Jn Plevris

Evidence of hydrogen ion secretion from the human gall bladder in vitro.

Gall bladder bile is more acid that hepatic bile and this has been attributed to bicarbonate absorption by the gall bladder epithelium. The aim of this study was to investigate in vitro the acid base changes that occur across the human gall bladder mucosa. Fresh gall bladder tissue was obtained at cholecystectomy and placed in an Ussing Chamber and perfused with Ringer-Krebs glucose bicarbonate solution. The viability of the gall bladder was assessed by measuring the potential differences across the epithelium and by the morphology of the epithelial cells at the end of the experiments. Aliquots from the solutions were taken at two, 45 and 70 minutes and pCO2, hydrogen ion and bicarbonate concentrations were measured. In the mucosal side of the chamber a consistent and significant decrease was observed from two minutes to 70 minutes in bicarbonate concentration while pCO2 and hydrogen ion concentrations significantly increased. The degree of inflammation correlated well with the ability for acidification, the more inflamed the tissue the less its ability to acidify. When the gall bladder was exposed to amiloride or sodium free solution acidification was abolished in the mucosal side. When tissue metabolism was irreversibly inhibited by exposure to formaldehyde, hydrogen ion concentration and pCO2 were significantly decreased in the mucosal side of the chamber compared with the viable gall bladder. The human gall bladder is capable of secreting acid and this may be an important mechanism for preventing calcium precipitation and gall stone formation.

PTH-068 The use of Oesophageal Capsule Endoscopy in Patients with Haemophilia; Experience from a Tertiary Centre

Introduction A great proportion of haemophiliacs are considered at risk of being co-infected with hepatitis C (HCV) and variant Creutzfeldt-Jakob Disease (vCJD). 1 Chronic hepatitis C leads to liver cirrhosis, which in turn causes portal hypertension and varices. 2 Alternative endoscopic modalities have been developed for the investigation of the upper gastrointestinal (GI) tract, such as oesophageal capsule endoscopy (OCE). However, OCE is widely accepted and its indications are still under evaluation. 3 Our aim was to evaluate the use of OCE in a tertiary referral centre for GI problems in Lothian, Southeast Scotland, giving a special focus on OCE in haemophiliacs. Methods A retrospective review of the OCE database from May 2005 to March 2012. Electronic case notes and OCE reports were reviewed. Demographics and clinical background, in particular haemophilia, hepatitis C, HIV and cirrhosis, reason for referral and OCE findings were abstracted. Results A total of 65 OCEs (50 patients; 27 M/23 F; mean age: 52.7 ± 13.7 years) were carried out in the aforementioned period. 32% pts had haemophilia (16/50 patients/all male; mean age: 51.6 ± 9.8 years; range 31–78 years; 28 OCEs); 5 pts had repeat OCEs (1 pt: 1 repeat, 2 pts: 2 repeat, 1 pt: 3 repeat & 1 patient: 4 repeat OCEs). All haemophiliacs were infected with HCV; 2 pts were co-infected with HIV. 3/16 (18.75%) of haemophiliacs had established cirrhosis, 5/16 (31.25%) probable cirrhosis. In haemophiliacs, indications for OCE were: variceal surveillance (OCEs group A: 17/28; 60.7%) and/or other upper GI symptoms (OCEs group B: 11/28; 39.3%). PillCam®ESO1 was used in 15/28 (53.6%) occasions and PillCam®ESO2 for the rest (13/28; 46.4%). The overall diagnostic yield (DY) of OCE in haemophiliacs was 78% (21/28). The DY was similar in OCEs group A: 64.7% (findings in 11/17) and OCEs group B: 54.5% (findings in 6/11 ), P = 1.0. Oesophageal transit times were mean: 166s; range: 3–1171s. All capsules reached the stomach, but only 8/28 (28.5%) capsules entered the duodenum. Conclusion OCE is a useful and acceptable alternative to conventional endoscopy in selected groups of patients. In particular, OCE in haemophiliacs has a high DY and should be considered a first line investigation to guide further endoscopic intervention. Disclosure of Interest None Declared. References Meijer K, et al. HCV-related liver cancer in people with haemophilia. Haemophilia 2012; 18:17–24 de Franchis R. Non-invasive (and minimally invasive) diagnosis of oesophageal varices. J Hepatol 2008; 49:520–7 Guturu P, et al. Capsule endoscopy with PILLCAM ESO for detecting esophageal varices: a meta-analysis. Minerva Gastroenterol Dietol 2011; 57:1–11

