The review presents evidence of the participation of low-density lipoproteins (LDL) modified by low molecular weight dicarbonyl compounds formed during freeradical oxidation of lipids (malondialdehyde) and carbohydrates in the development of endothelial dysfunction and atherosclerotic vascular lesions. The authors believe that it is they, and not oxidized (hydroperoxide-containing) LDL, that are the main factors of pathogenesis. The role of dicarbonyl-modified LDL in LOX-1 dependent induction of processes leading to the development of endothelial dysfunction is discussed. The results of studies proving that damage to the glycocalyx (a layer of macromolecules that prevent the development of endothelial dysfunction) covering the luminal surface of the endothelium is caused by hyperproduction of reactive oxygen species. Ways of pharmacological correction of free-radical oxidation processes are discussed, due to which inhibition of atherogenesis and diabetogenesis can be achieved.

Oxidatively modified plasma lipoproteins play an important role in the molecular mechanisms of vascular wall damage in atherosclerosis and diabetes mellitus. It was found that the oxidizability of low-density lipoproteins (LDLs) is more than one order of magnitude higher than that of high-density lipoproteins (HDLs). It was shown that acylhydroperoxy derivatives of phospholipids of oxidized biomembranes are predominantly captured by LDL particles, but not by HDLs, which refutes the hypothesis about the involvement of HDLs in the reverse transport of lipohydroperoxides (LOOH). The results suggest the possibility of different mechanisms of LOOH accumulation in LDLs: due to the increased oxidizability of LDLs and due to the effective transmembrane transport of LOOH by LDL particles.

Background. In view of the worsening forecast for global temperature rise worldwide, it seems relevant to study the effects of abnormal heat waves on systemic regulatory processes in people with chronic diseases, in particular coronary artery disease (CAD).Aims. This study aimed to investigate the effect of hyperthermia on oxidative stress parameters in patients with various severity of CAD and in healthy subjects.Materials and methods. We studied the level of malonic dialdehyde (MDA) and the activity of Cu,Zn-containing superoxide dismutase (Cu,Zn-SOD) in healthy subjects under conditions of 30-day long simulated hyperthermia and in patients with different severity of CAD after the summer heat wavesResults. We revealed signs of oxidative stress in healthy volunteers during model hyperthermia that manifested as an increase in content of MDA in blood plasma. At the same time we observed increasing activity of Cu,Zn-SOD in erythrocytes that utilizes reactive oxygen species. The increase of Cu,Zn-SOD activity started with a certain latency what also can be explained by de novo enzyme biosynthesis induction. We also studied oxidative stress parameters in patients at high and moderate cardiovascular risk according to the SCORE risk chart with uncomplicated CAD course and in patients with complicated CAD with severe coronary damage according to angiography during the summer heat waves. We observed accumulation of MDA in blood plasma and increasing activity of erythrocyte Cu,Zn-SOD in patients with uncomplicated CAD. At the same time we noted that accumulation of MDA in blood plasma was not followed by any increase in activity of red blood cell Cu,Zn-SOD in patients with severe complicated CAD. This fact indicates dysregulation of free radical processes in patients with severe course of CAD during the heat waves.Conclusions. The dysregulation of free-radical processes in patients with a severe clinical course of CAD has been revealed.

Aim. To study the effect of oxidative stress and telomere length in the chromosomes of blood leukocytes in patients with coronary heart disease (CHD) on the development of cardiovascular complications. Materials and methods. In 119 patients with CHD, the level of oxidatively modified low-density lipoproteins (ox-LDL) in blood plasma and the length of telomeres in nuclear blood cells were determined during the examination. After 5 years, a telephone survey of patients (or their relatives) was conducted to obtain data on the presence of cardiovascular complications. Telomere length was determined using quantitative real-time PCR, and the level of ox-LDL was determined by immunochemical method. Results. It was found that reducing the length of telomeres in patients with CHD increases the risk of subsequent development of cardiovascular complications. A strong negative correlation was found between the level of ox-LDL and telomere length in the group of examined CHD patients who had cardiovascular complications after 5 years. Conclusion. CHD patients with short telomere length and high levels of ox-LDL have an increased risk of cardiovascular complications during 5 years.

