1-Methylnicotinamide (MNA), a primary metabolite of nicotinamide, exerts anti-thrombotic activity mediated by a cyclooxygenase-2/prostacyclin pathway.

BACKGROUND AND PURPOSE: 1-methylnicotinamide (MNA) has been considered to be an inactive metabolite of nicotinamide. Here we assessed the anti-thrombotic activity of MNA in vivo. EXPERIMENTAL APPROACH: Antithrombotic action of MNA was studied in normotensive rats with extracorporeal thrombus formation (thrombolysis), in renovascular hypertensive rats with intraarterial thrombus formation (arterial thrombosis) and in a venous thrombosis model in rats (venous thrombosis). KEY RESULTS: MNA (3-100 mg kg(-1)) induced a dose-dependent and sustained thrombolytic response, associated with a rise in 6-keto-PGF(1alpha) in blood. Various compounds structurally related to MNA were either inactive or weaker thrombolytics. Rofecoxib (0.01-1 mg kg(-1)), dose-dependently inhibited the thrombolytic response of MNA, indomethacin (5 mg kg(-1)) abolished it, while L-NAME (5 mg kg(-1)) were without effect. MNA (3-30 mg kg(-1)) also reduced arterial thrombosis and this effect was abrogated by indomethacin (2.5 mg kg(-1)) as well as by rofecoxib (1 mg kg(-1)). MNA, however, did not affect venous thrombosis. In vitro MNA did not modify platelet aggregation nor induce vasodilation. CONCLUSIONS AND IMPLICATIONS: MNA displayed a profile of anti-thrombotic activity in vivo that surpasses that of closely related compounds. MNA inhibited platelet-dependent thrombosis by a mechanism involving cyclooxygenase-2 and prostacyclin. Our findings suggest that endogenous MNA, produced in the liver by nicotinamide N-methyltransferase, could be an endogenous activator of prostacyclin production and thus may regulate thrombotic as well as inflammatory processes in the cardiovascular system.

Note: Flow mediated skin fluorescence--A novel technique for evaluation of cutaneous microcirculation.

This note describes a newly developed technique for evaluation of cutaneous microcirculation. The technique called Flow Mediated Skin Fluorescence (FMSF) is based on monitoring of NADH fluorescence intensity emitted from the skin tissue cells of a forearm. The changes in fluorescence intensity as a function of time in response to blocking and releasing of blood flow in a forearm are used as a measure of oxygen transport with blood to the tissue, which directly correlates with the skin microcirculation status. Preliminary results collected for healthy volunteers and patients experiencing serious cardiovascular problems indicated a usefulness of FMSF technique for evaluation of health related perturbations in cutaneous microcirculation.

A pulse radiolysis investigation of the reactions of myeloperoxidase with superoxide and hydrogen peroxide.

Using pulse radiolysis, the rate constant for the reaction of ferric myeloperoxidase with O2- to give compound III was measured at pH 7.8, and values of 2.1.10(6) M-1.s-1 for equine ferric myeloperoxidase and 1.1.10(6) M-1.s-1 for human ferric myeloperoxidase were obtained. Under the same conditions, the rate constant for the reaction of human ferric myeloperoxidase with H2O2 to give compound I was 3.1.10(7) M-1.s-1. Our results indicate that although the reaction of ferric myeloperoxidase with O2- is an order of magnitude slower than with H2O2, the former reaction is sufficiently rapid to influence myeloperoxidase-dependent production of hypochlorous acid by stimulated neutrophils.

Radical initiated alpha-tocopherol depletion and lipid peroxidation in mitochondrial membranes.

The relationships between antioxidant status, lipid peroxidation and membrane protein integrity have been studied in an isolated mitochondrial membrane system. Tocopherol was shown to be present in both the outer and inner membrane of normal rat liver mitochondria; 77.3 and 22.3% of the total alpha-tocopherol was present in the outer and inner membranes, respectively. The endogenous alpha-tocopherol was depleted in a time-dependent manner by low levels of ferrous iron and by irradiation in the presence or absence of ferrous iron. This antioxidant depletion was followed by the appearance of lipid hydroperoxides. Fragmentation of monoamine oxidase, an integral outer membrane protein, was observed at irradiation doses that caused by antioxidant depletion and peroxide generation.

