M. Jover

Preparation of crayfish chitin by in situ lactic acid production

Abstract Lactic acid production by batch-fed fermentation of deproteinized whey, using immobilized cells of Lactobacillus pentosus-4023 in a spirally wound fibrous-sheet packed-column reactor, has been investigated. This fermentation process has been studied to produce low-cost lactic acid ‘in situ’ and to investigate its direct use for the demineralization of crayfish chitinous fraction (exoskeleton). Chitin obtained by this procedure was chemically and spectrometrically (FT-IR and 13C-NMR) characterized.

Variations in the Promoter Region of the Glutaminase Gene and the Development of Hepatic Encephalopathy in Patients With Cirrhosis: A Cohort Study

BACKGROUND Hepatic encephalopathy is a major complication of cirrhosis and is associated with a poor prognosis. OBJECTIVE To identify mutations in the gene sequence for glutaminase in humans that could be responsible for the development of hepatic encephalopathy in patients with cirrhosis. DESIGN Cohort study. SETTING Outpatient clinics in 6 Spanish hospitals. PATIENTS 109 consecutive patients with cirrhosis in the estimation cohort, 177 patients in the validation cohort, and 107 healthy control participants. MEASUREMENTS Patients were followed every 3 or 6 months until the development of hepatic encephalopathy or liver transplantation, death, or the end of the study. RESULTS The genetic analyses showed that glutaminase TACC and CACC haplotypes were linked to the risk for overt hepatic encephalopathy. Mutation scanning of the glutaminase gene identified a section in the promoter region where base pairs were repeated (a microsatellite). Over a mean follow-up of 29.6 months, hepatic encephalopathy occurred in 28 patients (25.7%) in the estimation cohort. Multivariable Cox models were used to determine the following independent predictors: Child-Turcotte-Pugh stage (hazard ratio [HR], 1.6 [95% CI, 1.29 to 1.98]; P = 0.001), minimal hepatic encephalopathy (HR, 3.17 [CI, 1.42 to 7.09]; P = 0.006), and having 2 long alleles of the microsatellite (HR, 3.12 [CI, 1.39 to 7.02]; P = 0.006). The association between 2 long alleles of the microsatellite and overt hepatic encephalopathy was confirmed in a validation cohort (HR, 2.1 [CI, 1.17 to 3.79]; P = 0.012). Functional studies showed higher luciferase activity in cells transfected with the long form of the microsatellite, which suggests that the long microsatellite enhances glutaminase transcriptional activity. LIMITATION Other genes and allelic variants might be involved in the clinical expression of hepatic encephalopathy. CONCLUSION This study identifies a genetic factor that is associated with development of hepatic encephalopathy in patients with cirrhosis. PRIMARY FUNDING SOURCE Instituto de Salud Carlos III, Spanish Ministry of Health.

Gut ammonia production and its modulation.

Systemic hyperammonemia has been largely found in patients with cirrhosis and hepatic encephalopathy, and ammonia plays a major role in the pathogenesis of hepatic encephalopathy. However, controversial points remain: a) the correlation between plasma ammonia levels and neurophysiological impairment. The lack of correlation between ammonia levels and grade of hepatic encephalopathy in some cases has been considered a weakness of the ammonia hypothesis, but new methods for ammonia measurements and the implication of systemic inflammation in the modulation of ammonia neurotoxicity could explain this gap; b) the source of ammonia production. Hyperammonemia has been considered as derived from urea breakdown by intestinal bacteria and the majority of treatments were targeted against bacteria-derived ammonia from the colon. However, some data suggest an important role for small intestine ammonia production: 1) the hyperammonemia after porto-caval shunted rats has been found similar in germ-free than in non-germ-free animals. 2) In cirrhotic patients the greatest hyperammonemia was found in portal drained viscera and derived mainly from glutamine deamination. 3) The amount of time required to increase of ammonia (less than one hour) after oral glutamine challenge supports a small intestine origin of the hyperammonemia. As the main source of ammonia in cirrhotics derives from portal drained viscera owing to glutamine deamidation, increased glutaminase activity in the intestine seems to be responsible for systemic hyperammonemia. Lastly, some genetic alterations in the glutaminase gene such as the haplotype TACC could modulate intestinal ammonia production and the risk of overt hepatic encephalopathy in cirrhotics.

