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Patients with nonalcoholic fatty liver disease (NAFLD) may show mild cognitive impairment (MCI). The mechanisms involved remain unclear. The plasma concentrations of several cytokines and chemokines were measured in 71 NAFLD patients (20 with and 51 without MCI) and 61 controls. Characterization and activation of leukocyte populations and CD4+ sub-populations were carried out and analyzed by flow cytometry. We analyzed the cytokines released from CD4+ cell cultures and the mRNA expression of transcription factors and receptors in peripheral blood mononuclear cells. The appearance of MCI in NAFLD patients was associated with increased activation of CD4+ T lymphocytes, mainly of the Th17 subtype, increased plasma levels of pro-inflammatory and anti-inflammatory cytokines such as IL-17A, IL-23, IL-21, IL-22, IL-6, INF-γ, and IL-13, and higher expression of the CCR2 receptor. Constitutive expression of IL-17 was found in cultures of CD4+ cells from MCI patients, reflecting Th17 activation. High IL-13 plasma levels were predictive of MCI and could reflect a compensatory anti-inflammatory response to the increased expression of pro-inflammatory cytokines. This study identified some specific alterations of the immune system associated with the appearance of neurological alterations in MCI patients with NAFLD that could be the basis to improve and restore cognitive functions and quality of life in these patients.

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.

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.

Patients with cirrhosis may show minimal hepatic encephalopathy (MHE), for which rifaximin is effective. Metabolic syndrome may be associated with cognitive impairment. Our aims were to evaluate the influence of metabolic syndrome features on response to rifaximin for neurological and inflammatory alterations in MHE. A prospective cohort study was conducted in 63 cirrhotic patients and 30 controls from two tertiary centres recruited between 2015 and 2019. Metabolic syndrome was defined according to the Adult Treatment Panel-III. Patients were classified into 31 without and 32 with MHE according to the Psychometric Hepatic Encephalopathy Score (PHES). All participants performed specific psychometric tests, and inflammatory parameters were studied. Patients with MHE received rifaximin (400 mg/8 h). Response was evaluated by PHES at 3 and 6 months. Response according to metabolic syndrome manifestations was compared. The response rate was 66%. Older age (p = 0.012) and all metabolic syndrome diseases (p p p = 0.011), working memory (p = 0.005), visual coordination (p = 0.013) and lower proportion of activated CD4+ lymphocytes (p = 0.039) at baseline, as well as worse concentration (p = 0.030), bimanual coordination (p = 0.004) and higher levels of intermediate monocytes (p = 0.026), CX3CL1 (p p = 0.022), AHR (p = 0.010) and IgG (p

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.

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.

Minimal hepatic encephalopathy (MHE) is associated with mild cognitive impairment and frailty. This study aims to identify cognitive and motor differences in cirrhotic patients with and without MHE, and the correlations between motor signs and cognitive performance. Gait, balance, hand strength and motor speed performance were evaluated in 66 cirrhotic patients (38 without and 28 with MHE, according to the Psychometric Hepatic Encephalopathy Score (PHES). Cognitive performance was measured with the Mini-Mental State Examination, Verbal Fluency Test, Aprendizaje Verbal España-Complutense Test (TAVEC), Wechsler Adult Intelligence Scale III, Hamilton Depression and Anxiety Rating Scale and Functioning Assessment Short Test (FAST). MHE patients performed worse than patients without MHE in cognitive and autonomous functioning, learning and long-term memory, and verbal fluency. The same pattern was found in gait, center of pressure movement, variability of hand strength performance and hand motor speed. In MHE patients, high correlations were found between balance and FAST test, gait velocity and verbal skills, hand strength variability and anxiety and depression, and motor speed and FAST and TAVEC. MHE patients showed worse motor and cognitive performance than patients without MHE. MHE patients could have impaired movement control expressed as bradykinesia, and this reduced motor performance could correlate with cognitive performance.

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.

