Azobis 2-amidinopropane Hydrochloride Research Articles

Guideline for screening antioxidant against lipid‐peroxidation by spectrophotometer

AbstractLipid peroxidation (LPO), an oxidative chain reaction often occurring in the human body and food, is harmful to human health. Due to its convenience and low cost, this guideline highlights the benefits and drawbacks of LPO models by spectrophotometer in vitro and in vivo. The preparation process and reaction mechanism of lipid substrates, oxidation triggers and detection methods are discussed in this paper to better screen antioxidants. The lipid substrates mostly consist of rat liver/brain homogenate, microsomal, low‐density lipoprotein (LDL), and linoleic acid (LA), are appropriate for modeling oxidative damage in a variety of situations. Transition metals, ascorbic acid, 2,2′‐azobis (2‐amidinopropane) hydrochloride (AAPH), sodium Nitroprusside (SNP), 3‐morpholinosydnonimine (SIN‐1) and nicotinamide adenine dinucleotide phosphate (NADPH) make up the majority of the oxidizing triggers. The following LPO quantification techniques have been reviewed: (a) thiobarbituric acid reactive substances (TBARS) assay, (b) measurement of conjugated‐diene (CV), (c) β‐carrot bleaching assay, (d) thiocyanate method, (e) ferrous oxidation‐xylenol orange (FOX‐2) assay. It can be concluded that more than one test system should be applied to obtain comprehensive results. In a word, this guideline provides a scientific basis for future research on antioxidants screening in food and body systems.

Hybridization of Curcumin Analogues with Cinnamic Acid Derivatives as Multi-Target Agents Against Alzheimer's Disease Targets.

The synthesis of the new hybrids followed a hybridization with the aid of hydroxy-benzotriazole (HOBT) and 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI.HCL) in dry DMF or thionyl chloride between curcumin analogues and cinnamic acid derivatives. IR, 1H-NMR, 13C-NMR, LC/MS ESI+, and elemental analysis were used for the confirmation of the structures of the novel hybrids. The lipophilicity values of compounds were calculated theoretically and experimentally via the reversed chromatography method as RM values. The novel derivatives were studied through in vitro experiments for their activity as antioxidant agents and as inhibitors of lipoxygenase, cyclooxygenase-2, and acetyl-cholinesterase. All the compounds showed satisfying anti-lipid peroxidation activity of linoleic acid induced by 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH). Hybrid 3e was the most significant pleiotropic derivative, followed by 3a. According to the predicted results, all hybrids could be easily transported, diffused, and absorbed through the blood–brain barrier (BBB). They presented good oral bioavailability and very high absorption with the exception of 3h. No inhibition for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 was noticed. According to the Ames test, all the hybrids induced mutagenicity with the exception of 3d. Efforts were conducted a) to correlate the in vitro results with the most important physicochemical properties of the structural components of the molecules and b) to clarify the correlation of actions among them to propose a possible mechanism of action. Docking studies were performed on soybean lipoxygenase (LOX) and showed hydrophobic interactions with amino acids. Docking studies on acetylcholinesterase (AChE) exhibited: (a) hydrophobic interactions with TRP281, LEU282, TYR332, PHE333, and TYR336 and (b) π-stacking interactions with TYR336.

Dual Effect of Histidine on Polysorbate 20 Stability: Mechanistic Studies.

L-Histidine (L-His) and polysorbate 20 (PS20) are two excipients frequently included in parenteral products to stabilize biotherapeutics. The objective of the current work was to investigate the impact of L-His on PS20 stability in aqueous solutions when subjected to forced oxidation and accelerated stability testing. The stability of PS20 in L-His buffer was compared with that in acetate buffer. Forced oxidation of PS20 in these two buffer systems was initiated by a free radical generator, 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH), while accelerated stability tests were carried out at 40°C. Ultra-performance liquid chromatography mass spectrometry was utilized to monitor intact PS20 and to analyze degradation products. Our results demonstrate a dual effect of L-His on PS20 stability. During exposure to AAPH, L-His protects PS20 from oxidation. Stable isotope labeling of L-His with 13C was employed for mechanistic investigations. The protection of L-His was abrogated when acetate was added to L-His buffer, implying that the anti-oxidative activity of L-His may be compromised by specific counter ions. The replacement of L-His by various His derivatives led to significant changes in the protection of PS20 against AAPH-induced degradation. In contrast to forced degradation, the addition of L-His promoted oxidative PS20 degradation during accelerated storage at 40°C in solution, generating mainly short chain POE-laurates. L-His exhibits a dual effect on the stability profile of PS20, protecting against AAPH-induced oxidation but promoting oxidative degradation during accelerated stability testing.

