Ultrasound-assisted hydration of finger millet (Eleusine Coracana) and its effects on starch isolates and antinutrients.

Finger millet (Eleusine Coracana) is rich in nutrients and minerals. The iron and calcium contents are comparatively higher than other cereal crops. Finger millet also has some antinutrients such as tannins and phytates, that needs to be removed for maximum health benefits. Traditionally, these antinutrients are removed by the hydration process. The conventional hydration process is time cumbersome and often results in poor quality grains. Ultrasonication during hydration of finger millet could reduce the processing time and antinutrient content in finger millet. The ultrasound amplitude, treatment time, and grain to water ratio during hydration were optimized. An ultrasound amplitude of 66%, treatment time of 26 min, and a grain to water ratio of 1:3 resulted in best desirability parameters with a reduction in phytate and tannin contents of the finger millet by 66.98 and 62.83%, respectively. Ultrasonication during hydration increased the water binding capacity and solubility of the finger millet starch. XRD study of the starch isolates confirmed the increased crystallinity of the particles. FESEM of the starch isolates also confirmed that ultrasound-assisted hydration of finger millet resulted in the desired size reduction and homogeneous distribution of starch particles. The optimized ultrasound-assisted hydration could be adopted and scaled up for bulk processing of finger millets.

Rapid recovery of phytic acid from rice brans using chitosan nanofiber-based porous hydrogels.

Phytic acid (PA) is a new type of naturally occurring pharmaceutical for afflictions such as cancer, diabetes, and renal calculi. The efficient, low-cost extraction of PA from biowaste is much sought after. Herein, highly pure PA was obtained from rice bran by adsorption at low pH onto porous chitosan nanofiber hydrogels. Due to the large surface area of the chitosan nanofiber-based porous hydrogels, the adsorption equilibrated within 60 min. Adsorption of PA was influenced by the buffer pH, temperature, and the ratio of chitosan in the hydrogel. PA was recovered by soaking the hydrogel in alkaline solution. After concentrating the solution and washing the residue with ethanol, highly pure sodium phytate was obtained with 32.2%-38.7% yield, as confirmed by Fourier transform infrared and high-performance liquid chromatography. To our knowledge, this is the first report on the recovery of pure PA in high yield without using toxic solvents.

Harvesting of Arthrospira platensis by flocculation with phytic acid from rice bran.

The recovery of algal biomass is one of the critical steps involved in the commercial production of beneficial metabolites from Arthrospira platensis. Efficient and safe harvesting methods that do not sacrifice quality of final product are important for commercial application. Phytic acid (PA) is a natural non-toxic phytochemical widely distributed in plant tissues. Effect of PA from rice bran on the growth, trichome morphology such as spiral number and algal filament length, and harvesting efficiency of A. platensis were investigated. Cells aggregated into large cell flocs after the addition of PA in the medium, and algal spiral number and filament length increased. UV-vis spectra indicated the interactions between PA and algal cells. Adding PA at stationary growth phase is a good strategy for harvesting, since no adverse effect to biomass growth and harvesting efficiency. Harvesting efficiency of 95.69% at 0.5% (v/v) PA was superior to other conventional harvesting methodologies. ABBREVIATIONS: PA: Phytic acid; PUFAs: Polyunsaturated fatty acids; FAO: Food and Agriculture Organization; γ-PGA: Poly (γ-glutamic acid); CNF: Cellulose nanofibrils; NIES: National Institute for Environmental Studies; SOT: Spirulina-Ogawa-Terui; CG: Control group; pI: Isoelectric point.

Isolation of Inositol Hexakisphosphate from Soils by Alkaline Extraction and Hypobromite Oxidation.

Inositol hexakisphosphates are extracted from soil in strong alkali and isolated from other organic phosphates by hypobromite oxidation. The procedure yields the four stereoisomeric forms of inositol hexakisphosphate in a form suitable for spectroscopic or chromatographic identification.

Lab-scale preparation and QC of phytase assay substrate from rice bran.