Cirrhotic patients may experience alterations in the peripheral nervous system and in somatosensory perception. Impairment of the somatosensory system could contribute to cognitive and motor alterations characteristic of minimal hepatic encephalopathy (MHE), which affects up to 40% of cirrhotic patients. We assessed the relationship between MHE and alterations in thermal, vibration, and/or heat pain sensitivity in 58 cirrhotic patients (38 without and 20 with MHE according to Psychometric Hepatic Encephalopathy Score) and 39 controls. All participants underwent attention and coordination tests, a nerve conduction study, autonomic function testing, and evaluation of sensory thresholds (vibration, cooling, and heat pain detection) by electromyography and quantitative sensory testing. The detection thresholds for cold and heat pain on the foot were higher in patients with, than those without MHE. This hyposensitivity was correlated with attention deficits. Reaction times in the foot were longer in patients with, than without MHE. Patients with normal sural nerve amplitude showed altered thermal sensitivity and autonomic function, with stronger alterations in patients with, than in those without MHE. MHE patients show a general decrease in cognitive and sensory abilities. Small fibers of the autonomic nervous system and thermal sensitivity are altered early on in MHE, before large sensory fibers. Quantitative sensory testing could be used as a marker of MHE.

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.

Patients with minimal hepatic encephalopathy (MHE) show increased oxidative stress in blood. We aimed to assess whether MHE patients show alterations in different types of blood cells in (a) basal reactive oxygen and nitrogen species levels; (b) capacity to metabolise these species. To assess the mechanisms involved in the altered capacity to metabolise these species we also analysed: (c) peroxynitrite formation and d) peroxynitrite reaction with biological molecules. Levels of reactive oxygen and nitrogen species were measured by flow cytometry in blood cell populations from cirrhotic patients with and without MHE and controls, under basal conditions and after adding generators of superoxide (plumbagin) or nitric oxide (NOR-1) to assess the capacity to eliminate them. Under basal conditions, MHE patients show reduced superoxide and peroxynitrite levels and increased nitric oxide (NO) and nitrotyrosine levels. In patients without MHE plumbagin strongly increases cellular superoxide, moderately peroxynitrite and reduces NO levels. In MHE patients, plumbagin increases slightly superoxide and strongly peroxynitrite levels and affects slightly NO levels. NOR-1 increases NO levels much less in patients with than without MHE. These data show that the mechanisms and the capacity to eliminate cellular superoxide, NO and peroxynitrite are enhanced in MHE patients. Superoxide elimination is enhanced through reaction with NO to form peroxynitrite which, in turn, is eliminated by enhanced reaction with biological molecules, which could contribute to cognitive impairment in MHE. The data show that basal free radical levels do not reflect the oxidative stress status in MHE.

Patients with minimal hepatic encephalopathy (MHE) show mild cognitive impairment associated with alterations in attentional and executive networks. There are no studies evaluating the relationship between memory in MHE and structural and functional connectivity (FC) changes in the hippocampal system. This study aimed to evaluate verbal learning and long-term memory in cirrhotic patients with (C-MHE) and without MHE (C-NMHE) and healthy controls. We assessed the relationship between alterations in memory and the structural integrity and FC of the hippocampal system. C-MHE patients showed impairments in learning, long-term memory, and recognition, compared to C-NMHE patients and controls. Cirrhotic patients showed reduced fimbria volume compared to controls. Larger volumes in hippocampus subfields were related to better memory performance in C-NMHE patients and controls. C-MHE patients presented lower FC between the L-presubiculum and L-precuneus than C-NMHE patients. Compared to controls, C-MHE patients had reduced FC between L-presubiculum and subiculum seeds and bilateral precuneus, which correlated with cognitive impairment and memory performance. Alterations in the FC of the hippocampal system could contribute to learning and long-term memory impairments in C-MHE patients. This study demonstrates the association between alterations in learning and long-term memory and structural and FC disturbances in hippocampal structures in cirrhotic patients.

Background and aims Minimal hepatic encephalopathy (MHE) is associated with cognitive alterations and changes in connectivity. We assessed the relationship of the abnormalities of resting-state functional connectivity (rs-FC) and gray matter (GM) volume with different cognitive alterations and biochemical parameters associated to MHE. Methods Thirty-nine cirrhotic patients (26 without and 13 with MHE) and 24 controls were widely cognitive assessed with a battery of psychometric tests. Atrophy was determined using Voxel-Based Morphometry and rs-FC was assessed by independent component analysis. Receiver operating characteristic (ROC) curves was performed to assess the diagnostic utility of rs-FC and GM reduction for the discrimination of patients with and without MHE. Blood ammonia, cGMP, and levels of pro-inflammatory interleukins were measured. Results MHE patients showed significant decrease of GM volume and lesser degree of rs-FC in different networks related to attention and executive functions as compared to controls and patients without MHE. There is a progressive reduction in rs-FC in the default mode network with the progression of cognitive impairment. MHE patients showed GM reduction in theright frontal lobe, right insula and right cerebellum compared to patients without MHE. Alterations in GM volume and rs-FC correlated with the scores of different cognitive tests. Conclusions Decreased cognitive performance is associated by reduced rs-FC and GM atrophy in MHE patients. These changes could have predictive value for detecting MHE.

