Synthesis of Deuterium-Labeled Pyrrolylcarnosine.

The effect of temperature on the effectiveness of the incorporation of deuterium into pyrrolylcarnosine (PC) was studied. Deuterium gas and heavy water were used as a source of deuterium. Isotope exchange was carried out using solid-phase and liquid-phase methods. It was found that it is better to use isotope exchange with deuterated water to obtain preparative amounts of labeled pyrrolylcarnosine. When using y solid-phase method, the main label is in pyrrole. The incorporation of deuterium at a higher temperature occurs more evenly. In addition, the use of deuterated water made it possible to reduce the amount of unlabeled isotopomer to almost 0% and to obtain a product with a yield of 70% and a content of more than seven deuterium atoms. It was established that the content of deuterium in the compound can be increased by pretreating the reaction mixture with deuterium gas. This approach opens up additional opportunities for the synthesis of labeled compounds.

Mechanisms of Neuroprotective Action of Hesperetin and Carnosine in Focal Ischemia of the Brain in Rats.

We compared the direct neuroprotective effect of minor food components, antioxidants hesperetin and carnosine, and analyzed their influence on the parameters of the oxidative status of the penumbra zones in the cerebral cortex during focal ischemia (1 h) of with reperfusion in Wistar rats. The animals received hesperetin and carnosine included in the diet in daily doses of 50 and 150 mg/kg, respectively, for 7 days before ischemia induction. The neuroprotective effect of hesperetin manifested in reduction of the ischemic lesion size by 30%, which was comparable with the effect of carnosine. Both hesperetin and carnosine reduced the level of MDA in the penumbra zone of the cerebral cortex and increased the total antioxidant activity of the brain tissue. Hesperetin also increased SOD activity to a level observed in the sham-operated control group.

Carnosine as an effective neuroprotector in brain pathology and potential neuromodulator in normal conditions.

Carnosine (b-alanyl-L-histidine) is an endogenous dipeptide widely distributed in excitable tissues, such as muscle and neural tissues-though in minor concentrations in the latter. Multiple benefits have been attributed to carnosine: direct and indirect antioxidant effect, antiglycating, metal-chelating, chaperone and pH-buffering activity. Thus, carnosine turns out to be a multipotent protector against oxidative damage. However, the role of carnosine in the brain remains unclear. The key aspects concerning carnosine in the brain reviewed are as follows: its concentration and bioavailability, mechanisms of action in neuronal and glial cells, beneficial effects in human studies. Recent literature data and the results of our own research are summarized here. This review covers studies of carnosine effects on both in vitro and in vivo models of cerebral damage, such as neurodegenerative disorders and ischemic injuries and the data on its physiological actions on neuronal signaling and cerebral functions. Besides its antioxidant and homeostatic properties, new potential roles of carnosine in the brain are discussed.

Comparative Action of Cardiotonic Steroids on Intracellular Processes in Rat Cortical Neurons.

Binding to Na+,K+-ATPase, cardiotonic steroids (CTS) activate intracellular signaling cascades that affect gene expression and regulation of proliferation and apoptosis in cells. Ouabain is the main CTS used for studying these processes. The effects of other CTS on nervous tissue are practically uncharacterized. Previously, we have shown that ouabain affects the activation of mitogen-activated protein kinases (MAP kinases) ERK1/2, p38, and JNK. In this study, we compared the effects of digoxin and bufalin, which belong to different subclasses of CTS, on primary culture of rat cortical cells. We found that CTS toxicity is not directly related to the degree of Na+,K+-ATPase inhibition, and that bufalin and digoxin, like ouabain, are capable of activating ERK1/2 and p38, but with different concentration and time profiles. Unlike bufalin and ouabain, digoxin did not decrease JNK activation after long-term incubation. We concluded that the toxic effect of CTS in concentrations that inhibit less than 80% of Na+,K+-ATPase activity is related to ERK1/2 activation as well as the complex profile of MAP kinase activation. A direct correlation between Na+,K+-ATPase inhibition and the degree of MAP kinase activation is only observed for ERK1/2. The different action of the three CTS on JNK and p38 activation may indicate that it is associated with intracellular signaling cascades triggered by protein-protein interactions between Na+,K+-ATPase and various partner proteins. Activation of MAP kinase pathways by these CTS occurs at concentrations that inhibit Na+,K+-ATPase containing the α1 subunit, suggesting that these signaling cascades are realized via α1. The results show that the signaling processes in neurons caused by CTS can differ not only because of different inhibitory constants for Na+,K+-ATPase.

