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 as bioreactors to decrease excess ammonium concentration in blood.

Increased blood ammonium concentrations cause neurological complications. Existing drugs are not always sufficiently effective. Alternatively, erythrocytes-bioreactors (EBRs) loaded with enzymes utilizing ammonium, were suggested for ammonium removal from blood. However all they worked only for a short period of time. The reasons for this were not investigated. In this study, EBR mathematical models were developed and analysed based on the reactions of glycolysis and different enzymes utilizing ammonium, which showed that the efficiency and duration of EBRs' functioning could be limited due to low permeability of the cell membrane for some key substrates and products. A new enzyme system including glutamate dehydrogenase and alanine aminotransferase was proposed and realised experimentally, which was not limited by cell membrane permeability for glutamate and α-ketoglutarate due to creating metabolic pathway where these metabolites were produced and consumed cyclically. New bioreactors removed ammonium in vitro at the rate of 1.5 mmol/h × lRBCs (for human bioreactors) and in vivo in a model of hyperammoniemia in mice at the rate of 2.0 mmol/h × lRBCs (for mouse bioreactors), which correlated with model calculations. Experimental studies proved the proposed mathematical models are correct. Mathematical simulation of erythrocyte-bioreactors opens new opportunities for analysing the efficiency of any enzyme included in erythrocytes.

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.

Rapid Elimination of Blood Alcohol Using Erythrocytes: Mathematical Modeling and In Vitro Study.

Erythrocytes (RBCs) loaded with alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALD) can metabolize plasma ethanol and acetaldehyde but with low efficiency. We investigated the rate-limiting factors in ethanol oxidation by these enzymes loaded into RBCs. Mathematical modeling and in vitro experiments on human RBCs loaded simultaneously with ADH and ALD (by hypoosmotic dialysis) were performed. The simulation showed that the rate of nicotinamide-adenine dinucleotide (NAD+) generation in RBC glycolysis, but not the activities of the loaded enzymes, is the rate-limiting step in external ethanol oxidation. The rate of oxidation could be increased if RBCs are supplemented by NAD+ and pyruvate. Our experimental data verified this theoretical conclusion. RBCs loaded with the complete system of ADH, ALD, NAD+, and pyruvate metabolized ethanol 20-40 times faster than reported in previous studies. The one-step procedure of hypoosmotic dialysis is the optimal method to encapsulate ADH and ALD in RBCs after cell recovery, encapsulation yield, osmotic resistance, and RBC-indexes. Consequently, transfusion of the RBCs loaded with the complete metabolic system, including ADH, ALD, pyruvate, and NAD+ in the patients with alcohol intoxication, may be a promising method for rapid detoxification of blood alcohol based on metabolism.

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.

Relationship between chronic disturbance of 2,3-diphosphoglycerate metabolism in erythrocytes and Alzheimer disease.

Alzheimer disease (AD) is one of the most common neurodegenerative disorders widely occurring among the elderly. The pathogenic mechanisms involved in the development of this disease are still unknown. In AD, in addition to brain, a number of peripheral tissues and cells are affected, including erythrocytes. In this study, we analyzed glycolytic energy metabolism, antioxidant status, glutathione, adenylate and proteolytic systems in erythrocytes from patients with AD and compared with those from age-matched controls and young adult controls. Glycolytic enzymes hexokinase, phosphofructokinase, bisphosphoglycerate mutase and bisphosphoglycerate phosphatase displayed lower activities in agematched controls, and higher activities in AD patients, as compared to those in young adult control subjects. In both aging and AD, oxidative stress is increased in erythrocytes whereas elevated concentrations of hydrogen peroxide and organic hydroperoxides as well as decreased glutathione/glutathione disulfide ratio and glutathione transferase activity can be detected. These oxidative disturbances are also accompanied by reductions in ATP levels, adenine nucleotide pool size and adenylate energy charge. Caspase-3 and calpain activities in age-matched controls and AD patients were about three times those of young adult controls. 2,3-diphosphoglycerate levels were significantly decreased in AD patients. Taken together these data suggest that AD patients are associated with chronic disturbance of 2,3-diphosphoglycerate metabolism in erythrocytes. These defects may play a central role in pathophysiological processes predisposing elderly subjects to dementia.

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.

Age-related defects in erythrocyte 2,3-diphosphoglycerate metabolism in dementia.

