Oxidative stress: a systemic factor implicated in the pathogenesis of hepatic encephalopathy

Although ammonia is considered the main factor involved in the pathogenesis of hepatic encephalopathy (HE), it correlates well with the severity of HE in acute liver failure, but not in chronic liver disease. Oxidative stress is another factor believed to play a role in the pathogenesis of this syndrome; it represents an imbalance between the production and neutralization of reactive oxygen species, which leads to cellular dysfunction. In the setting of liver disease, oxidative stress represents a systemic phenomenon induced by several mechanisms: decreased antioxidant synthesis, increased systemic release of oxidant enzymes, generation of reactive oxygen species, and impaired neutrophil function. High ammonia concentrations induce cerebral oxidative stress, thus contributing to severe hepatic encephalopathy, as observed in acute liver failure. In chronic liver disease, significantly lower degrees of hyperammonemia (<500 μM) do not induce cerebral nor systemic oxidative stress. Data from both animal and human studies sustain that there is a synergistic effect between systemic oxidative stress, and ammonia that is implicated in the pathogenesis of hepatic encephalopathy.     

Efficacy of liver assisting in patients with hepatic encephalopathy with special focus on plasma exchange

Severe liver injury result in development of hepatic encephalopathy (HE) and often also in brain edema that is a potentially fatal complication. HE and brain edema are correlated to the level and persistence of hyperammonemia and the presence of systemic inflammation. Treatment of HE and brain edema is based on restoring and keeping normal physiological variables including tonicity, blood gasses, lactate, temperature and vascular resistance by a wide variety of interventions. In addition liver support devices improve the stage of HE, cerebral metabolic rate for oxygen and glucose, and are used either as a bridge to liver transplantation or liver recovery in patients with fulminant hepatic failure and in patients with acute-on-chronic liver failure. This short review will mainly focus on the management and efficacy of doing plasma exchange on HE in patients with acute HE.     

Is it time to target gut dysbiosis and immune dysfunction in the therapy of hepatic encephalopathy?

The development of overt hepatic encephalopathy (HE) in a patient with cirrhosis confers a damning prognosis with a 1-year mortality approaching 64%. This complex neuropsychiatric syndrome arises as a consequence of a dysfunctional gut–liver–brain axis. HE has been largely neglected over the past 30 years, with the reliance on therapies aimed at lowering ammonia production or increasing metabolism following the seminal observation that the hepatic urea cycle is the major mammalian ammonia detoxification pathway and is key in the pathogenesis of HE. The relationship with ammonia is more clear-cut in acute liver failure; but in cirrhosis, it has become apparent that inflammation is a key driver and that a disrupted microbiome resulting in gut dysbiosis, bacterial overgrowth and translocation, systemic endotoxemia and immune dysfunction may be more important drivers. Therefore, it is important to re-focus our efforts into developing therapies that modulate the disrupted microbiome or alleviating its downstream consequences.     

Ornithine phenylacetate revisited

In patients with liver failure hyperammonemia is associated with the development of hepatic encephalopathy (HE) and immune impairment. Treatment of hyperammonemia is an unmet clinical need. Ornithine phenylacetate (OP) is a novel drug that is targeted at reducing ammonia concentration in patients with liver disease and therefore a potential treatment for HE. This review describes the mechanism of action of OP and its effect on plasma ammonia levels, brain function and inflammation of OP in both acute and chronic liver failure. Ammonia levels could shown to be reduced for up to 24 h in animal models until 120 h in patients with repeated dosing of the drug. Reduction of plasma ammonia levels is due to the stimulation of ammonia removal in the form of glutamine (through glutamine synthetase), the direct excretion of ammonia in the form phenylacetylglutamine and to a normalisation of glutaminase activity in the gut. Administration of OP is associated with a reduction of brain oedema in rats with chronic bile duct ligation and diminution of intracranial hypertension in a pig model of ALF. Studies to date have indicated that it is safe in humans and trials in overt HE are underway to establish OP as a treatment for this major complication of liver disease.     

