Ammoniagenic Action of Valproate without Signs of Hepatic Dysfunction in Rats: Possible Causes and Supporting Evidence.

(1) Background: Valproic acid (VPA) is one of the frequently prescribed antiepileptic drugs and is generally considered well tolerated. However, VPA neurologic adverse effects in the absence of liver failure are fairly common, suggesting that in the mechanism for the development of VPA-induced encephalopathy, much more is involved than merely the exposure to hyperammonemia (HA) caused by liver insufficiency to perform detoxification. Taking into account the importance of the relationship between an impaired brain energy metabolism and elevated ammonia production, and based on the ability of VPA to interfere with neuronal oxidative pathways, the current study intended to investigate a potential regional ammoniagenic effect of VPA on rats' brains by determining activities of the enzymes responsible for ammonia production and neutralization. (2) Methods: Rats received a single intraperitoneal injection of VPA (50, 100, 250, 500 mg/kg). Plasma, the neocortex, the cerebellum, and the hippocampus were collected at 30 min after injection. The levels of ammonia, urea, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured in blood plasma. The activities of glutaminase and glutamate dehydrogenase (GDH) in mitochondria and the activities of AMP deaminase (AMPD), adenosine deaminase (ADA), and glutamine synthetase (GS) in cytosolic fractions isolated from rat brain regions were measured. Ammonia, ALT, and AST values were determined in the mitochondrial and cytosolic fractions. (3) Results: Multi-dose VPA treatment did not significantly affect the plasma levels of ammonia and urea or the ALT and AST liver enzymes. Significant dose-independent increases in the accumulation of ammonia were found only in the cytosol from the cerebellum and there was a strong correlation between the ammonia level and the ADA activity in this brain structure. A significant decrease in the AMPD and AST activities was observed, while the ALT activity was unaffected. Only the highest VPA dose (500 mg/kg) was associated with significantly less activity of GS compared to the control in all studied brain structures. In the mitochondria of all studied brain structures, VPA caused a dose-independent increases in ammonia levels, a high concentration of which was strongly and positively correlated with the increased GDH and ALT activity, while glutaminase activity remained unchanged, and AST activity significantly decreased compared to the control in all studied brain structures. (4) Conclusions: This study highlights the rat brain region-specific ammoniagenic effects of VPA, which may manifest themselves in the absence of hyperammonemia. Further research should analyze how the responsiveness of the different brain regions may vary in VPA-treated animals that exhibit compromised energy metabolism, leading to increased ammoniagenesis.

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.

Erythrocytes Functionality in SARS-CoV-2 Infection: Potential Link with Alzheimer's Disease.

Coronavirus disease 2019 (COVID-19) is a rapidly spreading acute respiratory infection caused by SARS-CoV-2. The pathogenesis of the disease remains unclear. Recently, several hypotheses have emerged to explain the mechanism of interaction between SARS-CoV-2 and erythrocytes, and its negative effect on the oxygen-transport function that depends on erythrocyte metabolism, which is responsible for hemoglobin-oxygen affinity (Hb-O2 affinity). In clinical settings, the modulators of the Hb-O2 affinity are not currently measured to assess tissue oxygenation, thereby providing inadequate evaluation of erythrocyte dysfunction in the integrated oxygen-transport system. To discover more about hypoxemia/hypoxia in COVID-19 patients, this review highlights the need for further investigation of the relationship between biochemical aberrations in erythrocytes and oxygen-transport efficiency. Furthermore, patients with severe COVID-19 experience symptoms similar to Alzheimer's, suggesting that their brains have been altered in ways that increase the likelihood of Alzheimer's. Mindful of the partly assessed role of structural, metabolic abnormalities that underlie erythrocyte dysfunction in the pathophysiology of Alzheimer's disease (AD), we further summarize the available data showing that COVID-19 neurocognitive impairments most probably share similar patterns with known mechanisms of brain dysfunctions in AD. Identification of parameters responsible for erythrocyte function that vary under SARS-CoV-2 may contribute to the search for additional components of progressive and irreversible failure in the integrated oxygen-transport system leading to tissue hypoperfusion. This is particularly relevant for the older generation who experience age-related disorders of erythrocyte metabolism and are prone to AD, and provide an opportunity for new personalized therapies to control this deadly infection.

Is NMDA-Receptor-Mediated Oxidative Stress in Mitochondria of Peripheral Tissues the Essential Factor in the Pathogenesis of Hepatic Encephalopathy?