Phytases are involved in the phosphate acquisition and remobilization in plants, microbes and animals. They have become important technical enzymes in the feed industry and are used to make phosphate, present in animal feed as phytate, available for monogastric animal nutrition. Phytases may also be beneficial to human nutrition because phytate is known to interfere with the uptake of important micronutrients. Accordingly, phytases attract considerable research attention and phytate substrate lacking contaminants that interfere with commonly used phosphate-release assays is essential for this field of science. A procedure to prepare suitable sodium phytate from rice bran is presented. Extracted phytate is precipitated with barium hydroxide and re-dissolved in methanol after washing steps and sulphuric acid treatment. Remaining impurities are precipitated before the dissolved phytate is recovered as the sodium salt by addition of sodium hydroxide. In order to make the substrate widely available for research communities, the procedure relies solely on basic laboratory equipment and materials. Methods for quality control and monitoring of the purified sodium phytate or commercial alternatives are also presented.

Fast determination of bioactive phytic acid and pyrophosphate in walnuts using microwave accelerated extraction.

Bioactive compounds phytic acid (IP6) and pyrophosphate (PPi) are minor components of walnuts with the ability of being inhibitors of urolithiasis, among others. Since simultaneous analysis of IP6 and PPi have known drawbacks, a new method to determine their content in walnuts has been developed with emphasis on their extraction from walnuts by microwave-assisted extraction (MAE). Acid content of extracting solvent, extraction time and temperature were optimized. After extraction, compounds were purified by selective adsorption/desorption on an anion exchange solid phase extraction and analyzed by inductive coupled plasma/mass spectrometry. A mixture of H2SO4 and HCl as solvent to extract both, IP6 and PPi, provided results slightly higher than those determined by conventional extraction with no statistical difference. The possible hydrolysis of phytic acid by MAE was analyzed. Compared with the conventional acid extraction method, significant improvement is achieved by the MAE method reducing extraction time from 3h to 10min.

Increase of protein extraction yield from rapeseed meal through a pretreatment with phytase.

BACKGROUND: Rapeseed meal is a good source of high-quality vegetal protein but contains antinutritional compounds that limit its use for human and animal feed. The aim of this study was to develop a methodology to enhance alkaline protein extraction of rapeseed meal and to produce protein-rich products with low levels of phytic acid. Different phytase dosages and operating conditions were used for rapeseed meal pretreatment followed by alkaline extraction at different temperatures, time, pH and solid/liquid ratios (S/L). RESULTS: The highest protein extraction yield attained was 72.1%, for 2 h at 55 °C, with a phytase dosage of 0.8 U g-1 when the alkaline extraction was performed at 75 °C, pH 12.5 and 60 min for an S/L ratio of 10 g 100 mL-1 water. The extraction yields were higher than those previously obtained without enzymatic pretreatment. CONCLUSION: Phytase pretreatment enhanced alkaline extraction yield of proteins from rapeseed meal. This procedure allowed also the production of rapeseed protein concentrates with very low levels of phytic acid, ∼1 g kg-1 , improving their nutritional properties and commercial value. Moreover, after the pretreatment, the amount of phytic acid in the remaining rapeseed meal decreases about 25%. © 2016 Society of Chemical Industry.

Isolated Conglutin γ from Lupin, but not Phytate, Lowers Serum Cholesterol Without Influencing Vascular Lesion Development in the ApoE-deficient Mouse Model.

Conglutin γ and phytate are considered as potential biofunctional compounds of lupin protein isolate, but their impact on vascular health is unknown. This study aimed to investigate the effect of conglutin γ and phytate, respectively, on circulating levels of sterols, markers of cholesterol biosynthesis and minerals, and on the development and progression of aortic lesions in apoE-deficient mice. To this end, mice were fed a western diet with either casein (200 g/kg; served as a control), conglutin γ from L. angustifolius (200 g/kg) or casein (200 g/kg) supplemented with phytate (5 g/kg) for 16 weeks. Here we found that conglutin γ but not phytate was capable of reducing the circulating concentration of cholesterol. Plasma levels of desmosterol and lathosterol as markers of the cholesterol synthesis were not affected, and 7-dehydrocholesterol was even higher in mice fed conglutin γ than in mice fed casein or casein + phytate. All mice developed pronounced aortic lesions, but histological characterization of plaque area and composition showed no differences between the three groups of mice. Conclusively, conglutin γ exerts cholesterol-lowering effects but appears to have no anti-atherosclerotic properties in the apoE-deficient mice. Phytate neither affected plasma cholesterol nor aortic lesion development.

