Protocol for the Production of Concentrated Extracts of Food Folate for Use in Human Bioavailability Studies DEREK J. MCKILLOP, † KRISTINA D. PENTIEVA,* ,† JOHN M. SCOTT, ‡ J. J. STRAIN, † RICHARD MCCREEDY, § JOY ALEXANDER, § KAREN PATTERSON, § JOAN HUGHES, † AND HELENE MCNULTY † Northern Ireland Centre for Food and Health, University of Ulster, Coleraine, Northern Ireland BT52 1SA; Biochemistry Department, Trinity College, Dublin 2, Ireland; and The Food Centre, Department of Agriculture and Rural Development, Loughry College, Cookstown, Northern Ireland BT80 9AA To provide a tool to study folate bioavailability under controlled conditions, a methodology was developed to produce extracts representative of natural food folates but removed from their matrix and sufficiently concentrated so as to elicit a response in biomarkers of folate status without distorting usual dietary intake patterns. Egg, spinach, and yeast were selected to represent the wide range in extent of folate conjugation found in foods (0, 60, and 100% polyglutamyl folate, respectively). The protocol, which was based on extracting food folates using only reagents safe for human consumption, was optimized in the laboratory (thermal extraction for 10 min in a 2% ascorbate solution at pH 5) and then adapted for industrial scale production in a food-processing facility. Results showed that the extracts were 2.3-12 times more concentrated in folate compared with their corresponding food sources. Neither the mono- to polyglutamate ratio nor the distribution of the main folate derivatives was altered during processing, making these extracts suitable for use in human bioavailability studies. KEYWORDS: Food folate; bioavailability; polyglutamate; monoglutamate INTRODUCTION Folate has attracted major interest in recent years as having an established role in the prevention of neural tube defects (NTD) (1, 2) and possible preventive roles against cardiovascular disease (3) and certain cancers (4-7). For the prevention of NTD, various official bodies worldwide (8-10) recommend an additional 400 µg of folate/day for women prior to conception and in early pregnancy. Because of the public health difficulties in achieving this recommendation through supplementation of the target group, mandatory fortification of grain foods with folic acid has recently been introduced in the United States (11). Similar policies are under consideration elsewhere, but universal fortification is controversial for various reasons, in particular, the concern that it may mask the hematological changes associated with vitamin B 12 deficiency, thereby allowing the concomitant irreversible nerve degeneration to go undetected (12). The third approach to increase folate status, which does not have the health concerns associated with food fortification, is to increase consumption of foods naturally rich in folate. The effectiveness of this strategy as a means of optimizing folate status has, however, been shown to be limited (13-15), primarily as a result of the poor bioavailability of natural food folates compared with the synthetic vitamin, folic acid. Although food folate bioavailability is considered to be on average 50% that of folic acid (16), a great deal of uncertainty exists in our knowledge of folate bioavailability from natural food sources. The bioavailability of folates from various foods is considered to be dependent on the food matrix, the relative content of monoglutamyl and polyglutamyl folates, and the presence of components that could inhibit both intestinal folate deconjugation and specific transport processes of folate (17). However, the relative influence of each of these factors in determining the overall bioavailability of food folates is unknown. In particular, although disruption of the food matrix was shown to enhance the bioavailability of micronutrients such as -carotene and lycopene (18), few studies have addressed the role of the food matrix as a determinant of folate bioavail- ability from natural food sources. In addition, previous bio- availability studies in free-living subjects involving the provision of folate-rich foods may be confounded by factors including the poor stability of food folates during cooking, resulting in variable folate losses prior to ingestion (19), the poor compliance of subjects with demanding intervention protocols in long-term studies, or dietary displacement of usual food folate sources with intervention foods, all of which may result in considerably less food folate being consumed than that targeted. In contrast, the provision of folic acid in tablet form (against which * Author to whom correspondence should be addressed (telephone +44- 28-70324675; fax +44-28-70324965; e-mail k.pentieva@ulster.ac.uk). † Northern Ireland Centre for Food and Health. ‡ Trinity College Dublin. § Loughry College. 4382 J. Agric. Food Chem. 2003, 51, 4382-4388 10.1021/jf0262312 CCC: $25.00 © 2003 American Chemical Society Published on Web 06/17/2003