Antioxidant activity of the pyridoindole stobadine in liposomal and microsomal lipid peroxidation.

Stobadine, a pyridoindole derivative, is an efficient inhibitor of lipid peroxidation in phosphatidylcholine liposomes and in rat liver microsomes treated with iron/ADP/NADPH as pro-oxidant. Accumulation of thiobarbituric acid-reactive substances (TBARS) or low-level chemiluminescence were taken as a measure of lipid peroxidation and 5 microM stobadine doubled the duration of the lag phase preceding the onset of rapidly increasing chemiluminescence. Inhibition of lipid peroxidation was not observed with tocopherol-deficient microsomes, suggesting that the antioxidant effect of stobadine depends on vitamin E in the membrane. The cis(-) isomer was most effective, with the cis(+) and trans(rac) as well as dehydro- or acetyl derivatives being less active. In liposomes, the presence of reductant (NADPH or ascorbate) protects from the loss of stobadine.     

Structural aspects of antioxidant activity of substituted pyridoindoles

Stobadine and its two structural analogues, dehydrostobadine and N-acetylated stobadine were used to examine how structural alteration in the close proximity of the indolic nitrogen would influence the antioxidant activity of the substituted pyridoindoles. The compounds were tested for their efficiency to scavenge stable free radicals of alpha,alpha'-diphenyl-beta-picrylhydrazyl as well as for their ability to prevent 2,2'-azobis-(2-amidinopropane)hydrochloride induced peroxidation of dioleoyl phosphatidylcholine liposomes. The results proved that the substituted pyridoindoles can act as potent scavengers of peroxyl radicals both in aqueous and lipid phases, the antioxidant activity being comparable with that of Trolox. Structural changes in the proximity of the indolic nitrogen were found crucial for the radical scavenging efficiency: aromatisation of the pyridoindole skeleton in dehydrostobadine lowered the antioxidant activity, while acetylation of the indolic nitrogen completely abolished the ability to scavenge peroxyl radicals. The results are in agreement with the notion that the antioxidant activity of stobadine and of the related pyridoindoles may be mediated via the indolic nitrogen centre. When stobadine and Trolox were present simultaneously in liposomal incubations, Trolox spared stobadine in a dose-dependent manner; a direct interaction of Trolox with stobadinyl radical appears to be a plausible explanation with possible consequences for the antioxidant capacity of stobadine under in vivo conditions, where re-cycling of stobadine by vitamin E might occur.     

Black tea increases the resistance of human plasma to lipid peroxidation in vitro, but not ex vivo.

A number of in vitro studies have shown that polyphenols and flavonoids in tea exert significant antioxidant activity. However, epidemiologic and experimental studies have produced conflicting results. The purpose of the present study was to compare the antioxidant activity of black tea in vitro with that ex vivo. Black tea polyphenols (BTP), black tea extract (BTE), or their major polyphenolic antioxidant constituent, epigallocatechin gallate (EGCG), were added to human plasma and lipid peroxidation was induced by the water-soluble radical generator 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). Following a lag phase, lipid peroxidation was initiated and occurred at a rate that was lowered in a dose-dependent manner by BTP. Similarly, EGCG and BTE added to plasma in vitro strongly inhibited AAPH-induced lipid peroxidation. The lag phase preceding detectable lipid peroxidation was due to the antioxidant activity of endogenous ascorbate, which was more effective at inhibiting lipid peroxidation than the tea polyphenols and was not spared by these compounds. In contrast, when eight healthy volunteers consumed the equivalent of six cups of black tea, the resistance of their plasma to lipid peroxidation ex vivo did not increase over the next 3 h. These data suggest that, despite antioxidant efficacy in vitro, black tea does not protect plasma from lipid peroxidation in vivo. The striking discrepancy between the in vitro and ex vivo data is most likely explained by the insufficient bioavailability of tea polyphenols in humans.     

