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Objective: The aim of our study was to determine the relation of particular genetic variants in selected genes (GSTM1, GSTT1 null genotypes; rs1695 GSTP1; rs10735781 EVI5) to the risk of multiple sclerosis (MS) development and find out the possible association with disease disability progression rate. Material and methods: Our study included 202 MS patients and 174 healthy control volunteers. MS patients were divided according to disability progression rate to three groups - slowly progressing, mid-rate progressing and rapidly progressing. All DNA samples were isolated from venous blood. Genotyping was performed by PCR-RFLP and multiplex PCR. Results: Our analysis showed that GSTT1 null genotype (OR 0.56; 95%CI 0.33 -0.95; p=0.04) and GSTM1, GSTT1 double null genotype (OR 0.32; 95%CI 0.14 - 0.74; p=0.006) are potentially protective in relation to MS. We observed similar result in GSTT1 null genotype in association with mid-rate progression (OR 0.48; 95%CI 0.24 - 0.97; p=0.05). Frequency of GSTM1 and GSTT1 double null genotype is significantly lower in subgroup of MS patients with progression rate defined as slow (OR 0.22; 95%CI 0.05 - 0.98; p=0.05) and middle (OR 0.33; 95%CI 0.11 - 0.99; p=0.045). We did not show any significant association of genetic changes rs1695 in GSTP1 and rs10735781 in EVI5 with MS or rate of disease progression. Conclusions: Genetic basis of multiple sclerosis is still not fully elucidated. Further research may clarify our results and confirm the value of studied factors for clinical practice.

Abstract Objective Oxygen therapy is used for the treatment of various diseases, but prolonged exposure to high concentrations of O2 is also associated with formation of free radicals and oxidative damage. Methods In the present study we compared α-ketoglutarate dehydrogenase (KGDH) activity and mitochondrial oxidative damage in the hearts of guinea pigs after long-term (17 and 60 h) oxygenation with 100% normobaric O2 and with partially negatively (O2 neg) or positively (O2 posit) ionized oxygen. Results Inhalation of O2 led to significant loss in KGDH activity and thiol group content and accumulation of bityrosines. Inhalation of O2 neg was accompanied by more pronounced KGDH inhibition, possibly due to additional formation of protein-lipid conjugates. In contrast, O2 posit prevented loss in KGDH activity and diminished mitochondrial oxidative damage. Conclusions These findings suggest that oxygen treatment is associated with impairment of heart energy metabolism and support the view that inhalation of O2 posit optimizes the beneficial effects of oxygen therapy.

Elevated homocysteine (Hcy) level is a well known risk factor for cardiovascular and neuropsychiatric diseases. In this study, we investigated metabolic changes in blood plasma in Hcy-treated rats. In combination with Hcy injections to induce hyperhomocysteinemia-like state, we used an animal model of global cerebral ischemia to investigate metabolic changes after 24 h reperfusion in rats. We also focused on the endogenous phenomenon known as ischemic tolerance induced by the preischemic treatment. The experiments were carried out on blood plasma samples as they are easily available and metabolically reflect the overall changes in injured organism. We observed significant changes in plasma metabolite levels of: pyruvate, citrate, acetate implicating alterations in energy metabolism, and increase in triacylglycerols, arginine and lysine, in Hcy-treated rats compared with naive animals. Ischemic insult with 24 reperfusion in Hcy-treated rats led to increase in plasma lactate, and decrease in plasma glucose, pyruvate, citrate and acetate. Complementary, an increase in ketone body 3-hydroxybutyrate was observed. The plasma metabolites: alanine, lactate, valine, glucose, leucine, isoleucine, acetate, citrate and 3-hydroxybutyrate were considered to reflect the response induced by ischemic preconditioning in Hcy rats, where the extent of postischemic damage was not as high as in the non-preconditioned rats. Our results provide evidence that nuclear magnetic resonance (NMR) spectra analysis can identify a specific group of metabolites present in plasma with the capability of discriminating between individual groups of animals. Regarding the effect of elevated Hcy level on plasma metabolome, we showed, that acetate, pyruvate and glucose had the excellent discriminatory power between Hcy-treated and naive rats plasma. Concerning ischemic insult in Hcy-treated animals, we also document the ideal discrimination of ischemic from non-ischemic rats by various groups of metabolites, that can be considered as a potential plasma biomarkers. [ABSTRACT FROM AUTHOR]

