Theoretical analysis of a site-specific chemiluminescence reaction and its application to quantitation of lipid hydroperoxides

A system was designed for chemiluminescent measurement of lipid hydroperoxides by their site-specific reaction in sodium dodecylsulfate micelles. Ferrous ion-induced decomposition of lipid hydroperoxides in the sodium dodecylsulfate micelles resulted in strong chemiluminescence of the Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one (CLA). After addition of ferrous sulfate to the micelles containing lipid hydroperoxide and luciferin, the chemiluminescence intensity reached a maximum rapidly and then decreased. The sequence of this reaction was elucidated by theoretical analysis, which demonstrated that the maximum chemiluminescence intensity is proportional to the initial concentration of hydroperoxide. Good linear relationships were observed between the maximum counts of chemiluminescence and the amounts of hydroperoxides of linoleic acid, phosphatidylcholine, choresterol (5 alpha), cumene and tert-butyl and hydrogen peroxide. This chemiluminescence method was simple and sensitive enough to detect picomole levels of linoleic acid and phosphatidylcholine hydroperoxides.     

Determination of thiobarbituric acid-reactive substances in oxidized lipids by high-performance liquid chromatography with a postcolumn reaction system

A new high-performance liquid chromatography procedure with a postcolumn reaction system for determination of free malondialdehyde (MDA) and other thiobarbituric acid-reactive substances (TBA-RS) in oxidized lipids in vitro has been developed. Using this procedure, both thermally oxidized methyl linoleate and the degradation products of methyl linoleate hydroperoxides revealed many kinds of lipophilic TBA-RS, but no free MDA was detected on the high-performance liquid chromatography. Similarly, oxidized methyl arachidonate also produced certain kinds of TBA-RS in the lipophilic phase and a small amount of free MDA in the hydrophilic phase. These results indicate that lipophilic TBA-RS produced in oxidized lipids in vitro are major TBA-RS and that the production of free MDA is small, even though the degree of lipid oxidation has previously been estimated as an MDA equivalent measured by the TBA colorimetric test.     

The Photo-oxidation of Epinephrine by Spinach Chloroplasts and Its Inhibition by Superoxide Dismutase: Evidence for the Formation of Superoxide Radicals in Chloroplasts

The thiobarbituric acid (TBA) test for detecting lipid hydroperoxides does not require for fomation of TBA reacting compounds from hydroperoxides, but oxygen has an unfavorable effect, that is, it forms new hydroperoxides during the reaction when unoxidized lipids co-exist. Therefore, a method using a vacuum reaction tube was proposed.     

Lipid peroxidation and lipid peroxide detected by chemiluminescence

This article emphasizes the advantages of using a luminescence spectrometer based on photon counting techniques for the detection of lipid peroxidation. An overview is presented of how chemiluminescence can be stimulated in the luminol-cytochrome c heme peptide system as an assay for lipid hydroperoxides. This method is used for finding antioxidant drugs. The specificity and advantages of the chemiluminescent method for detecting lipid hydroperoxides is reviewed.     

Inhibition of the system luminol-H2O2-Fe(CN)63− chemiluminescence by the Mn(II) indirect determination of isoniazid in a pharmaceutical formulation

A flow injection procedure for the indirect chemiluminescent determination of isoniazid is proposed. The method is performed in a flow-injection manifold provided with a solid-phase reactor. The reactor was made from manganese dioxide physically entrapped by polymerization; the redox reaction isoniazid–manganese dioxide released Mn(II) which was monitored through its inhibitory effect on the reaction between luminol and hydrogen peroxide in presence of potassium hexacyanoferrate(III). The procedure resulted in a linear calibration graph over the range 5–15 mg/L of isoniazid with a sample throughput of 43 samples/h. The influence of foreign compounds was studied and the method was applied to determination of the drug in a pharmaceutical formulation. © 1998 John Wiley & Sons, Ltd.     

