[...]... low, and can be detected only with particularly sensitive methods, which in most of the cases detect the protein from its enzymatic activity Only the enzyme immunoassay method (2) From: Methods in Molecular Biology, vol 186: Oxidative Stress Biomarkers and Antioxidant Protocols Edited by: D Armstrong © Humana Press Inc., Totowa, NJ 3 4 Battelli and Musiani has the advantage of measuring both active and. .. hydroxy derivative (LOH) LHP and LOH represent the primary stable end products of lipid peroxidation (2) Since biological samples are comprised many different LPO products can vary in carbon chain length and degree of unsaturation as well as regioisomerism of the position of the hydroperoxy From: Methods in Molecular Biology, vol 186: Oxidative Stress Biomarkers and Antioxidant Protocols Edited by: D Armstrong... 1 LHP and LOH standards can be purchased or synthesized The synthesis of standards is described in detail in volume 108 of this series (8) 2 If LOH standards are synthesized by methanolic sodium borohydried reduction, rather than purchased in purified form, it is necessary to perform calibrations of 18 Browne and Armstrong Fig 1 Simultaneous chromatograms of LHP and LOH standards at 236 nm (A) and native... filtered, and degassed under vacuum sonication The mobile phase is continuously sparged with helium during analysis 2 System flow rate is 1.3 mL/min and pressure of 175 Kg/cm3 3 The diode array monitored the column effluent from 200–300 nm with specific analysis channels of 236 and 215 nm with 8 nm bandwidth for the LHP/LOH and PUFA, respectively 16 Browne and Armstrong Table 1 Nomenclature, Retention Times and. .. basis to adjust for retention time fluctuations 3.2 LOH Standards LOH standards are prepared from LHP standards by methanolic sodium borohydried reduction as previously described (6) Following chloroform reextraction the LOH standards are dissolved in ethanol and calibration solutions prepared and stored as described for LHP Following individual standard peak identification a hydroxy HPLC mixture is used... extremely broad peaks and poor resolution 5 It should be noted that a photodiode array is not necessary for this methodology and a simple two-channel UV detector could be used Integration of peak areas is performed at 236 nm with an 8 nm bandwidth for LOH and LHP This wavelength and bandwidth are chosen to encompass the wavelength of maximum absorbance (Imax) of the ODEs at 234 nm and ETE at 237 nm References... onto a silver chloride window, and allowed to oxidize in atmospheric oxygen Products of lipid oxidation were measured vs time using infrared spectroscopy 2 Materials 2.1 Equipment 1 Infrared spectrometer with digital output interfaced with a computer 2 Centrifuge 3 Microprobe sonicator From: Methods in Molecular Biology, vol 186: Oxidative Stress Biomarkers and Antioxidant Protocols Edited by: D Armstrong... 3600, 3050, and 3050, 24 Borchman and Sinha Fig 2 (A) (Upper trace) Infrared CH and OH stretching region of an anhydrous films of bovine rod outer segment lipids exposed to air and light for 20 h (Lower trace) curve-fit spectrum of upper trace using peak information from (B) Fourier self-deconvolution spectrum and (C) second derivative spectrum of (A) 2750 cm–1 for the CH and OH stretching bands, respectively... of 40 h is shown in Fig 1 The intensity of the OH stretching bands (3600–3100 cm–1), reflects the degree of lipid oxidation (5) and the amount of hydroxyl containing lipids such as sphingolipids and cholesterol (see Note 5) To quantify the increase in OH band intensity as a result of lipid oxidation, the areas of the OH and CH stretching bands are measured using the integration program provided by Grams... regioisomeric species of LHP and LOH derived from the four major PUFA found in human plasma; linoleic, arachidonic, linolenic, and docosahexaenoic acid (6) Following total lipid extraction, alkaline hydrolysis and reextraction of the liberated fatty acids, two separate systems with different mobile-phase conditions and analytical columns were used, one for LOH and LHP and the second for the native . Culture Protocols, edited by Clare Wise, 2002 187. PCR Mutation Detection Protocols, edited by Bimal D. M. Theophilus and Ralph Rapley, 2002 186. Oxidative Stress Biomarkers and Antioxidant Protocols, edited. vol. 186: Oxidative Stress Biomarkers and Antioxidant Protocols Edited by: D. Armstrong © Humana Press Inc., Totowa, NJ 4 Battelli and Musiani has the advantage of measuring both active and inactive. x0 y0 w1 h0" alt="" Oxidative Stress Biomarkers and Antioxidant Protocols M E T H O D S I N M O L E C U L A R B I O L O G Y TM John M. Walker, Series Editor 215. Cytokines and Colony Stimulating