Preventive effect of several antioxidants after oxidative stress on rat brain homogenates

Brain homogenate was used as a model system to study antioxidant properties of several natural and synthetic antioxidants under oxidative stress. Oxidative stress was induced by Fe/ascorbate system and lipid peroxidation as well as protein modification were studied. Thiobarbituric acid reactive substances (TBARS) were used as a marker of lipid peroxidation. The preventive effect concerning lipid peroxidation decreased in the order: buthylated hydroxytoluene (BHT) (3.5), stobadine (ST) (35), serotonin (54), trolox (98), U 74389G (160), melatonin (3100), (the numbers in the brackets represent IC50 in micromol/l). Methylprednisolone had no effect, and spin traps interfered with TBARS determination. Concerning creatine kinase (CK) activity as a selected marker of oxidative modification of proteins, the preventive effect of antioxidants (30 micromol/l) decreased in the order: BHT (30), trolox (75), stobadine (ST) (77), alpha-phenyl-N-tert-buthylnitrone (PBN) (87), sodium salt of N-tert-buthyl-C-(phenyl-2-sulfone) nitrone (SPBN) (90), (the numbers in the brackets represent the loss of CK activity in percentages, when 100% was the loss of CK activity in the absence of any antioxidant). The nonglucocorticoid steroid U 74389G, methylprednisolone and serotonin had no preventive effects, while melatonin had antioxidant effect only in a higher concentration (1 mmol/l).     

Stobadine inhibits doxorubicin-induced apoptosis through a caspase-9 dependent pathway in P815 mastocytoma cells

Doxorubicin (DOXO), a widely used chemotherapeutic agent, induces apoptosis in transformed and non-transformed cells. The apoptotic effect of DOXO has been linked to the generation of reactive oxygen species (ROS). Antioxidants may be effective in the prevention of DOX-induced apoptosis. In the present study we investigated the effects of stobadine, a pyridoindole antioxidant in a DOXO-induced apoptosis model of P815 cells by flow cytometric analyses and by measuring caspase-3 and caspase-9 activities. Pretreating cells with stobadine significantly increased cell viability and decreased apoptosis rate. Inhibition in apoptosis was observed at maximum levels following treatment of cells with 10(-7)M stobadine as evident from flow cytometric analyses. The antiapoptotic effect of stobadine was further confirmed by inhibition of caspase-3 and caspase-9 activities. We found that the antioxidative effects of stobadine were comparable to the effects of a well known antioxidant, N-acetyl l-cysteine (NAC).     

Antioxidant activity of palm oil carotenes in peroxyl radical-mediatedperoxidation of phosphatidyl choline liposomes

Abstract

The antioxidant efficacy of α-carotene and comparison with β-carotene in multilamellar liposomes prepared from egg yolk phosphatidyl choline (EYPC) exposed to the lipid soluble 2,2′-azobis (2,4-dimethyl valeronitrile) (AMVN) was investigated. Lipid peroxidation was measured as thiobarbituric acid reacting substances (TBARS)at 532 nm or as hydroperoxide formation at 234 nm after separation of phosphatidyl choline hydroperoxide (PCOOH) by high-pressure liquid chromatography (HPLC). Lutein and zeaxanthin, the hydroxyl derivatives of α- and β-carotenes, and the chain breaking antioxidant α-tocopherol were also included in the study.AMVN being a lipid soluble, non polar azo initiator penetrates into the hydrophobic interior of the phospholipid bilayer, forming peroxyl radicals which peroxidate the phospholipid leading to PCOOH accumulation. All the carotenoids tested at 1 mol% relative to EYPC significantly suppressed the formation of PCOOH compared to control samples.In this system, α-carotene retarded PCOOH formation better than β-carotene. Similarly, lutein was a better antioxidant than is zeaxanthin. But lutein and zeaxanthin were more effective antioxidants than α- and β-carotenes, respectively. After 1 h of incubation of the carotenoid with AMVN, α-, β-carotene, lutein and zeaxanthin limited PCOOH formation by 77%, 68%, 85%and 82%, respectively, while α-tocopherol elicited 90%reduction.AMVN incubated with EYPC for 2 h induced the formation of TBARS compared to control (P <0.001). α-Carotene significantly suppressed the TBARS formation by 78% whilst β-carotene, lutein, zeaxanthin and α-tocopherol elicited 60%, 91%and 80% reductions, respectively. Increasing the concentration of the carotenoid >1 mol% to EYPC did not significantly increase protection of the membrane against free radical attack.Our findings suggest that α-carotene is a better antioxidant than is β-carotene in phosphatidyl choline vesicles. It may, therefore, be useful in limiting free radical mediated peroxidative damage against membrane phospholipids _{in vivo}.     