Antioxidant activities of novel galloyl phytosterols evaluated by human erythrocytes with the aid of confocal microscopy imaging

Notable protective effect of the novel galloyl phytosterol antioxidant on human erythrocytes was disclosed in this study. 2,2′-Azo-bis (2-amidinopropane) hydrochloride (AAPH) induced haemolysis of erythrocytes, and depletion of cellular glutathione were effectively inhibited by the galloyl phytosterol antioxidant. It was revealed that the level of glutathione was a more sensitive indicator than haemolysis for cellular oxidative stress. The morphology of erythrocytes obtained through laser scanning confocal microscopy (LSCM) imaging provided structural details for haemolysis and glutathione investigation and was more sensitive than the level of glutathione. The liposome model exhibited that the novel antioxidant preferred to anchor in the membrane and had better performance for membrane protection than gallic acid. The strategy of integrated use of biochemical and visual methods developed in this study is valuable for investigation on mechanistic behaviours of antioxidants.

Protective effect of Scutellaria species on AAPH-induced oxidative damage in human erythrocyte.

Scutellaria baicalensis is a well-known plant in traditional Chinese medicine. Recently, several Scutellaria species with therapeutic potential have been recognized worldwide. Scutellaria colebrookiana and Scutellaria violacea, native to the Western Ghats of India, are reported to possess free radical scavenging efficacy. At present, the protective effect of these Scutellaria spp. against 2,2' azobis (2-amidinopropane) hydrochloride (AAPH)-induced oxidative damage in human erythrocytes has been analyzed. Oxidative stress in erythrocyte was induced by AAPH. The inhibition of hemolysis, membrane lipid peroxidation, and protein damage by chloroform extracts of Scutellaria spp. was assessed biochemically. Phytochemicals of the extracts were analyzed by Fourier transform infrared spectrophotometer (FTIR). Approximately 95% of erythrocytes were lysed by AAPH over 3 h of incubation. Significant reduction in hemolysis was observed by the extracts, and the IC50 values were 18.3 and 23.5 μg/mL for S. colebrookiana and S. violacea, respectively. Both the extracts were found to inhibit AAPH-induced lipid peroxidation in ghost membrane with IC50 92±2.8 and 70±5.6 μg/mL. In the analysis of the membrane proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the AAPH-induced degradation of actin was found reduced by both the extracts. The FTIR spectrum revealed the presence of polyphenols, carboxylic acids, alkanes, and aromatic compounds in extracts. In quantitative analysis, the total polyphenolic content estimated was 380±0.23 and 203.7±1.4 mg of gallic acid equivalent per gram extract of S. colebrookiana and S. violacea. Results indicate that S. colebrookiana and S. violacea are capable of protecting erythrocytes from oxidative damage. This cytoprotective effect of the extract is possibly by its antioxidant property.

0347 : Diuretic, antihypertensive and antioxidant effect of olea europaea leaves extract, in rats

