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.     

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.     

Possible treatment of end-stage hyperammonemic encephalopathy by inhibition of glutamine synthetase

Glutamine synthetase (GS) is highly active in astrocytes, and these cells are physiologically and morphologically compromised by hyperammonemia. Hyperammonemia in end-stage acute liver failure (ALF) is often associated with cerebral edema and astrocyte pathology/swelling. Many studies of animal models of hyperammonemia, and, more recently, nuclear magnetic resonance studies of liver disease patients, have shown that cerebral glutamine is elevated in hyperammonemia, contributing to the edema and encephalopathy. The GS inhibitor L-methionine-S,R-sulfoximine (MSO) is protective in animal models against acute ammonia intoxication. MSO is also an inhibitor of glutamate cysteine ligase, is converted to metabolic products, and causes convulsions at high doses. However, the susceptibility to MSO-induced convulsions is species dependent, with primates being relatively resistant. Moreover, it is possible to chronically maintain cerebral GS activity in mice at low levels by MSO treatment without any obvious untoward effects. Furthermore, MSO is protective in a mouse model of ALF. Extreme caution would be needed in administering MSO to patients. Nevertheless, inhibition of brain GS by MSO (or other GS inhibitors) may have therapeutic benefit in ALF.     

Clinical significance of the latency period of abnormal ammonia metabolism in chronic liver disease: Proposal of a new concept

The liver is a key organ in regulating metabolism, and chronic liver disease is associated with several metabolic disorders. In the later stages of liver cirrhosis, the urea cycle is impaired, which disrupts of ammonia detoxification and eventually causes hyperammonemia and hepatic encephalopathy. Although hyperammonemia is not detected during the period between the late stage of chronic hepatitis and the early stage of liver cirrhosis, hepatic albumin synthesis capacity decreases as the fibrosis progresses. Increased ammonia levels are associated with a decreased capacity of the liver to synthesize albumin as well as activation of hepatic stellate cells, which promote fibrosis. Herein, we discuss the possibility that abnormal ammonia metabolism might play an important role in the pathogenesis of liver diseases even without hyperammonemia. We consider the disease period without hyperammonemia as the latency period of abnormal ammonia metabolism and discuss its clinical significance.     

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.     

Control of acute, chronic, and constitutive hyperammonemia by wild-type and genetically engineered Lactobacillus plantarum in rodents

Hyperammonemia is a common complication of acute and chronic liver diseases. Often accompanied with side effects, therapeutic interventions such as antibiotics or lactulose are generally targeted to decrease the intestinal production and absorption of ammonia. In this study, we aimed to modulate hyperammonemia in three rodent models by administration of wild-type Lactobacillus plantarum, a genetically engineered ammonia hyperconsuming strain, and a strain deficient for the ammonia transporter. Wild-type and metabolically engineered L. plantarum strains were administered in ornithine transcarbamoylase-deficient Sparse-fur mice, a model of constitutive hyperammonemia, in a carbon tetrachloride rat model of chronic liver insufficiency and in a thioacetamide-induced acute liver failure mice model. Constitutive hyperammonemia in Sparse-fur mice and hyperammonemia in a rat model of chronic hepatic insufficiency were efficiently decreased by Lactobacillus administration. In a murine thioacetamide-induced model of acute liver failure, administration of probiotics significantly increased survival and decreased blood and fecal ammonia. The ammonia hyperconsuming strain exhibited a beneficial effect at a lower dose than its wild-type counterpart. Improved survival in the acute liver failure mice model was associated with lower blood ammonia levels but also with a decrease of astrocyte swelling in the brain cortex. Modulation of ammonia was abolished after administration of the strain deficient in the ammonium transporter. Intestinal pH was clearly lowered for all strains and no changes in gut flora were observed. CONCLUSION: Hyperammonemia in constitutive model or after acute or chronic induced liver failure can be controlled by the administration of L. plantarum with a significant effect on survival. The mechanism involved in this ammonia decrease implicates direct ammonia consumption in the gut.     