Serum metabolic signature of minimal hepatic encephalopathy by (1)H-nuclear magnetic resonance.

Minimal hepatic encephalopathy (MHE) reduces quality of life of cirrhotic patients, predicts overt hepatic encephalopathy, and is associated with poor prognosis. We hypothesized that MHE arises once metabolic alterations derived from the liver reach a particular threshold. Our aim was to assess whether metabolic profiling of serum samples by high-field (1)H-nuclear magnetic resonance spectroscopy ((1)H NMR) and subsequent multivariate analyses would be useful to characterize metabolic perturbations associated with MHE and to identify potential metabolic biomarkers. Metabolic serum profiles from controls (n = 69) and cirrhotic patients without MHE (n = 62) and with MHE (n = 39) were acquired using high field NMR. Supervised modeling of the data provided perfect discrimination between healthy controls and cirrhotic patients and allowed the generation of a predictive model displaying strong discrimination between patients with and without MHE (R(2)Y = 0.68, Q(2)Y = 0.63). MHE patients displayed increased serum concentrations of glucose, lactate, methionine, TMAO, and glycerol, as well as decreased levels of choline, branch amino acids, alanine, glycine, acetoacetate, NAC, and lipid moieties. Serum metabonomics by (1)H NMR offers a useful approach for characterizing underlying metabolic differences between patients with and without MHE. This procedure shows great potential as a diagnostic tool of MHE as it objectively reflects measurable biochemical differences between the patient groups and may facilitate monitoring of both disease progression and effects of therapeutic treatments.

Isolation and characterization of carotenoproteins from crayfish (Procambarus clarkii)

Abstract Crayfish (Procambarus clarkii) were separated by solid filtration into a proteinaceous fraction (PF-1) and a chitinous fraction (CF). The PF-1 and CF were used as starting materials for carotenoprotein-1 and carotenoprotein-2 preparations, respectively. Crawfish carotenoprotein-1 was obtained by controlled enzymatic autodigestion of FP-1 and carotenoprotein-2 by in situ repeated batch lactic acid fermentation. Carotenoprotein-1 has a high content of essential amino acids, ω-3- and ω-6-fatty acids and carotene (mainly astaxanthin), and constitutes an excellent nutritional source for patients with malnutrition. Carotenoprotein-2, of lower nutritional quality but with a substantial carotene content, can be used as a good protein source for animal nutrition where coloration is required, such as for poultry or for salmonid fish bred by aquaculture.

Experimental models for hepatic encephalopathy

Although there are many models for animal testing, the ideal model for chronic liver disease involving hepatic encephalopathy has not been described yet. Different problems associated with the models have led researchers to develop their own, sometimes unique experimental models, which makes difficult the comparison between the results of the conducted studies (1). Hepatic encephalopathy (HE) definition, nomenclature, diagnosis and quantification consensus were published in 2002 (2) where three types of HE were considered: type A, associated with acute liver failure, type B, associated with the existence of porto-systemic communication (by-pass) without intrinsic liver disease and type C, associated to liver cirrhosis. HE type C, in turn, is classified according to their form of presentation as spontaneous or episodic HE, in relation to precipitating factors, persistent HE is subdivided into mild (HE grade I), severe (HE II-IV) or treatment-dependent (early developed after abolition of treatment) and finally, the minimal HE, as the first manifestation of HE. HE type C is the most common form and from a clinical point of view, the episodic HE type because of liver cirrhosis decompensation is the most typical and relevant. The ideal model of HE should reproduce most of the clinical features of this syndrome in humans, as occurring mainly in patients with chronic liver disease, being precipitated by defined factors, it should be reversible with the correction of precipitating factors and improved using rifaximin or ammonia lowering-drugs, being strongly associated with altered nitrogen metabolism and showing a wide spectrum of severity. Because most patients with HE also suffer from chronic liver disease, porto-systemic shunts, developed hyperammonemia and systemic inflammation it is highly desirable that animal models would include these facts (Fig. 1).

