Determination of Subcellular Localization of Flavonol in Cultured Cells by Laser Scanning

Abstract

Flavonoids are widely distributed in the plant kingdom including in edible plants such as vegetables and fruits. They are polyphenolic compounds comprising of fifteen carbons, with two aromatic rings connected by a three-carbon bridge (C6-C3-C6). Flavonoids are divided into seven subclasses; the flavones, flavonols, flavanones, catechins, anthocyans, isoflavones and the chalcones, according to their basic skeletal structure (Fig. 1). They are known to have various beneficial pharmacological effects such as anti-cancer, anti-obesity, antiinflammatory and anti-oxidative activities (Ahn, et al. 2008; Boots, et al. 2008; Chu, et al. 2007; Hsu and Yen 2006; Kuzuhara, et al. 2008; Murakami, et al. 2008). To investigate the beneficial effects of trace food components, it is important to understand their metabolism, absorption, tissue distribution, and subcellular localization. Recently, the metabolism, including absorption and tissue distribution, of trace food components has been investigated using cultured cells and experimental animals (de Boer, et al. 2005; Urpi-Sarda, et al. 2008; Wang, et al. 2003). To determine the physiological concentrations and chemical structures of flavonoid metabolites, high-performance liquid chromatography (HPLC) or HPLC combined with mass spectrometry (MS) is conventionally used (de Boer, et al. 2005; Mullen, et al. 2006; Urpi-Sarda, et al. 2008; Wang, et al. 2003; Yanez, et al. 2008). The subcellular localization of the flavonoids is currently not fully understood, although the preparation of subcellular fractions using centrifugation, followed by HPLC analysis to determine the cellular localization of trace food components, including flavonoids, has been described (Gagne, et al. 2006). Radioactive isotope labeling has also been used to estimate tissue localization of trace food components in animals (Hirosawa and Yamada 1981), and this method could be applied to estimating subcellular localization in cultured cells. However, cross contamination between fractions during the preparation of subcellular fractions can limit the accuracy of such techniques. In this chapter, we demonstrate the value of confocal laser scanning fluorescence microscopy in the subcellular localization of flavanoids, by the detection of autofluorescence in intact culture cells. During this study, we focused on the flavonol subclass, as this subclass features stronger autofluorescence properties than other flavonoid subclasses.