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.     

NMDA receptors in hyperammonemia and hepatic encephalopathy

The NMDA type of glutamate receptors modulates learning and memory. Excessive activation of NMDA receptors leads to neuronal degeneration and death. Hyperammonemia and liver failure alter the function of NMDA receptors and of some associated signal transduction pathways. The alterations are different in acute and chronic hyperammonemia and liver failure. Acute intoxication with large doses of ammonia (and probably acute liver failure) leads to excessive NMDA receptors activation, which is responsible for ammonia-induced death. In contrast, chronic hyperammonemia induces adaptive responses resulting in impairment of signal transduction associated to NMDA receptors. The function of the glutamate-nitric oxide-cGMP pathway is impaired in brain in vivo in animal models of chronic liver failure or hyperammonemia and in homogenates from brains of patients died in hepatic encephalopathy. The impairment of this pathway leads to reduced cGMP and contributes to impaired cognitive function in hepatic encephalopathy. Learning ability is reduced in animal models of chronic liver failure and hyperammonemia and is restored by pharmacological manipulation of brain cGMP by administering phosphodiesterase inhibitors (zaprinast or sildenafil) or cGMP itself. NMDA receptors are therefore involved both in death induced by acute ammonia toxicity (and likely by acute liver failure) and in cognitive impairment in hepatic encephalopathy.     

Animal Models in the Study of Episodic Hepatic Encephalopathy in Cirrhosis

The availability of an animal model is crucial in studying the pathophysiological mechanisms of disease and to test possible therapies. Now, there are several models for the study of liver diseases, but there still remains a lack of a satisfactory animal model of chronic liver disease with hepatic encephalopathy (HE) and abnormalities in nitrogen metabolism, as seen in humans. In rats, two models of chronic HE are widely used: rats after portacaval anastomosis (PCA) and rats with chronic hyperammonemia. The first one mimics the situation induced in cirrhosis by collateral circulation, and has the problem of the absence of hepatocellular injury. The model of hyperammonemia is useful to study the effect of ammonia as a brain toxic substance, but also lacks liver failure. Bile-duct ligation has been used to induce cirrhosis and could also be a model of HE, probably with the addition of a precipitant factor. An ideal model of HE in chronic liver disease must have liver cirrhosis and a precipitant factor of HE; it must also show neuropathological characteristic findings of HE, neurochemical alterations in the main pathways impaired in these complications of cirrhosis, and low-grade brain edema.     

Recent insights into the pathogenesis of hepatic encephalopathy and treatments

Hepatic encephalopathy (HE) encompasses a spectrum of neuropsychiatric disorders related to liver failure. The development of HE can have a profound impact on mortality as well as quality of life for patients and carers. Ammonia is central in the disease process contributing to alteration in neurotransmission, oxidative stress, and cerebral edema and astrocyte swelling in acute liver failure. Inflammation in the presence of ammonia coactively worsens HE. Inflammation can result from hyperammonemic responses, endotoxemia, innate immune dysfunction or concurrent infection. This review summarizes the current processes implicated in the pathogenesis of HE, as well as current and potential treatments. Treatments currently focus on reducing inflammation and/or blood ammonia levels and provide varying degrees of success. Optimization of current treatments and initial testing of novel therapies will provide the basis of improvement of care in the near future.     

