Results: In livers from obese, diabetic mice with NASH, FC co-localised to plasma membrane, mitochondria and, to lesser extent, endoplasmic reticulum (ER). This pattern was replicated 5-Fluoracil cell line in primary hepatocytes incubated with LDL, which dose-dependently increased hepatocyte FC. Such FC loading caused dose-dependent increases in LDH

leakage, apoptosis (Höechst 33342) and necrosis (propidium iodide; release of high mobility group box1 [HMGB1]). At 40 μM LDL, cell death was associated with JNK1 activation (c-Jun phosphorylation), mitochondrial outer membrane pore transition (reduced tetramethyl rhodamine methyl ester fluorescence) resulting in cyt c release into cytoplasm, cellular oxidative stress (increased GSSG:GSH ratio) and ATP depletion. JNK inhibition by 1–2 μM CC-401 or CC-930 ameliorate FC-induced apoptosis and necrosis. Similarly, JNK1–/– primary hepatocytes

were refractory to FC-induced injury. Cyclosporine A (10 μM) and caspase-3 find more inhibition also protected WT hepatocytes from FC-mediated injury/cell death, but 500 μM 4-phenylbutyric acid (ER chaperone) had no effect and there was no evidence of ER stress in in vitro or in intact livers. FC deposition reduced plasma membrane fluidity (by pyrene eximer-to-monomer fluorescence emission), while blebbing and fragmentation/release of MPs from the surface of FC-injured hepatocytes was evident on TEM and SEM. Finally, addition of HMGB1-enriched culture medium or MP fractions from FC-loaded hepatocytes activated resting

KCs, as assessed by nuclear translocation of NF-κB, release of IL-1β, TNF-α and ultra-structural changes. Conclusions: These highly novel findings demonstrate how FC deposition in mitochondria and plasma membrane causes apoptosis and necrosis, confirm the centrality of JNK-1 activation for hepatocyte lipotoxic injury, and reveal direct links (via HMGB1 and MPs) between cholesterol lipotoxicity and engagement of KC activation/inflammatory recruitment in the transition of steatosis to NASH. 1. Van Rooyen DM, Larter CZ, Farrell GC et al. Hepatic Free Cholesterol Accumulated in Obese, Diabetic Mice and Causes Nonalcoholic Steatohepatitis. Gastroenterology 2011, 141(4), 1393–1403. 2. DM van Rooyen, Gan LT, Farrell 上海皓元医药股份有限公司 GC et al. Pharmacological cholesterol lowering reverses fibrotic NASH in obese diabetic mice. J Hepatol 2013;Mar 7. doi:pii: S0168-8278(13)00146-3. 10.1016/j.jhep.2013.02.024. KR MULLER,1 NS EYRE,1 KH VAN DER HOEK,1 K LI,2 MR BEARD1 1Hepatitis C Research Laboratory, University of Adelaide, South Australia, 2Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN, USA Introduction: Only a small proportion of cells in the hepatitis C virus (HCV)-infected liver harbour replicating HCV.

After 60 min of incubation at room temperature, the plates were washed three times and 100 μL of ortho-phenylenediamine substrate (OPD; Dako) containing H2O2, (4 OPD tablets were dissolved in 12 mL Tanespimycin concentration water and 5 μL 25% H2O2 was added just before use) was added to each well. After incubation for 18 min at room temperature, the reaction was stopped by adding 100 μL of 0.5 m H2SO4 (Merck, Whitehouse Station, NJ, USA) to each well. The optical density (OD)

in the wells was read at 490 nm wavelength using a Tecan Infinity F200 plate reader (Tecan, Männedorf, Switzerland). Samples from 10 healthy individuals, a positive control sample and two blank controls (quench buffer instead of sample) were analysed on each plate. All samples were run in duplicate and the mean OD was calculated after subtraction of the mean OD of the blank control. The cut-off for positivity was calculated based on the mean and +3 standard deviation (SD) of the 10 healthy individuals. Thus, on each plate, the OD readings are expressed as no. of SD above the mean of the 10 healthy individuals, AZD3965 molecular weight which results in a plate-specific cut-off that defines a positive sample. This is a necessary measure to avoid differences in classifications, as the absolute optical density