The elimination kinetics of carbonyl-modified low density lipoproteins (LDL) from rabbit bloodstream was studied using isolated LDL of rabbits and humans after preliminary biotinylation or labeling with FITC. Rabbit or human blood plasma LDL were isolated using differential ultracentrifugation in a density gradient; after labeling by biotinylation or by FITC, LDL were modified with various low molecular weight natural dicarbonyls: malondialdehyde (MDA), glyoxal or methylglyoxal. Native (control) and dicarbonyl-modified biotinylated or FITC-labeled LDL were injected into the ear vein of rabbits, and blood samples were taken at certain time intervals. The content of biotinylated LDL in blood plasma was determined by an enzyme immunoassay method; FITC-labeled LDL was determined from the fluorescence spectra. It has been found that glyoxal- and methylglyoxal-modified rabbit and human LDL circulate in the bloodstream of rabbits for almost the same period as native (unmodified) LDL. In contrast to this, MDA-modified rabbit and human LDL were very quickly eliminated from the rabbit bloodstream. Dicarbonyl-modified LDL from human blood plasma is not associated with red blood cells or endothelial cells. We found that using the Oxidized LDL ELISA kits (Mercodia, Sweden) it was possible to identify mainly MDA-modified LDL. The level of MDA-modified LDL in the blood plasma of CHD patients sharply decreased during therapy with evolocumab, the hypocholesterolemic inhibitor of PCSK9 (proprotein convertase of subtilisin/kexin type 9), which activates LDL reutilization in the liver cells. These results explain the extremely rapid clearance of MDA-modified LDL in our experiments by their increased utilization in hepatocytes. The results obtained indicate a high atherogenicity of glyoxal- and methylglyoxal-modified LDL, long-term circulating in the bloodstream.

Aim To study the oxidative modification of red blood cell Cu,Zn superoxide dismutase (SOD) in patients with ischemic heart disease (IHD) in vivo and in vitro to substantiate the use of a new oxidative stress marker.Material and methods Red blood cell Cu,Zn SOD was measured by depression of nitrotetrazolium blue reduction by the superoxide anion generated in xanthine oxidase xanthine oxidation. Red blood cell Cu,Zn SOD was measured immunochemically. The biochemical study was performed in the control group (patients with low extremity fracture without known history of cardiovascular diseases and hyperlipidemia) and in groups of patients with acute myocardial infarction, stable angina, and decompensated heart failure. For evaluation of oxidative stress intensity in IHD patients, an empirical SOD oxidative modification coefficient (OMCSOD) was proposed, which is a Cu,Zn SOD activity / Cu,Zn SOD content ratio.Results The red blood cell Cu,Zn SOD activity was significantly decreased in all IHD groups compared to the control group. Furthermore, OMCSOD was also considerably decreased in IHD patients, which warrants the use of this biochemical index as an oxidative stress marker.Conclusion It was shown that the Cu,Zn SOD modification was induced by interaction of the enzyme molecules with a natural dicarbonyl, malonic dialdehyde, and OMCSOD can be used for evaluation of oxidative stress intensity in IHD patients.