Prevention of osteoporosis by oral administration of phytate-removed and deamidated soybean ß-conglycinin.

Phytate-removed and deamidated soybean ß-conglycinin (PrDS) prepared by ion-exchange resins was supplemented to be 4% in the diet administered to ovariectomized rats to investigate its preventive effect on osteoporosis. The apparent calcium absorption rate decreased following ovariectomy and was not replenished by oral administration of phytate-removed soybean ß-conglycinin (PrS) or casein. On the other hand, administration of PrDS restored the calcium absorption rate to the same level as the sham group. Markers of bone resorption, such as serum parathyroid hormone (PTH) and urinary deoxypyridinoline (DPD), increased, and the bone mineral density and breaking stress decreased following ovariectomy. However, PrDS supplementation suppressed the changes caused by the decrease in calcium absorption from the small intestine. Therefore, PrDS supplementation shows promise for the prevention of postmenopausal osteoporosis.

Mechanism of myo-inositol hexakisphosphate sorption on amorphous aluminum hydroxide: spectroscopic evidence for rapid surface precipitation.

Inositol hexakisphosphates are the most abundant organic phosphates (OPs) in most soils and sediments. Adsorption, desorption, and precipitation reactions at environmental interfaces govern the reactivity, speciation, mobility, and bioavailability of inositol hexakisphosphates in terrestrial and aquatic environments. However, surface complexation and precipitation reactions of inositol hexakisphosphates on soil minerals have not been well understood. Here we investigate the surface complexation-precipitation process and mechanism of myo-inositol hexakisphosphate (IHP, phytate) on amorphous aluminum hydroxide (AAH) using macroscopic sorption experiments and multiple spectroscopic tools. The AAH (16.01 µmol m(-2)) exhibits much higher sorption density than boehmite (0.73 µmol m(-2)) and α-Al2O3 (1.13 µmol m(-2)). Kinetics of IHP sorption and accompanying OH(-) release, as well as zeta potential measurements, indicate that IHP is initially adsorbed on AAH through inner-sphere complexation via ligand exchange, followed by AAH dissolution and ternary complex formation; last, the ternary complexes rapidly transform to surface precipitates and bulk phase analogous to aluminum phytate (Al-IHP). The pH level, reaction time, and initial IHP loading evidently affect the interaction of IHP on AAH. In situ ATR-FTIR and solid-state NMR spectra further demonstrate that IHP sorbs on AAH and transforms to surface precipitates analogous to Al-IHP, consistent with the results of XRD analysis. This study indicates that active metal oxides such as AAH strongly mediate the speciation and behavior of IHP via rapid surface complexation-precipitation reactions, thus controlling the mobility and bioavailability of inositol phosphates in the environment.

Pro-apoptotic effect of rice bran inositol hexaphosphate (IP6) on HT-29 colorectal cancer cells.

Inositol hexaphosphate (IP6), or phytic acid is a natural dietary ingredient and has been described as a "natural cancer fighter", being an essential component of nutritional diets. The marked anti-cancer effect of IP6 has resulted in our quest for an understanding of its mechanism of action. In particular, our data provided strong evidence for the induction of apoptotic cell death, which may be attributable to the up-regulation of Bax and down-regulation of Bcl-xl in favor of apoptosis. In addition, the up-regulation of caspase-3 and -8 expression and activation of both caspases may also contribute to the apoptotic cell death of human colorectal adenocarcinoma HT-29 cells when exposed to IP6. Collectively, this present study has shown that rice bran IP6 induces apoptosis, by regulating the pro- and anti-apoptotic markers; Bax and Bcl-xl and via the activation of caspase molecules (caspase-3 and -8).

Preventive inositol hexaphosphate extracted from rice bran inhibits colorectal cancer through involvement of Wnt/ß-catenin and COX-2 pathways.