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.

Amyloid β25-35 (Aβ25-35) represents a neurotoxic fragment of Aβ1-40 or Aβ1-42, and is implicated in the progressive neurodegeneration in cases of the Alzheimer disease (AD). Amyloid β25-35 was shown to lyse rat erythrocytes (RBCs) of all ages, and the extent of the RBC toxicity is directly correlated with Aβ25-35 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, Aβ25-35 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 Aβ25-35 toxicity may contribute to the ultimate death of the RBCs in patients with AD.

Aβ 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-β 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-β, systemic ammonia, erythrocyte energy metabolism and the role of 2,3-diphosphoglycerate in AD pathogenesis.

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.

Background: Accumulating evidence implicates a role for altered energy and proteolytic metabolism in the pathogenesis of Alzheimer disease (AD), but the involvement of erythrocytes (RBC) remains to be completely understood. The objective stands to test a hypotheses that erythrocytic glycolytic and proteolytic metabolism is disturbed in the elderly and in AD patients. Methods: This pilot study analyzed the levels of the key glycolytic and proteolytic enzymes and metabolites in RBC from patients with AD and non-Alzheimer’s dementia (NA), from age-matched (AC) and young adult (YC) controls. Results: Glycolytic enzymes displayed lower activities in AC, and similar or higher activities in AD and NA, as compared to those in YC. Caspase-3 and calpain activities in AC were about 3 times those of YC. There were no differences between AC, AD and NA in proteolytic enzymes. Conclusion: Among glycolytic parameters studied in four subject groups, only hexokinase activity specifically increases in AD. Changes in other glycolytic and proteolytic enzyme activities and metabolite concentrations in RBC were not specific to dementias but characteristics for aging.

The first human transfusion was performed by the pioneer Dr Jean-Baptiste Denis in France in 1667 and now, three centuries later, around 50 millions blood units are transfused every year, saving millions of lives. Today, there is a new application for red blood cells (RBCs) in cellular therapy: the effective use of erythrocytes as vehicles for chemical or biological drugs. Using this approach, the therapeutic index of RBC-entrapped molecules can be significantly improved with increased efficacy and reduced side effects. This cell-based medicinal product can be manufactured at an industrial scale and is now used in the clinic for different therapeutic applications. A seminar dedicated to this field of research, debating on this inventive formulation for drugs, was held in Lyon (France) on 28 January 2011. Drs KC Gunter and Y Godfrin co-chaired the meeting and international experts working on the encapsulation of drugs within erythrocytes met to exchange knowledge on the topic ‘The Red Blood Cells as Vehicles for Drugs’. The meeting was composed of oral presentations providing the latest knowledge and experience on the preclinical and clinical applications of this technology. This Meeting Highlights article presents the most relevant messages given by the speakers and is a joint effort by international experts who share an interest in studying erythrocyte as a drug delivery vehicle. The aim is to provide an overview of the applications, particularly for clinical use, of this innovative formulation. Indeed, due to the intrinsic properties of erythrocytes, their use as a drug carrier is one of the most promising drug delivery systems investigated in recent decades. Of the different methods developed to encapsulate therapeutic agents into RBCs [1,2,] the most widely used method is the lysis of the RBCs under tightly controlled hypotonic conditions in the presence of the drug to be encapsulated, followed by resealing and annealing under normotonic conditions (Figure 1). This results in uniform encapsulation of the material into the cells and a final product with good stability, reproducibility and viability. This process, which has now been developed to an industrial scale, is the technique chosen by the majority of the experts presenting their work in this seminar (by R Franco). Open in a separate window Figure 1 The process of reversible hypotonic lysis of RBCs to entrap molecules RBCs are submitted to a hypotonic stress creating pores in the erythrocyte membrane. Drug can pass through the pores and be permanently entrapped after a resealing step using a hypertonic solution. RBC: Red blood cell.

