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We proposed a procedure for the IR spectrometric determination of lipid hydroperoxides in biological systems. The main bands in the IR absorption spectra of linoleic acid and its hydroperoxide were identified, and analytical bands suitable for the determination of both compounds in their mixtures were selected. It was demonstrated that tert-butyl hydroperoxide can be used as an external standard for determining fatty acid hydroperoxides. Using the external standard method (calibration curve) for tert-butyl hydroperoxides, we calculated the concentration of linoleic acid hydroperoxide in its mixture with linoleic acid; it agreed with the specified values. Using the developed procedure, we estimated the concentration of hydroperoxide groups in natural cardiolipin. The results were compared to those obtained by an independent method (activated chemiluminescence).

Oxidative stress acts as a pathogenetic factor in many diseases; estimating its level is important for early diagnosis and therapy adjustment. The antioxidant status was evaluated for the blood plasma. A set of chemiluminescence (CL) kinetic-curve parameters (latent period τlat and analytical signal increment ΔI CL) in a 2,2’-azo-bis(2-amidinopropane)dihydrochloride–luminol system were proposed for estimating the oxidative stress level. Uric acid and albumin were identified as major components that are responsible for the changes in the plasma CL kinetic curve. UV light caused oxidative modification of serum albumin in a dose-dependent manner, thus enhancing its antioxidant properties. Changes in plasma CL kinetics were proposed as a means to measure oxidative stress in the human body.

Mitochondrial dysfunctions are an underlying cause of many human diseases including degenerative diseases. One of the consequences of mitochondrial dysfunctions is apoptosis of functionally active cells. During the initial stage of apoptosis, increased production of superoxide anion-radical (SAR) is observed. A promising method of SAR detection in cells and tissues is chemiluminescence (CL), primarily, in the presence of lucigenin, a SAR specific amplifier of CL. In this study a means of improving CL was proposed, and its effectiveness in detecting SAR level in living tissues of laboratory animals in hypoxia and parkinsonism models was evaluated. Aerobic (O2 — 15 %, CO2 — 5 %, N2 — 80 %) and anaerobic (CO2 — 5 %, N2 — 95 %) gas mixtures proposed for samples aeration, maintained a constant pH of 7.4, necessary for accurate recording of CL. Using the studied method, a statistically significant increase (1.8 and 2.0 times) in SAR production level in rat heart tissue was detected with hypoxia duration of 150 to 240 minutes. In the parkinsonian model SAR production in mouse brain tissue samples of striatum and substantia nigra was 1.7 and 1.3 times higher after administration of the final dose of proneurotoxin, as compared to the control group.

Functional activity of neutrophils was evaluated by the chemiluminescent method with successive double stimulation by soluble stimuli with different mechanisms of action: phorbol-12-myristate-13-acetate (PMA) and phormyl-methionyl-leucyl-phenilalanine (fMLP). The study was carried out in 26 patients receiving oral sugar-reducing therapy. In addition to the functional activity of neutrophils, the levels of TBA reactive products, inflammation markers, blood clotting values, and biochemical parameters were measured. The results showed mainly reduction of the granulocytic component of the immune system in the patients.

The effect of existing anti-cancer therapies is based mainly on the stimulation of apoptosis in cancer cells. Here, we have demonstrated the ability of a catalytically-reactive nanoparticle-based complex of cytochrome c with cardiolipin (Cyt-CL) to induce the apoptosis and killing of cancer cells in a monolayer cell culture. Cyt-CL nanoparticles were prepared by complexing CytC with different molar excesses of CL. Following characterization, cytotoxicity and apoptosis inducing effects of nanoparticles were investigated. In an attempt to identify the anticancer activity mechanism of Cyt-CL, pseudo-lipoxygenase and lipoperoxidase reaction kinetics were measured by chemiluminescence. Using chemiluminescence, we have demonstrated that the Cyt-CL complex produces lipoperoxide radicals in two reactions: by decomposition of lipid hydroperoxides, and by lipid peroxidation under the action of H2O2. Antioxidants inhibited the formation of lipid radicals. Cyt-CL nanoparticles, but not the CytC alone, dramatically enhanced the level of apoptosis and cell death in two cell lines: drug-sensitive (A2780) and doxorubicin-resistant (A2780-Adr). The proposed mechanism of the cytotoxic action of Cyt-CL involves either penetration through the cytoplasm and outer mitochondrial membrane and catalysis of lipid peroxidation reactions at the inner mitochondrial membrane, or/and activation of lipid peroxidation within the cytoplasmic membrane. Here we propose a new type of anticancer nano-formulation, with an action based on the catalytic action of Cyt-CL nanoparticles on the cell membrane and and/or mitochondrial membranes that results in lipid peroxidation reactions, which give rise to activation of apoptosis in cancer cells, including multidrug resistant cells.

Oxidative stress is a pathogenetic factor of many diseases. The control of its level is important for early diagnosis and therapy adjustment. In this work, antioxidant status was estimated in blood plasma. In the system of 2,2'-azo-bis(2-amidinopropane)dihydrochloride-luminol a set of chemiluminescence kinetic curve parameters is proposed for oxidative stress level estimation (the latent period τ(lat) and the increasing of analytical signal ΔI(CL)). Uric acid and albumin were shown as the main components that responsible for changes in chemiluminescence kinetic curve of plasma. Serum albumin undergoes oxidative modification in dose-depend manner under the action of UV irradiation, it causes the enhancement of antioxidant properties. Changes in plasma chemiluminescence kinetics are proposed as a measure of oxidative stress in human body.

