=Cell-based antioxidant protection in erythrocytes (CAP-e)=
The Cell-based Antioxidant Protection in Erythrocytes (CAP-e) bioassay evaluates whether antioxidants are capable of penetrating and protecting live cells from oxidative damage.
Background
The CAP-e assay arose from a need for a cell-based assay that could answer the simple question of bioavailability at the cellular level: “Does a natural product contain antioxidants capable of entering into and protecting live cells?” The CAP-e assay uses red blood cells (erythrocytes). Many natural products contain compounds that may provoke cellular signaling in other, more complex cell types. Red blood cells (RBC) do not engage in complex inflammatory reactions and are not capable of producing reactive oxygen species (ROS) as a result of stress or death. Furthermore, unlike all other cells, RBCs do not contain mitochondria. Mitochondria engage in the production of ROS, which are pro-oxidant compounds. Testing the antioxidant capacity in a RBC model eliminates confounding factors that in other cellular models makes data interpretation difficult.
Method
Methods for testing the bioactivity of natural products have progressed over a number of years. Many researched natural products have been shown to have similar antioxidant properties as measured, for example, by the ORAC test, but have different effects on human cells. [Honzel D, Carter SG, Redman KA, Schauss AG, Endres JR, Jensen GS. [http://pubs.acs.org/cgi-bin/sample.cgi/jafcau/asap/pdf/jf800401d.pdf "Comparison of chemical and cell-based antioxidant methods for evaluation of foods and natural products: Generating multifaceted data by parallel testing using erythrocytes and polymorphonuclear cells."] J Agric. Food Chem. Aug 22,2008 (epub ahead of print).] Some products reduce inflammatory reactions, whereas other products enhance the response due to a potent immunostimulatory effect. Using complex cellular models, the effects go far beyond what would be anticipated based on antioxidant activity alone.]Another cell-based assay, the polymorphonuclear (PMN) cell type is a useful model for assessment of overall anti-inflammatory properties versus immune supportive properties of natural products. However, due to the complexity of the cell, it is not a good model for obtaining a baseline understanding of antioxidant bioavailability. The ROS PMN assay is an example of a more complex cell-based assay where a natural product may alter the ROS formation by at least three different mechanisms: Direct antioxidant neutralizing of the produced ROS; pro-inflammatory signaling leading to an increase of ROS production; and anti-inflammatory signaling leading to a reduction of ROS formation. Some complex natural products contain compounds that trigger all three mechanisms, so the data from the ROS PMN assay is a superimposition of three different types of reactions. No single test can provide comprehensive answers for complex natural products. A sequential strategy is necessary. In terms of anti-oxidant, anti-inflammatory, and immunomodulatory testing, the progression from analytical chemistry antioxidant testing to more complex biological testing systems is a big leap. As part of a sequential strategy, data from a simple cell-based assay can help bridge analytical chemistry (including antioxidant testing such as ORAC) with more complex cellular assays.