Oxidative Status in Different Areas of the Cerebral Cortex of Wistar Rats during Focal Ischemia and Its Modulation with Carnosine.

Oxidative status was assessed in different areas of the cerebral cortex of male Wistar rats under normal condition and during permanent 24-h focal ischemia. In intact animals, the level of lipid hydroperoxides in the frontal lobes of both hemispheres was by 36% higher than in other cortical areas, while total antioxidant activity was by 25% higher than in other areas. During ischemia, changes in oxidative status were localized only in the ischemic focus and penumbra zone and did not involve other cortical areas. We demonstrated for the first time a neuroprotective effect of therapeutic administration of carnosine in low doses (50 mg/kg) on parameters of the oxidative status under conditions of focal ischemia comparable to its effect of high doses (500 mg/kg) as well as its local effect in the penumbra zone. A dose-dependent effect of carnosine on antioxidant activity in the penumbra zone during ischemia was also demonstrated. These findings confirm effectiveness of not only preventive carnosine administration, but also its application in the postischemic period of the stroke.

Assessment of Oxidative Status of the Brain and Blood Plasma in Rats with Modeled Focal Cerebral Ischemia/Reperfusion Injury.

Parameters of the oxidative status of the brain and blood plasma were measured in rats 24 h after 1-h focal cerebral ischemia. In the brain of rats exposed to cerebral ischemia, activities of superoxide dismutase and catalase were elevated. Ischemia reduced the total antioxidant activity of the brain and the levels of malonic dialdehyde and protein carbonyl derivatives. In the blood plasma of experimental rats, superoxide dismutase activity and malonic dialdehyde level increased and total antioxidant activity decreased, i.e. the shifts were similar to those in the brain. The ischemia-induced changes in the brain and blood were not always co-directed.

[Nocturnal pulse oximetry indicators in the evaluation of obstructive sleep apnea syndrome in outpatients with concomitant diseases of the upper respiratory tract and overweight]. / Pokazateli nochnoi pul'soksimetrii v otsenke sindroma obstruktivnogo apnoé vo sne u ambulatornykh patsientov s soputstvuiushchei patologiei verkhnikh dykhatel'nykh putei i izbytochnoi massoi tela.

AIM: By using mathematical modeling, to evaluate the impact of upper respiratory tract diseases, retro- and micrognathia, and body mass index (BMI) on nocturnal pulse oximetry indicators (oxygen saturation level and oxygen desaturation index) in outpatients examined for suspected obstructive sleep apnea syndrome (OSAS). SUBJECTS AND METHODS: The study enrolled 260 subjects with a mean age of 47.8±12.0 years. All the examinees underwent outpatient pulse oximetry screening during nocturnal sleep because of suspected OSAS. Multislice spiral computed tomography was carried out to assess the paranasal sinuses and nasal septum. BMI was calculated. Variance factor analysis using an original programming application intended to create binary and ternary dispersion complexes was employed as a main mathematical tool. RESULTS: There were statistically significantly sets of risk factors for OSAS: nasal septum deviation + increased BMI + male gender = 68.66%; chronic allergic rhinitis + increased BMI + male gender = 63.09%; retromicrognathia + increased BMI + male ganger = 59.48%; and chronic tonsillitis + increased BMI + male gander = 60.88%. Higher BMI and male gender are a most statistically significant set of risk factors. CONCLUSION: Pulse oximetry screening during nocturnal sleep in snoring patients with suspected OSAS in combination with an assessment of age, sex, BMI, ENT comorbidity, retro- and micrognathia can predict the severity of the disease and serve as a basis for elaborating an OSAS screening program.

Neuroprotective Effect of Carnosine on Primary Culture of Rat Cerebellar Cells under Oxidative Stress.

