Scalable Chemoenzymatic Synthesis of Inositol Pyrophosphates.

The inositol pyrophosphates (PP-InsPs) are an important group of cellular messengers that influence a broad range of biological processes. To elucidate the functions of these high-energy metabolites at the biochemical level, access to the purified molecules is required. Here, a robust and scalable strategy for the synthesis of various PP-InsPs [5PP-InsP5, 1PP-InsP5, and 1,5(PP)2-InsP4] is reported, relying on the highly active inositol hexakisphosphate kinase A from Entamoeba histolytica and the kinase domain of human diphosphoinositol pentakisphosphate kinase 2. A facile purification procedure using precipitation with Mg2+ ions and an optional strong anion exchange chromatography on an FPLC system afforded PP-InsPs in high purity. Furthermore, the newly developed protocol could be applied to simplify the synthesis of radiolabeled 5PP-InsP5-ß32P, which is a valuable tool for studying protein pyrophosphorylation. The chemoenzymatic method for obtaining PP-InsPs is readily amenable to both chemists and biologists and will thus foster future research on the multiple signaling functions of PP-InsP molecules.

Determination of d-myo-inositol phosphates in 'activated' raw almonds using anion-exchange chromatography coupled with tandem mass spectrometry.

BACKGROUND: Activated almonds are raw almonds that have been soaked in water for 12-24 h at room temperature, sometimes followed by a 24 h drying period at low temperature (50 ± 5 °C). This treatment is thought to enhance the nutrient bioavailability of almonds by degrading nutrient inhibitors, such as phytic acid or d-myo-inositol hexaphosphate (InsP6 ), through the release of phytase or passive diffusion of InsP6 into the soaking water. Over a wide pH range, InsP6 is a negatively charged compound that limits the absorption of essential nutrients by forming insoluble complexes with minerals such as iron and zinc. It is hypothesized that hydrating the seed during soaking triggers InsP6 degradation into lower myo-inositol phosphates with less binding capacity. RESULTS: Anion-exchange chromatography coupled with tandem mass spectrometry was used to quantify myo-inositol mono-, di-, tris-, tetra-, penta-, and hexaphosphates (InsP1-6 ) in raw pasteurized activated almonds. At least 24 h of soaking at ambient temperature was required to reduce InsP6 content from 14.71 to 14.01 µmol g-1 . CONCLUSIONS: The reduction in InsP6 is statistically significant (P < 0.05) after 24 h of activation, but only represents a 4.75% decrease from the unsoaked almonds. © 2018 Society of Chemical Industry.

Synthesis of inositol phosphate ligands of plant hormone-receptor complexes: pathways of inositol hexakisphosphate turnover.

Reduction of phytate is a major goal of plant breeding programs to improve the nutritional quality of crops. Remarkably, except for the storage organs of crops such as barley, maize and soybean, we know little of the stereoisomeric composition of inositol phosphates in plant tissues. To investigate the metabolic origins of higher inositol phosphates in photosynthetic tissues, we have radiolabelled leaf tissue of Solanum tuberosum with myo-[2-3H]inositol, undertaken a detailed analysis of inositol phosphate stereoisomerism and permeabilized mesophyll protoplasts in media containing inositol phosphates. We describe the inositol phosphate composition of leaf tissue and identify pathways of inositol phosphate metabolism that we reveal to be common to other kingdoms. Our results identify the metabolic origins of a number of higher inositol phosphates including ones that are precursors of cofactors, or cofactors of plant hormone-receptor complexes. The present study affords alternative explanations of the effects of disruption of inositol phosphate metabolism reported in other species, and identifies different inositol phosphates from that described in photosynthetic tissue of the monocot Spirodela polyrhiza. We define the pathways of inositol hexakisphosphate turnover and shed light on the occurrence of a number of inositol phosphates identified in animals, for which metabolic origins have not been defined.

Cloning, expression, purification, crystallization and X-ray analysis of inositol monophosphatase from Mus musculus and Homo sapiens.

