Azobis 2-amidinopropane Hydrochloride Research Articles

Acid Phosphatase Reaction with Peroxyl Radicals: Inactivation Mechanism and Behavior of the Partially Modified Ensemble

Acid phosphatase (AP) is readily inactivated when exposed to the free radicals generated in the pyrolysis of 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH) under aerobic conditions. On average, a large number of tryptophan groups are modified by each protein molecule that loses its catalytic activity. In spite of this, the enzyme inactivation takes place without induction times, a result that indicates either that damage is progressive or that damage of a critical target is needed to inactivate the enzyme (all-or-nothing mechanism). A Lineweaver–Burk plot of the enzyme activity measured at pH 4.8 is not compatible with an all-or-nothing mechanism, showing that after exposure of the native protein ensemble to the free radical source there are partially damaged molecules whose affinity for the substrate is widely different from that of the native molecules. On the other hand, the partially damaged ensemble shows a normal Michaelis–Menten behavior when the activity is measured at pH 7.0, with only a reduced value of VM, relative to that of the unmodified ensemble. These results show that the native protein and modified proteins that remain active constitute different populations, with different responses to pH changes. Comparative heat denaturation studies of the native and pretreated proteins support this proposal.

Lidocaine inhibits potassium efflux and hemolysis in erythrocytes during oxidative stress in vitro

Lidocaine is a widely used local anesthetic agent. The aim of this work was to study the action of lidocaine on human red blood cells exposed to an oxidative stress in vitro. Blood was obtained from healthy volunteers. After separation from plasma, the erythrocytes were suspended in phosphate buffer. Oxidative stress was induced by incubation with a free radical generator, the 2,2′ azobis (2-amidinopropane) hydrochloride (AAPH). Erythrocytes were incubated with or without lidocaine at two concentrations (36.93 and 73.85 μM) and with or without AAPH (20 mM). Electron paramagnetic resonance (EPR) spectroscopy was performed to identify the free radical species generated by AAPH using the spin trap 5-5′-dimethyl- l-pyroline- N-oxide (DMPO). Different sets of experiments were run. Potassium efflux was measured by flame photometry in each group at time 0 min and every 30 min of the experiment for 2 h. Hemolysis was studied by the Drabkin method at increasing concentrations of AAPH (20, 50, and 100 mM) and with or without lidocaine (36.93 μM). The oxygen radical absorbance capacity (ORAC) was measured by using allophycocyanin (APC) as a fluorescent indicator protein, and the antioxidant capacity of lidocaine (36.93 μM) was studied by the analysis of fluorescence of the APC. AAPH was shown to produce alkoxyl free radicals. Oxidative stress induced a marked increase in the potassium efflux and the hemolysis that was AAPH dose-dependent. Lidocaine inhibited the potassium efflux and delayed the occurrence of hemolysis. Lidocaine did not show any antioxidant properties for the free radical species generated by AAPH. In this model, lidocaine protects erythrocytes against oxidative stress. This effect is not explained by a free radical scavenging property. The results may be of great interest in clinical practice such as intravenous regional anesthesia or the prevention of ischemia–reperfusion injury.

The effect of copper supplementation on red blood cell oxidizability and plasma antioxidants in middle-aged healthy volunteers

A multicenter European study (FoodCue) was undertaken to provide data on the significance of increased dietary copper as a pro-oxidant or antioxidant in vivo. The present work describes the effect of Cu supplementation on (2,2′-azo-bis(2-amidinopropane) hydrochloride (AAPH)-induced red blood cell oxidation in middle-aged people. Double-blinded copper supplementation was achieved in 26 healthy volunteers (50–70 years) with pills containing 3 mg CuSO 4, 3 mg Cu glycine chelate (CuG) and 6 mg CuG. Each 6 week supplementation period was preceded and followed by 6 weeks of washout (WO) on placebo. The results show significant increases in time necessary to achieve 50% hemolysis (LT 50) after 3CuSO 4 and 6CuG compared with values after WO periods. Cu supplementation did not increase the levels of (Cu,Zn)SOD activity in red blood cells. Resistance to hemolysis was significantly and positively correlated (r = .30, p < .01) with α- and β-carotene content in the plasma. Together, these data suggest that intake of copper as high as 7 mg/d has no pro-oxidant activity and may rather result in protection of red blood cells against oxidation. The decreased oxidizability of red blood cells did not result from increased (Cu,Zn)SOD activity and may occur through other mechanisms such as changes in membrane antioxidant content.

