Yu. A. Vladimirov

Photobiological Principles of Therapeutic Applications of Laser Radiation

Laser therapy based on the stimulating and healing action of light of low-intensity lasers (LIL), along with laser surgery and photodynamic therapy, has been lately widely applied in the irradiation of human tissues in the absence of exogenous photosensitizers. Besides LIL, light-emitting diodes are used in phototherapy (photobiostimulation) whose action, like that of LIL, depends on the radiation wavelength, dose, and distribution of light intensity in time but, according to all available data, does not depend on the coherence of radiation.

Free radicals and cell chemiluminescence

Application of chemiluminescence (CL) for study of free-radical reactions in human and animal cells and tissues is considered in this review. Historically, the study of intrinsic (ultraweak) luminescence gave place to the measurement of CL in the presence of chemical activators (CL probes) and physical activators (sensitizers) of luminescence, which made the method much more sensitive and specific. The methods of CL and EPR are direct methods of radical investigation, though the advantage of the CL method consists in the fact that CL intensity is directly proportional to a steady-state concentration of the radicals responsible for luminescence (first of all, lipid and oxygen radicals) irrespective the activity of these radicals. The mechanisms of CL reactions in the absence of activators and in the presence of luminol and lucigenin are considered. Examples of various applications of the CL method in medical, biological, and clinical investigations are given including those for estimation of the phagocytic activity of cells, antioxidant activity, determination of toxicity, and other purposes.

THE ACTION OF HYPOCHLOROUS ACID ON PHOSPHATIDYLCHOLINE LIPOSOMES IN DEPENDENCE ON THE CONTENT OF DOUBLE BONDS. STOICHIOMETRY AND NMR ANALYSIS

Kinetics of the consumption of hypochlorous acid in its reaction with double bonds of unsaturated phospholipids and fatty acids were measured using luminol chemiluminescence. Stoichiometry ratios between the consumption of HOCl/OCl- and the loss of double bonds vary from 2:1 to 1:1. Highest values were found in DMPC liposomes containing 5 mol% oleic acid or OPPC. With increasing content of double bonds or higher numbers of double bonds in a fatty acid acyl chain due to incorporated unsaturated fatty acids or phospholipids in DMPC liposomes the stoichiometry ratio falls continuously to 1:1. A ratio of about 1:1 was observed in multilamellar and unilamellar liposomes composed of egg yolk phosphatidylcholine. Products of the reaction of oleic acid with hypochlorous acid were analyses by 1H-NMR spectroscopy. Chlorohydrins were formed in both DMPC liposomes containing 5 or 40 mol% oleic acid.

Cardiolipin activates cytochrome c peroxidase activity since it facilitates H2O2 access to heme

In this work, the effect of liposomes consisting of tetraoleyl cardiolipin and dioleyl phosphatidylcholine (1: 1, mol/mol) on the rate of three more reactions of Cyt c heme with H2O2 was studied: (i) Cyt c (Fe2+) oxidation to Cyt c (Fe3+), (ii) Fe···S(Met80) bond breaking, and (iii) heme porphyrin ring decomposition. It was revealed that the rates of all those reactions increased greatly in the presence of liposomes containing cardiolipin and not of those consisting of only phosphatidylcholine, and approximately to the same extent as peroxidase activity. These data suggest that cardiolipin activates specifically Cyt c peroxidase activity not only because it promotes Fe···S(Met80) bond breaking but also facilitates H2O2 penetration to the reaction center.

Dihydroquercetin (taxifolin) and other flavonoids as inhibitors of free radical formation at key stages of apoptosis.

Formation of free radicals in mitochondria plays a key role in the development of apoptosis, which includes formation of superoxide by the respiratory chain, formation of radicals by cytochrome c-cardiolipin complex in the presence of hydrogen peroxide or lipids, and chain lipid peroxidation resulting in cytochrome c release from mitochondria and initiation of the apoptotic cascade. In this work the effect of taxifolin (dihydroquercetin) and some other antioxidants on these three radical-producing reactions was studied. Peroxidase activity of the complex of cytochrome c with dioleyl cardiolipin estimated by chemiluminescence with luminol decreased by 50% with quercetin, taxifolin, rutin, Trolox, and ionol at concentrations 0.7, 0.7, 0.8, 3, and 10 μM, respectively. The lipid radical production detected by coumarin C-525-activated chemiluminescence decreased under the action of rutin and taxifolin in a dose-dependent manner, so that a 50% inhibition of chemiluminescence was observed at the antioxidant concentrations of 3.7 and 10 μM, respectively. Thus, these two radical-producing reactions responsible for apoptosis onset are inhibited by antioxidants at rather low concentrations. Experiments performed on liver slices and mash showed that taxifolin, quercetin, naringenin, and Trolox have low inhibitory effect on the lucigenin-dependent chemiluminescence in the tissue only at concentrations higher than 100 μM.

