Inositol pyrophosphate profiling reveals regulatory roles of IP6K2-dependent enhanced IP7 metabolism in the enteric nervous system.

Inositol pyrophosphates regulate diverse physiological processes; to better understand their functional roles, assessing their tissue-specific distribution is important. Here, we profiled inositol pyrophosphate levels in mammalian organs using an originally designed liquid chromatography-mass spectrometry (LC-MS) protocol and discovered that the gastrointestinal tract (GIT) contained the highest levels of diphosphoinositol pentakisphosphate (IP7) and its precursor inositol hexakisphosphate (IP6). Although their absolute levels in the GIT are diet dependent, elevated IP7 metabolism still exists under dietary regimens devoid of exogenous IP7. Of the major GIT cells, enteric neurons selectively express the IP7-synthesizing enzyme IP6K2. We found that IP6K2-knockout mice exhibited significantly impaired IP7 metabolism in the various organs including the proximal GIT. In addition, our LC-MS analysis displayed that genetic ablation of IP6K2 significantly impaired IP7 metabolism in the gut and duodenal muscularis externa containing myenteric plexus. Whole transcriptome analysis of duodenal muscularis externa further suggested that IP6K2 inhibition significantly altered expression levels of the gene sets associated with mature neurons, neural progenitor/stem cells, and glial cells, as well as of certain genes modulating neuronal differentiation and functioning, implying critical roles of the IP6K2-IP7 axis in developmental and functional regulation of the enteric nervous system. These results collectively reveal an unexpected role of mammalian IP7-a highly active IP6K2-IP7 pathway is conducive to the enteric nervous system.

A myo-inositol bioassay utilizing an auxotrophic strain of S. cerevisiae.

Myo-inositol is a six­carbon sugar that is essential for the growth of mammalian cells and must be obtained through either extracellular uptake or de novo biosynthesis. The physiological importance of myo-inositol stems from its incorporation into phosphoinositides and inositol phosphates, which serve a variety of signaling, regulatory, and structural roles in cells. To study myo-inositol metabolism and function, it is essential to have a reliable method for assaying myo-inositol levels. However, current approaches to assay myo-inositol levels are time-consuming, expensive, and often unreliable. This article describes a simple new myo-inositol bioassay that utilizes an auxotrophic strain of S. cerevisiae to measure myo-inositol concentration in solutions. The accuracy of this method was confirmed by comparing assay values to those obtained by tandem mass spectrometry (LC-MS/MS). It is easy to perform, inexpensive, does not require sophisticated equipment, and is specific for myo-inositol.

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.

An ATP-responsive metabolic cassette comprised of inositol tris/tetrakisphosphate kinase 1 (ITPK1) and inositol pentakisphosphate 2-kinase (IPK1) buffers diphosphosphoinositol phosphate levels.

Inositol polyphosphates are ubiquitous molecular signals in metazoans, as are their pyrophosphorylated derivatives that bear a so-called 'high-energy' phosphoanhydride bond. A structural rationale is provided for the ability of Arabidopsis inositol tris/tetrakisphosphate kinase 1 to discriminate between symmetric and enantiomeric substrates in the production of diverse symmetric and asymmetric myo-inositol phosphate and diphospho-myo-inositol phosphate (inositol pyrophosphate) products. Simple tools are applied to chromatographic resolution and detection of known and novel diphosphoinositol phosphates without resort to radiolabeling approaches. It is shown that inositol tris/tetrakisphosphate kinase 1 and inositol pentakisphosphate 2-kinase comprise a reversible metabolic cassette converting Ins(3,4,5,6)P4 into 5-InsP7 and back in a nucleotide-dependent manner. Thus, inositol tris/tetrakisphosphate kinase 1 is a nexus of bioenergetics status and inositol polyphosphate/diphosphoinositol phosphate metabolism. As such, it commands a role in plants that evolution has assigned to a different class of enzyme in mammalian cells. The findings and the methods described will enable a full appraisal of the role of diphosphoinositol phosphates in plants and particularly the relative contribution of reversible inositol phosphate hydroxykinase and inositol phosphate phosphokinase activities to plant physiology.

Novel gluten-free formulations from lentil flours and nutritional yeast: Evaluation of extrusion effect on phytochemicals and non-nutritional factors.

