HPLC Separation of Inositol Polyphosphates

  • Christopher J. Barker
  • Christopher Illies
  • Per-Olof Berggren
Part of the Methods in Molecular Biology book series (MIMB, volume 645)


High performance liquid chromatography (HPLC) is an essential analytical tool in the study of the large number of inositol phosphate isomers. This chapter focuses on the separation of inositol polyphosphates from [3H]myo-inositol labeled tissues and cells. We review the different HPLC columns that have been used to separate inositol phosphates and their advantages and disadvantages. We describe important elements of sample preparation for effective separations and give examples of how changing factors, such as pH, can considerably improve the resolving ability of the HPLC chromatogram.

Key words:

High performance liquid chromatography Inositol phosphates Acid extraction HPLC columns HPLC gradients 



The authors would like to thank Prof. R.H. Michell and Dr. C.J. Kirk for permission to use data obtained while C.J.B was a research fellow in their laboratory. The authors own work was supported by grants from Karolinska Institutet, Novo Nordisk Foundation, the Swedish Research Council, the Swedish Diabetes Association, EFSD, The Family Erling-Persson Foundation, Berth von Kantzow’s Foundation, and EuroDia (LSHM-CT-2006-518153).


  1. 1.
    Irvine, R.F., and Schell, M.J. (2001) Back in the water: the return of the inositol phosphates. Nat. Rev. Mol. Cell. Biol. 2, 327–338.CrossRefPubMedGoogle Scholar
  2. 2.
    York, J.D. (2006) Regulation of nuclear processes by inositol polyphosphates. Biochim. Biophys. Acta. 1761, 552–559.PubMedGoogle Scholar
  3. 3.
    Shears, S.B. (2007) Understanding the biological significance of diphosphoinositol polyphosphates (‘inositol pyrophosphates’). Biochem. Soc. Symp. 74, 211–221.CrossRefPubMedGoogle Scholar
  4. 4.
    Berggren, P.O., and Barker C.J. (2008) A key role for phosphorylated inositol compounds in pancreatic beta-cell stimulus-secretion coupling. Adv. Enzyme Regul. 48, 276–294.CrossRefPubMedGoogle Scholar
  5. 5.
    Wong, N.S., Barker, C.J., Morris, A.J, Craxton, A., Kirk, C.J., and Michell R.H. (1992) The inositol phosphates in WRK1 rat mammary tumour cells. Biochem. J. 286, 459–468.PubMedGoogle Scholar
  6. 6.
    Palmer S., Hughes, K.T., Lee, D.Y, and Wakelam, M.J. (1989) Development of a novel, Ins(1,4,5)P3-specific binding assay. Its use to determine the intracellular concentration of Ins(1,4,5)P3 in unstimulated and vasopressin-stimulated rat hepatocytes. Cell. Signal. 1, 147–156.CrossRefPubMedGoogle Scholar
  7. 7.
    Mayr, G.W. (1988) A novel metal-dye detection system permits picomolar-range h.p.l.c. analysis of inositol polyphosphates from non-radioactively labelled cell or tissue specimens. Biochem. J. 254, 585–591.PubMedGoogle Scholar
  8. 8.
    Maccallum, S.H., Barker, C.J., Hunt, P.A., Wong, N.S., Kirk, C.J., and Michell, R.H. (1989) The use of cells doubly labelled with [14C]inositol and [3H]inositol to search for a hormone-sensitive inositol lipid pool with atypically rapid metabolic turnover. J. Endocrinol. 122, 379–389.CrossRefPubMedGoogle Scholar
  9. 9.
