Impaired Enzymatic Antioxidant Defense in Erythrocytes of Rats with Ammonia-Induced Encephalopathy: Role of NMDA Receptors.

Hepatic encephalopathy (HE), a neuropsychiatric disorder developing in patients with severe hepatic dysfunction, has been known for more than a century. However, pathogenetic mechanisms of cerebral dysfunction associated with liver disease are still poorly understood. There is a consensus that the primary cause of HE is accumulation of ammonia in the brain as a result of impaired liver detoxification capacity or the portosystemic shunt. Current evidence suggests that ammonia toxicity is mediated by hyperactivation of glutamate receptors, mainly N-methyl-D-aspartate receptors (NMDARs), and affects brain aerobic metabolism, which provides energy for multiple specific functions and neuronal viability. Recent reports on the presence of functional NMDARs in erythrocytes and the data on the deviations of blood parameters from their normal ranges indicate impaired hemodynamics and reduced oxygen-carrying capacity of erythrocytes in most patients with HE, thus suggesting a relationship between erythrocyte damage and cerebral dysfunction. In order to understand how hyperammonemia (HA)-induced disturbances in the energy metabolism in the brain (which needs a constant supply of large amounts of oxygen in the blood) lead to encephalopathy, it is necessary to reveal ammonia-induced impairments in the energy metabolism and antioxidant defense system of erythrocytes and to explore a potential role of ammonia in reduced brain oxygenation. To identify the said missing link, the activities of antioxidant enzymes and concentrations of reduced glutathione (GSH), oxidized glutathione (GSSG), and H2O2 were measured in the erythrocytes of rats with HA that were injected with the noncompetitive NMDAR antagonist MK-801. We found that in rats with HA, ammonia was accumulated in erythrocytes (cells lacking ammonia removal enzymes), which made them more susceptible to the prooxidant environment created during oxidative stress. This effect was completely or partially inhibited by MK-801. The data obtained might help to identify the risk factors in cognitive disorders and facilitate prediction of unfavorable outcomes of hypoperfusion in patients with a blood elevated ammonia concentration.

Portacaval shunting causes differential mitochondrial superoxide production in brain regions.

The portacaval shunting (PCS) prevents portal hypertension and recurrent bleeding of esophageal varices. On the other hand, it can induce chronic hyperammonemia and is considered to be the best model of mild hepatic encephalopathy (HE). Pathogenic mechanisms of HE and dysfunction of the brain in hyperammonemia are not fully elucidated, but it was originally suggested that the pathogenetic defect causes destruction of antioxidant defense which leads to an increase in the production of reactive oxygen species (ROS) and the occurrence of oxidative stress. In order to gain insight into the pathogenic mechanisms of HE in the brain tissue, we investigated the effects of PCS in rats on free radicals production and activity levels of antioxidant and prooxidant enzymes in mitochondria isolated from different brain areas. We found that O2·- production, activities of Mn-superoxide dismutase (Mn-SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione transferase (GT), nitric oxide synthase (NOS), and levels of carbonylated proteins differed between the four brain regions both in the amount and response to PCS. In PCS rats, Mn-SOD activity in the cerebellum was significantly decreased, and remained unchanged in the neocortex, hippocampus and striatum compared with that in sham-operated animals. Among the four brain regions in control rats, the levels of the carbonyl groups in mitochondrial proteins were maximal in the cerebellum. 4 weeks after PCS, the content of carbonylated proteins were higher only in mitochondria of this brain region. Under control conditions, O2·- production by submitochondrial particles in the cerebellum was significantly higher than in other brain regions, but was significantly increased in each brain region from PCS animals. Indeed, the production of O2·- by submitochondrial particles correlated with mitochondrial ammonia levels in the four brain regions of control and PCS-animals. These findings are the first to suggest that in vivo levels of ammonia in the brain directly affect the rate of mitochondrial O2·- production.