will vary between test runs. To approve a test run, the positive control sample had to be within

specified limits (95 percentile based on repeated measurements). To be classified as NNA in this study, a positive sample has to be positive on at least two independent test runs. To control for antibody specificity, the positive samples were mixed with excess antigen prior to the analysis. This was performed by pre-incubating the 1/100 diluted plasma sample for 2 h at 37°C with 0.5 μg of recombinant FVIII in a volume 200 μL. After incubation, the samples were re-analysed with ELISA assay according to the protocol described above. For four samples with a remaining positive, although significantly medchemexpress weaker, signal after pre-incubation with soluble FVIII, and for seven samples not assayed with pre-incubation with soluble FVIII, Protein G Sepharose adsorption was performed to decide if the positive signal was dependent on IgG in the sample or not. Five-hundred microlitre of Protein G Sepharose (Fast Flow; GE Healthcare, Uppsala, Sweden) was mixed with 100 μL plasma and 400 μL of quench buffer (see above) and incubated at room temperature for 2 h on a platform rotator. After incubation, the solution was diluted in quench buffer to reach a final plasma concentration of 1/100. The ELISA assay was then performed as described above.

567 CHIR99021 (range, −0.984-2.233) and 0.106 log IU/mL/year (range, −0.375-1.189), respectively (P < 0.001). The optimal HBsAg annual log reduction to predict HBsAg seroclearance was 0.5 log (Youden's index, 5.15; sensitivity, 62.8%; specificity, 88.7%). One hundred and seven patients with HBsAg seroclearance (52.7%) achieved ≥0.5 log reduction from 3 to 2 years, significantly more than 17 (8.4%) patients in the control group (P < 0.001). We further examined patients with serum HBsAg ≥200 IU/mL at 3 years (n = 33 and 150 for patients with HBsAg

seroclearance and controls, respectively). In this subgroup of patients, the AUC for HBsAg log reduction was 0.867 (P < 0.001; 95% confidence interval [CI]: 0.778-0.956), with a 0.5-log reduction most optimal in predicting HBsAg seroclearance (Youden's index, 6.35; sensitivity, 74.1%; specificity, 89.4%). For

patients with serum HBsAg <200 IU/mL (n = 170 and 53, respectively), the AUC for HBsAg log reduction was comparably lower at 0.796 (P < 0.001; 95% CI: 0.724-0.868). We also examined whether the addition of HBV DNA into HBsAg levels could improve the AUC for predicting HBsAg seroclearance. We found that there was no increase in AUCs using different combinations of HBsAg and HBV DNA in terms of their absolute levels and reductions (data not shown). Analyzing HBsAg among patients with undetectable HBV DNA levels produced an AUC of only 0.648 (P = 0.013; 95% CI: 0.538-0.823). SCH727965 Among the subgroups of patients with HBsAg ≥200 IU/mL, HBV DNA log reduction also produced an AUC of only 0.735 上海皓元 (P < 0.001; 95% CI: 0.623-0.848). Our current study demonstrated the kinetics of serum HBsAg and HBV DNA levels preceding HBsAg seroclearance in a large population of CHB patients with HBsAg seroclearance. To our knowledge, this is a study with the largest number of patients with HBsAg seroclearance to date (n = 203). Our present study outlines the changes in HBsAg kinetics before spontaneous HBsAg seroclearance. The enrollment of age- and

sex-matched controls would allow us to optimally delineate the differences in serologic and virologic kinetics between the two patient groups. With 3 years of serial data, we were able to show a marked difference in HBsAg levels between patients with HBsAg seroclearance and controls. In our study, the median HBsAg levels of controls were between 366 and 846 IU/mL at different time points, levels which were similar to those reported in other studies on serum HBsAg levels in HBeAg-negative CHB.13-15 The results of our control group also provide additional insight into the natural history of HBsAg levels in HBeAg-negative CHB. Serum HBsAg levels decreased gradually over time and appears to be a much more stable marker than HBV DNA levels, which are known for their fluctuating nature.25 Our study confirms that serum HBsAg measurements can be an important tool for physicians in weighing the chances of HBsAg seroclearance in the long term.

