A novel method for the purification of inositol phosphates from biological samples reveals that no phytate is present in human plasma or urine

Inositol phosphates are a large and diverse family of signalling molecules. While genetic studies have discovered important functions for them, the biochemistry behind these roles is often not fully characterized. A key obstacle in inositol phosphate research in mammalian cells has been the lack of straightforward techniques for their purification and analysis. Here we describe the ability of titanium dioxide (TiO(2)) beads to bind inositol phosphates. This discovery allowed the development of a new purification protocol that, coupled with gel analysis, permitted easy identification and quantification of InsP(6) (phytate), its pyrophosphate derivatives InsP(7) and InsP(8), and the nucleotides ATP and GTP from cell or tissue extracts. Using this approach, InsP(6), InsP(7) and InsP(8) were visualized in Dictyostelium extracts and a variety of mammalian cell lines and tissues, and the effects of metabolic perturbation on these were explored. TiO(2) bead purification also enabled us to quantify InsP(6) in human plasma and urine, which led to two distinct but related observations. Firstly, there is an active InsP(6) phosphatase in human plasma, and secondly, InsP(6) is undetectable in either fluid. These observations seriously question reports that InsP(6) is present in human biofluids and the advisability of using InsP(6) as a dietary supplement.