    Menniti, F.S., Oliver, K.G., Nogimori, K., Obie, J.F., Shears, S.B., and Putney, J.W. Jr. (1990) Origins of myo-inositol tetrakisphosphates in agonist-stimulated rat pancreatoma cells. Stimulation by bombesin of myo-inositol 1,3,4,5,6-pentakisphosphate breakdown to myo-inositol 3,4,5,6-tetrakisphosphate. J. Biol. Chem. 265, 11167–11176.PubMedGoogle Scholar
  10. 10.
    Barker, C.J., Wright, J., Hughes, P.J., Kirk, C.J., and Michell, R.H. (2004) Complex changes in cellular inositol phosphate complement accompany transit through the cell cycle. Biochem. J. 380, 465–473.CrossRefPubMedGoogle Scholar
  11. 11.
    Barker, C.J., French, P.J., Moore, A.J., Nilsson, T., Berggren, P. O., Bunce, C.M., et al. (1995) Inositol 1,2,3-trisphosphate and inositol 1,2- and/or 2,3-bisphosphate are normal constituents of mammalian cells. Biochem. J. 306, 557–564.PubMedGoogle Scholar
  12. 12.
    Wong, N.S., Barker, C.J., Shears, S.B., Kirk, C.J., and Michell, R.H. (1988) Inositol 1:2(cyclic),4,5-trisphosphate is not a major product of inositol phospholipid metabolism in vasopressin-stimulated WRK1 cells. Biochem. J. 252, 1–5.PubMedGoogle Scholar
  13. 13.
    Sekar, M.C., Dixon, J.F., and Hokin, L.E. (1987) The formation of inositol 1,2-cyclic 4,5-trisphosphate and inositol 1,2-cyclic 4-bisphosphate on stimulation of mouse pancreatic minilobules with carbamylcholine. J. Biol. Chem. 262, 340–344.PubMedGoogle Scholar
  14. 14.
    Stephens, L.R., Hawkins, P.T., Morris, A.J., and Downes, P.C. (1988) L-myo-inositol 1,4,5,6-tetrakisphosphate (3-hydroxy)kinase. Biochem. J. 249, 283–292.PubMedGoogle Scholar
  15. 15.
    Shears S.B. (1998) The versatility of inositol phosphates as cellular signals. Biochim. Biophys. Acta. 1436, 49–67.PubMedGoogle Scholar
  16. 16.
    Irvine, R.F., Anggård, E.E., Letcher, A.J., and Downes, C.P. (1985) Metabolism of inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate in rat parotid glands. Biochem. J. 229, 505–511.PubMedGoogle Scholar
  17. 17.
    Stephens, L.R., Hawkins, P.T., Barker, C.J., and Downes, C.P. (1988) Synthesis of myo-inositol 1,3,4,5,6-pentakisphosphate from inositol phosphates generated by receptor activation. Biochem. J. 253, 721–733.PubMedGoogle Scholar
  18. 18.
    Balla, T., Guillemette, G., Baukal, A.J., and Catt, K.J. (1987) Metabolism of inositol 1,3,4-trisphosphate to a new tetrakisphosphate isomer in angiotensin-stimulated adrenal glomerulosa cells. J. Biol. Chem. 262, 9952–9955.PubMedGoogle Scholar
  19. 19.
    Hughes, P.J., Hughes, A.R., Putney, J.W. Jr., and Shears, S.B. (1989) The regulation of the phosphorylation of inositol 1,3,4-trisphosphate in cell-free preparations and its relevance to the formation of inositol 1,3,4,6-tetrakisphosphate in agonist-stimulated rat parotid acinar cells. J. Biol. Chem. 264, 19871–19878.PubMedGoogle Scholar
  20. 20.
    Stephens, L.R., Hughes, K.T., and Irvine, R.F. (1991) Pathway of phosphatidylinositol(3,4,5)-trisphosphate synthesis in activated neutrophils. Nature 351, 33–39.CrossRefPubMedGoogle Scholar
  21. 21.
    Heslop, J.P., Irvine, R.F., Tashjian, A.H. Jr., and Berridge, M.J. (1985) Inositol tetrakis- and pentakisphosphates in GH4 cells. J. Exp. Biol. 119, 395–401.PubMedGoogle Scholar
  22. 22.