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.

Evaluation of antioxidant activities of rosehip ethanol extracts in different test systems

Rosehip extracts of 18 samples representing six taxa in the genus Rosa were evaluated for antioxidant activities by use of different test systems. The ferric-reducing antioxidant power (FRAP) and Trolox-equivalent antioxidant capacity (TEAC) of the crude extracts ranged from 983.4 to 2187.1 µmol FRAP g−1 dry matter and from 457.2 to 626.2 µmol TEAC g−1 dry matter. The high antioxidant capacity was related to high contents of phytonutrients. The overall mean of antioxidants was 23.23 mg g−1 total carotenoids and 76.26 mg g−1 total phenolics. The phenolic component made a major contribution to the total antioxidant activities in both assays (overall mean was 90.5% and 75.7%), whereas the ascorbate made a minor contribution (8.6% and 16.9%) and the lipophilic component made an even smaller one (0.9% and 7.3%). However, the lipophilic component was the most effective when the comparison was based on the ratio of antioxidant activity to content of antioxidants. The crude extracts exhibited 50.9% (46.6–60.3%) inhibitory effect against the lipid peroxidation induced by 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN) and 85.0% (80.1–90.2%) inhibition in 2,2′-azobis(2-amidinopropane)hydrochloride (AAPH) assay at a concentration of 250 µg ml −1. Ascorbate acted as an antioxidant in both peroxy radical-induced lipid peroxidations, but as a pro-oxidant in the metal ion-induced lipid peroxidation. The crude extracts showed a large inhibitory effect in the ferric ion-induced lipid peroxidation and caused 83.7% inhibition at a concentration of 25 µg ml −1 dried rosehip powder. © 2000 Society of Chemical Industry

Comparison of the effect of α-lipoic acid and α-tocopherol supplementation on measures of oxidative stress

In vitro studies have shown that α-lipoic acid (LA) is an antioxidant. There is a paucity of studies on LA supplementation in humans. Therefore, the aim of this study was to assess the effect of oral supplementation with LA alone and in combination with α-tocopherol (AT) on measures of oxidative stress. A total of 31 healthy adults were supplemented for 2 months either with LA (600 mg/d, n = 16), or with AT (400 IU/d, n = 15) alone, and then with the combination of both for 2 additional months. At baseline, after 2 and 4 months of supplementation, urine for F 2-isoprostanes, plasma for protein carbonyl measurement and low-density lipoprotein (LDL) oxidative susceptibility was collected. Plasma oxidizability was assessed after incubation with 100 mM 2,2′-azobis (2-amidinopropane) hydrochloride (AAPH) for 4 h at 37°C. LDL was subjected to copper- and AAPH-catalyzed oxidation at 37°C over 5 h and the lag time was computed. LA significantly increased the lag time of LDL lipid peroxide formation for both copper-catalyzed and AAPH-induced LDL oxidation ( p < .05), decreased urinary F 2-isoprostanes levels ( p < .05), and plasma carbonyl levels after AAPH oxidation ( p < .001). AT prolonged LDL lag time of lipid peroxide formation ( p < .01) and conjugated dienes ( p < .01) after copper-catalyzed LDL oxidation, decreased urinary F 2-isoprostanes ( p < .001), but had no effect on plasma carbonyls. The addition of LA to AT did not produce an additional significant improvement in the measures of oxidative stress. In conclusion, LA supplementation functions as an antioxidant, because it decreases plasma- and LDL-oxidation and urinary isoprostanes.