Inositol monophosphatase (IMPase) catalyses the hydrolysis of inositol monophosphate to inositol and is crucial in the phosphatidylinositol (PI) signalling pathway. Lithium, which is the drug of choice for bipolar disorder, inhibits IMPase at therapeutically relevant plasma concentrations. Both mouse IMPase 1 (MmIMPase 1) and human IMPase 1 (HsIMPase 1) were cloned into pRSET5a, expressed in Escherichia coli, purified and crystallized using the sitting-drop method. The structures were solved at resolutions of 2.4 and 1.7 Å, respectively. Comparison of MmIMPase 1 and HsIMPase 1 revealed a core r.m.s. deviation of 0.516 Å.

Direct LC-MS/MS Analysis of Extra- and Intracellular Glycerophosphoinositol in Model Cancer Cell Lines.

Glycerophosphoinositols (GPIs) are water-soluble bioactive phospholipid derivatives of increasing interest as intracellular and paracrine mediators of eukaryotic cell functions. The most representative compound of the family is glycerophosphoinositol (GroPIns), an ubiquitous component of mammalian cells that participates in cell proliferation, cell survival and cell response to stimuli. Levels and activity of this compound vary among cell types and deciphering these functions requires accurate measurements in in vitro and in vivo models. The conventional approaches for the analysis of GroPIns pose several issues in terms of sensitivity and product resolution, especially when the product is in the extracellular milieu. Here we present an UPLC-MS study for the quantitative analysis of this lipid derivative in cells and, for the first time, culture supernatants. The method is based on a solid-phase extraction that allows for fast desalting and analyte concentration. The robustness of the procedure was tested on the simultaneous measurements of intra- and extracellular levels of GroPIns in a number of human cell lines where it has been shown that the non-transformed cells are characterized by high extracellular level of GroPIns, whereas the tumor cells tended to have higher intracellular levels.

Over-expression of Brassica napus phosphatidylinositol-phospholipase C2 in canola induces significant changes in gene expression and phytohormone distribution patterns, enhances drought tolerance and promotes early flowering and maturation.

Phosphatidylinositol-specific phospholipase C (PtdIns-PLC2) plays a central role in the phosphatidylinositol-specific signal transduction pathway. It catalyses the hydrolysis of membrane-bound phosphatidylinositol 4,5-bisphosphate to produce two second messengers, sn-1,2-diacylglycerol and inositol 1,4,5-trisphosphate. The former is a membrane activator of protein kinase C in mammalian systems, and the latter is a Ca(2+) modulator which induces distinctive oscillating bursts of cytosolic Ca(2+), resulting in regulation of gene expression and activation of proteins. Sustained over-expression of BnPtdIns-PLC2 in transgenic Brassica napus lines brought about an early shift from vegetative to reproductive phases, and shorter maturation periods, accompanied by notable alterations in hormonal distribution patterns in various tissues. The photosynthetic rate increased, while stomata were partly closed. Numerous gene expression changes that included induction of stress-related genes such as glutathione S-transferase, hormone-regulated and regulatory genes, in addition to a number of kinases, calcium-regulated factors and transcription factors, were observed. Other changes included increased phytic acid levels and phytohormone organization patterns. These results suggest the importance of PtdIns-PLC2 as an elicitor of a battery of events that systematically control hormone regulation, and plant growth and development in what may be a preprogrammed mode.

Intercellular communication between follicular angiotensin receptors and Xenopus laevis oocytes: medication by an inositol 1,4,5-trisphosphate-dependent mechanism.

In Xenopus laevis oocytes, activation of angiotensin II (AII) receptors on the surrounding follicular cells sends a signal through gap junctions to elevate cytoplasmic calcium concentration ([Ca2+]i) within the oocyte. The two major candidates for signal transfer through gap junctions into the oocyte during AII receptor stimulation are Ins(1,4,5)P3 and Ca2+. In [3H]inositol-injected follicular oocytes, AII stimulated two- to fourfold increases in phosphoinositide hydrolysis and production of inositol phosphates. Injection of the glycosaminoglycan, heparin, which selectively blocks Ins(1,4,5)P3 receptors, prevented both AII-stimulated and Ins(1,4,5)P3-induced Ca2+ mobilization in Xenopus follicular oocytes but did not affect mobilization of Ca2+ by ionomycin or GTP. These results indicate that the AII-regulated process of gap junction communication between follicular cells and the oocyte operates through an Ins(1,4,5)P3-dependent mechanism rather than through transfer of Ca2+ into the ooplasm and subsequent Ca(2+)-induced Ca2+ release.

