Mélanie Tremblay

AST-120 (spherical carbon adsorbent) lowers ammonia levels and attenuates brain edema in bile duct-ligated rats.

The pathogenesis of hepatic encephalopathy is multifactorial, involving gut‐derived toxins such as ammonia, which has been demonstrated to induce oxidative stress. Therefore, a primary hepatic encephalopathy treatment target is reducing ammonia production in the gastrointestinal tract. AST‐120, an oral adsorbent of engineered activated carbon microspheres with surface areas exceeding 1600 m2/g, acts as a sink for neurotoxins and hepatotoxins present in the gut. We evaluated the capacity of AST‐120 to adsorb ammonia in vitro and to lower blood ammonia, oxidative stress and brain edema in cirrhotic rats. Cirrhosis was induced in rats by bile duct ligation for 6 weeks. AST‐120 was administered by gavage preventively for 6 weeks (0.1, 1, and 4 g/kg/day). In addition, AST‐120 was evaluated as a short‐term treatment for 2 weeks and 3 days (1 g/kg/day) and as a sink to adsorb intravenously infused ammonium acetate. In vitro, AST‐120 efficiently adsorbed ammonia. Ammonia levels significantly decreased in a dose‐dependent manner for all AST‐120–treated bile duct‐ligated rats (nontreated: 177.3 ± 30.8 μM; AST‐120, 0.1 g/kg/day: 121.9 ± 13.8 μM; AST‐120, 1 g/kg/day: 80.9 ± 30.0 μM; AST‐120, 4 g/kg/day: 48.8 ± 19.6 μM) and significantly correlated with doses of AST‐120 (r = −0.6603). Brain water content and locomotor activity normalized after AST‐120 treatments, whereas arterial reactive oxygen species levels remained unchanged. Furthermore, AST‐120 significantly attenuated a rise in arterial ammonia after ammonium acetate administration (intravenously). Conclusion:AST‐120 treatment decreased arterial ammonia levels, normalized brain water content and locomotor activity but did not demonstrate an effect on systemic oxidative stress. Also, AST‐120 acts as an ammonia sink, efficiently removing blood‐derived ammonia. Additional studies are warranted to evaluate the effects of AST‐120 on hepatic encephalopathy in patients with advanced liver disease. (HEPATOLOGY 2011;)

Increased brain lactate is central to the development of brain edema in rats with chronic liver disease

BACKGROUND & AIMS The pathogenesis of brain edema in patients with chronic liver disease (CLD) and minimal hepatic encephalopathy (HE) remains undefined. This study evaluated the role of brain lactate, glutamine and organic osmolytes, including myo-inositol and taurine, in the development of brain edema in a rat model of cirrhosis. METHODS Six-week bile-duct ligated (BDL) rats were injected with (13)C-glucose and de novo synthesis of lactate, and glutamine in the brain was quantified using (13)C nuclear magnetic resonance spectroscopy (NMR). Total brain lactate, glutamine, and osmolytes were measured using (1)H NMR or high performance liquid chromatography. To further define the interplay between lactate, glutamine and brain edema, BDL rats were treated with AST-120 (engineered activated carbon microspheres) and dichloroacetate (DCA: lactate synthesis inhibitor). RESULTS Significant increases in de novo synthesis of lactate (1.6-fold, p<0.001) and glutamine (2.2-fold, p<0.01) were demonstrated in the brains of BDL rats vs. SHAM-operated controls. Moreover, a decrease in cerebral myo-inositol (p<0.001), with no change in taurine, was found in the presence of brain edema in BDL rats vs. controls. BDL rats treated with either AST-120 or DCA showed attenuation in brain edema and brain lactate. These two treatments did not lead to similar reductions in brain glutamine. CONCLUSIONS Increased brain lactate, and not glutamine, is a primary player in the pathogenesis of brain edema in CLD. In addition, alterations in the osmoregulatory response may also be contributing factors. Our results suggest that inhibiting lactate synthesis is a new potential target for the treatment of HE.

Mechanism of Kex2p inhibition by its proregion.

Many proteases are produced as zymogens bearing an N‐terminal proregion acting both as intramolecular chaperone and as enzyme inhibitor. We studied here the inhibition mechanism of the yeast proprotein convertase Kex2p by its proregion. A recombinant secreted and soluble form of Kex2p was produced in Pichia pastoris and its enzymatic properties toward a fluorogenic synthetic peptide were characterized. Recombinant Escherichia coli‐produced Kex2p proregion specifically and potently inhibited the enzyme, with an IC50 of 160 nM. Exploration of the inhibition mechanism revealed that the proregion behaved as a mixed inhibitor.