Although, there are known the beneficial effects of the fruits and the olive oil, lately a special attention was given to extracts obtained from olive leaves. The studies have reported that oleuropein (the main active component of olive leaves) has antioxidant, vasodilator, anti-inflammatory properties. 30 male Sprague-Dawley rats were randomized in 3 groups (n=10) that received orally for seven days: 1. distillate water (control group), 2. Furosemide (10mg/kg), and 3. Leaves extract of Olea europaea (OE). After the first and last dose of vegetal extract, the diuresis and saluresis (urinary Na+ and K+ concentration) were determined. At the end of the study the systolic blood pressure was recorded, by a non-invasive method. Human erythrocytes were subjected to an oxidative stress induced by 2,2’-azobis (2-amidinopropane) hydrochloride (AAPH) in the presence or absence of increasing concentrations of OE, lipoic acid and trolox (the last two substances were used like reference antioxidants). Furosemide and OE leaves extract induced a significant (p<0.05) increase in rats diuresis and saluresis as compared to control group. For OE extract, the same effect intensity was recorded after the first and the last doses. Also after seven days of treatment, the rats’ systolic blood pressure was significantly reduced in OE group. In vitro conditions, in the presence of OE extract, extracellular potassium efflux, the signs of erythrocyte membranes early degradation by oxidative stress, was decreased significantly and dosedependent manner. In our experimental conditions it appears that, OE leaves extract presented diuretic and antihypertensive effects. Also erythrocyte membranes are very significantly protected by the antioxidant compound from OE. Our results could open new perspectives for the use of Olea europaea leaves extract like adjuvant therapy in diseases associated with hypertension and oxidative stress.

English

Oxidative stress, specii¬cally lipid peroxidation, contributes to the pathogenesis of asthma. A natural antioxidant could be a potential therapeutic intervention. Hydro-alcoholic extract of Ixora coccinea (ICE) exhibit the anti-asthmatic activity in an ovalbumin (OVA) induced asthmatic rat model. These facts led us to examine their antioxidant activities. The free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity and the intracellularly antioxidant activity of ICE were determined. The protective effect of ICE against 2,2′ azobis (2-amidinopropane) hydrochloride (AAPH)-induced red blood cell lysis was also evaluated. It was found that ICE could scavenge DPPH with an IC50 of 283.3 µg/ml and protected red blood cell against AAPH-induced hemolysis with an IC50 of 72.92 versus 52.08 µg/ml for ascorbic acid. Erythrocytes obtained from the ICE-administrated rats showed an enhanced resistance to hemolysis. In OVA-induced asthma, rats were sensitized and challenged with ovalbumin. The effect of ICE at 1500 mg/kg per os on malondialdehyde (MDA) production and lung catalase activity were determined. ICE significantly reduced the lipid peroxidation and enhanced catalase activity in lung (p < 0.05). In conclusion, the hydro-alcoholic extract of I. coccinea possesses an antioxidant activity and protective effect against free-radical-induced hemolysis. This may explain the traditional use of this plant as a remedy against asthma and other diseases. Key words: Asthma, oxidative stress, antioxidants, Ixora coccinea.

In vitro lipid peroxidation of intestinal bile salt-based nanoemulsions: Potential role of antioxidants

Over the last decades, oxidative stress has been described as a deleterious phenomenon contributing to numerous noncommunicable diseases such as cardiovascular disease, diabetes, and cancers. As many authors ascribed the healthy effect of fruit and vegetable consumption mainly to their antioxidant contents, it has been hypothesized that their protection could occur from the gut. Therefore, the aim of this study was to develop an original and physiological model of nanoemulsions to study lipid peroxidation within the intestine and to assess the properties of potential antioxidants in this setting.Several nanoemulsions were compared in terms of physical characteristics and reactivity to 2,2’-azobis-(2-amidinopropane) hydrochloride (AAPH)-induced oxidation. Formulations included different types of lipids, a detergent (a conjugated bile salt or sodium dodecyl sulfate) and, finally, lipophilic antioxidants. Hemin and myoglobin were also tested as relevant potential oxidants. Fatty acid (FA) peroxidation was monitored by gas chromatography while malondialdehyde and antioxidant contents were measured by HPLC.Investigated nanoemulsions were composed of spherical or cylindrical mixed micelles, the latter being the least resistant to oxidation. In the experimental conditions, AAPH was the only efficient oxidant. Alpha-tocopherol and lutein significantly slowed FA degradation from 4 to 1 μM, respectively. On the contrary, beta-carotene did not show any protective capacity at 4 μM.In conclusion, the tested nanoemulsions were appropriate to assess antioxidant capacity during the intestinal phase of digestion.

