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The CAP-e assay: testing antioxidant bioavailability at the cellular level and creating a foundation for the biological testing of natural products.

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At the recent natural products trade show, Supply Side West, a novel bioassay, CAP-e, specifically developed to evaluate antioxidant bioavailability in vitro and in vivo, was recognized with the presentation of the NPI Scientific Excellence Award 2008. This article presents recent advances in the CAP-e (cell-based antioxidant protection) assay regarding its role in natural products testing, its application as a QC tool and the importance of controlling inter-assay variability between batches of cells used for the assay. The CAP-e assay is a novel, accelerated method for the evaluation of cell-based antioxidant protection using erythrocytes. (1,2) It was designed to help the natural products industry move away from analytical chemistry testing towards more complex biological testing. The method uses assay principles comparable with the Oxygen Radical Absorbance Capacity (ORAC) assay. The CAP-e assay involves applying natural products to intact erythrocytes, and specifically measuring antioxidants capable of penetrating into and protecting the cells from free radical damage. The assay can be applied to test the antioxidant availability of live cells in vitro and document antioxidant uptake in vivo. (2,3) During the past year, we have made progress in several areas, particularly regarding quality control of the cells used for this assay and also controlling inter-assay variability.

Free Radical Research

Free radicals are generated as a result of normal metabolism, as well as by disease-induced stress and environmental pollution. As part of the growing interest in nutritional strategies for antiageing and disease prevention, antioxidants in foods and nutritional supplements have become a major area of focus. (4,5) Antioxidant testing has become a cornerstone of the natural products industry. One of the most frequently utilized methods is the ORAC assay. Neither the ORAC assay nor other chemical-based antioxidant tests provide information regarding the bioavailability of antioxidants to living cells, organs or organisms. To prove that antioxidants in a product are absorbed by a living organism (whether animal or human) requires a clinical bioavailability trial. Such studies are highly complex, costly and must take into account the effects of the digestive process on the chemical nature of compounds. Another method of clinically proving antioxidant uptake is to test whether consumption of a product actually contributes to antioxidant protection in a whole living being. Once a product is characterized by the ORAC test panel, further information is needed to examine various aspects of its bioavailability. In other words, do the antioxidants have any meaningful impact for those who consume them? This includes addressing the following questions:

* Is the antioxidant absorbed upon consumption?

* What are the local effects in gut tissue?

* Is there entry into circulating blood?

* Is there entry into living cells?

* Can the antioxidant protect cells from oxidative damage?

The current investigative options/experimental models include the following:

* A full clinical trial focused on assessment of inflammation/pathology

* A clinical trial to evaluate the antioxidant capacity and/or inflammatory markers in serum

* Animal testing

* Cell-based testing.

Before entering into clinical testing, it may be prudent to conduct cell-based assays. Numerous cell types are used by laboratories; but, most cell-based assays are too complex to provide answers regarding antioxidant uptake into live cells. NIS Labs has developed an assay that addresses one simple, yet very important question: can antioxidants enter and protect a living cell? (1-3)

The CAP-e Assay

The CAP-e assay is based on a methodology similar to the ORAC test, but is performed in cells of a very simple composition. The assay makes use of erythrocytes (predominantly human, but dog and horse cells have also been used). In contrast to other cell-based models, the CAP-e assay utilizes erythrocytes (red blood cells) because they do not contribute to oxidative damage, and the assay specifically measures those antioxidants capable of crossing the plasma membrane into the intracellular space. In this assay, the cells are exposed to test products in physiological saline and are given enough time to absorb compounds from the test product. Any compounds that are not absorbed by the cells during that period are removed by centrifugation and subsequent washing. The cells are exposed to a precursor dye that becomes fluorescent when exposed to oxidative damage. Subsequently, the cells are subjected to an oxidative challenge such as [H.sub.2][O.sub.2] or AAPH. The fluorescence intensity reflects the amount of oxidative damage. As a positive control, cells are exposed to oxidative challenge without any antioxidant protection. This serves as a measure of maximal oxidative damage. Any reduction of oxidative damage to the cells pretreated with the test product reflects antioxidant protection from the test product. The CAP-e assay is qualitative in principle, but does allow for some semiquantitative comparisons with standards such as gallic acid, Trolox, MSM, quercetin and ascorbic acid.