Radiation-induced lipid peroxidation and the fluidity of erythrocyte membrane lipids.

The effect of radiation-induced peroxidation on the fluidity of the phospholipids of the erythrocyte membrane was studied using both erythrocyte ghosts and liposomes formed from the polar lipids of erythrocytes. In liposomes, the oxidation of the phospholipids increased with radiation dose, but there was no change in the fluidity of the lipids as measured by spin-label motion. Under the same conditions of irradiation, no oxidation of phospholipid was detected in erythrocyte ghosts, although changes occurred in the motion of spin labels intercalated with the membrane. These changes were attributed to radiation-induced alterations in the membrane proteins. It is concluded that alterations in motion of spin labels, observed with intact membranes after irradiation, are most likely the result of changes in the structure of membrane proteins rather than the lipids.

The role of lipid peroxidation and antioxidants in oxidative modification of LDL.

The purpose of this study is to provide a comprehensive survey on the compositional properties of LDL (e.g., lipid classes, fatty acids, antioxidants) relevant for its susceptibility to oxidation, on the mechanism and kinetics of LDL oxidation, and on the chemical and physico-chemical properties of LDL oxidized by exposure to copper ions. Studies on the occurrence of oxidized LDL in plasma, arteries, and plaques of humans and experimental animals are discussed with particular focus on the use of poly- and monoclonal antibodies for immunochemical demonstration of apolipoprotein B modifications characteristic for lipid peroxidation. Apart from uptake of oxidized LDL by macrophages, studies describing biological effects of heavily or minimally oxidized LDL are only briefly addressed, since several reviews dealing with this subject were recently published. This article is concluded with a section on the role of natural and synthetic antioxidants in protecting LDL against oxidation, as well as some previously unpublished material from our laboratories.

Effect of amino acid peroxides on the erythrocyte.

The effect of amino acid peroxides, relatively stable products of irradiation of amino acid solutions, on erythrocyte components was studied. Interaction of proline, lysine, valine, and leucine peroxides (100-300 mu M) with erythrocyte membranes brought about a decrease of membrane protein -SH group content and of activities of (Na+, K+)-ATPase and Ca2+ -ATPase, and induced aggregation of membrane proteins, due mainly to the formation of interpeptide disulfides. Interaction of amino acid peroxides with hemoglobin brought about hemoglobin oxidation to methemoglobin. The effects of amino acid peroxides are similar to those of t-butyl hydroperoxide. These results indicate that peroxides of amino acid and proteins, which can also be formed under physiological conditions, may be mediators of the cellular action of reactive oxygen species.

Modification of horseradish peroxidase induced by hydroxyl radicals. The influence of oxygen.

Reactions of hydroxyl radicals with horseradish peroxidase (HRP) have been studied by means of pulse radiolysis technique in the absence and presence of oxygen. Hydroxyl radicals, produced in excess towards enzyme, react exclusively with the protein part of HRP with the rate constant k = 1.1 x 10(11) M-1 s-1. Activity loss induced by OH. is connected with such an enzyme modification that causes both the interference with substrate binding and partial blocking of the channel used by peroxide. It is shown that in the presence of oxygen the loss of activity is ca 10% higher, mainly due to restrictions in the formation of compound I, ie ferryl [Fe(IV) = O] pi-radical cation.

Vitamin E content and low density lipoprotein oxidizability induced by free radicals.