Management of hepatic encephalopathy in patients with cirrhosis

The term hepatic encephalopathy encompasses a spectrum of neuropsychiatric abnormalities seen in patients with liver dysfunction. Distinct syndromes are identified in acute liver failure and cirrhosis. Rapid deterioration in consciousness level and increased intracranial pressure that may result in brain herniation and death are a feature of acute liver failure whereas manifestations of hepatic encephalopathy in cirrhosis include psychomotor dysfunction, impaired memory, increased reaction time, sensory abnormalities, poor concentration and in severe forms, coma. In patients with acute-on-chronic liver failure the pathophysiology remains undefined. Ammonia has been considered central to its pathogenesis. In the brain, the astrocyte is the main site for ammonia detoxification, during the conversion of glutamate to glutamine. An increased ammonia level raises the amount of glutamine within astrocytes, causing an osmotic imbalance resulting in cell swelling and ultimately brain oedema. Recent studies suggest that inflammation and it modulators may play a synergistic role with ammonia in the pathogenesis of hepatic encephalopathy. Therapy of hepatic encephalopathy is directed primarily at reducing ammonia generation and increasing its detoxification. The currently accepted regimens to treat hepatic encephalopathy such as lactulose and protein restricted diets need further clinical trials and therefore placebo controlled clinical trials in hepatic encephalopathy are justified. In liver failure, ammonia metabolism involves multiple organs and therefore ammonia reduction will require simultaneous targeting of these organs. The present review describes the pathophysiological basis of hepatic encephalopathy and evaluates the available therapies.     

Systemic inflammatory response exacerbates the neuropsychological effects of induced hyperammonemia in cirrhosis

BACKGROUND/AIMS: Studies in acute liver failure show correlation between evidence of a systemic inflammatory response syndrome (SIRS) and progression of hepatic encephalopathy (HE). We tested the hypothesis that SIRS mediators, such as nitric oxide and proinflammatory cytokines, may exacerbate the neuropsychological effects of hyperammonemia in cirrhosis. METHODS: Ten patients with cirrhosis were studied, 24-36 h after admission with clinical evidence of infection, and following its resolution. Hyperammonemia was induced by oral administration of an amino-acid (aa) solution mimicking hemoglobin composition. Inflammatory mediators, nitrate/nitrite, ammonia, aa profiles and a battery of neuropsychological tests were measured. RESULTS: The hyperammonemia generated in response to the aa solution was similar prior to, and after resolution, of the inflammation (P=0.77). With treatment of the infection there were significant reductions in white blood cell count (WBC), C-reactive protein (CRP), nitrate/nitrite, interleukin-6, interleukin-1beta and tumour necrosis factor alpha. Induced hyperammonemia resulted in significant worsening of the neuropsychological scores when patients showed evidence of SIRS but not after its resolution. CONCLUSIONS: The significant deterioration of neuropsychological test scores following induced hyperammonemia during the inflammatory state, but not after its resolution, suggests that the inflammation and its mediators may be important in modulating the cerebral effect of ammonia in liver disease.     

Elevated cerebral lactate: Implications in the pathogenesis of hepatic encephalopathy

Hepatic encephalopathy (HE), a complex neuropsychiatric syndrome, is a frequent complication of liver failure/disease. Increased concentrations of lactate are commonly observed in HE patients, in the systemic circulation, but also in the brain. Traditionally, increased cerebral lactate is considered a marker of energy failure/impairment however alterations in lactate homeostasis may also lead to a rise in brain lactate and result in neuronal dysfunction. The latter may involve the development of brain edema. This review will target the significance of increased cerebral lactate in the pathogenesis of HE.     

Branched-chain amino acids and muscle ammonia detoxification in cirrhosis

Branched-chain amino acids (BCAA) are used as a therapeutic nutritional supplement in patients with cirrhosis and hepatic encephalopathy (HE). During liver disease, the decreased capacity for urea synthesis and porto-systemic shunting reduce the hepatic clearance of ammonia and skeletal muscle may become the main alternative organ for ammonia detoxification. We here summarize current knowledge of muscle BCAA and ammonia metabolism with a focus on liver cirrhosis and HE. Plasma levels of BCAA are lower and muscle uptake of BCAA seems to be higher in patients with cirrhosis and hyperammonemia. BCAA metabolism may improve muscle net ammonia removal by supplying carbon skeletons for formation of alfa-ketoglutarate that combines with two ammonia molecules to become glutamine. An oral dose of BCAA enhances muscle ammonia metabolism but also transiently increases the arterial ammonia concentration, likely due to extramuscular metabolism of glutamine. We, therefore, speculate that the beneficial effect of long term intake of BCAA on HE demonstrated in clinical studies may be related to an improved muscle mass and nutritional status rather than to an ammonia lowering effect of BCAA themselves.     

