Muscle dipeptides--natural inhibitors of lipid peroxidation

The effects of carnosine (beta-alanyl-L-histidine) and anserine (beta-alanyl-1-methyl-L-histidine) on ascorbate-dependent lipid peroxidation in frog skeletal muscle sarcoplasmic reticulum were studied. It was found that the dipeptides (10-50 mM) cause a 25-90% inhibition of ascorbate-dependent lipid peroxidation and decrease the reaction rate and the amount of end products. The nature of lipid peroxidation primary products in the presence of the dipeptides changes which can be evidenced from changes in their spectral properties. Unlike other known natural antioxidants, skeletal muscle dipeptides do not only inhibit lipid peroxidation but also decrease the level of accumulated lipid peroxidation products. Histidine and beta-alanine, similar to imidazole, glycyl-glycine, arginyl-phenyl alanine and alpha-alanyl-D-histidine do not inhibit lipid peroxidation. At the same time, the carnosine stereoisomer D-carnosine which does not exist in nature exhibits a far greater inhibiting effect as compared to its natural counterpart. It is assumed that the skeletal muscle dipeptides carnosine and anserine are highly effective as natural antioxidants.     

Formation of alpha-tocopherol radical and recycling of alpha-tocopherol by ascorbate during peroxidation of phosphatidylcholine liposomes. An electron paramagnetic resonance study

The events accompanying the inhibitory effect of alpha-tocopherol and/or ascorbate on the peroxidation of soybean L-alpha-phosphatidylcholine liposomes, which are an accepted model of biological membranes, were investigated by electron paramagnetic resonance, optical and polarographic methods. The presence of alpha-tocopherol radical in the concentration range 10(-8)-10(-7) M was detected from its EPR spectrum during the peroxidation of liposomes, catalysed by the Fe3+-triethylenetatramine complex. The alpha-tocopherol radical, generated in the phosphatidylcholine bilayer, is accessible to ascorbic acid, present in the aqueous phase at physiological concentrations. Ascorbic acid regenerates from it the alpha-tocopherol itself. A kinetic rate constant of about 2 X 10(5) M-1 X s-1 was estimated from the reaction as it occurs under the adopted experimental conditions. The scavenging effect of alpha-tocopherol on lipid peroxidation is maintained as long a ascorbic acid is present.     

Ganglioside-dependent factor inhibiting lipid peroxidation in synaptosomal membranes

The inhibitory effect of exogenous monosialoganglioside GM1 on lipid peroxidation was studied in synaptosomal membranes from rat brain. When this effect was studied over a wide GM1 concentration range, the biphasic kinetics was observed, the highest per cent of inhibition (70%) was found at GM1 concentration of 10(-9)- 10(-8) M. In liposomes made from lipids isolated from rat synaptosomal membranes the inhibition of lipid peroxidation by exogenous GM1 was much less pronounced (25% at maximum) it reached the saturation at ganglioside concentration of 10(-8)-10(-6) M. The thermal denaturation (90 degrees C), storage at 0 degrees C, addition of polymyxin B result in considerable decrease of inhibitory effect of GM1 on lipid peroxidation in synaptosomal membranes. On the contrary phorbol-12-myristate-13-acetate (10(-6)M) or Ca2+ (2.10(-3)M) inhibit lipid peroxidation in synaptosomal membranes, the presence of exogenous GM1 in incubation medium having additional inhibitory effect. Possible mechanisms of ganglioside participation in regulation of functional activity of excitatory membranes are discussed.     

