Lipid-soluble Radical Initiator Research Articles

Highly Unsaturated Fatty Acid might Act as an Antioxidant in Emulsion System Oxidized by Azo Compound

Now it is recognized that DHA is oxidatively stable fatty acid compared with linoleic acid (LA) in emulsified system, although DHA is oxidatively unstable in a bulk system. In fact, an emulsified mixture of DHA and LA behaves as in a bulk system, namely the oxidative stability of DHA becomes lower than that of LA. Therefore, in this study, tridocosahexaenoate (DDD) and glycerol trilinoleate (LLL) were separately emulsified using TritonX-100 as an emulsifier and DDD emulsion was mixed with the oxidizing LLL emulsion using a water-soluble radical initiator, 2,2'-azobis(2-aminopropane) dihydrochloride. As a result, DHA suppressed the oxidation of LA, while DHA was not significantly oxidized. This suppression ability was examined using glycerol trieicosapentaenoate, glycerol trilinolenate, or glycerol trioleate instead of DDD and it was found that this activity was increased with the increasing number of double bonds in the structure. Furthermore, the same type of experiment was carried out using a lipid-soluble radical initiator, 2,2'-azobisisobutyronitrile and the similar result was obtained. These results indicated that a highly polyunsaturated fatty acid might act as an antioxidant in an emulsion system oxidized by an azo compound.

Protection by Exogenously Added Coenzyme Q9 against Free Radical-Induced Injuries in Human Liver Cells

Reduced coenzyme Q10 (CoQ10H2) is known as a potent antioxidant in biological systems. However, it is not yet known whether CoQ9H2 could act as an antioxidant in human cells. The aim of this study is to assess whether exogenously added CoQ9 can protect human liver cells against injuries induced by a water-soluble radical initiator, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and a lipid-soluble radical initiator, 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN). CoQ9-enriched cells were obtained by treatment of HepG2 cells with 10 µM CoQ9 liposomes for 24 h. CoQ9-enriched cells were exposed to 10 mM AAPH and 500 µM AMVN over 4 h and 24 h, respectively. The loss of viability after treatment with AAPH or AMVN was much less in CoQ9-enriched cells than in naive HepG2 cells. The decrease in glutathione and the increase in thiobarbituric acid-reactive substance after treatment with AAPH or AMVN were also suppressed in CoQ9-enriched cells. The incubation of CoQ9-enriched cells with AAPH or AMVN led to a decrease in cellular CoQ9H2 and reciprocal increase in cellular CoQ9 resulting from its antioxidant function. Taken together, it was demonstrated for the first time that exogenously added CoQ9 could prevent oxidative stress-mediated damage to human cells by virtue of its antioxidant activity.

ESR Study on the Antioxidant Activity of TAK-218 in Biological Model Membranes.

TAK-218 has a 2,3-dihydrobenzofuran-5-amine (coumaran) structure which resembles alpha-tocopherol, and is a promising candidate as an agent for central nervous system (CNS) trauma and ischemia. The radical scavenging activity of TAK-218 was studied using electron spin resonance (ESR) spectroscopy. TAK-218 exhibited a more potent scavenging activity towards the hydroxyl radical than did the well-known hydroxyl radical scavengers, mannitol and dimethylsulfoxide. Towards the superoxide radical, TAK-218 showed equal potency to glutathione. TAK-218 reacted rapidly with stable radicals, such as galvinoxyl and 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH), and gave the quinone as a two-electron oxidized product in analogy with alpha-tocopherol. To exhibit an excellent antioxidative activity in living systems, the compounds should not only have the intrinsic radical scavenging activity but also good distribution in the biological lipid-bilayer membrane. To examine the antioxidant activity of TAK-218, the inhibition of lipid peroxidation by alpha-tocopherol and TAK-218 in liposomal membranes was studied using an ESR spin-label technique. Both alpha-tocopherol and TAK-218 completely inhibited lipid peroxidation by radicals generated in an aqueous layer using a water-soluble radical initiator, 2,2'-azobis-(2-amidinopropane) hydrochloride (AAPH). At a high incubation temperature (45 degrees C), alpha-tocopherol scavenged radicals more effectively than TAK-218 on the surface of the membrane, while TAK-218 scavenged radicals more effectively in the interior of the membrane. The difference between TAK-218 and alpha-tocopherol for radical scavenging in the membrane system derives from the different distribution pattern of these compounds. TAK-218 can penetrate the membrane freely and can scavenge the radical in the membrane interior. Furthermore, TAK-218 was shown to inhibit lipid peroxidation initiated by a lipid soluble radical initiator, 2,2'-azobis-(2,4-dimethylvaleronitrile) (AMVN), in a membrane more effectively than alpha-tocopherol.

