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A straightforward method for analysis of highly phosphorylated inositols in blood cells by high-performance liquid chromatography.

Soluble inositol polyphosphates are found in many cells. The trisphosphate isomers, mainly inositol-1,4,5-trisphosphate, have been extensively studied because of their involvement in signal transduction. However, higher phosphorylated inositols are less frequently studied and their physiological role is poorly understood. Among these, only the myo-inositol-1,3,4,5,6-pentakisphosphate (Ins1,3,4,5,6P5), an important component of bird erythrocytes, has been intensively studied in comparative studies because it is a potent allosteric effector of hemoglobin and decreases its affinity to oxygen. We have developed a procedure for the analysis of inositol polyphosphates and other phosphate compounds in vertebrate blood cells based on a quick and accurate HPLC separation coupled to metal-dye detection. The procedure includes acid extraction of cellular phosphates, acid elimination and concentration of the extract, HPLC separation of phosphate compounds, and quantification by coupled highly sensitive metal-dye detection. The method is especially useful for analyses of highly phosphorylated inositols and for red cell comparative studies. Using the described method we have quantified Ins1,3,4,5,6P5 and the low quantities of InsP6 found in bird erythrocytes. We also identified traces of Ins3,4,5,6P4 and Ins1,3,4,6P4. Moreover, by applying the method in cultured murine macrophages, we have found changes of highly phosphorylated inositols when these cells are activated by lipopolysaccharide.