The food industry is increasingly innovating and applying new processing technologies and ingredients to develop novel food products that meet the consumers' demand. In this study, the effect of extrusion (at 140 °C and 160 °C) was evaluated in different lentil flours formulations enriched with nutritional yeast, in terms of α-galactosides (raffinose, stachyose, verbascose), inositol phosphates (IPs), trypsin inhibitors and lectins content. The content of α-galactosides and IPs was determined by high performance liquid chromatography. Trypsin inhibitor activity (TIA) was evaluated using a small-scale quantitative assay. The lectin content was analyzed using a haemagglutination assay and a Competitive Indirect Enzyme-linked immunosorbent assay. Extrusion promoted a significant increase, up to 85% in total α-galactosides content. After extrusion, IPs content was significantly decreased and TIA as well as lectins content had a reduction higher than 90%. Extrusion demonstrated to have a beneficial effect by increasing desirable prebiotic compounds and decreasing non-nutritional factors.

Analytical Methods for Determination of Phytic Acid and Other Inositol Phosphates: A Review.

From the early precipitation-based techniques, introduced more than a century ago, to the latest development of enzymatic bio- and nano-sensor applications, the analysis of phytic acid and/or other inositol phosphates has never been a straightforward analytical task. Due to the biomedical importance, such as antinutritional, antioxidant and anticancer effects, several types of methodologies were investigated over the years to develop a reliable determination of these intriguing analytes in many types of biological samples; from various foodstuffs to living cell organisms. The main aim of the present work was to critically overview the development of the most relevant analytical principles, separation and detection methods that have been applied in order to overcome the difficulties with specific chemical properties of inositol phosphates, their interferences, absence of characteristic signal (e.g., absorbance), and strong binding interactions with (multivalent) metals and other biological molecules present in the sample matrix. A systematical and chronological review of the applied methodology and the detection system is given, ranging from the very beginnings of the classical gravimetric and titrimetric analysis, through the potentiometric titrations, chromatographic and electrophoretic separation techniques, to the use of spectroscopic methods and of the recently reported fluorescence and voltammetric bio- and nano-sensors.

Quantitation of Inositol Phosphates by HPLC-ESI-MS.

The coupling of anion exchange high-pressure liquid chromatography (HPLC) with electrospray ionization mass spectrometry (ESI-MS) allows for the simultaneous detection of the six forms of inositol phosphate (InsP). Here we describe a rapid quantitative analysis of InsPs by HPLC-ESI-MS, which can be applied to a wide array of sample types. With this method, InsPs could be separated and detected within 20 min of sample injection. The detection limit was as low as 25 pmol (i.e., ca. 2 nmol/g sample) for each type of InsP, which is particularly important for analytes that are often present at low abundance in nature.

Detection of Inositol Phosphates by Split PH Domains.

The pleckstrin homology (PH) domain is a family of structurally conserved proteins which can bind inositol phosphate derivatives. Some proteins involved in cellular signaling and cytoskeletal organization possess split PH domains that assemble into a structure which can bind specific inositol phosphates. Here we describe the design of split PH domain from a structurally well-characterized PH domain of phospholipase C (PLC) δ1 and Bruton's tyrosine kinase (Btk), which selectively bind Ins(1,4,5)P3 and Ins(1,3,4,5)P4, respectively. The PH domains fold into a functional structure when the split halves are brought to close proximity, and can be utilized to detect specific inositol phosphate of interest.

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry.

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal transduction, metabolism, and development. These metabolites perform this regulatory activity by binding to proteins, thereby changing protein conformation, catalytic activity, and/or interactions. The method described here uses affinity chromatography coupled to mass spectrometry or Western blotting to identify proteins that interact with inositol phosphates or phosphoinositides. Inositol phosphates or phosphoinositides are chemically tagged with biotin, which is then captured via streptavidin conjugated to agarose or magnetic beads. Proteins are isolated by their affinity of binding to the metabolite, then eluted and identified by mass spectrometry or Western blotting. The method has a simple workflow that is sensitive, non-radioactive, liposome-free, and customizable, supporting the analysis of protein and metabolite interaction with precision. This approach can be used in label-free or in amino acid-labelled quantitative mass spectrometry methods to identify protein-metabolite interactions in complex biological samples or using purified proteins. This protocol is optimized for the analysis of proteins from Trypanosoma brucei, but it can be adapted to related protozoan parasites, yeast or mammalian cells.

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.

Hydrophilic interaction liquid chromatography-tandem mass spectrometry for the quantitative analysis of mammalian-derived inositol poly/pyrophosphates.

