dinoprost and 2-2--azobis(2-amidinopropane)

An improved method for the measurement of lipid peroxidation in vivo has been recently developed, where total hydroxyoctadecadienoic acid (HODE) and 7-hydroxycholesterol (FCOH) were determined by GC/MS analysis from physiological samples after reduction with sodium borohydride and saponification by potassium hydroxide. In this method, both free and ester forms of hydroperoxides and ketones as well as hydroxides of linoleic acid and cholesterol are measured as HODE and FCOH, respectively. The ratio of stereo-isomer, (Z, E)-HODE/(E, E)-HODE, could be also measured. In the present study, in order to examine the effect of continuous, slow flux of free radicals in vivo, a water-soluble radical generator was administered to rats and mice and the amounts of HODE and 8-isoprostane in plasma and liver were measured. It was found that the administration of free radical-generating azo compound increased the level of HODE and decreased the (Z, E)-HODE/(E, E)-HODE ratio in both plasma and liver. The level of HODE was much higher than 8-isoprostane.

Topics: Amidines; Animals; Azo Compounds; Biomarkers; Dinoprost; Drinking; Fatty Acids, Unsaturated; Free Radicals; Gas Chromatography-Mass Spectrometry; Hydroxycholesterols; Imidazoles; Lipid Peroxidation; Liver; Male; Mice; Rats; Specific Pathogen-Free Organisms

It is essential to generate free radicals at a controled and constant rate for specific duration and at specific site to study the dynamics of oxidation and also antioxidation. Both hydrophilic and lipophilic azo compounds have been used for such purpose. In the present work, the action of 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH) was examined and compared with those of 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)-propionamide] (AMHP). The rate constant of free radical formation (ek(d)) for AIPH was 2.6 x 10(-6)/s at 37 degrees C in PBS (pH 7.4) solution, indicating that AIPH gives 3.8 times more free radicals than AAPH under the same conditions. It was found that the dynamics of oxidation and antioxidation induced by AIPH can be studied satisfactorily in the oxidation in micelles, LDL and erythrocyte suspensions, plasma, and cultured cells. The extent of cell death induced by AIPH and AAPH was directly proportional to the total free radicals formed. Interestingly, it was found that rats would not drink water containing AAPH, but they drank water containing AIPH. The levels of 8-iso-prostaglandin F2alpha (8-isoPs), 7-hydroxycholesterol (FCOH), lysophosphatidylcholine in the plasma of rats given water containing 50 mM AIPH for 1 month increased compared with those of control rats which drank water without AIPH. It may be concluded that AIPH is useful for kinetic and mechanistic studies on oxidative stress to membranes, lipoproteins, cells, and even animal models.

Topics: Adult; Amidines; Animals; Cells, Cultured; Chromans; Culture Media, Serum-Free; Dinoprost; Dose-Response Relationship, Drug; Erythrocytes; Free Radicals; Humans; Hydroxycholesterols; Jurkat Cells; Kinetics; Lipid Metabolism; Lipoproteins, LDL; Lysophosphatidylcholines; Male; Micelles; Models, Chemical; Oxidants; Oxidative Stress; Oxygen; Rats; Rats, Wistar; Temperature; Time Factors; Water

It has been questioned whether carotenoids can act as antioxidants in biological membranes. Biological membranes can be modeled for studies of lipid peroxidation using unilamellar liposomes. Both carotenoid depletion and lipid peroxidation were increased with increasing oxygen tension in unilamellar liposomes. Carotenoids in such liposomes were found to be very sensitive to degradation by free radicals generated from iron and 2,2'-azobis(2-amidinopropane) dihydrochloride, but they were not protective against lipid peroxidation. Lycopene and beta-carotene were more sensitive to free radical attack than lutein, zeaxanthin, and beta-cryptoxanthin.

Topics: Amidines; Antioxidants; beta Carotene; Carotenoids; Cryptoxanthins; Dinoprost; F2-Isoprostanes; Free Radicals; Hydrogen-Ion Concentration; Iron; Lipid Peroxidation; Liposomes; Lutein; Lycopene; Oxygen; Thiobarbituric Acid Reactive Substances; Xanthophylls