Electronic-nose breath print distinguishes non-alcoholic fatty liver disease from healthy lean control: A pilot study

Sensor 1, Sensor 2, Sensor 3 and Sensor 4 identified NAFLD cirrhosis patients with AUC 0.96 (standard error=0.043; p<0.001), 0.89 (standard error=0.046; p<0.001), 0.98 (standard error =0.016; p<0.001) and 0.96 (standard error=0.022; p<0.001) respectively eNose was able to differentiate between healthy from; non-cirrhotic NAFLD (p<0.001, CVV 96.8%) and NAFLD cirrhotic (p<0.001, CVV 95.1%). This method, designed to reflect the generalization property of the k-nearest neighbour’s (k-NN) classifier, scored a classification rate of 96%. METHODS

PWE-144 Small Bowel Capsule Endoscopy In Patients With Cirrhosis: The Edinburgh Experience

Introduction Portal hypertensive enteropathy (PHE) remains difficult to diagnose in patients with cirrhosis and portal hypertension. Limited test choices exist for the inspection of the small bowel in these patients. Small bowel capsule endoscopy (SBCE) would be ideal in this situation but it is rarely performed.1–3 Aim We aimed to determine the prevalence of PHE using SBCE in a cirrhotic patient population from our centre. Methods This was a retrospective study using the SBCE data base of our unit. We searched through 1,477 patients that had SBCE between 2005 and 2013. Patients with cirrhosis who underwent SBCE were identified, data retrieved and abstracted. The Fischer’s exact or the chi-square tests were used to compare between groups. A two-tailed P value of <0.05 was considered statistically significant. Results We identified 53 patients with cirrhosis who underwent SCBE. We used PillCam®SB (Given®Imaging Ltd, Israel) system on 36 patients and the MiroCam® capsule (IntroMedic Co, Korea) on 17 patients. Thirty patients were referred for iron deficiency anaemia, 15 for obscure gastrointestinal bleeding, and 4 for other indications. Four data sets were not available for review at the time of the study, leaving 49 patients to be reviewed. Mean age was 61.19 ± 14.54 years (M/F=27/22). Table 1 shows the aetiologies of liver disease in these patients. Six SBCE examinations were incomplete. Thirty three patients had evidence of portal hypertensive gastropathy (PHG) and 17 patients had evidence of oesophageal varices. In total, 29 patients had SCBE evidence of PHE (67%). 28/29 (96.5%) of patients with PHE had also evidence of PHG. 13/17 (76.4%) patients with oesophageal varices had also evidence of PHE. Our mean follow up was 58.0 ± 13.7 months. Twenty patients died during the follow up period. There was no correlation between the presence of PHE and aetiology of liver disease (P = 0.4261) or subsequent death (P= 0.2145). Abstract PWE-144 Table 1 ALD 15 NAFLD 9 HepC 7 Cryptogenic 6 PBC 6 Other 6 Conclusion The prevalence of PHE in our study was 67%. SBCE is a useful tool in evaluating PHE in cirrhotic patients irrespective of aetiology. References Rondonotti E, et al. Capsule endoscopy in portal hypertension. Clin Liver Dis 2010;14:209–20. Krystallis C, et al. Update of endoscopy in liver disease: more than just treating varices. World J Gastroenterol 2012;18:401–11 Sidhu R, et al. Does small bowel capsule endoscopy alter management in patients with liver disease? Scand J Gastroenterol 2011;46:123–4 Disclosure of Interest None Declared.

