Quantification of folate in fruits and vegetables: A fluorescence-based homogeneous assay

A high-throughput, homogeneous, fluorescence polarization, and fluorescence intensity assay has been developed for the measurement of folate in fruits and vegetables. This assay is based on the competitive displacement of the fluorescent folate ligands Alexa Fluor (Alexa) 594-folate and Alexa 660-folate from bovine milk folate-binding protein by folates in fruit and vegetable extracts. These fluorescent ligands are employed because their excitation and emission maxima are in regions of the spectrum with minimal autofluorescence in many extracts. Folate-binding protein and Alexa-folate were typically used at concentrations of 0.5 μg/ml and 5 nM, respectively, in 20-μl volumes in 384-well microplates. The assay is complete within 100 min. The folate estimate is unaffected by the heterogeneity of polyglutamyl residues that complicates the liquid chromatography-mass spectrometry (LC-MS)-based methods of quantification. In this assay, folic acid had an apparent affinity 2.5-fold greater than 5-methyltetrahydrofolate (5MTHF); therefore, it cannot be used to quantify folate when both natural and synthetic folate are present. 5MTHF-equivalent values were measured in broccoli (240 μg/100 g), strawberry (113 μg/100 g), white grape (32 μg/100 g), orange (44 μg/100 g), tomato (12 μg/100 g), raspberry (31 μg/100 g), banana (29 μg/g), and kiwifruit (36 μg/100 g). These data are similar to published values. However, the assay will not detect 5-formyltetrahydrofolate which is a significant constituent of the total folate in lettuce, spinach, carrot, and peppers.