It is well known that the brain is quite vulnerable to oxidative stress, initiating neuronal loss after ischemia-reperfusion (IR) injury. A potent protective mechanism is ischemic preconditioning (IPC), where proteins are among the primary targets. This study explores redox-active proteins' role in preserving energy supply. Adult rats were divided into the control, IR, and IPC groups. Protein profiling was conducted to identify modified proteins and then verified through activity assays, immunoblot, and immunohistochemical analyses. IPC protected cortex mitochondria, as evidenced by a 2.26-fold increase in superoxide dismutase (SOD) activity. Additionally, stable core subunits of respiratory chain complexes ensured sufficient energy production, supported by a 16.6% increase in ATP synthase activity. In hippocampal cells, IPC led to the downregulation of energy-related dehydrogenases, while a significantly higher level of peroxiredoxin 6 (PRX6) was observed. Notably, IPC significantly enhanced glutathione reductase activity to provide sufficient glutathione to maintain PRX6 function. Astrocytes may mobilize PRX6 to protect neurons during initial ischemic events, by decreased PRX6 positivity in astrocytes, accompanied by an increase in neurons following both IR injury and IPC. Maintained redox signaling via astrocyte-neuron communication triggers IPC's protective state. The partnership among PRX6, SOD, and glutathione reductase appears essential in safeguarding and stabilizing the hippocampus.

The study of an organism's response to cerebral ischemia at different levels is essential to understanding the mechanism of the injury and protection. A great interest is devoted to finding the links between quantitative metabolic changes and post-ischemic damage. This work aims to summarize the outcomes of the most studied metabolites in brain tissue-lactate, glutamine, GABA (4-aminobutyric acid), glutamate, and NAA (N-acetyl aspartate)-regarding their biological function in physiological conditions and their role after cerebral ischemia/reperfusion. We focused on ischemic damage and post-ischemic recovery in both experimental-including our results-as well as clinical studies. We discuss the role of blood glucose in view of the diverse impact of hyperglycemia, whether experimentally induced, caused by insulin resistance, or developed as a stress response to the cerebral ischemic event. Additionally, based on our and other studies, we analyze and critically discuss post-ischemic alterations in energy metabolites and the elevation of blood ketone bodies observed in the studies on rodents. To complete the schema, we discuss alterations in blood plasma circulating amino acids after cerebral ischemia. So far, no fundamental brain or blood metabolite(s) has been recognized as a relevant biological marker with the feasibility to determine the post-ischemic outcome or extent of ischemic damage. However, studies from our group on rats subjected to protective ischemic preconditioning showed that these animals did not develop post-ischemic hyperglycemia and manifested a decreased metabolic infringement and faster metabolomic recovery. The metabolomic approach is an additional tool for understanding damaging and/or restorative processes within the affected brain region reflected in the blood to uncover the response of the whole organism via interorgan metabolic communications to the stressful cerebral ischemic challenge.

Multifactorial interactions, including nutritional state, likely participate in neurodegeneration's pathogenesis and evolution. Dysregulation in methionine (Met) metabolism could lead to the development of hyperhomocysteinaemia (hHcy), playing an important role in neuronal dysfunction, which could potentially lead to the development of Alzheimer's disease (AD)-like pathological features. This study combines proton magnetic resonance spectroscopy ( 1 H MRS) with immunohistochemical analysis to examine changes in the metabolic ratio and histomorphological alterations in the dorsal rat hippocampus (dentate gyrus-DG) subjected to a high Met diet. Male Wistar rats (420-480 g) underwent hHcy evoked by a Met-enriched diet (2 g/kg of weight/day) lasting four weeks. Changes in the metabolic ratio profile and significant histomorphological alterations have been found in the DG of hHcy rats. We have detected increased morphologically changed neurons and glial cells with increased neurogenic markers and apolipoprotein E positivity parallel with a diminished immunosignal for the N-Methyl-D-Aspartate receptor 1 in hHcy animals. A Met diet induced hHcy, likely via direct Hcy neurotoxicity, an interference with one carbon unit metabolism, and/or epigenetic regulation. These conditions lead to the progression of neurodegeneration and the promotion of AD-like pathological features in the less vulnerable hippocampal DG, which presents a plausible therapeutic target.