PTU-023 The use of Prokinetics in Small-Bowel Capsule Endoscopy: a Systematic Review and Meta-Analysis

Introduction Small-bowel capsule endoscopy (SBCE) is often limited by incomplete small-bowel transit. Although there are available meta-analysis data on the use of purgatives in SBCE, there is no similar data or consensus regarding the regular use of prokinetics for capsule ingestion. Our aim was to systematically review existing literature on the use of prokinetics in SBCE. Methods Thorough and extensive, recursive search of PubMed/MEDLINE, Embase and Scopus databases for studies, published to the end of Nov 2012, was performed. No language, time or age limits were used. Abroad search strategy was employed, using the MeSH term “capsule endoscopy” connected with the following keywords by “AND”: “prokinetic”, “promotility”, “metoclopramide”, “domperidone”, “erythromycin”, “antiemetic”, “ondensetron”, “completion”, “gastric emptying”, “transit”, “ingestion”, “preparation”, “oral/liquid”, “intramuscular” & “retention”. Additionally, the reference list of all the selected articles was manually checked for potentially suitable references that were not identified by the initial search. Studies were selected based on title and/or abstract. Eligible studies were included if the met all of the following criteria: (1)published as full articles of randomised control trials, (2)contained information on the type of the SBCE system used, (3)used prokinetics in (at least) one of the reported study arms/groups, (4)specified the type and dose of prokinetics used & (5)contained data on the rate of SBCE completion to caecum (CR). Data were extracted by the first author using a predifined Excel sheet. Primary end-point: the effect of prokinetics to SBCE CR. Results A total of 13 studies (all prospective, randomised-controlled, single-centre; total of 1439 subjects) was selected for final review and analysis. In 11 of them, PillCam® (Given®Imaging Ltd) was used; 2 studies were performed with OMOM® (Chongqingjinshan Science & Tech Co, Ltd). 6 studies were designed to look at the value of metoclopramide vs control. In the remainder, other type of prokinetic factors (Erythromycin, Mosapride, Lubiprostone, Deikenchuto or chewing gum) was administered. Using random effects model analysis, the use of prokinetics seem to improve CR in SBCE (OR = 1.888, 95% C.I. = 1.178, 3.02; I2 = 52.5%, P = 0.014). Moreover, in the sub-analysis for metoclopramide studies using fixed effect model, the results were similar (OR = 1.711 95% C.I. = 1.138, 2.573; I2 = 42.3%, P = 0.123). Conclusion Pooled data show that in comparison to no prokinetic, any type of administered prokinetic factor, before SBCE, improves the SBCE completion rate. Furthermore, most data to present are behind the use of Metoclopramide. Disclosure of Interest None Declared

PMO-130 Altered acetyl-coa metabolism in hepatic mitochondrial impairment in in vitro models of hepatic cellular steatosis

Introduction Increased ketogenesis, in the presence of unaltered β-oxidation, is a feature of human steatohepatitis. This is thought to be attributable to decreased acetyl-coA entry to tricarboxylic acid cycle with mitochondrial impairment. In this study, we examined the diversion of acetyl-coA towards free fatty acid (FFA) biosynthesis and mevalonate pathways (including vitamin D3, steroids hormones and bile acids) in the presence of mitochondrial dysfunction and triglyceride accumulation. Methods Human hepatoblastoma C3A cells were treated with; oleate or various combinations of octanoate (O), lactate (L), pyruvate (P) and ammonia (N) for 72 h. Metabolites that correspond to the intermediates of FFA biosynthesis, mevalonate pathways were measured using metabolomics study. Results We have previously shown that LPON treatment, but not oleate, affected mitochondrial function as evidenced by decreased respiration and ROS formation with concomitant enhanced ketogenesis despite the similarities in triglyceride accumulation. Using metabolomics analysis, we identified three metabolites that correspond to FFA biosynthesis, three were bile acids and three were the derivatives of steroid hormones and vitamin D3 synthesis. We also identified mevalonate and 7-dehydrodemosterol, the intermediates of cholesterol biosynthesis. The concentrations of FFA biosynthesis intermediates were higher with LPON compared with oleate (3-oxo-tetradecanoate (p=0.005) and 3-oxo-hexadecanoate (p=0.02)). Although mevalonate (p=0.37) and 7-dehydrodesmosterol (p=0.46) levels were higher with oleate than that seen with LPON, these differences did not reach statistical significance. In contrast, bile acids were significantly elevated with oleate than LPON ((taurocholate (p=0.002), glycocholate (p=0.001), (6RS)-22-oxo-23,24,25,26,27-pentanorvitamin D3 6,19-sulphur dioxide adduct (p=0.04) and 1,25-dihydroxy-2,4-dinor-1,3-secovitamin D3 (p=0.0006). Conclusion These data suggest that, aside from enhanced ketogenesis, impaired mitochondrial function is also associated with acetyl-coA diversion towards FFA synthesis, but not mevalonate pathways. These differences are likely to reflect cellular demand in the presence of decreased ATP formation with mitochondrial dysfunction. Competing interests None declared.