Although myo-inositol pyrophosphates such as diphosphoinositol pentakisphosphate (InsP7) are important in biology, little quantitative information is available regarding their presence in mammalian organisms owing to the technical difficulties associated with accurately detecting these materials in biological samples. We have developed an analytical method whereby InsP7 and its precursor inositol hexakisphosphate (InsP6) are determined directly and sensitively using tandem mass spectrometry coupled with hydrophilic interaction liquid chromatography (HILIC). InsP6 and InsP7 peak symmetry is influenced greatly by the buffer salt composition and pH of the mobile phase used in HILIC analysis. The use of 300 mM ammonium carbonate (pH 10.5) as an aqueous mobile phase resolves InsP6 and InsP7 on a polymer-based amino HILIC column with minimal peak tailing. Method validation shows that InsP6 and InsP7 can be quantitated from 20-500 pmol with minimal intra-day/inter-day variance in peak area and retention time. The concentration of InsP6 in C57BL/6J mouse brain (40.68 ± 3.84 pmol/mg wet weight) is successfully determined. HILIC‒MS/MS analysis using HEK293 culture cells confirms previous observations that InsP7 is induced by NaF treatment and ectopic expression of InsP6K2, a primary kinase for InsP7 synthesis. Furthermore, this analysis reveals the abundance of InsP6 (50.46 ± 18.57 pmol/106 cells) and scarcity of InsP7 in human blood cells. The results demonstrate that HILIC‒MS/MS analysis can quantitate endogenous InsP6 and InsP7 in mouse and human samples, and we expect that the method will contribute to further understanding of InsP7 functions in mammalian pathobiology.

Effect of enzymatic treatment on phytate content and mineral bioacessability in soy drink.

The objective of this study was to evaluate in vitro bioaccessibility of calcium (Ca), iron (Fe) and zinc (Zn) in soy drink after phytase treatment and correlate it with the content of myo-inositol phosphates. Samples of commercial soy drink products and one sample produced in the laboratory by maceration were evaluated. Phytase was applied using 300 U per liter in 60 min considering the phosphate release. The content of myo-inositol tris-, tetrakis-, pentakis and hexakisphosphate was not observed after phytase treatment. The solubility assay showed an increase from 2.0% to 20.8% for Ca, 2.2% to 37.1% for Fe and 38.8% to 67.4% for Zn after phytase treatment with significant differences (p ≤ 0.05) for most samples. Dialysis assay demonstrated 1.0% to 9.5% for Ca after phytase treatment (p ≤ 0.05) except for one commercial sample. The phytase treatment is a valuable alternative process for improving mineral natural availability in soy drink and decreased the use of salts in the fortification.

FSH receptor binding inhibitor influences estrogen production, receptor expression and signal pathway during in vitro maturation of sheep COCs.

Follicle-stimulating hormone (FSH) promotes secretion of follicle fluid and follicle development. FSH acts via cognate FSH receptor (FSHR). It remains unknown whether the supplement of FSH-receptor binding inhibitor (FRBI) into the in vitro maturation (IVM)medium influence the estrogen receptor expression and signal pathway of oocytes in sheep. The present study aimed to investigate FRBI effects on inositol trisphosphate (IP3) of oocytes and protein kinase A (PKA) of sheep granulosa cells, further to elucidate the signal pathway of FRBI effects. Cumulus-oocyte complexes (COCs) were recovered from antral follicles. COCs were cultured for 24 h in the IVM medium supplemented with varying concentrations of FRBI (0, 10, 20, 30 and 40 µg/mL) and FSH (10IU/mL). ELISA was used to measure the concentrations of estradiol (E2) and IP3 in the IVM medium. Western blotting was utilized to detect protein expression of ERß of COCs and protein kinase A (PKA) of granulosa cells. The results showed IP3 concentrations of FRBI-3 and FRBI-4 groups were less than that of CG and FSH groups at 22 h and 24 h (P < 0.05). PKA levels of FRBI-3 and FRBI-4 groups were significantly less than that of CG and FSH group (P < 0.05 or P < 0.01). Expression levels of ERß mRNA and protein of FRBI-treated groups were gradually decreased in comparison to CG and FSH group. The minimum value was detected in the FRBI-4 group. ERß protein level of the FRBI-4 group was significantly less than that of FSH group (P < 0.05). E2 concentrations of FRBI-treated groups were elevated as compared to CG, with the highest increment of FRBI-2 group (P < 0.05). Our results revealed a higher dose of FRBI reduced IP3 production. FRBI could suppress slightly expression levels of ERß mRNA and protein of COCs and PKA of granulosa cells, additionally increased E2 production of sheep COCs.