Portacaval anastomosis-induced hyperammonemia does not lead to oxidative stress

Ammonia is neurotoxic and believed to play a major role in the pathogenesis of hepatic encephalopathy (HE). It has been demonstrated, in vitro and in vivo, that acute and high ammonia treatment induces oxidative stress. Reactive oxygen species (ROS) are highly reactive and can lead to oxidization of proteins resulting in protein damage. The present study was aimed to assess oxidative status of proteins in plasma and brain (frontal cortex) of rats with 4-week portacaval anastomosis (PCA). Markers of oxidative stress, 4-hydroxy-2-nonenal (HNE) and carbonylation were evaluated by immunoblotting in plasma and frontal cortex. Western blot analysis did not demonstrate a significant difference in either HNE-linked or carbonyl derivatives on proteins between PCA and sham-operated control rats in both plasma and frontal cortex. The present study suggests PCA-induced hyperammonemia does not lead to systemic or central oxidative stress.

Induction of systemic oxidative stress leads to brain oedema in portacaval shunted rats

The pathogenesis of hepatic encephalopathy (HE) is multifactorial and often associated with the development of brain oedema. In addition to ammonia playing a central role, systemic oxidative stress is believed to aggravate the neuropsychological effects of ammonia in patients with chronic liver disease (CLD). The aim of this study was to (i) induce systemic oxidative stress in hyperammonaemic portacaval anastomosed (PCA) rats by inhibiting the antioxidant glutathione using Dimethyl maleate (DEM) and (ii) investigate whether a synergistic relationship between ammonia and oxidative stress contributes to the pathogenesis of brain oedema in CLD.

Elevated FABP1 serum levels are associated with poorer survival in acetaminophen‐induced acute liver failure

Acetaminophen (APAP)‐induced acute liver failure (ALF) is associated with significant mortality. Traditional prognostic scores lack sensitivity. Serum liver‐type fatty acid binding protein (FABP1) early (day 1) or late (day 3‐5) levels are associated with 21‐day mortality in the absence of liver transplant. Serum samples from 198 APAP‐ALF patients (nested case–control study with 99 survivors, 99 nonsurvivors) were analyzed by enzyme‐linked immunosorbent assay with clinical data from the US Acute Liver Failure Study Group registry (1998‐2014). APAP‐ALF survivors had significantly lower serum FABP1 levels early (238.6 versus 690.8 ng/mL, P < 0.0001) and late (148.4 versus 612.3 ng/mL, P < 0.0001) compared with nonsurvivors. FABP1 > 350 ng/mL was associated with significantly higher risk of death at early (P = 0.0004) and late (P < 0.0001) time points. Increased serum FABP1 early (log FABP1 odds ratio = 1.31, P = 0.027) and late (log FABP1 odds ratio = 1.50, P = 0.005) were associated with significantly increased 21‐day mortality after adjusting for significant covariates (Model for End‐Stage Liver Disease, vasopressor use). Areas under the receiver operating characteristic curve for early and late multivariable models were 0.778 and 0.907, respectively. The area under the receiver operating characteristic curve of the Kings College criteria (early, 0.552 alone, 0.711 with FABP1; late, 0.604 alone, 0.797 with FABP1) and the Acute Liver Failure Study Group prognostic index (early, 0.686 alone, 0.766 with FABP1; late, 0.711 alone, 0.815 with FABP1) significantly improved with the addition of FABP1 (P < 0.002 for all). Conclusion: In patients with APAP‐ALF, FABP1 may have good potential to discriminate survivors from nonsurvivors and may improve models currently used in clinical practice; validation of FABP1 as a clinical prediction tool in APAP‐ALF warrants further investigation. (Hepatology 2017;65:938‐949)

The bile duct ligated rat: A relevant model to study muscle mass loss in cirrhosis

Muscle mass loss and hepatic encephalopathy (complex neuropsychiatric disorder) are serious complications of chronic liver disease (cirrhosis) which impact negatively on clinical outcome and quality of life and increase mortality. Liver disease leads to hyperammonemia and ammonia toxicity is believed to play a major role in the pathogenesis of hepatic encephalopathy. However, the effects of ammonia are not brain-specific and therefore may also affect other organs and tissues including muscle. The precise pathophysiological mechanisms underlying muscle wasting in chronic liver disease remains to be elucidated. In the present study, we characterized body composition as well as muscle protein synthesis in cirrhotic rats with hepatic encephalopathy using the 6-week bile duct ligation (BDL) model which recapitulates the main features of cirrhosis. Compared to sham-operated control animals, BDL rats display significant decreased gain in body weight, altered body composition, decreased gastrocnemius muscle mass and circumference as well as altered muscle morphology. Muscle protein synthesis was also significantly reduced in BDL rats compared to control animals. These findings demonstrate that the 6-week BDL experimental rat is a relevant model to study liver disease-induced muscle mass loss.