Hyperammonemia in gene-targeted mice lacking functional hepatic glutamine synthetase

Urea cycle defects and acute or chronic liver failure are linked to systemic hyperammonemia and often result in cerebral dysfunction and encephalopathy. Although an important role of the liver in ammonia metabolism is widely accepted, the role of ammonia metabolizing pathways in the liver for maintenance of whole-body ammonia homeostasis in vivo remains ill-defined. Here, we show by generation of liver-specific Gln synthetase (GS)-deficient mice that GS in the liver is critically involved in systemic ammonia homeostasis in vivo. Hepatic deletion of GS triggered systemic hyperammonemia, which was associated with cerebral oxidative stress as indicated by increased levels of oxidized RNA and enhanced protein Tyr nitration. Liver-specific GS-deficient mice showed increased locomotion, impaired fear memory, and a slightly reduced life span. In conclusion, the present observations highlight the importance of hepatic GS for maintenance of ammonia homeostasis and establish the liver-specific GS KO mouse as a model with which to study effects of chronic hyperammonemia.Hepatic ammonia and Gln metabolism are embedded into a structural/functional organization in the liver acinus, which allows efficient ammonia detoxification by the liver (1, 2). Hepatic urea synthesis in periportal hepatocytes is dependent on the activity of carbamoylphosphate synthetase, the rate-controlling enzyme of the urea cycle requiring ammonia as a substrate (3). The affinity of carbamoylphosphate synthetase for ammonia is low, and adequate flux through the urea cycle requires the establishment of high ammonia concentrations in periportal hepatocytes, which is achieved through ammonia amplification by periportal glutaminase activity (1–3). Excess ammonia not used by urea synthesis is taken up with high affinity by a small perivenous hepatocyte population (so-called “perivenous scavenger cells”) (1), which detoxifies ammonia by amidation of Glu (4). This reaction is catalyzed by Gln synthetase (GS), which is specifically expressed in this small population of hepatocytes surrounding the terminal hepatic venule but not in other hepatocytes (4–6). This high-affinity ammonia removal by perivenous hepatocytes thus prevents spillover of hepatic ammonia into the systemic circulation. The acinar compartmentation of urea synthesis, Gln hydrolysis, and Gln synthesis therefore allows the generation of sizable ammonia concentrations in hepatic tissue needed for urea formation without the risk of toxic ammonia concentrations in systemic circulation (1).Ammonia is toxic, particularly to the brain, where it can trigger hepatic encephalopathy (HE). HE is seen as the clinical manifestation of a low-grade cerebral edema with oxidative/nitrosative stress and subsequent derangements of signal transduction, neurotransmission, synaptic plasticity, and oscillatory networks in the brain (7–9). In particular, the oxidative/nitrosative stress response results in protein Tyr nitration (PTN) and RNA oxidation (9, 10). Astrocytes in close proximity to the blood–brain barrier exhibit strong PTN, possibly affecting its permeability (11). Data derived from HE animal models suggest a relationship between impaired functions of brain regions involved in cognition, learning, memory formation, and motor control, as well as elevated markers for oxidative stress in the hippocampus (12, 13), cerebral cortex (14), and cerebellum (15, 16). Consistently, increased markers for oxidative stress, such as PTN and RNA oxidation, have also been shown in postmortem human brain tissue of patients with liver cirrhosis and HE (17). In mice, ammonia can also inhibit potassium buffering by astrocytes, which results in increased extracellular potassium and may contribute to neuronal depolarization and dysfunction, and, consequently, altered behavior (18).Liver damage can trigger defects in the ammonia metabolizing pathways, which consequently increase ammonia levels in circulating blood (19, 20). For example, LPS-induced liver injury results in PTN of hepatic GS, which inactivates the enzyme (21). The impact of the urea cycle for ammonia metabolism can be seen in children with urea cycle defects, who exhibit hyperammonemia and cognitive symptoms (22). In turn, hereditary GS deficiency in humans is a rare disorder leading to Gln deficiency and severe disease with a lethal outcome (23). Although destruction of perivenous hepatocytes by carbon tetrachloride intoxication impairs ammonia detoxification in perfused rat liver (24), the in vivo significance of the GS in perivenous hepatocytes has remained elusive. Because Gln can be synthesized in a wide variety of tissues (4), specific deletion of hepatic GS would not be expected to result in Gln deficiency. However, lack of hepatic GS may disrupt perivenous ammonia metabolism, thus leading to increased ammonia concentrations in systemic blood.To test this hypothesis, gene-targeted mice lacking functional hepatic GS were generated and analyzed. As a result of liver-specific GS deletion, mice developed systemic hyperammonemia, which was accompanied by cerebral RNA oxidation, PTN, motoric and behavioral abnormalities, and a reduced life span.     