A simple and sensitive method for lipoprotein and lipids profiles analysis of individual micro-liter scale serum samples

A simple and sensitive method to determine lipoprotein and lipids profiles in micro-liter scale individual serum sample is not presently available. Traditional lipoprotein separation techniques either by ultra-centrifugation or by liquid chromatography methods have their disadvantages in both lipoprotein separation and lipids component quantification. In this study we used small volume needing size-exclusion fast protein liquid chromatography to separate different lipoprotein subclasses in 50μL serum. And lipids contents, such as cholesterol, cholesterol ester and triacylglycerol, were measured by using two different fluorescence-based lipid detection methods. With this method, very low density lipoprotein, low density lipoprotein and high density lipoprotein could be easily separated, and follow-up lipid detection was completed by simple kinds of reactions. Serum lipoprotein and lipids profiling from C57BL/6 mice (n=5) and human (n=5) were analyzed. The elution profiles of five individuals were highly reproducible, and there were lipoprotein and lipids distribution variations between C57BL/6 mice and human beings. In conclusion, this method which combined small volume needing size-exclusion fast protein liquid chromatography and fluorescence-based lipids measurement, provided a simple, efficient, integrity and reproducible procedure for determining serum lipoprotein and lipids profiles in micro-liter scale levels. It becomes possible that determination of lipoprotein profiles and gaining information of lipids in different lipoproteins can be accomplished simultaneously.     

The reactions of hypochlorous acid, the reactive oxygen species produced by myeloperoxidase, with lipids

Myeloperoxidase (MPO), an abundant enzyme in phagocytes, has been implicated in the pathogenesis of various inflammatory diseases including atherosclerosis. The major oxidant produced by MPO, hypochlorous acid (HOCl), is able to modify a great variety of biomolecules by chlorination and/or oxidation. In this paper the reactions of lipids (preferentially unsaturated fatty acids and cholesterol) with either reagent HOCl or HOCl generated by the MPO-hydrogen peroxide-chloride system are reviewed. One of the major issues has been whether the reaction of HOCl with lipids of low density lipoprotein (LDL) yields predominantly chlorohydrins or lipid hydroperoxides. Electrospray mass spectrometry provided direct evidence that chlorohydrins rather than peroxides are the major products of HOCl- or MPO-treated LDL phosphatidylcholines. Nevertheless lipid peroxidation is a possible alternative reaction of HOCl with polyunsaturated fatty acids if an additional radical source such as pre-formed lipid hydroperoxides is available. In phospholipids carrying a primary amino group such as phosphatidylethanolamine chloramines are the preferred products compared to chlorohydrins. Cholesterol can be converted by HOCl to great variety of oxysterols besides three isomers of chlorohydrins. For the situation in vivo it appears that the type of reaction occurring between HOCl and lipids would very much depend on the circumstances, e.g. the pH and the presence of radical initiators. The biological effects of lipid chlorohydrins are not yet well understood. It has been shown that chlorohydrins of both unsaturated fatty acids as well as of cholesterol may cause lysis of target cells, possibly by disruption of membrane structures.     

Oxidative degradation of model lipids representative for main paper pulp lipophilic extractives by the laccase–mediator system

Different model lipids-alkanes, fatty alcohols, fatty acids, resin acids, free sterols, sterol esters, and triglycerides-were treated with Pycnoporus cinnabarinus laccase in the presence of 1-hydroxybenzotriazole as mediator, and the products were analyzed by gas chromatography. The laccase alone decreased the concentration of some unsaturated lipids. However, the most extensive lipid modification was obtained with the laccase-mediator system. Unsaturated lipids were largely oxidized and the dominant products detected were epoxy and hydroxy fatty acids from fatty acids and free and esterified 7-ketosterols and steroid ketones from sterols and sterol esters. The former compounds suggested unsaturated lipid attack via the corresponding hydroperoxides. The enzymatic reaction on sterol esters largely depended on the nature of the fatty acyl moiety, i.e., oxidation of saturated fatty acid esters started at the sterol moiety, whereas the initial attack of unsaturated fatty acid esters was produced on the fatty acid double bonds. In contrast, saturated lipids were not modified, although some of them decreased when the laccase-mediator reactions were carried out in the presence of unsaturated lipids suggesting participation of lipid peroxidation radicals. These results are discussed in the context of enzymatic control of pitch to explain the removal of lipid mixtures during laccase-mediator treatment of different pulp types.     

Highly Sensitive Flow Injection Analysis of Lipid Hydroperoxides in Foodstuffs

Lipid hydroperoxides in oils and foods were measured by a flow injection analysis system with high sensitivity and selectivity. After sample injection, lipid hydroperoxides were reacted with diphenyl-1-pyrenylphosphine (DPPP) in a stainless steel coil, then the fluorescence intensity of DPPP oxide, that was produced by the reaction, was monitored. By this method, trilinolein hydroperoxide showed good linearity between 0.4 and 79pmol and their detection limits were 0.2pmol (signal-to-noise ratio = 3). The method made it possible to inject samples at 2-min intervals. There was a good agreement of the amounts of lipid hydroperoxides in oils and foods between by the batch method with DPPP and by the proposed method (coefficient of correlation: r = 0.999; n = 21; peroxide value = 0.09–167 meq/g). With this method, the calibration graph of trilinolein hydroperoxide was useful for all samples tested.     