Statins are widely used and well tolerated cholesterol-lowering drugs, and when used for therapy purposes reduce morbidity and mortality from coronary heart disease. Simvastatin is one of nine known statins, specific inhibitors of hepatic enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting step of cholesterol biosynthesis, and is believed to reduce plasma cholesterol levels by decreasing the activity of this enzyme. Statin drugs represent the major improvement in the treatment of hypercholesterolemia that constitutes the main origin of atherosclerosis, leading to coronary heart disease. Although statins are generally safe, minor and severe adverse reactions are well known complications of statin use. Adverse events associated with simvastatin therapy are uncommon, but potentially serious. In this review some details about statins including their adverse effects in humans and animals, the effects of simvastatin on various intracellular and mitochondrial processes, and molecular mechanisms underlying simvastatin cytotoxicity are discussed.

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.

In hyperammonemia, a decrease in brain ATP can be a result of adenine nucleotide catabolism. Xanthine dehydrogenase (XD) and xanthine oxidase (XO) are the end steps in the purine catabolic pathway and directly involved in depletion of the adenylate pool in the cell. Besides, XD can easily be converted to XO to produce reactive oxygen species in the cell. In this study, the effects of acute ammonia intoxication in vivo on brain adenine nucleotide pool and xanthine and hypoxanthine, the end degradation products of adenine nucleotides, during the conversion of XD to XO were studied. Injection of rats with ammonium acetate was shown to lead to the dramatic decrease in the ATP level, adenine nucleotide pool size and adenylate energy charge and to the great increase in hypoxanthine and xanthine 11 min after the lethal dose indicating rapid degradation of adenylates. Conversion of XD to XO in hyperammonemic rat brain was evidenced by elevated XO/XD activity ratio. Injection of MK-801, a NMDA receptor blocker, prevented ammonia-induced catabolism of adenine nucleotides and conversion of XD to XO suggesting that in vivo these processes are mediated by activation of NMDA receptors. The in vitro dose-dependent effects of sodium nitroprusside, a NO donor, on XD and XO activities are indicative of the direct modification of the enzymes by nitric oxide. This is the first report evidencing the increase in brain xanthine and hypoxanthine levels and adenine nucleotide breakdown in acute ammonia intoxication and NMDA receptor-mediated prevention of these alterations.

Mitochondrial enzyme monoamine oxidase A (MAO-A) generates hydrogen peroxide (H2O2) and is up-regulated by Ca2+ and presumably by ammonia. We hypothesized that MAO-A may be under the control of NMDA receptors in hyperammonemia. In this work, the in vivo effects of single dosing with ammonia and NMDA receptor antagonist MK-801 and the in vitro effect of Ca2+ on MAO-A activity in isolated rat brain mitochondria were studied employing enzymatic procedure. Intraperitoneal injection of rats with ammonia led to an increase in MAO-A activity in mitochondria indicating excessive H2O2 generation. Calcium added to isolated mitochondria stimulated MAO-A activity by as much as 84%. MK-801 prevented the in vivo effect of ammonia, implying that MAO-A activation in hyperammonemia is mediated by NMDA receptors. These data support the conclusion that brain mitochondrial MAO-A is regulated by the function of NMDA receptors. The enzyme can contribute to the oxidative stress associated with hyperammonemic conditions such as encephalopathy and Alzheimer’s disease. The attenuation of the oxidative stress highlights MAO-A inactivation and NMDA receptor antagonists as sources of novel avenues in the treatment of mental disorders.

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.

This study investigated the effect of antihypertensive therapy with lisinopril on plasma cholesterol concentration and erythrocyte catalase activity in hypertensive patients. We observed, for the first time, significant inverse correlations between systolic blood pressure (BP) and erythrocyte catalase activity and between diastolic BP and erythrocyte catalase activity. Plasma total and low density lipoprotein (LDL) cholesterol as well as triglyceride levels were similar between baseline and 1-, 3-, and 6-month treatment values in the same patients; however, systolic and diastolic BP levels were expectedly reduced after the therapy. Thus, there was no association between BP and lipid-cholesterol metabolism. These findings confirm antihypertensive effect of lisinopril and suggest that erythrocyte catalase is involved in BP control in hypertension and antihypertensive therapy.