Quantitative real-time RT-PCR was performed using one-step SYBR Green PCR Master Mix (Takara) as per the manufacturer’s instructions. qRT-PCR was performed for the genes of interest and 16S rRNA selleck inhibitor in an iCycler IQ5 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). From threshold cycle (CT) values, the relative levels of expression of the genes of interest were calculated using the 2−ΔΔCT method [33], with 16S rRNA as an internal reference. The experiments were repeated at least thrice, and statistical significance of the observed differences

was calculated using the two-sample t-test. p-value of <.05 was considered significant. The genes whose expression was examined and the sequences of the primers used for each gene are given in Table S1. To introduce deletion mutation into the fur gene of H. pylori, two pairs of primers designated P1-P2 and P3-P4 (Table S1) were used to PCR amplify the up-and downstream regions, respectively, of the fur gene producing 306-bp and 310-bp products flanking the region Alisertib to be deleted. A chloramphenicol resistance gene (cat) was also PCR-amplified using primers designated C1 and C2. Primers P2 and P3 contained

21 base complementary regions at the 5′ region to C1 and C2, respectively (Table S1). A template mixture containing 200 ng to 400 ng of each of the three purified PCR products was then PCR-amplified using primers P1 and P4 in a single reaction, to generate a linear construct in which the up- and downstream fragments of fur were fused to the cat gene in the center. PCR conditions used were as follows: 10 cycles of 94 °C for 1 minute, 42 °C for 5 minutes and 72 °C for 4 minutes, followed by 40 cycles of 94 °C for 1 minute, 50 °C for 1 minute and 72 °C for 2.5 minute, and finally 72 °C for 10 minutes. The resulting allelic replacement construct was confirmed by sequencing

and introduced into H. pylori 26695 by natural transformation. Briefly, H. pylori cells from 48-hour confluent plates were suspended in 0.2 mL of BHI broth, about 3 μg PCR-amplified DNA was added, and the culture was spotted on GC agar plates. After incubation for 12 hours, the transformed cells were spread over the plates and incubated for a further 24 hours. The cells were then scraped onto selective GC agar plates containing chloramphenicol (20 μg per mL) and incubated for 3 to 7 days. Mutation 上海皓元医药股份有限公司 was confirmed by sequencing using primers P5 and P6. Two independent experiments were performed, and one mutant from each experiment was selected. Expression of the major virulence genes cagA, vacA, and ureA was examined in H. pylori strain 26695 following adherence to AGS cells and compared with that in unadhered bacteria grown under identical conditions without cell line. RNA was isolated from unadhered and adhered bacteria at different times after adherence, and expression of cagA, vacA and ureA was quantitated by real-time RT-PCR.

Pri-miRNAs are cleaved to precursor miRNAs (pre-miRNAs) of about 50–150 nucleotides by Drosha, an endoribonuclease III (RNAse III), and its cofactor RNA-binding protein Pasha (DGCR8). The pre-miRNAs are then exported to the cytoplasm by exportin 5.