The accumulation of UV photolysis products of amino acids tyrosine and tryptophan, which possess antioxidant activity, has been studied by the method of luminol-dependent chemiluminescence. The amount of antioxidant products was judged by the value of the total antioxidant potential of a UV-irradiated solution, the measure of which was the distance between the peaks of the chemiluminescence curve in the system 2,2′-azo-bis(2-amidinopropane) hydrochloride + luminol with a UV-irradiated and an unirradiated sample (induction period, τ i ). Simultaneously, the absorption and fluorescence spectra of unirradiared and UV-irradiated amino acid solutions were recorded. It was shown that exposure of a tryptophan solution to radiation led to accumulation of a fluorescent product N-formyl kynurenine (λem = 325 nm, λmax = 440 nm), and the curve of its accumulation was similar to the growth of antioxidant potential. When a tyrosine solution was irradiated, the main fluorescent product was dityrosine (λem = 310 nm, λmax = 415 nm). Nevertheless, the dose dependences of the formation of dityrosine and the total antioxidant potential were completely different. It was found that another product of tyrosine UV photolysis, dihydroxyphenylalanine, possessed pronounced antioxidant activity. It was concluded that the main antioxidant produced under UV irradiation of tryptophan is formyl kynurenine, and under irradiation of tyrosine it is dihydroxyphenylalanine.

Among the distinct molecular signatures present in the mitochondrion is the tetra-acylated anionic phospholipid cardiolipin, a lipid also present in primordial, single-cell bacterial ancestors of mitochondria and multiple bacterial species today. Cardiolipin is normally localized to the inner mitochondrial membrane; however, when cardiolipin becomes externalized to the surface of dysregulated mitochondria, it promotes inflammasome activation and stimulates the elimination of damaged or nonfunctional mitochondria by mitophagy. Given the immunogenicity of mitochondrial and bacterial membranes that are released during sterile and pathogen-induced trauma, we hypothesized that cardiolipins might function as “eat me” signals for professional phagocytes. In experiments with macrophage cell lines and primary macrophages, we found that membranes with mitochondrial or bacterial cardiolipins on their surface were engulfed through phagocytosis, which depended on the scavenger receptor CD36. Distinct from this process, the copresentation of cardiolipin with the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide dampened TLR4-stimulated production of cytokines. These data suggest that externalized, extracellular cardiolipins play a dual role in host-host and host-pathogen interactions by promoting phagocytosis and attenuating inflammatory immune responses.

Exposure to rotenone in vivo results in selective degeneration of dopaminergic neurons and development of neuropathological features of Parkinson's disease. As rotenone acts as an inhibitor of mitochondrial respiratory complex I, we employed oxidative lipidomics to assess oxidative metabolism of a mitochondria-specific phospholipid, cardiolipin, in substantia nigra of exposed animals. We found a significant reduction of oxidizable PUFA-containing cardiolipin molecular species. We further revealed increased contents of mono-oxygenated cardiolipin species at late stages of the exposure. Notably, linoleic acid in sn-1 position was the major oxidation substrate yielding its mono-hydroxy- and epoxy-derivatives whereas more readily “oxidizable” fatty acid residues (arachidonic, docosahexaenoic acids) – remained non-oxidized. Elevated levels of PUFA cardiolipins were detected in plasma of rats exposed to rotenone. Characterization of oxidatively modified cardiolipin molecular species in substantia nirga and detection of PUFA-containing cardiolipin species in plasma may contribute to better understanding of the Parkinson's disease pathogenesis and lead to the development of new biomarkers of mitochondrial dysfunction associated with this disease.

We propose a new approach to evaluation of oxidative stress based on kinetic chemiluminescence: measurement of antioxidant and pro-oxidant activities of the plasma. The study included 50 patients with type 2 diabetes mellitus receiving peroral hypoglycemic therapy. In addition to the above parameters, the levels of TBA-reactive products, inflammation markers, clotting parameters, and biochemical values were studied. The new method provides information on oxidative stress in patients with type 2 diabetes mellitus irrespective of the clinical and laboratory values. The use of this method in complex with the clinical, laboratory, and instrumental studies allows comprehensive evaluation of patient's status for the diagnosis and choice of therapy.

The accumulation of UV photolysis products of amino acids tyrosine and tryptophan, which possess an antioxidant activity, has been studied by the method of luminol-activated chemiluminescence. The amount of antioxidant products was judged by the value of the total antioxidant potential of a UV-irradiated solution, the measure of which was the distance between the peaks of the chemiluminescence curve in the system 2,2'-azo-bis(2-amidinopropane)hydrochloride + luminol in a UV-irradiated and an unirradiated samples (induction period, tau(i)). Simultaneously, the absorption and fluorescence spectra of unirradiared and UV-irradiated amino acid solutions were recorded. It was shown that, upon the exposure of a tryptophan solution to radiation, the accumulation of the fluorescent product N-formyl kynurenine (lambda(em) = 325 nm, lambda(max) = 440 nm) occures, and the curve of its accumulation was similar to the curve of growth of tau(i) photoproducts produced during UV-radiation. When a tyrosine solution was irradiated, the main fluorescent product was dityrosine (lambda(em) = 310 nm, lambda(max) = 415 nm). Nevertheless, the dose dependencies of the formation of dityrosine, and the total antioxidant potential (tau(i)) were completely different. It was found that another product of tyrosine UV-photolysis, dioxyphenylalanine, possessed a pronounced antioxidant activity. It was concluded that the main antioxidants produced under UV-irradiation of tryptophan is formyl kynurenine, and under the irradiation of tyrosine, dioxyphenylalanine.