Lipid hydroperoxide, detection

Nourooz-Zadeh J. 1999. Ferrous ion oxidation in presence of xylenol orange for detection of lipid hydroperoxides in plasma. Methods Enzymol 300 58-62. 

Lipid Peroxidation Chemistry, 105, 273 overview of methods used for detecting lipid peroxidation, 105, 283 chemical methods for the detection of lipid hydroperoxides, 105, 293 comparative studies on different methods of malonaldehyde determination, 105, 299 concentrating ethane from breath to monitor lipid peroxidation in vivo, 105, 305. 

The following spectrophotometric methods are conceptually related to the POV however, they are intended for other purposes. HPLC-UVD at 234 nm was applied to detect lipid hydroperoxides, obtained in a set of experiments for assessing the effectiveness of lipoxygenase enzymes extracted from various microorganisms. The absorbance. A, measured at three different wavelengths is linearly correlated to the concentrations in xM units of lipid hydroperoxides, Clh, 7-oxocholesterol, Cqc and dienals, Cde according to the set of simultaneous equations 60. This method was used to track the Cu(II)-induced oxidation of LDL" °. 

Reviews appeared on the following subjects Analysis of lipid hydroperoxides , the difficulties encountered for hydroperoxide analysis in a plasma matrix , post-column derivatization after GLC of lipid hydroperoxides and methods for detection and characterization of hydroperoxy groups in oxidized polyolefins . 

Hydroperoxides may be determined by measuring at 290 nm (e = 44100 M cm ) or 360 nm (e = 28000 cm ) the concentration of 13 formed in the presence of a large excess of ions. The reaction may be too slow for practical purposes, unless a catalyst is present. For example, an assay for lipid hydroperoxides conducted without a catalyst may require several measurements every 6 min until the absorbance reaches a maximum. Exclusion of air from the sample cuvette is important. The method is about 1000-fold more sensitive than thiosulfate titration The iodometric method with UVD at 360 was adopted for detecting the presence of hydroperoxides derived from protein, peptide or amino acid substrates subjected to y-radiation, after destroying the generated H2O2 with catalase. ... 

A procedure for determination of lipid hydroperoxides in human plasma is based on kinetic measurement of the CL of luminol (124) with hemin (75a) catalysis . CLD of microperoxidase-catalyzed oxidation of luminol (124) or isoluminol (190) was applied to detection and determination of amino acid hydroperoxides after exposure to UV and y-irradiation A method for determination of hydroperoxides in the phospholipids of cultured cells uses isoluminol (190) and microperoxidase as catalyst " . Simultaneous determination of phosphatidylcholine hydroperoxides and cholesteryl ester hydroperoxides in human serum is carried out by quantitative extraction of the lipids, HPLC separation by column switching and CLD using isoluminol (190) with microperoxidase catalysis . ... 

Cyt c is associated with the outer surface of the inner mitochondrial membrane. Phospholipids induce conformational changes in the protein and, in certain instances, the haem can convert to the high spin (S = 5/2) form, indicative of a weakening of the ligand field caused by displacement of the sixth ligand (Met-80). This has been associated with the detection of lipid radicals by direct EPR (at 11 K).65 Indeed, peroxidase-type activity is also evident in the reaction of cyt c with lipid hydroperoxides, as studied by spin trapping in conjunction with HPLC and MS.66... 

Fig. 9. Concentrations of cutaneous lipophilic antioxidants after a single large dose (25J/cm2) of simulated sunlight. Total quinol/one = ubiquinol + ubiquinone, n = 6. Inset (A) concentration of lipid hydroperoxides in the same skin samples, irradiated and non-irradiated (none detectable in non-irradiated samples). Statistically different from unirradiated controls double asterisks, p < 0.01 ...

Fig. 10. Lipid hydroperoxides in skin of hairless mice, detected by HPLC with chemiluminescent detection. Representative chromatograms are shown for one mouse. Top non-irradiated middle irradiated bottom irradiated, treated with triphenyl phosphine.

Yamamoto, Y., Brodsky, M.H., Baker, J.C., and Ames, B.N. (1987) Detection and characterization of lipid hydroperoxides at picomole levels by high-performance liquid chromatography, Anal. Biochem. 160, 7-13. 

The advantage of this method lies in its sensitivity (the limit of detection is 6 pmol/assay) but the disadvantage is the need for a purified lipid hydroperoxide standard which is a time consuming and difficult preparation. 

It should be noted that the tetroxide intermediate proposed as the mechanism for peroxyl radical disproportionation remains somewhat controversial. If it exists, it has been argued that the oxygen should be released as O2 to avoid spin restrictions (291). Some studies claim to have detected O2 from lipid hydroperoxides (366), but the evidence has not been conclusive. One of the difficulties in determining when 02 is produced is that 02 reduces singlet oxygen when water sufficient to provide a hydration shell of five water molecules is present (373). 

Ferric Ion Complexes Other chemical methods based on the oxidation of ferrous ion (Fe ) to ferric ion (Fe ) in an acidic medium and the formation of iron complexes have also been widely accepted. These methods spectrophotometri-cally measure the abihty of lipid hydroperoxides to oxidize ferrous ions to ferric ions, which are complexed by either thiocyanate or xylenol orange (23, 28, 29). Ferric thiocyanate is a red-violet complex that shows strong absorption at 500-510 nm (8). The method of determining PV by coloremetric detection of ferric thiocyanate is simple, reproducible, and more sensitive than the standard iodometric assay, and has been used to measure hpid oxidation in milk products, fats, oils, and liposomes (8, 23). 

There is considerable body of (indirect) evidence which makes oxidative stress one of the best accepted hypothesis for explaining the cause of Parkinson s disease. For example, the Fe(II)/Fe(III) ratio in the substantia nigra is shifted from 2 1 in the normal brain to 1 2 in Parkinsonian brain.131,132 In the Parkinsonian brain several enzymes which constitute the antioxidative defence mechanisms (glutathione peroxidase, catalase) have a decreased activity, while the activity of superoxide dismutase is increased, relative to the normal brain.133 Furthermore, specific products of radical damage, such as lipid hydroperoxides, were detected at a 10-fold increased level in the Parkinsonian brain.134... 

The application of UV alone to monitor substrate oxidation product formation is limited to studies on polyunsaturated fats with conjugated oxidation products. Reverse-phase HPLC elution of conjugated and non-conjugated lipid hydroperoxides (LOOH) can be followed electrochemically (concurrently with UV detection) so long as the eluent contains a supporting electrolyte (such as sodium chloride " or tetraethylammonium perchlorate ). 

Pryor W.A., Castle L., Chemical methods for the detection of lipid hydroperoxides, Meth. Enzymol., 1984,105,293-299. 

Akasaka, K., Ohrui, H., and Meguro, H. (1993) Normal-phase high-performance liquid chromatography with a fluorimetric postcolumn detection system for lipid hydroperoxides, J. Chromatogr., 628, 31-35. 

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