Chemical Modification of Lysozyme, Glucose 6-Phosphate Dehydrogenase, and Bovine Eye Lens Proteins Induced by Peroxyl Radicals: Role of Oxidizable Amino Acid Residues

Chemical and structural alterations to lysozyme (LYSO), glucose 6-phosphate dehydrogenase (G6PD), and bovine eye lens proteins (BLP) promoted by peroxyl radicals generated by the thermal decomposition of 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) under aerobic conditions were investigated. SDS-PAGE analysis of the AAPH-treated proteins revealed the occurrence of protein aggregation, cross-linking, and fragmentation; BLP, which are naturally organized in globular assemblies, were the most affected proteins. Transmission electron microscopy (TEM) analysis of BLP shows the formation of complex protein aggregates after treatment with AAPH. These structural modifications were accompanied by the formation of protein carbonyl groups and protein hydroperoxides. The yield of carbonyls was lower than that for protein hydroperoxide generation and was unrelated to protein fragmentation. The oxidized proteins were also characterized by significant oxidation of Met, Trp, and Tyr (but not other) residues, and low levels of dityrosine. As the dityrosine yield is too low to account for the observed cross-linking, we propose that aggregation is associated with tryptophan oxidation and Trp-derived cross-links. It is also proposed that Trp oxidation products play a fundamental role in nonrandom fragmentation and carbonyl group formation particularly for LYSO and G6PD. These data point to a complex mechanism of peroxyl-radical mediated modification of proteins with monomeric (LYSO), dimeric (G6PD), and multimeric (BLP) structural organization, which not only results in oxidation of protein side chains but also gives rise to radical-mediated protein cross-links and fragmentation, with Trp species being critical intermediates.

Oxidative stress parameters in serum and low density lipoproteins of Hashimoto's thyroiditis patients with subclinical and overt hypothyroidism

BackgroundAlthough prooxidant and antioxidant status were reported to be changed in clinical and experimental hypothyroidism, obtained results are conflicting. In addition, in subclinical hypothyroidism, scarced and controversial data are available about oxidative stress. Therefore, we aimed to investigate prooxidant–antioxidant status only in Hashimoto's thyroiditis (HT) patients with subclinical (sHT) and overt hypothyroidism (oHT). Subjects and methodsThirty sHT and 18 oHT patients and 30 healthy control subjects were included in the study. Endogenous and prooxidant 2,2′-azobis-(2-amidinopropane) hydrochloride (AAPH)-induced malondialdehyde (MDA), diene conjugate (DC), protein carbonyl (PC) and nitrotyrosine (NT) levels as well as ferric reducing antioxidant power (FRAP) were determined in serum. In addition, endogenous DC and copper-induced MDA levels were measured in low density lipoprotein (LDL) fraction. ResultsAlthough there were no significant difference in serum endogenous MDA and DC levels, AAPH-induced MDA levels were significantly increased in sHT patients. All these parameters increased in oHT patients. Serum PC levels were detected to be increased in both sHT and oHT patients. Serum FRAP values did not alter in sHT patients, but they lowered in oHT patients. Endogenous DC and copper-induced MDA levels in LDL fraction did not change in sHT patients. However, these parameters were detected to be increased significantly in oHT patients as compared to controls and sHT patients. ConclusionIn conclusion, there were significant increases in oxidative stress parameters in serum and LDL-fraction in oHT patients. However, oxidative stress was detected to stimulate partly in serum, but not LDL fraction in sHT patients.

Comparative kinetics of thiol oxidation in two distinct free-radical generating systems: SIN-1 versus AAPH

To study oxidative stress in biological systems, chemical compounds capable of producing free radicals have been widely used. Here, we compared two free-radical generators, 3-morpholinosydnonimine (SIN-1) and 2,2′-azo-bis(2-amidinopropane) hydrochloride (AAPH), by measuring the thiol oxidation kinetics of various thiols. We found that SIN-1 is > 30 times potent in causing thiol oxidation than AAPH. Kinetic simulations revealed that in the SIN-1 system (0.1 mM), superoxide, nitrogen dioxide and carbonate radicals are the major reactive species which, in combination, induce ∼50% of thiol molecules to undergo one-electron oxidation, thereby forming the thiyl radical which propagates further thiol oxidation by direct coupling with thiolates. Similarly, the alkyl peroxyl radical derived from AAPH (3 mM) initiates comparable extent of one-electron oxidation and formation of the thiyl radical. In conclusion, our study provides experimental and theoretical evidence that SIN-1 is mainly an one-electron oxidizing agent that can be functionally mimicked by AAPH.

