Quantitative determination of total lipid hydroperoxides by a flow injection analysis system

A flow injection analysis (FIA) system coupled with a fluorescence detection system using diphenyl-1-pyrenylphosphine (DPPP) was developed as a highly sensitive and reproducible quantitative method of total lipid hydroperoxide analysis. Fluorescence analysis of DPPP oxide generated by the reaction of lipid hydroperoxides with DPPP enabled a quantitative determination of the total amount of lipid hydroperoxides. Use of 1-myristoyl-2-(12-((7-nitro-2-1,3-benzoxadiazol-4-yl)amino) dodecanoyl)-sn-glycero-3-phosphocholine as the internal standard improved the sensitivity and reproducibility of the analysis. Several commercially available edible oils, including soybean oil, rapeseed oil, olive oil, corn oil, canola oil, safflower oil, mixed vegetable oils, cod liver oil, and sardine oil were analyzed by the FIA system for the quantitative determination of total lipid hydroperoxides. The minimal amounts of sample oils required were 50 μg of soybean oil (PV=2.71 meq/kg) and 3 mg of sardine oil (PV=0.38 meq/kg) for a single injection. Thus, sensitivity was sufficient for the detection of a small amount and/or low concentration of hydroperoxides in common edible oils. The recovery of sample oils for the FIA system ranged between 87.2±2.6% and 102±5.1% when PV ranged between 0.38 and 58.8 meq/kg. The CV in the analyses of soybean oil (PV=3.25 meq/kg), cod liver oil (PV=6.71 meq/kg), rapeseed oil (PV=12.3 meq/kg), and sardine oil (PV=63.8 meq/kg) were 4.31, 5.66, 8.27, and 11.2%, respectively, demonstrating sufficient reproducibility of the FIA system for the determination of lipid hydroperoxides. The squared correlation (r ²) between the FIA system and the official AOCS iodometric titration method in a linear regression analysis was estimated at 0.9976 within the range of 0.35−77.8 meq/kg of PV (n=42). Thus, the FIA system provided satisfactory detection limits, recovery, and reproducibility. The FIA system was further applied to evaluate changes in the total amounts of lipid hydroperoxides in fish muscle stored on ice.     

Coloring conditions of thiobarbituric acid test for detecting lipid hydroperoxides

The coloring reaction of the thiobarbituric acid test for hydroperoxides was completely inhibited by the addition of EDTA. Therefore, it was necessary to add a metal salt to the reaction mixture to complete the reaction and also to add an antioxidant to prevent autoxidation when unoxidized unsaturated fatty acids co-exist. The optimal pH of the reaction was found at 3.6 using glycine-hydrochloric acid buffer.     

Characterization of lipid hydroperoxides generated by photodynamic treatment of leukemia cells

A new technique, high-performance liquid chromatography with reductive mode electrochemical detection on a mercury drop (HPLC-EC), has been used for analyzing lipid hydroperoxide (LOOH) formation in photooxidatively stressed L1210 leukemia cells. Highly specific and sensitive for peroxides (detection limits <0.5 pmol for cholesterol hydroperoxides and <50 pmol for phospholipid hydroperoxides), this approach allows different classes of LOOH to be separated and determined in minimally damaged cells. L1210 cells in serum-containing growth medium were irradiated in the presence of merocyanine 540 (MC540), a lipophilic photosensitizing dye. Lipid extracts from cells exposed to a light fluence of 0.11 J/cm² (which reduced clonally assessed survival by 30%) showed 12–15 well-defined peaks in HPLC-EC. None of these peaks was observed when cells were irradiated without MC540 or when dye/light-treated samples were reduced with triphenylphosphine prior to analysis. Three peaks of relatively low retention time (<12 min) were assigned to the following species by virtue of comigration with authentic standards: 3β-hydroxy-5α-cholest-6-ene-5-hydroperoxide (5α-OOH), 3β-hydroxycholest-4-ene-6β-hydroperoxide (6β-OOH), and 3β-hydroxycholest-5-ene-7α/7β-hydroperoxide (7α/7β-OOH). Formation of 5α-OOH and 6β-OOH (singlet oxygen adducts) was confirmed by subjecting [¹⁴C]cholesterol-labeled cells to relatively high levels of photooxidation and analyzing extracted lipids by HPLC with radiochemical detection. Material represented in a major peak at 18–22 min on HPLC-EC was isolated in relatively large amounts by semipreparative HPLC and shown to contain phospholipid hydroperoxides (predominantly phosphatidylcholine species, PCOOH) according to the following criteria: (i) decay of 18–22 min peak during Ca²⁺/phospholipase A₂ treatment, with reciprocal appearance of fatty acid hydroperoxides; (ii) reduction of peroxide during treatment with reduced glutathione and phospholipid hydroperoxide glutathione peroxidase, but not glutathione peroxidase; and (iii) comigration with PCOOH standards in thin-layer chromatography. HPLC-EC analysis revealed quantifiable amounts ofPCOOH and ChOOH at a light fluence that clonally inactivated <10% of the cells, which allows for the possibility that photoperoxidative damage plays a causal role in cell killing. This paper is based on a dissertation submitted by G.J. Bachowski in partial fulfillment of the requirements for a Ph.D. degree in Biochemistry at the Medical College of Wisconsin (Milwaukee, WI).     

