Analytical MethodsPhytoestrogen content of fruits and vegetables commonly consumed in the UK based on LC–MS and 13C-labelled standards
Introduction
Phytoestrogens are a group of non-steroidal polyphenolic plant metabolites that induce biological responses and can mimic or modulate the action of endogenous oestrogens, often by binding to oestrogen receptors (Committee on Toxicity of Chemicals in Food & the Environment, 2003). The bioactivity of these compounds is based on their structural similarity with 17β-oestradiol (Branham et al., 2002, Martin et al., 1978, Setchell and Adlercreutz, 1988, Verdeal et al., 1980) and their ability to bind to oestrogen receptors (Shutt & Cox, 1972). Apart from their effect on oestrogen receptors, phytoestrogens can also act as antioxidants (Wei, Bowen, Cai, Barnes, & Wang, 1995) and inhibitors of enzymes such as tyrosine kinase (Akiyama et al., 1987) and DNA topoisomerase (Markovits et al., 1989). As a result of their bioactivity, these compounds have received increasing attention for potentially beneficial effects for a wide range of human conditions such as cancer (Adlercreutz, 2002, Duffy et al., 2007, Peeters et al., 2003, Stark and Madar, 2002), cardiovascular disease (Anthony, 2002, Stark and Madar, 2002), osteoporosis (Dang and Lowik, 2005, Stark and Madar, 2002) menopausal symptoms (Krebs et al., 2004, Stark and Madar, 2002), male infertility (Phillips & Tanphaichitr, 2008), obesity and type 2 diabetes (Bhathena & Velasquez, 2002). However, elevated endogenous sex hormone levels are generally associated with an increased risk of breast cancer in women (The Endogenous Hormones and Breast Cancer Collaborative, 2002) and not all studies have shown a beneficial effect on breast cancer risk associated with increased exposure to phytoestrogens in Western societies (Grace et al., 2004, Ward et al., 2008). There are also strong gene–nutrient interactions between phytoestrogens and oestrogen receptor polymorphisms (ESR1 and NR1I2) (Low et al., 2005b, Low et al., 2007), polymorphisms in the gene for the sex-hormone binding globulin (SHBG) (Low et al., 2006) and probably polymorphisms in the gene encoding aromatase (CYP19) (Low et al., 2005a) which influence their bioactivity. Despite the large number of studies conducted, there is still no clear evidence whether phytoestrogen intake has a beneficial or detrimental effect on human health and the UK Committee on Toxicity (COT) has recommended further research (Committee on Toxicity of Chemicals in Food, 2003).
Exposure to phytoestrogens can be determined either directly by measuring diet or indirectly by using biomarkers in plasma or urine (Grace et al., 2004). Although biomarkers are often more reliable due to the limitations in dietary assessment (Day et al., 2001, Kipnis et al., 2003), their use is often not feasible, particularly in larger studies, and intake has to be either calculated from dietary information provided by participants or determined by a combination of biomarkers and dietary information. Accurate information on the phytoestrogen content in foods is therefore crucial for the investigation of effects on health; and to determine population levels for surveillance purposes.
The main dietary sources of phytoestrogens are plant-based foods such as fruits and vegetables. In plants, where these compounds occur predominantly as glycosides, they act as antioxidants, screen against light and most importantly act as defensive agents against predators (Mazur & Adlercreutz, 1998a). The principal phytoestrogen-classes are isoflavones (found mainly in legumes, e.g. chickpeas and soybean), lignans (e.g. in cereals, linseed and other fruits and vegetables) and coumestans (e.g. in young sprouting legumes like clover or alfalfa sprouts) (Committee on Toxicity of Chemicals in Food, 2003).
Several detailed studies have been conducted to determine the phytoestrogen content of food previously, amongst others in the UK (Liggins et al., 1998a, Liggins et al., 1998b, Liggins et al., 2000, Liggins et al., 2002, Liggins et al., 2000), Finland (Dwyer et al., 1994, Mazur, 1998, Mazur et al., 1996, Mazur et al., 1998b, Valsta et al., 2003), and the US (US US Department of Agriculture, 2002); however, these studies provide only data for approximately 12% of the UK diet (Mulligan, Welch, McTaggart, Bhaniani, & Bingham, 2007) and had methodological limitations (Adlercreutz et al., 1993, Wähälä et al., 1995, Wähälä and Rasku, 1997). Previously, we have developed a sensitive LC/MS/MS method using 13C3-labelled standards to analyse phytoestrogens in plasma and urine (Grace et al., 2003). We have adapted this method to be used for food samples and have measured the phytoestrogen content (isoflavones: biochanin A, daidzein, formononetin, genistein, glycitein; lignans: matairesinol, secoisolariciresinol; coumestrol) in more than 240 foods based on fruits and vegetables commonly consumed in the UK. This is one of the most comprehensive analysis of plant-based phytoestrogens in the UK and elsewhere.
Section snippets
Chemicals
Biochanin A, daidzein, genistein, glycitein, formononetin, secoisolariciresinol, matairesinol and coumestrol were purchased from Plantech (Reading, Berkshire, UK). 13C3-biochanin A 13C3-daidzein, 13C3-genistein, 13C3-glycitein, 13C3-formononetin, 13C3-matairesinol, 13C3-secosiolariciresinol and 13C3-enterolactone were obtained from Dr. Nigel botting (University of St. Andrews, Fife, UK) (Fryatt and Botting, 2005, Haajanen and Botting, 2006, Whalley et al., 1998, Whalley et al., 2000).
Results
In all foods analysed, with the exception of microwaved mushrooms and unheated tinned sweet-corn, phytoestrogens were detected (Table 1). In most foods, the phytoestrogen content was below 100 μg/100 g wet weight (median: 20 g/100 g; IQR (inter-quartile range): 7–66 μg/100 g) with less isoflavones (median: 2 μg/100 g; IQR: 1–8 μg/100 g) than lignans (median: 12 μg/100 g; IQR 3–47 μg/100 g) and a low amount of coumestrol (median: <1 μg/100 g). However, 5% of foods analysed contained more than 400 μg/100 g
Discussion
Phytoestrogens are formed as secondary metabolites by most plants and are therefore ubiquitous in plant products (Mazur & Adlercreutz, 1998a). In this study, we have analysed more than 240 foods based on fruit and vegetables for their phytoestrogen content to provide a comprehensive database for the assessment of dietary intake and exposure. The results are expressed per 100 g wet weight to facilitate the use in epidemiological studies and diet composition databases. Phytoestrogens were found in
Acknowledgements
This work was funded by the UK Food Standards Agency (FSA), Contract No. T05028 and the Medical Research Council (MRC).
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