Variability in amino acid digestibility and metabolizable energy of corn studied in cecectomized laying hens1.

To optimize the use of corn grain in diets for laying hens, differences in amino acid (AA) digestibility and metabolizable energy among different corn samples should be considered in feed formulation. The present study investigated the variability of AA digestibility and AMEn concentration of 20 corn samples in cecectomized laying hens. Corn grains were characterized based on their physical properties (thousand seed weight, test weight, grain density, and extract viscoelasticity), chemical composition (proximate nutrients, AA, minerals, and inositol phosphates), gross energy concentration, and in vitro solubility of nitrogen to study any relationship with AA digestibility or AMEn. The animal study comprised 4 Latin squares (6 × 6) distributed between 2 subsequent runs. Cecectomized LSL-Classic hens were individually housed in metabolism cages and fed either a basal diet containing 500 g/kg cornstarch or one of 20 corn diets, each replacing the cornstarch with one corn batch, for 8 days. During the last 4 d, feed intake was recorded and excreta were collected quantitatively. A linear regression approach was used to calculate AA digestibility of the corn. The digestibility of all AA differed significantly between the 20 corn batches, including Lys (digestibility range 64 to 85%), Met (86 to 94%), Thr (72 to 89%), and Trp (21 to 88%). The AMEn of the corn batches ranged between 15.7 and 17.1 MJ/kg DM. However, consistent correlations between AA digestibility or AMEn and the physical and chemical characteristics of the grains were not detected. Equations to predict AA digestibility or AMEn based on the grain's physical and chemical characteristics were calculated by multiple linear regressions. The explanatory power (adjusted R2;) of prediction equations was below 0.6 for the majority of AA and AMEn, and, thus, was not sufficiently precise for practical use. Possible explanations for the variation in AA digestibility and AMEn beyond the determined characteristics are discussed. In conclusion, AA digestibility and AMEn of corn grain is high in laying hens, but varies among different corn samples, with physical and chemical characteristics not suitable for explaining these variations.

Separation of inositol phosphate isomers in environmental samples by ion-exchange chromatography coupled with electrospray ionization tandem mass spectrometry.

A method for isomeric separation of inositol phosphates (InsPn) in environmental samples originating from different sources such as soil, manure/compost, and aquatic sediments has been developed. The method includes a single NaOH-EDTA extraction step, centrifugation and direct injection of a particle free solution into an ion chromatographic column. Isomeric liquid chromatographic separation was achieved with an ammonium carbonate gradient compatible with electrospray ionization tandem mass spectrometric detection (LC-ESI-MS/MS). The detection limits of the LC-ESI-MS/MS method were between 0.03-0.16µM for the different InsPn, corresponding to 37-99ng P/g sample DW. The method has shown to be able to analyze more than 200 samples from soil, manure and sediment without any severe matrix effects. This will allow future studies of the fate of inositol phosphates in the environment.

Profile and bioavailability analysis of myo-inositol phosphates in rye bread supplemented with phytases: a study using an in vitro method and Caco-2 monolayers.

Commercial preparations of 6-phytase A alone and in combination with phytase B were used in rye breadmaking. Determination of bioavailability of myo-inositol phosphates from bread was performed by an in vitro digestion method followed by the measurement of an uptake by Caco-2 cells in culture. In bread supplemented with a combination of 6-phytase A and phytase B, a significant reduction in phytate content was observed from 3.62 µmol/g in the control to 0.7 µmol/g. Bioavailability of phytate estimated by an in vitro method simulating digestion in the human alimentary tract was 9% in the bread supplemented with phytase B, 7% (6-phytase A) and 50% in the control bread. In cell culture, the bioaccessibilities of inositol triphosphates from bread baked with the addition of 6-phytase A was higher by 36% as compared to the samples baked with phytase B and by 32% in breads baked with combination of both phytases.

Comparative Metabolomic and Lipidomic Analysis of Phenotype Stratified Prostate Cells.