The protective effect on Cu 2+- and AAPH-mediated oxidation of human low-density lipoproteins depends on glycosaminoglycan structure

The effect of various glycosaminoglycans on Cu 2+- and AAPH-induced oxidation of human low-density lipoprotein (LDL) was studied by monitoring conjugated diene formation. Heparin (Hep) increased the lag phase (t lag) of LDL oxidation, and fast moving and slow moving Hep species modified the kinetics of LDL oxidation to the same extent. Beef spleen heparan sulfate (HS) sample produced a significant increase of the t lag and a decrease of the conjugated diene formation of LDL whilst beef kidney HS species modified LDL oxidation kinetics to a lower extent. Dermatan sulfate (DS) from different sources caused a significant increase of the t lag and a decrease of the conjugated diene formation of LDL. Hyaluronic acid had no effect. Chondroitin sulfate (CS) from beef trachea produced a very strong protective antioxidant effect evaluated by increasing of the t lag and decreasing of the conjugated diene formation. Hep was completely ineffective in protecting LDL from 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH)-mediated oxidation, whilst DS was moderately effective. Beef trachea CS showed a very strong ability to protect LDL oxidation induced by 1 mM AAPH. The different protective effect on Cu 2+- and AAPH-induced LDL oxidation by glycosaminoglycans is discussed considering their various structures and properties, and their capacity to interact to different extents with hydrophobic regions of LDL protein is confirmed by measuring the LDL-tryptophan fluorescence kinetics.

Antioxidant properties of aminoguanidine.

It is well known that aminoguanidine (AG) can diminish advanced glycosylation of proteins, which might be beneficial in preventing chronic diabetic complications. Recent reports suggested an inter-relationship between glycosylation of protein and free radical damage. In the present study, we examined the free radical scavenging properties of AG. Electron paramagnetic resonance using the spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was performed to determine the superoxide and hydroxyl radical scavenging abilities of AG. These experiments revealed that AG was an effective hydroxyl radical scavenger even though it expressed a direct inhibitory effect on the xanthine oxidase activity at high concentrations (AG > or = 5 mM). In the second part of the study, allophycocyanin was used as an indicator of free radical mediated protein damage. In the assay, 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) was used as a peroxyl radical generator, and the loss of allophycocyanin fluorescence was monitored. The antioxidant effect of AG was expressed in oxygen-radical absorbing capacity (ORAC), where one ORAC unit equals the net protection produced by 1 microM Trolox (a water soluble analogue of vitamin E) as a control standard. AG exhibited a significant dose-dependent effect against free radical damage. These radical scavenging properties of AG may contribute to protective effects during glycation and explain the prevention of diabetic complications.

ESR study on the structure–antioxidant activity relationship of tea catechins and their epimers