The influence of semotiadil, a novel benzothiazine calcium antagonist on in-vitro copper-, 2,2àzo-bis-(2,4 dimethylvaleronitrile)[AMVN]-, and 2,2àzo-bis-(2-amidinopropane) [AAPH]-induced low-density lipoprotein (LDL) oxidation was assessed. The following parameters were measured: lag-time of oxidation, maximal rate of oxidation, dienes formed through continuous monitoring of developing conjugated dienes, thiobarbituric acid reactive substances, isoprostane(8-iso-PGF2alpha)-generation and relative electrophoretic mobility. The effect was compared with nifedipine, amlodipine and diltiazem. Besides the influence on isoprostane (8-iso-PGF2alpha)-generation where nifedipine was equipotent with semotiadil at 10(-3) M, semotiadil demonstrated a strong and significant effect in attenuating the indicated indices of LDL-oxidation, in particular, dose-dependently (10(-3) M to 10(-7) M). These results indicate that semotiadil may have the strongest antioxidant activity on LDL among the calcium antagonists examined.

Topics: Adult; Amidines; Azo Compounds; Calcium Channel Blockers; Copper; Dinoprost; F2-Isoprostanes; Humans; Lipoproteins, LDL; Male; Middle Aged; Nitriles; Oxidants; Oxidation-Reduction; Thiazoles; Thiobarbituric Acid Reactive Substances

8-Epi PGF2alpha, a potent vasocontrictor, is a specific product of non-enzymatic peroxidation of arachidonic acid. It seems likely that similar products could arise from other polyunsaturated fatty acids (PUFAs) and might be useful biomarkers of their peroxidation in vivo. This was investigated using eicosapentaenoic acid (EPA). EPA liposomes (1 mg/ml PBS) were exposed at 37 degrees C to either 2,2'-azobis-(2-amidinopropane) dichloride (AAPH) or copper ions at final concentrations of 1 mM and 10 microM, respectively. Sample processing involved solid-phase extraction on a C18-followed by an NH2 cartridge. After conversion to pentafluorobenzyl ester/trimethylsilyl derivatives, F3-isoprostanes were analysed by negative ion-chemical ionisation mass spectrometry (GC-MS/NICI) using tetradeuterated PGF2alpha (PGF2-d4) as the internal standard. Quantitative analysis was carried out by selected ion monitoring of the carboxylated anion [M-180] at m/z 567 and 573 for the PGF3-like compounds and PGF2-d4, respectively. EPA oxidised by AAPH or by copper ions gave rise to a family of F3-isoprostanes with 8-epi PGF3alpha as a minor product. Formation of F3-isoprostanes correlated well with other indices of lipid peroxidation (hydroperoxides and thiobarbituric acid reactive substances). The possibility of analysing specific lipid peroxidation products from individual fatty acids should facilitate nutritional and biomedical studies.

Topics: Amidines; Copper; Dinoprost; Eicosapentaenoic Acid; Gas Chromatography-Mass Spectrometry; Lipid Peroxides; Liposomes; Prostaglandins F

F2-isoprostanes are prostaglandin F2-like compounds that are known to be formed in vivo by free radical oxidation of arachidonyl-containing lipids, and their plasma levels have been suggested as indicators of in vivo oxidative stress. As oxidation of LDL, a likely causal factor in atherosclerosis, involves lipid peroxidation, we investigated whether F2-isoprostanes are formed in plasma and LDL exposed to oxidative stress, and how F2-isoprostane formation is related to endogenous antioxidant status. In plasma exposed to aqueous peroxyl radicals, lipid hydroperoxides and esterified F2-isoprostanes were formed simultaneously after endogenous ascorbate and ubiquinol-10 had been exhausted, despite the continued presence of urate, alpha-tocopherol, beta-carotene, and lycopene. In isolated LDL exposed to aqueous peroxyl radicals or Cu2+, consumption of endogenous ubiquinol-10 and alpha-tocopherol was followed by rapid formation and subsequent breakdown of lipid hydroperoxides and esterified F2-isoprostanes, and a continuous increase in LDL's electronegativity, indicative of atherogenic modification. In Cu(2+)-exposed LDL, the decrease in esterified F2-isoprostane levels was paralleled by the appearance of free F2-isoprostanes, suggesting that hydrolysis by an LDL-associated activity had occurred. Our data suggest that F2-isoprostanes are useful markers of LDL oxidation in vivo. As F2-isoprostanes are potent vasoconstrictors and can modulate platelet aggregation, their formation in LDL demonstrated here may also have important implications for the etiology of cardiovascular disease.

Topics: Amidines; Antioxidants; Copper; Dinoprost; Humans; In Vitro Techniques; Lipid Peroxidation; Lipoproteins, LDL; Male; Oxidation-Reduction; Prostaglandins