During aging, heart structure and function gradually deteriorate, which subsequently increases susceptibility to ischemia-reperfusion (IR). Maintenance of Ca 2+ homeostasis is critical for cardiac contractility. We used Langendorff's model to monitor the susceptibility of aging (6-, 15-, and 24-month-old) hearts to IR, with a specific focus on Ca 2+ -handling proteins. IR, but not aging itself, triggered left ventricular changes when the maximum rate of pressure development decreased in 24-month-olds, and the maximum rate of relaxation was most affected in 6-month-old hearts. Aging caused a deprivation of Ca 2+ -ATPase (SERCA2a), Na + /Ca 2+ exchanger, mitochondrial Ca 2+ uniporter, and ryanodine receptor contents. IR-induced damage to ryanodine receptor stimulates Ca 2+ leakage in 6-month-old hearts and elevated phospholamban (PLN)-to-SERCA2a ratio can slow down Ca 2+ reuptake seen at 2-5 μM Ca 2+ . Total and monomeric PLN mirrored the response of overexpressed SERCA2a after IR in 24-month-old hearts, resulting in stable Ca 2+ -ATPase activity. Upregulated PLN accelerated inhibition of Ca 2+ -ATPase activity at low free Ca 2+ in 15-month-old after IR, and reduced SERCA2a content subsequently impairs the Ca 2+ -sequestering capacity. In conclusion, our study suggests that aging is associated with a significant decrease in the abundance and function of Ca 2+ -handling proteins. However, the IR-induced damage was not increased during aging.

Metabolomics is a relatively new research area that focuses mostly on the profiling of selected molecules and metabolites within the organism. A SARS-CoV-2 infection itself can lead to major disturbances in the metabolite profile of the infected individuals. The aim of this study was to analyze metabolomic changes in the urine of patients during the acute phase of COVID-19 and approximately one month after infection in the recovery period. We discuss the observed changes in relation to the alterations resulting from changes in the blood plasma metabolome, as described in our previous study. The metabolome analysis was performed using NMR spectroscopy from the urine of patients and controls. The urine samples were collected at three timepoints, namely upon hospital admission, during hospitalization, and after discharge from the hospital. The acute COVID-19 phase induced massive alterations in the metabolic composition of urine was linked with various changes taking place in the organism. Discriminatory analyses showed the feasibility of successful discrimination of COVID-19 patients from healthy controls based on urinary metabolite levels, with the highest significance assigned to citrate, Hippurate, and pyruvate. Our results show that the metabolomic changes persist one month after the acute phase and that the organism is not fully recovered., Competing Interests: The authors declare no conflict of interest.

The ever-present risk of brain ischemic events in humans and its full prevention make the detailed studies of an organism's response to ischemia at different levels essential to understanding the mechanism of the injury as well as protection. We used the four-vessel occlusion as an animal model of forebrain ischemia to investigate its impact on the metabolic alterations in both the hippocampus and the blood plasma to see changes on the systemic level. By inducing sublethal ischemic stimuli, we focused on the endogenous phenomena known as ischemic tolerance. NMR spectroscopy was used to analyze relative metabolite levels in tissue extracts from rats' hippocampus and blood plasma in three various ischemic/reperfusion times: 3 h, 24 h, and 72 h. Hippocampal tissues were characterized by postischemically decreased glutamate and GABA (4-aminobutyrate) tissue content balanced with increased glutamine level, with most pronounced changes at 3 h reperfusion time. Glutamate (as well as glutamine) levels recovered towards the control levels on the third day, as if the glutamate re-synthesis would be firstly preferred before GABA. These results are indicating the higher feasibility of re-establishing of glutamatergic transmission three days after an ischemic event, in contrast to GABA-ergic. Tissue levels of N-acetylaspartate (NAA), as well as choline, were decreased without the tendency to recover three days after the ischemic event. Metabolomic analysis of blood plasma revealed that ischemically preconditioned rats, contrary to the non-preconditioned animals, did not show hyperglycemic conditions. Ischemically induced semi-ketotic state, manifested in increased plasma ketone bodies 3-hydroxybutyrate and acetoacetate, seems to be programmed to support the brain tissue revitalization after the ischemic event. These and other metabolites changes found in blood plasma as well as in the hippocampus were observed to a lower extent or recovered faster in preconditioned animals. Some metabolomic changes in hippocampal tissue extract were so strong that even single metabolites were able to differentiate between ischemic, ischemically preconditioned, and control brain tissues., (Copyright © 2022. Published by Elsevier Ltd.)