Lipoxygenase-induced lipid peroxidation of isolated cardiac microsomes modulates their calcium-transporting function

We demonstrated previously that products of linoleic and arachidonic acids, arising from enzymatic or non-enzymatic oxidation, inhibit ATP-dependent calcium accumulation into and promote release of calcium from vesicles derived from sarcoplasmic reticulum of guinea-pig heart. In the present study, direct enzymatic peroxidation of cardiac membrane lipids was performed and the effect on calcium transport was examined. Vesicles were preincubated at 37 degrees C with soybean lipoxygenase-1 (linoleate:oxygen oxidoreductase, EC 1.13.11.12) for up to 1 h prior to the initiation of calcium accumulation. The extent of membrane peroxidation was assessed by monitoring the production of malondialdehyde. Pretreatment of vesicles with lipoxygenase for 40 and 60 min markedly depressed calcium accumulation. The lipoxygenase-induced suppression of calcium transport was completely antagonized by nordihydroguaiaretic acid (1 microM), not at all by indomethacin (1 microM), and only partially by 5,8,11,14-eicosatetraynoic acid (0.3 microM). Low concentrations of calcium (10(-5)-5 X 10(-5) M) enhanced, and a high concentration (10(-3) M) inhibited lipoxygenase-induced peroxidation of membrane lipids. The calcium-accumulating ability of the vesicles was inversely related to the extent of membrane peroxidation. The vesicles which showed the highest degree of peroxidation in the presence of 5 X 10(-5) M calcium, accumulated the lowest amount of calcium. In contrast, calcium at 10(-3) M suppressed lipid peroxidation, resulting in higher calcium uptake than in vesicles peroxidized in the absence of calcium. Thus, calcium transport is depressed in microsomes undergoing lipoxygenase-induced peroxidation, a process which in turn is modulated by calcium.     

The characteristics of the effect of dipalmitoylphosphatidylcholine and its structural fragments on the lipid peroxidation of biological membranes]

The effect of dipalmitoyl phosphatidylcholine (DPPC) and its structural fragments--phosphatidic acid (PA) and choline chloride--on the ascorbate-dependent mice liver microsomal lipid peroxidation (LP) has been studied at the different LP rate. At DPPC, PA and choline chloride introduction into the incubation medium before the onset of peroxidation induction DPPC causes the more considerable inhibition of LP than PA each separate fragment. The inhibition effect of DPPC is lower than PA or choline chloride when DPPC, PA and choline chloride added on the background of developing process of peroxidation (e.i. after LP induction). A specific regulatory role of PC in the normal cell membrane LP process and during pathologic development is under discussion.     

Protection of liposomal lipids against radiation induced oxidative damage.

Liposomes were prepared from phospholipids extracted from biological membranes. A comparison was made between the peroxidation rate in handshake liposomes and in sonicated liposomes. The smaller sonicated liposomes were more vulnerable to peroxidation, probably because of the smaller radius of curvature, which results in a less dense packing of lipid molecules in the bilayer and a facilitated action of water radicals produced by the X-irradiation. High oxygen enhancement ratios were obtained, especially at low dose rates, suggesting the operation of slowly progressing chain reactions initiated by ionizing radiation. Three compounds were tested for their ability to protect the liposomal membranes against lipid peroxidation. The naturally occurring compounds reduced glutathione (GSH) and vitamin E(alpha-T) and the powerful radiation protector cysteamine (MEA). All three molecules could protect the liposomes against peroxidation. The membrane-soluble compound vitamin E was by far the most powerful. About 50 per cent protection was achieved by using 5 X 10(-6) M alpha-T, 10(-4) M GSH and 5 X 10(-4) M MEA. The fatty acid composition of the lipids altered drastically as a result of the irradiation. Arachidonic acid and docosahexanoic acid were the most vulnerable of the fatty acids. Very efficient protection of these polyunsaturated fatty acids could be obtained with relatively low concentrations of vitamin E built into the membranes.     