Dipeptide carnosine (ß-alanyl-L-histidine) is a natural antioxidant, but its protective effect under oxidative stress induced by neurotoxins is studied insufficiently. In this work, we show the neuroprotective effect of carnosine in primary cultures of rat cerebellar cells under oxidative stress induced by 1 mM 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), which directly generates free radicals both in the medium and in the cells, and 20 nM rotenone, which increases the amount of intracellular reactive oxygen species (ROS). In both models, adding 2 mM carnosine to the incubation medium decreased cell death calculated using fluorescence microscopy and enhanced cell viability estimated by the MTT assay. The antioxidant effect of carnosine inside cultured cells was demonstrated using the fluorescence probe dichlorofluorescein. Carnosine reduced by half the increase in the number of ROS in neurons induced by 20 nM rotenone. Using iron-induced chemiluminescence, we showed that preincubation of primary neuronal cultures with 2 mM carnosine prevents the decrease in endogenous antioxidant potential of cells induced by 1 mM AAPH and 20 nM rotenone. Using liquid chromatography-mass spectrometry, we showed that a 10-min incubation of neuronal cultures with 2 mM carnosine leads to a 14.5-fold increase in carnosine content in cell lysates. Thus, carnosine is able to penetrate neurons and exerts an antioxidant effect. Western blot analysis revealed the presence of the peptide transporter PEPT2 in rat cerebellar cells, which suggests the possibility of carnosine transport into the cells. At the same time, Western blot analysis showed no carnosine-induced changes in the level of apoptosis regulating proteins of the Bcl-2 family and in the phosphorylation of MAP kinases, which suggests that carnosine could have minimal or no side effects on proliferation and apoptosis control systems in normal cells.

[Experimental study of the basic pharmacokinetic characteristics of dipeptide carnosine and its efficiency of penetration into brain tissues].

We have used an original chromatography/mass spectrometry technique to study the pharmacokinetics of dipeptide carnosine in C57 Black/6 mice after intra-peritoneal administration of the drug at a dose of 1 g/kg. The basic pharmacokinetic characteristics of carnosine were measured the in the blood and brain. The obtained concentration-time curve has a biexponential character. It is shown that the maximum concentration of carnosine in the blood plasma is Cmax = 1081.75 ± 124.24 µg/mL and it is achieved in a time interval of Tmax = 0.25 h. We showed that i.p. administration of exogenous carnosine could significantly increase the concentration of that substance in the brain. Tissue availability of dipeptide carnosine for brain tissue is relatively good and constitutes 59% from the total amount of blood carnosine. It was found that the maximum concentration of carnosine in the brain occurs at the sixth hour after i.p. administration when the concentration of drug in the blood is minimal.

Effects of carnosine on polyamine levels in red blood cells of patients with hypertonic discirculatory encephalopathy.

The levels of polyamines (putrescine, spermine, spermidine) in erythrocytes from patients with hypertonic discirculatory encephalopathy are reduced (by 37, 45, and 50%, respectively) in comparison with the corresponding parameters in the control group. Addition of carnosine to the treatment protocol for chronic brain ischemia normalized the content of putrescine and spermine. The mechanisms of carnosine influence on polyamine metabolism are discussed: trapping of acrolein, end-product of polyamine oxidation, and compensation of NMDA-receptor excitotoxicity.

[Mexidant increases the effectiveness of levadopa treatment of Parkinson's disease].

The efficiency of mexidant therapy in patients with Parkinson's disease (PD) has been evaluated. The study included 49 patients aged 58-65 with a trembling-rigid and trembling forms of PD at an disease duration of 6.5 +/- 3.8 years. All patients were treated with levadopa-containing drugs, dopamine receptor agonists and/or amantadine. In addition, 27 patients received mexidant at a dose of 200 mg/day (i.v.) for the first 10 days, followed by intramuscular injections of 100 mg (twice a day) for 10 days. The dynamics of symptoms in the group of patients receiving mexidat showed that the inclusion of this drug into the therapeutic regime significantly decreased the degree of levadopa therapy side effects. Mexidant reduced the oxidative damages of blood plasma lipoproteins by neutralizing the growth of lipid hydroperoxide and increased the endogenous antioxidant status. The presented data show that mexidant enhances the efficiency of PD therapy.

[Determination of morphine and codeine in forensic chemical studies with the use of a single quadrupole mass-selective detector coupled to the HPLC system].

It was shown that a single quadrupole mass-selective detector coupled to the HPLC system can be used in forensic chemical practice for the detection and quantitative measurement of morphine and codeine in forensic in various biological objects. An algorithm for the reliable identification of opiates in a concentration range starting from 0.002 mg% with a relative error below 20% (mean ca 9.5%) is proposed.

