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 Dietary Supplements: Antioxidant Methodology: A Critique 
Robert Jenkins R. PhD ©

It has been estimated that some 3 x 109 years ago, blue-green algae appeared and there went the environment, so to speak. Up to that time it is likely that oxygen existed only in oxides. We now live in an atmosphere consisting of a 21 percent oxygen radical or diradical, to be correct. Since the time of Lavoisier, Priestly, and Scheele, the pioneers in oxygen research, a vast literature related to oxygen has emerged. For instance, there have been nearly 131,000 papers on oxygen published since 1968. That was the year that McCord and Fridovich1 demonstrated that certain enzymes liberated the superoxide radical. Within the next decade, McCord2 demonstrated that radicals induce damage and scavenging enzymes protect against such damage; Chance, Sies and Boveris3 published a seminal review on hydroperoxide metabolism; and Vladimirov et al.4 summarized their work on radicalinduced membrane peroxidation. Such breakthroughs attracted new workers from a wide array of disciplines to the emerging topic of oxidative stress and antioxidant protection. The enormous amount of subsequent research caused Forster and Estabrook5 to suggest that it is proper to conceive of oxygen as an essential nutrient that presents us with problems of malnutrition and overnutrition.

Analysis Methods
The principal difficulty in trying to study free radical events in biological systems rests in the fleetingly short life times of radicals and reactive species. Chance et al.3 quote Warburg as saying,"Wieland has processed whole dogs and not found one drop of H2O2!" The problem was not the inability to detect H2O2 but the failure to realize how rapidly it was degraded in biological systems. It is essential to keep that point in mind when designing research related to oxidative stress.

Radicals themselves exist for only a brief time, and their observation and characterization are limited to various techniques involving a special spectrometer capable of determining electron spin resonance (ESR), also known as electron paramagnetic resonance. The application of ESR to biological samples is fraught with complications. For instance, aqueous solutions have a high dielectric absorption of microwave energy. Investigators have resorted to low-temperature techniques or the use of compounds known as spin traps in an attempt to overcome this problem. Furthermore, tissue preparation itself can generate radicals. These problems coupled with the method's inapplicability to in viva systems has resulted in its infrequent use in exercise-related radical studies.

Lacking a suitable method for direct observation of radicals, most investigators have resorted to various "fingerprint" approaches to establishing that oxidative stress reactions have occurred. One of the most frequently employed techniques involves reacting samples with thiobarbituric acid (TBA) in fluorometric or spectrophotometric assays to establish what are often called TBARS for thiobarbituric reaction products. Some investigators persist in referring to this reaction as a measure of malondialdehyde (MDA), a reaction product of lipid peroxidation. However, it is well known that TBA will react with molecules other than MDA. MDA can be assayed directly by high-pressure liquid chromatography (HPLC)6 or by gas chromatography.7 Often, investigators have failed to detect a rise in plasma thiobarbituric acid reactive substances (TBARS3 subsequent to exercise. Such an observation does not necessarily indicate that oxidative stress has not occurred. We have shown that TEARS is rapidly cleared from electrically stimulated muscle and that rats fatigued by running showed a significant increase in urinary TBARS.8

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