Linoleic acid hydroperoxide determination

The value for e is only approximate here, as the extinction coefficient was based on iodometric PV determinations of an ethanolic solution of linoleic acid hydroperoxide (O Brien, 1969). 

Enzyme Assays. Cytochrome P-450 peroxidase activity was determined by the method of O Brien and Rahimthula (27) as modified by Reddy, et al, with cumene hydroperoxide (CHP), linoleic acid hydroperoxide (LHP) and 15-HPETE as substrates and tetramethyl-P-phenylene diamine (TMPD) as electron donor. Prostaglandin H synthase activity was measured as previously described (19). 

Nerland, D.E., M.M. Iba, and G.J. Mannering (1981). Use of linoleic acid hydroperoxide in the determination of absolute spectra of membrane-bound cytochrome P450. Mol. Pharmacol. 19, 162-167. 

The first FI-CL system for lipid hydroperoxide determination, published in 1993, was based on luminol as the CL reagent and microperoxidase as the catalyst. The optimized system was used for measuring lipid hydroperoxides in native low-density lipoprotein (LDL) (Cominacini et al. 1993). The FI-CL system consisted of two pumps, an autosampler, and a chemiluminescent detector with a T-mixing coil. Samples were injected by using an autosampler and mixed with luminescent reagent (3 (iM luminol and 1 xM microperoxidase in 0.1 M carbonate buffer, pH 10). The calibration curve was obtained using linoleic acid, and a detection limit of 3 pmol linoleic acid hydroperoxide was reached. 

RP-HPLC with nonaqueous solvents and UVD at 246 nm was developed for the determination of low level POVs of vegetable oils. These measurements are specific for conjugated diene peroxides derived from vegetable oils with relatively high linoleic acid content. These measurements may be supplemented by nonspecific UVD at 210 nm and ELSD for detection of all eluted species. The elution sequence of the triglycerides in a nonaqueous RP-HPLC is linearly dependent on the partition number of each species, Vp, which is defined as = Nq — 2Ni, where Nq is the carbon number and is the double bond number. In the case of hydroperoxides = Nq — 2Nd — Vhpo, where Vhpo is the number of hydroperoxyl groups in the molecule (usually 1 for incipient POV). For... 

CZE-ELD, with a An microelectrode at —0.6 V vs. SCSE and a Pt wire as auxiliary electrode, using sodium borate buffer and dodecyltrimethylammonium bromide for dynamic coating of the capillary internal surface, can be applied for separation and determination of hydroperoxides in ultra-trace amounts. Thus, various hydroperoxides derived from linoleic acid undergo total dissociation to carboxylates in borate buffer however, due to their similar molecular masses, in order to resolve the ELD signals, it is necessary to add /3 -cyclodextrin (83) to form complexes with the analytes and reduce their mobility, in accordance with the value of the complexation equilibrium constants . 

Localization of double bonds in unknown compounds has frequently been determined by ozonolysis. Unsaturated fatty acids of biological membranes are susceptible to ozone attack, but there are some important differences from autoxidation reactions. These include the fact that malonaldehyde is produced from linoleate by ozonolysis (53) but not autoxidation and also that ozonolysis does not cause double bond conjugation as judged by absorption at 233 nm (52). Reactions with the polyunsaturated fatty acids produce several possibilities for toxic reactions direct disruption of membrane integrity and toxic reactions caused by fatty acid hydroperoxides, hydrogen peroxide, and malonaldehyde. 

When the Fe enzyme is treated with an excess of the product hydroperoxide or a combination of linoleic acid and O2, a transient purple intermediate having a chromophore at 585 nm ( 535 1300M cm ) is formed. " The structure of this species has been determined by X-ray crystallography... 