The purpose of this study is to examine the relationship between the free radical scavenging activities and the chemical structures of tea catechins ((−)-epigallocatechin gallate (EGCG), (−)-epigallocatechin (EGC) and (−)-epicatechin (EC)) and their corresponding epimers ((−)-gallocatechin gallate (GCG), (−)-gallocatechin (GC) and (+)-catechin ((+)-C)). With electron spin resonance (ESR) we investigated their scavenging effects on superoxide anions (O −· 2) generated in the irradiated riboflavin system, singlet oxygen( 1O 2) generated in the photoradiation–hemoporphyrin system, the free radicals generated from 2,2′-azobis(2-amidinopropane)hydrochloride (AAPH) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. The results showed that the scavenging effects of galloylated catechins (EGCG and GCG) on the four free radicals were stronger than those of nongalloylated catechins (EGC, GC, EC, (+)-C), and the scavenging effects of EGC and GC were stronger than those of EC and (+)-C. Thus, it is suggested that the presence of the gallate group at the 3 position plays the most important role in their free radical-scavenging abilities and an additional insertion of the hydroxyl group at the 5′ position in the B ring also contributes to their scavenging activities. Moreover, the corresponding phenoxyl radicals formed after the reaction with O −· 2 were trapped by DMPO and the ESR spectra of DMPO/phenoxyl radical adducts were observed ( a N=15.6 G and a H β =21.5 G). No significant differences were found between the scavenging effects of the catechins and their epimers when their concentrations were high. However, significant differences were observed at relatively low concentrations, and the lower their concentrations, the higher the differences. The scavenging abilities of GCG, GC and (+)-C were stronger than those of their corresponding epimers (EGCG, EGC and EC). The differences between their sterical structures played a more important role in their abilities to scavenge large free radicals, such as the free radicals generated from AAPH and the DPPH radical, than to scavenge small free radicals, such as O −· 2 and 1O 2, especially in the case with EGCG and GCG with more bulky steric hindrance.

Spin trapping of lipid peroxyl radical reassignment of secondary alkoxyl radical adducts: [formula omitted] and Ronald P. Mason〈BR〉National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709

This study deals with the activity of various vitamins against the radical-mediated oxidative damage in human whole blood. We have used a biological method that allows both the evaluation of plasma and that of red blood cell resistance against the free radicals induced by 2,2′-azobis (2-amidinopropane) hydrochloride (AAPH). Spin trapping measures using mainly 5-(diethoxyphosphoryl)-5-methyl-1-pyrolline N-oxide nitrone (DEPMPO) were carried out under several conditions to identify the free radicals implicated in this test. Only the oxygenated-centred radical generated from AAPH was found highly reactive to initiate red blood cell lysis. With DEPMPO only alkoxyl radicals were observed and no evidence was found for alkylperoxyl radicals. The antioxidant activity of several lipid- and water-soluble vitamins has been assessed by the biological assay and through two chemical methods. We have noticed high antioxidant activities for tocopherols (in the order δ>γ>α) in the biological test but not through chemical methods. At 1 μM, the δ-tocopherol efficiency in inhibiting radical-induced red blood cell hemolysis was three times as high as the α-tocopherol efficiency. For β-carotene no significant activity even in whole blood was shown. Highly surprising antioxidant activities were observed for acid folic and pyridoxine, compared to ascorbic acid. At 10 μM, the effectiveness of folic acid was almost three times as high as vitamin C. The biological test seems clinically more relevant than most other common assays because it can detect several classes of antioxidants.

Mutations at G:C base pairs predominate after replication of peroxyl radical-damaged pSP189 plasmids in human cells

The mutagenicity of peroxyl radicals, important participants in lipid peroxidation cascades, was investigated using a plasmid-based mutational assay system. Double-stranded pSP189 plasmids were incubated with a range of concentrations of the water-soluble peroxyl radical generator 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH). Following replication in human Ad293 cells, the plasmids were screened for supF mutations in indicator bacteria. Exposure to peroxyl radicals caused strand nicking and a decrease in transfection efficiency, which was accompanied by a significant increase in supF mutants. Each of these effects was abolished in the presence of the water-soluble vitamin E analogue Trolox. Automated sequencing of 76 AAPH-induced mutant plasmids revealed that substitutions at G:C base pairs were the most common changes, accounting for 85.5% of all identified mutations. Of these, most comprised G:C-->T:A transversions (53.5%), with lesser contributions by G:C-->A:T transitions (23.9%) and G:C-->C:G transversions (22.5%). Collectively, these data confirm our previous findings concerning the spectrum of mutations produced upon bacterial replication of peroxyl radical-damaged phage DNA and extend them by showing that such damage has mutagenic consequences during replication in more complex eukaryotic systems.