Detection of Picomole Levels in Lipid Hydroperoxides by a Chemiluminescence Assay

A highly sensitive and simple chemiluminescent method for the quantitation of lipid hydroperoxides at the picomole level is described. The method is based on detecting the chemiluminescence generated during the oxidation of luminol by the reaction with hydroperoxide and cytochrome c under mild conditions. A semilogarithmic relationship was observed between the hydroperoxide added and the chemiluminescence produced. For lipid hydroperoxides, cytochrome c was a most favorable catalyst for generating the chemiluminescence, rather than cytochrome c heme peptide and horseradish peroxidase. This method had high sensitivity to methyl linoleate hydroperoxide, arachidonic acid hydroperoxide and cholesterol hydroperoxide, but low to /-butyl hydroperoxide, J-butyl perbenzoate, diacyl peroxides (lauroyl peroxode and benzoyl peroxide) and dialkyl peroxides (di-/-butyl peroxide and dicumyl peroxide).     

Antioxidant defenses in rat intestine and mesenteric lymph

Abstract

Dietary oxysterols can reach the circulation and this may contribute to atherosclerosis, where lipid oxidation is thought to be important. There is also evidence that, in rats,peroxidized lipids are absorbed and transported into lymph [Aw TY, Williams MW, Gray L. Absorption and lymphatic transport of peroxidized lipids by rat small intestine in vivo: role of mucosal GSH. Am J Physiol 1992; 262: G99–G106], although the method used to detect lipid peroxides lacked specificity. We tested whether intragastric administration of vegetable oils containing triglyceride hydroperoxides (TG-OOH) to rats resulted in detectable lipid hydroperoxides in mesenteric lymph. Using sensitive HPLC with postcolumn chemiluminescence detection, we were unable to detect hydroperoxides of triglycerides, cholesterylesters or phospholipids during the course of lipid absorption, and lymph levels of ascorbate, urate, α-tocopherol and ubiquinol-9 did not change significantly. By contrast, we observed a striking reducing activity judged by the efficient reduction of administered ubiquinones-9 and -10 to the corresponding ubiquinols. Exposure of rat lymph and isolated chylomicrons to aqueous peroxyl radicals revealed patterns of antioxidant consumption and lipid hydroperoxide formation similar to those described previously for human extravascular fluids and isolated lipoproteins, respectively. In particular, rates of TG-OOH formation in lymph and chylomicrons were very low to undetectable as long as ascorbate and/or ubiquinols were present, but subsequently proceeded in a chain reaction despite the presence of α-tocopherol. These studies demonstrate that rat intestine and mesenteric lymph possess efficient antioxidant defenses against preformed lipid hydroperoxides and (peroxyl) radical mediated lipid oxidation. We conclude that dietary lipid hydroperoxides or postprandial oxidation of lipids are not likely to contribute to these particular forms of oxidized lipids in circulation and aortic tissue.     

Thin-layer chromatography blotting for the fluorescence detection of phospholipid hydroperoxides and cholesteryl ester hydroperoxides

A blotting technique was developed to specifically detect lipid hydroperoxides in thin-layer chromatography. Phosphatidylcholine hydroperoxides and cholesteryl linoleate hydroperoxides ranging from 0.1 to 0.5 nmol, which were prepared by reaction with soybean lipoxygenase, were visualized as fluorescent spots on the blotted membrane by immersing the plate into a blotting solvent containing 0.01% (w/v) diphenyl-1-pyrenylphosphine. This technique was applied successfully to monitor lipid peroxidation in human low-density lipoprotein in vitro.     