PMO-122 Eicosapentaenoic acid is effective at reducing hepatocyte triglyceride content of untreated C3A cells but is not effective in two models of cellular steatosis

Introduction Eicosapentaenoic acid (EPA), one of the major physiologically active constituents of Omega-3 fatty acids, has been suggested as a treatment for non-alcoholic fatty liver disease (NAFLD). The aim of these experiments was to assess the effects of EPA on intrahepatic triglyceride content in cell culture models of steatosis. Methods Human C3a hepatocytes were incubated in MEME (standard media) and two models of cellular steatosis: oleate (a model of isolated steatosis) and LPON (a model of steatosis and mitochondrial dysfunction containing the gluconeogenic substrates Lacate, Pyruvate, Octanoate and ammoNia). Test media was either unsupplemented, or supplemented with 50 μM or 250 μM EPA. Hepatocyte triglyceride accumulation was assessed both by microscopy (using oil red staining) and by quantifying the intracellular triglyceride concentration of cells incubated in culture media for 3 and 7 days. Each cell culture experiment was performed in triplicate. Results MEME When quantified by oil red staining a 73.1% (95% CI 63% to 83%) reduction in cell triglyceride content with 250 μM EPA compared with untreated cells was seen (7659 vs 28 564 pixels; p<0.001). This was confirmed in cell culture experiments as 250 μM EPA was associated with reduced intrahepatocyte triglyceride content after both 3 (74.1 vs 94.9 mmol/gTP; p<0.05) and 7 days (62.2 vs 80.9 mmol/gTP; p<0.05) incubation compared with untreated cells equating to a 21.9% (95% CI 9% to 35%) and 23.1% (95% CI 5% to 41%) reduction respectively. For both experiments a linear trend between increasing EPA concentration and reduced triglyceride content was confirmed. Oleate Here reduced triglyceride content with both 50 μM EPA (p<0.01) and 250 μM EPA (p<0.05) was seen with oil red staining and equated to reductions of 27.6% (95% CI 16% to 39%) and 22.5% (95% CI 9% to 36%) compared with untreated cells. However these results were not reproduced in cell culture experiments although on post test analysis there was a significant linear trend between increasing EPA concentration and reduced triglyceride content (p=0.04). LPON Although incubation with 250 μM EPA reduced triglyceride content in the LPON model when quantified with oil red staining (60 308 vs 79 219 pixels in 250 μM EPA vs untreated cells; p<0.05) this was not confirmed in cell culture experiments. On post hoc analysis no trend was demonstrated between EPA concentration and triglyceride content. Conclusion These results suggest that EPA is effective at reducing triglyceride accumulation in untreated hepatocytes but is not effective in either oleate or LPON models of cellular steatosis. It is therefore possible that the presence of steatosis and mitochondrial dysfunction in NAFLD may limit the efficacy of EPA as a treatment. Competing interests None declared.

Tissue engineering of vascularised human liver organoids: study of morphology phenotypic expression and metabolomics of transitional co-cultures of hepatic/endothelial progenitors