Mechanism of lower oxidizability of eicosapentaenoate than linoleate in aqueous micelles. II. Effect of antioxidants.

We have reported that the peroxyl radicals derived from methyl eicosapentaenoate (20:5n-3) are more polar than those from methyl linoleate (18:2n-6) since the former peroxyl radicals have at least two molecules of oxygen in a molecule while the latter peroxyl radical has one. This lowers the oxidizability for 20:5n-3 in aqueous Triton X-100 micelles by enhancing the termination reaction rate for peroxyl radicals and by reducing the rate of propagation since there may be more polar peroxyl radicals derived from 20:5n-3 at the surface than within the micelle core. In this study, we measured the effect of three antioxidants, di-tert-butyl-4-methylphenol (BHT), 2,2,5,7,8-pentamethyl-6-chromanol (PMC) and 2-carboxy-2,5,7,8-tetramethyl-6-chromanol (Trolox), on the oxidation of lipids in aqueous micelle. Antioxidants give a clear induction period during oxidation of 18:2n-6 initiated with a water-soluble radical initiator, and its induction length decreases in the order of BHT > PMC > Trolox. This is consistent with the proposed location of three antioxidants: being in the core of micelle, at the surface, or in aqueous phase, respectively. However, BHT does not inhibit the oxidation of 20:5n-3 efficiently, and its rate of oxidation is slower than that observed in the oxidation of 18:2n-6, supporting the idea that polar peroxyl radicals derived from 20:5n-3 are preferentially located at the surface of the micelle. Similar results were obtained when oxidation was initiated with a lipid-soluble radical initiator except antioxidants had lesser effect on the oxidation rate of 20:5n-3.

Mechanism of free radical-induced hemolysis of human erythrocytes. II. Hemolysis by lipid-soluble radical initiator.

Hemolysis of human erythrocytes induced by free radicals initiated from lipid-soluble 2,2'-azobis(2,4-dimethylvaleronitrile) (ADVN) was examined under various conditions. From the ESR spectra of the spin-labeled erythrocytes, it was found that the fluidity of the membrane did not change during the radical-induced hemolysis. The curves of the time courses of the extent of oxidation and the conformational change of band 3 proteins were hyperbolic, though the hemolysis curves were sigmoidal. In spite of the necessity of lipid peroxidation, the peroxidation did not seem to relate directly to the hemolysis. It was observed that the hemolytic holes were formed by a lateral clustering of band 3, an anion exchange protein in erythrocytes. The competitive reaction model between lipid peroxidation and the redistribution of oxidized band 3 proteins, which was previously presented for the hemolysis initiated from water-soluble, 2,2'-azobis(amidinopropane)dihydrochloride (AAPH), could explain well the curves for the hemolysis by ADVN. Further, the rates of lipid peroxidation at various concentrations of ADVN and AAPH were calculated on the basis of the hemolysis curves, and they were compared with the experimental values estimated from the curves for the lipid peroxidation. The curves which showed a dependence of the calculated rate constant on the concentration of radical initiators were similar to those of the experimental values. These results indicate that the competitive reaction model is appropriately represents the hemolysis induced by free radicals which also originated from lipid-soluble initiators.

