Ammonia toxicity: from head to toe?

Ammonia is diffused and transported across all plasma membranes. This entails that hyperammonemia leads to an increase in ammonia in all organs and tissues. It is known that the toxic ramifications of ammonia primarily touch the brain and cause neurological impairment. However, the deleterious effects of ammonia are not specific to the brain, as the direct effect of increased ammonia (change in pH, membrane potential, metabolism) can occur in any type of cell. Therefore, in the setting of chronic liver disease where multi-organ dysfunction is common, the role of ammonia, only as neurotoxin, is challenged. This review provides insights and evidence that increased ammonia can disturb many organ and cell types and hence lead to dysfunction

Sarcopenia from mechanism to diagnosis and treatment in liver disease

Sarcopenia or loss of skeletal muscle mass is the major component of malnutrition and is a frequent complication in cirrhosis that adversely affects clinical outcomes. These include survival, quality of life, development of other complications and post liver transplantation survival. Radiological image analysis is currently utilized to diagnose sarcopenia in cirrhosis. Nutrient supplementation and physical activity are used to counter sarcopenia but have not been consistently effective because the underlying molecular and metabolic abnormalities persist or are not influenced by these treatments. Even though alterations in food intake, hypermetabolism, alterations in amino acid profiles, endotoxemia, accelerated starvation and decreased mobility may all contribute to sarcopenia in cirrhosis, hyperammonemia has recently gained attention as a possible mediator of the liver-muscle axis. Increased muscle ammonia causes: cataplerosis of α-ketoglutarate, increased transport of leucine in exchange for glutamine, impaired signaling by leucine, increased expression of myostatin (a transforming growth factor beta superfamily member) and an increased phosphorylation of eukaryotic initiation factor 2α. In addition, mitochondrial dysfunction, increased reactive oxygen species that decrease protein synthesis and increased autophagy mediated proteolysis, also play a role. These molecular and metabolic alterations may contribute to the anabolic resistance and inadequate response to nutrient supplementation in cirrhosis. Central and skeletal muscle fatigue contributes to impaired exercise capacity and responses. Use of proteins with low ammoniagenic potential, leucine enriched amino acid supplementation, long-term ammonia lowering strategies and a combination of resistance and endurance exercise to increase muscle mass and function may target the molecular abnormalities in the muscle. Strategies targeting endotoxemia and the gut microbiome need further evaluatio

Hyperinsulinism-hyperammonaemia syndrome: novel mutations in the GLUD1 gene and genotype-phenotype correlations

Background: Activating mutations in the GLUD1 gene (which encodes for the intra-mitochondrial enzyme glutamate dehydrogenase, GDH) cause the hyperinsulinism–hyperammonaemia (HI/HA) syndrome. Patients present with HA and leucine-sensitive hypoglycaemia. GDH is regulated by another intra-mitochondrial enzyme sirtuin 4 (SIRT4). Sirt4 knockout mice demonstrate activation of GDH with increased amino acid-stimulated insulin secretion. Objectives: To study the genotype–phenotype correlations in patients with GLUD1 mutations. To report the phenotype and functional analysis of a novel mutation (P436L) in the GLUD1 gene associated with the absence of HA. Patients and methods: Twenty patients with HI from 16 families had mutational analysis of the GLUD1 gene in view of HA (n=19) or leucine sensitivity (n=1). Patients negative for a GLUD1 mutation had sequence analysis of the SIRT4 gene. Functional analysis of the novel P436L GLUD1 mutation was performed. Results: Heterozygous missense mutations were detected in 15 patients with HI/HA, 2 of which are novel (N410D and D451V). In addition, a patient with a normal serum ammonia concentration (21 µmol/l) was heterozygous for a novel missense mutation P436L. Functional analysis of this mutation confirms that it is associated with a loss of GTP inhibition. Seizure disorder was common (43%) in our cohort of patients with a GLUD1 mutation. No mutations in the SIRT4 gene were identified. Conclusion: Patients with HI due to mutations in the GLUD1 gene may have normal serum ammonia concentrations. Hence, GLUD1 mutational analysis may be indicated in patients with leucine sensitivity; even in the absence of HA. A high frequency of epilepsy (43%) was observed in our patients with GLUD1 mutations

Inducible arginase 1 deficiency in mice leads to hyperargininemia and altered amino acid metabolism

Arginase deficiency is a rare autosomal recessive disorder resulting from a loss of the liver arginase isoform, arginase 1 (ARG1), which is the final step in the urea cycle for detoxifying ammonia. ARG1 deficiency leads to hyperargininemia, characterized by progressive neurological impairment, persistent growth retardation and infrequent episodes of hyperammonemia. Using the Cre/loxP-directed conditional gene knockout system, we generated an inducible Arg1-deficient mouse model by crossing "floxed" Arg1 mice with CreER(T2) mice. The resulting mice (Arg-Cre) die about two weeks after tamoxifen administration regardless of the starting age of inducing the knockout. These treated mice were nearly devoid of Arg1 mRNA, protein and liver arginase activity, and exhibited symptoms of hyperammonemia. Plasma amino acid analysis revealed pronounced hyperargininemia and significant alterations in amino acid and guanidino compound metabolism, including increased citrulline and guanidinoacetic acid. Despite no alteration in ornithine levels, concentrations of other amino acids such as proline and the branched-chain amino acids were reduced. In summary, we have generated and characterized an inducible Arg1-deficient mouse model exhibiting several pathologic manifestations of hyperargininemia. This model should prove useful for exploring potential treatment options of ARG1 deficiency