A variety of compounds such as hydrocarbons, alcohols, furans, aldehydes, ketones, and acid compounds are formed as secondary oxidation products and are responsible for the undesirable flavors and odors associated with rancid fat. The off-flavor properties of these compounds depend on the structure, concentration, threshold values, and the tested system. Aliphatic aldehydes are the most important volatile breakdown products because they are major contributors to unpleasant odors and flavors in food products. The peroxidation pathway from linoleic acid to various volatiles is determined in several researchs, - by using various techniques (Gas chromatography mass spectrometry, GC-MS, and electron spin resonance spectroscopy, ESR), identified the volatile aldehydes that are produced during the oxidation of sunflower oil. In both cases, hexanal was the major aldehyde product of hydroperoxide decomposition, whereas pentanal, 2-heptenal, 2-octenal, 2-nonenal, 2,4-nonadienal, and 2,4-decadienal were also identified. 

Henderson, D. E., Slickman, A. M., and Henderson, S. K. 1999. Quantitative HPLC determination of the antioxidant activity of capsaicin on the formation of lipid hydroperoxides of linoleic acid a comparative study against BHT and melatonin. J. Agric. Food Chem. 47 2563-2570. 

In our study, the reusability of LOX immobilized in calcium-alginate beads was determined by repeatedly using the same beads for five successive reactions with linoleic acid (LA) (Fig. 2). The LOX activity was measured after each cycle, and the beads were recovered and washed with sodium borate buffer before reuse. To initiate the next cycle of oxidation, LA was added to the incubation mixture containing the recovered beads. The data (Fig. 2) demonstrates that LOX immobilized in beads can be reused at least five times without substantial loss in enzyme activity. In contrast, free lipoxygenase is typically inactivated by hydroperoxide accumulation and the partial anaerobic condition that develops in the reaction mixture (8). 

Hydroperoxide lyase assay. Hydroperoxide lyase was determined by measurement of the formation of hexanal from 13-hydroperoxlde of linoleic acid at pH 6.3.(1 ) The substrate (6 pmol) dissolved In diethyl ether was pipetted Into a 50-ml flask and the solvent was evaporated 1 vacuo. Then, 10 ml of chloroplast suspension or leaf homogenate was added. The mixture was sealed In the flask with a rubber stopper and Incubated at 35°C for 10 mLn. The Cg-aldehydes formed were measured by the headspace method with GLC. 

Hydroperoxide dehydrase activity was determined at 25 C by monitoring the decrease in absorbance at 234 nm [5] with 10 fjM of 13-hydroperoxide of linoleic acid as substrate. Hydroperoxide lyase activity was determined by a specific spectrophotometric assay [7], using 40 /iM of 13-hydroperoxide of linoleic acid as substrate. The solution of 13-hydroperoxide of... 

Reactive aldehydes derived from lipid peroxidation, which are able to bind to several amino acid residues, are also capable of generating novel amino acid oxidation products. By means of specific polyclonal or monoclonal antibodies, the occurrence of malonaldehyde (MDA) and 4-hydroxynonenal (4-HNE) bound to cellular protein has been shown. Lysine modification by lipid peroxidation products (linoleic hydroperoxide) can yield neo-antigenic determinants such as N-c-hexanoyl lysine. Both histidine and lysine are nucleophilic amino acids and therefore vulnerable to modification by lipid peroxidation-derived electrophiles, such as 2-alkenals, 4-hydroxy-2-alkenals, and ketoaldehydes, derived from lipid peroxidation. Histidine shows specific reactivity toward 2-alkenals and 4-hydroxy-2-alkenals, whereas lysine is an ubiquitous target of aldehydes, generating various types of adducts. Covalent binding of reactive aldehydes to histidine and lysine is associated with the appearance of carbonyl reactivity and antigenicity of proteins [125]. 

Hydroperoxy fatty acids as such cannot be separated by GC as they decompose at high temperatures, and HPLC is probabiy the preferred method for their anaiysis [168], Nonetheiess, there are times when it is advantageous to convert them to the hydroxy derivatives by means of sodium borohydride reduction and then to the TMS ethers for GC anaiysis, for exampie for identification by GC-mass spectrometry products derived from linoleic [223,341,911,946,1005], arachidonic [124,407,568, 1005] and docosahexaenoic acids [948] have been examined in this way (the list is not intended to be comprehensive). Woollard and Mallet [1005], in particular, have presented a comprehensive list of ECL data for compounds of this type. In addition, GC methods were used for the determination of the absolute configuration of hydroperoxides formed by lipoxygenase reaction [124,568,947]. 

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