Bile acids content in brain of common duct ligated rats

Introduction. Cholestasis leads to liver cell death, fibrosis, cirrhosis, and eventually liver failure. Bile duct ligated rats constitute an interesting model to study the mechanism of cholestasis, and its action on several organs and tissues, including the brain.Aim. To analyze brain bile acids individually in ligated rats to evaluate if its profile is altered towards a more toxic condition in cholestasis.Material and methods. Male Wistar rats were used and separated in two groups: bile duct ligated rats and sham operated rats (n = 5 in each group). Bile acid profile was assessed in brain homogenates. Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase determinations, bilirubin and ammonia plasma concentration were also measured in both groups.Results. Although the total amount of bile acids in control animal brains showed a higher concentration than in bile duct ligated rats, the bile acid profile in this group was found more toxic composition than in controls. Lithocholic acid was present in brain in higher concentration (87.4 % of total brain bile acids) in ligated rats and absent in controls. Alkaline phosphatase, bilirubin and ammonia were significantly higher in bile duct ligated rats than in control group.Conclusion. It was found a toxic brain bile acid profile during hepatic cholestasis which could be one of the explanations of hepatic encephalopathy observed in cholestatic diseases.     

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.     

Role of ammonia and inflammation in minimal hepatic encephalopathy

Background: Minimal hepatic encephalopathy (MHE) is common in cirrhosis but its pathophysiologic basis remains undefined. We evaluated whether the presence of MHE was associated with severity of liver disease, ammonia levels or the presence of inflammation and assessed factors determining neuropsychological deterioration accompanying induction of hyperammonemia. Methods: Eighty four cirrhotics were studied. A neuropsychological test battery was performed and blood taken for ammonia, WCC, CRP, nitrate/nitrite, IL-6 and amino acids, before and after, induction of hyperammonemia by administration of a solution mimicking the amino acid composition of haemoglobin (60) or placebo (24). Results: The presence and severity of MHE were independent of severity of liver disease and ammonia concentration but markers of inflammation were significantly higher in those with MHE compared with those without. Induction of hyperammonemia produced deterioration in one or more neuropsychological tests by ≥1 SD in 73.3%. This was independent of the magnitude of change in plasma ammonia and severity of liver disease but was significantly greater in those with more marked inflammation. Conclusion: Our data show that inflammation is an important determinant of the presence and severity of MHE. The change in neuropsychological function following induced hyperammonemia is greater in those with more severe inflammation.     