Alterations of G-protein coupling function in phosphoinositide signaling pathways of cells transformed by ras and other membrane-associated and cytoplasmic oncogenes.

We showed previously that transformation by cytoplasmic and membrane-associated oncogenes including ras results in uncoupling between surface stimulation by platelet-derived growth factor, bombesin, and serum and activation of intracellular phospholipase C (PLC); this uncoupling does not involve alterations at the receptor or effector enzyme levels (T. Alonso, R. O. Morgan, J. C. Marvizon, H. Zarbl, and E. Santos, Proc. Natl. Acad. Sci. USA 85:4271-4275, 1988). In this study, we stimulated normal and oncogene-transformed NIH 3T3 cells with fluoroaluminate (AIF4-), thus directly activating PLC-associated G protein(s) and bypassing the receptor step. A1F4(-)-elicited PLC responses were significantly impaired in transformed cells when compared with those in their normal counterparts, suggesting that the uncoupling of PLC is the result, at least in part, of functional impairment at the G-protein level. Transformation by ras oncogenes has also been reported to result in enhanced PLC response to bradykinin resulting from increased receptor numbers (G. Parries, R. Hoebel, and E. Racker, Proc. Natl. Acad. Sci. USA 84:2648-2652, 1987; J. Downward, J. de Gunzburg, R. Riehl, and R. Weinberg, Proc. Natl. Acad. Sci. USA 85:5774-5778, 1988). We demonstrate here that transformation by other membrane-associated and cytoplasmic oncogenes also results in increased responsiveness to bradykinin ("supercoupling") and enhanced receptor numbers. However, there is no direct correlation between the number of receptors and the enhancement in responsiveness, suggesting that other factors besides receptor number are also involved in the enhanced responses. We propose that a common effect of transformation by cytoplasmic and membrane-associated oncogenes is functional alteration of coupling G proteins and that a similar modification of different kinds of G proteins may account for the pleiotropic alterations of signal transduction (uncoupling and supercoupling) observed.

Thrombin-dependent association of phosphatidylinositol-3 kinase with p60c-src and p59fyn in human platelets.

Recent studies have shown that ligand-activated growth factor receptors as well as transforming versions of nonreceptor protein-tyrosine kinases physically associate with phosphatidylinositol-3 kinase (PI-3 kinase). Reasoning that PI-3 kinase might also play a role in the normal functions of nonreceptor kinases, we sought to determine whether association with PI-3 kinase might serve as a measure of nonreceptor protein-tyrosine kinase activation under physiological conditions. We found that p60c-src as well as p59fyn, the product of another member of the src family of proto-oncogenes, physically associated with a PI kinase activity within 5 s after exposure to thrombin. Furthermore, PI kinase reaction products generated in p60v-src, p60c-src or p59fyn containing immunoprecipitates were indistinguishable, demonstrating the identity of the associated enzyme as PI-3 kinase. These findings demonstrate a thrombin-dependent interaction between p60c-src or p59fyn and PI-3 kinase and suggest a role for nonreceptor protein-tyrosine kinases in human platelet signal transduction.

Gastrin and CCK-8 induce inositol 1,4,5-trisphosphate formation in rabbit gastric parietal cells.