91 TOLL LIKE RECEPTOR 4 ANTAGONIST PREVENTS ACETAMINOPHEN INDUCED ACUTE LIVER FAILURE IN MICE: A NOVEL THERAPEUTIC STRATEGY

Introduction Without transplantation, about 40% of patients with acute liver failure (ALF) die. Its treatment is an unmet need. Unregulated inflammation plays an important role in the pathogenesis. Our hypothesis is that Toll like receptor 4 (TLR 4) is critical in the progression of inflammation in ALF. The aims of the study were to determine whether (1) administration of a novel TLR4 antagonist to an acetaminophen (APAP) model of ALF in mice would prevent liver injury. (2) TLR4 antagonist in ALF prolongs survival. (3) TLR4 KO are protected from the liver injury induced by APAP. Methods Study 1 : 3 groups of CD1 mice were studied (n=6 in each group) Naive, APAP, 500 mg/kg single dose IP after overnight fasting). APAP+TLR4 antagonist, STM28 (Osaka, Japan); 20 μg IP, 1 h prior to administration of APAP and 6 h late). Study 2 : 3 groups of C57BL/6 mice were studied (n=6) in each group. Naive, APAP (500 mg/kg single dose IP), APAP+TLR4 antagonist; IAXO (Innaxon) 3 mg/kg IP, 1 h prior to administration of APAP and 6 h later. Study 3: C57BL/6 TLR4 KO were administered APAP 500 mg/kg and the respective naive controls were administered 500 mg/kg of saline. Biochemistry was measured using COBAS integra, Cytokines in plasma and tissue homogenate of liver, kidney and brain were measured using ELISA bead array. Brain water was measured using the dry-wet weight method. Results Both the TLR4 antagonist9s (STM28 and IAXO compound) reduced the plasma liver enzymes, ammonia and creatinine to the control level. The increase in the plasma TNF-α induced by APAP (45±3.2) was attenuated following TLR4 antagonist (20±2.3) (p Conclusion These data provides evidence for an important of TLR4 in APAP induced ALF and provide the rationale for a clinical trial of this strategy in ALF. Competing interests None declared.

248 EVIDENCE REFUTING THE TROJAN-HORSE HYPOTHESIS OF BRAIN SWELLING IN ACUTE LIVER FAILURE: L-TYPE GLUTAMINASE IS EXCLUSIVELY NEURONAL

Introduction Astrocytic swelling is the characteristic feature of hyperammonemia and acute liver failure (ALF) which is thought to result from accumulation of glutamine due to the action of astrocytic glutamine synthetase (GS). It has been suggested that glutamine may not be a benign amino acid and may act as “Trojan horse” which leads to astrocytic apoptosis as it is metabolised by Glutaminase (GLN) yielding glutamate and ammonia. In vivo proof for this hypothesis is lacking. In health, GLN is mainly neuronal and generates glutamate and GABA. The aims of the study were to define the expression of the ammonia metabolising enzymes, GS and GLN in the brain of ALF animals. Methods Two groups of CD1 mice were studied, sham: n=6; paracetamol (500 mg/kg IP): n=7. The animals were maintained normothermic and resuscitated with fluid and glucose and sacrificed at 8 h after injection of APAP or before development of coma. Arterial ammonia (COBAS) and frontal cortex brain water (dry weight technique) were measured. The brain sections were stained for GS and K and L-type GLN. Results Arterial ammonia was significantly higher in the ALF group compared with controls (345±32 vs 132±11 p=0.002) and brain water did not reach significance (83.6±2.3 vs 76.3±2.6 p=0.05). GS protein expression was observed in the astrocytes in the dentate fascia in both groups and was not different but was also seen in the oligodendrocytes only in ALF group. L-type GLN was expressed only in the neurons and not in the astrocytes and was significantly higher in the ALF animals (++++) compared with controls (+). The most marked areas were the striatum and dentate fascia and interestingly the staining was mainly cytoplasmic. K-type GLN was not different between groups and limited to brain capillaries. Conclusion Conclusion: The results of this study refute the Trojan-horse hypothesis and show for the first time increased protein expression of L-type GLN which is exclusively neuronal. From the pathophysiological perspective, this may function to generate excessive ammonia in the neuron thereby producing neuronal cell death. Competing interests None declared.