N-acyl dehydroalanines scavenge oxygen radicals and inhibit in vitro free radical mediated processes

N-substituted dehydroalanines react with and scavenge oxygen radicals. One of those compounds, the para-methoxyphenylacetyl dehydroalanine derivative, indexed as AD-5, inhibits the reduction of ferricytochrome c by superoxide anion (O2-.). It can also inhibit the oxidation of linolenic acid, another chemical process, which is mediated by hydroxyl radical (HO.). Furthermore, microsomal lipid peroxidation induced by iron salts was also inhibited by AD 5, but with a different degree of efficacy. In fact, lipid peroxidation initiated by a ferrous-oxygen complex (as in iron/NADPH-dependent peroxidation) was inhibited by AD 5 in a range of concentration of 2-4 mM. On the contrary, iron/NADPH-independent lipid peroxidation, where alkoxy radicals (RO.) have principally been involved, was inhibited in a range of concentration of 6-10 mM. The ESR studies by using the spin trapping agent DMPO, show that AD-5 reacts with HO. with a second order constant of 2.8 X 10(9)-4.5 X 10(9) M-1 s-1.     

Arsenite-induced lipid peroxidation in Saccharomyces cerevisiae

The ability of sodium arsenite at concentrations of 10(-2), 10(-4), and 10(-6) M to induce lipid peroxidation in Saccharomyces cerevisiae cells was studied. Arsenite at the concentrations 10(-2) and 10(-4) M enhanced lipid peroxidation and inhibited the growth of yeast cells. Enhanced lipid peroxidation likely induced oxidative damage to various cellular structures, which led to suppression of the metabolic activity of cells. Arsenite at the concentration 10(-6) M did not activate lipid peroxidation in cells. All of the tested arsenite concentrations inhibited the activity of alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase in cells. The inference is made that the toxicity of arsenite may be related to its stimulating effect on intracellular lipid peroxidation.     

Inhibition of liver microsomal lipid peroxidation by 13-cis-retinoic acid

The effects of 13-cis-retinoic acid on iron/ascorbate-dependent lipid peroxidation were investigated with rat liver microsomes. 13-cis-retinoic acid effectively inhibited malondialdehyde generation and molecular oxygen consumption associated with lipid peroxidation. Under the conditions employed, inhibition was complete at concentrations as low as 25 microM and the IC50 was 10 microM. Evidence for concomitant retinoid oxidation by microsomal unsaturated fatty acid-derived peroxyl radicals was demonstrated by detection of several retinoid-derived metabolites, including 5,8-oxy-13-cis-retinoic acid, generated during lipid peroxidation. The data indicate that 13-cis-retinoic acid inhibits lipid peroxidation by scavenging lipid peroxyl radicals with its conjugated polyene system. Its antioxidant properties may contribute to the pharmacological activities of this and related retinoids.     

Effect of lecithin on liver microsomal lipid peroxidation]

The effect of exogeneous (egg) lecithin on peroxidation of microsomal lipids was studied with the view of elucidating the role of various components of lipid substrate in the overall oxidation rate of the lipids. The following processes were studied a) NADPH-dependent microsomal lipid peroxidation in the presence of lecithin; b) ascorbate-dependent microsomal lipid peroxidation in the presence of lecithin; c) oxidation of lipid mixture, isolated from the microsomes, and that of lecithin in the presence of the Fe2+ + ascorbate system; 4) oxidation of lecithin induced by the Fe2+ + ascorbate system. It was found that in the presence of exogeneous lecithin the oxidation of microsomal lipids in inhibited, which is probably due to the peculiarities of lecithin oxidation. It was shown that the specific rate of lecithin oxidation is decreased with an increase in lecithin concentration. Possible mechanisms of lecithin effect on microsomal lipid peroxidation are discussed.     

Efficient scavenging of hydroxyl radicals and inhibition of lipid peroxidation by novel analogues of 1,3,7-trimethyluric acid.

