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Mechanisms and Biomarkers (WG 4) page 25
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changes usually occurring with more or less long delay. It should be emphasized that such
biomarkers are very useful to investigate the interactions of diet components on antioxidant
status in short-term experiments. Thus, based on assumptions that oxidised DNA and
lipoproteins are respectively involved in cancer and cardiovascular diseases, measurement of
oxidised DNA bases and level of peroxidised lipids in LDL were used as surrogate
biomarkers for the latter diseases. The methods and the limits of these biomarkers were
recently discussed by Halliwell (1999). Other parameters are now considered to evaluate
antioxidant status. The latest investigations were designed to be used for human studies
implying minimally invasive methods and focused mainly on products resulting from free
radicals as biomarkers of free radical damage (De Zwart et al., 1999) and on the determination
of plasma antioxidant capacity (Prior and Cao, 1999).
Among the products resulting from free radical damage, the most representatives of lipid
peroxidation products are exhaled pentanes, plasma and urine isoprostanes and aldehydic
products. Hydroxylated DNA products (mainly Hydroxylated guanosine, 8-OH-dG) and
carbonyls groups are the most usual markers for DNA and protein damage, respectively.
More recently, a concept of antioxidant capacity has been developed and can be defined as an
individual measure reflecting the sum of available endogenous and exogenous defence
mechanisms which ensure the oxidative balance (Fürst, 1996). Measurement of antioxidant
capacity is mostly developed in the plasma assuming as representative of in vivo whole-body
antioxidants. Such a measurement avoids the measurement of each antioxidant separately and
takes into account the possible interactions between them. The methodology consists on using
a prooxidant, generally a free radical, and an oxidizable substrate. The prooxidant induces
oxidative damage to the substrate which is inhibited by the antioxidants contained in the
biological samples to be tested. In these inhibition methods many techniques were developed
and the most usual one is oxygen radical absorbance capacity also known as ORAC assay
(Cao et al., 1993). It uses phycoerythrin as oxidizable protein substrate and AAPH (an azo
compound) as a peroxyl radical generator or copper-hydrogen peroxide as hydroxyl generator.
Protein oxidation induced a quenching of fluorescence which is retarded by the antioxidants
present in the tested samples. The quantitation is made by area-under-curve determination
after completion of the reaction. A single value is thus determined for both inhibition
percentage and length of inhibition time (the lag time during which the antioxidants from the
sample protected oxidation of the protein). The ORAC assay has been used in several
laboratories to determine the antioxidant of biological samples but also for pure compounds
(melatonine, flavonoids) and complex matrices (tea, fruits and vegetables, and tissues). Other