This is further excised to double-stranded duplices of 20–23 nucleotides by Dicer, an RNAse III enzyme. The miRNA duplex later separates into single-stranded mature miRNA, and incorporates into the RNA-induced silencing complex (RISC), which is composed of Argonaute proteins. This complex binds to the 3′-untranslated region (3′-UTR) of its target transcript Vemurafenib and negatively regulates protein translation by a mechanism that depends on the complementarity between the miRNA and target messenger RNA. Partial complementarity results in translational repression, while complete complementarity triggers mRNA degradation. An important feature of miRNA is that a single miRNA can regulate multiple target

mRNAs. This pleiotropic property enables miRNAs to exert wide control see more on a network of genes. It has been demonstrated that overexpression of a single miRNA can downregulate over 100 mRNAs.5 Further, bioinformatic analysis predicts miRNAs can affect up to 30% of all human genes.6 It is therefore not surprising that miRNAs are involved in diverse physiological and developmental processes. These range from cell survival, differentiation, responses to external

stress and morphogenesis. In cancer, an imbalance of miRNA expression was first described in B-cell chronic lymphocytic leukemia,7 and by now virtually all examined tumor types display abnormal miRNA expression patterns. Studies on cancer miRNA have shown that they are often downregulated in tumor tissues, irrespective of cell type.8 This general phenomenon may be exemplified by the observation of common downregulation of Dicer, a key enzyme in the miRNA biogenesis, in many human neoplasms, including hepatocellular carcinoma (HCC),9 ovarian cancer,10 gastric cancer,11 and lung adenocarcinoma.12 In addition, reduced Dicer expression has been shown to correlate with shorter postoperative survival of patients with non-small-cell lung carcinoma.13 A study correlating the medchemexpress expression of precursor and mature miRNAs further showed that a vast number of miRNAs was transcribed but not processed to mature forms in cancer cell lines.14 Taken together, these findings seem to indicate that defective miRNA processing promotes transformation of normal cells to cancer, and that the miRNAome, as a whole, plays a critical role in tumor suppression. This review will focus on the miRNA dysregulation in HCC. The first part summarizes the involvement of miRNAs in the pathogenicity of HCC risk factors.

Sixteen percent of CH patients state that oxygen is unaffordable while 12% are getting

welder grade oxygen because of costs of medical grade oxygen, and this form of oxygen could be potentially dangerous to the individual user. Conclusions.— Oxygen is underutilized LDE225 purchase by CH patients living in the United States. Current recommended oxygen treatment regime is not meeting the needs of many CH patients. Prescribed oxygen flow rates are too low for efficacy. Oxygen can be expensive and very difficult to obtain. Physicians need to be better educated on the use of inhaled oxygen for CH. “
“Background.— The brain of migraineurs is hyperexcitable, particularly the occipital cortex, which is probably hypersensitive to light. Photophobia or hypersensitivity to light may be accounted for by an increased excitability of trigeminal, the visual pathways, and the occipital cortex. Objective.— To study light sensitivity and photophobia by assessing the response to light stimuli with functional magnetic selleck products resonance imaging–blood oxygenation level dependent (fMRI-BOLD) of the occipital cortex in migraineurs and in controls. Also, to try to decipher the contribution of the occipital cortex to photophobia and whether the cortical reactivity of migraineurs may be part of a constitutional (defensive) mechanism or represents an acquired (sensitization) phenomenon. Methods.— Nineteen patients with migraine

(7 with aura and 12 without aura) and 19 controls were studied with fMRI-BOLD during 4 increasing