We studied the effect of human blood low density lipoproteins (LDL) modified by reactive halogen species (HOCl, HOBr) or reactive carbonyl species (glyoxal, methylglyoxal) on NETosis. A 2-h incubation of blood with LDL modified by reactive halogen species led to a significant increase in the number of neutrophil extracellular traps (NETs), as observed in blood smears, indicating the induction of NETosis. Such an effect was not found for LDL modified by carbonyl compounds (glyoxal or methylglyoxal). Examination of blood smears showed that the number and morphology of neutrophils were not changed after incubation with native LDL or LDL modified by the above compounds. The addition to blood of LDL modified by reactive halogen species, as well as by carbonyls, caused no significant increase in blood luminol-dependent chemiluminescence. When following modified LDL, a cell activator (phorbol-12-myristate-13-acetate) was added to the blood, the chemiluminescence signal increased but did not differ significantly from that for control (native) LDL. At an earlier time (30 min) of blood incubation with LDL, neither native nor modified LDL showed activating effect on NETosis. Consequently, NETosis induced by LDL modified by reactive halogen species is apparently a slow, oxygen-independent process not accompanied by neutrophil death. An elevated level of circulating NETs in response to halogenative stress increases the likelihood of microthrombus formation and disturbance of the microcirculation.

The elimination kinetics of carbonyl-modified low density lipoproteins (LDL) from rabbit bloodstream was studied using isolated LDL of rabbits and humans after preliminary biotinylation or labeling with FITZ. LDL from rabbit or human blood plasma were isolated using differential ultracentrifugation in a density gradient, and then LDL were labeled using biotinylation or FITZ, after which they were modified with various low molecular weight natural dicarbonyls: malondialdehyde (MDA), glyoxal or methylglyoxal. Native and dicarbonyl-modified biotinylated or FITZ-labeled LDL were injected into the ear vein of rabbits and blood samples were taken at certain intervals. To determine the content of biotinylated LDL in blood plasma, an enzyme immunoassay was performed; FITZ-labeled LDL were determined by spectra fluorescence. It is shown that glyoxal- and methylglyoxal-modified LDL in rabbits and humans circulated in the bloodstream for almost the same time as native (unmodified) LDL. At the same time, MDA-modified rabbit and human LDL were extremely quickly eliminated from the rabbit bloodstream. Dicarbonyl-modified LDL from the donors blood plasma were not associated with the red blood cells and endothelial cells. It has been shown that using the kits Oxidized LDL ELISA (“Mercodia”, Sweden), it is possible to identify mainly MDA-modified LDL. The level of MDA-modified LDL in the blood plasma of CHD patients sharply decreases during therapy with the hypocholesterolemic drug the PCSK9 inhibitor (evulokumab), which activates LDL reutilization in the liver cells. These results explain the extreme drop in the level of MDA-modified LDL by their increased utilization in hepatocytes. The results obtained indicate a high atherogenicity of glyoxal- and methylglyoxal-modified LDL, long-term circulating in the bloodstream.

The paper explores the formation of a-oxoaldehydes during the interaction of glucose metabolites with hydroxyl or alkoxyl radicals. Hydroxyl radicals were generated under radiolysis of aqueous solutions, and alkoxyl radicals (t-BuO) were obtained in the model system tert-butyl hydroperoxide/Fe2+. High-performance liquid chromatography revealed that methylglyoxal was one of the organic products resulting from t-BuO-induced transformations of fructose-1,6-bisphosphate under hypoxic conditions. The interaction of lysine and methylglyoxal one of the main targets of a-oxoaldehydes in proteins was also studied. As chemiluminescence and EPR spectroscopy demonstrated, this reaction generates a methylglyoxal anion radical, a cation-radical of methylglyoxal dialkylamine and a superoxide anion radical. EPR signal of methylglyoxal-derived free radicals was observed in hypoxia, whereas only the trace amounts of these free radicals were recorded in the aerated reaction medium.

It was established that recombinant human peroxiredoxins (Prx1, Prx2, Prx4, and Prx6) inhibit natural dicarbonyls formed during free radical peroxidation of unsaturated lipids (malonic dialdehyde) and oxidative transformations of glucose (glyoxal and methylglyoxal). A possible role of the decrease in the activity of peroxiredoxins under oxidative and carbonyl stress is discussed as an important factor that triggers the molecular mechanisms of vascular wall damage in atherosclerosis and diabetes mellitus.