Biological activity of novel synthetic derivatives of carnosine.

Two novel derivatives of carnosine--(S)-trolox-L-carnosine (STC) and (R)-trolox-L-carnosine (RTC) are characterized in terms of their antioxidant and membrane-stabilizing activities as well as their resistance to serum carnosinase. STC and RTC were synthesized by N-acylation of L-carnosine with (S)- and (R)-trolox, respectively. STC and RTC were found to react more efficiently with 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and protect serum lipoproteins from Fe(2+)-induced oxidation more successfully than carnosine and trolox. At the same time, STC, RTC and trolox suppressed oxidative hemolysis of red blood cells (RBC) less efficiently than carnosine taken in the same concentration. When oxidative stress was induced in suspension of cerebellum granule cells by their incubation with N-methyl-D-aspartate (NMDA), or hydrogen peroxide (H(2)O(2)), both STC and RTC more efficiently decreased accumulation of reactive oxygen species (ROS) than carnosine and trolox. Both STC and RTC were resistant toward hydrolytic degradation by human serum carnosinase. STC and RTC were concluded to demonstrate higher antioxidant capacity and better ability to prevent cerebellar neurons from ROS accumulation than their precursors, carnosine and trolox.

[Efficiency of complex rehabilitation in chronic obstructive pulmonary disease and occupational bronchitis].

AIM: To evaluate the efficiency of the rehabilitation in patients with obstructive pulmonary disease (COPD) and occupational bronchitis (OB) and the disabled due to these diseases, which was based on the authors' developed procedure and criteria for evaluating the efficiency of rehabilitative measures. MATERIALS AND METHODS: The efficiency of implementation of 55 individual rehabilitation programs (RP) elaborated by the Medicosocial Examination Bureau for the disabled due to COPD and OB was evaluated. RESULTS: The authors have determined qualitative criteria for evaluating the efficiency of rehabilitation: the extent to which the recommended rehabilitation measures being implemented, the trend in the magnitude of various vital activity categories and social inadequacy and in the disability group on regular examination, the results of implementation of some parts of an individual RP, the conformity of the achieved results to the rehabilitation potential of a disabled patient. The evaluation procedure supposes a unified quantitative gradation of each criterion. Overall, rehabilitation is more effective in COPD than in OB. CONCLUSION: The developed criteria and procedures for evaluating the efficiency of rehabilitation in patients with COPD and OB show a great informative value and practical content richness, which permits one to make corrections into the rehabilitation process. Functional impairments, the degree of vital activity restrictions and social inadequacy, as well as the rehabilitation potential and rehabilitation prognosis are concomitantly evaluated, which allows minor changes to be assessed in the clinical, personality, and social status of a patient.

[Neuroprotective mechanisms of the ubiquinol action in experimental focal ischemia]. / Issledovanie neiroprotektornykh mekhanizmov deistviia ubikhinola pri éksperimental'noi fokal'noi ishemii.

Ischemic stroke is one of the most socially important diseases characterized by impaired cerebral circulation with focal damage of the brain tissue and decreased functionality. Despite the successes of modern pharmacology, possibilities of pharmacotherapy for stroke remain limited, and the research for new drugs with neuroprotective effects that can prevent brain cell death is still relevant. In this study we have investigated the neuroprotective activity of ubiquinol as a part of an innovative form on a rat model of irreversible 24 h-cerebral ischemia with evaluation of the mechanisms of its neuroprotective effect. Ubiquinol (30 mg/kg), administered intravenously in the acute period of irreversible 24 h focal cerebral ischemia, had a direct neuroprotective effect, characterized by a decrease in the volume of brain tissue necrosis. The protective effect of ubiquinol is due to its ability to inhibit the development of oxidative stress by the direct anti-radical action, preventing the increase in the lipid hydroperoxide content in the brain tissue adjacent to the focus of necrosis, lowering the lipid oxidation rate in plasma against under conditions of increased total antioxidant activity in the brain and blood of experimental animals. In vitro experiments have shown the ability of ubiquinol to prevent cell death in primary culture of cerebral neurons of rat brain under 4 h oxygen/glucose deprivation followed by 20 h reoxygenation.

[Clinical aspects of rehabilitation potential in patients with chronic obstructive pulmonary disease].

