Antioxidant inhibition of oxygen radicals for measurement of total antioxidant capacity in biological samples.
Few methods for assessing total antioxidant capacity (TAC) include both the percentage of inhibition and the length of inhibition in the measurement. Available methods require above ambient constant temperature incubation, reaction preheating, and/or separate assays for testing hydrophilic and hydrophobic samples. We describe a high-throughput method, antioxidant inhibition of oxygen radicals (AIOR), that overcomes these difficulties. AIOR uses peroxyl radicals to trigger a decrease in fluorescence of the indicator molecule, uroporphyrin I, which is delayed by the presence of antioxidants. The area under the curve is measured by a fluorescence spectrophotometer in a 96-well microplate format, and TAC results are expressed as millimole/liter Trolox equivalents. AIOR is performed at ambient temperature and is applicable to samples in either aqueous or common organic solvents. The reaction between uroporphyrin I and the peroxyl radicals generated from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) was found to be of first-order kinetics with a mean rate constant (k) of 0.0254. Applications to measure antioxidant capacity are demonstrated on individual chemicals and biological samples. The method has good linearity, within- and between-assay precision, and recovery.
PathWest Laboratory Medicine WA, Queen Elizabeth II Medical Centre, Nedlands, Western Australia 6009, Australia. simon.ching@health.wa.gov.au
Simon Y L
SY
Ching
Jon
J
Hall
Kevin
K
Croft
John
J
Beilby
Enrico
E
Rossi
Emilio
E
Ghisalberti
D000975:Antioxidants / Q000032:analysis* / Q000378:metabolism; D001628:Beverages / Q000032:analysis*; D006801:Humans; D007700:Kinetics; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000032:analysis / Q000097:blood; D014527:Uric Acid / Q000032:analysis / Q000097:blood; D014578:Uroporphyrins / Q000737:chemistry / Q000378:metabolism; D014815:Vitamins / Q000032:analysis / Q000097:blood
D000975:Antioxidants; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds; D014578:Uroporphyrins; D014815:Vitamins; D014527:Uric Acid; C034103:uroporphyrin I