Interaction of tert-butyl hydroperoxide with hypochlorite induces peroxyl radicals. A chemiluminescence study

The interaction of hypochlorite (HOCl/OCl-) with tert-butyl hydroperoxide ((CH3)3COOH) was investigated by chemiluminescence. It was shown that the addition of HOCl/OCl- to (CH3)3COOH induces a fast chemiluminescent flash. The intensity of this flash increases with the increase in both HOCl/OCl- and (CH3)3COOH concentration. The chemiluminescence is quenched in a concentration-dependent manner in the presence of free radical spin traps N-tert-butyl nitrone and alpha-(4-pyridyl-1-oxyl)-N-tert-butyl nitrone. This fact proves that free radicals take part in the interaction of HOCl/OCl- and (CH3)3COOH. Hypochlorite yielded a very similar chemiluminescence spectrum in its reaction with (CH3)3COOH as Ce4+. It differed considerably from the spectrum in the system H2O2 and HOCl/OCl-. It is well known that the interaction of Ce4+ and (CH3)3COOH produces peroxyl radicals. These results confirm the hyothesis that the interaction of HOCl/OCl- and (CH3)3COOH is mediated by peroxyl radicals. Thus, organic hydroperoxides always present in unsaturated lipids can induce lipid peroxidation processes in the reaction with HOCl/OCl-.     

The stomach as a bioreactor: dietary lipid peroxidation in the gastric fluid and the effects of plant-derived antioxidants.

Atherosclerosis may result partly from processes that occur following food consumption and that involve oxidized lipids in chylomicrons. We investigated reactions that could occur in the acidic pH of the stomach and accelerate the generation of lipid hydroperoxides and co-oxidation of dietary constituents. The ability of dietary polyphenols to invert catalysis from pro-oxidation to antioxidation was examined. The acidic pH of gastric fluid amplified lipid peroxidation catalyzed by metmyoglobin or iron ions. Metmyoglobin catalyzed peroxidation of edible oil, resulting in 8-fold increase of hydroperoxide concentration. The incubation of heated muscle tissue in simulated gastric fluid for 2 h enhanced hydroperoxides accumulation by 6-fold to 1200 microM. In the presence of catechin or red wine polyphenols, metmyoglobin catalyzed the breakdown of hydroperoxides to zero, totally preventing lipid peroxidation and beta-carotene cooxidation. We suggest that human gastric fluid may be an excellent medium for enhancing the oxidation of lipids and other dietary constituents. The results indicate the potentially harmful effects of oxidized fats intake in the presence of endogenous catalysts found in foods, and the major benefit of including in the meal plant dietary antioxidants.     

Lipoxygenase-dependent degradation of storage lipids

Oilseed germination is characterized by the mobilization of storage lipids as a carbon source for the germinating seedling. In spite of the importance of lipid mobilization, its mechanism is only partially understood. Recent data suggest that a novel degradation mechanism is initiated by a 13-lipoxygenase during germination, using esterified fatty acids specifically as substrates. This 13-lipoxygenase reaction leads to a transient accumulation of ester lipid hydroperoxides in the storage lipids, and the corresponding oxygenated fatty acid moieties are preferentially removed by specific lipases. The free hydroperoxy fatty acids are subsequently reduced to their hydroxy derivatives, which might in turn undergo beta-oxidation.     

High performance thin-layer chromatography and densitometry of synaptic plasma membrane lipids

Previously, it has been shown that phospholipids, cholesterol, and glycolipids could be quantitated using the same high performance thin-layer chromatography (HPTLC) method. Here we examined that method in terms of linearity of standards in the nanogram range, recovery of nonacidic and acidic lipids after Sephadex column chromatography, and quantitation of lipids in mouse synaptic plasma membranes (SPM) where lipid content is low. Nonacidic and acidic fractions were separated by Sephadex column chromatography, applied to plates using contact spotting, chromatographed, visualized with cupric acetate, and quantitated using in situ densitometry. Recovery of nonacidic and acidic fractions off the columns was determined with radiolabeled phospholipids. Standards for each lipid class were linear in the nanogram range. Quantitation of SPM lipid classes could be made with as little as 1.5 micrograms of total lipid. Recovery of the nonacidic fraction after Sephadex column chromatography was approximately 100% whereas the acidic fraction was approximately 91%. Phospholipids, cholesterol, and glycolipids could be determined in nanogram amounts using the same method. This method is an efficient method for examining different lipid classes and in samples where lipid content is low.     

