The separation of [ 32P] inositol phosphates by ion-pair chromatography: Optimization of the method and biological applications

Abstract

We have developed an ion-pair reverse-phase HPLC method to measure inositol phosphates in 32P-labeled cells. The different chromatographic parameters were analyzed to optimize the resolution of the 32P-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)P 3. 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 [ 3H] inositol labeling: (i) 32P labeling is less expensive and more efficient than 3H 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.