Effects of beta-carotene and alpha-tocopherol on radical-initiated peroxidation of microsomes.

Rat liver microsomal membranes were exposed to either beta-nicotinamide adenine dinucleotide phosphate (NADPH), adenosine 5'-diphosphate (ADP), and Fe+3 or to azocompounds, and the antioxidant activities of beta-carotene and alpha-tocopherol were studied. Lipid peroxidation was monitored either by malondialdehyde (MDA) formation in the thiobarbituric acid assay at 535 nm or by hydroperoxide formation at 234 nm, after high-pressure liquid chromatography (HPLC) separation of phospholipid hydroperoxides. The radical initiators, water-soluble 2,2'-azobis(2-amidinopropane) (AAPH) and lipid-soluble 2,2'-azobis(2,4-dimethylvaleronitrile (AMVN), when thermally decomposed at 37 degrees C under air, produced a constant rate of lipid peroxidation in microsomes and lag times inversely related to their concentrations. Using 25 mM AAPH, beta-carotene suppressed lipid peroxidation at a concentration of 50 nmol/mg protein; using 24 mM AMVN, an inhibition of MDA formation was observed at a concentration of only 5 nmol/mg protein. Inhibition by beta-carotene did not produce a clearly defined lag phase. During AAPH-induced lipid peroxidation, beta-carotene was consumed linearly, and high levels of the antioxidant were still present at the end of 45 min of incubation. Using NADPH/ADP/Fe+3, protection by beta-carotene was observed at 10 nmol/mg protein. alpha-Tocopherol effectively suppressed both MDA and hydroperoxide formation in a dose-dependent manner when either NADPH/ADP/Fe+3 or azocompounds were used. These effects were observed at very low concentrations of the added alpha-tocopherol, ranging from 2 to 3 nmol/mg protein. When the lag times were measurable (AAPH and AMVN), they were directly proportional to the concentration of alpha-tocopherol and revealed the presence of endogenous antioxidants in the microsomal membranes. Different temporal relationships between the loss of alpha-tocopherol and lipid peroxidation were observed in relation to the prooxidant used. A substantial depletion of about 70% of endogenous alpha-tocopherol preceded the propagation phase when induced by the azocompounds, while only 20% of antioxidant disappeared at the beginning of the peroxidation when induced by NADPH/ADP/Fe+3. Although our results show that both beta-carotene and alpha-tocopherol suppress the peroxidation of microsomal membranes, their antioxidant efficacy is influenced by several factors, including the type of radical initiator involved and the site and rate of radical production.     

The crocin assay for the determination of relative rate constants of alkoxyl radical reactions with the pyridoindole stobadine and with other antioxidants

Summary

The water-soluble carotenoid crocin is bleached in aqueous solutions by tert-butyloxy radicals (t-BuO^.) photolytically generated from tert-butyl hydroperoxide. Crocin bleaching as measured at 440 nm can be competitively inhibited by antioxidants. This method therefore allows the determination of the relative reaction rates of these compounds with the t-BuO^. radicals. In this test system the scavenging effect of the pyridoindole stobadine, a new antioxidant with potential for the treatment of tissue injury caused by oxidative stress, was compared with other known antioxidants. Generally good agreement was observed in two different approaches for the evaluation of the experimental data. The relative rate constants of the antioxidants tested increased in the order: stobadine, chlorpromazine, isoascorbic acid, trolox, ascorbic acid. Since it has been shown previously that stobadine can scavenge alkoxyl radicals also in a non-polar environment, this antioxidant property may be important in the inhibition of lipid peroxidation.     