New water-soluble analogues of 1,3,7-trimethyluric acid with N-1 methyl replaced by various groups were prepared and evaluated for their ability to scavenge hydroxyl radicals as well as their protective potential against lipid peroxidation in erythrocyte membranes. The deoxyribose degradation method indicates that all the analogues tested effectively scavenge hydroxyl radicals and some of them show better activity than uric acid and methyluric acids. These effects are shown to be concentration dependent and are more potent at low concentrations (10-50 microM). Among the analogues tested, 1-butenyl-, 1-propargyl- and 1-benzyl-3,7-dimethyluric acids show high hydroxyl radical scavenging property with a reaction rate constant (Ks) of 3.2-6.7 x 10(10) M(-1) S(-1), 2.3-3.7 x 10(10) M(-1) S(-1) and 2.4-3.7 x 10(10) M(-1) S(-1), respectively. The effectiveness of these analogues as hydroxyl radical scavengers appears to be better than mannitol (Ks, 1.9-2.5 x 10(9) M(-1) S(-1)). With the exception of 1-pentyl- and 1-(2'-oxopropyl)-3,7-dimethyluric acids, all other analogues tested are effective inhibitors of tert-butylhydroperoxide-induced lipid peroxidation in human erythrocyte membranes. All the analogues tested are susceptible to peroxidation in the presence of hemoprotein and hydrogen peroxide. The present study has pointed out that it is possible to significantly enhance the antioxidant property of 1,3,7-trimethyluric acid by structural modification at N-1 position. Such compounds may be useful as antioxidants in vivo.     

Liver lipid peroxidation in the ontogeny of rats during perinatal exposure to polychlorinated biphenyls

We studied the activity of NADPH-cytochrome P-450 reductase, NADPH- and ascorbate-dependent systems of lipid peroxidation in liver microsomes, the activity of superoxide dismutase in the supernatant and the level of malonic acid dialdehyde in liver tissue of rats of various age. The activity of lipid peroxidation system and the malonic dialdehyde content in the early postnatal period increased to the adult level. The NADPH-cytochrome P-450 reductase activity increased during the first four months of animals life while that of superoxide dismutase increased until the animals were seven months old. A single administration of polychlorinated diphenyls at a dose of 500 mg/kg (1/10 LD50) to pregnant rats drastically stimulated and changed the pattern of the studied activities in their offspring. The role of lipid peroxidation in modification of microsomal membranes after the monooxygenase system induction by polychlorinated diphenyls in early ontogenesis is discussed.     

Modifications of cellular lipids induce insulin resistance in cultured hepatoma cells

We altered the cellular lipid composition of an insulin sensitive rat hepatoma cell line through supplementation of the culture medium with linoleic acid (18:2) or 25-hydroxycholesterol, and we studied the effects on insulin stimulation of aminoacid transport system A and glycogen synthesis. The basal rate of sodium-dependent aminoisobutyric acid uptake was slightly reduced in hydroxysterol-treated cells and increased in 18:2-enriched cells. Maximal insulin stimulation of transport was decreased by about 40% in both 18:2 and 25-hydroxycholesterol modified cells, as compared to control cells. In addition to reduced responsiveness, the hydroxysterol-treated cells also showed a diminished sensitivity to insulin, as revealed by a right-shift of the dose-response curve leading to a ED50 of 1.2 X 10(-8) M (P less than 0.02), as compared to 2.45 X 10(-9) M in control cells and 2.13 X 10(-9) M in 18:2 enriched cells. Concerning glycogen synthesis, the basal rate was unaffected by 25-hydroxycholesterol supplementation and slightly reduced in cells enriched in 18:2. Maximal insulin stimulation of glycogen synthesis was reduced by about 40% in both types of lipid modified cells. 25-Hydroxycholesterol again provoked a decrease in sensitivity to insulin: the ED50 was enhanced to 4.9 X 10(-9) M (P less than 0.05), as compared to 1.25 X 10(-9) M in control cells and 1.57 X 10(-9) M in 18:2-supplemented cells. Taken together with the previously reported changes of insulin binding to lipid modified hepatoma cells (Bruneau et al. (1987) Biochim. Biophys. Acta 928, 287-296) our results demonstrate an influence of alterations of the cellular lipid composition on both binding and biological actions of insulin, leading to an insulin-resistant state. Divergences between insulin binding and action were obtained and it was suggested that post-binding events may be responsible for the observed changes. Our findings may be relevant to experimental and clinical states of insulin resistance.     