AIM: To reveal and evaluate clinical signs influencing rehabilitation potential in COPD patients. MATERIAL AND METHODS: Clinical findings in COPD patients were analysed at medical and social expert examination (MSEE). RESULTS: Progression of the pathological process is associated with aggravation of chest pain, fever, sputum discharge at coughing. Cough and dyspnea occurred in all the examinees irrespective of the disease etiology. Severity of respiratory failure correlated with severity of COPD. Respiratory and circulatory failure aggravate quality of life. The study determined clinical signs in patients with COPD of various etiology which influence rehabilitation potential of COPD patients. This helped specification of disability criteria and certification. Conclusion. In MSEE of COPD patients it is necessary to base on the patient's complaints, duration of the disease, severity of functional respiratory and circulation failure, complications, social problems.

[Study of the neuroprotective effects of carnosine in an experimental model of focal cerebral ischemia/reperfusion]. / Issledovanie neiroprotektornykh mekhanizmov deistviia karnozina pri éksperimental'noi fokal'noi ishemii/reperfuzii.

Oxidative stress is one of the key factors in brain tissue damage in ischemia, which indicates the appropriateness of using antioxidants under these conditions. One of the promising antioxidants for the therapy of ischemic stroke is the natural dipeptide carnosine. The neuroprotective effect of dietary carnosine administration was investigated in an experimental model of focal cerebral ischemia/reperfusion in Wistar rats. Animals received carnosine with a diet at a daily dose of 150 mg/kg for 7 days before temporary occlusion of the middle cerebral artery (MCA), performed for 60 min. At 24 h after the onset of ischemia the effect of carnosine on the area of the necrotic core was evaluated in animals. In brain tissue of animals the content of malondialdehyde (MDA), protein carbonyls (PC), total antioxidant capacity (TAC), total activity of superoxide dismutase (SOD), glutathione peroxidase (GP), catalase (CAT) and glutathione transferase (GT), content of isoprostanes and cytokines were measured. Carnosine significantly reduced the infarct size. Carnosine also increased TAC and reduced the level of MDA and isoprostanes in brain tissue. Influence of carnosine on other parameters was not detected. Thus carnosine consumed prophylactically with the diet for 7 days before the induction of ischemia by means of MCA occlusion in rats provides the direct neuroprotective effect, retains high antioxidant activity of brain tissue, reduces the level of oxidative damage markers (MDA and isoprostanes) but does not have any effect on the activity of antioxidant enzyme systems and production of cytokines in brain tissue.

[Lipoilcarnosine: synthesis, study of physico-chemical and antioxidant properties, biological activity]. / Lipoilkarnozin: sintez, izuchenie fiziko-khimicheskikh i antioksidantnykh svoistv, biologicheskaia aktivnost'.

Synthesis of lipoilcarnosine (LipC) - a conjugated molecule based on two natural antioxidants, carnosine and a-lipoic acid, is described. Its physico-chemical, antioxidant properties and biological activity are characterized. According to reversed-phase HPLC with a UV detector, purity of the final product was 89.3%. The individuality of the obtained sodium salt of LipC was confirmed by tandem HPLC-mass spectrometry. High resistance of LipC to hydrolysis with serum carnosinase was demonstrated. The antioxidant activity of LipC measured by reaction with the formation of thiobarbituric acid reacting substances and kinetic parameters of iron-induced chemiluminescence was higher than that of carnosine and lipoic acid. LipC did not affect viability of SH-SY5Y human neuroblastoma culture cells, differentiated towards the dopaminergic type, at concentrations not exceeding 5 mM. At the concentration range of 0.1-0.25 mM LipC protected neuronal cells against 1-methyl-4-phenylpyridinium (MPP + )-induced toxicity.

[Carnosine: endogenous physiological corrector of antioxidative system activity].

The results of research of camosine as an antioxidative system corrector in conditions of oxidative stress caused by the action of damaging factors (y-rays, overcooling, hypobaric hypoxia, brain ischemia, neurotoxin impact) are summarized in the present review. The effects of carnosine are characterized not only at the level of the whole organism but also in "in vitro" models with use of a whole series of enzymatic systems. The results of the experiments conducted displayed the ability of carnosine to protect animals from oxidative stress based on the combination of direct antioxidative effects and a modulation of enzymes' activities which participate in controlling of reactive oxygen species level in tissues.