Effect of Hydroxytyrosol Found in Extra Virgin Olive Oil on Oxidative DNA Damage and on Low-Density Lipoprotein Oxidation

Hydroxytyrosol found in extra virgin olive oil strongly inhibited low-density lipoprotein oxidation stimulated by 2,2‘-azobis(2-amidinopropane) hydrochloride (AAPH), suggesting the ability to scavenge the AAPH-derived peroxyl radicals. Hydroxytyrosol inhibited iron-dependent phospholipid liposome peroxidation at low concentrations (IC50 = 50 ± 1.3 μM). In similar experiments, the calculated, IC50 values for other antioxidants compared are 1.5 ± 0.05 μM (carnosol), 2.25 ± 0.08 μM (carnosic acid), 65 ± 2.6 μM (Trolox C), and 250 ± 10 μM (vitamin E). Hydroxytyrosol and ascorbate reduced copper(II) ions to their copper(I) prooxidant form, but this was not reflected by their abilities to induce oxidative DNA damage in the complex copper−phenanthroline. Only high, nonphysiological, millimolar concentrations of pure hydroxytyrosol weakly stimulated copper-dependent chemical modification to DNA bases. The prooxidant (redox actions on metal ions) concentrations in vitro may never be achieved in vivo (following con...

Atorvastatin and gemfibrozil metabolites, but not the parent drugs, are potent antioxidants against lipoprotein oxidation

Increased atherosclerosis risk in hyperlipidemic patients may be a result of the enhanced oxidizability of their plasma lipoproteins. We have previously shown that hypocholesterolemic drug therapy, including the 3-hydroxy-3-methyl-glutaryl CoenzymeA (HMG-CoA) reductase inhibitors, and the hypotriglyceridemic drug bezafibrate, significantly reduced the enhanced susceptibility to oxidation of low density lipoprotein (LDL) isolated from hyperlipidemic patients. Although this antioxidative effect could not be obtained in vitro with all of these drugs, the active drug metabolites, which are formed in vivo, could affect lipoprotein oxidizability. We thus sought to analyze the effect of atorvastatin and gemfibrozil, as well as specific hydroxylated metabolites, on the susceptibility of LDL, very low density lipoprotein (VLDL), and high density lipoprotein (HDL) to oxidation. LDL oxidation induced by either copper ions (10 microM CuSO4), by the free radical generator system 2'-2'-azobis 2-amidino propane hydrochloride (5 mM AAPH), or by the J-774A.1 macrophage-like cell line, was not inhibited by the parent forms of atorvastatin or gemfibrozil, but was substantially inhibited (57-97%), in a concentration-dependent manner, by pharmacological concentrations of the o-hydroxy and the p-hydroxy metabolites of atorvastatin, as well as by the p-hydroxy metabolite (metabolite I) of gemfibrozil. On using the atorvastatin o-hydroxy metabolite and gemfibrozil metabolite I in combination an additive inhibitory effect on LDL oxidizability was found. Similar inhibitory effects (37-96%) of the above metabolites were obtained for the susceptibility of VLDL and HDL to oxidation in the oxidation systems outlined above. The inhibitory effects of these metabolites on LDL, VLDL, and HDL oxidation could be related to their free radical scavenging activity, as well as (mainly for the gemfibrozil metabolite I) to their metal ion chelation capacities. In addition, inhibition of HDL oxidation was associated with the preservation of HDL-associated paraoxonase activity. We conclude that atorvastatin hydroxy metabolites, and gemfibrozil metabolite I possess potent antioxidative potential, and as a result protect LDL, VLDL, and HDL from oxidation. We hypothesize that in addition to their beneficial lipid regulating activity, specific metabolites of both drugs may also reduce the atherogenic potential of lipoproteins through their antioxidant properties.

Direct evidence of caeruloplasmin antioxidant properties.