Orotic Aciduria

Orotic acid is an intermediate found in the pathway for pyrimidine synthesis. The mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) catalyzes the production of orotic acid by the conversion of the compound dihydroorotate to orotic acid. Orotic acid is commonly produced by this reaction in erythrocytes, hepatocytes, and kidney cells. Chemical modification of orotic acid in the pyrimidine pathway will generate nucleotides involved in DNA and RNA synthesis. Orotic aciduria can occur as a secondary manifestation due to a defect in an enzyme or transporter within the urea cycle, due to competitive inhibition by anti-cancer drugs such as allopurinol and 6-azauridine, or due to diseases such as Reye’s syndrome and mitochondrial disorders. The primary cause of orotic aciduria is a rare, autosomal recessive disorder in the family of inborn errors in metabolism, and can be categorized into three groups. Type I, II, and III orotic aciduria occur due to a genetic defect in the bifunctional enzyme uridine monophosphate synthase (UMPS). Neonatal patients diagnosed with this disorder commonly exemplify symptoms such as lethargy, difficulty feeding, and low birth weight and height. Neural and developmental deficits, megaloblastic anemia, and crystalluria may also occur. Laboratory findings typically include extremely high urinary orotic acid levels, megaloblastic anemia, and very low UMPS activity. Since early treatment with Xuriden may cause remission of symptoms, future studies should focus on improving early detection screening methods. Early diagnosis and treatment is clinically significant because impaired pyrimidine synthesis can lead to severe symptoms such as mental retardation, coma, or death

Mathematical models and hepatology; oil and vinegar?

Mathematical models are increasingly being used in medicine to study physiological, pathophysiological and therapeutic pathways [1–3]. In hepatology, mathematical abstractions have been beneficial to predict the viral load of hepatitis C following treatment [4], outcome after acetaminophen overdose [5], or to quantify porto-systemic shunting and inter-organ ammonia metabolism at different stages of cirrhosis [6]. However, in many medical fields (including hepatology), there is much resistance to include theoretical models in their traditional tool set and furthermore a lack of confidence in the generated theoretical results

Low myo-inositol and high glutamine levels in brain are associated with neuropsychological deterioration after induced hyperammonemia

The neuropsychological effect of hyperammonemia is variable. This study tests the hypothesis that the effect of ammonia on the neuropsychological function in patients with cirrhosis is determined by the ability of the brain to buffer ammonia-induced increase in glutamine within the astrocyte by losing osmolytes like myo-inositol (mI) and not by the magnitude of the induced hyperammonemia. Fourteen cirrhotic patients with no evidence of overt hepatic encephalopathy were given a 75-g amino acid (aa) solution mimicking the hemoglobin molecule to induce hyperammonemia. Measurement of a battery of neuropsychological function tests including immediate memory, ammonia, aa, and short-echo time proton magnetic resonance spectroscopy were performed before and 4 h after administration of the as solution. Eight patients showed deterioration in the Immediate Memory Test at 4 h. Demographic factors, severity of liver disease, change in plasma ammonia, and as profiles after the as solution were similar in those that showed a deterioration compared with those who did not. In patients who showed deterioration in the memory test, the mI-to-creatine ratio (mI/Cr) was significantly lower at baseline than those that did not deteriorate. In contrast, the glutamate/glutamine-to-Cr ratio was significantly greater in the patients that deteriorated. The observation that deterioration in the memory test scores was greater in those with lower mI/Cr supports the hypothesis that the neuropsychological effects of induced hyperammonemia is determined by the capacity of the brain to handle ammonia-induced increase in glutamine

Targeting the muscle for the treatment and prevention of hepatic encephalopathy

Muscle mass loss or sarcopenia is a principle component of malnutrition which prevails in 65–90% of patients with end-stage liver disease [1]. Intuitively, the roots of malnutrition play a precipitating role in muscle catabolism. Undernutrition frequently occurs in cirrhosis since an inadequate diet is compounded by a hypermetabolic energy demand. However, multiple other factors contribute to the pathogenesis of malnutrition including malabsorption of nutrients, metabolic alterations, increased intestinal protein losses, reduced protein synthesis, increased protein catabolism and disturbance of substrate utilization [2,3]

Glutamate induces autophagy via the two-pore channels in neural cells

NAADP (nicotinic acid adenine dinucleotide phosphate) has been proposed as a second messenger for glutamate in neuronal and glial cells via the activation of the lysosomal Ca2+ channels TPC1 and TPC2. However, the activities of glutamate that are mediated by NAADP remain unclear. In this study, we evaluated the effect of glutamate on autophagy in astrocytes at physiological, non-toxic concentration. We found that glutamate induces autophagy at similar extent as NAADP. By contrast, the NAADP antagonist NED-19 or SiRNA-mediated inhibition of TPC1/2 decreases autophagy induced by glutamate, confirming a role for NAADP in this pathway. The involvement of TPC1/2 in glutamate-induced autophagy was also confirmed in SHSY5Y neuroblastoma cells. Finally, we show that glutamate leads to a NAADP-dependent activation of AMPK, which is required for autophagy induction, while mTOR activity is not affected by this treatment. Taken together, our results indicate that glutamate stimulates autophagy via NAADP/TPC/AMPK axis, providing new insights of how Ca2+ signalling glutamate-mediated can control the cell metabolism in the central nervous system