573: BRAIN-FABP SERUM LEVELS AND CLINICAL OUTCOMES IN ACETAMINOPHEN-INDUCED ACUTE LIVER FAILURE.

Crit Care Med 2016 • Volume 44 • Number 12 (Suppl.) the blood products used after patients were assessed as receiving probably inappropriate and inappropriate treatment. Results: Of 1136 critically ill patients, physicians assessed 98 (8.6%) as receiving probably inappropriate treatment and 123 (11%) receiving inappropriate treatment. Fifty-nine (48%) of the 123 patients received blood products after they were assessed to be receiving inappropriate treatment during the study period: 242 units of PRBCs, which was 7.6% of all PRBC units transfused in the health system; 161 (9.9%) units of plasma, 137 (12.1%) units of apheresis platelets, and 21 (10.5%) units of prepooled cryoprecipitate. Conclusions: Blood products are administered to patients receiving inappropriate treatment. More careful use in critical care is warranted for this limited, donated resource.

573 ATTENUATION OF OXIDATIVE STRESS PROTECTS THE BRAIN IN RATS WITH “ACUTE-ON-CHRONIC” LIVER FAILURE

related to hyperammonemia is based on studies of cell cultures, animal models and clinical studies where the actual tissue or plasma concentrations of ammonium range from clinical relevant levels in the micromolar range to more than 5 mM. To assess the significance of the ammonium concentration we studied the extracellular release of lactate, glutamate, and lactate in cerebral cortex of rat brain slices in a dose–response study. Methods: We applied concentrations of ammonium from 0.15 mM to 10 mM to 29 brain slices in a perfusion chamber with exposure times up to 90 minutes. We measured the extracellular changes in lactate, adenosine and glutamate by the use of enzymatic biosensors inserted into cerebral cortex. Results:We found a consistent reduction in the extracellular lactate concentration ranging from 4 to 400 micromolar independent of the ammonium concentration (R=0.006, ammonium vs. lactate). The reduction in lactate was not affected by inhibition of the neuronal lactate transporter MCT-2 by adding alpha-cyano-4hydroxycinnamic acid. We found a positive correlation between the ammonium concentration and the glutamate increase (R=0.43, p < 0.05) with a marked release of glutamate (up to 55 micromolar) with exposure to 10 mM of ammonium. We also observed a positive correlation between the ammonium level and the change in adenosine (R=0.68, p < 0.05) where ammonium levels above 500 mM were associated with adenosine release up to 18 micromolar.. The peak in glutamate preceded the peak in adenosine release by 18±7 minutes (p < 0.05). Conclusion: Cortical tissue exposed ammonium displayed a linear dose–response-like relationship between ammonium concentration and the changes in glutamate and adenosine. Interestingly, we found that ammonium induced a reduction in the extracellular lactate concentration independent of the ammonium concentration within the studied range. The reduction appeared not to be related to increased neuronal uptake of lactate.

574 SYSTEMIC OXIDATIVE STRESS INDUCTION AGGRAVATES HEPATIC ENCEPHALOPATHY IN HYPERAMMONEMIC PORTACAVAL-SHUNTED RATS

related to hyperammonemia is based on studies of cell cultures, animal models and clinical studies where the actual tissue or plasma concentrations of ammonium range from clinical relevant levels in the micromolar range to more than 5 mM. To assess the significance of the ammonium concentration we studied the extracellular release of lactate, glutamate, and lactate in cerebral cortex of rat brain slices in a dose–response study. Methods: We applied concentrations of ammonium from 0.15 mM to 10 mM to 29 brain slices in a perfusion chamber with exposure times up to 90 minutes. We measured the extracellular changes in lactate, adenosine and glutamate by the use of enzymatic biosensors inserted into cerebral cortex. Results:We found a consistent reduction in the extracellular lactate concentration ranging from 4 to 400 micromolar independent of the ammonium concentration (R=0.006, ammonium vs. lactate). The reduction in lactate was not affected by inhibition of the neuronal lactate transporter MCT-2 by adding alpha-cyano-4hydroxycinnamic acid. We found a positive correlation between the ammonium concentration and the glutamate increase (R=0.43, p < 0.05) with a marked release of glutamate (up to 55 micromolar) with exposure to 10 mM of ammonium. We also observed a positive correlation between the ammonium level and the change in adenosine (R=0.68, p < 0.05) where ammonium levels above 500 mM were associated with adenosine release up to 18 micromolar.. The peak in glutamate preceded the peak in adenosine release by 18±7 minutes (p < 0.05). Conclusion: Cortical tissue exposed ammonium displayed a linear dose–response-like relationship between ammonium concentration and the changes in glutamate and adenosine. Interestingly, we found that ammonium induced a reduction in the extracellular lactate concentration independent of the ammonium concentration within the studied range. The reduction appeared not to be related to increased neuronal uptake of lactate.