Fluorescent products of lipid peroxidation: II. Methods for analysis and characterization

The effects of pH and of the metal chelator, europium (Tric[2,2,6,6-tetra-methyl-3,5-heptanedionate]), upon fluorescence of lipid peroxidation products were tested, and fluorescence decay times and fluorescence polarization values were determined. Measurements of fluorescence intensity showed that the fluorescence of these compounds was quenched at basic pH and that it was restored by adjustment of pH to neutrality. Metal chelator decreased the fluorescence intensity 8–15%. pH Effects and metal coordination effects are useful for analysis and characterization of these fluorescent products. Fluorescence polarization and fluorescence decay times are also useful analytical techniques for characterization of fluorescent products.     

Fluorescent products of phospholipids during lipid peroxidation

During peroxidation, phosphatidyl ethanolamine and phosphatidyl serine formed fluorescent chromophores with maximum emission at 435 nm and maximum excitation at 365 nm. The development of fluorescence was related to formation of thiobarbituric acid reactive substance during lipid peroxidation. This relationship was studied by reacting dipalmityl phosphatidyl ethanolamine with the oxidation products of the methyl esters of arachidonic, linolenic and linoleic fatty acids. Reaction parameters affecting the development of lipid-extractable fluorescent chromophores are: the production of peroxidation products, especially malonaldehyde, from autoxidation of polyunsaturated fatty acids; the length of time these products react; and the availability of reactive amino groups on the phospholipids.     

Formation of dityrosine and other fluorescent amino acids by reaction of amino acids with lipid hydroperoxides

Formation of fluorescence by the reaction of various amino acids with lipid hydroperoxides,i.e., linoleic acid 13-monohydroperoxide, methyl linoleate 13-monohydroperoxide and phosphatidylcholine hydroperoxide, in the presence of methemoglobin was investigated. Two types of fluorescence were produced: fluorescent dityrosine (3,3′-dityrosine) from tyrosine, and unidentified fluorophores with α- and ε-amino groups of various amino acids. While the former was stable after treatment with borohydride, the latter fluorophores were readily destroyed. The rate of dityrosine formation was rapid, and the yield of dityrosine was dependent on the concentrations of tyrosine and the lipid hydroperoxides. Butylated hydroxytoluene and tocopherol inhibited the formation of dityrosine, but did not affect the formation of fluorophores on the amino groups. Dityrosine appears to be formed by radical reaction of the lipid hydroperoxides, while the other fluorophores seem to be created by nonradical mechanisms.     

A highly sensitive method for the micro-determination of lipid hydroperoxides by potentiometry

A modified method for peroxide value (POV) determination of lipids was developed through the application of potentiometry to conventional POV tests such as the official method of the Japan Oil Chemists’ Society (JOCS). The new method permits a simple and reliable determination of low hydroperoxide levels in the initial stages of lipid autoxidation when only very small amounts of sample are available, even when those levels are measured on less than 10 mg of lipid. Using the present method, hydroperoxide levels as low as 20 nanoequivalents (neq) were determined with reasonable precision. This method is applicable to all lipids tested including oils and fats, free fatty acids, phospholipids, glycolipids and cholesterol esters.     

Preparation and purification of lipid hydroperoxides from arachidonic and γ-linolenic acids

Commercial soybean lipoxygenase may be used under carefully controlled reaction conditions to give high yields of lipid hydroperoxides. Lipid hydroperoxides so derived from γ-linolenic or arachidonic acid may be purified by high pressure liquid chromatography. Thus, commercial lipoxygenase serves as a viable source for 100 mg quantities of lipid hydroperoxides.     

Reducing intra‐sample variability of muscle lipid analysis

Intra‐sample variability of intramyocellular triacylglycerols (imcTG) in skeletal muscle has been examined using two different tissue processing sequences assisted by tracer and solvent extraction techniques. Aliquoting after homogenization of muscle biopsies significantly reduced intra‐sample variability compared to the aliquoting before homogenization approach. The results suggested that skeletal muscle samples be homogenized before aliquoting, if necessary, in order to reduce variability in imcTG measurements. Intramyocellular non‐esterified fatty acids have a lower variability than triacylglycerols.     

