Abstract 4797: The antioxidation microsphere flow cytometer assay.

Abstract

Abstract Increased production of reactive oxygen species is a feature of most, if not all, human disease, including cardiovascular disease and cancer. Dietary antioxidants may be especially important in protecting against human diseases associated with free radical damage to cellular DNA, lipids, and proteins. Yet, present data are not sufficient to quantify micronutrient requirements needed to protect against oxidative damage. The antioxidant roles of many food constituents, including herbs and spices, have not been clarified. The AMFC assay measures the oxidation of R-Phycoerythrin attached to microspheres via a protein coupling linker. A 25mM stock solution of ABTS.+ [2,2’-Azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) free radical], prepared during a 12 hour, 4 °C incubation with potassium persulfate to cause removal of one electron generating a metastable ABTS. This stock solution was diluted to 100μM in 150μl, which resulted in 80% quenching of 15μl of 104 PE microspheres. The antioxidant effects were measured by first incubating 50μl the 80 % quenching ABTS.+ with 100μl portions of serial diluted antioxidants for 30 min. at 22 °C to determine the antioxidants neutralize the ABTS.+. Subsequently, 15μl of PE-tagged microspheres were added to each sample and incubated one hour at 22 °C. The peak fluorescent channel number was determined for 5,000 microspheres. The typical percentage protection by the antioxidant standard at 500 ng/ml for Trolox (water soluble Vitamin E derivative) was 8, while 500 ng/ml of Vitamin C restored 78% The range of 500-5,000 ng/ml showed the protective effects of Trolox and Vitamin C remained constant. On the average, equivalent percentage protection for both the hydrophilic and lipid soluble fractions of 14 spices (courtesy of McCormick Science Institute) was not achieved until the 20-50 μg/ml concentration of the spices with Turmeric, Cinnamon, Oregano and Cloves, showing the highest antioxidant protection. Interestingly, Rosemary, Sage, Thyme and Turmeric revealed a significant reduction in antioxidant activity at 200 μg/ml. These are still significant antioxidant levels that show a secondary reason besides flavoring to use spices for achieving clinically important antioxidant activity. The AMFC assay is very sensitive to the effects of oxidants, functions under physiological conditions, requires little sample and allows application to the study of serum samples. Thus, one may monitor antioxidant activity in healthy volunteers taking compounds to boost the antioxidant activity to offset the long-term effects of oxidative radiation and chemotherapy. Citation Format: Jerry T. Thornthwaite, Hare R. Shah. The antioxidation microsphere flow cytometer assay. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4797. doi:10.1158/1538-7445.AM2013-4797 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.