light intensities. Eight axial image sections of 0.5 cm that covered the occipital cortex were acquired for each intensity. We measured the extension and the intensity of activation for every light stimuli. Photophobia was estimated according to a 0 to 3 semiquantitative scale of light discomfort. Results.— Migraineurs had a significantly higher number of fMRI-activated voxels at low (320.4 for migraineurs [SD = 253.9] and 164.3 for controls [SD = 102.7], P = .027) and medium-low luminance levels (501.2 for migraineurs [SD = 279.5] and 331.1 for controls [SD = 194.3], P = .034) but not at medium-high (579.5 for migraineurs [SD = 201.4] and 510.2 for controls [SD = 239.5], P = .410) and high light stimuli (496.2 for migraineurs [SD = 216.2] and 394.7 for controls [SD = 240], P = .210). No differences were found with respect 上海皓元医药股份有限公司 to the voxel activation intensity (amplitude of the BOLD wave) between migraineurs and controls (8.98 [SD = 2.58] vs 7.99 [SD = 2.57], P = .25; 10.82 [SD = 3.27] vs 9.81 [SD = 3.19], P = .31; 11.90 [SD = 3.18] vs 11.06 [SD = 2.56], P = .62; 11.45 [SD = 2.65] vs 10.25 [SD = 2.22], P = .16). Light discomfort was higher in the group of migraineurs at all the intensities tested, but there was no correlation with the number of activated voxels in the occipital cortex and photophobia. Repetitive light stimuli failed to demonstrate a lack of habituation in migraineurs. Conclusions.

1A and 1B. In these figures, it is demonstrated that many C282Y homozygotes have normal ALT levels, but also that patients with an elevated ALT level are unlikely to be C282Y homozygotes. The correlation between ALT and ferritin was stronger in C282Y homozygotes than in nonhomozygotes, which is consistent with an inflammatory cause of the hyperferritinemia in nonhomozygotes. Daporinad datasheet The proportion of male C282Y homozygotes with ALT and AST levels <40 IU/L was 71% and 87%, respectively. The proportion of female C282Y homozygotes with ALT and AST levels <40 IU/L was 87%

and 95%, respectively. The decreasing probability of being a C282Y homozygote across groups in men and women with increasing ALT levels is shown in Fig. 2. Similar results were determined for AST. P values for chi-square tests for trends in proportions for ALT were 0.036 for men and 0.00017 for women. Mantel-Haenszel chi-square adjusted for gender was <0.0001. An unanticipated observation was that the probability of being a C282Y homozygote decreased as serum ALT and AST levels increased. The results of the subgroup analysis limited to Caucasians were similar. It is widely believed that the probability of diagnosing many liver diseases increases as serum transaminases increase. In the present study of subjects with

hyperferritinemia, the probability of being a C282Y homozygote decreased with increasing ALT and AST levels. This probably occurs

because the deposition of excessive iron alone in hepatocytes of persons with hemochromatosis is not inflammatory. “Silent” hepatic fibrosis PD 332991 occurs in some subjects with hemochromatosis and normal serum transaminases.6, 7 On the other hand, some patients with hemochromatosis and HFE C282Y homozygosity have both hepatic iron overload and an inflammatory liver condition. For example, approximately 15% of C282Y homozygotes diagnosed in medical care 上海皓元医药股份有限公司 have severe hepatic steatosis proven by liver biopsy. These patients had higher median serum ALT and ferritin levels than C282Y homozygotes without hepatic steatosis or other inflammatory liver disorder.8 In contrast, patients referred for evaluation of elevated serum ferritin levels usually have hyperferritinemia resulting from inflammatory liver disease, rather than iron overload resulting from HFE hemochromatosis.9 In prospective analyses of subjects with chronic elevation of serum transaminases, hepatic steatosis associated with or without excessive ethanol consumption was the predominant cause of elevated serum transaminases.10-13 Hemochromatosis was rare in these case series.9 In the present study, there was a potential bias wherein HEIRS Study non-C282Y homozygous participants were deliberately selected for postscreening clinical examinations because they had elevated serum transferrin saturation and ferritin measures.