Prostate cancer (PCa) is the most prevalent cancer amongst men and the second most common cause of cancer related-deaths in the USA. Prostate cancer is a heterogeneous disease ranging from indolent asymptomatic cases to very aggressive life threatening forms. The goal of this study was to identify differentially expressed metabolites and lipids in prostate cells with different tumorigenic phenotypes. We have used mass spectrometry metabolomic profiling, lipidomic profiling, bioinformatic and statistical methods to identify, quantify and characterize differentially regulated molecules in five prostate derived cell lines. We have identified potentially interesting species of different lipid subclasses including phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), glycerophosphoinositols (PIs) and other metabolites that are significantly upregulated in prostate cancer cells derived from distant metastatic sites. Transcriptomic and biochemical analysis of key enzymes that are involved in lipid metabolism demonstrate the significant upregulation of choline kinase alpha in the metastatic cells compared to the non-malignant and non-metastatic cells. This suggests that different de novo lipogenesis and other specific signal transduction pathways are activated in aggressive metastatic cells as compared to normal and non-metastatic cells.

Novel fiber-rich lentil flours as snack-type functional foods: an extrusion cooking effect on bioactive compounds.

Novel snack-type functional foods based on extruded lentil flours could convey the related health benefit of their bioactive compounds, provide a gluten-free alternative to consumers, and potentially increase the consumption of pulses. Extrusion treatment promoted an increase in galactopinitol, ciceritol, raffinose, stachyose and total α-galactoside content, in most lentil flours. As α-galactosides may act as prebiotics, they could convey beneficial effects to human and monogastric animals. Conversely, extrusion significantly (p < 0.05) reduced the inositol hexaphosphate content to less phosphorylated phytates (inositol pentaphosphate and inositol tetraphosphate), which provide health effects. The gluten-free formulation (control formulation #3) presented the highest significant (p < 0.05) drop in the inositol hexaphosphate of 14.7-fold decrease, but had a large increase in inositol pentaphosphate, due to extrusion processing. These two results are desirable in the finished product. Extrusion also caused a significant (p < 0.05) reduction in the trypsin content and completely inactivated lectin, in all processed samples.

Quantitative analysis of ³¹P NMR spectra of soil extracts--dealing with overlap of broad and sharp signals.

Solution (31)P NMR analysis following extraction with a mixture of sodium hydroxide and ethylenediaminetetraacetic acid is the most widely used method for detailed characterization of soil organic P. However, quantitative analysis of the (31)P NMR spectra is complicated by severe spectral overlap in the monoester region. Various deconvolution procedures have been developed for the task, yet none of these are widely accepted or implemented. In this mini-review, we first describe and compare these varying approaches. We then review approaches to similar issues of spectral overlap in biomedical science applications including NMR-based metabolic profiling and analyzing (31)P magnetic resonance spectra of ex vivo and in vivo intact tissues. The greater maturity and resourcing of this biomedical research means that a wider variety of approaches has been developed. Of particular relevance are approaches to dealing with overlap of broad and sharp signals. Although the existence of this problem is still debated in the context of soil analyses, not only is it well-recognized in biomedical applications, but multiple approaches have been developed to deal with it, including T2 editing and time-domain fitting. Perhaps the most transferable concept is the incorporation of 'prior knowledge' in the fitting of spectra. This is well established in biomedical applications but barely touched in soil analyses. We argue that shortcuts to dealing with overlap in the monoester region (31)P NMR soil spectra are likely to be found in the biomedical literature, although some degree of adaptation will be necessary.

Speciation of inositol phosphates in lake sediments by ion-exchange chromatography coupled with mass spectrometry, inductively coupled plasma atomic emission spectroscopy, and 31P NMR spectroscopy.

A method for the detection and speciation of inositol phosphates (InsP(n)) in sediment samples was tested, utilizing oxalate-oxalic acid extraction followed by determination by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) using electrospray ionization (ESI) in negative mode. The chromatographic separation was carried out using water and ammonium bicarbonate as mobile phase in gradient mode. Data acquisition under MS/MS was attained by multiple reaction monitoring. The technique provided a sensitive and selective detection of InsP(n) in sediment samples. Several forms of InsP(n) in the oxalate-oxalic acid extracted sediment were identified. InsP6 was the dominating form constituting 0.250 mg P/g DW (dry weight); InsP5 and InsP4 constituted 0.045 and 0.014 mg P/g DW, respectively. The detection limit of the LC-ESI-MS/MS method was 0.03 µM InsP(n), which is superior to the currently used method for the identification of InsP(n), (31)P nuclear magnetic resonance spectroscopy ((31)P NMR). Additionally sample handling time was significantly reduced.