Control of lipid oxidation in fish oil with various antioxidative compounds

Samples of a commercial fish oil were separately treated with various chemical compounds and then studied for their susceptibility to rancidity by means of an accelerated oxidation test at 60°C.α-Tocopherol acetate and ascorbyl palmitate showed the lowest antioxidative effects among the group of seven chemicals. Anoxomer, a synthetic phenolic polymer, had an antioxidative power comparable to that of ethoxyquin, butylated hydroxytoluene or butylated hydroxyanisole when all were applied to the oil in the concentration of 0.02%. However, the most powerful antioxidant was tertiary-butylhydroquinone (TBHQ), with an antioxidant efficiency twice that of the above-mentioned phenolic compounds when used at only 0.01% concentration in the oil. Although TBHQ and Anoxomer proved to be potential compounds for preventing rancidity in fish oils, their use is still hindered by the limited acceptance from the appropriate authorities.     

Structural analysis of hydroperoxides formed by oxidation of phosphatidylcholine with singlet oxygen

Soybean phosphatidylcholine (PC) and dilinoleoyl PC (di-18∶2 PC) were oxidized with singlet molecular oxygen using methylene blue as the photosensitizer. The oxidation products, PC monohydroperoxides (PC-MHP) and PC dihydroperoxides (PC-DHP), were isolated by reverse phase liquid chromatography, and their structures were analyzed by nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS). Signals for the hydroperoxy proton appeared downfield in NMR spectra of PC-MHP and PC-DHP. Soybean PC-MHP and di-18∶2 PC-MHP were converted to trimethylsilyl (TMS) derivatives of hydrogenated diglycerides when treated with phospholipase C and hydrogenated. Thetert-butyldimethylsilyl (TBDMS) derivatives of hydrogenated diglycerides were also prepared from di-18∶2 PC-MHP. Fragmentation of the TMS and TBDMS derivatives was obtained in electron impact mass spectra. The isomeric composition of hydroperoxylinoleate component in di-18∶2 PC-MHP was determined by methanolysis of the hydrogenated diglyceride and mass chromatographic analysis of the resulting isomeric hydroxy octadecanoates.     

Limitations of the method using peroxidase activity of hemoglobin for detecting lipid hydroperoxides

The method using peroxidase activity of hemoglobin (Hb) for the determination of lipid peroxides was examined by using pure methyl linoleate hydroperoxides, trilinoleoylglycerol hydroperoxides and egg yolk phosphatidylcholine hydroperoxides as substrates and tetramethyl benzidine as electron donor for the peroxidase reaction of Hb. The reactivities of these substrates were quite varied. Furthermore, some electron donors were tested for peroxidase activity of Hb, but none showed a complete reduction of methyl linoleate hydroperoxides. From these results, it seems the Hb method needs to be carefully applied to biological materials that contain mixtures of different types of lipid classes.     

Development of a flow injection chemiluminescent assay for the quantification of lipid hydroperoxides

An automated flow injection chemiluminescence (FICL) system for measuring lipid hydroperoxide (LOOH) concentrations in oils was developed. Initially, a crude oil-in-water emulsion (formed by mixing solvent-diluted oil with the aqueous-based CL compound, luminol, and the catalyst for the reaction, cytochrome c) was tested. The assay was rapid (60 samples per hour), reproducible (CV no greater than 10%, n=3) and had a low sample requirement (1 mg of oil) because of its high sensitivity (0.5 nmol LOOH). CL intensity was influenced by the amount and type of oil under analysis. Owing to these factors, quantitative data were attainable only with a uniform oil concentration and with a calibrant derived from an oil equivalent to that under analysis. This method yielded quantitative data in good agreement with an iodometric titration assay for LOOH (r=0.9204). A refinement of the first method consisted of replacing the luminol and cytochrome c CL compounds with lucigenin, resulting in an assay insensitive to α-tocopherol. A monophasic reaction solution was devised to remove the effect of turbidity; however, the CL signal was still influenced by oil type. Therefore, quantitative data were still attainable only when the same type of oil was used for calibration.     

Fluorescent modification of serum albumin by lipid peroxidation

Peroxidation of fatty acids bound to human serum albumin results in the production of fluorescent chromophores in the protein when it is stored in the liquid, powdered or crystalline state. Peroxidizing polyunsaturated fatty acid esters, and carbonyls derived from peroxidizing lipids react with amino groups of protein to give products that have fluorescence spectra very similar to those observed for stored commercial preparations of serum albumin.     

