To investigate the reactivity of these systems, gallic acid (GA) was used as a model system for the polyphenols present in foods products (Chvátalová et al., 2008 and Fazary et al., 2009). The formation of the Fe3+–GA complex can be followed over time using spectrophotometry, as the complex has a dark small molecule library screening blue colour (Chvátalová et al., 2008 and Mellican et al., 2003). This increase in absorption was used as an indication for the reactivity of the iron contained in the particles. However, the analysis is complicated by the ability of polyphenols to reduce Fe3+, resulting

in a Fe2+–quinone complex that is also blue. Although various possible pathways are known for this reaction (Arif Kazmi et al., 1987, Funabiki et al., 1986 and Powell and Taylor, 1982), the most probable one under physiological conditions is described by Hynes (2001). Once the quinone has been formed, the Fe2+ can be oxidised to form a new complex with free gallic acid. As will be shown here, the oxidation reaction is much slower than the initial complex formation and the cyclisation of the reaction MAPK inhibitor can be limited

by sealing the sample air tight. The difference between the two complexes can be distinguished using spectrophotometry, since they have different absorption maxima, although it does interfere with the quantification of the complexation reaction. Due to the side reactions and the complexity of the system, only the initial reactivity during the first 5 h after addition was analysed and only qualitative comparisons between identically prepared samples were made. FeCl3·6H2O (ACS reagent grade, 97%) and zein protein were obtained from Sigma Aldrich. Na4P2O7·10H2O (ACS reagent grade), CaCl2·2H2O (ACS reagent grade, ⩾99%) and NaCl (p.a., ⩾99.5%) were purchased from Merck and MgCl2·6H2O (puriss. p.a., ⩾99%) from Fluka. Gallic acid (extra pure, ⩾99.5%) was obtained from Scharlau Chemie. All

chemicals were used as received; aqueous solutions were prepared using water deionised by a Millipore Synergy water purification system. Systems were dialysed using Spectra/Por 2 Dialysis Membrane, molecular weight cut-off (MWCO) 12–14 Da, corresponding to roughly a 1.5 nm pore size. Iron pyrophosphate was prepared as described previously (van Leeuwen et al., 2012a and van Leeuwen Phosphoribosylglycinamide formyltransferase et al., 2012c). Briefly, nanoparticles were prepared by coprecipitation of Na4P2O7 with FeCl3. 0.86 mmol iron chloride dissolved in 50 ml water was added drop wise, over about 15 min to 0.64 mmol sodium pyrophosphate in 100 ml. A turbid white precipitate formed in the final 5 min of the addition (van Leeuwen et al., 2012c), the resulting dispersion had a pH of 4. The pH-dependent preparation comprised of two steps: first, the precipitation and washing of the intermediate pyrophosphate salt, which was subsequently redissolved in acid and then precipitated in an alkaline solution. For the intermediate, 50 ml 1 M M2+ Cl2 solution was added drop wise over 1 h to 800 ml 0.