Human LDL, HDL and lipoprotein deficient plasma isolated from 15 normal subjects was exposed to oxygen free radicals generated by gamma rays and the formation of peroxides and changes in levels of LDL alpha-tocopherol were measured. LDL exhibited an initial resistance against oxidation stress when compared to HDL. The results obtained for different individuals showed that there was no correlation between the initial levels of vitamin E in LDL or plasma and the amount of peroxide formed after exposure of the LDL to a standard quantity of oxygen radicals. Kinetic experiments with original LDL and LDL containing incorporated alpha-tocopherol demonstrated that the vitamin performed its antioxidant role by conferring some early protection to the lipids, being consumed in the process, but it was clear that additional factors are also instrumental in determining the total antioxidant potential of the human LDL.

Increased oxidizability of plasma lipoproteins in diabetic patients can be decreased by probucol therapy and is not due to glycation.

Atherosclerosis is considered to be the major complication of diabetes mellitus. Since diabetic patients have increased blood levels of lipid peroxidation products we investigated whether the susceptibility of blood components to oxidation is altered in this disease. We analysed the parameters characterizing the extent of oxidative change and the antioxidant status of low density lipoprotein (LDL) and high density lipoprotein (HDL) in a group of diabetic patients and in a control population. LDL oxidizability was significantly higher for patients (P = 0.001) than for individuals in the control group. There were no significant differences in the alpha-tocopherol content or levels of performed peroxides in LDL samples isolated from diabetic patients and control individuals which could account for this effect. Similarly, LDL glycation, common in diabetes mellitus, was not responsible, since LDL glycated in vitro was more rather than less resistant to oxidation. Even the presence of unbound glucose at normal or elevated physiological concentrations had a delaying effect on the oxidation of LDL. The increased oxidizability of LDL isolated from diabetic patients could be reduced to control levels by a 6-week standard treatment with Probucol, originally applied to reduce their blood cholesterol.

Hydrogen peroxide modulation of the respiratory burst of human neutrophils.

Addition of micromolar concentrations of hydrogen peroxide (H2O2) to human neutrophils resulted in a dose-dependent luminol-enhanced chemiluminescent response. Pretreatment of neutrophils with micromolar concentrations of H2O2 altered their response to the surface acting stimulants serum-treated zymosan (STZ) and formyl-methionyl-leucyl-phenylalanine (fMLP), but not to the intracellular stimulant phorbol myristate acetate (PMA). The alterations were partially reversible by catalase, but exacerbated by superoxide dismutase. These results suggest a modulatory role for H2O2 in the respiratory burst of neutrophils.

The radical cation of anti-tricyclooctadiene and its rearrangement products

The anti dimer of cyclobutadiene (anti-tricyclo[4.2.0.0(2.5)]octa-3,7-diene, TOD) is subjected to ionization by gamma-irradiation in Freon matrices, pulse radiolysis in hydrocarbon matrices, and photoinduced electron transfer in solution. The resulting species are probed by optical and ESR spectroscopy (solid phase) as well as by CIDNP spectroscopy (solution). Thereby it is found that ionization of anti-TOD invariably leads to spontaneous decay to two products, that is bicyclo[4.2.0]octa-2,4,7-triene (BOT) and 1,4-dihydropentalene (1,4-DHP), whose relative yield strongly depends on the conditions of the experiment. Exploration of the C8H8*+ potential energy surface by the B3LYP/6-31G* density functional method leads to a mechanistic hypothesis for the observed rearrangements which involves a bifurcation between a pathway leading to the simple valence isomer, BOT*+, and another one leading to an unprecedented other valence isomer, the anti form of the bicyclo[3.3.0]octa-2,6-diene-4,8-diyl radical cation (anti-BOD*+). The latter product undergoes a very facile H-shift to yield the radical cation of 1,3a-dihydropentalene (1,3a-DHP*+) which ultimately rearrranges by a further H-shift to the observed product, 1,4-DHP*+.