BACKGROUND: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome of increased ammonia-mediated brain dysfunction caused by impaired hepatic detoxification or when the blood bypasses the liver. Ammonia-activated signal transduction pathways of hyperactivated NMDA receptors (NMDAR) are shown to trigger a cascade of pathological reactions in the brain, leading to oxidative stress. NMDARs outside the brain are widely distributed in peripheral tissues, including the liver, heart, pancreas, and erythrocytes. To determine the contribution of these receptors to ammonia-induced oxidative stress in peripheral tissues, it is relevant to investigate if there are any ammonia-related changes in antioxidant enzymes and free radical formation and whether blockade of NMDARs prevents these changes. METHODS: Hyperammonemia was induced in rats by ammonium acetate injection. Oxidative stress was measured as changes in antioxidant enzyme activities and O2•- and H2O2 production by mitochondria isolated from the tissues and cells mentioned above. The effects of the NMDAR antagonist MK-801 on oxidative stress markers and on tissue ammonia levels were evaluated. RESULTS: Increased ammonia levels in erythrocytes and mitochondria isolated from the liver, pancreas, and heart of hyperammonemic rats are shown to cause tissue-specific oxidative stress, which is prevented completely (or partially in erythrocyte) by MK-801. CONCLUSIONS: These results support the view that the pathogenesis of HE is multifactorial and that ammonia-induced multiorgan oxidative stress-mediated by activation of NMDAR is an integral part of the disease and, therefore, the toxic effects of ammonia in HE may be more global than initially expected.

A Look into Liver Mitochondrial Dysfunction as a Hallmark in Progression of Brain Energy Crisis and Development of Neurologic Symptoms in Hepatic Encephalopathy.

BACKGROUND: The relationship between liver disease and neuropathology in hepatic encephalopathy is well known, but the genesis of encephalopathy in liver failure is yet to be elucidated. Conceptually, the main cause of hepatic encephalopathy is the accumulation of brain ammonia due to impaired liver detoxification function or occurrence of portosystemic shunt. Yet, as well as taking up toxic ammonia, the liver also produces vital metabolites that ensure normal cerebral function. Given this, for insight into how perturbations in the metabolic capacity of the liver may be related to brain pathology, it is crucial to understand the extent of ammonia-related changes in the hepatic metabolism that provides respiratory fuel for the brain, a deficiency of which can give rise to encephalopathy. METHODS: Hepatic encephalopathy was induced in starved rats by injection of ammonium acetate. Ammonia-induced toxicity was evaluated by plasma and freeze-clamped liver and brain energy metabolites, and mitochondrial, cytoplasmic, and microsomal gluconeogenic enzymes, including mitochondrial ketogenic enzymes. Parameters of oxidative phosphorylation were recorded polarographically with a Clark-type electrode, while other measures were determined with standard fluorometric enzymatic methods. RESULTS: Progressive impairment of liver mitochondrial respiration in the initial stage of ammonia-induced hepatotoxicity and the subsequent energy crisis due to decreased ATP synthesis lead to cessation of gluconeogenesis and ketogenesis. Reduction in glucose and ketone body supply to the brain is a terminal event in liver toxicity, preceding the development of coma. CONCLUSIONS: Our study provides a framework to further explore the relationship between hepatic dysfunction and progression of brain energy crisis in hepatic encephalopathy.

The Erythrocytic Hypothesis of Brain Energy Crisis in Sporadic Alzheimer Disease: Possible Consequences and Supporting Evidence.

Alzheimer's disease (AD) is a fatal form of dementia of unknown etiology. Although amyloid plaque accumulation in the brain has been the subject of intensive research in disease pathogenesis and anti-amyloid drug development; the continued failures of the clinical trials suggest that amyloids are not a key cause of AD and new approaches to AD investigation and treatment are needed. We propose a new hypothesis of AD development based on metabolic abnormalities in circulating red blood cells (RBCs) that slow down oxygen release from RBCs into brain tissue which in turn leads to hypoxia-induced brain energy crisis; loss of neurons; and progressive atrophy preceding cognitive dysfunction. This review summarizes current evidence for the erythrocytic hypothesis of AD development and provides new insights into the causes of neurodegeneration offering an innovative way to diagnose and treat this systemic disease.

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.

Metabolic Abnormalities of Erythrocytes as a Risk Factor for Alzheimer's Disease.