Установлено, что рекомбинантные пероксиредоксины человека (Prx1, Prx2, Prx4 и Prx6) ингибируют природные дикарбонилы, образующиеся при свободнорадикальном окислении ненасыщенных липидов (малоновый диальдегид) и окислительных превращениях глюкозы (глиоксаль, метилглиоксаль). Обсуждается возможная роль снижения активности пероксиредоксинов при окислительном и карбонильном стрессе в качестве важного фактора, включающего молекулярные механизмы повреждения стенки сосудов при атеросклерозе и сахарном диабете.

Aim. To assess the influence of coronary heart disease patients (CHD) therapy with statins or PCSK9 inhibitors influence on the level of oxidatively modified low density lipoproteides (LDL) and activity of erythrocyte Se-glutathione peroxidase (GSH-Px).Material and methods. To the study, CHD patients were included (9-10 males per group), who during 6 months were undergoing statin therapy — 40 mg per day of pravastatin (group 1) or 0,4 mg per day of cerivastatin (group 2), as the therapy with PCSK9 inhibitor — 420 mg per month evolocumab (group 3) during 1 year. The level of lipohydroperoxide in LDL (LOOH-LDL) was measured in the groups 1 and 2 with the modified method and usage of Fe-xilenolorange; content of oxidized LDL (oxLDL) in the group 3 — with immune chemistry method (assays Mercodia, Sweden). Activeness of GSH-Px in all groups was assessed with the modified methods bound with glutathione reductase system and tret-buthyl hydroperoxide as a substrate.Results. Simultaneously with the decrease of LDL cholesterol in the groups 1 and 2 there was significant increase (in the group 2 — 6-7 times in 3-6 months of therapy) of the level of LOOH-LDL. In the group 2 there was marked significant decrease of GSH-Px activity beginning from the month 3. In the group 3, with decreased LDL cholesterol there was significant decline in oxLDL with changed activity of GSH-Px.Conclusion. Statins, effectively decreasing the level of LDL cholesterol, simultaneously induce the oxidation of LDL and decrease the activity of GSH-Px. Inhibitor PCSK9 not only does effectively decrease the level of LDL cholesterol, but also the content of oxLDL, not leading to decreased GSH-Px activity.

AIM:We study the dynamics of oxidatively modified low-density lipoprotein (ox-LDL) content in blood plasma, as well as changes in the activity of key antioxidant enzymes such as Se-containing glutathione peroxidase (GSH-Px) Cu,Zn-superoxide dismutase (SOD) and catalase in erythrocytes of patients with coronary artery disease during treatment with PCSK9 inhibitor (ewolocumab).MATERIALS AND METHODS:The study included 9 men (59 ± 10 years) with coronary artery disease with atherosclerotic lesion at least one main coronary artery according to coronary angiography. Patients took standard therapy before taking the study, everyone took the maximum tolerated dose of statins. Since the target cholesterol levels of low-density lipoprotein cholesterol (LDL-C) were not achieved during the statin therapy, patients were prescribed lipid-lowering therapy with the inclusion of the inhibitor PCSK9-emocoucumab from Amgen 420 mg once a month. The content of lipid metabolism indices was determined by standard biochemical methods. The level of ox-LDL in the blood plasma was determined by the immunochemical method. The activity of antioxidant enzymes was determined in blood erythrocytes using biochemical techniques.RESULTS:Cholesterol-lowering drug of the new type - inhibitor protein convertase subtilisin/kexin type 9 (PCSK9) evolocumab (Amgen) not only effectively lowers the level of cholesterol in low density lipoprotein (LDL), but also significantly reduces the content of oxdatively modified LDL in blood plasma. Unlike statins, the inhibitor of PCSK9 does not cause a decrease in the activity of antioxidant enzymes of the blood.CONCLUSION:PCSK9 inhibitor has no effect on the parameters of oxidative stress.

