The separation of [32P]inositol phosphates by ion-pair chromatography: optimization of the method and biological applications.
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)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 [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.
Physiologie de la Nutrition, CNRS UA 646, Orsay, France.
J C
JC
Sulpice
P
P
Gascard
E
E
Journet
F
F
Rendu
D
D
Renard
J
J
Poggioli
F
F
Giraud
D001792:Blood Platelets / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004912:Erythrocytes / Q000378:metabolism; D005635:Fructosediphosphates / Q000302:isolation & purification; D005958:Glucosephosphates / Q000302:isolation & purification; D006801:Humans; D066298:In Vitro Techniques; D007295:Inositol Phosphates / Q000302:isolation & purification*; D008099:Liver / Q000166:cytology; D009711:Nucleotides / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D013403:Sugar Phosphates / Q000302:isolation & purification*; D013997:Time Factors; D014667:Vasopressins / Q000494:pharmacology
D005635:Fructosediphosphates; D005958:Glucosephosphates; D007295:Inositol Phosphates; D009711:Nucleotides; D010761:Phosphorus Radioisotopes; D013403:Sugar Phosphates; D014667:Vasopressins