Alzheimer's disease (AD) is a slowly progressive, neurodegenerative disorder of uncertain etiology. According to the amyloid cascade hypothesis, accumulation of non-soluble amyloid ß peptides (Aß) in the Central Nervous System (CNS) is the primary cause initiating a pathogenic cascade leading to the complex multilayered pathology and clinical manifestation of the disease. It is, therefore, not surprising that the search for mechanisms underlying cognitive changes observed in AD has focused exclusively on the brain and Aß-inducing synaptic and dendritic loss, oxidative stress, and neuronal death. However, since Aß depositions were found in normal non-demented elderly people and in many other pathological conditions, the amyloid cascade hypothesis was modified to claim that intraneuronal accumulation of soluble Aß oligomers, rather than monomer or insoluble amyloid fibrils, is the first step of a fatal cascade in AD. Since a characteristic reduction of cerebral perfusion and energy metabolism occurs in patients with AD it is suggested that capillary distortions commonly found in AD brain elicit hemodynamic changes that alter the delivery and transport of essential nutrients, particularly glucose and oxygen to neuronal and glial cells. Another important factor in tissue oxygenation is the ability of erythrocytes (red blood cells, RBC) to transport and deliver oxygen to tissues, which are first of all dependent on the RBC antioxidant and energy metabolism, which finally regulates the oxygen affinity of hemoglobin. In the present review, we consider the possibility that metabolic and antioxidant defense alterations in the circulating erythrocyte population can influence oxygen delivery to the brain, and that these changes might be a primary mechanism triggering the glucose metabolism disturbance resulting in neurobiological changes observed in the AD brain, possibly related to impaired cognitive function. We also discuss the possibility of using erythrocyte biochemical aberrations as potential tools that will help identify a risk factor for AD.

Critical analysis of Alzheimer's amyloid-beta toxicity to mitochondria.

Amyloid-beta peptide (Abeta) is believed to be a central player in the Alzheimer disease (AD) pathogenesis. However, its mechanisms of toxicity to the central nervous system are unknown. To explore this area, investigators have recently focused on Abeta-induced cellular dysfunction. Extensive research has been conducted to investigate Abeta monomers and oligomers, and these multiple facets have provided a wealth of data from specific models. Abeta appears to be accumulated in neuronal mitochondria and mediates mitochondrial toxicity. Mitochondrial dysfunction became a hallmark of Abeta-induced neuronal toxicity. Mitochondria are currently considered as primary targets in the pathobiology of neurodegeneration. This review provides an overview of the Abeta toxicity to isolated mitochondria, mitochondria in different tissues and cells in vitro and in vivo. Full texts and abstracts from all 530 biomedical articles listed in PubMed and published before January 2014 were analysed. The mechanisms underlying the interaction between Abeta and mitochondrial membranes and resulting mitochondrial dysfunction are most disputed issues. Understanding and discussing this interaction is essential to evaluating Abeta effects on various intracellular metabolic processes.

Differential up-regulation of ammonia detoxifying enzymes in cerebral cortex, cerebellum, hippocampus, striatum and liver in hyperammonemia.

In order to gain insight into the ammonia-detoxification mechanisms in the brain and liver tissues, we have investigated the effects of hyperammonemia in rats, in vivo, on the activity levels of a number of ammonia- and glutamate-metabolizing enzymes in mitochondria and the cytosolic fractions of the cerebral cortex, cerebellum, hippocampus, striatum and liver. In general, the ammonia metabolizing enzymes - glutaminase, glutamine synthetase, glutamate dehydrogenase, AMP deaminase, adenosine deaminase, as well as aspartate aminotransferase and alanine aminotransferase - are differentially upregulated in various brain and liver regions of the hyperammonemic rats, indicating that divergent ammonia-detoxification mechanisms are involved in the various brain regions and liver in acute hyperammonemia.

Impact of amyloid ß25-35 on membrane stability, energy metabolism, and antioxidant enzymes in erythrocytes.

Amyloid ß25-35 (Aß25-35) represents a neurotoxic fragment of Aß1-40 or Aß1-42, and is implicated in the progressive neurodegeneration in cases of the Alzheimer disease (AD). Amyloid ß25-35 was shown to lyse rat erythrocytes (RBCs) of all ages, and the extent of the RBC toxicity is directly correlated with Aß25-35 concentration and cell age. Activities of glycolytic, antioxidant, and Na(+)/K(+)-adenosine triphosphatase (ATPase) enzymes, in vivo, are significantly decreased in older RBCs as compared to the young RBCs. In vitro, Aß25-35 reduced activities of hexokinase, phosphofructokinase, pyruvate kinase, glutathione peroxidase, and glutathione transferase and increased Na(+)/K(+)-ATPase activity; these effects are significantly greater in aged RBCs as compared to those of the younger cells. The diminution in activity of certain enzymes may determine the life span of the RBCs in vivo and may be relevant to the human AD; higher sensitivity of older RBCs to Aß25-35 toxicity may contribute to the ultimate death of the RBCs in patients with AD.