The chain-breaking antioxidant potential of caeruloplasmin and bovine serum albumin (BSA) has been investigated in comparison with other well-established antioxidants. Their Oxygen Radical Absorbing Capacity (ORAC), was measured by using beta-phycocyanin (beta-PC) as a fluorescent indicator protein, 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) as a peroxyl radical generator and the water soluble vitamin E analogue, Trolox, as a reference standard. The relative peroxyl absorbing capacities/mole for Trolox, caeruloplasmin, heat-denatured caeruloplasmin (hCP), catalase, bovine serum albumin (BSA), superoxide dismutase (SOD), and deferoxamine were 1; 2.6; 3.3; 3.7; 1.2; 0.1; 0.2, respectively. Caeruloplasmin was far more effective as a peroxyl radical scavenger than SOD, deferoxamine and BSA, but slightly less effective than catalase. The peroxyl radical absorbing capacity of caeruloplasmin was enhanced by heat-denaturation of the protein. Electron paramagnetic resonance (EPR) spectroscopy using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin-trap, was applied in order to measure the scavenger abilities of caeruloplasmin on superoxide radical and hydroxyl radical production and the concentration required to inhibit by 50% oxygen free radical formation (IC50) was determined. The IC50 values of caeruloplasmin, hCP, and BSA for the superoxide radical were 12, 2, 260 microM and for the hydroxyl radical 15, 2, 200 microM. These results show that caeruloplasmin is an effective chain-breaking antioxidant for a variety of radicals, independently of its catalytic ferroxidase activity.

Antioxidant properties of indapamide, 5-OH indapamide and hydrochlorothiazide evaluated by oxygen-radical absorbing capacity and electron paramagnetic resonance.

The aim of these experiments was to investigate the radical scavenging properties of three diuretics: indapamide (IND) and its major metabolite, 5-OH indapamide (5-OH IND), compared to a reference diuretic, hydrochlorothiazide (HTZ). Electron Paramagnetic Resonance (EPR) was used to determine the scavenging abilities of these compounds on enzymatically produced superoxide radical anion, with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) used as a spin-trap. These experiments revealed that IND and specially 5-OH IND were effective superoxide radical anion scavengers at 0.2 mg/ml. In the second part of these studies, allophycocyanin was used as an indicator of free radical mediated protein damage. In the assay, 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) was used as a peroxyl radical generator, Trolox (a water-soluble analogue of vitamin E) as a control standard, and the loss of allophycocyanin fluorescence was monitored. The antioxidant effects of the diuretics were expressed in oxygen-radical absorbing capacity (ORAC), where one ORAC unit equals the net protection produced by 1 microM Trolox. HTZ showed no protection up to 100 microM final concentration, whereas IND and 5-OH IND showed linear correlation with respect to concentration when expressed in ORAC units: 5-OH IND induced the highest protection against peroxyl radical. The above observations suggested that IND and 5-OH IND are potent radical scavengers, with the metabolite 5-OH IND having a superior antioxidant potency than IND. By contrast, HTZ had no effect. These radical scavenging properties of 5-OH IND may be of clinical interest for vascular protection and may help to protect the heart from oxidative injury.

F4-Isoprostanes: A Novel Class of Prostanoids Formed during Peroxidation of Docosahexaenoic Acid (DHA)