The role of phosphoinositide turnover in the mediation of acid secretion was examined in an enriched preparation of isolated rabbit parietal cells (75%). Both gastrin and CCK-8 (octapeptide of cholecystokinin) stimulated [14C]aminopyrine (AP) uptake by cells (EC50 0.07 +/- 0.03 nM (gastrin) and 0.093 +/- 0.065 nM (CCK-8] and increased [3H]inositol phosphates cellular contents (EC50 0.142 +/- 0.016 nM (gastrin) and 0.116 +/- 0.027 nM (CCK-8] in a parallel fashion. In addition, the EC50 values for both phenomenon were quite similar to the Kd values obtained from binding experiments. HPLC analysis of the different [3H]inositol phosphates produced under gastrin or CCK-8 stimulation showed a 2-fold increase in [3H]Ins(1,4,5)P3 levels within 5 s with a concomitant increase in [3H]Ins(1,4)P2 content within 15 s. A low but significant rise in [3H]Ins(1,3,4,5)P4 and [3H]Ins(1,3,4)P3 cellular contents was also observed. No difference between gastrin- and CCK-8-induced inositol phosphates production could be shown. We can conclude that gastrin and CCK-8 display an identical profile of action, suggesting that they stimulate the acid secretory function of parietal cells through the same receptor site coupled to the Ins(1,4,5)P3 production.

Interactions between inositol phosphates and cytosolic free calcium following bradykinin stimulation in cultured human skin fibroblasts.

The inositol triphosphate (IP3) that results from hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is generally accepted to be responsible for the mobilization of intracellular calcium. However, some studies suggest that low concentrations of agonists elevate cytosolic free calcium concentration ([Ca2+]i) without IP3 formation. Thus, in the present studies, a comparison of the temporal response of inositol phosphates (IP3, IP2 and IP) and [Ca2+]i to a wide range of bradykinin concentrations was used to examine the relation of these two signal transduction events in cultured human skin fibroblasts (GM3652). In addition, the effects of alterations in internal or external calcium on the response of these second messengers to bradykinin were determined. Bradykinin stimulated accumulation of inositol phosphates and a rise of [Ca2+]i in a time- and dose-dependent manner. Decreasing the bradykinin concentration from 1 microM to 0.1 microM increased the time until the IP3 peak, and when the bradykinin concentration was reduced to 0.01 microM IP3 was not detected. [Ca2+]i was examined under parallel conditions. As the bradykinin concentration was reduced from 1 microM to 0.01 microM, the time to reach the peak of [Ca2+]i increased progressively, but the magnitude of the peak was unaltered. These two second messengers were variably dependent on external calcium. Although the bradykinin-stimulated initial spike of [Ca2+]i did not depend on extracellular calcium, the subsequent sustained levels of [Ca2+]i were abolished in calcium free medium. The bradykinin-stimulated inositol phosphate formation was not dependent on the extracellular calcium nor on the elevation of [Ca2+]i that was produced with Br-A23187. These results demonstrate that bradykinin-induced IP3 formation can be independent of [Ca2+]i and of external calcium, whereas changes in [Ca2+]i are partially dependent on external calcium.

Functional cholecystokinin receptors are distinguished kinetically by biotinyl-Tyr-Gly-(Thr28,Nle31)CCK(25-33) in rat pancreatic acini.

Biotinyl-tyrosine-glycine(Thr28,Nle31)CCK(25-33) (BTG-TN-CCK-9) promoted amylase secretion and phosphatidylinositol (PI) metabolism with the same potency and efficacy as TN-CCK-9 in dispersed rat pancreatic acini. A 1 min preincubation of the ligand with a 20-fold excess of streptavidin completely suppressed this biological activity. On the other hand, amylase secretion and PI metabolism prestimulated with BTG-TN-CCK-9 were blocked within 1-5 min after streptavidin addition. [125I]BTG-TN-CCK-9 bound to high (Kd 0.17 nM) and low (Kd 13 nM) affinity receptors. Its dissociation, in the presence of either streptavidin or TN-CCK-9, showed a rapid component and a slow component. The proportion of tracer dissociating slowly increased with increasing preincubation time as did the proportion of tracer that could not be washed away quickly by acidic treatment, in parallel experiments. This phenomenon occurred less readily at 4 degrees C or in the presence of 1 mM CCCP. In acini preincubated for 30 min with 0.3 nM [125I]BTG-TN-CCK-9 and various concentrations of unlabelled BTG-TN-CCK-9, then washed at neutral pH (in order to eliminate rapidly dissociating ligand preferentially), the tracer displacement curve was shifted leftward, suggesting that rapidly dissociating receptors corresponded to low affinity receptors. When acini were preincubated for 1 min with BTG-TN-CCK-9, then washed at neutral pH with buffer only, we observed residual stimulated secretion over the next 30 min period, that correlated with the BTG-TN-CCK-9 concentration offered during the short preincubation period. This phenomenon was inhibited by streptavidin suggesting that intracellularly accumulated intact BTG-TN-CCK-9 (as shown, by radio-HPLC) promoted residual secretion when free to bind again to cell surface receptors in the absence of streptavidin. Taken collectively, these data suggest the coexistence of at least 2 types (or states) of CCK receptors.