The radical cation of syn-tricyclooctadiene and its rearrangement products

The syn dimer of cyclobutadiene (tricyclo[4.2.0.0(2.5)]octa-3,7-diene, TOD) is subjected to ionization under different conditions and the resulting species are probed by optical and ESR spectroscopy. By means of quantum chemical modelling of the potential energy surfaces and the optical spectra, it is possible to assign the different products that arise spontaneously after ionization or after subsequent warming or illumination of the samples. Based on these findings, we propose a mechanistic scheme which involves a partitioning of the incipient radical cation of TOD between two electronic states. These two states engage in (near) activation-less decay to the more stable valence isomers, cyclooctatetraene (COT*+) and a bis-cyclobutenylium radical cation BCB*+. The latter product undergoes further rearrangement, first to tetracyclo[4.2.0.0(2,4).0(3,5]oct-7-ene (TCO*+) and eventually to bicyclo[4.2.0]octa-2,4,7-triene (BOT*+) which can also be generated photochemically from BCB*+ or TCO*+. The surprising departure of syn-TOD*+ from the least-motion reaction path leading to BOT*+ can be traced to strong vibronic interactions (second-order Jahn-Teller effects) which prevail in both possible ground states of syn-TOD*+. Such effects seem to be more important in determining the intramolecular reactivity of radical cations than orbital or state symmetry rules.

Scavenging of oxygen radicals by heme peroxidases.

The reactions of two heme peroxidases, horseradish peroxidase and lactoperoxidase and their compounds II (oxoferryl heme intermediates, Fe(IV) = O or ferric protein radical Fe(III)R.) and compounds III (resonance hybrids [Fe(III)-O2-.<--> Fe(II)-O2] with superoxide radical anion generated enzymatically or radiolytically, and with hydroxyl radicals generated radiolytically, were investigated. It is suggested that only the protein radical form of compound II of lactoperoxidase reacts with superoxide, whereas compound II of horseradish peroxidase, which exists only in oxoferryl form, is unreactive towards superoxide. Compound III of the investigated peroxidases does not react with superoxide. The lactoperoxidase activity loss induced by hydroxyl radicals is closely related to the loss of the ability to form compound I (oxoferryl porphyrin pi-cation radical, Fe(IV) = O(Por+.) or oxoferryl protein radical Fe(IV) = O(R.)). On the other hand, the modification of horseradish peroxidase induced by hydroxyl radicals has been reported to cause also restrictions in substrate binding (Gebicka, L. & Gebicki, J.L., 1996, Biochimie 78, 62-65). Nevertheless, it has been found that only a small fraction of hydroxyl radicals generated homogeneously does inactivate the enzymes.

Decomposition of lipid hydroperoxides enhances the uptake of low density lipoprotein by macrophages.

This study examined the roles of low-density lipoprotein (LDL) lipid oxidation and peroxide breakdown in its conversion to a form rapidly taken up by mouse peritoneal macrophages. Oxidation of the LDL without decomposition of the hydroperoxide groups was performed by exposure to gamma radiation in air-saturated solutions. Virtually complete decomposition of the hydroperoxides was achieved by treatment of the irradiated LDL with Cu2+ under strictly anaerobic conditions. No uncontrolled LDL uptake by macrophages occurred when the lipoprotein contained less than 150 hydroperoxide groups per particle. More extensively oxidized LDL was taken up and degraded by mouse macrophages significantly faster than the native lipoprotein. The uptake was greatly enhanced by treatment of the oxidized LDL with Cu2+. A significant proportion of the LDL containing intact or copper-decomposed LDL hydroperoxide groups accumulated within the macrophages without further degradation. Treatment of the radiation-oxidized LDL with Cu2+ was accompanied by aggregation of the particles. Competition studies showed that the oxidized LDL was taken up by macrophages via both the LDL and the scavenger receptors, whereas the copper-treated lipoprotein entered the cells only by the scavenger pathway. Phagocytosis also played an important role in the metabolism of all forms of the extensively modified LDL. Our results suggest that minimally-oxidized LDL is not recognized by the macrophage scavenger receptors unless the lipid hydroperoxide groups are decomposed to products able to derivatize the apo B protein.