The effects of toxic doses of ammonia on the induction of pro-apoptotic markers in rat brain cells were studied. It was found that neither induction of caspase-9 and caspase-3, nor changes in the membrane potential in nonsynaptic brain mitochondria, release of cytochrome c from mitochondria to cytosol, or apoptosis-inducing signals in the cell nuclei, were responsible for the ammonia toxicity in vivo. Thus, no evidence was found for any functional significance of mitochondria in cell death associated with acute hyperammonemia. The resistance of cell nuclei to ammonia-induced apoptosis could be caused either by a disordered translocation of the p53 protein from the cytosol into the nucleus or by translocation of its inactive form. The nonsynaptic brain mitochondria were also shown to be more resistant to the formation of nonspecific permeability pores and to Ca2+-induced swelling than cardiac and hepatic mitochondria and, as well, supported their membrane potential even under conditions of ammonia-stimulated production of active oxygen metabolites, excessive accumulation of Ca2+, oxidative stress, and impaired energy metabolism.

Background: Cyclic GMP (cGMP) concentration is increased in plasma of patients with liver cirrhosis. Three possible mechanisms may contribute: increased cGMP synthesis by soluble (activated by nitric oxide), or particulate (activated by atrial natriuretic peptide (ANP)) guanylate cyclase or increased release from cells. Aim: The aim of this work was to analyze the possible contributors to increased plasma cGMP and to assess whether changes in the parameters of the system vary with the degree of liver disease (Child Pugh score) or by the presence of ascites. Methods: We measured cGMP in plasma and lymphocytes, soluble guanylate cyclase activation by nitric oxide in lymphocytes, nitrates and nitrites and ANPs (activator of particulate guanylate cyclase) in plasma. We analyzed the correlation between changes in different parameters to discern which parameters contribute to increased plasma cGMP. Results: The plasma content of nitrates+nitrites, ANP and cGMP are increased. Activation of soluble guanylate cyclase by nitric oxide is increased in patients while basal cGMP in lymphocytes is decreased. Conclusions: Both increased ANP and increased activation of soluble guanylate cyclase by nitric oxide contribute to increased plasma cGMP in patients. The concentrations of ANP and cGMP in plasma increase with the degree of disease and are higher in patients with ascites.

Long-term potentiation (LTP) is a long-lasting enhancement of synaptic transmission efficacy and is considered the base for some forms of learning and memory. Nitric oxide (NO)-induced formation of cGMP is involved in hippocampal LTP. We have studied in hippocampal slices the effects of application of a tetanus to induce LTP on cGMP metabolism and the mechanisms by which cGMP modulates LTP. Tetanus application induced a transient rise in cGMP, reaching a maximum at 10 s and decreasing below basal levels 5 min after the tetanus, remaining below basal levels after 60 min. Soluble guanylate cyclase (sGC) activity increased 5 min after tetanus and returned to basal levels at 60 min. The decrease in cGMP was due to sustained tetanus-induced increase in cGMP-degrading phosphodiesterase activity, which remained activated 60 min after tetanus. Tetanus-induced activation of PDE and decrease of cGMP were prevented by inhibiting protein kinase G (PKG). This indicates that the initial increase in cGMP activates PKG that phosphorylates (and activates) cGMP-degrading PDE, which, in turn, degrades cGMP. Inhibition of sGC, of PKG or of cGMP-degrading phosphodiesterase impairs LTP, indicating that proper induction of LTP involves transient activation of sGC and increase in cGMP, followed by activation of cGMP-dependent protein kinase, which, in turn, activates cGMP-degrading phosphodiesterase, resulting in long-lasting reduction of cGMP content. Hyperammonemia is the main responsible for the neurological alterations found in liver disease and hepatic encephalopathy, including impaired intellectual function. Hyperammonemia impairs LTP in hippocampus by altering the modulation of this sGC-PKG-cGMP-degrading PDE pathway. Exposure of hippocampal slices to 1 mM ammonia completely prevents tetanus-induced decrease of cGMP by impairing PKG-mediated activation of cGMP-degrading phosphodiesterase. This impairment is responsible for the loss of the maintenance of LTP in hyperammonemia, and may be also involved in the cognitive impairment in patients with hyperammonemia and hepatic encephalopathy.