The effect of detergents on firefly luciferase reactions

The reaction rate of ATP-limited firefly luciferase-catalysed reactions, is affected by the presence of detergents. Anionic detergents inhibit luciferase activity without causing significant enzyme inactivation during the reaction. Cationic detergents increase reaction rate several-fold with a sharply defined optimum concentration of detergent for the effect. However, cationic detergents inactivate firefly luciferase during the reaction, resulting in a continuously decreasing reaction rate. Under such conditions, peak light intensity must be used as an indication of initial reaction rate. The inactivation rate increases with increasing detergent concentration. Non-ionic and zwitterionic detergents increase reaction rate over a broad range of detergent concentrations. Enzyme stability during the reaction is not affected by non-ionic detergents and only affected by zwitterionic detergents at high detergent concentration. Cyclodextrins, which can increase reaction rates of some chemiluminescent reactions, have little effect on firefly luciferase activity. Assays for ATP using firefly luciferase must be internally standardized by the constant addition technique in which a known amount of ATP is added to the test sample, since external calibration of such assays, by reference to a previously prepared standard curve, can lead to imprecision when detergents are present.     

Detection of lipid hydroperoxides and hydrogen peroxide at picomole levels by an HPLC and isoluminol chemiluminescence assay

An isoluminol assay is utilized for the detection of hydrogen peroxide and lipid hydroperoxides in biological samples. The combination of this assay as a post-column detection for HPLC avoids interference of antioxidants and enables characterization of hydroperoxides at picomole levels. Two useful HPLC conditions for the separation of hydrogen peroxide, lipid hydroperoxides, antioxidants, and unoxidized lipids are described.     

Photooxidation of cell membranes in the presence of hematoporphyrin derivative: reactivity of phospholipid and cholesterol hydroperoxides with glutathione peroxidase

The susceptibility of photodynamically-generated lipid hydroperoxides to reductive inactivation by glutathione peroxidase (GPX) has been investigated, using hematoporphyrin derivative as a photosensitizing agent and the human erythrocyte ghost as a target membrane. Photoperoxidized ghosts were reactive in a glutathione peroxidase/reductase (GPX/GRD)-coupled assay only after phospholipid hydrolysis by phospholipase A2 (PLA2). However, enzymatically determined lipid hydroperoxide values were consistently approx. 40% lower than iodometrically determined values throughout the course of photooxidation. Moreover, when irradiated ghosts were analyzed iodometrically during PLA2/GSH/GPX treatment, a residual 30-40% of non-reactive lipid hydroperoxide was observed. The possibility that cholesterol product(s) account for the non-reactive lipid hydroperoxide was examined by tracking cholesterol hydroperoxides in [14C]cholesterol-labeled ghosts. The sum of cholesterol hydroperoxides and GPX/GRD-detectable lipid hydroperoxides was found to agree closely with iodometrically determined lipid hydroperoxide throughout the course of irradiation. Thin-layer chromatography of total lipid extracts indicated that cholesterol hydroperoxide was unaffected by PLA2/GSH/GPX treatment, whereas most of the phospholipid peroxides were completely hydrolyzed and the released fatty acid peroxides were reduced to alcohols. It appears, therefore, that the GPX-resistant lipid hydroperoxides in photooxidized ghosts were derived primarily from cholesterol. Ascorbate plus Fe3+ produced a burst of free-radical lipid peroxidation in photooxidized, PLA2-treated ghosts. As expected for fatty acid hydroperoxide inactivation, the lipid peroxidation was inhibited by GSH/GPX, but only partially so, suggesting that cholesterol hydroperoxide-derived radicals play a major role in the reaction.     

Kinetic study of the reaction between vitamin E radical and alkyl hydroperoxides in solution

Kinetic study of the reaction between vitamin E radical and alkyl hydroperoxides has been performed, as a model for the reactions of lipid hydroperoxides with vitamin E radical in biological systems. The rates of reaction of hydroperoxides (n-butyl hydroperoxide 1, sec-butyl hydroperoxide 2, and tert-butyl hydroperoxide 3) with vitamin E radical (5,7-diisopropyl-tocopheroxyl 4) in benzene solution have been determined spectrophotometrically. The second-order rate constants, k-1, obtained are 1.34 x 10(-1) M-1s-1 for 1, 2.42 x 10(-1) M-1s-1 for 2, and 3.65 x 10(-1) M-1s-1 for 3 at 25.0 degrees C. The result indicates that the rate constants increase as the total electron donating capacity of the alkyl substituents at alpha-carbon atom of hydroperoxides increases. The above rates, k-1, are about seven order of magnitude lower than those, k1, for the reaction of vitamin E with peroxyl radical.