Role of Phosphate-Activated Glutaminase in the Pathogenesis of Hepatic Encephalopathy

Disturbed body nitrogen homeostasis due to impaired hepatic urea synthesis leads to an alteration in interorgan ammonia trafficking, resulting in hyperammonemia. Glutamine (Gln) synthase is the alternative pathway for ammonia detoxification. Gln taken up by several organs is split by the intramitochondrial phosphate-activated enzyme glutaminase (PAG) into glutamate (Glu) and ammonia. In cirrhotic patients with portosystemic intrahepatic shunt, the main source of systemic hyperammonemia is the small intestine, and ammonia derives mainly from Gln deamidation. Recently, PAG has been found increased in cirrhotics showing minimal hepatic encephalopathy and, therefore, could be implicated in the production of systemic hyperammonemia in these patients. Intestinal PAG activity correlates with psychometric test and magnetic resonance spectroscopy findings. Moreover, nitric oxide and tumor necrosis factor seem to be the major factors regulating intestinal ammonia production in cirrhotics. In the brain, PAG localized into the astrocytes is responsible for ammonia and free-radical production. The blockade of PAG, using 6-oxo-5-norleucine, avoids the toxic effects of Gln accumulation in the brain. These data support an important role for intestinal and brain glutaminase in the pathogenesis of hepatic encephalopathy and could be a new target for future therapies.     

Pro-inflammatory cytokines are raised in extrahepatic portal venous obstruction, with minimal hepatic encephalopathy

Background and Aims: Minimal hepatic encephalopathy (MHE) and hyperammonemia are seen in patients with extrahepatic portal venous obstruction (EHPVO). Inflammation has been shown to play an important role in the pathogenesis of hepatic encephalopathy in cirrhotics. This study assessed serum pro‐inflammatory cytokines and their correlation with hyperammonemia, ¹H‐magnetic resonance (MR) spectroscopy‐derived brain glutamine, and diffusion tensor imaging (DTI)‐derived metrics in patients with EPHVO, with and without MHE. Methods: Neuropsychological tests, DTI, ¹H‐MR spectroscopy, and estimation of blood ammonia and pro‐inflammatory cytokines (tumor necrosis factor‐α[TNF‐α] and interleukin‐6 [IL‐6]) were done in 20 patients with EHPVO and eight healthy controls. Results: Pro‐inflammatory cytokines (TNF‐α and IL‐6), blood ammonia, brain glutamine, and mean diffusivity were increased in both patient groups, as compared to controls. Patients with MHE (n‐12) had significantly higher TNF‐α, IL‐6, blood ammonia, brain glutamine, and mean diffusivity, signifying brain edema, than controls. A significant, positive correlation was seen between TNF‐α and IL‐6 and between blood ammonia and TNF‐α, IL‐6, and brain glutamine. Significant, positive correlations of TNF‐α, IL‐6, and blood ammonia with mean diffusivity values were seen in various brain regions, including spectroscopy voxel‐derived mean diffusivity. Conclusion: Patients with extrahepatic portal vein obstruction have inflammation and hyperammonemia made evident by higher blood TNF‐α, IL‐6, ammonia, and brain glutamine levels. A significant correlation between hyperammonemia, pro‐inflammatory cytokines, and cerebral edema on DTI in various brain regions suggests that both these factors play a role in the pathogenesis of MHE in these patients.     