Acute ammonia toxicity is mediated by excessive activation of NMDA receptors. Activation of NMDA receptors leads to activation of poly(ADP-ribose) polymerase (PARP) which mediates NMDA excitotoxicity. PARP is activated following DNA damage and may lead to cell death via NAD+ and ATP depletion. The aim of the present work was to assess whether acute ammonia intoxication in vivo leads to increased PARP in brain cells nuclei and to altered NAD+ and superoxide metabolism and the contribution of NMDA receptors to these alterations. Acute ammonia intoxication increases PARP content twofold in brain cells nuclei.NAD+ content decreased by 55% in rats injected with ammonia. This was not due to decreased NAD+ synthetase nor increased NAD+ hydrolase activities and would be due to increased NAD+ consumption by PARP. Superoxide radical formation increased by 75% in nuclei of brains of rats injected with ammonia, that also induced protein nitrotyrosylation and DNA damage. Blocking NMDA receptors prevented ammonia-induced PARP, superoxide and nitrotyrosylation increase, DNA damage and NAD+ decrease. These results show that acute ammonia intoxication in vivo leads to activation of NMDA receptors, leading to increased superoxide formation and PARP content and depletion of NAD+ in brain cells nuclei that contribute to ammonia toxicity.

Background/Aims : Patients with liver disease show increased plasma cGMP and decreased intracellular cGMP in lymphocytes. The initial aim of this work was to assess whether decreased intracellular cGMP and increased plasma cGMP may be due to increased ATP-dependent release of cGMP from cells. The results obtained led to a new aim: to identify and quantify a protein responsible for the increased cGMP binding found in erythrocyte membranes from patients with liver disease. Methods : ATP-dependent cGMP transport was determined in inside-out vesicles from erythrocyte membranes. cGMP-binding proteins were isolated from the membranes and identified by MALDI-TOF peptide mass fingerprint. Protein kinase A was quantified by immunoblotting. Results : ATP-independent cGMP binding is increased in erythrocyte membranes from patients. There is a significant increase in the membrane content of a cGMP-binding protein with Mr 48,000, which was identified as the regulatory subunit of protein kinase A. Conclusions : The content of the regulatory subunit of protein kinase A is significantly increased (twice) in erythrocyte membranes from patients with liver cirrhosis. This protein binds cGMP strongly and may be responsible for the decrease in intracellular cGMP in liver disease.

In primary cultures of cerebellar neurons glutamate neurotoxicity is mainly mediated by activation of the NMDA receptor, which allows the entry of Ca2+ and Na+ into the neuron. To maintain Na+ homeostasis, the excess Na+ entering through the ion channel should be removed by Na+,K(+)-ATPase. It is shown that incubation of primary cultured cerebellar neurons with glutamate resulted in activation of the Na+,K(+)-ATPase. The effect was rapid, peaking between 5 and 15 min (85% activation), and was maintained for at least 2 h. Glutamate-induced activation of Na+,K(+)-ATPase was dose dependent: It was appreciable (37%) at 0.1 microM and peaked (85%) at 100 microM. The increase in Na+,K(+)-ATPase activity by glutamate was prevented by MK-801, indicating that it is mediated by activation of the NMDA receptor. Activation of the ATPase was reversed by phorbol 12-myristate 13-acetate, an activator of protein kinase C, indicating that activation of Na+,K(+)-ATPase is due to decreased phosphorylation by protein kinase C. W-7 or cyclosporin, both inhibitors of calcineurin, prevented the activation of Na+,K(+)-ATPase by glutamate. These results suggest that activation of NMDA receptors leads to activation of calcineurin, which dephosphorylates an amino acid residue of the Na+,K(+)-ATPase that was previously phosphorylated by protein kinase C. This dephosphorylation leads to activation of Na+,K(+)-ATPase.

Acute administration of large doses of ammonia leads to the rapid death of animals. This article reviews the role of excessive activation of N-methyl-D-aspartate (NMDA) receptors in the mediation of ammonia-induced mortality. The studies reviewed here show that acute intoxication with large doses of ammonia leads to the activation of NMDA receptors in brain in vivo. Moreover, excessive activation of NMDA receptors is responsible for ammonia-induced death of animals, which is prevented by different antagonists of NMDA receptors. This article also reviews the studies showing that activation of NMDA receptors is also responsible for the following effects of acute ammonia intoxication: (1) depletion of brain ATP, which, in turn, leads to release of glutamate; (2) activation of calcineurin and dephosphorylation and activation of Na+/K+-ATPase in brain, thus increasing ATP consumption; (3) impairment of mitochondrial function and calcium homeostasis at different levels, thus decreasing ATP synthesis; (4) activation of calpain that degrades the microtubule-associated protein MAP-2, thus altering the microtubular network; (5) increased formation of nitric oxide (NO) formation, which, in turn, reduces the activity of glutamine synthetase, thus reducing the elimination of ammonia in brain.