PMO-142 Evidence refuting the Trojan-horse hypothesis of brain swelling in acute liver failure: l-type glutaminase is exclusively neuronal: Abstract PMO-142 Figure 1

Introduction Astrocytic swelling is the characteristic feature of hyperammonemia and acute liver failure (ALF) which is thought to result from accumulation of glutamine due to the action of astrocytic glutamine synthetase (GS). It has been suggested that glutamine may not be a benign amino acid and may act as “Trojan horse” which leads to astrocytic apoptosis as it is metabolised by Glutaminase (GLN) yielding glutamate and ammonia. In vivo proof for this hypothesis is lacking. In health, GLN is mainly neuronal and generates glutamate and GABA. The aims of the study were to define the expression of the ammonia metabolising enzymes, GS and GLN in the brain of ALF animals. Methods Two groups of CD1 mice were studied, sham: n=6; paracetamol (500 mg/kg IP): n=7. The animals were maintained normothermic and resuscitated with fluid and glucose and sacrificed at 8 h after injection of APAP or before development of coma. Arterial ammonia (COBAS) and frontal cortex brain water (dry weight technique) were measured. The brain sections were stained for GS and K and L-type GLN. Results Arterial ammonia was significantly higher in the ALF group compared with controls (345±32 vs 132±11 p=0.002) and brain water did not reach significance (83.6±2.3 vs 76.3±2.6 p=0.05). GS protein expression was observed in the astrocytes in the dentate fascia in both groups and was not different but was also seen in the oligodendrocytes only in ALF group. L-type GLN was expressed only in the neurons and not in the astrocytes and was significantly higher in the ALF animals (++++) compared with controls (+). The most marked areas were the striatum and dentate fascia and interestingly the staining was mainly cytoplasmic. K-type GLN was not different between groups and limited to brain capillaries. Conclusion Conclusion: The results of this study refute the Trojan-horse hypothesis and show for the first time increased protein expression of L-type GLN which is exclusively neuronal. From the pathophysiological perspective, this may function to generate excessive ammonia in the neuron thereby producing neuronal cell death.Abstract PMO-142 Figure 1 Competing interests None declared.

155 THDP-17 INHIBITS SELECTIVELY THE K-TYPE INTESTINAL GLUTAMINASE, BUT NOT THE TYPE-L IN VITRO AND IN VIVO

Background: Avid water retention is a pathophysiological hallmark of ascites and dilutional hyponatremia in cirrhosis. This is partly due to excessive stimulation of vasopressin 2 receptors in the renal collecting duct. A number of selective V2 receptor antagonists, vaptans, have been developed. These drugs increase free water clearance and may correct hypervolaemic hyponatriemic and potentially mobilize ascites. Methods: Systematic review of randomised controlled trials (RCTs) on vaptans versus placebo, no intervention or diuretics for patients with cirrhosis and ascites or hyponatraemia. Electronic searches were performed in Medline, Embase, Web of Science and the Cochrane Library (November 2010). The primary outcomes measured were mortality and complications to cirrhosis. Secondary outcomes included serum sodium levels and adverse events. Random effects meta-analyses were performed and results presented as relative risks (RR) or standardised mean differences (SMD) with 95% confidence intervals (CI). Results: From 1965 potential references, 20 RCTs were indentified. Eight RCTs fulfilled our inclusion criteria (6 full papers and 2 abstracts). Random effects meta-analyses suggested that vasopressin antagonists had no effect on mortality (RR: 0.78, 95%CI: 0.25, 2.42) or complications to cirrhosis (RR: 0.71, 95%CI: 0.17, 3.02). Vaptans had no apparent influence on the number of serious adverse events (RR: 1.12, 95%CI: 0.82, 1.53) or adverse events in general (RR: 1.06, 95%CI: 0.93, 1.22). However, the number of patients with thirst (RR: 3.97, 95%CI: 1.78, 8.83) and urine volume >5 L/day (RR: 9.96, 95%CI: 1.38, 71.68) increased and in ascitic patients the body weight decreased (SMD: −0.46 kg, 95%CI: −0.74, −0.17). Vasopressin antagonists increased significant serum sodium levels (SMD: 0.36, 95%CI: 0.13, 0.59) as well as vasopressin (SMD: 2.59, 95%CI: 1.92, 3.26) and renin (SMD: 0.46, 95%CI: 0.19, 0.73) levels. Conclusion: Current evidence suggest that vaptans does not affect any hard clinical endpoints but only surrogate markers. Serum sodium levels increases, but also the risk of some adverse events. More research is needed before treatment recommendations can be made.