Total antioxidant performance: a validated fluorescence assay for the measurement of plasma oxidizability.
The antioxidant capacity of human plasma was determined by following the oxidation kinetics of the lipid-soluble fluorescent marker BODIPY using 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN) as the lipophilic radical initiator. The results are expressed as a total antioxidant performance (TAP) value based on the inhibition of BODIPY oxidation, as determined by the appearance of green fluorescence, with respect to a control sample (phosphatidylcholine with or without delipidized human serum). The suitability of the assay was evaluated on the basis of its precision, reproducibility, and specificity. The intra- and interassay coefficients of variation both were less than 5%. The addition of a representative substrate of plasma peroxidation, phosphatidylcholine, up to 750mug/ml did not induce significant changes in the TAP value. Also, BODIPY photooxidation was not observed during the experimental time course (220min). The TAP values of 6 plasma samples from healthy donors were measured and correlated with the main plasma water- and lipid-soluble antioxidants (uric acid and ascorbic acid, alpha-tocopherol, and carotenoids) and lipid profiles. Significant correlations were found between TAP and uric acid (R=0.97, P<0.05) and cholesterol-adjusted alpha-tocopherol (R=0.93, P<0.01). The results confirm that the TAP assay is suitable to measure the antioxidant activity of plasma antioxidants localized in both the lipophilic and hydrophilic compartments.
Beretta
Giangiacomo
G
Istituto Chimico Farmaceutico Tossicologico, Faculty of Pharmacy, University of Milan, 20143 Milan, Italy. giangiacomo.beretta@unimi.it
Aldini
Giancarlo
G
Facino
Roberto Maffei
RM
Russell
Robert M
RM
Krinsky
Norman I
NI
Yeum
Kyung-Jin
KJ
D000328:Adult; D000368:Aged; D000975:Antioxidants / Q000378:metabolism*; D001391:Azo Compounds; D001774:Blood Chemical Analysis / Q000379:methods*; D001896:Boron Compounds; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D066298:In Vitro Techniques; D008875:Middle Aged; D009570:Nitriles; D010084:Oxidation-Reduction; D010777:Photochemistry; D010949:Plasma / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D000975:Antioxidants; D001391:Azo Compounds; D001896:Boron Compounds; D005456:Fluorescent Dyes; D009570:Nitriles; C109522:V 70