Retinoic acid blocks potassium channels in human lymphocytes

Using the whole-cell variation of the patch-clamp technique, we have determined that retinoic acid, an active metabolite of natural vitamin A that possesses potent immunomodulating activity, reduces the K+ current in human T lymphocytes and natural killer cells in a dose-dependent manner: acute treatment with 5 X 10(-5) M caused over a 70% reduction while concentrations less than 1 X 10(-5) M caused less than 30% inhibition. Natural killer activity and T cell mitogenesis was inhibited by RA at concentrations that reduced the K+ conductance and correlated with the ability of a variety of classical ion-channel blockers to inhibit the functional activity of these cells. Thus, the reported inhibitory effects on natural killer activity and T cell mitogenesis by high concentrations of retinoic acid can be explained by its effect on the K channel.     

Inhibition of Lipid Peroxidation by Dihydroquinoline-Type Antioxidant (CH 402)

The in vitro effect of a non-toxic, water soluble, low molecular weight, stable dihydroquinoline-type antioxidant, CH 402 (Na (2,2-dimethyl-1,2-dihydroquinoline-4-yl) –- methane sulphonic acid) was studied on free radical reactions in brain subcellular fractions. Experiments were performed using rat and mouse brain homogenate and microsomal fractions. Non … enzymatically induced lipid peroxidation by ascorbic acid was studied in correlation with ascorbic acid and CH 402 concentrations and incubation time. Malondialdehyde production during lipid peroxidation was measured by the thiobarbituric acid test. In a concentration range of 10^?2–10^?5 M CH 402 dose - dependently inhibited the ascorbic acid induced in vitro lipid peroxidation in mouse and rat brain subcellular fractions.     

Importance of two iron-reducing systems in lipid peroxidation of rat brain: implications for oxygen toxicity in the central nervous system

The mechanism of lipid peroxidation in the central nervous system has been studied using oxygen-flushed rat brain homogenates at pH 7.4. Brain lipid peroxidation was monitored by chemiluminescence and by determination of thiobarbituric acid-reactive substances. Less involvement of O2-., H2O2 and probably .OH in the initiation of lipid peroxidation was indicated. Deferroxamine was an extremely potent inhibitor of lipid peroxidation, suggesting that lipid peroxidation was catalyzed by endogenous iron. Brain tissues were shown to contain at least two iron-reducing systems for promotion of lipid peroxidation. One was ascorbate-dependent and the other NADPH-dependent. The former was much more potent than the latter with respect to iron-reducing activity.     

Modification of the fatty acid composition of cultured human fibroblasts.

The fatty acid composition of human skin fibroblasts grown in 10% dialyzed fetal calf serum can be modified considerably by adding supplemental fatty acids to the culture medium. The degree of modification was dependent on the concentration of added fatty acid over the range tested, 2.5 X 10(-5) to 1 X 10(-4) M. At the higher concentration, the extent of the modifications was as those which can be produced in nonhuman or malignant cell lines. Although the greatest changes were produced in the neutral lipid fraction, the cellular phospholipids also exhibited appreciable modifications. The phospholipids isolated from a microsomal fraction prepared from the cell homogenate exhibited similar changes in fatty acyl composition. These findings indicate that the human fibroblast can tolerate considerable variability in fatty acid composition, even in membrane phospholipids. The triglyceride content of the cells increased when they were grown in the presence of added fatty acids, but the phospholipid and cholesterol content remained unchanged. Growth was not affected by either oleic or linoleic acids, but it was reduced up to 50% when palmitic linolenic, or arachidonic acid was added in concentrations of 5 X 10(-5) M or above. Extensive modifications in phospholipid fatty acid composition also were produced in confluent monolayers of these fibroblasts. This suggest that some membrane lipid turnover occurs even when the cultures are not rapidly growing. Fatty acid modifications also were produced in the commercially available IMR-90 strain of human lung fibroblasts, suggesting that the ability to tolerate considerable differences in fatty acid composition is not a special property of the skin fibroblast line that was isolated locally.     