The sequential use of several methods serves to bridge analytical and biological testing methods.[ For example, if a product is suspected to contain antioxidants, analytical chemistry methods, such as ORAC, can be used to verify the presence of antioxidants. Subsequently, the CAP-e assay can be used to assess whether these antioxidants can enter and protect living cells. Then, various complex cellular models can be applied to test for immunomodulatory effects. The data from the CAP-e assay serves an important role in this sequence and allows for a more definitive analysis of data from more complex cellular assays. ]Example 1: Blueberries contain antioxidants and score well in the ORAC test. However, conflicting data arise when testing blueberries in more complex cellular assays, such as immortalized (tumor) cell lines. Consumption of blueberries has been shown to increase RBC resistance to oxidative stress. [Youdim KA, Shukitt-Hale B, MacKinnon S, Kalt W, Joseph JA. [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T1W-419BDYX-H&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=10&md5=9b2bf7ec6278f0168c0722c97e315184 "Polyphenolics enhance red blood cell resistance to oxidative stress: in vitro and in vivo."] Biochim Biophys Acta. 2000 Sep 1;1523(1):117-22.] Furthermore, it is biologically important that blueberries contain compounds that induce apoptosis in many types of tumor cells. [Boivin D, Blanchette M, Barrette S, Moghrabi A, Béliveau R. [http://www.ncbi.nlm.nih.gov/pubmed/17465224 "Inhibition of cancer cell proliferation and suppression of TNF-induced activation of NFkappaB by edible berry juice."] Anticancer Res. 2007 27:937-48.] [Seeram NP, Adams LS, Zhang Y, Lee R, Sand D, Scheuller HS, Heber D. [http://pubs.acs.org/cgi-bin/abstract.cgi/jafcau/2006/54/i25/abs/jf061750g.html "Blackberry, black raspberry, blueberry, cranberry, red raspberry, and strawberry extracts inhibit growth and stimulate apoptosis of human cancer cells in vitro."] J Agric Food Chem. 2006 54:9329-39.] [Srivastava A, Akoh CC, Fischer J, Krewer G. [http://pubs.acs.org/cgi-bin/abstract.cgi/jafcau/2007/55/i08/abs/jf062915o.html "Effect of anthocyanin fractions from selected cultivars of Georgia-grown blueberries on apoptosis and phase II enzymes."] J Agric Food Chem. 2007 Apr 18;55(8):3180-5. Epub 2007 Mar 24.] However, this makes the data difficult to interpret when tumor cell lines, such as the liver cancer cell line HepG2, are used to measure the antioxidant capacity of blueberries. [Wolfe KL, Liu RH. [http://pubs.acs.org/cgi-bin/abstract.cgi/jafcau/2007/55/i22/abs/jf0715166.html "Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements."] J Agric Food Chem. 2007 55:8896-907. ] It seems questionable to use tumor cell lines to study the antioxidant capacity of products that also contains compounds that induce programmed cell death (apoptosis) in the tumor cells. During apoptosis, ROS are formed in the dying cells. The same natural product may contain antioxidant compounds that will neutralize some of the ROS, and the data cannot be interpreted conclusively, since the end result is a combination of production versus neutralization of ROS. In contrast, using erythrocytes in the CAP-e assay to measure the antioxidant capacity of blueberries, such confounding effects do not cloud the data interpretation.
Example 2: The Açai palm berry has an ORAC value of over 1,000 uM TE/gram. However, it performed beyond expectation in the ROS PMN assay [Schauss AG, Wu X, Prior RL, Ou B, Huang D, Owens J, Agarwal A, Jensen GS, Hart AN, Shanbrom E. [http://pubs.acs.org/cgi-bin/abstract.cgi/jafcau/2006/54/i22/abs/jf0609779.html "Antioxidant capacity and other bioactivities of the freeze-dried Amazonian palm berry, Euterpe oleraceae mart. (Açai)."] J Agric Food Chem. 2006 54:8604-10.] where inhibition of ROS formation was measured at extremely low concentrations of Açai aqueous extract. When Açai was tested in the CAP-e assay, and dose responses were carefully compared, it was verified that the anti-inflammatory effect of Açai in the polymorphonuclear (PMN) cell-based assay was a result of anti-inflammatory cellular signaling and not only a result of antioxidants present in the aqueous extract.
The CAP-e assay has shown applicability to in vivo testing to help evaluate antioxidant uptake upon oral consumption. [Jensen GS, Wu X, Patterson KM, Barnes J, Carter SG, Scherwitz L, Beaman R, Endres JR, Schauss AG. [http://pubs.acs.org/cgi-bin/sample.cgi/jafcau/asap/pdf/jf8016157.pdf "In vitro and in vivo antioxidant and anti-inflammatory capacity of an antioxidant-rich fruit and berry juice blend. Results of a pilot and randomized, double-blinded, placebo-controlled, crossover study."] J Agric. Food Chem. Aug 22,2008 (epub ahead of print).] When blood samples are collected before and after consumption of antioxidant-rich foods, both serum and RBCs can be evaluated in a modified CAP-e assay, demonstrating whether consumption resulted in increased antioxidant capacity of serum and/or increased antioxidant protection of RBCs. Both aspects are clinically important, since some antioxidants are only transiently apparent in serum and are rapidly absorbed into cells.
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