Mechanism of activation of cytochrome c peroxidase activity by cardiolipin

In this work, the actions of bovine heart cardiolipin, synthetic tetraoleyl cardiolipin, and a nonspecific anionic detergent sodium dodecyl sulfate (SDS) on cytochrome c (Cyt c) peroxidase activity recorded by chemiluminescence in the presence of luminol and on the Fe···S(Met80) bond whose presence was estimated by a weak absorption band amplitude with peak at 695–700 nm (A695) were compared. A strict concurrency between Fe···S(Met80) breaking (A695) and cytochrome peroxidase activity enhancement was shown to exist at cardiolipin/Cyt c and SDS/Cyt c molar ratios of 0:1 to 50:1 (by chemiluminescence). Nevertheless, when A695 completely disappeared, Cyt c peroxidase activity under the action of cardiolipin was 20 times more than that under the action of SDS, and at low ligand/protein molar ratios (≤4), SDS failed to activate peroxidase activity while cardiolipin enhanced Cyt c peroxidase activity 16–20-fold. A695 did not change on Cyt c binding with liposomes consisting of tetraoleyl cardiolipin and phosphatidylcholine (1:10:10), while peroxidase activity was enhanced by a factor of 8. Breaking of 70% of the Fe···S(Met80) bonds resulted in only threefold enhancement of peroxidase activity. Cardiolipin-activated Cyt c peroxidase activity was reduced by high ionic strength solution (1 M KCl). The aggregated data suggest that cardiolipin activating action is caused, first, by a nonspecific effect of Fe···S(Met80) breaking as the result of conformational changes in the prote in globule caused by the protein surface electrostatic recharging by an anionic amphiphilic molecule, and second, by a specific acceleration of the peroxidation reaction which is most likely due to enhanced heme accessibility for H2O2 as a result of the hydrophobic interaction between cardiolipin and cytochrome.

Activation of lipid peroxidation in liver mitochondria of hyperthyroid rabbits

If thyroxine is added to medium containing liver mitochondria or homogenates it inhibits lipid peroxidation (LPO) in these systems [2, 5, 6, 9, I0]. Since the doses of the hormone used in these systems were close to physiological (10-6-10 -7 M) and since the antioxidant activity of the hormone was comparable with the ability of ~-tocopherol to slow the rate of LPO processes [2, I0], it was suggested that thyroxine may have a specific function as a natural antioxidant [6]. It is not known, however, whether the fall in the thyroid hormone level leads to activation of LPO reactions or, conversely, to their inhibition at the expense of lipid metabolism. It has been shown that the concentration of a-tocopherol in the serum and liver rises in hyperthyroidism [12]. Meanwhile liver homogenates from hyperthyroid animals in the course of incubation accumulate products reacting with thiobarbituric acid with higher velocities than samples prepared from the liver of normal and hypothyroid animals [13]. Acceleration of LPO reactions in fragments of the sarcop!asmic reticulum in hyperthyroid rabbits also was observed by the present writers previously [3]. Direct addition of thyroxine or other derivatives of the thyroid gland, moreover, is not always accompanied by inhibition of LPO and, on the contrary, it may actually activate LPO, as has been shown in the case of isolated erythrocytes [Ii]. Relations between thyroid hormones and LPO are further complicated by the fact that deiodination of the hormone, considered to be essential for the realization of its biological activity, is increased when the diet is deficient in vitamin E [8], whereas elevation of the bodys thyroid levels leads to an increase in the concentration of this vitamin in the serum and liver.

Biological activity of hemoprotein nitrosyl complexes

Chemical and biological functions of hemoprotein nitrosyl complexes as well as their photolysis products are discussed in this review. Chemical properties of nitric oxide are discussed, and major chemical reactions such as interaction with thiols, free radicals, and transition metals are considered. Specific attention is paid to the generation of hemoprotein nitrosyl complexes. The mechanisms of nitric oxide reactions with hemoglobin and cytochrome c and physicochemical properties of their nitrosyl complexes are discussed. A review of photochemical reactions of nitrosyl complexes with various ligands is given. Finally, we observe physiological effects of visible radiation on hemoprotein nitrosyl complexes: smooth muscle relaxation and reactivation of mitochondrial respiration.

Mechanism of therapeutic effect of low-intensity infrared laser radiation.

The effect of infrared low-intensity laser irradiation on functional activity of blood polymorphonuclear leukocytes was studied in vitro. A dose-dependent priming of polymorphonuclear leukocytes induced by infrared low-intensity laser irradiation was demonstrated. Similar effects were also observed in the presence of the photosensitizer photosense.

Evidence for a direct interaction of superoxide anion radical with carnosine

Using photochemiluminescence, the interaction between carnosine and superoxide anion was measured directly. Carnosine at physiological concentrations decreased the amplitude of luminol chemiluminescence like superoxide dismutase (SOD) did, and prolonged the lag‐period of the chemiluminescence similar to the effect of ascorbic acid. From the interaction of nitro blue tetrazolium with superoxide anion generated by the xanthine oxidase system, the constant for interaction of carnosine with O2* was calculated to be 105 M‐1. sec‐1. The possible biological significance of the quenching of superoxide anion by carnosine is discussed.