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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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Visualizing InsP6, InsP7 and InsP8from mammalian cells and organs. (a) Cells from sixdifferent human lines were collected and washed in PBS. A smallaliquot was taken for determination of protein concentration, whilethe rest was PA extracted and subjected to TiO2 beadenrichment. Extracts relative to equivalent amounts of protein foreach cell line (approx. 35 mg) were loaded on two parallel gelssubsequently stained by DAPI (top) and toluidine blue (bottom).(b) Densitometry of toluidine blue-stained gelfrom three independent experiments was used to calculate ratios ofInsP7 and InsP8 over their precursorInsP6. (c) Mouse brain and liver(approximately 0.5 g) were homogenized and extracted with PA. AfterTiO2 purification the inositol phosphates wereresolved by PAGE and stained with DAPI. (d) Two 14cm dishes of 80% confluent HCT116 cells were pre-treated inglucose-free medium for 30 min before addition of 5 µMoligomycin for 3 h. The TiO2 extracts were then resolvedby PAGE and stained with toluidine blue. The gels presented arerepresentative of experiments performed three times.
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RSOB150014F3: Visualizing InsP6, InsP7 and InsP8from mammalian cells and organs. (a) Cells from sixdifferent human lines were collected and washed in PBS. A smallaliquot was taken for determination of protein concentration, whilethe rest was PA extracted and subjected to TiO2 beadenrichment. Extracts relative to equivalent amounts of protein foreach cell line (approx. 35 mg) were loaded on two parallel gelssubsequently stained by DAPI (top) and toluidine blue (bottom).(b) Densitometry of toluidine blue-stained gelfrom three independent experiments was used to calculate ratios ofInsP7 and InsP8 over their precursorInsP6. (c) Mouse brain and liver(approximately 0.5 g) were homogenized and extracted with PA. AfterTiO2 purification the inositol phosphates wereresolved by PAGE and stained with DAPI. (d) Two 14cm dishes of 80% confluent HCT116 cells were pre-treated inglucose-free medium for 30 min before addition of 5 µMoligomycin for 3 h. The TiO2 extracts were then resolvedby PAGE and stained with toluidine blue. The gels presented arerepresentative of experiments performed 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)

Visualizing InsP6, InsP7 and InsP8from mammalian cells and organs. (a) Cells from sixdifferent human lines were collected and washed in PBS. A smallaliquot was taken for determination of protein concentration, whilethe rest was PA extracted and subjected to TiO2 beadenrichment. Extracts relative to equivalent amounts of protein foreach cell line (approx. 35 mg) were loaded on two parallel gelssubsequently stained by DAPI (top) and toluidine blue (bottom).(b) Densitometry of toluidine blue-stained gelfrom three independent experiments was used to calculate ratios ofInsP7 and InsP8 over their precursorInsP6. (c) Mouse brain and liver(approximately 0.5 g) were homogenized and extracted with PA. AfterTiO2 purification the inositol phosphates wereresolved by PAGE and stained with DAPI. (d) Two 14cm dishes of 80% confluent HCT116 cells were pre-treated inglucose-free medium for 30 min before addition of 5 µMoligomycin for 3 h. The TiO2 extracts were then resolvedby PAGE and stained with toluidine blue. The gels presented arerepresentative of experiments performed three times.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB150014F3: Visualizing InsP6, InsP7 and InsP8from mammalian cells and organs. (a) Cells from sixdifferent human lines were collected and washed in PBS. A smallaliquot was taken for determination of protein concentration, whilethe rest was PA extracted and subjected to TiO2 beadenrichment. Extracts relative to equivalent amounts of protein foreach cell line (approx. 35 mg) were loaded on two parallel gelssubsequently stained by DAPI (top) and toluidine blue (bottom).(b) Densitometry of toluidine blue-stained gelfrom three independent experiments was used to calculate ratios ofInsP7 and InsP8 over their precursorInsP6. (c) Mouse brain and liver(approximately 0.5 g) were homogenized and extracted with PA. AfterTiO2 purification the inositol phosphates wereresolved by PAGE and stained with DAPI. (d) Two 14cm dishes of 80% confluent HCT116 cells were pre-treated inglucose-free medium for 30 min before addition of 5 µMoligomycin for 3 h. The TiO2 extracts were then resolvedby PAGE and stained with toluidine blue. The gels presented arerepresentative of experiments performed 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