1 for all outcomes). No significant publication bias with regard to LTP was observed using the funnel selleckchem plot. Conclusions: To

our knowledge, this is the first meta-analysis comparing the two modalities in treating primary HCC. Based on the available evidence, both RFA and MWA are equally safe and effective. However, the results should be interpreted with caution because of the different types of generators and antennas used in these studies. Further well designed randomized controlled trials using current generators with high power output and with larger sample sizes are warranted to confirm these findings. MA CHINNARATHA,1 R MCCORMICK,2 R WUNDKE,2 RJ WOODMAN,1 AJ WIGG1,2 1School of Medicine, Flinders University of South Australia, 2Gastroenterology/Hepatology, Flinders Medical Centre, Bedford Park, SA Background and Aims: Bone

disease (BD) is a major complication of cirrhosis. Previous studies investigating the prevalence of BD in cirrhosis have focused on patients with a single etiology or those awaiting liver transplant. Our aim was to determine the prevalence of BD (both osteopenia and osteoporosis) in an unselected cohort of cirrhotic patients with mixed etiology and severity; and to determine risk factors for BD in cirrhosis. Methods: A single center review of prospectively collected data for buy MK-1775 consecutive patients newly diagnosed with cirrhosis between Sep 2009 and Dec 2012. All patients underwent Bone Mineral Density (BMD) assessment using Dual Energy X-ray Absorptiometry (DEXA) within 3 months of diagnosis. Relevant clinical and biochemical data were collected on diagnosis and with follow-up patient survey. Osteoporosis was defined as a T-score 上海皓元医药股份有限公司 <−2.5 SD. Binary logistic regression was used to determine

risk factors associated with BD (T-score <−1 SD or a Z-score <−2 SD). Results: Data was collected for a total of 406 subjects (67% males) with a mean (±SD) age of 56.2 (±10.9) years. Alcohol (41.1%), hepatitis C (HCV) + alcohol (16.5%) and HCV (16%) were the most common etiologies. 84% of patients were either Childs-Pugh class A or B with a mean MELD (±SD) score of 12.4 (±5.7). The prevalence of BD was 56% of the total cohort. Osteoporosis was present in 20.5% (66/320) of patients with a T-score measurement. Moderate or severe vitamin D deficiency (≤50 nmol/L) was present in 54% (212/389) and fragility fractures in 3.3% (12/362) of patients. In multivariate analysis, only older age and lower BMI were significant independent risk factors for BD (Table) with both displaying a linear trend. Amongst females, high serum FSH level, irrespective of menopausal status, was associated with BD in univariate analysis [OR (95%CI) = 1.01 (1.00–1.03), p = 0.04] but was no longer associated with BD after adjustment for age and BMI. Conclusion: This is the largest study of bone disease in unselected cirrhotic subjects with mixed etiology and disease severity.

In the JFH-1cc–infected chimpanzee, genome sequence of predominant infecting virus at week 2 was identical to JFH-1 wild-type

(JFH-1/wt [in this study, this abbreviation was used instead of JFH-1 to distinguish it from other variant strains]), and the infecting virus has four synonymous and seven nonsynonymous mutations at week 7. In the JFH-1 patient serum–infected chimpanzee, 19 synonymous and six nonsynonymous mutations were observed in predominantly circulating virus at week 2, and this number increased to 35 synonymous and 17 nonsynonymous mutations at the later stage of infection course (week 23).11 From these observations, we presumed that the isolates evolved in each chimpanzee at later stages of infection might have some advantage over the viruses isolated at earlier time points for survival in infected animals. Thus, in this study, we generated JFH-1 variants containing the mutations observed in these animals and assessed their effect on replication selleck and infectious virus production Sorafenib price in cell culture. Furthermore, we examined the effects of infection of these strains to tumor necrosis factor α (TNF-α)– or Fas ligand (FasL)–mediated

apoptosis. Ag, antigen; CTL, cytotoxic T lymphocytes; FasL, Fas ligand; HCV, hepatitis C virus; JFH-1cc, cell culture–generated JFH-1 virus; JFH-1/wt, JFH-1 wild-type; MFI, mean fluorescence intensity; NK, natural killer, NS, nonstructural; PARP, poly(adenosine diphosphate ribose) polymerase; TNF-α, tumor necrosis factor α; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling. The complete Materials