End-stage kidney disease is preferably treated by kidney transplantation. The suboptimal function of the allograft often results in misbalances in kidney-controlled processes and requires long-term monitoring of allograft function and viability. As the kidneys are organs with a very high metabolomic rate, a metabolomics approach is suitable to describe systematic changes in post-transplant patients and has great potential for monitoring allograft function, which has not been described yet. In this study, we used blood plasma samples from 55 patients after primary kidney transplantation identically treated with immunosuppressants with follow-up 50 months in the mean after surgery and evaluated relative levels of basal plasma metabolites detectable by NMR spectroscopy. We were looking for the correlations between circulating metabolites levels and allograft performance and allograft rejection features. Our results imply a quantitative relationship between restricted renal function, insufficient hydroxylation of phenylalanine to tyrosine, lowered renal glutamine utilization, shifted nitrogen balance, and other alterations that are not related exclusively to the metabolism of the kidney. No link between allograft function and energy metabolism can be concluded, as no changes were found for glucose, glycolytic intermediates, and 3-hydroxybutyrate as a ketone body representative. The observed changes are to be seen as a superposition of changes in the comprehensive inter-organ metabolic exchange, when the restricted function of one organ may induce compensatory effects or cause secondary alterations. Particular differences in plasma metabolite levels in patients with acute cellular and antibody-mediated allograft rejection were considered rather to be related to the loss of kidney function than to the molecular mechanism of graft rejection since they largely follow the alterations observed by restricted allograft function. In the end, we showed using a simple mathematical model, multilinear regression, that the basal plasmatic metabolites correlated with allograft function expressed by the level of glomerular filtration rate (with creatinine: p -value = 4.0 × 10 -26 and r = 0.94, without creatinine: p -value = 3.2 × 10 -22 and r = 0.91) make the noninvasive estimation of the allograft function feasible.

Increased concentration of plasma homocysteine (Hcy) is an independent risk factor of cardiovascular disease, yet the mechanism by which hyperhomocysteinemia (HHcy) causes cardiac dysfunction is largely unknown. The aim of present study was to investigate the contribution of sarcoplasmic reticulum to impaired cardiac contractile function in HHCy. HHcy-induced by subcutaneous injection of Hcy (0.45 μmol/g of body weight) twice a day for a period of 2 weeks resulted in significant decrease in developed left ventricular pressure and maximum rate of ventricular relaxation. Our results show that abundances of SR Ca 2+ -handling proteins, Ca 2+ -ATPase (SERCA2), calsequestrin and histidine-rich calcium-binding protein are significantly reduced while the content of phospholamban is unchanged. Moreover, we found that increased PLN:SERCA2 ratio results in the inhibition of SERCA2 activity at low free Ca 2+ concentrations. We further discovered that HHcy is not associated with increased oxidative stress in SR. Taken together, these findings suggest that disturbances in SR Ca 2+ handling, caused by altered protein contents but not oxidative damage, may contribute to impaired cardiac contractility in HHcy., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

1 H Nuclear Magnetic Resonance (NMR) metabolomics is one of the fundamental tools in the fast-developing metabolomics field. It identifies and quantifies the most abundant metabolites, alterations of which can describe energy metabolism, activated immune response, protein synthesis and catabolism, neurotransmission, and many other factors. This paper summarizes our results of the 1 H NMR metabolomics approach to characterize the distribution of relevant metabolites and their alterations induced by cerebral ischemic injury or its combination with hyperhomocysteinemia in the affected tissue and blood plasma in rodents. A decrease in the neurotransmitter pool in the brain tissue likely follows the disordered feasibility of post-ischemic neurotransmission. This decline is balanced by the increased tissue glutamine level with the detected impact on neuronal health. The ischemic injury was also manifested in the metabolomic alterations in blood plasma with the decreased levels of glycolytic intermediates, as well as a post-ischemically induced ketosis-like state with increased plasma ketone bodies. As the 3-hydroxybutyrate can act as a likely neuroprotectant, its post-ischemic increase can suggest its supporting role in balancing ischemic metabolic dysregulation. Furthermore, the 1 H NMR approach revealed post-ischemically increased 3-hydroxybutyrate in the remote organs, such as the liver and heart, as well as decreased myocardial glutamate. Ischemic preconditioning, as a proposed protective strategy, was manifested in a lower extent of metabolomic changes and/or their faster recovery in a longitudinal study. The paper also summarizes the pre- and post-ischemic metabolomic changes in the rat hyperhomocysteinemic models. Animals are challenged with hyperglycemia and ketosis-like state. A decrease in several amino acids in plasma follows the onset and progression of hippocampal neuropathology when combined with ischemic injury. The 1 H NMR metabolomics approach also offers a high potential for metabolites in discriminatory analysis in the search for potential biomarkers of ischemic injury. Based on our results and the literature data, this paper presents valuable findings applicable in clinical studies and suggests the precaution of a high protein diet, especially foods which are high in Met content and low in B vitamins, in the possible risk of human cerebrovascular neuropathology.