Pathogenesis of Alzheimer disease: role of oxidative stress, amyloid-ß peptides, systemic ammonia and erythrocyte energy metabolism.

Aß exerts prooxidant or antioxidant effects based on the metal ion concentrations that it sequesters from the cytosol; at low metal ion concentrations, it is an antioxidant, whereas at relatively higher concentration it is a prooxidant. Thus Alzheimer disease (AD) treatment strategies based solely on the amyloid-ß clearance should be re-examined in light of the vast accumulating evidence that increased oxidative stress in the human brains is the key causative factor for AD. Accumulating evidence indicates that the reduced brain glucose availability and brain hypoxia, due to the relatively lower concentration of ATP and 2,3-diphosphoglycerate, may be associated with increased concentration of endogenous ammonia, a potential neurotoxin in the AD brains. In this review, we summarize the progress in this area, and present some of our ongoing research activities with regard to brain Amyloid-ß, systemic ammonia, erythrocyte energy metabolism and the role of 2,3-diphosphoglycerate in AD pathogenesis.

Antioxidant status and energy state of erythrocytes in Alzheimer dementia: probing for markers.

Subject age and brain oxidative stress play pivotal roles in Alzheimer disease (AD) pathology. Erythrocytes (red blood cells: RBC) are considered as passive "reporter cells" for the oxidative status of the whole organism, not active participants in mechanisms of AD pathogenesis and are not well studied in AD. The aim of this work is to assess whether the antioxidant status and energy state of RBC from elderly people change in AD. We measured levels of key products and enzymes of oxidative metabolism in RBC from AD (n = 12) and non-Alzheimer dementia (NA, n = 13) patients, as well as in cells from age-matched controls (AC, n = 14) and younger adult controls (YC, n = 14). Parameters of the adenylate system served to evaluate the energy state of RBC. In both aging and dementia, oxidative stress in RBC increased and exhibited elevated concentrations of H2O2 and organic hydroperoxides, decreased the GSH/GSSG ratio and glutathione-S-transferase activity. Reductions in the ATP levels, adenine nucleotide pool size (AN) and adenylate energy charge accompanied these oxidative disturbances. The patterns of changes in these indices between groups strongly correlated with each other, Spearman rank correlation coefficients being r(s) = 1.0 or -1.0 (p < 0.01). Alterations of the RBC parameters of oxidative stress and adenylate metabolism were nonspecific and interpreted as age-related abnormalities. Decreased glutathione peroxidase activity in RBC may be considered as a new peripheral marker for AD.

Impacts of lisinopril and lisinopril plus simvastatin on erythrocyte and plasma arginase, nitrite, and nitrate in hypertensive patients.

Angiotensin-converting enzyme inhibitors are effective at reducing blood pressure, whereas statins decrease plasma cholesterol, impeding atherosclerosis. The authors hypothesize that these medications may improve blood pressure by modifying the arginase-nitric oxide synthase system of erythrocytes. In this study, the effects of lisinopril alone versus lisinopril + simvastatin on erythrocyte and plasma arginase enzyme and nitric oxide metabolites are compared. Patients with atherosclerosis and hypertension are randomly assigned to receive lisinopril 10 to 20 mg/d or lisinopril 10 to 20 mg/d plus simvastatin 20 mg/d for 24 weeks. Higher arginase activity is observed in erythrocytes from 100% of patients and mainly recovered after 12 and 24 weeks of treatment with lisinopril or lisinopril + simvastatin. Plasma arginase activity is 3 orders of magnitude lower than erythrocyte arginase activity in all participants, suggesting a lack of its clinical significance. Both treatments cause the increase in plasma $$\hbox{ N }{\hbox{ O }}_{2}^{-}$$ , $$\hbox{ N }{\hbox{ O }}_{3}^{-}$$ , and $$\hbox{ N }{\hbox{ O }}_{2}^{-}$$ + $$\hbox{ N }{\hbox{ O }}_{3}^{-}$$ in 100% of patients. Erythrocyte $$\hbox{ N }{\hbox{ O }}_{2}^{-}$$ + $$\hbox{ N }{\hbox{ O }}_{3}^{-}$$ concentration is greatly decreased in hypertensive patients but recovers after monotherapy and combined therapy. The results show for the first time that lisinopril monotherapy and combined lisinopril + simvastatin therapy exhibit pronounced and equipotential normalizing effects on erythrocyte arginase and nitric oxide synthase activities.