Peroxidation of proteins and lipids in suspensions of liposomes, in blood serum, and in mouse myeloma cells.

There is growing evidence that proteins are early targets of reactive oxygen species, and that the altered proteins can in turn damage other biomolecules. In this study, we measured the effects of proteins on the oxidation of liposome phospholipid membranes, and the formation of protein hydroperoxides in serum and in cultured cells exposed to radiation-generated hydroxyl free radicals. Lysozyme, which did not affect liposome stability, gave 50% protection when present at 0.3 mg/ml, and virtually completely prevented lipid oxidation at 10 mg/ml. When human blood serum was irradiated, lipids were oxidized only after the destruction of ascorbate. In contrast, peroxidation of proteins proceeded immediately. Protein hydroperoxides were also generated without a lag period in hybrid mouse myeloma cells, while at the same time no lipid peroxides formed. These results are consistent with the theory that, under physiological conditions, lipid membranes are likely to be effectively protected from randomly-generated hydroxyl radicals by proteins, and that protein peroxyl radicals and hydroperoxides may constitute an important hazard to biological systems under oxidative stress.

Preparation and properties of vesicles enclosed by fatty acid membranes.

Stable preparations of microscopic particles were obtained from long-chain fatty acids by mechanical agitation of evaporated films in presence of buffer solutions. Oleic and linoleic acids were used. Studies of osmotic swelling and shrinking of the particles indicated that they are enclosed by semipermeable membranes. The particles, which were named ufasomes, are also capable of entrapping glucose in spaces inaccessible to enzymes. It was concluded that the ufasomes closely resemble phospholipid liposomes in their structure and properties.

Protein hydroperoxides as new reactive oxygen species.

There is convincing evidence for the involvement of Reactive Oxygen Species (ROS) in the initiation and development of various forms of damage in living organisms. Attempts to prevent or limit such damage have been largely unsuccessful, principally because most of the pathways linking the formation of ROS with the end-point pathology are unknown. Evidence summarized in this review suggests that proteins are the most likely initial targets of ROS in cells and that protein hydroperoxides are major products of this interaction. Recent research has shown that the protein hydroperoxides can in turn generate new free radicals, inactivate enzymes, destroy antioxidants, and crosslink with DNA. This suggests that protein hydroperoxides may constitute an important intermediate stage in the development of ROS-induced biological damage, and that they should therefore be regarded as a new form of reactive oxygen species.

Reactions of hydrated electrons with horseradish peroxidase: a pulse radiolysis study.

Kinetics of the reactions of hydrated electrons with horseradish peroxidase (HRP) have been studied by pulse radiolysis. Hydrated electrons reduce HRP to its ferrous form with a yield of 40%. At higher dose (250 Gy) the formation of compound I followed by its reduction to compound II was also observed.

Continuous measurement of oxygen consumption by linoleic acid membranes exposed to free radicals generated by gamma-radiation.

Vesicles enclosed by membranes prepared from linoleic acid were exposed in the chamber of an oxygen electrode to free radicals generated by 60Co gamma-rays. Oxidation was observed by oxygen consumption, conjugated diene formation, and tri-iodide assay for hydroperoxides. There was a dose-dependent lag period before the onset of rapid peroxidation. The radiation chemical yields (G-values) ranged from 4.45 to 19.37 mu-mol J-1 for maximum rates of oxygen consumption and from 2.18 to 16.37 mumol J-1 for maximum rates of hydroperoxide production when the radiation dose-rate was varied between 5.39 and 0.14 Gy min-1. The magnitudes of these G-values and the linear relationship between yield of hydroperoxide and (dose-rate)1/2 were indicative of a chain mechanism for peroxidation operating in membranes. The lack of congruence between the amount of oxygen consumed and hydroperoxide formed suggested that the oxygen consumed in membrane oxidation led to the formation of oxidized derivatives of linoleic acid additional to the hydroperoxides.