Antioxidant Methodology: A Critique

Background

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

The use of a hydrocarbon breath test has been underutilized in exercise research. The peroxidation of n-3 and n-6 fatty acid families results in the production of the volatile alkanes ethane and pentane. The compounding problems with this approach include the fact that the atmosphere breathed may be contaminated by hydrocarbons and our gastrointestinal tract bacteria also make these compounds. Snider et al.⁹ have found that the contamination problems can be overcome by including a wash out period, during which the subjects breathed hydrocarbon-free air prior to the experimental period.

During radical reactions, there is an electron jump from ground state to an
excited state. When the electron falls back to ground state, a low level of
light energy is emitted; this is called chemiluminescence. We employ a single
photon counting system similar to that described by Boveris et al.¹⁰ to
determine a tissue s ability to defend itself against oxidative stress.
The tissue can be challenged by various organic or inorganic hydroperoxides. When a variety of antioxidants are systematically added to the incubation
mixture, the procedure may yield evidence to implicate the radical species
that may be involved.

Recently we have begun to couple assays of mitochondrial oxygen consumption and the chemiluminescence analysis, with the intent of developing a model system for the study of oxidative stress. Robinson and co-workers¹¹ have demonstrated that an increased electron flux capacity is necessary to achieve the increased peak oxygen consumption derived by training. Furthermore, Kehrer and Lund¹² have provided an excellent review on the relationship between cellular reducing equivalents and oxidative stress. We employ myxothiazol, a tight inhibitor of complex III, in mitochondrial suspensions. This results in an inhibition of state III respiration and a subsequent increase in chemiluminescence. Although still in the preliminary stages of development, this approach offers some promise, since reducing equivalents and antioxidants can easily be added to the system.

Obviously, in viva studies involving antioxidant supplementation should be accompanied by marker assays to demonstrate that the dosage regimen has altered the antioxidant concentration. The Methods in Enzymology series^13-16 end Evans et al.¹⁷ text provide a convenient source of reliable assays. Since antioxidants often are involved in an interrelated chemistry, it is useful to attempt to understand how the protocol of interest has influenced the total milieu. Cao and co-workers have developed a technique suitable for that purpose.¹⁸

Needed Research

To date, the preponderance of studies related to exercise and oxidative stress have centered on determining evidence of stress and the influence of one antioxidant at a time. Literature related to a multiple supplement approach is sparse. While youth sport leagues in the United States expose children to high aerobic doses, there are virtually no oxidative stress-related data available on children younger than age 18. Furthermore, just as some members of the general population are apt to receive orthopedic trauma from certain types of intense aerobic activity, we might assume that some genetic sets are unable to cope with oxidative stress. Oxidative stress research is needed to detect aberrant responses to exercise rather than to focus on group means. Finally, we are beginning to understand that radicals play many vital roles in our biochemistry. We must question whether massive doses of antioxidants are violating that beneficial chemistry.

1. McCord JM, Fridovich I. The reduction of cytochrome c by milk xanthine oxidase. J Biol Chem 1968;243:5753 60.

2.McCord JM. Free radicals and inflammation: protection of synovial fluid by superoxide dismutase. Science 1974; 185:529-30.

3. Chance B. Sies H. Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev 1979;59:527 605.

4. Vladimirov YA, Oleenev VI, Suslova TB, Cheresmisnina ZP. Lipid peroxidation in mitochondrial membrane. Adv Lipids Res 1980;17:173-249.

5.Forster RE, Estabrook RW. Is oxygen an essential nutrient? Annu Rev Nutr 1993;13:383-403.

6.Bird RP, Hung SO, Hadley M, Drapper HH. Determination of malondialdehyde in biological materials by high-pressure liquid chromatography. Anal Biochem 1983;128:240-4.

7.Ichinose T. Miller MG, Shibamoto T. Gas chromatographic analysis of free and bound malondialdehyde in rat liver homogenates. Lipids 1989;24:895-8.

8. Jenkins RR, Krause K, Schofield LS. Influence of exercise on clearance of oxidant stress products and loosely bound iron. Med Sci Sports Exerc 1993;25:213-7.

9.Snider MT, Balke PO, Oerter KE, Francalancia NA, Pasko KA, Robbins ME, et al. Methods for measuring lipid peroxidation products in the breath of man. Life Chem Reports 1985;3:168-73.

10. Boveris A, Cadenas E, Reiter R. Filipkowski M, Nakase Y. Chance B. Organ chemiluminescence: noninvasive assay for oxidative radical reactions. Proc Natl Acad Sci U S A 1980;77:347-51.

11. Robinson DM, Ogilvie RW, Tullson PC, Terjung R. Increased peak oxygen consumption of trained muscle requires increased electron flux capacity. J Appl Physiol 1994;77(4):1941-52.

12.Kehrer JP, Lund LG. Cellular reducing equivalents and oxidative stress. Free Radic Biol Med 1994;17:65-75.

13. Colowick SP, Kaplan NO, eds. Methods in enzymology. New York: Academic Press, 1984:105.
14.

15. 14. Abelson JN, Simon Ml, eds. Methods in enzymology. New York: Academic Press, 1990:186.

15. Abelson JN, Simon Ml, eds. Methods in enzymology. New York: Academic Press, 1994:233.

16. Abelson JN, Simon MI, eds. Methods in enzymology. New York: Academic Press, 1994:234.

17. Rice-Evans CA, Diplock AT, Symons MCR. Techniques in free radical research. Amsterdam: Elsevier, 1991.

18. Cao GC, Alessio HM, Cutler RG. Oxygen-radical absorbance capacity assay for antioxidants. Free Radic Biol Med 1993; 14:303-11.