Glutamate is the main excitatory neurotransmitter in mammals. Glutamatergic neurotransmission involves several steps, beginning with release of glutamate from the presynaptic neuron. Glutamate in the extracellular space activates glutamate receptors present in the synaptic membranes, leading to activation of signal transduction pathways associated with these receptors. To avoid continuous activation of glutamate receptors, glutamate is removed from the synaptic cleft by specific glutamate transporters located mainly on astrocytes. All these steps are tightly modulated under physiological conditions, and alterations of any of the above steps may result in impairment of glutamatergic neurotransmission, leading to neurological alterations. There are studies in the literature reporting alterations in all these steps in hyperammonemia and/or hepatic failure. Glutamatergic neurotransmission modulates important cerebral processes. Some of these processes are altered in patients with liver disease and hepatic encephalopathy, who show altered sleep-wake patterns, neuromuscular coordination, and decreased intellectual capacity. The alterations in glutamatergic neurotransmission may be responsible for some of these neurological alterations found in hepatic encephalopathy. The effects of hyperammonemia and liver failure on different steps of glutamatergic neurotransmission including alterations of glutamate concentration in the extracellular fluid in brain, transport and transporters of glutamate, the content and function of different types of glutamate receptors and signal transduction pathways. Alterations induced by hyperammonemia and liver failure on the glutamate-nitric oxide-cGMP pathway in brain may result in changes in long-term potetiation and learning ability.

The aim of the present work was to assess the effects of activation of NMDA receptors in rat brain in vivo on calcium homeostasis in isolated non-synaptic brain mitochondria. We have shown recently that acute intoxication with large doses of ammonia leads to activation of NMDA receptors in rat brain in vivo. In the present work we injected rats with ammonium acetate to activate NMDA receptors in vivo and isolated non-synaptic mitochondria to assess calcium homeostasis. We also tested whether blocking NMDA receptors with MK-801 prevents effects on calcium homeostasis induced by ammonium injection. It is shown that activation of NMDA receptors in rat brain in vivo leads to a rapid increase in intramitochondrial calcium content followed by a reduction in the calcium capacity and calcium uptake rate in rat brain mitochondria. Activation of NMDA receptors resulted in increased spontaneous calcium efflux from rat brain mitochondria and in a strong inhibition of Na-induced and tert-butylhydroperoxide-induced calcium efflux. All these effects were prevented by previous blocking of NMDA receptors by injection of MK-801. Cyclosporin A did not affect any of the above parameters, indicating that the mitochondrial permeability transition pore does not play a role in calcium efflux under any of the conditions studied. The results reported indicate that ammonia-induced activation of NMDA receptors in rat brain in vivo alters mitochondrial calcium homeostasis at several different steps.

Acute ammonia intoxication diminishes the activities of antioxidant enzymes and increases superoxide formation in brain. These effects could play a role in the mechanism of ammonia toxicity. It has been shown that ammonia toxicity is mediated by activation of NMDA receptors. The aim of this work was to assess whether ammonia-induced changes in antioxidant enzymes and in superoxide formation are mediated by activation of NMDA receptors. It is shown that MK-801, an antagonist of NMDA receptors prevents ammonia-induced changes in superoxide dismutase, glutathione peroxidase and catalase. Ammonia intoxication also induces a depletion of glutathione and an increase in lipid peroxidation. Both effects, as well as ammonia-induced increase in superoxide formation are prevented by MK-801. These results indicate that ammonia-induced oxidative stress in brain is mediated by excessive activation of NMDA receptors and support the idea that oxidative stress can play a role in the mechanism of ammonia toxicity.

Injection of large doses of ammonium salts leads to the rapid death of animals. However, the molecular mechanisms involved in ammonia toxicity remain to be clarified. We reported that injecting ammonium acetate (7 mmol/kg) to rats increases the production of superoxide and reduces the activities of some antioxidant enzymes in rat liver and brain. We proposed that these effects induced by ammonia intoxication would be mediated by formation of nitric oxide. To test this possibility we tested whether injection of nitroarginine, an inhibitor of nitric oxide synthase, prevents the effects of ammonia intoxication on antioxidant enzymes and superoxide formation. Following injection of ammonia, glutathione peroxidase, superoxide dismutase and catalase activities were decreased in liver by 42%, 54% and 44%, respectively. In brain these activities were reduced by 35%, 46% and 65%, respectively. Glutathione reductase remained unchanged. Superoxide production in submitochondrial particles from liver and brain was increased by more than 100% in both tissues. Both reduction of activity of antioxidant enzymes and increased superoxide radical production were prevented by previous injection of 45 mg/kg of nitroarginine, indicating that ammonia induces increased formation of nitric oxide, which in turn reduces the activity of antioxidant enzymes, leading to increased formation of superoxide.

