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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

Absence of InsP6 and presence of inositol phosphatephosphatase in human plasma. All the extracts were resolved by PAGEand visualized with toluidine blue staining. (a)EDTA was added to 20 ml of commercial human plasma from male (MP)and female (FP); InsP6 was added to the spiked aliquot(InsP6; 2 nmol). The samples were then acidified andsubjected to TiO2 enrichment. (b) Plasmafrom healthy anonymous donors (D1 to D4) was prepared as describedin Material and methods, with spiking (InsP6; 1 nmol),and subjected to TiO2 extraction. (c) 4nmol of InsP6 was added to 1 ml of human plasma andincubated at 37°C for the indicated time before acidificationand extraction of inositol phosphates with the TiO2procedure. Standards: InsP6 (4 nmol); InsP5 (6nmol of Ins(1,3,4,5,6)P5); InsP4 (5 nmol ofIns(1,4,5,6)P4); InsP3 (20 nmol ofIns(1,4,5)P3). (d) 4 nmol ofInsP6 was added to 1 ml of a different source ofhuman plasma (HP) and bovine plasma (BP) before incubation at37°C for the indicated time, followed by acidification of thesamples and TiO2 extraction. The gels presented arerepresentative of experiments performed two to four times.
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RSOB150014F4: Absence of InsP6 and presence of inositol phosphatephosphatase in human plasma. All the extracts were resolved by PAGEand visualized with toluidine blue staining. (a)EDTA was added to 20 ml of commercial human plasma from male (MP)and female (FP); InsP6 was added to the spiked aliquot(InsP6; 2 nmol). The samples were then acidified andsubjected to TiO2 enrichment. (b) Plasmafrom healthy anonymous donors (D1 to D4) was prepared as describedin Material and methods, with spiking (InsP6; 1 nmol),and subjected to TiO2 extraction. (c) 4nmol of InsP6 was added to 1 ml of human plasma andincubated at 37°C for the indicated time before acidificationand extraction of inositol phosphates with the TiO2procedure. Standards: InsP6 (4 nmol); InsP5 (6nmol of Ins(1,3,4,5,6)P5); InsP4 (5 nmol ofIns(1,4,5,6)P4); InsP3 (20 nmol ofIns(1,4,5)P3). (d) 4 nmol ofInsP6 was added to 1 ml of a different source ofhuman plasma (HP) and bovine plasma (BP) before incubation at37°C for the indicated time, followed by acidification of thesamples and TiO2 extraction. The gels presented arerepresentative of experiments performed two to four times.

Mentions: Since large volumes of acidified fluid can be subject to TiO2 beadextraction, this gave us the opportunity to assay InsP6 in biofluids.Initially, we used commercially available serum from bovine, equine and humansources. We extracted 20 ml of serum with TiO2 beads and analysed theextracts by PAGE. While we were able to detect an almost completeInsP6 recovery in the spiked samples, we did not recover anyInsP6 in non-spiked serum (electronic supplementary material,figure S2A,B). We next analysed human plasma from a commercial source. Similarto serum, TiO2 extraction and PAGE analysis showed thatInsP6 could not be recovered from non-spiked samples of humanplasma (figure4a). The lower limit of InsP6 standarddetection on PAGE is about 0.25 nmol (figures 4a,b and 5), thereforeTiO2-extracting 20 ml of plasma with a recovery of approximately85% (figure1e) indicates that the lower limit of plasmaInsP6 we are able to extract and detect is approximately 15 nM.Consequently, we conclude that substantially less than 15 nM InsP6 ispresent in human plasma, in agreement with the enzymatic radio-assay previouslyreported [8]. Figure 4.


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)

Absence of InsP6 and presence of inositol phosphatephosphatase in human plasma. All the extracts were resolved by PAGEand visualized with toluidine blue staining. (a)EDTA was added to 20 ml of commercial human plasma from male (MP)and female (FP); InsP6 was added to the spiked aliquot(InsP6; 2 nmol). The samples were then acidified andsubjected to TiO2 enrichment. (b) Plasmafrom healthy anonymous donors (D1 to D4) was prepared as describedin Material and methods, with spiking (InsP6; 1 nmol),and subjected to TiO2 extraction. (c) 4nmol of InsP6 was added to 1 ml of human plasma andincubated at 37°C for the indicated time before acidificationand extraction of inositol phosphates with the TiO2procedure. Standards: InsP6 (4 nmol); InsP5 (6nmol of Ins(1,3,4,5,6)P5); InsP4 (5 nmol ofIns(1,4,5,6)P4); InsP3 (20 nmol ofIns(1,4,5)P3). (d) 4 nmol ofInsP6 was added to 1 ml of a different source ofhuman plasma (HP) and bovine plasma (BP) before incubation at37°C for the indicated time, followed by acidification of thesamples and TiO2 extraction. The gels presented arerepresentative of experiments performed two to four times.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB150014F4: Absence of InsP6 and presence of inositol phosphatephosphatase in human plasma. All the extracts were resolved by PAGEand visualized with toluidine blue staining. (a)EDTA was added to 20 ml of commercial human plasma from male (MP)and female (FP); InsP6 was added to the spiked aliquot(InsP6; 2 nmol). The samples were then acidified andsubjected to TiO2 enrichment. (b) Plasmafrom healthy anonymous donors (D1 to D4) was prepared as describedin Material and methods, with spiking (InsP6; 1 nmol),and subjected to TiO2 extraction. (c) 4nmol of InsP6 was added to 1 ml of human plasma andincubated at 37°C for the indicated time before acidificationand extraction of inositol phosphates with the TiO2procedure. Standards: InsP6 (4 nmol); InsP5 (6nmol of Ins(1,3,4,5,6)P5); InsP4 (5 nmol ofIns(1,4,5,6)P4); InsP3 (20 nmol ofIns(1,4,5)P3). (d) 4 nmol ofInsP6 was added to 1 ml of a different source ofhuman plasma (HP) and bovine plasma (BP) before incubation at37°C for the indicated time, followed by acidification of thesamples and TiO2 extraction. The gels presented arerepresentative of experiments performed two to four times.
Mentions: Since large volumes of acidified fluid can be subject to TiO2 beadextraction, this gave us the opportunity to assay InsP6 in biofluids.Initially, we used commercially available serum from bovine, equine and humansources. We extracted 20 ml of serum with TiO2 beads and analysed theextracts by PAGE. While we were able to detect an almost completeInsP6 recovery in the spiked samples, we did not recover anyInsP6 in non-spiked serum (electronic supplementary material,figure S2A,B). We next analysed human plasma from a commercial source. Similarto serum, TiO2 extraction and PAGE analysis showed thatInsP6 could not be recovered from non-spiked samples of humanplasma (figure4a). The lower limit of InsP6 standarddetection on PAGE is about 0.25 nmol (figures 4a,b and 5), thereforeTiO2-extracting 20 ml of plasma with a recovery of approximately85% (figure1e) indicates that the lower limit of plasmaInsP6 we are able to extract and detect is approximately 15 nM.Consequently, we conclude that substantially less than 15 nM InsP6 ispresent in human plasma, in agreement with the enzymatic radio-assay previouslyreported [8]. Figure 4.

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