Nutritional or dietary factors have drawn attention due to their potential as an effective chemopreventive agent, which is considered a more rational strategy in cancer treatment. This study was designed to evaluate the effect of IP6 extracted from rice bran on azoxymethane- (AOM-) induced colorectal cancer (CRC) in rats. Initially, male Sprague Dawley rats were divided into 5 groups, with 6 rats in each group. The rats received two intraperitoneal (i.p.) injections of AOM in saline (15 mg/kg body weight) over a 2-week period to induce CRC. IP6 was given in three concentrations, 0.2% (w/v), 0.5% (w/v), and 1.0% (w/v), via drinking water for 16 weeks. The deregulation of the Wnt/ß-catenin signaling pathway and the expression of cyclooxygenase (COX)-2 have been implicated in colorectal tumorigenesis. ß-Catenin and COX-2 expressions were analysed using the quantitative RT-PCR and Western blotting. Herein, we reported that the administration of IP6 markedly suppressed the incidence of tumors when compared to the control. Interestingly, the administration of IP6 had also markedly decreased ß-catenin and COX-2 in colon tumors. Thus, the downregulation of ß-catenin and COX-2 could play a role in inhibiting the CRC development induced by IP6 and thereby act as a potent anticancer agent.

Quantifying phytate in dairy digesta and feces: alkaline extraction and high-performance ion chromatography.

Development of an analytical method with appropriate combination of extraction and quantification approaches for undigested phytate in ruminant feces and digesta will advance knowledge of phytate degradation in ruminants and help to reduce phosphorus excretion. Established quantification methods give satisfactory results for feedstuffs and nonruminant manure but recovery of phytate is incomplete for ruminant feces and digesta because of their complex sample matrix and low ratio of phytate to inorganic P. The objective was to develop a robust, accurate, sensitive, and inexpensive method to extract and quantify phytate in feeds, ruminant feces, and digesta. Diets varying in phytate content were fed to dairy heifers, dry cows, and lactating cows to generate digesta and fecal samples of varying composition to challenge extraction and quantification methods. Samples were extracted with 0.5 M HCl or 0.25 M NaOH + 0.05 M EDTA. Acid extracts were mixed with 20% NaCl, alkaline extracts were acidified to final pH < 2, and then both extracts were clarified with C18 cartridges and 0.2-µm filters. High-performance ion chromatography (HPIC) was used to quantify phytate. In feed samples, the measured phytate was comparable in alkaline and acid extracts (2,965 vs. 3,085 µg/g of DM). In digesta and fecal samples, alkaline extraction yielded greater estimates of phytate content than did acid extraction (40.7 vs. 33.6 and 202.9 vs. 144.4 µg/g of DM for digesta and fecal samples, respectively). Analysis of alkaline extracts by HPIC is usually not possible because of sample matrix interferences; acidification and C(18)-cartridge elution of alkaline extracts prevented this interference. Pure phytate added to dry samples before extraction was almost completely recovered (88 to 105%), indicating high extraction efficiency, no adverse effect of extract clean-up procedures, and accurate quantification of phytate. The proposed method is rapid, inexpensive, robust, and combines the extraction power of NaOH-EDTA with the precision and sensitivity of HPIC quantification, allowing accurate quantification of phytate in feeds, ruminant digesta, and fecal samples.

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.

An investigation of the mode of sorption of inositol hexaphosphate to goethite.

Adsorption of inositol hexaphosphate (IP(6)) on goethite has been studied as a function of pH and concentration, and by use of Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR). While adsorption was highest at low pH, a significant amount remained adsorbed above pH 10 where, in the absence of IP(6), the surface is expected to have a net negative charge. The adsorption isotherm at pH 5.5 indicated strong binding to the surface with each adsorbed species occupying about 2.5 nm(2). ATR-FTIR spectra of IP(6) solutions in the pH range from 2 to 12 were fitted with a single set of IR bands which were assigned primarily by analogy with phosphate spectra. From its variation in intensity with pH the band at 1040 cm(-1) was assigned to the effect of hydrogen bonding on the PO vibration. No additional bands were required to fit the spectra of IP(6) adsorbed to goethite, indicating that adsorption occurs by outer-sphere complexation in this system. At all pH values studied the band associated with hydrogen bonding was more intense for the adsorbed species than in solution at the corresponding pH indicating that hydrogen bonding plays an important role in binding IP(6) to goethite.