Cyclic GMP pathways in hepatic encephalopathy. Neurological and therapeutic implications

Cyclic GMP (cGMP) modulates important cerebral processes including some forms of learning and memory. cGMP pathways are strongly altered in hyperammonemia and hepatic encephalopathy (HE). Patients with liver cirrhosis show reduced intracellular cGMP in lymphocytes, increased cGMP in plasma and increased activation of soluble guanylate cyclase by nitric oxide (NO) in lymphocytes, which correlates with minimal HE assessed by psychometric tests. Activation of soluble guanylate cyclase by NO is also increased in cerebral cortex, but reduced in cerebellum, from patients who died with HE. This opposite alteration is reproduced in vivo in rats with chronic hyperammonemia or HE. A main pathway modulating cGMP levels in brain is the glutamate-NO-cGMP pathway. The function of this pathway is impaired both in cerebellum and cortex of rats with hyperammonemia or HE. Impairment of this pathway is responsible for reduced ability to learn some types of tasks. Restoring the pathway and cGMP levels in brain restores learning ability. This may be achieved by administering phosphodiesterase inhibitors (zaprinast, sildenafil), cGMP, anti-inflammatories (ibuprofen) or antagonists of GABAA receptors (bicuculline). These data support that increasing cGMP by safe pharmacological means may be a new therapeutic approach to improve cognitive function in patients with minimal or clinical HE.     

Factors contributing to the development of overt encephalopathy in liver cirrhosis patients

The aim of this study was to clarify the relationships among psychometric testing results, blood ammonia (NH₃) levels, electrolyte abnormalities, and degree of inflammation, and their associations with the development of overt hepatic encephalopathy (HE) in liver cirrhosis (LC) patients. The relationships between covert HE and blood NH₃, sodium (Na), and C-reactive protein (CRP) were examined in 40 LC patients. The effects of elevated NH₃, hyponatremia, and elevated CRP on the development of overt HE were also investigated. The covert HE group had significantly lower serum Na levels and significantly higher serum CRP levels. During the median observation period of 11 months, 10 patients developed overt HE, and the results of multivariate analysis showed that covert HE and elevated blood NH₃ were factors contributing to the development of overt HE. Electrolyte abnormalities and mild inflammation are involved in the pathogenesis of HE. Abnormal psychometric testing results and hyperammonemia are linked to subsequent development of overt HE.     

Efficacy of oral L-ornithine L-aspartate in cirrhotic patients with hyperammonemic hepatic encephalopathy

Hyperammonemia and associated cerebral edema cause neurological abnormalities in liver disease patients. Although only 15% of ammonia production originates in the colon, management strategies for hepatic encephalopathy (HE) have focused on reducing ammonia generation from the bowel rather than on manipulating systemic mechanisms involved in ammonia metabolism. Administration of L-ornithine L-aspartate (LOLA) improves mental status and decreases serum and spinal fluid ammonia levels by stimulating both the urea cycle and glutamine (Gln) synthesis, which are key metabolic pathways in ammonia detoxification. LOLA was shown to be superior to a placebo for management of HE, and the results of several clinical trials suggest that its effectiveness could be higher with the more severe grades of this syndrome. Compared with the standard treatment, LOLA is effective not only in reducing hyperammonemia and the severity of this disease, but also in improving the patient's perceived quality of life. Therefore, LOLA is a promising alternative for the management of HE.     

Serotonin Brain Circuits with a Focus on Hepatic Encephalopathy

Despite enormous strides in our understanding of human disease, the precise mechanisms of hepatic encephalopathy (HE), a potential long-term complication of liver failure, are still unclear. Brain serotonin, a neurotransmitter with widespread distribution in the CNS, plays a role in the regulation of a wide range of physiological behaviors and functions. In addition, it has been implicated in the pathogenesis of several pathological processes, including HE. This work reviews the relationship between brain serotonergic dysfunction and hepatic encephalopathy in cirrhotic patients and experimental animals. The existing changes in the synthesis, metabolism, storage, and release of neuronal serotonin in HE point to a serotonergic synaptic deficit in this condition. Given the established role of the brain serotonergic system in the regulation of sleep, circadian rhythmicity, and locomotion, selective alterations of this system could be an important part of the neurophysiological background responsible for the behavioral changes in rats with portacaval anastomosis and may contribute to the pathogenesis of HE in cirrhotic patients. The findings that serotoninergic turnover is exquisitely and selectively sensitive to the degree of porto-systemic shunting and hyperammonemia suggest a role for serotonin in early neuropsychiatric symptoms of HE. Pharmacological manipulation of the brain serotonergic system may be beneficial in the prevention and treatment of HE in cirrhotic patients.     

