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

Wilson MS, Bulley SJ, Pisani F, Irvine RF, Saiardi A - Open Biol (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory for Molecular Cell Biology, University College London, London, UK.

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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Related in: MedlinePlus

TiO2 beads purify nucleotides and inositol phosphates frommammalian cells. (a) PA extracts from vegetativeD. discoideum (4 × 106 cells)and the human HCT116 cell line (80 × 106 cells)were subjected to TiO2 enrichment. After resolving theextract with PAGE, phosphate-rich molecules were visualized bytoluidine blue staining for comparison with InsP6, ATPand GTP nucleotide standards. (b)TiO2-purified HCT116 extract and nucleotide standardswere subjected to apyrase treatment, before resolution by PAGE andstaining with toluidine blue. (c) Two 14 cm dishesof 80% confluent HeLa, HCT116 and MCF7 cells were treatedwith 10 mM sodium fluoride (NaF) for 1 h, before purification ofinositol phosphates with TiO2 beads and resolution byPAGE with toluidine blue staining. The gels presented arerepresentative of experiments performed at least three times.
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RSOB150014F2: TiO2 beads purify nucleotides and inositol phosphates frommammalian cells. (a) PA extracts from vegetativeD. discoideum (4 × 106 cells)and the human HCT116 cell line (80 × 106 cells)were subjected to TiO2 enrichment. After resolving theextract with PAGE, phosphate-rich molecules were visualized bytoluidine blue staining for comparison with InsP6, ATPand GTP nucleotide standards. (b)TiO2-purified HCT116 extract and nucleotide standardswere subjected to apyrase treatment, before resolution by PAGE andstaining with toluidine blue. (c) Two 14 cm dishesof 80% confluent HeLa, HCT116 and MCF7 cells were treatedwith 10 mM sodium fluoride (NaF) for 1 h, before purification ofinositol phosphates with TiO2 beads and resolution byPAGE with toluidine blue staining. The gels presented arerepresentative of experiments performed at least three times.

Mentions: We tested TiO2 enrichment on extracts from HCT116 cells (human coloncancer cell line). PAGE analysis (figure2a) of the phosphate-rich molecules extractedrevealed the presence of three inositol phosphate bands that co-migrate withD. discoideum-extracted InsP6, InsP7and InsP8 [10].Interestingly, unlike D. discoideum extracts, mammalian cellextracts revealed extra bands that co-migrate with the nucleotide standards ATPand GTP. We confirmed the identity of the bands presumed to be ATP and GTP bytreating the TiO2-purified samples with apyrase, an enzyme thatspecifically hydrolyses nucleotides (figure 2b). We also detected a faint, slowermigrating band of unknown nature (labelled Unk), which is particularly abundantin liver extract (figure3c). This band does not represent anInsP9 species since it is not fully degraded after phytasetreatment unlike the InsP6, InsP7 and InsP8bands (data not shown). The partial action of phytase on this unknown bandsuggests a complex molecule containing an inositol phosphate group. Inositolpyrophosphates in mammalian cells are known to be dramatically regulated bysodium fluoride (NaF) [26]. Toconfirm the identity of the observed InsP7 and InsP8bands, we incubated three human cell lines with NaF: firstly, MCF7 cells, whichusually have undetectable levels of inositol pyrophosphates; secondly, HeLacells, which have detectable levels of InsP7; and thirdly, HCT116cells, which have detectable levels of InsP7 and InsP8.PAGE analysis after TiO2 extraction revealed that NaF treatmentincreases inositol pyrophosphate levels, and decreased the level of theirprecursor InsP6, in all three cell lines (figure 2c). Figure 2.


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

Wilson MS, Bulley SJ, Pisani F, Irvine RF, Saiardi A - Open Biol (2015)

TiO2 beads purify nucleotides and inositol phosphates frommammalian cells. (a) PA extracts from vegetativeD. discoideum (4 × 106 cells)and the human HCT116 cell line (80 × 106 cells)were subjected to TiO2 enrichment. After resolving theextract with PAGE, phosphate-rich molecules were visualized bytoluidine blue staining for comparison with InsP6, ATPand GTP nucleotide standards. (b)TiO2-purified HCT116 extract and nucleotide standardswere subjected to apyrase treatment, before resolution by PAGE andstaining with toluidine blue. (c) Two 14 cm dishesof 80% confluent HeLa, HCT116 and MCF7 cells were treatedwith 10 mM sodium fluoride (NaF) for 1 h, before purification ofinositol phosphates with TiO2 beads and resolution byPAGE with toluidine blue staining. The gels presented arerepresentative of experiments performed at least three times.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4389793&req=5

RSOB150014F2: TiO2 beads purify nucleotides and inositol phosphates frommammalian cells. (a) PA extracts from vegetativeD. discoideum (4 × 106 cells)and the human HCT116 cell line (80 × 106 cells)were subjected to TiO2 enrichment. After resolving theextract with PAGE, phosphate-rich molecules were visualized bytoluidine blue staining for comparison with InsP6, ATPand GTP nucleotide standards. (b)TiO2-purified HCT116 extract and nucleotide standardswere subjected to apyrase treatment, before resolution by PAGE andstaining with toluidine blue. (c) Two 14 cm dishesof 80% confluent HeLa, HCT116 and MCF7 cells were treatedwith 10 mM sodium fluoride (NaF) for 1 h, before purification ofinositol phosphates with TiO2 beads and resolution byPAGE with toluidine blue staining. The gels presented arerepresentative of experiments performed at least three times.
Mentions: We tested TiO2 enrichment on extracts from HCT116 cells (human coloncancer cell line). PAGE analysis (figure2a) of the phosphate-rich molecules extractedrevealed the presence of three inositol phosphate bands that co-migrate withD. discoideum-extracted InsP6, InsP7and InsP8 [10].Interestingly, unlike D. discoideum extracts, mammalian cellextracts revealed extra bands that co-migrate with the nucleotide standards ATPand GTP. We confirmed the identity of the bands presumed to be ATP and GTP bytreating the TiO2-purified samples with apyrase, an enzyme thatspecifically hydrolyses nucleotides (figure 2b). We also detected a faint, slowermigrating band of unknown nature (labelled Unk), which is particularly abundantin liver extract (figure3c). This band does not represent anInsP9 species since it is not fully degraded after phytasetreatment unlike the InsP6, InsP7 and InsP8bands (data not shown). The partial action of phytase on this unknown bandsuggests a complex molecule containing an inositol phosphate group. Inositolpyrophosphates in mammalian cells are known to be dramatically regulated bysodium fluoride (NaF) [26]. Toconfirm the identity of the observed InsP7 and InsP8bands, we incubated three human cell lines with NaF: firstly, MCF7 cells, whichusually have undetectable levels of inositol pyrophosphates; secondly, HeLacells, which have detectable levels of InsP7; and thirdly, HCT116cells, which have detectable levels of InsP7 and InsP8.PAGE analysis after TiO2 extraction revealed that NaF treatmentincreases inositol pyrophosphate levels, and decreased the level of theirprecursor InsP6, in all three cell lines (figure 2c). Figure 2.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory for Molecular Cell Biology, University College London, London, UK.

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.

Show MeSH
Related in: MedlinePlus