1. Previous results suggest that glutamine synthesis in brain could be modulated by nitrix oxide. The aim of this work was to assess this possibility.

Injection of large doses of ammonium salts lead to the rapid death of animals. However, the molecular mechanisms involved in ammonia toxicity remain to be clarified. We have tested the effect of injecting 7 mmol/kg of ammonium acetate on the production of superoxide and on the activities of some antioxidant enzymes in rat liver, brain, erythrocytes and plasma. Glutathione peroxidase, superoxide dismutase and catalase activities were decreased in liver and brain (both in cytoso-lic and mitochondrial fractions) and also in blood red cells, while glutathione reductase activity remained unchanged. Superoxide production in submitochon-drial particles from liver and brain was increased by more than 100% in both tissues. Both diminished activity of antioxidant enzymes and increased superoxide radical production could lead to oxidative stress and cell damage, which could be involved in the mechanism of acute ammonia toxicity.

We have proposed that acute ammonia toxicity is mediated by activation of the N-methyl-D-aspartate type of glutamate receptors. MK-801, a selective antagonist of these receptors, prevents death of animals induced by acute ammonia intoxication as well as ammonia-induced depletion of ATP. It seems therefore that, following activation of the N-methyl-D-aspartate receptors, the subsequent events in ammonia toxicity should be similar to those involved in glutamate neurotoxicity. As it has been shown that inhibitors of nitric oxide synthetase such as nitroarginine prevent glutamate toxicity, we have tested whether nitroarginine prevents ammonia toxicity and ammonia-induced alterations in brain energy and ammonia metabolites. It is shown that nitroarginine prevents partially (approximately 50%), but significantly death of mice induced by acute ammonia intoxication. Nitroarginine also prevents partially ammonia-induced depletion of brain ATP. It also prevents completely the rise in glucose and pyruvate and partially that in lactate. Injection of nitroarginine alone, in the absence of ammonia, induces a remarkable accumulation of glutamine and a decrease in glutamate. The results reported indicate that nitroarginine attenuates acute ammonia toxicity and ammonia-induced alterations in brain energy metabolites. The effects of MK-801 and of nitroarginine are different, suggesting that ammonia can induce nitric oxide synthetase by mechanisms other than activation of N-methyl-D-aspartate receptors.

Amyloid β-protein (Aβ) is the major amyloid component of toxic amyloid senile plaques inducing slow neuronal degeneration in brains of Alzheimer's patients. It can induce proteolysis of some cytoskeletal proteins in the neuron; however, studies of proteolytic enzyme activity in different brain regions and their subcellular compartmentalization were not carried out. In this work, the effects of chronic intracerebroventricular administration of Aβ 25–35 on proteolytic enzymes in subcellular fractions from rat brain regions were studied. Mitochondrial and cytosolic caspase-9 and caspase-3 activities in neocortex, cerebellum, and hippocampus were shown to be increased during infusion of Aβ 25–35 . In Aβ 25–35 -treated rats, cytosolic calcium-dependent thiol proteases calpain-1 and calpain-2 appeared in mitochondria and lysosomes, causing apparent release of lysosomal cathepsins B and D to mitochondria and of β-galactosidase to the cytosol. The increase in all proteolytic activities in brain subcellular fractions under the influence of administered Aβ suggests that these enzymes could be transferred across intracellular membranes and involved in neurodegeneration.

Cytosolic enzymes AMP deaminase and adenosine deaminase (ADA) catalyze AMP and adenosine deamination, constitute rate-limiting steps of adenine nucleotide catabolism and play important roles in cellular energy metabolism. In this study, AMP deaminase and ADA activities of rat liver, neocortex, cerebellum, striatum and hippocampus were investigated in acute ammonia intoxication and subacute CCl(4)-induced hepatitis. Activities of both AMP deaminase and ADA in the liver were elevated by 2.4-4.2-fold (p