How to diagnose hepatic encephalopathy in the emergency department

Introduction. Blood ammonia-measurements are often performed in the emergency departments to diagnose or rule out hepatic encephalopathy (HE). However, the utility and correct interpretation of ammonia levels is a matter of discussion. At this end the present prospective study evaluated whether blood ammonia levels coincide with HE which was also established by the West Haven criteria and the critical flicker frequency, respectively.Material and methods. In 59 patients with known cirrhosis ammonia-levels were determined and patient were additionally categorized by the West-Haven criteria and were also evaluated psychophysiologically using the critical flicker frequency, CFF for the presence of HE.Results. When false positive and false negative results were collapsed the determination of blood ammonia levels alone resulted in 40.7% in a misdiagnoses of HE compared to the West-Haven criteria (24/59 when using West-Haven criteria, 95% confidence interval [CI], 28.1% to 54.3%) and 49.2% when compared with the results of the CFF (29/59, when using CFF, 95% CI, 35.9% to 62.5%).Discussion. Ammonia blood levels do not reliably detect HE and the determination of blood ammonia can not be regarded a useful screening test for HE. Its use as sole indicator for a HE in the Emergency Department may frequently result in frequent misinterpretations.     

Covert hepatic encephalopathy: elevated total glutathione and absence of brain water content changes

Recent pathophysiological models suggest that oxidative stress and hyperammonemia lead to a mild brain oedema in hepatic encephalopathy (HE). Glutathione (GSx) is a major cellular antioxidant and known to be involved in the interception of both. The aim of this work was to study total glutathione levels in covert HE (minimal HE and HE grade 1) and to investigate their relationship with local brain water content, levels of glutamine (Gln), myo-inositol (mI), neurotransmitter levels, critical flicker frequency (CFF), and blood ammonia. Proton magnetic resonance spectroscopy (¹H MRS) data were analysed from visual and sensorimotor cortices of thirty patients with covert HE and 16 age-matched healthy controls. Total glutathione levels (GSx/Cr) were quantified with respect to creatine. Furthermore, quantitative MRI brain water content measures were evaluated. Data were tested for links with the CFF and blood ammonia. GSx/Cr was elevated in the visual (mHE) and sensorimotor (mHE, HE 1) MRS volumes and correlated with blood ammonia levels (both P < 0.001). It was further linked to Gln/Cr and mI/Cr (P < 0.01 in visual, P < 0.001 in sensorimotor) and to GABA/Cr (P < 0.01 in visual). Visual GSx/Cr correlated with brain water content in the thalamus, nucleus caudatus, and visual cortex (P < 0.01). Brain water measures did neither show group effects nor correlations with CFF or blood ammonia. Elevated total glutathione levels in covert HE (< HE 2) correlate with blood ammonia and may be a regional-specific reaction to hyperammonemia and oxidative stress. Brain water content is locally linked to visual glutathione levels, but appears not to be associated with changes of clinical parameters. This might suggest that cerebral oedema is only marginally responsible for the symptoms of covert HE.     

Changes in glutamate-cycle enzyme mRNA levels in a rat model of hepatic encephalopathy

To detect possible changes in the regulation of glutamate/-aminobutyric acid (GABA) enzymes at the level of gene expression in a thioacetamide-induced rat model of acute hepatic encephalopathy, we have examined changes in the mRNAs of four glutamate/GABA enzymes by quantitative RNA blot hybridization analysis. Such changes could reflect cell adaptation to excess ammonia or some other associated metabolic stress. The mRNA levels of glutamate dehydrogenase (GDH) decreased similarly in three different brain regions, whereas those of glutamine synthetase (GS) and glutaminase (GA) increased. The mRNA levels of glutamate decarboxylase (GAD) were unchanged. The results indicate that some effect of liver damage, presumably hyperammonemia, affected the expression of some, but not all, genes associated with ammonia and glutamate metabolism in the brain. This adaptation of gene expression to secondary effects of ammonia on brain amino acid neurotransmitter metabolism or brain energy metabolism could play a role in the physiological changes observed in hepatic encephalopathy.     

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.     