Antioxidant activity of palm oil carotenes in peroxyl radical-mediated peroxidation of phosphatidyl choline liposomes

The antioxidant efficacy of alpha-carotene and comparison with beta-carotene in multilamellar liposomes prepared from egg yolk phosphatidyl choline (EYPC) exposed to the lipid soluble 2,2'-azobis (2,4-dimethyl valeronitrile) (AMVN) was investigated. Lipid peroxidation was measured as thiobarbituric acid reacting substances (TBARS) at 532 nm or as hydroperoxide formation at 234 nm after separation of phosphatidyl choline hydroperoxide (PCOOH) by high-pressure liquid chromatography (HPLC). Lutein and zeaxanthin, the hydroxyl derivatives of alpha- and beta-carotenes, and the chain breaking antioxidant alpha-tocopherol were also included in the study. AMVN being a lipid soluble, non polar azo initiator penetrates into the hydrophobic interior of the phospholipid bilayer, forming peroxyl radicals which peroxidate the phospholipid leading to PCOOH accumulation. All the carotenoids tested at 1 mol% relative to EYPC significantly suppressed the formation of PCOOH compared to control samples. In this system, alpha-carotene retarded PCOOH formation better than beta-carotene. Similarly, lutein was a better antioxidant than is zeaxanthin. But lutein and zeaxanthin were more effective antioxidants than alpha- and beta-carotenes, respectively. After 1 h of incubation of the carotenoid with AMVN, alpha-, beta-carotene, lutein and zeaxanthin limited PCOOH formation by 77%, 68%, 85% and 82%, respectively, while alpha-tocopherol elicited 90% reduction. AMVN incubated with EYPC for 2 h induced the formation of TBARS compared to control (P < 0.001). alpha-Carotene significantly suppressed the TBARS formation by 78% whilst beta-carotene, lutein, zeaxanthin and alpha-tocopherol elicited 60%, 91% and 80% reductions, respectively. Increasing the concentration of the carotenoid > 1 mol% to EYPC did not significantly increase protection of the membrane against free radical attack. Our findings suggest that alpha-carotene is a better antioxidant than is beta-carotene in phosphatidyl choline vesicles. It may, therefore, be useful in limiting free radical mediated peroxidative damage against membrane phospholipids in vivo.     

Aggregation of human blood platelets in the presence of the pyridoindole stobadine

The antiarrhythmic and cardioprotective drug stobadine, possessing antioxidant and neuroprotective properties, was studied as to its in vitro effect on aggregation of human blood platelets. Pretreatment of platelets with stobadine for 30 s inhibited stimulated platelet aggregation in a dose-dependent way. Depending on the aggregation stimulus used, the minimal effective concentrations of the drug were 1 micromol/l (adrenaline), 200 micromol/l (ADP), and 1,000 micromol/l (PMA). Aggregation induced with thrombin or Ca2+-ionophore A23187 was not changed in the presence of stobadine even in the concentration of 1,000 micromol/l. Addition of stobadine 30 s after adrenaline was also effective and terminated aggregation (100 and 1,000 micromol/l) or prolonged onset of its second phase (10 micromol/l). The presented experiments showed stobadine as a potent inhibitor of adrenaline-induced aggregation, indicating its involvement in the observed antithrombotic and cytoprotective activity.     

Seasonal variation in copper-mediated low-density lipoprotein oxidation in vitro is related to varying plasma concentration of oxidised lipids in summer and winter

The seasonal variation of CuCl2-mediated low density lipoprotein (LDL) oxidation (10 microM Cu2+, lag phase, rate of oxidation and maximum absorbance at 234 nm) were measured in 43 men and women on 4-6 occasions (mean 5.7 +/- 0.5) over a 12-month period. The lag phase averaged 52.7 +/- 0.6 min and did not differ by gender. Lag phase and rate of the rapid propagation phase of LDL oxidation showed a sinusoidal pattern over the year (increased and reduced oxidative susceptibility during January and June-July, respectively; both p < 0.001). Changes in plasma alpha-tocopherol, ascorbic acid, lycopene or beta-carotene concentrations did not explain seasonal differences in oxidative susceptibility of LDL in vitro. Nor did plasma lipid content of linoleic acid, the main substrate of lipid peroxidation, vary. However, the amount of hydroperoxy- plus hydroxy-fatty acids in plasma lipids varied according to season (p < 0.024) and was related to the lag phase (r = -0.26, p < 0.001). Seasonal variation in oxidative susceptibility was not significant after adjusting for hydroperoxy- plus hydroxy-fatty acids (p = 0.506). Isolated LDL is more vulnerable to Cu2+-induced lipid peroxidation during the winter and this may be due to the higher amount of oxidised lipids during that period.     