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The Advantages of Sequential Testing

For the natural products industry, the CAP-e assay serves as a bridge when moving from analytical to biological testing. (2) Antioxidant capacity is only one limited aspect of most natural products. For example, juices can be made from fruits and berries, but the complex biological activities differ from fruit to fruit, independent of their antioxidant capacity. The CAP-e assay provides data that enables more complex biological assays to be more correctly interpreted. Many natural products will induce the formation of reactive oxygen species (ROS) in more complex cell-based assays. Such assays are necessary to document other biological effects, but cannot be used to document antioxidant capacity in a biological system.

By contrast, many natural products possess potent anti-inflammatory properties, even at doses wherein the antioxidant capacity is undetectable. An example is the Amazonian palm berry, Acai. Freeze-dried Acai has a very high antioxidant capacity according to ORAC and performs well in the CAP-e assay. (2,6) Acai also has potent anti-inflammatory effects, which are not detectable at doses when its antioxidant capacity is best demonstrated. The anti-inflammatory property is clearly independent of the antioxidant content of the product.

Testing of Solid Versus Liquid Products

CAP-e antioxidant protection units are calculated differently for dry powders and fresh foods compared with liquids (Figures 1 and 2). When reporting per weight, we must report the data in GAE (Gallic Acid Equivalents). When reporting on liquids, we must report in Mol GA/L. There is no simple direct conversion factor from juices to solids; it can depend on how thick a juice is, how much pulp it has, whether extracts were added, how those extracts were made, etc. For example, liquid green tea can be tested using the CAP-e assay. The green tea liquid itself represents a hot water extract of a certain amount of dry leaves into a certain amount of liquid. The CAP-e value of the tea could either be related to the amount of crude tea leaves and hot water that were used to make the liquid extract or to the amount of solids in the tea extract. This is similar to ORAC. This may go unnoticed by many people and lead to confusion when comparing different products. ORAC on powders is reported in Trolox Equivalents per gram product. Liquids are reported in Mol Trolox/L. There is no simple and direct conversion factor from liquids to solids.

Applications of the CAP-e Assay

The CAP-e assay has three distinct uses:

Antioxidant bioavailability in vitro: Testing of natural products for antioxidants available to enter and protect live cells.

Antioxidant bioavailability in vivo (I): Testing of serum samples obtained from human subjects before and after consuming antioxidant-rich products. (3)

Antioxidant bioavailability in vivo (II): Testing of red blood cells obtained from human subjects before and after consuming antioxidant-rich products.

Quality of Cells and Inter-Assay QC

The CAP-e assay is useful tool for the comparison of raw ingredients, blends and processed products, as well for comparing similar ingredients from different suppliers. It also lends itself to finished product quality control (QC). To offer CAP-e testing for QC, it was necessary to establish a protocol to reduce inter-assay variability. For those reasons, QC of the source of cells used for each assay is an important factor for reducing inter-assay variability and standardizing the procedure. When erythrocytes are removed from the blood, they contain varying amounts of redox enzymes and antioxidants obtained from the donor's diet. Even within the same donor, day-to-day variations exist as a result of diet and inflammatory conditions. In female donors, the menstrual cycle will affect the antioxidant capacity of freshly isolated erythrocytes.

We have shown that when erythrocytes are allowed to incubate at 4[degrees]C for several weeks, the innate antioxidant capacity gets depleted and variations between different samples are reduced. In each assay, we use data from the antioxidant gallic acid to determine satisfactory assay performance. By comparing data from more than 60 experiments performed with erythrocytes that were either fresh or had aged for various times, we have established protocols for generating consistency between assays when testing natural products, extracts, foods and beverages in vitro.