The study of an organism's response to ischemia at different levels is essential to understand the mechanism of the injury as well as protection. We used the occlusion of four vessels as an animal model of global cerebral ischemia to investigate metabolic alterations in cerebral cortex, hippocampus, blood plasma, as well as in a remote organ, the heart, in rats undergoing 24 h postischemic reperfusion. By inducing sublethal ischemic stimuli, we focused on endogenous phenomena known as ischemic tolerance that is currently the best known and most effective way of protecting against ischemic injury. NMR spectroscopy was used to analyze relative metabolite levels in homogenates from rats' cerebral cortex, hippocampus, and heart together with deproteinized blood plasma. In individual animals subjected to global cerebral ischemia, relative concentrations of the essential amino acids isoleucine, valine, phenylalanine, and tyrosine in cerebral cortex correlated with those in blood plasma ( p < 0.05, or boundary significant p < 0.09). This did not apply for the hippocampus, suggesting a closer relation between ischemic cortex and metabolomic blood components. Hippocampal non-participation on correlation with blood components may emphasize the observed partial or full normalization the post-ischemically altered levels of a number of metabolites in the preconditioned animals. Remarkably, that was observed for cortex to a lesser extent. As a response to the global cerebral ischemia in heart tissue, we observed decreased glutamate and increased 3-hydroxybutyrate. Ischemically induced semi-ketotic state and other changes found in blood plasma partially normalized when ischemic preconditioning was introduced. Some metabolomic changes were so strong that even individual metabolites were able to differentiate between ischemic, ischemically preconditioned, and control brain tissues.

Targeting metabolomic pathways is a promising strategy for cancer treatment. Alterations in the metabolomic state have also an epigenetic impact, making the metabolomic studies even more interesting. We explored metabolomic changes in the blood plasma of patients with primary and secondary lung cancer and tried to explore their origin. We also applied a discrimination algorithm to the data. In the study, blood samples from 132 patients with primary lung cancer, 47 with secondary lung cancer, and 77 subjectively healthy subjects without any cancer history were used. The samples were measured by NMR spectroscopy. PCA and PLS-DA analyses did not distinguish between patients with primary and secondary lung tumors. Accordingly, no significantly changed levels of plasmatic metabolites were found between these groups. When comparing with healthy controls, significantly increased glucose, citrate, acetate, 3-hydroxybutyrate, and creatinine balanced with decreased pyruvate, lactate, alanine, tyrosine, and tryptophan were found as a common feature of both groups. Metabolomic analysis of blood plasma showed considerable proximity of patients with primary and secondary lung cancer. The changes observed can be partially explained as cancer-derived and also as changes showing ischemic nature. Random Forrest discrimination based on the relative concentration of metabolites in blood plasma performed very promising with AUC of 0.95 against controls; however noticeable parts of differencing metabolites are overlapping with those observed after ischemic injury in other studies.

L-methionine, an essential amino acid, plays a critical role in cell physiology. High intake and/or dysregulation in methionine (Met) metabolism results in accumulation of its intermediate(s) or breakdown products in plasma, including homocysteine (Hcy). High level of Hcy in plasma, hyperhomocysteinemia (hHcy), is considered to be an independent risk factor for cerebrovascular diseases, stroke and dementias. To evoke a mild hHcy in adult male Wistar rats we used an enriched Met diet at a dose of 2 g/kg of animal weight/day in duration of 4 weeks. The study contributes to the exploration of the impact of Met enriched diet inducing mild hHcy on nervous tissue by detecting the histo-morphological, metabolomic and behavioural alterations. We found an altered plasma metabolomic profile, modified spatial and learning memory acquisition as well as remarkable histo-morphological changes such as a decrease in neurons' vitality, alterations in the morphology of neurons in the selective vulnerable hippocampal CA 1 area of animals treated with Met enriched diet. Results of these approaches suggest that the mild hHcy alters plasma metabolome and behavioural and histo-morphological patterns in rats, likely due to the potential Met induced changes in "methylation index" of hippocampal brain area, which eventually aggravates the noxious effect of high methionine intake.

Elevated concentration of homocysteine (Hcy) in the blood plasma, hyperhomocysteinemia (HHcy), has been implicated in various disorders, including cardiovascular and neurodegenerative diseases. Accumulating evidence indicates that pathophysiology of these diseases is linked with mitochondrial dysfunction. In this review, we discuss the current knowledge concerning the effects of HHcy on mitochondrial homeostasis, including energy metabolism, mitochondrial apoptotic pathway, and mitochondrial dynamics. The recent studies suggest that the interaction between Hcy and mitochondria is complex, and reactive oxygen species (ROS) are possible mediators of Hcy effects. We focus on mechanisms contributing to HHcy-associated oxidative stress, such as sources of ROS generation and alterations in antioxidant defense resulting from altered gene expression and post-translational modifications of proteins. Moreover, we discuss some recent findings suggesting that HHcy may have beneficial effects on mitochondrial ROS homeostasis and antioxidant defense. A better understanding of complex mechanisms through which Hcy affects mitochondrial functions could contribute to the development of more specific therapeutic strategies targeted at HHcy-associated disorders.