Antioxidant activity of dihydrolipoate against microsomal lipid peroxidation and its dependence on alpha-tocopherol

The antioxidant effect of dihydrolipoate and lipoate was examined in microsomal fractions obtained from normal and alpha-tocopherol-deficient animals after initiation of lipid peroxidation with an NADPH/iron/ADP system. Dihydrolipoate prolonged the lag phase before the onset of low-level chemiluminescence and before the rapid accumulation of thiobarbituric acid-reactive substances in normal but not in vitamin E-deficient microsomes. Lipoate did not show such an antioxidant effect. It is concluded that the dihydrolipoate-mediated protection against lipid peroxidation by prolonging the lag phase is dependent on alpha-tocopherol. Likewise, dihydrolipoate prolonged the lag phase before the onset of the rapid loss of vitamin E during lipid peroxidation. Dihydrolipoate, like other biological thiols such as GSH, also affects the peroxidative process after the lag period. The effects included a smaller slope of the chemiluminescence increase, a lower maximal level of chemiluminescence, a slower loss of alpha-tocopherol and a slower accumulation, but unchanged maximal levels, of thiobarbituric acid-reactive substances. The biological significance may be most prominent in the mitochondrial matrix space, where lipoamide-containing ketoacid dehydrogenases are located. A potential pharmacological use of this biological dithiol in conditions associated with oxidative stress could be based on the antioxidant activity of dihydrolipoate.     

Influence of local anesthetics on the phosphatidylcholine model membrane: small-angle synchrotron X-ray diffraction and neutron scattering study

The phase preferences of egg yolk phosphatidylcholine (EYPC) have been examined in the presence of tertiary amine anesthetics [2-(propyloxy)phenyl]-2-(1-piperidinyl)ethyl ester of carbamic acid (C3A) and [2-(heptyloxy)phenyl]-2-(1-piperidinyl)ethyl ester of carbamic acid (C7A, heptacaine). Using the synchrotron small-angle X-ray diffraction (SAXD), it is shown that the C3A anesthetic induces the cubic and hexagonal (H(I)) phases at 2 > or = C3A:EYPC > 0.5 and H2O:EYPC < or = 40 molar ratios. In contrast, longer alkyloxy chain homolog C7A has no effect on the bilayer arrangement of EYPC at C7A:EYPC < = 1 molar ratios as observed by SAXD in C7A + EYPC mixtures hydrated at H2O:EYPC < = 40 molar ratios, as well as in sonicated C7A + EYPC mixtures hydrated in excess water as proved by the small-angle neutron scattering (SANS). The bilayer thickness d(L) decreases and the bilayer C7A surface area SC7A increases with the increase of C7A:EYPC molar ratio. It is suggested that the ability of tertiary amine local anesthetics to influence the dL and SC7A values and EYPC polymorphism is caused by their effective molecular shape and by charge. The possibility that anesthetic molecules may exert some of their biological effects by virtue of these properties is discussed.     

Stobadine: bellwether of a broader view of drug actions

Stobadine was recognized early in its development as having antioxidant properties. A number of laboratories found associations between the antioxidant properties of stobadine and its potential beneficial effects. We found that stobadine acted as an antioxidant in a modification of an oxygen radical absorbance capacity (ORAC) assay. Similar results were observed with other drugs, including tirilazad and pramipexole. We suggest that stobadine and certain other drugs exhibit antioxidant properties in both hydrophilic and hydrophobic environments. Other drugs have been developed for their antioxidant properties and some currently marketed drugs have antioxidant properties. Although they may not have been explicitly sought during development, at least some of the beneficial effects may be related to antioxidant properties and/or scavenging of free radicals. Because stobadine was one of the first drugs for which useful properties were associated with its antioxidant actions, stobadine may be seen as a bellwether of a broader view of pharmacological actions--a view that encompasses antioxidant properties as a useful basis of therapeutic effects.     

