The separation of ( sup 32 P)inositol phosphates by ion-pair chromatography: Optimization of the method and biological applications

Journal Article · Mon May 15 00:00:00 EDT 1989 · Analytical Biochemistry; (USA)

DOI:https://doi.org/10.1016/0003-2697(89)90206-6· OSTI ID:5673421

Sulpice, J C; Gascard, P; Journet, E; Rendu, F; Renard, D; Poggioli, J; Giraud, F ^[1]

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1. CNRS UA 646, Orsay (France)

We have developed an ion-pair reverse-phase HPLC method to measure inositol phosphates in {sup 32}P-labeled cells. The different chromatographic parameters were analyzed to optimize the resolution of the {sup 32}P-labeled metabolites. Analysis of inositol phosphates in biological samples was improved by a single charcoal pretreatment which eliminated interfering nucleotides without removing inositol phosphates. The kinetics of production of inositol phosphates in calcium-activated erythrocytes, vasopressin-stimulated hepatocytes, and thrombin-activated platelets were analyzed. Original data on the activation of phosphoinositide phospholipase C were obtained in intact erythrocytes by direct measurement of inositol (1,4,5)P3. Data from agonist-stimulated hepatocytes and platelets were consistent with those from previous studies. In conclusion, this technique offers many advantages over the methodologies currently employed involving anion-exchange chromatography and ({sup 3}H)inositol labeling: (i) {sup 32}P labeling is less expensive and more efficient than {sup 3}H labeling and can be used with all types of cells without permeabilization treatments and (ii) ion-pair HPLC gives good resolution of inositol phosphates from nucleotides with shorter retention times, and long reequilibration periods are not required.

• OSTI ID: 5673421
• Journal Information: Analytical Biochemistry; (USA), Vol. 179:1; ISSN 0003-2697
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
PHOSPHOLIPIDS
LIQUID COLUMN CHROMATOGRAPHY
BIOSYNTHESIS
BLOOD PLATELETS
ERYTHROCYTES
IN VITRO
ION EXCHANGE CHROMATOGRAPHY
LIVER
NUCLEOTIDES
PHOSPHORUS 32
TRACER TECHNIQUES
VASOPRESSIN
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY
BODY FLUIDS
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
ESTERS
GLANDS
HORMONES
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
LIPIDS
MATERIALS
NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
ORGANS
PEPTIDE HORMONES
PHOSPHORUS ISOTOPES
PITUITARY HORMONES
RADIOISOTOPES
SEPARATION PROCESSES
SYNTHESIS
550201* - Biochemistry- Tracer Techniques