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

Wilson, Miranda S. C.; Bulley, Simon J.; Pisani, Francesca M.; Irvine, Robin F.; Saiardi, Adolfo

doi:10.1098/rsob.150014

Cited by 121 publications

(150 citation statements)

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“…However, a recent analysis of an HCT116 human colonic carcinoma cell line revealed it to contain substantially higher levels of InsP 8 than those found in some other mammalian cell lines [19]. We now demonstrate that there is considerable variability in the cellular levels of InsP 8 within two divergent HCT116 cell lines in our two laboratories.…”

Section: Introductionmentioning

confidence: 48%

“…This method does not match the sensitivity of HPLC, but by using TiO 2 beads to concentrate PP-InsPs prior to analysis [19], the cellular levels of total InsP 7 can be readily monitored. This methodology can even resolve 5-InsP 7 from 1-InsP 7 [20], but to date gel electrophoresis has not detected 1-InsP 7 in any mammalian cell line [19], perhaps because its levels are below the limits of sensitivity. Thus, we have developed an alternative HPLC technique that, for the first time, can directly measure 1-InsP 7 levels in intact cells.…”

Section: Introductionmentioning

confidence: 99%

“…The assay of cellular InsP 8 has also proved to be challenging for gel electrophoresis, at least when using an experimentally convenient number of cells [19]. However, a recent analysis of an HCT116 human colonic carcinoma cell line revealed it to contain substantially higher levels of InsP 8 than those found in some other mammalian cell lines [19].…”

Section: Introductionmentioning

confidence: 99%

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Inositol Pyrophosphate Profiling of Two HCT116 Cell Lines Uncovers Variation in InsP8 Levels

Wilson

Jessen

et al. 2016

PLoS ONE

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The HCT116 cell line, which has a pseudo-diploid karotype, is a popular model in the fields of cancer cell biology, intestinal immunity, and inflammation. In the current study, we describe two batches of diverged HCT116 cells, which we designate as HCT116NIH and HCT116UCL. Using both gel electrophoresis and HPLC, we show that HCT116UCL cells contain 6-fold higher levels of InsP8 than HCT116NIH cells. This observation is significant because InsP8 is one of a group of molecules collectively known as ‘inositol pyrophosphates’ (PP-InsPs)—highly ‘energetic’ and conserved regulators of cellular and organismal metabolism. Variability in the cellular levels of InsP8 within divergent HCT116 cell lines could have impacted the phenotypic data obtained in previous studies. This difference in InsP8 levels is more remarkable for being specific; levels of other inositol phosphates, and notably InsP6 and 5-InsP7, are very similar in both HCT116NIH and HCT116UCL lines. We also developed a new HPLC procedure to record 1-InsP7 levels directly (for the first time in any mammalian cell line); 1-InsP7 comprised <2% of total InsP7 in HCT116NIH and HCT116UCL lines. The elevated levels of InsP8 in the HCT116UCL lines were not due to an increase in expression of the PP-InsP kinases (IP6Ks and PPIP5Ks), nor to a decrease in the capacity to dephosphorylate InsP8. We discuss how the divergent PP-InsP profiles of the newly-designated HCT116NIH and HCT116UCL lines should be considered an important research opportunity: future studies using these two lines may uncover new features that regulate InsP8 turnover, and may also yield new directions for studying InsP8 function.

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Section: Introductionmentioning

confidence: 48%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Inositol Pyrophosphate Profiling of Two HCT116 Cell Lines Uncovers Variation in InsP8 Levels

Wilson

Jessen

et al. 2016

PLoS ONE

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“…PolyP extraction not only purifies polyP and RNA but also inositol phosphates (Fig. 2D), nucleotides (28), and likely free phosphate. The abundance of these phosphate-containing molecules influences these polyP quantification methods (8).…”

Section: Ppk1 the Only Enzyme Responsible For Polyp Synthesis Controlmentioning

confidence: 99%

Developmental accumulation of inorganic polyphosphate affects germination and energetic metabolism in Dictyostelium discoideum

Livermore

Chubb

Saiardi

2016

Proc. Natl. Acad. Sci. U.S.A.

