Next generation antioxidant assay takes testing inside cells

The new test, named the cellular antioxidant activity (CAA) assay, is dubbed the 'next step' in quantifying antioxidant activity, and marks an advance in the understanding of these compounds.

"The CAA assay is a more biologically relevant method than the popular chemistry antioxidant activity assays because it accounts for some aspects of uptake, metabolism, and location of antioxidant compounds within cells," wrote Kelly L. Wolfe and Rui Hai Liu in the Journal of Agricultural and Food Chemistry .

Previously, the antioxidant activity of selected compounds has been measured using a range of lab-based assays, including the ferric reducing ability of plasma (FRAP) assay, the oxygen radical absorbance capacity (ORAC) and Trolox equivalent antioxidant capacity (TEAC).

The CAA centres on dichlorofluorescin, a probe molecule trapped within cells that can be easily oxidised to produce fluorescence.

The test uses 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP)-generated peroxyl radicals to oxidise dichlorofluorescin, and the ability of antioxidant compounds to inhibit this process.

"The decrease in cellular fluorescence when compared to the control cells indicates the antioxidant capacity of the compounds," explained the authors.

The advance does not make the other tests redundant, they said.

Commenting on the ORAC test, Liu noted its value to scientists.

"What we've done is advance the research to see how these compounds react with cells.

We believe this is a stronger measure of how antioxidant compounds could potentially react in the body," he said.

The developer of the ORAC test, Ron Prior, Ph.D., a scientist with the USDA, welcomed the new advance: "The CAA assay provides information regarding cellular levels of antioxidants which is important to our understanding in this area of antioxidant research," he said.

Proof in the blueberry pudding As a proof of concept, Wolfe and Liu applied the new technique to a small range of fruit extracts.

They report that wild blueberries topped the CAA rankings, followed by cranberry, apple, red grape, and finally green grape.

Not satisfied to have tested only the extracts, the researchers turned their attention to a series of phytochemicals, individual compounds from plants reported to have health benefits.

Amongst the chemicals tested, the researchers reported that quercetin had the highest CAA value, followed by kaempferol, epigallocatechin gallate (EGCG), myricetin, and luteolin, respectively.

The research does add to a growing body of evidence of the potential health benefits of fruit, and in particular berries, that has filtered through to the consumers and has seen demand increase.

Indeed, sales of blueberries, for example, are reported to have rocketed by 130 per cent, raspberry sales are said to have grown by 62 per cent in the last two years, and strawberry sales in the UK are reported to have increased by 34 per cent during the last two years.

Source: Journal of Agricultural and Food Chemistry Volume 55, Issue 22, Pages 8896-8907, doi: 10.1021/jf0715166 "Cellular Antioxidant Activity (CAA) Assay for Assessing Antioxidants, Foods, and Dietary Supplements" Authors: K.L. Wolfe and R.H. Liu