Synthesis of 4-methylcoumarin derivatives containing 4,5-dihydropyrazole moiety to scavenge radicals and to protect DNA

A series of 4-methylcoumarin derivatives containing 4,5-dihydropyrazole moiety were synthesized and their antioxidant activities were evaluated in AAPH (2,2′-azobis(2-amidinopropane hydrochloride))-induced oxidation of DNA, and in trapping DPPH (2,2′-diphenyl-1-picrylhydrazyl) and ABTS+• (2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical), respectively. Among coumarin derivatives, 3a–d and 4a–c exhibited the termination of radical propagation-chains in AAPH-induced oxidation of DNA. The ortho dihydroxyphenyl substitution at 5 position and 1-unsubstitution of the 4,5-dihydroxylpyrazole was found enhancing the antioxidant activities of these coumarin derivatives.

Evaluation of in vitro antioxidant and antitumour activities of Astragalus membranaceus aqueous extract

Aqueous extracts of Astragalus membranaceus (AMEE) were prepared in this study. In vitroantioxidant and antitumour activities of aqueous extracts of A. membranaceus were evaluated. The aqueous extracts exhibited could effectively scavenge superoxide anion, Hydrogen peroxide, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and reduce 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH)-induced human erythrocytes hemolysis. Moreover, aqueous extracts of A. membranaceus could still effectively dose-dependently inhibit against the human Gastric cancer cell line SGC-7901 and human hepatoma SMMC-7721 cells growth at the given dose range. The results indicate that the aqueous extracts of A. membranaceus have a very potent antioxidant and antitumour activity. Key words: Astragalus membranaceus aqueous extracts, antioxidant, antitumour, SGC-7901, SMMC-7721.

Potential Health Implication of in Vitro Human Low-Density Lipoprotein–Vitamin E Oxidation Modulation by Polyphenols Derived from Côte d’Ivoire’s Oil Palm Species

Antioxidant activities of polyphenolic compounds extracted (PPEs) from ripe fruits of oil palms are investigated by studying their in vitro effects on human low-density lipoprotein (LDL) oxidation. Four oil palm species ( Elaeis guineensis ) are issued from the National Centre of Agronomic Research of Côte d'Ivoire, of which two are parental varieties (HP1 and HP2), while the other two are crossing varieties (HP3 and HP4). The main identified compounds were rutin (HP3 and HP4) and caffeic and chlorogenic (5-caffeoyl quinic) acids (HP1, HP3, and HP4). The highest total phenolic content was found for HP4, while it was significantly lower for HP2. Antioxidative effects were monitored by Cu(2+)- or 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH)-induced generation of conjugated dienes (lag time and oxidation rate). The highest PPE specific antioxidant activity (SAA) values were obtained with crossing varieties (HP3 and HP4) in the copper-oxidation assay. In the AAPH-oxidation assay, SAA values were comparable for all four varieties. PPEs were effective at preventing LDL-vitamin E depletion in vitro. They could exert direct beneficial antioxidant effects on vitamin E and other antioxidants contained in food and beverages in vivo, within the gastrointestinal (GI) tract. These data could also be of particular importance for a healthier nutrition or the management of chronic diseases by a polyphenol-rich diet.

The biliverdin–bilirubin antioxidant cycle of cellular protection: Missing a wheel?