Studies of ascorbate-dependent, iron-catalyzed lipid peroxidation

We have previously observed that both Fe(II) and Fe(III) are required for lipid peroxidation to occur, with maximal rates of lipid peroxidation observed when the ratio of Fe(II) to Fe(III) is approximately one (J. R. Bucher et al. (1983) Biochem. Biophys. Res. Commun. 111, 777-784; G. Minotti and S. D. Aust (1987) J. Biol. Chem. 262, 1098-1104). Consistent with the requirement for both Fe(II) and Fe(III), ascorbate, by reducing Fe(III) to Fe(II), stimulated iron-catalyzed lipid peroxidation but when the ascorbate concentration was sufficient to reduce all of the Fe(III) to Fe(II), ascorbate inhibited lipid peroxidation. The rates of lipid peroxidation were unaffected by the addition of catalase, superoxide dismutase, or hydroxyl radical scavengers. Exogenously added H2O2 also either stimulated or inhibited ascorbate-dependent, iron-catalyzed lipid peroxidation apparently by altering the ratio of Fe(II) to Fe(III). Thus, it appears that the prooxidant effect of ascorbate is related to the ability of ascorbate to promote the formation of a proposed Fe(II):Fe(III) complex and not due to oxygen radical production. The antioxidant effect of ascorbate on iron-catalyzed lipid peroxidation may be due to complete reduction of iron.     

Mechanisms of the effects of Ca2+ ions on lipid peroxidation

Mechanisms underlying Ca2+ effects on lipid peroxidation (LPO) induced in liposomes (from egg yolk lecithin) and UFsomes (from linolenic acid, methyl linolenate) with the aid of O2- -system (Fe2+ + ascorbate) were studied. It was shown that stimulation of lipid peroxidation by low Ca2+ concentrations (10(-6)-10(-5) M) was due to its ability to release Fe2+-ions bound to negatively charged (phosphate, carboxylic) lipid groups (of licethin, linolenic acid), thus increasing the concentration of catalytically active Fe2+. The inhibitory effect of high Ca2+ concentrations was caused by its interaction with superoxide anion-radicals and was not observed in LPO-systems, independent of O2- generation (e. g. Fe2+ + cumol hydroperoxide).     

Both somatostatin and the caudal neuropeptide, urotensin II, stimulate lipid mobilization from coho salmon liver incubated in vitro

The direct effects of somatostatin-14 (SRIF; synthetic ovine) and the fish caudal neuropeptide, urotensin II (UII; synthetic Gillichthys), on fatty acid (FA) release and on lipolytic enzyme (triacylglycerol lipase) activity were determined on coho salmon liver slices incubated in vitro. FA release was continuously measured by pH-stat titration. Additionally, gas chromatographic analysis of the incubation medium was performed to determine the type and relative composition of medium fatty acid constituents. SRIF and UII both stimulated FA release in a dose-dependent manner; the two peptides appeared to stimulate FA release in an equimolar manner. Maximal response was obtained at 1 X 10(-5) M; ED50 was approximately 2 X 10(-7) M. SRIF-stimulated FA release did not result in differential secretion of any particular FA type. Tissue triacylglycerol lipase activity was significantly enhanced by addition of UII or SRIF (2 X 10(-6) M). Dibutyryl cAMP and IBMX both stimulated FA release and lipase activity; dbcAMP stimulated FA release in dose-dependent manner. These results indicate that SRIF and UII directly enhance lipid mobilization from salmon liver slices and suggest that SRIF- and UII-stimulated lipid mobilization from salmon liver slices is mediated through cAMP.