Myo-inositol hexakisphosphate, isolated from female gametophyte tissue of loblolly pine, inhibits growth of early-stage somatic embryos.

• Myo-inositol hexakisphosphate (InsP(6)), abundant in animals and plants, is well known for its anticancer activity. However, many aspects of InsP(6) function in plants remain undefined. We now report the first evidence that InsP(6) can inhibit cellular proliferation in plants under growth conditions where phosphorus is not limited. • A highly anionic molecule inhibitory to early-stage somatic embryo growth of loblolly pine (LP) was purified chromatographically from late-stage LP female gametophytes (FGs), and then characterized structurally using mass spectrometry (MS) and nuclear magnetic resonance (NMR) analyses. • Exact mass and mass spectrometry-mass spectrometry (MS-MS) fragmentation identified the bioactive molecule as an inositol hexakisphosphate. It was then identified as the myo-isomer (i.e. InsP(6)) on the basis of (1)H-, (31)P- and (13)C-NMR, (1)H-(1)H correlation spectroscopy (COSY), (1)H-(31)P heteronuclear single quantum correlation (HSQC) and (1)H-(13)C HSQC. Topical application of InsP(6) to early-stage somatic embryos indeed inhibits embryonic growth. • Recently evidence has begun to emerge that InsP(6) may also play a regulatory role in plant cells. We anticipate that our findings will help to stimulate additional investigations aimed at elucidating the roles of inositol phosphates in cellular growth and development in plants.

Experimental assessment of toxic phytochemicals in Jatropha curcas: oil, cake, bio-diesel and glycerol.

BACKGROUND: Jatropha curcas seed is a rich source of oil; however, it can not be utilised for nutritional purposes due to presence of toxic and anti-nutritive compounds. The main objective of the present study was to quantify the toxic phytochemicals present in Indian J. curcas (oil, cake, bio-diesel and glycerol). RESULTS: The amount of phorbol esters is greater in solvent extracted oil (2.8 g kg⁻¹) than in expeller oil (2.1 g kg⁻¹). Liquid chromatography-mass spectroscopy analysis of the purified compound from an active extract of oil confirmed the presence of phorbol esters. Similarly, the phorbol esters content is greater in solvent extracted cake (1.1 g kg⁻¹) than in cake after being expelled (0.8 g kg⁻¹). The phytate and trypsin inhibitory activity of the cake was found to be 98 g kg⁻¹ and 8347 TIU g⁻¹ of cake, respectively. Identification of curcin was achieved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the concentration of curcin was 0.95 g L⁻¹ of crude concentrate obtained from cake. CONCLUSION: Higher amounts of phorbol esters are present in oil than cake but bio-diesel and glycerol are free of phorbol esters. The other anti-nutritional components such as trypsin inhibitors, phytates and curcin are present in cake, so the cake should be detoxified before being used for animal feed.

Isolation of phytate from Jatropha curcas kernel meal and effects of isolated phytate on growth, digestive physiology and metabolic changes in Nile tilapia (Oreochromis niloticus L.).