Bilirubin reportedly protects cultured cells from the toxicity of a 10,000-fold molar excess of H 2O 2. A bilirubin–biliverdin cycling mechanism has been proposed to explain this remarkable effect whereby bilirubin reacts with oxyradicals specifically generating biliverdin, which is then reduced back to bilirubin by NADPH/biliverdin reductase. Chemical evidence for this mechanism was formation of biliverdin during incubation of bilirubin–albumin with 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) in vitro and the assumption that biliverdin was formed by the reaction of peroxyl radicals with bilirubin. This paper describes spectroscopic studies on the reaction of bilirubin with AAPH in the presence and absence of human serum albumin. Reactions were run in air and also under oxygen-depleted and oxygen-saturated solutions, the former to inhibit peroxyl radical formation, the latter to augment it. The results confirm that degradation of bilirubin, rather than dehydrogenation to biliverdin, predominates in the reaction of bilirubin with peroxyl radicals generated by AAPH thermolysis. They also suggest that biliverdin produced in the presence of albumin is not formed by the reaction of bilirubin with alkyl peroxyl radicals, as previously assumed. The observations undermine the plausibility of the bilirubin–biliverdin recycling mechanism proposed to explain the reported hyperprotective effect of bilirubin on mammalian cells exposed to excess H 2O 2.

Inhibitory Effect of Gallic Acid and Its Esters on 2,2′-Azobis(2-amidinopropane)hydrochloride (AAPH)-Induced Hemolysis and Depletion of Intracellular Glutathione in Erythrocytes

The protective effect of gallic acid and its esters, methyl, propyl, and lauryl gallate, against 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH)-induced hemolysis and depletion of intracellular glutathione (GSH) in erythrocytes was studied. The inhibition of hemolysis was dose-dependent, and the esters were significantly more effective than gallic acid. Gallic acid and its esters were compared with regard to their reactivity to free radicals, using the DPPH and AAPH/pyranine free-cell assays, and no significant difference was obtained. Gallic acid and its esters not only failed to inhibit the depletion of intracellular GSH in erythrocytes induced by AAPH but exacerbated it. Similarly, the oxidation of GSH by AAPH or horseradish peroxidase/H(2)O(2) in cell-free systems was exacerbated by gallic acid or gallates. This property could be involved in the recent findings on pro-apoptotic and pro-oxidant activities of gallates in tumor cells. We provide evidence that lipophilicity and not only radical scavenger potency is an important factor regarding the efficiency of antihemolytic substances.

Effect of carboxymethylated pyridoindoles on free radical-induced haemolysis of rat erythrocytes in vitro.

Recently novel carboxymethylated pyridoindoles, analogues of the efficient chain-breaking antioxidant stobadine, have been designed, synthesised and characterised as bifunctional compounds with joint antioxidant/aldose reductase inhibitory activities with the potential of preventing diabetic complications. The critical property for the efficacy of the novel aldose reductase inhibitors in vivo is their ability to penetrate into target tissues. In this study, the issue was addressed by measuring the antioxidant activity of compounds 1 [(2-benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-yl)-acetic acid] and 2 [(+/-)-2-benzyl-(4a,9b)-cis-1,2,3,4,4a,9b-hexahydro-1H-pyrido[4,3-b] indole-8-yl acetic acid] in the cellular system of intact erythrocytes exposed to peroxyl radicals generated by thermal degradation of the azoinitiator 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) in vitro. Isolated washed rat erythrocytes were incubated in the presence of the azoinitiator AAPH and the compounds tested for increasing periods of time up to 4 h at 37 degrees C. The degree of haemolysis was determined by absorbance of the haemoglobin released. The onset of AAPH-induced haemolysis was found to be shifted from the starting zero point by the time interval assigned as a lag period. In the presence of the compounds studied the lag period was prolonged significantly. The free radical-initiated haemolysis was retarded by the compounds studied with decreasing efficiency: stobadine > compound 1 ~ Trolox > compound 2. The results have demonstrated an antioxidant activity of the novel carboxymethylated pyridoindoles developed as potential agents for multitarget pharmacology of diabetic complications.