Murine model to study brain,behavior and immunity during hepatic encephalopathy

AIM:To propose an alternative model of hepatic encephalopathy(HE) in mice,resembling the human features of the disease.METHODS:Mice received two consecutive intraperitoneal injections of thioacetamide(TAA) at low dosage(300 mg/kg).Liver injury was assessed by serum transaminase levels(ALT) and liver histology(hematoxylin and eosin).Neutrophil infiltration was estimated by confocal liver intravital microscopy.Coagulopathy was evaluated using prolonged prothrombin and partial thromboplastin time.Hemodynamic parameters were measured through tail cuff.Ammonia levels were quantified in serum and brain samples.Electroencephalography(EEG) and psychomotor activity score were performed to show brain function.Brain edema was evaluated using magnetic resonance imaging.RESULTS:Mice submitted to the TAA regime developed massive liver injury,as shown by elevation of serum ALT levels and a high degree of liver necrosis.An intense hepatic neutrophil accumulation occurred in response to TAA-induced liver injury.This led to mice mortality and weight loss,which was associated with severe coagulopathy.Furthermore,TAA-treated mice presented with increased serum and cerebral levels of ammonia,in parallel with alterations in EEG spectrum and discrete brain edema,as shown by magnetic resonance imaging.In agreement with this,neuropsychomotor abnormalities ensued 36 h after TAA,fulfilling several HE features observed in humans.In this context of liver injury and neurological dysfunction,we observed lung inflammation and alterations in blood pressure and heart rate that were indicative of multiple organ dysfunction syndrome.CONCLUSION:In summary,we describe a new murine model of hepatic encephalopathy comprising multiple features of the disease in humans,which may provide new insights for treatment.     

45CaCl2 autoradiography in brain from rabbits with encephalopathy from acute liver failure or acute hyperammonemia

In experimental hepatic encephalopathy and hyperammonemia, extracellular levels of glutamate are increased in hippocampus and cerebral cortex. It has been suggested that overstimulation of glutamate receptors causes a pathological entry of calcium into neurons via receptor-operated (NMDA- and AMPA-type) or voltage-dependent calcium channels leading to calcium overload and cell death. Neurodegeneration as a result of exposure to excitotoxins, including glutamate, can be localized and quantified using⁴⁵CaCl₂ autoradiography. This approach was used to study cerebral calcium accumulation in rabbits with acute liver failure and acute hyperammonemia. Acute liver failure was induced in 6 rabbits, acute hyperammonemia in 4 rabbits; 4 control rabbits received sodium-potassium-acetate. At the start of the experiment 500 µCi⁴⁵CaCl₂ was given intravenously. After development of severe encephalopathy, the animals were killed by decapitation. All rabbits with acute liver failure or acute hyperammonemia developed severe encephalopathy, after 13.2±1.7 and 19.3±0.5 hours respectively (mean±SEM). Plasma ammonia levels were 425±46 and 883±21 µmol/l, respectively (p<0.05). Control rabbits maintained normal plasma ammonia levels (13±5 µmol/l), demonstrated normal behaviour throughout the study and were sacrificed after 16 hours.⁴⁵Ca²⁺-autoradiograms of 40 µm brain sections were analyzed semiquantitatively using relative optical density and computerized image analysis. As compared to background levels⁴⁵Ca was not increased in hippocampus or any other brain area of rabbits with severe encephalopathy from acute liver failure or acute hyperammonemia. This suggests that, despite increased extracellular brain glutamate levels in these conditions, glutamate neurotoxicity was not important for the development of encephalopathy in these rabbits.