Source of Cells

The CAP-e constitutes a cell-based model for antioxidant testing that neither has the complexity of the neutrophil/monocyte assay nor the risk of misinterpretation possible with tumour cell-line-based assays. In the CAP-e assay, erythrocytes are used as a model because they represent a more inert type of body cell. Furthermore, unlike all other cells, erythrocytes do not contain mitochondria. Mitochondria engage in the production of ROS. Testing the antioxidant capacity in an erythrocyte model eliminates confounding factors that--in other cellular models--makes data interpretation difficult. It assists in creating a baseline of information upon which data from more complex cellular assays can be more correctly interpreted. (2) The choice of immortalized cell lines for the purpose of antioxidant testing is not a straightforward approach. Immortalized cell lines--which include many commonly used tumour cell lines--offer some consistency for assays that test natural products. However, although it may be argued that this eliminates the problem associated with minor variations in blood samples drawn from healthy donors for cell-based testing, many tumour cell lines proliferate in a highly deregulated manner. This can lead to a proportion of the cells in the culture being from derived asymmetrical cell division, which can result in apoptosis (programmed cell death) and thus introduce inherent variability in the cell population.

The most serious problem with using tumour cell lines for antioxidant testing is that ROS are produced by tumour cells as a result of programmed cell death. A reduction in ROS formation may actually reflect a reduction in cell death in these cell lines in the presence of a test product. Of course, increased survival or proliferation of a tumour cell line is not an ideal marketing claim--especially because it may have little clinical relevance. Despite their poor suitability for antioxidant capacity testing, tumour cell lines lend themselves to other types of natural products testing. The use of such cell lines to evaluate tumour-suppressive effects is more straightforward and can include the following assays, either separately or in combination: inhibition of cell proliferation; effects on mitochondrial functioning; and the induction of apoptosis.

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Clinical Application of the CAP-e Assay

We recently applied the CAP-e assay methodology to serum samples collected from a randomized, double-blinded, placebo-controlled, crossover pilot study of 12 healthy adult subjects. (3) The purpose of this study was to investigate the in vitro and in vivo antioxidant capacity of MonaVie Active, a juice primarily consisting of the Amazonian palm berry, Acai. Blood samples were obtained at baseline (T = 0) as well as at one and two hours following the consumption of either 4 oz (120 mL) of the juice or a placebo. The CAP-e assay demonstrated an increase in serum antioxidant level, which at two hours post-consumption showed a correlation with another method of measuring oxidative stress, namely TBA[R.sub.S]--a measure of MDA (malondialdehyde) that is proportional to the level of serum lipid peroxidation. Consumption of the juice resulted in an increase in serum antioxidants at one hour (p<0.03) and at two hours (p<0.015), as well as a reduction in serum lipid peroxidation (TBA[R.sub.S]) within two hours (p<0.01).

Conclusion

The CAP-e assay is a useful tool in antioxidant research. The assay helps to assess bioavailability at the cellular level and may help to make rational decisions during formulation and product design. Also, CAP-e is applicable to the evaluation of antioxidant uptake in clinical studies.

For more information

Gitte S. Jensen

Research Director

NIS Labs

1437 Esplanade

Klamath Falls

Oregon 97601, USA.

Tel. +1 541 884 0112

www.nislabs.com

References

(1.) G.S. Jensen, "Cell-Based Antioxidant Protection Assay," US Patent Application No. 60/985,166.

(2.) D. Honzel, et al., "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. 56(18), 8319-8325 (2008).

(3.) G.S. Jensen, et al., "In Vitro and In Vivo Antioxidant and Anti-Inflammatory Capacities 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. 56(18), 8326-8333 (2008).

(4.) R.L. Prior, X. Wu and K. Schaich, "Standardized Methods for the Determination of Antioxidant Capacity and Phenolics in Foods and Dietary Supplements," J. Agric. Food Chem. 53, 4290-4302 (2005).

(5.) R.L. Prior, et al., "Plasma Antioxidant Capacity Changes Following a Meal as a Measure of the Ability of a Food to Alter In Vivo Antioxidant Status," J. Am. Coll. Nutr. 26, 170-181 (2007).

(6.) A.G. Schauss, et al., "Antioxidant Capacity and Other Bioactivities of the Freeze-Dried Amazonian Palm Berry, Euterpe oleracea Mart . (Acai)," J. Agric. Food Chem. 54, 8604-8610 (2006).
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Title Annotation:antioxidants
Author:Jensen, Gitte S.
Publication:Nutraceutical Business & Technology
Geographic Code:1USA
Date:Mar 1, 2009
Words:2269
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