Cardiac arrest is one of the major causes of death and disability. The aim of the study was to identify dynamic time-dependent metabolomic changes reflected in rat plasma induced by cerebral ischemia and reperfusion with the focus on the protective effect of ischemic preconditionig. Global cerebral ischemia in rats was induced by the four-vessel occlusion. Blood plasma was collected in three reperfusion times: an early post-acute 3 hr, then 24 hr, as an incipient time for delayed neuronal death induction and 72 hr as prolonged reperfusion period. The metabolomic measurements were conducted via untargeted nuclear magnetic resonance spectroscopy. Plasma of ischemized rats manifested dynamic metabolomic changes over the reperfusion time, such as increased levels of ketone bodies, decreased levels of pyruvate, alanine, and citrate. All three branched chain amino acids showed common pattern during reperfusion time: a decrease in 3 hr compared to sham, then a highest level in 24 hr and decrease in 72 hr reperfusion time, similar to their corresponding ketoacids. The protective effect of ischemic preconditioning was demonstrated by a faster tendency of plasma metabolites to normalize. Results also proved the remarkable metabolomic differences between the control (naïve) and sham-operated anesthetized animals, what warrants for critical evaluation of surgery/anaesthesy in the algorithm of metabolomic animal studies., (© 2020 International Union of Biochemistry and Molecular Biology.)

Hyperhomocysteinemia (hHcy) represents a strong risk factor for atherosclerosis-associated diseases, like stroke, dementia or Alzheimer's disease. A methionine (Met)-rich diet leads to an elevated level of homocysteine in plasma and might cause pathological alterations across the brain. The hippocampus is being constantly studied for its selective vulnerability linked with neurodegeneration. This study explores metabolic and histo-morphological changes in the rat hippocampus after global ischemia in the hHcy conditions using a combination of proton magnetic resonance spectroscopy and magnetic resonance-volumetry as well as immunohistochemical analysis. After 4 weeks of a Met-enriched diet at a dose of 2 g/kg of animal weight/day, adult male Wistar rats underwent 4-vessel occlusion lasting for 15 min, followed by a reperfusion period varying from 3 to 7 days. Histo-morphological analyses showed that the subsequent ischemia-reperfusion insult (IRI) aggravates the extent of the sole hHcy-induced degeneration of the hippocampal neurons. Decreased volume in the grey matter, extensive changes in the metabolic ratio, deeper alterations in the number and morphology of neurons, astrocytes and their processes were demonstrated in the hippocampus 7 days post-ischemia in the hHcy animals. Our results suggest that the combination of the two risk factors (hHcy and IRI) endorses and exacerbates the rat hippocampal neurodegenerative processes.

Hyperhomocysteinemia (hHcy) is regarded as an independent and strong risk factor for cerebrovascular diseases, stroke, and dementias. The hippocampus has a crucial role in spatial navigation and memory processes and is being constantly studied for neurodegenerative disorders. We used a moderate methionine (Met) diet at a dose of 2 g/kg of animal weight/day in duration of four weeks to induce mild hHcy in adult male Wistar rats. A novel approach has been used to explore the hippocampal metabolic changes using proton magnetic resonance spectroscopy (1H MRS), involving a 7T MR scanner in combination with histochemical and immunofluorescence analysis. We found alterations in the metabolic profile, as well as remarkable histo-morphological changes such as an increase of hippocampal volume, alterations in number and morphology of astrocytes, neurons, and their processes in the selective vulnerable brain area of animals treated with a Met-enriched diet. Results of both methodologies suggest that the mild hHcy induced by Met-enriched diet alters volume, histo-morphological pattern, and metabolic profile of hippocampal brain area, which might eventually endorse the neurodegenerative processes.

Blood biomarkers are usually present in low concentration and can be masked by the high-abundance proteins, of which albumin is the predominant one. The purpose of this study was to compare four different albumin removal methods compatible with in-gel based proteomics, applicable for plasma, without requiring specific techniques and high financial input. Plasma underwent albumin depletion with ultrafiltration device Amicon Ultra, commercial ProteoPrep Blue Albumin and IgG Depletion Kit, acetonitrile precipitation method and precipitation with acetonitrile-methanol protocol. All samples were evaluated by 1-D and 2-D gel electrophoresis with subsequent mass spectrometry protein identification. Two of the tested methods (ProteoPrep BlueKit and acetonitrile-methanol precipitation) maintained sufficient protein content for further in-gel analyses. Their 2-D protein profiles were distinctively separated and overlapped with protein profile of crude plasma. Protein spot count showed significant increase in protein spots, compared to crude plasma, only with acetonitrile-methanol precipitation method. Precipitation with acetonitrile-methanol method significantly increased number of protein spots on 2-D protein profile and improved score of mass spectrometry identification. However, albumin was still present and found in number of protein spots.