Isoprostanes are prostaglandin (PG)-like compounds derived from free radical-catalyzed peroxidation of polyunsaturated fatty acids. F2-isoprostanes are produced in vivo by a non-cyclooxygenase mechanism involving free radical peroxidation of arachidonic acid. Peroxidation of eicosapentaenoic acid produces F3-isoprostanes. In this study, we explore the possibility of formation of F4-isoprostanes during peroxidation of docosahexaenoic acid (DHA) in vitro. DHA-liposomes were exposed at 37°C to either 2, 2′-azobis-(2-amidinopropane) hydrochloride (AAPH) or copper ions at final concentrations of 10 mM and 50 μM, respectively. Sample processing involved solid-phase extraction on a C18and an NH2- cartridge. After conversion to pentafluorobenzyl ester and trimethylsilyl derivatives, F4-isoprostanes were analysed by negative ion-chemical ionisation mass spectrometry using tetradeuterated PGF2α(PGF2-d4) as the internal standard. Quantitative analysis was carried out by selected ion monitoring (SIM) of the carboxylated anion [M-180]−at m/z 593 and 573 for the F4-isoprostanes and PGF2-d4, respectively. DHA oxidised by AAPH or by copper ions gave rise to a similar family of F4-isoprostanes. Formation of F4-isoprostanes increased throughout the oxidation period and was correlated with other indices of lipid peroxidation (hydroperoxides and thiobarbituric acid reactive substances). The possibility of analyzing F4-isoprostanes should provide new opportunities for studying the role of lipid peroxidation in nutritional studies and in the pathogenesis of neurodegenerative diseases.

Suitability of chemical in vitro models to investigate LDL oxidation: study with different initiating conditions in native and α-tocopherol-supplemented LDL

Isolated human LDL, used in the native form or supplemented with alpha-tocopherol (alpha T), were oxidized with Cu2+, 2,2'-azobis-(2-amidino propane) hydrochloride (AAPH), and H2O2 plus horseradish peroxidase (HRP). The oxidation kinetics were measured spectrophotometrically at 234 nm to follow the formation of conjugated dienes and evaluated as resistance to oxidation (lag phase, LP) and maximal oxidation rate (propagation rate, PR). The duration of LP in nonsupplemented LDL was different with the three prooxidant stimuli (LP, in min: 96 +/- 19 for Cu2+, 28.7 +/- 6.7 for HRP, and 67.1 +/- 11.2 for AAPH). No correlation was found between the values obtained with Cu2+ and AAPH or HRP, but a significant correlation was found with AAPH and HRP (r = 0.798, P < 0.002). In vitro alpha T supplementation prolonged the LP and decreased the PR with all the stimuli. The extent of increase in LP was highly correlated (r = 0.872, P < 0.001 for Cu2+ and HRP; r = 0.603, P < 0.03 for Cu2+ and AAPH; r = 0.749, P < 0.005 for AAPH and HRP). Although the evaluation of ex vivo LDL oxidation is dependent on the prooxidant stimulus, the three prooxidant conditions used detect equally well the efficiency of alpha T supplementation in preventing LDL oxidation.

Licorice extract and its major polyphenol glabridin protect low-density lipoprotein against lipid peroxidation: in vitro and ex vivo studies in humans and in atherosclerotic apolipoprotein E-deficient mice

Polyphenolic flavonoids are powerful antioxidants. In the present study we investigated the antioxidative activity against low-density-lipoprotein (LDL) oxidation of a not yet studied subclass of polyphenols, the isoflavans, which are present in licorice alcoholic extract. The study was performed in humans as well as in atherosclerotic apolipoprotein E-deficient mice (E zero), because their LDL is highly susceptible to oxidation. LDL oxidation was induced by incubating it with copper ions as well as with the aqueous or lipid-soluble free radical generators 2,2'-azobis'2-amidino propane hydrochloride (AAPH) and 2,2'-azobis 2,4-dimethylvaleronitrile (AMVN), respectively. The extent of LDL oxidation was determined by measuring the formation of conjugated dienes, thiobarbituric acid reactive-substances (TBARS), and lipid peroxides. By all methods in human studies, licorice ethanolic extract as well as a pure material, which was identified by gas chromatography-mass spectroscopy as the isoflavan glabridin, were shown to inhibit LDL oxidation by a mechanism involving scavenging of free radicals. In an ex vivo study, LDL isolated from the plasma of 10 normolipidemic subjects who were orally supplemented for 2 wk with 100 mg licorice/d was more resistant to oxidation than was LDL isolated before licorice supplementation. Dietary supplementation of each E zero mouse with licorice (200 micrograms/d) or pure glabridin (20 micrograms/d) for 6 wk resulted in a substantial reduction in the susceptibility of their LDL to oxidation along with a reduction in the atherosclerotic lesion area. These results could be related to the absorption and binding of glabridin to the LDL particle and subsequent protection of the LDL from oxidation by multiple modes as shown in humans and in E zero mice.

