A novel method for the purification of inositol phosphates from
biological samples reveals that no phytate is present in human plasma or
urine |
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Authors: | Miranda S C Wilson Simon J Bulley Francesca Pisani Robin F Irvine Adolfo Saiardi |
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Institution: | 1.Medical Research Council Laboratory for
Molecular Cell Biology, University College
London, London, UK;2.Department of Pharmacology, University of Cambridge, Tennis Court
Road, Cambridge CB2 1PD, UK;3.Department of Haematology, Cambridge University Hospitals NHS Trust, Hills Road, Cambridge CB2 0QQ, UK;4.Department of Biology, Ecology and Earth
Science, University of Calabria, Rende, Italy |
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Abstract: | 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 (TiO2) 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
InsP6 (phytate), its pyrophosphate derivatives InsP7
and InsP8, and the nucleotides ATP and GTP from cell or tissue
extracts. Using this approach, InsP6, InsP7 and
InsP8 were visualized in Dictyostelium extracts
and a variety of mammalian cell lines and tissues, and the effects of metabolic
perturbation on these were explored. TiO2 bead purification also
enabled us to quantify InsP6 in human plasma and urine, which led to
two distinct but related observations. Firstly, there is an active
InsP6 phosphatase in human plasma, and secondly, InsP6
is undetectable in either fluid. These observations seriously question reports
that InsP6 is present in human biofluids and the advisability of
using InsP6 as a dietary supplement. |
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Keywords: | phytic acid IP6 IP7 IP8 blood |
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