Myocardial ischemia reperfusion is associated with mitochondrial dysfunction and increased formation of reactive oxygen/nitrogen species. The main purpose of this study was to assess the role of tyrosine nitration of mitochondrial proteins in postischemic contractile dysfunction known as myocardial stunning. Isolated Langendorff-perfused rat hearts were subjected to 20-min global ischemia followed by 30-min reperfusion. The reperfused hearts showed marked decline in left ventricular developed pressure, maximal rate of contraction (+dP/dt), and maximal rate of relaxation (-dP/dt). Immunofluorescence and ELISA assays demonstrated enhanced protein tyrosine nitration in reperfused hearts. Using two-dimensional gel electrophoresis and MALDI-TOF/TOF mass spectrometry, eight mitochondrial proteins were identified to be nitrated after ischemia reperfusion. These proteins are crucial in mitochondrial electron transport, fatty acid oxidation, tricarboxylic acid cycle, ATP synthesis, and control of high-energy phosphates. The proteome data also indicated reduced abundance in several of nitrated proteins. The results suggest that these changes may contribute to inhibition of aconitase activity but are unlikely to affect electron transport chain activity. Whether tyrosine nitration of mitochondrial proteins can be considered the contributing factor of postischemic contractile dysfunction remains to be explored.

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder that results in massive hippocampal and neocortical neuronal loss leading to dementia and eventual death. The exact cause of Alzheimer's disease is not fully explored, although a number of risk factors have been recognized, including high plasma concentration of homocysteine (Hcy). Hyperhomocysteinemia (hHcy) is considered a strong, independent risk factor for stroke and dementia. However, the molecular background underlying these mechanisms linked with hHcy and ischemic stroke is not fully understood. Paper describes rat model of global forebrain ischemia combined with the experimentally induced hHcy. Global ischemia-reperfusion injury (IRI) was developed by 4-vessels occlusion lasting for 15 min followed by reperfusion period of 72 h. hHcy was induced by subcutaneous injection of 0.45 µmol/g of Hcy in duration of 14 days. The results showed remarkable neural cell death induced by hHcy in the brain cortex and neurodegeneration is further aggravated by global IRI. We demonstrated degeneration of cortical neurons, alterations in number and morphology of tissue astrocytes and dysregulation of oxidative balance with increased membrane protein oxidation. Complementary to, an immunohistochemical analysis of tau protein and β-amyloid peptide showed that combination of hHcy with the IRI might lead to the progression of AD-like pathological features. Conclusively, these findings suggest that combination of risk factor hHcy with IRI aggravates neurodegeneration processes and leads to development of AD-like pathology in cerebral cortex.

Cardiac arrest is one of the leading causes of death among adults in older age. Understanding mechanisms how organism responds to ischemia at global level is essential for the prevention and ischemic patient's treatment. In this study, we used a global cerebral ischemia induced by four-vessel occlusion as an established animal model for ischemic stroke to investigate metabolic changes after 24 h reperfusion, when transitions occur due to the onset of delayed neuronal death. We also focused on the endogenous phenomenon known as ischemic tolerance by the pre-ischemic treatment. The experiments were carried out on blood plasma samples as easily available and metabolically reflecting the overall changes in injured organism. Our results imply that disturbed glycolysis pathway, as a consequence of ischemic injury, leads to the increased level of ketone bodies (acetone, acetoacetate and β-hydroxybutyrate) along with increased utilization of triacylglycerols in plasma of ischemic and ischemically preconditioned rats. Complementary to, a decreased level of glycolytic intermediates (lactate, pyruvate, acetate) with increased level of glucose was found in ischemic and preconditioned animals. The protective effect of ischemic preconditioning on metabolome recovery was demonstrated by significantly increased level of creatine compared to ischemic, non-preconditioned rats. We also document that acetoacetate, pyruvate, lactate, and leucine have the best discriminatory power between ischemic and control plasma. Conclusively, our results provide evidence that NMR spectra analysis can identify specific group of metabolites present in plasma with the capability for discrimination between individual groups of animals. In addition, an excellent feasibility for the statistical discrimination among ischemic, preconditioned, and control rats can be applied regardless of native or deproteinated plasma and also regardless of noesy or cpmg NMR acquisition.