Stable Isotope Labeled 4-(Dimethylamino)benzoic Acid Derivatives of Glycerophosphoethanolamine Lipids

A set of four (D(0), D(4), D(6), and D(10)) deuterium enriched 4-(dimethylamino)benzoic acid (DMABA) N-hydroxysuccinimide (NHS) ester reagents was developed that react with the primary amine group of glycerophosphoethanolamine (PE) lipids to create derivatives where all subclasses of DMABA labeled PE are detected by a common precursor ion scan. The positive ion collision induced dissociation data from (D(0), D(4), D(6), and D(10))-DMABA labeled PE standards indicated that a precursor ion scan of m/z 191.1, 195.1, 197.1, and 201.1 could be used to selectively detect (D(0), D(4), D(6), and D(10))-DMABA modified PE, respectively, in a complex biological mixture. The PE lipids from a time course (0, 30, 60, and 300 min) of 2,2'-azobis-(2-amidinopropane) hydrochloride (AAPH) treatment of liposomes made of RAW 264.7 cell phospholipids were each labeled with the D(0)-, D(4)-, D(10)-, and D(6)-DMABA NHS ester reagents, respectively. The DMABA derivatives revealed loss of endogenous PE lipids and an increase in oxidized PE lipid throughout the time course of AAPH treatment. These DMABA NHS ester reagents provide a universal scan for diacyl, ether, and plasmalogen PE lipids that cannot be readily observed otherwise, enable differential labeling, and provide an internal standard for each PE lipid.

Oxidation of melatonin by AAPH-derived peroxyl radicals: Evidence of a pro-oxidant effect of melatonin

Background Melatonin is well-established as a powerful reducing agent of oxidant generated in the cell medium. We aimed to investigate how readily melatonin is oxidized by peroxyl radicals ROO⋅ generated by the thermolysis of 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH) and the role of glutathione (GSH) during the reaction course. Methods Chromatographic, mass spectroscopy, and UV–visible spectrometric techniques were used to study the oxidation of melatonin by ROO⋅ or horseradish peroxidase (HRP)/H 2O 2. Our focus was the characterization of products and the study of features of the reaction. Results We found that N 1-acetyl- N 2-formyl-5-methoxykynuramine (AFMK) and a monohydroxylated derivative of melatonin were the main products of the reaction between melatonin and ROO⋅. Higher pH or saturation of the medium with molecular oxygen increased the yield of AFMK but did not affect the reaction rate. Melatonin increased the depletion of intracellular GSH mediated by AAPH. Using the HRP/H 2O 2 as the oxidant system, the addition of melatonin promoted the oxidation of GSH to GSSG. Conclusions These results show, for the first time, that melatonin radical is able to oxidize GSH. General significance We propose that this new property of melatonin could explain or be related to the recently reported pro-oxidant activities of melatonin.

Antioxidant properties of alpha-lipoic acid: effects on red blood membrane permeability and adaptation of isolated rat heart to reversible ischemia

The aim of our work was to study (1) the antioxidant properties of lipoic acid (LA) and its reduced metabolite dihydrolipoic acid (DHLA) formed by reduction of LA and (2) the effects of treatment with LA and DHLA on (a) K(+) efflux from human red blood cells and (b) post-ischemic recovery and oxidative stress in isolated perfused rat hearts challenged with an ischemia-reperfusion (IR) sequence. In vitro, we used xanthine and xanthine oxidase to generate superoxide anion, which is not directly measurable by electron paramagnetic resonance (EPR), but specifically oxidizes the spin probe CPH into an EPR-detectable long lasting CP(*) nitroxide radical. While 5 mM of LA was ineffective in reducing the kinetics of CP(*) nitroxide formation, DHLA was shown to lessen this rate in a dose-dependent manner and at 30 mM was even more efficient than 300 UI/ml SOD. These results are in agreement with the fact that DHLA is able to directly scavenge superoxide anion. Red cells are a good model to investigate oxidative damage in biological membranes; hence, we used a suspension of erythrocytes incubated with 2,2(')-azobis(2-amidinopropane) hydrochloride (AAPH) which generates in vitro free radicals. DHLA provided more effective protection of red cells membranes than LA; DHLA was comparable to Trolox for its antioxidant potency. In vivo, treatment of rats (50 mg/kg/day i.p. for 7 days) with LA induced a slight increase in coronary flow (CF) in isolated perfused hearts, after 30 min of global total ischemia. This effect was not associated with an improvement in contractile function and reduction of myocardial oxidative stress. In conclusion, because of their ability to scavenge free radicals, LA and to an even greater degree DHLA were able to protect the membranes of red blood cells. This finding suggests that LA and DHLA might be useful in the treatment of diseases associated with oxidative stress such as diabetes.