Oxidation of Low Density Lipoprotein Particles Decreases Their Ability to Bind to Human Aortic Proteoglycans: DEPENDENCE ON OXIDATIVE MODIFICATION OF THE LYSINE RESIDUES

Oxidation of low density lipoprotein (LDL) leads to its rapid uptake by macrophages in vitro, but no detailed studies have addressed the effect of oxidation on the binding of LDL to proteoglycans. We therefore treated LDL with various substances: copper sulfate, 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH), soybean lipoxygenase, and mouse peritoneal macrophages, and determined the extent to which the oxidatively modified LDL bound to human aortic proteoglycans in an affinity column. Oxidation of LDL with copper, AAPH, or macrophages, all of which increased its electrophoretic mobility, was associated with reduced binding to proteoglycans, until strongly oxidized LDL was totally unable to bind to them. After treatment of LDL with soybean lipoxygenase, the change in electrophoretic mobility was small, and the amount of binding to proteoglycans was only slightly decreased. The increased electrophoretic mobility of oxidized LDL reflects modification of the lysine residues of apolipoprotein B-100 (apoB-100). To mimic the oxidative modification of lysines, we treated LDL with malondialdehyde. This treatment also totally prevented the binding of LDL to proteoglycans. In contrast, if the lysine residues of apoB-100 were methylated to shield them against oxidative modification, subsequent treatment of LDL with copper sulfate failed to reduce the degree of LDL binding to proteoglycans. Finally, the active lysine residues in the oxidized LDL particles, which are thought to be involved in this binding, were quantified with NMR spectroscopy. In oxidized LDL, the number of these residues was found to be decreased. The present results show that, after modification of the lysine residues of apoB-100 during oxidation, the binding of LDL to proteoglycans is decreased, and suggest that oxidation of LDL tends to lead to intracellular rather than extracellular accumulation of LDL during atherogenesis.

Influence of Follicular Maturation on the Susceptibility of Chicken Ovarian Granulosa Cells to Free Oxygen Radicals

The influence of ovarian follicular maturation on lipid peroxidation-induced changes in the fluidity of plasma membrane of granulosa cells was investigated using the fluorogenic polyunsaturated fatty acid cis-parinaric acid (cPNA). An increase in lipid peroxidation results in the decrease of fluorescence intensity of cPNA. A decrease in membrane fluidity results in the decrease of fluorescence polarization of cPNA. Granulosa cells isolated from the largest (F1; mature), and third largest (F3; developing) preovulatory follicles as well as from a pool of immature small yellow follicles (SYF) of the domestic hen ovary were pre-labelled with cPNA. The cPNA-labelled cells were exposed to a free oxygen radical generating agent 2,2′azobis-(2-amidinopropane)-hydrochloride (AAPH). AAPH enhanced the rate of decrease in fluorescence intensity of cPNA-labelled cells. This effect of AAPH was greater in the more differentiated granulosa cells obtained from mature follicles than in less diferentiated cells isolated from immature follicles. Similarly, the degree of change in fluorescence polarization was greater in more differentiated F1 granulosa cells than in the less differentiated ones. The analysis of fatty acid composition of phospholipids indicated that the plasma membrane of differentiated granulosa cells contained a higher proportion of the unsaturated fatty acids, oleic and linoleic acids. The fluorescence data show that the rigidity of the plasma membrane of chicken granulosa cells decreases with advancing follicular maturation presumably due to relative increase in the unsaturated fatty acid content of plasma membrane phospholipid.