Handbook of Vitamin C Research

12Kelsey H. Fisher-Wellman and Richard J. Bloomermagnetic field [27]. Due to the high cost of such equipment and the high degree of laborassociated with each direct method, the majority of free radial research related to exercise hasutilized indirect methods, rather than direct methods, for the assessment of resultant oxidativestress.Indirect assessment of oxidative stress involves the measurement of the more stablemolecular products formed via the reaction of radical species with certain biomolecules.Using indirect measurement techniques, free radical production is inferred based on thequantification of specific compounds of oxidatively modified biological molecules and/or adecrease in the antioxidant defense system. A variety of analysis procedures have been used[75], ranging from simple spectrophotometric assays to more complex assays using gaschromatography-mass spectroscopy (GC-MS) and high performance liquid chromatography(HPLC) coupled with electrochemical or chemiluminescence detection. The varioustechniques can be used in analyzing various body fluids (i.e., blood, urine), as well as muscleand organ tissue. Blood and urine are most commonly used for analysis in the majority ofoxidative stress research utilizing human subjects.Common Biomarkers of Oxidative StressLipidsSeveral methods exist for assessing lipid peroxidation, including measurement of thesusceptibility of certain isolated lipid fragments in bodily fluids (typically low densitylipoprotein (LDL) cholesterol) to oxidation in vitro, as well as in vivo measurements ofcertain oxidation products of lipid peroxidation. These end products include conjugateddienes, lipid hydroperoxides (LOOH), and malondialdehyde (MDA; a major aldehydeproduced during decomposition of a lipid hydroperoxide). Additionally, thiobarbituric acidreactive substances (TBARS) is an assay used to measure aldehyde products (primarilyMDA) formed via decomposition of lipid hydroperoxides. However, the TBARS assay lacksspecificity, for in addition to aldehydes, TBA also can react with several other biologicalmolecules (such as carbohydrates, sialic acid, or protoglandins), thus interfering with theassay [76]. Certain hydrocarbons, such as pentane and ethane can also be measured viabreath analysis, as an index of lipid peroxidation, as they are produced during peroxidation ofpolyunsaturated fatty acids. Finally, measurement of F 2 -isoprostanes, a prostaglandin-likecompound generated in vivo by non-enzymatic peroxidation of arachidonic acid (an omega-6fatty acid present in the phospholipids of cell membranes), is regarded as the most reliableapproach for the assessment of free radical mediated lipid peroxidation [28]. F 2 -isoprostanescan be measured using high performance liquid chromatography (HPLC), HPLCchemiluminescence(HPLC-CL), gas chromatography-mass spectrometry (GC-MS), andenzyme linked immunosorbent techniques.