Fluorescent products of lipid peroxidation of mitochondria and microsomes

Liver microsomes and mitochondria and heart sarcosomes from rats fed diets with varying α-tocopherol concentrations and lipid contents were peroxidized over a 6 hr time period. Lipid peroxidation was measured by absorption of oxygen, production of thiobarbituric acid (TBA) reactants and by development of fluorescence. The spectral characteristics of the fluorescent compounds were the same for all peroxidizing systems; the excitation maximum was 360 nm and the emission maximum was 430 nm. As time of peroxidation increased, uptake of oxygen and production of fluorescent compounds increased. These two parameters as well as production of TBA reactants were dependent upon dietary antioxidant and all three had an inverse relationship with the amount of dietary α-tocopherol. The relationship between absorption of oxygen and development of fluorescent compounds was also dependent upon dietary polyunsaturated fats (PUFA). Subcellular particles from animals fed higher levels of PUFA produced more fluorescent products per mole of oxygen absorbed than did those from animals on a diet with lower PUFA content. TBA reacting products increased with time during the initial phase of peroxidation: in the microsomal systems their production stabilized or decreased by 4–6 hr of peroxidation. Using the synthetic 1-amino-3-iminopropene derivative of glycine as standard for quantitation of fluorescence, the molar ratios of oxygen absorbed per fluorescent compound produced were calculated. This ratio for subcellular particles isolated from rats fed diets with PUFA ratios similar to those in the average American human diet was 393∶1. The fluorescent compounds had the same spectral characteristics as the lipofuscin pigment that accumulates in animal tissues as a function of age, oxidative stress or antioxidant deficiency. The fluorescent molecular damage represented by that accumulated in human heart age pigment by 50 years of age was calculated to have been caused by approximately 0.6 μmole of free radicals per gram of heart tissue.     

Modified ferrous oxidation‐xylenol orange method to determine lipid hydroperoxides in fried snacks

A modified ferrous oxidation‐xylenol orange (FOX) method was adapted to measure lipid hydroperoxides (LHP) in lipid extracts from snack foods fried in vegetable oils. First, a methanol‐based FOX reaction medium was assayed, but this became turbid upon addition of the lipid extracts dissolved in ethanol. To avoid the precipitation of lipids, the polarity of the reaction medium was reduced by lowering its water content and by replacing the methanol as the basis of the medium for less polar solvents. Some of the solvents used instead of methanol yielded a lower FOX reaction response. Of the reaction media assayed, the one based on dichloromethane/ethanol (3:2, vol/vol) was not turbid at high lipid extract concentrations (assayed at up to 25 mg of lipid extract/mL reaction medium) and provided the same response level as the methanol‐based medium. Thus, this FOX method shows high sensitivity and is particularly useful for lipid extracts with low LHP content. This method was also successfully applied to edible oils. Solvents such as 2‐propanol, ethyl acetate and butanol were discarded, because they easily produce hydroperoxides, which interfere in the FOX reaction. Xylenol orange preparations from a number of suppliers were tested, and some differences affecting the sensitivity of the reaction were observed.     

Effects of membrane charges and hydroperoxides on Fe(II)-supported lipid peroxidation in liposomes

The processes in producing a lag phase in Fe²⁺-supported lipid peroxidation in liposomes were investigated. Incorporation of phosphatidylserine (PS) or dicetyl phosphate (DCP) into phosphatidylcholine [PC(A)] liposomes, which have arachidonic acid, produced a marked lag phase in Fe²⁺-supported peroxidation, where PS was more effective than DCP. Phosphatidylcholine dipalmitoyl [PC(DP)] with a net-neutral charge was still effective in producing a lag phase, though weak. Increasing concentrations of PS, DCP, and PC(DP) prolonged the lag period. Initially after adding Fe²⁺, slight oxygen consumption occurred in PC(A)/PS liposomes including hydroperoxides, followed by a lag phase. An increase in the hydroperoxide resulted in a shortening of the lag period. The initial events of Fe²⁺ oxidation accompanied by oxygen consumption were dependent on the hydroperoxide content, but significant changes in diene conjugation and hydroperoxide levels at this stage were not found. The molar ratios of both dis-appeared Fe²⁺ and consumed O₂ to preformed hydroperoxide in liposomes with or withouttert-butylhydroxytoluene were constant, regardless of the different amounts of lipid hydroper-oxides. The antioxidant completely inhibited the propagation of lipid peroxidation in the lipid phase, following a lag phase. In a model system containing 2,2′-azobis (2-amidinopropane) dihydrochloride, Fe²⁺ were consumed. We suggest that Fe²⁺ retained at a high level on membrane surfaces play a role in producing a lag phase following the terminating behavior of a sequence of free radical reactions initiated by hydroperoxide decompositin, probably by intercepting peroxyl radicals.