as TGF-β 1 mRNA levels were significantly reduced by taurine in activated HSC. Conclusions Oral taurine administration enhances hepatic taurine accumulation, reduces oxidative stress and prevents progression of hepatic fibrosis in CCl 4 -induced HD rats, as well as inhibits transformation of the HSC.

We have introduced S1–S2 paradigm (task S) as well as oddball paradigm (task O) visual event-related potentials (ERPs) under different interstimulus intervals (ISIs) in Parkinson's disease (PD). ERP measurements were correlated with motor disability, WAIS-R, and regional cerebral blood flow (rCBF). The ‘group’ influence was characterized by longer latency for P300, N200, and reaction time and decreased P300 amplitude in PD. Both P300 latency and reaction time during task O showed significantly longer latency in longer ISI condition. Our results revealed ‘ISI’ influence on ERPs during task S and significant correlation between ERPs and rCBF in task S.

Fibrates, including bezafibrate (BF), upregulate the expression of ATP binding cassette protein B4 (ABCB4) through gene transcription in mice. To determine the effects of BF on the expression levels of ABCB4 and on the stimulation of biliary phosphatidylcholine (PC) transport in human HepG2 hepatoblastoma cells, mRNA and protein levels as well as subcellular localization were investigated in the cells treated with BF. The canalicular accumulation of a fluorescent PC was assessed by confocal laser scanning microscopy. Treatment with 300 micromol/l BF for 24 h increased levels of ABCB4 mRNA but not protein by up to 151%. BF caused redistribution of ABCB4 into pseudocanaliculi formed between cells. In association with this redistribution, BF accelerated the accumulation of fluorescent PC in bile canaliculi (up to 163% of that in nontreated cells). Suppression of peroxisome proliferator-activated receptor alpha (PPARalpha) expression by either a small interfering RNA duplex or morpholino antisense oligonucleotide attenuated the BF-induced redistribution of ABCB4. These findings suggest that BF may enhance the capacity of human hepatocytes to direct PC into bile canaliculi via PPARalpha-mediated redistribution of ABCB4 to the canalicular membrane. This provides a rationale for the use of BF to improve cholestasis and/or cholangitis that is attributable to hypofunction of ABCB4.

Great interest has been focused on the chemoprevention of colonic carcinogenesis by oral administration of ursodeoxycholic acid (UDCA) because its administration reportedly reduces the incidence of colon cancer in animal experiments. To elucidate the precise role of UDCA in the chemoprevention of azoxymethane-induced colon carcinogenesis, we examined the expression levels of group II phospholipase A 2 in the colonic tissue of UDCA-treated and untreated rats and correlated the levels with the findings of aberrant crypt foci, putative preneoplastic lesions. Twelve weeks after azoxymethane exposure, the total number of aberrant crypt foci in 0.4% UDCA-fed rats and 1% UDCA-fed rats was significantly decreased compared to the untreated animals. The mucosal concentrations of PGE 2 and 6-keto PGF1 α were significantly lower in the UDCA-treated rats than in untreated rats. In correlation with lowering, the enhanced activity, protein mass and mRNA levels of group II phospholipase A 2 were significantly attenuated in the UDCA-treated animals. The chemopreventive role of UDCA in colon carcinogenesis may lie in its modulation of the arachidonate metabolism in colonic mucosa.

Multiple cholesterol stones are associated with more biliary complications and show more rapid cholesterol nucleation than solitary stones. Group II phospholipase A2 (PLA2-II) may play a critical role in the process of mucosal inflammation, which in turn may produce pronucleating agents. PLA2-II concentrations in gallbladders and gallbladder bile from patients with different types of gallstone disease were assayed to correlate PLA2-II with alterations in biliary composition.PLA2-II protein concentrations were assayed immunoradiometrically using monoclonal antibodies against human splenic PLA2-II.Immunoreactive PLA2-II levels in gallbladder bile were significantly higher in patients with multiple cholesterol stones (68.2 +/- 6.3 ng/dL, mean +/- SEM; n = 24) than in those with solitary stones (24.9 +/- 2.8; n = 20; P0.01), those with multiple pigment stones (24.2 +/- 3.7; n = 18; P0.01), or control subjects (13.4 +/- 1.7; n = 19; P0.01). Increased biliary immunoreactive PLA2-II levels in multiple cholesterol stones were associated with a concomitant increase in the lysophosphatidylcholine to phosphatidylcholine ratio; free arachidonate, protein, and hexosamine concentrations; and gallbladder bile viscosity. The gallbladders showed an increased PLA2-II protein mass and steady-state messenger RNA levels, which was associated with increased prostaglandin E2 levels.Increased biliary PLA2-II may be of pathogenetic importance in multiple cholesterol stones, probably through potentiating gallbladder mucosal inflammation with associated biliary alterations favoring cholesterol crystal formation.

