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By Barbara J. King

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2. Preparation of Lipids 1. At this point, the level of conjugated dienes, indicative of the extent of lipid peroxidation, should be measured to assure the occurrence of in vivo peroxidation. 0 mL of ethanol (“photrex” grade, J. T. Baker, Phillipsburg, NJ) for UV absorption measurement. A dual-cavity spectrophotometer is used to record the absorption range of 200–400 nm. Absorbance is measured at 233 nm and a molar extinction coefficient of 25,200 is used to estimate the amount of conjugated dienes produced in the sample (5).

Multiple biosynthetic pathways exist for eicosanoid formation (2) and specific eicosanoids seem to be produced by specific cell types in response to various inflammatory stimuli (3). The multiple synthetic pathways suggest that multiple sites of inhibition may exist. Phospholipase A2 (PLA2) acts on cell membranes to release arachidonic acid (4). While numerous enzymes have demonstrated PLA2 activity, two groups of enzymes are primarily responsible for the arachidonic acid generated during inflammatory responses.

The area of the minor band at 3440 cm–1 relative to the area of the CH stretching band is indicative of the number of lipid-hydroperoxyl groups. We curve-fit the original spectrum (Fig. 2A) although often the Fourier self-deconvoluted spectrum (Fig. 2B) is curve-fit. Caution should be taken when interpreting the data obtained using the curve-fit algorithm and difference spectra (the spectrum of the control sample minus the spectrum of the suspected oxidized spectrum) should be used to confirm that changes in the minor bands detected using the curve-fit spectrum are real.

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