Spin-labelled lutein as a new antioxidant in protection against lipid peroxidation

A new potentially antioxidant compound, spin-labelled lutein (SL-lut), was synthesized and incorporated into egg yolk phosphatidylcholine (EYPC) liposome membrane. The approximate location of nitroxide free radical groups of SL-lut was determined based on electron paramagnetic resonance (EPR) spectra. Then the ability of SL-lut to protect EYPC liposomes against lipid peroxidation (LPO) was compared to the antioxidant effects of lutein and a nitroxide spin label 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy (3-CP). Two free radical generation systems were used—a thermal decomposition of 2,2'-azobis (2,4 dimethyl-valeronitrile) (AMVN) and a modified Fenton reaction using ferric-8-hydroxyquinoline (Fe(HQ)₃). Determination of the amount of thiobarbituric acid reactive species (TBARS) was used as a measure of LPO. SL-lut was the most powerful antioxidant, reducing LPO by about 6-times in AMVN-treated liposomes and 7-times in Fe(HQ)₃-treated liposomes. Lutein alone gave only a moderate protection in both systems, while 3-CP was as efficient as SL-lut in the presence of AMVN, but not efficient whatsoever in the presence of Fe(HQ)₃. The results suggest that a nitroxide part of SL-lut plays an important role in enhancing the antioxidant activity of lutein and makes SL-lut a powerful antioxidant efficient under different conditions.     

Spin-labelled lutein as a new antioxidant in protection against lipid peroxidation

A new potentially antioxidant compound, spin-labelled lutein (SL-lut), was synthesized and incorporated into egg yolk phosphatidylcholine (EYPC) liposome membrane. The approximate location of nitroxide free radical groups of SL-lut was determined based on electron paramagnetic resonance (EPR) spectra. Then the ability of SL-lut to protect EYPC liposomes against lipid peroxidation (LPO) was compared to the antioxidant effects of lutein and a nitroxide spin label 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy (3-CP). Two free radical generation systems were used—a thermal decomposition of 2,2′-azobis (2,4 dimethyl-valeronitrile) (AMVN) and a modified Fenton reaction using ferric-8-hydroxyquinoline (Fe(HQ)₃). Determination of the amount of thiobarbituric acid reactive species (TBARS) was used as a measure of LPO. SL-lut was the most powerful antioxidant, reducing LPO by about 6-times in AMVN-treated liposomes and 7-times in Fe(HQ)₃-treated liposomes. Lutein alone gave only a moderate protection in both systems, while 3-CP was as efficient as SL-lut in the presence of AMVN, but not efficient whatsoever in the presence of Fe(HQ)₃. The results suggest that a nitroxide part of SL-lut plays an important role in enhancing the antioxidant activity of lutein and makes SL-lut a powerful antioxidant efficient under different conditions.     

Iron-induced lipid peroxidation and protein modification in endoplasmic reticulum membranes. Protection by stobadine

Treatment with FeSO(4)/EDTA (0.2 micromol Fe(II) per mg of protein) was used to study the effect of oxidative stress on lipid peroxidation and structural properties of endoplasmic reticulum (ER) membranes isolated from rabbit brain. Oxidative stress resulted in conjugated diene formation and a decrease of 1-anilino-8-naphthalenesulfonate (ANS) fluorescence in a time-dependent manner. In contrast, fluorescence anisotropy of 1, 6-diphenyl-1,3,5-hexatriene was increased early after the initiation of lipid peroxidation and no further increase was observed after 1, 2 and 3 h of peroxidation. FeSO(4)/EDTA treatment was accompanied by formation of conjugates of lipid peroxidation products with membrane proteins, as detected by the increase in fluorescence excitation (350-360 nm) and emission (440-450 nm) maximum. Oxidative stress also induced a marked decrease of the intrinsic fluorescence of aromatic amino acids, suggesting modification or changes in the environment of these amino acid residue(s). The lipid antioxidant, stobadine, completely prevented the changes of ANS fluorescence and production of peroxidized lipid-protein conjugates whereas tryptophan fluorescence was only partially protected. These results suggest that Fe(II) induces both lipid-mediated- and lipid peroxidation independent-modification of ER membrane proteins. The study also demonstrates that stobadine is a potent inhibitor of Fe(II)-induced protein modification.     