Regulation of calcium influx across the plasma membrane of the human T-leukemic cell line, JURKAT: dependence on a rise in cytosolic free calcium can be dissociated from formation of inositol phosphates.

A rise in the cytosolic free Ca2+ concentration due to both mobilization of Ca2+ from internal stores and influx of extracellular Ca2+ across the plasma membrane through 'second messenger-operated Ca2+ channels' is one of the first transmembrane signals detected following activation of CD2 or CD3 receptors on T-cells. In this study, we have further elucidated the regulation of these channels in the human T-leukemic cell line, JURKAT. Stimulation with either OKT3 or PHA induced a prompt influx of Ca2+ as assessed by MN2+ quenching of intracellular fura-2 fluorescence. When cytosolic free Ca2+ transient was partially buffered by loading the cells with BAPTA, neither agonist could induce Ca2+ entry into the cells as depicted by the lack of quenching of the fluorescence signal by Mn2+. This is in good agreement with our previous data on agonist-induced 45Ca2+ influx demonstrating that a rise in cytosolic free Ca2+ due to agonist-induced mobilization of Ca2+ from intracellular stores, could, directly or indirectly via the inositol cycle, initiate Ca2+ influx in these cells. Further support of this idea comes from the data demonstrating that agonist-induced mobilization of Ca2+ precedes the influx of Ca2+ across the plasma membrane. The present findings show that agonist-stimulation significantly increased the levels of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 after only 5 s, indicating that one or both of these substances could play a role in the regulation of Ca2+ influx. However, when agonist-induced Mn2+ influx was totally abolished, by partially buffering the cytosolic free Ca2+ rise, the formation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 was not affected. Consequently, the dependence of an initial rise in cytosolic free Ca2+ for the subsequent regulation of Ca2+ influx across the plasma membrane, can be dissociated from the formation of both Ins(1,4,5)P3 and Ins(1,3,4,5)P4.

Inositol glycan phosphate derived from human erythrocyte acetylcholinesterase glycolipid anchor and inositol cyclic 1,2-phosphate antagonize glucagon activation of glycogen phosphorylase.

In this study we examine the hypothesis that an inositol glycan phosphate can act similarly to insulin on intact cells. The inositol glycan phosphate used in this study (glycan alpha) was isolated previously from the glycoinositol phospholipid anchor of human erythrocyte acetylcholinesterase and was shown to have the structure glycine-ethanolamine-PO4-Man-Man-(N,N-dimethylethanolamine-PO4)Man- (N,N-dimethyl)GlcN-inositol-PO4. The cellular response investigated was the glucagon-stimulated activation of glycogen phosphorylase in rat hepatocytes. When hepatocytes were incubated with 20 nM glucagon for 4 min, the ratio of phosphorylase a activity to total phosphorylase increased from a basal value of 0.49 +/- 0.02 to 0.82 +/- 0.03 (mean +/- SE, n = 15). Inclusion of either 100 nM insulin or 3-10 microM glycan alpha during the glucagon incubation significantly decreased the glucagon-stimulated activity ratio to 0.74 +/- 0.03 for either agent. Furthermore, hepatocyte preparations differed in their response to insulin and were divided into insulin-responsive and -resistant groups. Glycan alpha had a significant effect only in the insulin-responsive group for which the observed activity ratio for 10 microM glycan alpha plus glucagon (0.68 +/- 0.05) compared closely with that for insulin plus glucagon (0.70 +/- 0.04). For the insulin-resistant group, the activity ratio in the presence of 10 microM glycan alpha was 0.81 +/- 0.03, unchanged from the control with glucagon alone. Because glycan alpha contains an inositol phosphate group, the effect of inositol cyclic 1,2-phosphate on the glucagon-stimulated activity ratio was determined.(ABSTRACT TRUNCATED AT 250 WORDS)

The major surface antigens of Entamoeba histolytica trophozoites are GPI-anchored proteophosphoglycans.