Phospholipids Increase Radical-Scavenging Activity of Vitamin E in a Bulk Oil Model System

The effect of phospholipids on the radical-scavenging activity of vitamin E in a bulk oil model was studied to clarify the mechanism of their antioxidant synergism. Phospholipids retarded the consumption of vitamin E and elongated the induction period arising from vitamin E in the mixture of methyl linoleate and methyl laurate when exposed to a water-soluble radical initiator. However, they accelerated the consumption of vitamin E in methyl laurate alone. On the other hand, phospholipids little affected the consumption of vitamin E when lipid peroxidation was initiated by a lipid-soluble radical initiator. These results implied that phospholipids enhanced the accessibility of vitamin E toward the chain-initiating radicals in an aqueous microenvironment, resulting in the interruption of chain initiation. Phospholipids could act as neither the free radical scavengers nor the regenerators for vitamin E in this model system. Thus, it is concluded that the enhanced effect of phospholipids for vitamin E in bulk oil originated from the elevation of its radical-scavenging activity.

Antioxidant Action of Neuromelanin: The Mechanism of Inhibitory Effect on Lipid Peroxidation

Iron and lipid peroxidation are believed to be involved in the degeneration of pigmented neurons in Parkinson′s disease. Melanin-iron interaction is thought to play a role in iron accumulation and reactivity. The purpose of this study was to examine antioxidant properties of isolated natural and synthetic neuromelanin. Effect of neuromelanin from substantia nigra and its synthetic model, dopamine melanin, on Lipid peroxidation, induced by ferrous ions and free-radical initiators, has been studied in methyl linoleate aqueous dispersions, 2,2′-azobis(amidinopropane)dihydrochloride and 2,2′-azobis(2,4-dimethyl-valeronitrile) were used as water-soluble and lipid-soluble radical initiator, respectively. Rate of oxidation was followed quantitatively by measuring oxygen uptake and accumulation of lipid hydroperoxides. Melanin had a distinct protective effect on lipid peroxidation induced by ferrous ions or water-soluble free-radical initiator but was relatively inefficient when peroxidation was initiated with lipid-soluble compound. It also inhibited iron-catalyzed decomposition of methyl linoleate hydroperoxides in the presence of ascorbate. Extent of the inhibition depended on the ratio of melanin to iron. Taken together, these results provide strong support for the idea that neuromelanin of pigmented neurons can act as a natural antioxidant by sequestering redox-active metal ions.

Synthesis of a phosphatidyl derivative of vitamin E and its antioxidant activity in phospholipid bilayers

A novel phospholipid containing a chromanol structure at its polar head group was synthesized from egg yolk phosphatidylcholine and 2,5,7,8-tetramethyl-6-hydroxy-2-(hydroxyethyl)chroman by transphosphatidylation catalyzed by phospholipase D from Streptomyces lydicus. The structure of the product synthesized was shown by spectral analysis to be 1,2-diacyl-sn-glycero-3-phospho-2'-hydroxyethyl- 2',5',7',8'-tetramethyl-6'-hydroxychroman. The phosphatidylchromanol (PCh) showed antioxidant activity against radical chain oxidation of methyl linoleate in solution in a manner similar to that of d-alpha-tocopherol (alpha-Toc) and 2,2,5,7,8-pentamethyl-6-chromanol. However, PCh was less effective as a chain-breaking antioxidant than was alpha-Toc when unilamellar egg yolk phosphatidylcholine liposomes were exposed to either a water-soluble or a lipid-soluble radical initiator. It is likely that the phospholipid nature of PCh affects the location and the mobility of the chromanol moiety in the membrane bilayer resulting in a decrease in antioxidant activity. On the other hand, the antioxidant activity of PCh was little different from that of alpha-Toc in unilamellar liposomes when exposed to a lipid-soluble radical initiator in the presence of ascorbic acid. It appears that PCh in phospholipid bilayers can be regenerated by ascorbic acid in aqueous phase as can be alpha-Toc. The new phospholipid, phosphatidylchromanol, should prove useful as a chain-breaking antioxidant in phospholipid membranes.

