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.