Jatropha curcas seeds are rich in oil and protein. The oil is used for biodiesel production. The defatted Jatropha kernel meal obtained after oil extraction is rich in protein (58-66%) and phytate (9-11%). The phytate rich fraction was isolated from defatted kernel meal using organic solvents (acetone and carbon tetracholride). It had 66% phytate and 22% crude protein. The fingerlings (n=50, 16.2 ± 0.64 g) were randomly distributed into five groups containing 10 replicates and fed iso-nitrogenous diets (crude protein 36%): control diet containing casein and gelatin as proteins; control diet containing 1.5% and 3% Jatropha phytate (PWP(1.5) and PWP(3), respectively); and control diet containing 1.5% and 3% Jatropha phytate supplemented with phytase (1500 FTU/kg) (PWP(1.5+Phytase) and PWP(3+Phytase), respectively). Significantly lower (P<0.05) growth and feed utilization in PWP(1.5) and PWP(3) groups than for control and both phytase containing groups were observed; whereas feed gain ratio exhibited opposite trend. Protein and lipid digestibilities of the diets, amylase and protease enzyme activities in the intestine were significantly higher (P<0.05) in PWP(1.5+Phytase) and PWP(3+Phytase) groups than for PWP(1.5) and PWP(3) groups. Lowest red blood cell counts, and hemoglobin and hematocrit concentrations were observed in PWP(3) group which were not statistically different to those for PWP(1.5) group, but were significantly (P<0.05) lower than those for all other groups. Highest albumin, globulin and total protein concentrations were observed in PP(3+Phytase) group and lowest in PWP(1.5) group; and values for the latter were statistically similar to those for control group. Calcium, phosphorus and glucose concentrations in blood and cholesterol concentration in plasma were significantly lower (P<0.05) in the phytate enriched groups compared with control and phytase treated groups (PP(1.5+Phytase) and PP(3+Phytase)). Higher (P<0.05) alkaline phosphatase activity was observed in phytase supplemented groups compared with that in non-supplemented groups which (PP(1.5+Phytase)) was statistically similar to that in control group, whereas alanine transaminase activity in blood exhibited opposite trend. In conclusion, Jatropha phytate present in DJKM is an antinutrient and addition of phytase in the diet containing DJKM is recommended.

A femtomole-sensitivity mass assay for inositol hexakisphosphate.

Inositol hexakisphosphate (InsP(6)) is an important component of cells, and its mass levels are usually assayed by either (a) equilibrium labelling of cell cultures with radiolabelled inositol or (b) by a variety of mass assays of differing sensitivities and ambiguities. Here, we describe a mass assay for InsP(6) that is based on phosphorylating InsP(6) with [(32)P]-ATP to 5-(PP)InsP(5) using a recombinant Giardia InsP(6) kinase and quantification of the radiolabelled 5-[(32)P](PP)InsP(5) product by anion exchange HPLC with an internal [(3)H]-(PP)InsP(5) standard. Interference with the enzyme reaction by other factors in the tissue extract is corrected for by assay of identical aliquots of tissue spiked with known amounts of InsP(6). This assay only measures InsP(6) (and not other inositol phosphates), and although it is simple in principle and requires no dedicated or specialised equipment, it is quite time-consuming. But the assay is unambiguous and is capable of quantifying accurately as little as 10 fmol of InsP(6) in a cell extract.

Synthesis and nonradioactive micro-analysis of diphosphoinositol phosphates by HPLC with postcolumn complexometry.

A nonradioactive high-performance anion-exchange chromatographic method based on MDD-HPLC (Mayr Biochem. J. 254:585-591, 1988) was developed for the separation of inositol hexakisphosphate (InsP(6), phytic acid) and most isomers of pyrophosphorylated inositol phosphates, such as diphosphoinositol pentakisphosphate (PPInsP(5) or InsP(7)) and bis-diphosphoinositol tetrakisphosphate (bisPPInsP(4) or InsP(8)). With an acidic elution, the anion-exchange separation led to the resolution of four separable PPInsP(5) isomers (including pairs of enantiomers) into three peaks and of nine separable bisPPInsP(4) isomers into nine peaks. The whole separation procedure was completed within 20-36 min after optimization. Reference standards of all bisPPInsP(4) isomers were generated by a nonenzymatic shotgun synthesis from InsP(6). Hereby, the phosphorylation was brought about nonenzymatically when concentrated InsP(6) bound to the solid surface of anion-exchange beads was incubated with creatine phosphate under optimal pH conditions. From the mixture of pyrophosphorylated InsP(6) derivatives containing all theoretically possible isomers of PPInsP(5), bisPPInsP(4), and also some isomers of trisPPInsP(3), isomers were separated by anion-exchange chromatography and fractions served as reference standards of bisPPInsP(4) isomers for further investigation. Their isomeric nature could be partly assigned by comparison with position specifically synthesized or NMR-characterized purified protozoan reference compounds and partly by limited hydrolysis to PPInsP(5) isomers. By applying this nonradioactive analysis technique to cellular studies, the isomeric nature of the major bisPPInsP(4) in mammalian cells could be identified without the need to obtain sufficient material for NMR analysis.