Alzheimer disease (AD) is the most common dementing illness. Metabolic defects in the brain with aging contribute to the pathogenesis of AD. These changes can be found systematically and thus can be used as potential biomarkers. Erythrocytes (RBCs) are passive "reporter cells" that are not well studied in AD. In the present study, we analyzed an array of glycolytic and related enzymes and intermediates in RBCs from patients with AD and non-Alzheimer dementia (NA), age-matched controls (AC) and young adult controls (YC). AD is characterized by higher activities of hexokinase, phosphofructokinase, and bisphosphoglycerate mutase and bisphosphoglycerate phosphatase in RBCs. In our study, we observed that glycolytic and related enzymes displayed significantly lower activities in AC. However, similar or significantly higher activities were observed in AD and NA groups as compared to YC group. 2,3-diphosphoglycerate (2,3-DPG) levels were significantly decreased in AD and NA patients. The pattern of changes between groups in the above indices strongly correlates with each other. Collectively, our data suggested that AD and NA patients are associated with chronic disturbance of 2,3-DPG metabolism in RBCs. These defects may play a pivotal role in physiological processes, which predispose elderly subjects to AD and NA.

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.

Subcellular and metabolic examination of amyloid-beta peptides in Alzheimer disease pathogenesis: evidence for Abeta(25-35).

Amyloid-beta peptide (Abeta) is a central player in the pathogenesis and diagnosis of Alzheimer disease. It aggregates to form the core of Alzheimer disease-associated plaques found in coordination with tau deposits in diseased individuals. Despite this clinical relevance, no single hypothesis satisfies and explicates the role of Abeta in toxicity and progression of the disease. To explore this area, investigators have focused on mechanisms of cellular dysfunction, aggregation, and maladaptive responses. Extensive research has been conducted using various methodologies to investigate Abeta peptides and oligomers, and these multiple facets have provided a wealth of data from specific models. Notably, the utility of each experiment must be considered in regards to the brain environment. The use of Abeta(25-35) in studies of cellular dysfunction has provided data indicating that the peptide is indeed responsible for multiple disturbances to cellular integrity. We will review how Abeta peptide induces oxidative stress and calcium homeostasis, and how multiple enzymes are deleteriously impacted by Abeta(25-35). Understanding and discussing the origin and properties of Abeta peptides is essential to evaluating their effects on various intracellular metabolic processes. Attention will also be specifically directed to metabolic compartmentation in affected brain cells, including mitochondrial, cytosolic, nuclear, and lysosomal enzymes.

Encapsulation of glutamine synthetase in mouse erythrocytes: a new procedure for ammonia detoxification.

There are a number of pathological situations in which ammonia levels increase leading to hyperammonemia, which may cause neurological alterations and can lead to coma and death. Currently, there are no efficient treatments allowing rapid and sustained decrease of ammonia levels in these situations. A way to increase ammonia detoxification would be to increase its incorporation in glutamine by glutamine synthetase. The aim of this work was to develop a procedure to encapsulate glutamine synthetase in mouse erythrocytes and to assess whether administration of these erythrocytes containing glutamine synthetase (GS) reduce ammonia levels in hyperammonemic mice. The procedure developed allowed the encapsulation of 3 +/- 0.25 IU of GS / mL of erythrocytes with a 70% cell recovery. Most metabolites, including ATP, remained unaltered in glutamine synthetase-loaded erythrocytes (named ammocytes by us) compared with native erythrocytes. The glutamine synthetase-loaded ammocytes injected in mice survived and retained essentially all of their glutamine synthetase activity for at least 48 h in vivo. Injection of these ammocytes into hyperammonemic mice reduced ammonia levels in the blood by about 50%. The results reported indicate that ammocytes are able to keep their integrity, normal energy metabolism, the inserted glutamine synthetase activity, and can be useful to reduce ammonia levels in hyperammonemic situations.

Effects of amyloid-beta peptides on hydrogen peroxide-metabolizing enzymes in rat brain in vivo.

Amyloid-beta (Abeta) peptides are components of senile plaques initiating degeneration of brain neurons in Alzheimer's disease. They increase reactive oxygen species generation that may exceed the defensive capacity of cells. To test the hypothesis, this study investigated the in vivo effects of Abeta peptides on mitochondrial and non-mitochondrial enzymic sources of reactive oxygen species and antioxidant enzymes in rat brain. Continuous intracerebroventricular infusion of both Abeta(25-35) and Abeta(1-40) for up to 14 days stimulated the hydrogen peroxide (H2O2) generation in isolated neocortex mitochondria. Infusion of Abeta(1-40) led to an increase in Mn-superoxide dismutase activity and a decrease in activities of catalase and glutathione peroxidase in mitochondria, to elevation of activities of Cu,Zn-superoxide dismutase and aldehyde oxidase, forwarded the conversion of xanthine dehydrogenase to xanthine oxidase and corresponding increase in the rate of H2O2 formation in the cytosol. Thus, Abeta peptides increase H2O2-formation and H2O2-forming enzyme activities and inhibit H2O2-consuming enzyme activities in mitochondria and cytosol in vivo. These studies suggest that disbalance between H2O2-generating and H2O2-metabolizing enzyme activities can contribute to oxidative stress underlying neurodegeneration and neuronal death in Alzheimer's disease.