Normobaric hyperoxia is applied for the treatment of a wide variety of diseases and clinical conditions related to ischemia or hypoxia, but it can increase the risk of tissue damage and its efficiency is controversial. In the present study, we analyzed cardiac mitochondrial proteome derived from guinea pigs after 60 h exposure to 100% molecular oxygen (NBO) or O 2 enriched with oxygen cation (NBO+). Two-dimensional gel electrophoresis followed by MALDI-TOF/TOF mass spectrometry identified twenty-two different proteins (among them ten nonmitochondrial) that were overexpressed in NBO and/or NBO+ group. Identified proteins were mainly involved in cellular energy metabolism (tricarboxylic acid cycle, oxidative phosphorylation, glycolysis), cardioprotection against stress, control of mitochondrial function, muscle contraction, and oxygen transport. These findings support the viewpoint that hyperoxia is associated with cellular stress and suggest complex adaptive responses which probably contribute to maintain or improve intracellular ATP levels and contractile function of cardiomyocytes. In addition, the results suggest that hyperoxia-induced cellular stress may be partially attenuated by utilization of NBO+ treatment.

Background: The causes of the individual differences in the rate of disability progression in multiple sclerosis (MS) are still not completely clear. According to the long-term prognosis of MS patients, the search for new valuable prognostic markers of "benign" or "malign" MS is necessary., Objectives: Our aim was to assess the possible association of MS onset age with the disease disability progression rate in Slovak patients with MS., Methods: By the unique pattern of evaluation of disability progression rate using Multiple Sclerosis Severity Score (MSSS), each of 270 MS patients was defined as slow-progressing, mid-rate progressing or rapidly progressing., Results: We found a significant differences in the age at onset between MS patients with different rate of disability progression (p(K-W)<0,00005). The faster was a disability progression assessed by MSSS score, the higher was the MS onset age., Conclusion: We showed for the first time in Central European Slovak population that MS onset age is an early marker that is in the positive correlation with disease disability progression rate, evaluated by MSSS score. We conclude that relapsing-remitting MS patients older at clinical onset have a higher risk of unfavorable prognosis (Tab. 2, Fig. 1, Ref. 21).

One of the characteristic features of the aging is dysfunction of mitochondria. Its role in the regulation of metabolism and apoptosis suggests a possible link between these cellular processes. This study investigates the relationship of respiratory complex I with aging-related oxidative stress in the cerebral mitochondria. Deterioration of complex I seen in senescent (26-months old) mitochondria was accompanied by decline in total thiol group content, increase of HNE and HNE-protein adducts as well as decreased content of complex I subunits, GRIM-19 and NDUFV2. On the other hand, decline of complex I might be related with the mitochondrial apoptosis through increased Bax/Bcl-2 cascade in 15-month old animal brains. Higher amount of Bcl-2, Bcl-xL with the lower content of GRIM-19 could maintain to some extent elevated oxidative stress in mitochondria as seen in the senescent group. In the cortical M1 region increased presence of TUNEL+ cells and more than 20-times higher density of Fluoro-Jade C+ cells in 26-months old was observed, suggesting significant neurodegenerative effect of aging in the neuronal cells. Our study supports a scenario in which the age-related decline of complex I might sensitize neurons to the action of death agonists, such as Bax through lipid and protein oxidative stimuli in mitochondria. Although aging is associated with oxidative stress, these changes did not increase progressively with age, as similar extent of lesions was observed in oxidative stress markers of the both aged groups.

Hyperhomocysteinemia (HHcy) is an independent risk factor of cardiovascular disease, but the mechanisms of tissue injury are poorly understood. In the present study, we investigated the effect of HHcy on rat heart function, activities electron transport chain (ETC) complexes, mitochondrial protein expression, and protein oxidative damage. HHcy was induced by subcutaneous injection of Hcy (0.45 μmol/g of body weight) twice a day for a period of 2 weeks. Performance of hearts excised after the Hcy treatment was examined according to the Langendorff method at a constant pressure. Left ventricular developed pressure, as well as maximal rates of contraction (+dP/dt) and relaxation (-dP/dt), was significantly depressed in HHcy rats. HHcy was accompanied by significant inhibition of ETC complexes II-IV, whereas activity of the complex I was unchanged. The decline in ETC activities was not associated with elevated protein oxidative damage, as indicated by unchanged protein carbonyl, thiol, and dityrosine contents. Moreover, the level of protein adducts with 4-hydroxynonenal was decreased in HHcy rats. Additionally, 2D-gel electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry did not show alterations in contents of inhibited ETC complexes. However, mass spectrometry analyses identified 8 proteins whose expression was significantly increased by HHcy. These proteins are known to play important roles in the cellular stress response, bioenergetics, and redox balance. Altogether, the results suggest that oxidative damage and altered protein expression are not possible causes of ETC dysfunction in HHcy rats. Increased expression of the other mitochondrial proteins indicates a protective response to Hcy-induced myocardial injury.