Effects of non-ionic surfactants N-alkyl-N,N-dimethylamine-N-oxides on the structure of a phospholipid bilayer: small-angle X-ray diffraction study

Effects of non-ionic surfactants N-alkyl-N,N-dimethylamine-N-oxides (C(n)NO, n is the number of alkyl carbons) on the structure of egg yolk phosphatidylcholine (EYPC) bilayers in the lamellar fluid phase was studied by small-angle X-ray diffraction as a function of H(2)O:EYPC and C(n)NO:EYPC molar ratios. The bilayer thickness d(L) and the lipid surface area at the bilayer-aqueous interface S(L) were calculated from the repeat period, d of the lamellar phase, based on the model that water and EYPC + CnNO molecules form separated layers and that their molecular volumes are additive. In the studied range of m=CnNO:EYPC molar ratios up to 1:1, d(L) and S(L) change linearly. The slopes Delta L = delta dL/ delta m and Delta S= delta S L / delta m are equal to -0.876 +/- 0.027 nm and 0.347 +/- 0.006 nm2 for C(6)NO, -1.025+/-0.060 nm and 0.433+/-0.025 nm(2) for C(8)NO, -0.836+/-0.046 nm and 0.405+/-0.018 nm(2) for C(10)NO, -0.604+/-0.015 nm and 0.375+/-0.007 nm(2) for C(12)NO, -0.279+/-0.031 nm and 0.318+/-0.005 nm(2) for C(14)NO, -0.0865+/-0.070 nm and 0.2963 +/-0.014 nm(2) for C(16)NO, and -0.040+/-0.022 nm and 0.297+/- 0.002 nm(2) for C(18)NO, respectively, at full bilayer hydration. The peak-peak distance in the bilayer electron density profile, which relates to the P-P distance d(PP), obtained from the first four diffraction peaks by the Fourier transform also depends linearly on m, and the slope Delta PP = delta dPP/delta m is -0.528+/-0.065 nm for C(6)NO, -0.680+/-0.018 nm for C(8)NO, -0.573+/-0.021 nm for C(10)NO, -0.369+/-0.075 nm for C(12)NO, -0.190+/-0.015 for C(14)NO, -0.088+/-0.016 nm for C(16)NO and -0.094+/-0.016 nm for C(18)NO. The effects of C(n)NO on Delta(L), Delta(S) and Delta(PP) are the results of C(n)NO insertion into EYPC bilayers and depend on the hydrophobic mismatch between C(n)NO and EYPC hydrocarbon chains and on the lateral interactions of C(n)NO and EYPC in the bilayer.     

Lipid peroxidation of phosphatidylcholine liposomes depressed by the calcium channel blockers nifedipine and verapamil and by the antiarrhythmic-antihypoxic drug stobadine

Nifedipine, verapamil and stobadine were tested and compared with butylated hydroxytoluene (BHT) as possible free radical scavengers inhibiting lipid peroxidation in phosphatidylcholine liposomes. Liposomes were peroxidized by incubation in air at 50 degrees C. Verapamil less than nifedipine less than BHT less than stobadine depressed the lipid peroxidation as detected spectroscopically for conjugate diene and thiobarbituric acid product formation. Verapamil and stobadine were tested as OH radical scavengers in a Fenton-type reaction against spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO), as detected by ESR spectroscopy. The tested drugs competed with DMPO in trapping OH radicals, with stobadine being more effective than verapamil. ESR spectra of nifedipine in the incubated liposomes revealed that nifedipine could be involved in free radical reactions in the liposomes leading to nifedipine-stable radical(s) which were immobilized in the membrane. The obtained results suggest that some of the beneficial effects of the studied drugs can be mediated in disease by their ability to scavenge free radicals and by their protective effect on lipid peroxidation.     

The antioxidant action of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-α]pyrazin-3-one (MCLA), a chemiluminescence probe to detect superoxide anions

The antioxidant effect of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-α]pyrazin-3-one (MCLA), a Cypridina luciferin analog that acts as a chemiluminescence probe to detect O^⋅−₂, was investigated. MCLA produced a lag in oxygen consumption induced by cumene hydroperoxide in microsomes or by 2,2′-azobis (2-amidinopropane) dihydrochloride in liposomes and disappeared during the duration of the lag. MCLA profoundly inhibited the propagation reaction in Fe²⁺-dependent lipid peroxidation in liposomes, and MCLA disappearance accompanied by suppression of oxygen consumption markedly occurred in liposomes susceptible to peroxidation. Thiobarbituric acid-reactive substances in all systems used were also suppressed by MCLA dose dependently. These results indicate that MCLA has an antioxidant property through scavenging free radicals.