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Inorganic polyphosphate (polyP) is composed of linear chains of phosphate groups linked by high-energy phosphoanhydride bonds. However, this simple, ubiquitous molecule remains poorly understood. The use of nonstandardized analytical methods has contributed to this lack of clarity. By using improved polyacrylamide gel electrophoresis we were able to visualize polyP extracted from Dictyostelium discoideum. We established that polyP is undetectable in cells lacking the polyphosphate kinase (DdPpk1). Generation of this ppk1 null strain revealed that polyP is important for the general fitness of the amoebae with the mutant strain displaying a substantial growth defect. We discovered an unprecedented accumulation of polyP during the developmental program, with polyP increasing more than 100-fold. The failure of ppk1 spores to accumulate polyP results in a germination defect. These phenotypes are underpinned by the ability of polyP to regulate basic energetic metabolism, demonstrated by a 2.5-fold decrease in the level of ATP in vegetative ppk1. Finally, the lack of polyP during the development of ppk1 mutant cells is partially offset by an increase of both ATP and inositol pyrophosphates, evidence for a model in which there is a functional interplay between inositol pyrophosphates, ATP, and polyP.inorganic polyphosphate | phosphate | metabolism | inositol pyrophosphate | mitochondria T he simplest biological polymer is inorganic polyphosphate (polyP), which consists of a linear chain of phosphates linked by high-energy phosphoanhydride bonds (1). Regardless of its origin, polyP is ubiquitously present in today's living organisms. The synthesis, enzymology, and role of polyP are best characterized in the bacteria Escherichia coli (2). However, polyP is also present in archaea (3), eukaryotes (4), and human cells (5). Whilst polyP is present in virtually all cellular compartments (6), it accumulates in acidocalcisomes, organelles common to bacteria and mammalian cells (7).PolyP has been implicated in a range of cellular processes over recent years, but its most basic proposed function is to act as a buffer for the level of free phosphate (8). Phosphate buffering is essential for general metabolism, and the synthesis or degradation of polyP is able to safeguard the level of free phosphate in cells. In addition, the polyanionic nature of polyP makes it a strong chelator of bivalent cations; thus polyP plays a key role in cation homeostasis. The chelation of calcium by polyP might even play a role as a calcium sink, regulating calcium signaling (8). Besides these common roles, many specific functions have been attributed to polyP in eukaryotes (see reviews in refs. 9-11). Recently its ability to function as a protein-folding chaperone (12) and to drive a newly discovered posttranslational protein modification, protein polyphosphorylation (13), have provided further insight into the mechanism of action of this simple polymer.Little is known about the synthesis or metabolism of polyP in eukaryotes. The yeast Saccharomyces c...

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“…In the future, we hope to improve the detection of PP-IPs directly from patient cells and tissue samples. It will be possible for us to apply and further develop the techniques of Saiardi et al who showed that PP-IPs are efficiently enriched on titanium oxide beads and can be visualized using the fluorescent dye 4 ′ ,6-diamidino-2-phenylindole (DAPI) (Wilson et al 2015).…”

Section: Resultsmentioning

confidence: 99%

Inositol pyrophosphates as multifaceted metabolites in the regulation of mammalian signaling networks

Park

Lee

Kim

2017

Animal Cells and Systems

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Inositol pyrophosphates (PP-IPs) such as 5-diphosphoinositol pentakisphosphate (5-IP 7 ) are inositol metabolites with high-energy phosphoanhydride bonds. The formation of PP-IPs is catalyzed by two groups of enzymes, the IP6 kinases and the PPIP5 kinases, which both phosphorylate inositol hexakisphosphate (IP 6 ). In mammals, PP-IPs are implicated in diverse biological phenomena including cellular growth, vesicular trafficking, apoptosis, and metabolic homeostasis. Mechanistically, all the diverse actions of PP-IPs proceed in one of two ways: the PP-IPs modulate the activity of their target proteins either through allosteric binding or protein pyrophosphorylation. In this review, we highlight recent advances in our understanding of the pleiotropic functions of the mammalian PP-IPs and the metabolic enzymes that produce them. We also discuss some future challenges in the exploration of areas where PP-IPs play important but unknown roles in physiology and disease.ARTICLE HISTORY

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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

Cited by 121 publications

References 47 publications

Inositol Pyrophosphate Profiling of Two HCT116 Cell Lines Uncovers Variation in InsP8 Levels

Inositol Pyrophosphate Profiling of Two HCT116 Cell Lines Uncovers Variation in InsP8 Levels

Developmental accumulation of inorganic polyphosphate affects germination and energetic metabolism in Dictyostelium discoideum

Inositol pyrophosphates as multifaceted metabolites in the regulation of mammalian signaling networks

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