Background and Aim: Recent studies have implicated several important hepatic cellular processes and signaling pathways that are affected by abnormal lipid metabolism, resulting in specific biochemical, histological, and clinical changes associated with NAFLD. We established highly sensitive and specific analysis of serum biomarkers for comprehensive view of the change in cholesterol and fatty acid metabolism by HPLC-ESI-MS/MS. By using this method, we monitored lipid metabolism in NAFLD patients before and during administration of pitavastatin. Materials and Methods: Serum samples were obtained from patients with NAFLD enrolled in multicenter case control study (n=15) at baseline, 3 months (mo), and 12 mo from initiation of pitavastatin treatment(1~2mg/day). Sex and age matched control (CTL) samples were obtained from 36 healthy volunteers with none of obesity, hyperlipidemia, and liver dysfunction. Internal standards were added to 10μL of serum. After extraction and derivatization, the samples were analyzed by LC-MS/MS equipped with reversed C18 column. Results: Markers of intestinal cholesterol absorption, serum sitosterol (CTL vs. NAFLD: 4.04±1.79 vs 1.69±0.68 ng/mL, P

Background: Alcoholic chronic liver disease (ACLD) is one of the most common forms of acquired immunodeficiency. Aim: To evaluate ex vivo toll-like receptor (TLR) 2 and TLR4 innate immune response in stable ACLD. Methods: Blood was collected from 26 males with ACLD in an outpatient hepatology clinic and from 10 controls. Serum was used for lipopolysaccharide (LPS), sCD14, LPS-binding protein(LBP), tumour necrosis factor alpha (TNF-α) and interleukin 10 (IL-10) quantification. mRNA expression of TLR2, TLR4, MD2, CD14, TNF-α and IL-10 was evaluated in anti-CD11b positive selected monocytes. Monocyte primary cultures were stimulated with zymosan (TLR2 agonist) or LPS (TLR4 agonist), and TNF-α production analyzed. Results: ACLD patients presented increased circulating LPS (+22.5±4.1%), LBP (+60.6±12.2%) and sCD14 (+23.5±4.6%), but with no differences in TNF-α and IL-10. TNF-αmRNA expression was decreased in monocytes from ACLD patients (-50.1±8.0%), with no significant differences in the other studied genes. Zymosan, but not LPS, induced TNF-α production by monocytes was blunted in ACLD (-55.2±10.9%). Results were similar in Child-Pugh A and B/C patients. See figure *=p

Background: We have previously demonstrated that Thl type cytokine mRNA firstly increased and Th2 type cytokine mRNA elevated subsequently in liver infiltrating CD4 ÷ T cells of PBC-like hepatic lesion using murine GVHR (Gastroenterology, 110, A1219, 1996). A number of cell adhesion molecules are important for CD4 ÷ T ceils to infiltrate the liver. Moreover, we have also shown that antibodies against adhesion molecules such as LFA-1 and VLA-4 suppressed the formation of PBC-like lesions (Hepatology, 22 (4) pt.2, 1995, Gastroenterology 110 (4) A1235, 1996). However, the precise molecular mechanism of these suppressions by the administration of antibodies against adhesion molecules still remains unknown. Aim: To clarify whether Thl/Th2 balance is related to the suppression of PBC-like hepatic lesions by antibodies against adhesion molecules. Methods: Sex-matched 1 x 107 C57BL/6 (B6) T spleen cells were injected into (B6.C-H-2 bml2 x B6) F 1 mice intravenously. Each 50 lag of anti-mouse t~ chain of VLA-4 mAb (PS2.3), anti-mouse VCAM-1 mAb (MK/2) or normal rat IgG were administered intraperitoneally per mouse during cell transfer. All mice were sacrificed 2 weeks after the first cell transfer. 1 x 10 -s liver infiltrating Thy 1.2+CD4 ÷ lymphocytes were sorted using FACS vantage and the RNA was isolated by AGPC method. A semiquantitative RT-PCR was carried out using primers specific for IL-2, IFN~, IL-4, IL-10. Immunohistochemical, HE staining, and ELISA for AMA were examined. Results: 1) H.E. staining showed the grade of portal cellular infiltration in the group administered both anti-VLA-4 mAbs and anti-VCAM-1 mAbs was significantly suppressed compared with the control administered normal rat IgG. The grade in the group administered just anti-VLA-4 mAbs was also suppressed compared with the control. 2) The induction of GVHR and the elevation of AMA titer were not affected in these groups by administering mAbs. 3) The expressions of IL-2, IFN'/, IL-4 and IL-10 mRNA were not changed in these groups. 4) Immunohistochemically, CD4, CDS, B220 or Mac-1 positive cells were detected in these groups. Conclusion: Although the administration against adhesion molecules did not suppress the induction of GVHR and the elevation of AMA titer, the portal cellular infiltration is reduced by just the administration of antibodies against VLA-4 in this model. However, cytokine profile of liver infiltrating CD4 ÷ T ceils and the presence of infiltrating ceils such as CD4, CD8, B220 or Mac-1 positive cells were not changed. In conclusion, it is suggested that the administration of antibodies against adhesion molecule could suppress PBC-like lesions without affecting Th 1/Th2 balance.

IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis and a promising target for cancer chemotherapy, was purified 4860-fold to homogeneity from rat hepatoma 3924A by a method including affinity chromatography in which IMP is bound to epoxy-activated Sepharose 6B. This affinity gel provided a specific elution of the enzyme with 0.5 mM IMP. The final enzyme preparation gave a single band with a molecular weight of 60,000 +/- 1000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis.

A rapid microassay method for the accurate measurement of the activity of inosine 5'-monophosphate dehydrogenase in crude tissue extracts was described. [8-14C]IMP and the radioactive products were separated by high-voltage electrophoresis in 0.1 M potassium phosphate buffer, pH 7.0, for 45 min. This separation method provides an analysis of the possible interfering reactions such as the metabolic conversion of the substrate IMP to inosine and adenylosuccinate, and the loss of the product XMP to xanthosine or GMP and to other metabolites. Low blank values were consistently obtained with this method because the XMP spot moves faster than the IMP spot. The major advantages of this assay method are direct measurement of IMP dehydrogenase activity in crude extracts, high sensitivity (with a limit of detection of 5 pmol of XMP production), high reproducibility (less than +/- 3.6%), low blank values (60-80 cpm), speed (2 h per 30 assays), and capability to measure activity in small amounts of tissue (10-50 mg wet wt).