Antioxidant mechanism of Mn(II) in phospholipid peroxidation

Antioxidant action of Mn 2+ on radical-mediated lipid peroxidation without added iron in microsomal lipid liposomes and on iron-supported lipid peroxidation in phospholipid liposomers or in microsomes was investigated. High concentrations of Mn 2+ above 50 μM inhiibited 2,2′-azobis (2-amidinopropane) (ABAP)-supported lipid peroxidation without added iron at the early stage, while upon prolonged incubation, malondialdehyde production was rather enhanced as compared with the control in the absence of Mn 2+. However, in a lipid-soluble radical initiator, 2,2′-azobis, (2,4-dimethyl-valeronitrile) (AMVN)- supported lipid peroxidation of methyl linoleate in methanol Mn 2+ apparently did not scavenge lipid radicals and lipid peroxyl radicals, contrary to a previous report. At concentrations lower than 5 μM, Mn 2+ competitively inhibited Fe 2+-pyrophosphate-supported lipid peroxidation in liposomes consisting of phosphatidylcholine with arachidonic acid at the β-position and phosphatidylserine dipalmitoyl, and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-supported lipid peroxidation in the presence of iron complex in microsomes. Iron reduction responsible for lipid peroxidation in microsomes was not influenced by Mn 2+.

Inhibition of Methyl Linoleate Peroxidation by Maize Zein in Powder Model System at High Water Activity.

When stored at high relative water activity (Aw 0.9), zein has a prominent antioxidant activity against the peroxidation of methyl linoleate, while its papain hydrolysate does not. Using a lipid-soluble radical initiator, AMVN [2,2′-Azobis(2,4-dimethyl-valeronitrile)], to alter the induction period of peroxidation of methyl linoleate, we found a turning point just at 24 hr where the rate of oxygen absorption declined. The formation of a solid, tightly aggregated outer shield was observed by scanning electron microscopy. A rather amount of embedded substrate oil (methyl linoleate-methyl stearate, 7:3) was obtained when aggregations formed, and there was little change in the level of embedded oil for at least 16 days. On the other hand, a hydrolysate of zein had scarcely any embedding effect. The ratio of methyl linoleate to methyl stearate showed that embedded oil (hexane unextractable) was more protective to oxidation than hexane-extractable oil. These findings indicate that the substrate oil must have be...

The inhibition of radical-initiated peroxidation of microsomal lipids by both α-tocopherol and β-carotene

Rat liver microsomal lipids in hexane solution were exposed to the lipid-soluble radical initiator, azobis-isobutyronitrile (AIBN), and the antioxidant activities of α-tocopherol and β-carotene have been compared. Lipid peroxidation was monitored both by conjugated diene formation at 233 nm, and by malondialdehyde (MDA) formation in the thiobarbituric acid assay at 535 nm. Diene formation was continuous for at least 120 min in the presence of 85 μg/ml lipid and 4 mM AIBN. Both α-tocopherol and β-carotene acted as chain-breaking antioxidants, suppressing lipid peroxidation and producing an induction period at concentrations as low as 0.5 and 8 μM, respectively. When both of these lipid-soluble antioxidants were present together, the oxidation was strongly suppressed and the induction period was the sum of the individual antioxidants. α-Tocopherol and β-carotene also inhibited MDA generation. In the presence of 170 μg/ml lipid and 8 mM AIBN, β-carotene exhibited an IC 50 of 1.1 μM and inhibited completely at 15 μM. Using β-carotene, an induction period was observed, although much less pronounced than with α-tocopherol. Furthermore, β-carotene inhibited MDA production in a concentration-dependent manner and exhibited an IC 50 of 50 μM. In addition, added β-carotene delayed the radical-initiated destruction of the endogenous α-tocopherol and γ-tocopherol in this system.

CHARACTERIZATION OF RADICAL ADDUCTS FORMED DURING PHOTOCHEMICAL SPIN TRAPPING IN LIPOSOMES

There exists an urgent need to monitor radical reactions in biological membranes. With the exception of a few studies in model and natural membranes, most biological spin trapping reactions have been confined to homogeneous media. In the present work we have devised a methodology by which spin trapping reactions can be investigated in liposomes composed of a fully-saturated phospholipid, dimyristoylphosphatidylcholine. Using photochemical spin trapping, we have detected and characterized the formation and partitioning of spin adducts in liposomes. Benzophenone (a lipid-soluble photosensitizer) and benzoyl peroxide (a lipid-soluble radical initiator) have been used to generate free radicals from hydrogen donors during photolysis in liposomes. Both water-soluble and lipid-soluble spin traps have been used to trap the radicals.