    Yu, J., Leibiger, B., Yang, S.N., Caffery, J.J., Shears, S.B., Leibiger, I.B., Barker, C.J., and Berggren, P.O. (2003) Cytosolic multiple inositol polyphosphate phosphatase in the regulation of cytoplasmic free Ca2+ concentration. J. Biol. Chem. 278, 46210–46218.CrossRefPubMedGoogle Scholar
  23. 23.
    Illies, C., Gromada, J., Fiume, R., Leibiger, B., Yu, J., Juhl, K., et al. (2007) Requirement of inositol pyrophosphates for full exocytotic capacity in pancreatic beta cells. Science 318, 1299–1302.CrossRefPubMedGoogle Scholar
  24. 24.
    Naccarato WF, Ray RE, Wells WW. (1974) Biosynthesis of myo-inositol in rat mammary gland. Isolation and properties of the enzymes. Arch. Biochem. Biophys. 164, 194–201.Google Scholar
  25. 25.
    Allison, J.H., Blisner, M.E., Holland, W.H., Hipps, P.P., and Sherman, W.R. (1976) Increased brain myo-inositol 1-phosphate in lithium-treated rats. Biochem. Biophys. Res. Commun. 71, 664–670.CrossRefPubMedGoogle Scholar
  26. 26.
    Berridge, M.J., Downes, C.P., and Hanley, M.R. (1982) Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands. Biochem. J. 206, 587–595.PubMedGoogle Scholar
  27. 27.
    Singh, J., Hunt, P., Eggo, M.C., Sheppard, M.C., Kirk, C.J., and Michell, R.H. (1996) Thyroid-stimulating hormone rapidly stimulates inositol polyphosphate formation in FRTL-5 thyrocytes without activating phosphoinositidase C. Biochem. J. 316, 175–182.PubMedGoogle Scholar
  28. 28.
    Van Sande, J., Dequanter, D., Lothaire, P., Massart, C., Dumont, J.E., and Erneux, C. (2006) Thyrotropin stimulates the generation of inositol 1,4,5-trisphosphate in human thyroid cells. J. Clin. Endocrinol. Metab. 91, 1099–1107.CrossRefPubMedGoogle Scholar
  29. 29.
    Larsson, O., Barker, C.J., Sjöholm, A., Carlqvist, H., Michell, R.H., Bertorello, A., et al. (1997) Inhibition of phosphatases and increased Ca2+ channel activity by inositol hexakisphosphate Science 278, 471–474.Google Scholar
  30. 30.
    Stephens, L.R., Hawkins, P.T., Stanley, A.F., Moore, T., Poyner, D.R., Morris, P.J, et al. (1991) myo-inositol pentakisphosphates. Structure, biological occurrence and phosphorylation to myo-inositol hexakisphosphate. Biochem. J. 275, 485–499.PubMedGoogle Scholar
  31. 31.
    Menniti, F.S., Miller, R.N., Putney, J.W. Jr., and Shears, S.B. (1993) Turnover of inositol polyphosphate pyrophosphates in pancreatoma cells. J. Biol. Chem. 268, 3850–3856.PubMedGoogle Scholar
  32. 32.
    Stephens, L.R., Berrie, C.P., Irvine, R.F. (1990) Agonist-stimulated inositol phosphate metabolism in avian erythrocytes. Biochem. J. 269, 65–72.PubMedGoogle Scholar
  33. 33.
    Stephens, L., Radenberg, T., Thiel, U., Vogel, G., Khoo, K.H., Dell, A. et al. (1993) The detection, purification, structural characterization, and metabolism of diphosphoinositol pentakisphosphate(s) and bisdiphosphoinositol tetrakisphosphate(s). J. Biol. Chem. 268, 4009–4015.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Christopher J. Barker
    • 1
  • Christopher Illies
    • 1
  • Per-Olof Berggren
    • 1
  1. 1.The Rolf Luft Research Center for Diabetes and EndocrinologyKarolinska InstitutetStockholmSweden

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