Trophozoites of the parasitic protozoa, Entamoeba histolytica, synthesize a cell surface lipoglycoconjugate, termed lipophosphoglycan, which is thought to be an important virulence factor and potential vaccine candidate against invasive amebiasis. Here, we show that the E. histolytica lipophosphoglycans are in fact glycosylphosphatidylinositol (GPI)-anchored proteophosphoglycans (PPGs). These PPGs contain a highly acidic polypeptide component which is rich in Asp, Glu and phosphoserine residues. This polypeptide component is extensively modified with linear glycan chains having the general structure, [Glcalpha1-6](n)Glcbeta1-6Gal (where n=2-23). These glycan chains can be released after mild-acid hydrolysis with trifluoroacetic or hydrofluoric acid and are probably attached to phosphoserine residues in the polypeptide backbone. The PPGs are further modified with a GPI anchor which differs from all other eukaryotic GPI anchors so far characterized in containing a glycan core with the structure, Gal(1)Man(2)GlcN-myo-inositol, and in being heterogeneously modified with chains of alpha-galactose. Trophozoites of the pathogenic HM-1:IMSS strain synthesize two distinct classes of PPG which have polydisperse molecular masses of 50-180 kDa (PPG-1) and 35-60 kDa (PPG-2) and are modified with glucan side-chains of different average lengths. In contrast, the non-pathogenic Rahman strain synthesizes one class of PPG which is only elaborated with short disaccharide side-chains (i.e. Glcbeta1-6Gal). However, the PPGs are abundant in all strains (8x10(7) copies per cell) and are likely to form a protective surface coat.

An inositol phosphoglycan from Trypanosoma cruzi inhibits ACTH action in calf adrenocortical cells.

We describe the effect of an inositol phosphoglycan (IPG) purified from Trypanosoma cruzi on the stimulation of aldosterone and cAMP production by ACTH in calf adrenocortical cells. T. cruzi IPG has two galactofuranose residues (Galf) which are not frequent in other IPGs. The effect of IPG with galactofuranose residues (IPG Galf) and IPG without these residues (IPG) was investigated. It was found that IPG Galf slightly decreased the stimulation of aldosterone and cAMP production by ACTH, whereas IPG significantly inhibited ACTH-mediated accumulation of both aldosterone and cAMP. The inhibition of aldosterone content in ACTH-treated cells by IPG was dose dependent. It was also found that the pretreatment of calf adrenocortical cells with IPG inhibited the accumulation of aldosterone provoked by ACTH and dibutyryladenosine-3',5'-cyclic monophosphate (db-cAMP). On the other hand, the activation of a GPI (glycosyl phosphatidylinositol)-phospholipase C by ACTH was evaluated. First it was found that the release of ceramide from a GPI-like molecule: a glycoinositol-phosphoceramide (LPPG) purified from T. cruzi is increased in ACTH-treated cells. Second, the release of alkaline phosphatase, a GPI-anchored enzyme, to the extracellular medium was increased in these cells by ACTH. These data suggest that ACTH activates a phospholipase C in calf adrenocortical cells, releasing IPG, which in turn may inhibit, or modulate ACTH action.

Conundrum of IP6.

Here are comments on the recent paper on the determination of inositol hexaphosphate (IP6) in human plasma and on its efficacy.

There is no 'Conundrum' of InsP6.

Indirect assays have claimed to quantify phytate (InsP6) levels in human biofluids, but these have been based on the initial assumption that InsP6 is there, an assumption that our more direct assays disprove. We have shown that InsP6 does not and cannot (because of the presence of an active InsP6 phosphatase in serum) exist in mammalian serum or urine. Therefore, any physiological effects of dietary InsP6 can only be due either to its actions in the gut as a polyvalent cation chelator, or to inositol generated by its dephosphorylation by gut microflora.

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

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.