Metabolic fate of isoleucine in a rat model of hepatic encephalopathy and in cultured neural cells exposed to ammonia

Hepatic encephalopathy is a severe neuropathological condition arising secondary to liver failure. The pathogenesis is not well understood; however, hyperammonemia is considered to be one causative factor. Hyperammonemia has been suggested to inhibit tricarboxylic acid (TCA) cycle activity, thus affecting energy metabolism. Furthermore, it has been suggested that catabolism of the branched-chain amino acid isoleucine may help curb the effect of hyperammonemia by by-passing the TCA cycle block as well as providing the carbon skeleton for glutamate and glutamine synthesis thus fixating ammonia. Here we present novel results describing [U-¹³C]isoleucine metabolism in muscle and brain analyzed by mass spectrometry in bile duct ligated rats, a model of chronic hepatic encephalopathy, and discuss them in relation to previously published results from neural cell cultures. The metabolism of [U-¹³C]isoleucine in muscle tissue was about five times higher than that in the brain which, in turn, was lower than in corresponding cell cultures. However, synthesis of glutamate and glutamine was supported by catabolism of isoleucine. In rat brain, differential labeling patterns in glutamate and glutamine suggest that isoleucine may primarily be metabolized in the astrocytic compartment which is in accordance with previous findings in neural cell cultures. Lastly, in rat brain the labeling patterns of glutamate, aspartate and GABA do not suggest any significant inhibition by ammonia of TCA cycle activity which corresponds well to findings in neural cell cultures. Branched-chain amino acids including isoleucine are used for treating hepatic encephalopathy and the present findings shed light on the possible mechanism involved. The low turn-over of isoleucine in rat brain suggests that this amino acid does not serve the role of providing metabolites pertinent to TCA cycle function and hence energy formation as well as the necessary carbon skeleton for subsequent ammonia fixation in hyperammonemia. The higher metabolism of isoleucine in muscle could, however, contribute to ammonia fixation and thus likely be of value in the treatment of hepatic encephalopathy.     

Medium-chain triglycerides supplement therapy with a low-carbohydrate formula can supply energy and enhance ammonia detoxification in the hepatocytes of patients with adult–onset type II citrullinemia

Citrin, encoded by SLC25A13, constitutes the malate-aspartate shuttle, the main NADH-shuttle in the liver. Citrin deficiency causes neonatal intrahepatic cholestasis (NICCD) and adult–onset type II citrullinemia (CTLN2). Citrin deficiency is predicted to impair hepatic glycolysis and de novo lipogenesis, resulting in hepatic energy deficit. Secondary decrease in hepatic argininosuccinate synthetase (ASS1) expression has been considered a cause of hyperammonemia in CTLN2. We previously reported that medium–chain triglyceride (MCT) supplement therapy with a low–carbohydrate formula was effective in CTLN2 to prevent a relapse of hyperammonemic encephalopathy. We present the therapy for six CTLN2 patients. All the patients’ general condition steadily improved and five patients with hyperammonemic encephalopathy recovered from unconsciousness in a few days. Before the treatment, plasma glutamine levels did not increase over the normal range and rather decreased to lower than the normal range in some patients. The treatment promptly decreased the blood ammonia level, which was accompanied by a decrease in plasma citrulline levels and an increase in plasma glutamine levels. These findings indicated that hyperammonemia was not only caused by the impairment of ureagenesis at ASS1 step, but was also associated with an impairment of glutamine synthetase (GS) ammonia-detoxification system in the hepatocytes. There was no decrease in the GS expressing hepatocytes. MCT supplement with a low–carbohydrate formula can supply the energy and/or substrates for ASS1 and GS, and enhance ammonia detoxification in hepatocytes. Histological improvement in the hepatic steatosis and ASS1-expression was also observed in a patient after long-term treatment.     

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.     

