Date of this Version
DEVELOPMENT OF FOURIER TRANSFORM MID-INFRARED SPECTROSCOPY AS A METABOLOMIC TECHNIQUE FOR CHARACTERIZING THE PROTECTIVE PROPERTIES OF GRAIN SORGHUM AGAINST OXIDATION
Emily D. Sitorius, M.S. University of Nebraska, 2010 Advisor: Vicki L. Schlegel
Cellular oxidative damage has been linked to many chronic diseases including Alzheimer’s, diabetes, and cancer. Recent studies have shown that foods rich in antioxidants (i.e., grain sorghum) can protect against oxidative stress and thus reduce the risk of disease. However, a significant gap in knowledge exists in that dietary antioxidants are usually characterized by methods that determine free radical and reactive oxygen species’ scavenging capacity that are not strongly correlated to a biological system. To fully understand the health promoting properties of natural antioxidants, the study of the metabolome is essential as shifts in the biochemical phenotype, or function of a cell, will be generated. Therefore, the objective of this study was to develop a method using Fourier-Transform Mid-Infrared spectroscopy (FT-mIR) that would simultaneously monitor several cellular biochemicals in response to oxidation and dietary antioxidants. To achieve the cited research objective, sorghum polyphenols were extracted into water and characterized via Folin-Ciocalteau, aluminum chloride, and oxygen radical absorbance capacity (ORAC) methods resulting in: 109.6 ± 4.3 μg cinnamic / g flour; 22.6 ± 0.15 μg catechin/ g flour; 336 μmol Trolox/ 100 g flour. FTmIR analysis of human intestinal epithelial cells, Caco-2, exposed to 1-40 mM of hydrogen peroxide (HP) for 16 h indicated that concentrations > 5 mM induced protein structural changes and DNA mutations. Exposure to grain sorghum extract (SE) (1-40 mg/mL) for 8 h prior to treatment with 15 mM HP for 16 h revealed minimal protection of cells with the notable exception of the phosphate containing groups, i.e., phospholipids, DNA, and RNA. At the amide III band, principle component analysis indicated that treatment with 40 mg/mL SE may actually have damaged the cells. This study showed FT-mIR to be a powerful metabolomic tool to identify optimal safe and effective dietary options for protecting cells against oxidative stress.