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Figure 1 Detection of authentic ROOHs in plasma. PC-OOH, phospholipid hydroperoxides; 5-HPETE, 5-hydroperoxyeicotetraenoic acid.

it is unclear to what extent plasma lipoprotein peroxidation assessed by this method accounts for biological changes associated with oxidative stress.

We have used the ferrous oxidation with xylenol orange (FOX) assay coupled with triphenylphosphine (TPP) to determine plasma lipid hydroperoxide (ROOH) levels in health and disease (16,17). TPP is used to reduce ROOHs. This maneuver is necessary to generate a proper control for each individual plasma sample because plasma contains interfering components, mainly ferric ions that are detected by xylenol orange (Fig. 1). Other advantages of the FOX2 assay over existing techniques are kinetics of the reaction are independent of the chemical structure of the ROOHs and no extraction step is normally needed because the use of the 90% methanol-25 mM H2S04 denatures proteins sufficiently allow access of the ferrous ions to available ROOHs. The coefficient of variation for individual plasma samples using this method is typically less than 5%, whereas that for the interassay coefficient of variation is <10% (16-19).

I. DIURNAL VARIATION OF PLASMA ROOHs

No information is available on the effect of diurnal variation on plasma lipid peroxidation products. Using the FOX assay, we examined this issue in two subjects (one woman, one man, aged 30 and 36, respectively) under fasting

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