Lipopolysaccharide-induced tyrosine nitration and inactivation of hepatic glutamine synthetase in the rat

Glutamine synthetase (GS) in the liver is restricted to a small perivenous hepatocyte population and plays an important role in the scavenging of ammonia that has escaped the periportal urea-synthesizing compartment. We examined the effect of a single intraperitoneal injection of lipopolysaccharide (LPS) in vivo on glutamine synthesis in rat liver. LPS injection induced expression of inducible nitric oxide synthase, which was maximal after 6 to 12 hours but returned toward control levels within 24 hours. Twenty-four hours after LPS injection, an approximately fivefold increase in tyrosine-nitrated proteins in liver was found, and GS protein expression was decreased by approximately 20%, whereas GS activity was lowered by 40% to 50%. GS was found to be tyrosine-nitrated in response to LPS, and immunodepletion of tyrosine-nitrated proteins decreased GS protein by approximately 50% but had no effect on GS activity. Together with the finding via mass spectrometry that peroxynitrite-induced inactivation of purified GS is associated with nitration of the active site tyrosine residue, our data suggest that tyrosine nitration critically contributes to inactivation of the enzyme. In line with GS inactivation, glutamine synthesis from ammonia (0.3 mmol/L) in perfused livers from 24-hour LPS-treated rats was decreased by approximately 50%, whereas urea synthesis was not significantly affected. In conclusion, LPS impairs hepatic ammonia detoxification by both downregulation of GS and its inactivation because of tyrosine nitration. The resulting defect of perivenous scavenger cell function with regard to ammonia elimination may contribute to sepsis-induced development of hyperammonemia in patients who have cirrhosis.     

Contribution of hyperammonemia and inflammatory factors to cognitive impairment in minimal hepatic encephalopathy

To assess the contribution of hyperammonemia and inflammation to induction of mild cognitive impairment (or MHE). We analyzed the presence of mild cognitive impairment (CI) by using the PHES battery of psychometric tests and measured the levels of ammonia and of the inflammatory cytokines IL-6 and IL-18 in blood of patients with different types of liver or dermatological diseases resulting in different grades of hyperammonemia and/or inflammation. The study included patients with 1) liver cirrhosis, showing hyperammonemia and inflammation; 2) non-alcoholic fatty liver disease (NAFLD) showing inflammation but not hyperammonemia; 3) non-alcoholic steatohepatitis (NASH) showing inflammation and very mild hyperammonemia; 4) psoriasis, showing inflammation but not hyperammonemia; 5) keloids, showing both inflammation and hyperammonemia and 6) controls without inflammation or hyperammonemia. The data reported show that in patients with liver diseases, cognitive impairment may appear before progression to cirrhosis if hyperammonemia and inflammation are high enough. Five out of 11 patients with NASH, without liver cirrhosis, showed cognitive impairment associated with hyperammonemia and inflammation. Patients with keloids showed cognitive impairment associated with hyperammonemia and inflammation, in the absence of liver disease. Hyperammonemia or inflammation alone did not induce CI but the combination of certain levels of hyperammonemia and inflammation is enough to induce CI, even without liver disease.     

Exercise-induced hyperammonemia in patients with compensated chronic liver disease

Plasma levels of ammonia and amino acids were measured during and after graded physical exercise in seven ambulatory patients with well-compensated chronic liver disease and in seven healthy controls. Plasma ammonia was similar in both groups at rest but reached significantly higher peak values (124.0 +/- 29.3 (SD) versus 74.7 +/- 17.7 mumol/l) in the patients with liver disease during exercise. The return to base line during the recovery period was delayed in the patients (T1/2 9.9 +/- 5.5 versus 2.3 +/- 1.0 min). Except for plasma taurine, which was significantly lower in the patients at rest and which showed a significant decrease in the controls but not in the patients during exercise, changes in the plasma concentration of amino acids were similar in the two groups. The increased exposure of patients with chronic liver disease to ammonia while performing an identical workload results from an impaired clearance of ammonia plus, possibly, an increased generation of ammonia in muscle working at a higher intensity. Since hyperammonemia may be associated with the sensation of fatigue, increases in plasma ammonia during daily physical activities might in part explain the easy fatigability often reported by patients with chronic liver disease.