Aim. To study the oxidative damage of biopolymers (proteins and nucleic acids) in blood of patients with type 2 diabetes mellitus (DM). Materials and methods. In the blood of 50 patients with DM and 25 patients without disorders of carbohydrate metabolism were estimated: the level of oxidized low-density lipoprotein (oxLDL) by immunochemical method, the content of SH-groups in plasma proteins, the activity of Cu, Zn-superoxide dismutase (SOD) in erythrocytes, the length of telomere in leukocyte DNA, the level of 8-hydroxy-2'-deoxygunosine (8-oxo-dG) in plasma and urine. Results and discussion. It is shown that in DM patients the level of oxLDL increases and the content of SH-groups in proteins and peptides of the blood plasma decreases, which indicates the development of oxidative stress. In addition, a carbonyl-dependent modification of erythrocyte SOD was detected in DM patients, as well as oxidative DNA destruction (decrease in telomere length in leukocytes and an increase in the level of 8-oxo-dG in blood plasma and urine). Conclusion. On the basis of the definition of a complex of correct indicators, a multiple oxidative modification of biopolymers of blood (proteins and DNA) was detected in patients with DM.

The interaction of superoxide radical anion (O2 •−) with active dicarbonyls (methylglyoxal, glyoxal, and malonic dialdehyde) was studied. It was demonstrated that glyoxal and methylglyoxal inhibited superoxide-dependent accumulation of formazan; however, malonic dialdehyde stimulated this process. The formation of O2 •− in these experiments occurred during the decomposition of the SOTS-1 azo initiator. On the other hand, all of the studied dicarbonyls in this system of O2 •− generation competed for superoxide with the TIR ON spin trap. These compounds also inhibited luminal-dependent chemiluminescence during the AIBN azo initiator-induced peroxidation of liposomes from the egg phosphatidylcholine. A mechanism for the antiradical and antioxidant effects of the studied dicarbonyls, assuming the production of free radical intermediates in their reactions with O2 •− or its protonated form, is proposed.

Se-containing glutathione peroxidase (GSH-Px) is one of the key enzymes of the body's antioxidant system. The kinetic characteristics of GSH-Px (substrate is tert-butyl hydroperoxide) after modification of the enzyme by various concentrations of natural dicarbonyls (glyoxal, methylglyoxal, malonic dialdehyde) were studied. It was shown that dicarbonyls affected both K m and V max for GSH-Px. It is suggested that the effect of various dicarbonyls on GSH-Px depends on the molecular mechanisms of their interaction with the amino acid residues of the enzyme.

Low-molecular-weight dicarbonyls formed during free radical peroxidation of polyene lipids (malondialdehyde) and autooxidation (glyoxal) or other oxidative transformations of glucose (methylglyoxal) are able to modify the structure of lipid-protein supramolecular complexes of cells. We investigated changes in the erythrocyte membrane structure after an 18-h exposure of human red blood cells in the presence of various natural dicarbonyls. The changes in the mechanical properties of the membrane after membrane modification by carbonyls were evaluated by the susceptibility of erythrocytes to hypoosmotic hemolysis. It has been shown that treatment of red blood cells with malondialdehyde increases the resistance of these cells to hypoosmotic hemolysis, whereas the malondialdehyde isomer, methylglyoxal, in contrast, makes red blood cells more sensitive to the action of hypoosmotic solutions. Paradoxically, a homologue of malondialdehyde, glyoxal, has no effect on hemolysis of red blood cells in hypoosmotic solutions. The findings point to the possibility of the multidirectional effect of low-molecular-weight dicarbonyls with similar structures on the structure and function of biological membranes.

Se-содержащая глутатионпероксидаза (GSH-Px) является одним из ключевых ферментов антиоксидантной системы организма. Получены кинетические характеристики GSH-Px (субстрат - гидропероксид трет-бутила) после модификации фермента разными концентрациями природных дикарбонилов (глиоксаль, метилглиоксаль, малоновый диальдегид). Дикарбонилы влияют как на K, так и на V GSH-Px. Действие разных дикарбонилов на GSH-Px, возможно, зависит от молекулярных механизмов их взаимодействия с аминокислотными остатками фермента.