上海皓元医药股份有限公司 and Methods are provided in the Supporting Information. To investigate the effect of mutations on virus phenotype, we generated constructs containing the mutations observed in JFH-1 patient serum–infected chimpanzee and JFH-1cc–infected chimpanzee at various time points. The JFH-1 variants JFH-1/S1 and JFH-1/S2 contain the mutations observed in the patient serum–infected chimpanzee at week 2 and week 23, respectively, and JFH-1/C contains the mutations observed in the JFH-1cc–infected chimpanzee at week 7 (Supporting Table 1). The replication and virus production capacity of these variants in HuH-7 cells was compared with that of JFH-1/wt. After electroporation of in vitro–synthesized full-genome RNA of JFH-1/wt and variant strains, extracellular and intracellular HCV RNA and core antigen (Ag) were measured (Fig. 1). At day 5 posttransfection, all constructs displayed similar intracellular HCV RNA levels. However, extracellular HCV RNA level of JFH-1/C was 1.6 times higher than that of JFH-1/wt. Likewise, extracellular HCV RNA level of JFH-1/S2 was 3.4 times higher than that of JFH-1/S1 (Fig. 1A). Intracellular HCV core Ag levels of JFH-1/S2 and C were 240.9 ± 58.2 and 189.8 ± 42.1 fmol/mg protein, respectively, and were significantly lower (P < 0.005) than that of JFH-1/S1 (526.1 ± 58.2 fmol/mg protein) and JFH-1/wt (511.7 ± 32.

Moreover, experiments carried out with freshly isolated rat hepatocytes HSP inhibitor indicated that these cells can release NO species when they are incubated with UDCA. UDCA-induced

release of NO from isolated hepatocytes was not observed in the presence of the protein synthesis inhibitor cycloheximide, and this supports the involvement of increased production of iNOS in these liver cells. It has been shown that SNOs prolong the NO half-life and allow this molecule to be transported in biological fluids to fulfill biological functions at places distant from the point at which it is produced.15 The considerable abundance of glutathione in bile makes this compound an excellent carrier for the transport of functional NO along the biliary tree. In our study, we observed a great rise in SNOs upon UDCA infusion, mainly at the expense of SNOs with a molecular weight less than 10 kDa, and this strongly suggests that GSNO is the most likely NO carrier in bile. MS analysis confirmed that, upon UDCA infusion, there was a rise in GSNO and putative GSNO derivatives in bile. Moreover, when the rat liver was depleted of glutathione with BSO, the infusion of UDCA failed to induce any increase in biliary NO, and this

further reinforces the view that GSNO has an essential role as the NO carrier in bile. ABCC2/Mrp2 is a canalicular protein involved in the transport of glutathione and glutathione conjugates to bile (reviewed by Ballatori et al.33). In TR− rats, which have defective ABCC2 function,27 there is a marked MCE impairment in biliary secretion of glutathione, the concentration of which falls from the normal Atezolizumab millimolar range to a micromolar range.28 This impairment is associated with decreased biliary NO secretion in response to UDCA. The level of total biliary NO (which is normally excreted at concentrations in the micromolar range) falls to less than half of normal

values, and the level of biliary SNOs falls to about one-third of normal. This observation reveals a role of ABCC2 in mediating, at least in part, the canalicular efflux of NO species from hepatocytes to bile. Furthermore, this finding is consistent with the existence of a link between biliary NO secretion and biliary transport of glutathione. Although GSNO might be the prevalent NO species in canalicular bile, its catabolism or degradation to nitrites/nitrates within the bile ducts may create a gradient of decreasing concentration along the biliary tree. Also, a portion of the GSNO that remains intact in the bile duct lumen may enter the bile duct epithelial cells. It thus seems probable that the actual concentration of GSNO that is secreted at the canaliculi and drains into the intrahepatic bile ducts is substantially higher than that detected at the end of the common bile duct. Our data indicate that the secretion of GSNO to bile is critical for the hypercholeretic activity of UDCA.