Introduction Tissue architecture and hepatic cell morphology reflect the functional differentiation of the liver. Differentiated functions of hepatocytes, depend on complex, hetereotypic cell-cell/cell-matrix interactions mediated by cell adhesion molecules (CAMs) in a 3D microenvironment. Vascularisation of human hepatic tissue for therapeutic/pharmaceutical applications, requires knowledge of optimal trophic conditions to support different cell types and how these cells behave embedded in different biocompatible matrices. Methods The authors aimed to assess morphology, metabolic functionality and phenotypic expression of human progenitor EoCs (Endothelial outgrowth Cells) vascular microvessels, cocultured with hepatic C3A cells (C3As). EoC/C3A cocultures in different media and test biomatrices: Matrigel/MaxGel/Puramatrix (self-assembling nanofibres); and control standard 2D cultures (tissue culture plastic; TCP) were analysed following immunostaining using morphology (light/confocal microscopy); and flow cytometry. Metabolomics analysis of culture media provided a global picture of metabolic changes in response to hepatic/EoC coculture (vs reference C3A mono-cultures). Results Titration in standard 2D/TCP co-culture in various media showed a ratio of 3C3A:1EoC in Lonza EGM-2 medium was optimal. Cells retained phenotypic expression of differentiation markers: (1) Hepatic: Albumin, EpCAM, E-CAD; (2) EoC: CD146, CD31, CD105, VWF; as evidenced by flow cytometry/immunostaining. Metabolomics analysis of media (from 2D/TCP co-cultures), showed modulation of key hepatic metabolic intermediaries including: (1) Urea cycle: 50% enhancement of L-ornithine production; (2) Biosynthesis: Bile Acids: Enhanced Glycocholate; amino acid (eg, Taurine) utilisation; and Creatine production: (3) Antioxidants: co-culture ameliorated the requirement of high Glutathione antioxidant levels inherent to EoC monocultures. Test biomatrices under different 3D culture configurations, showed: (1) MaxGel sandwich culture promoted differentiated (cuboidal) morphology of C3As, but not EoCs; (2) Conversely, only EoCs overlaid on MaxGel formed differentiated (microvessel) structures; (3) Puramatrix supported 3D culture of C3As but not EoCs; (4) Matrigel supported only EoCs 3D microtubular structures. Conclusion This study proved informative for engineering of vascularised organoids for future clinical/pharmaceutical applications. Work in progress include comprehensive metabolomics analysis, flow cytometric profiling and combinatorial analysis of cell-supportive biomatrix configurations to further optimise 3D coculture.

P53 The contrasting effect of octanoate and oleate on phosphatase and tensin homologue expression in in vitro model of steatosis using HepG2/C3A cells

Introduction The tumour suppressor phosphatase and tensin homologue (PTEN) is mutated or deleted in several human cancers including hepatocellular carcinoma. PTEN-deficient mice demonstrated triglyceride accumulation, steatohepatitis, progressing to liver fibrosis and hepatocellular carcinoma. Similarly, reduced PTEN expression with free fatty acid (FFA) oleate has been shown to promote hepatic steatosis. In other cancer, mitochondrial respiration defect with enhanced glycolysis and NADH formation has been suggested to be a key event in PTEN downregulation. Aim Our aims were to examine whether i) medium chain FFA octanoate altered PTEN expression ii) PTEN downregulation with FFA was associated with hepatic mitochondrial dysfunction. Method Human hepatoblastoma cell line HepG2/C3A was pretreated for 3 days with oleate (0.25 mM) or octanoate (2 mM). PTEN expression was determined using quantitative real time PCR. Mitochondrial function was measured using BDTM oxygen biosensor in the presence of 2,4 dinitrophenol. Lactate and pyruvate concentrations were measured in the supernatant to determine glycolytic activity and NADH/NAD+ ratio. Intracellular lipid accumulation was confirmed with triglyceride concentrations. Experiments were done in triplicate to n=3. Results are expressed in mean±SEM. Differences between groups were analysed by one-way ANOVA. Results We have previously demonstrated that oleate and octanoate pretreatment resulted in a similar intracellular triglyceride accumulation. In this study, we have found that despite similarities in triglyceride concentration, PTEN expression was lower in octanoate pretreated cells (octanoate 0.84±0.06, oleate 1.18±0.12, untreated 1.19±0.12 fold change from b-actin, p=0.04). However, octanoate pretreatment was not associated with impaired respiration (octanoate 0.24±0.01, oleate 0.20±0.02, untreated 0.28±0.01 AFU/gTP (gram of total protein)/min). Nevertheless, reduced PTEN expression with octanoate was associated with increased glycolysis (octanoate 315.2±42.91, oleate 100.9±14.09, untreated 145.3±8.83 μmol/gTP/hr, p=0.0001) with raised NADH/NAD ratio (octanoate 17.3±1.4, oleate 13.8±2.9 untreated 17.3±1.4; p=0.007). Conclusion To our knowledge, the effect of octanoate on PTEN expression has not been previously shown. In contrast to the previous finding, our data demonstrate that octanoate, not oleate, downregulates PTEN expression. Differences in glycolysis hence redox potential may have influenced the disparity in PTEN expression between these FFA. Octanoate has recently been proposed to be beneficial in weight loss and diabetes. However, our findings suggest that it may not have a favourable effect on the progression of nonalcoholic fatty liver disease.