Quantitative analysis of inositol lipids and inositol phosphates in synaptosomes and microvessels by column chromatography: comparison of the mass analysis and the radiolabelling methods

Chromatographic methods that measure both the mass and the radiolabelling of various inositol lipids and inositol phosphates in tissues have been developed. The mass of phosphatidylinositol (PtdIns), phosphatidylinositol-4-monophosphate [PtdIns(4)P] and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂] was quantitated by measuring the inorganic phosphate, whereas inositol monophosphate (IP), inositol bisphosphate (IP₂), inositol trisphosphate (IP₃) and inositol tetrakisphosphate (IP₄) were quantitated by using an enzymic method. The radiolabelling of various inositol lipids and inositol phosphates was determined by incubating the tissue samples with [³H]myo-inositol, separating individual inositol lipids and inositol phosphates, and measuring the radioactivity in each compound. Although the mass analysis method was sensitive enough to measure low levels of inositol lipids or inositol phosphates, the method was laborious and time-consuming. Compared with the enzymic method, the radiolabelling method was simple and fast, but it gave variable results. This study demonstrated differences in inositol lipid and inositol phosphate levels by radiolabelling and mass measurements, and agonist-stimulated phosphatidylinositol turnover of synaptosomes versus the blood-brain barrier as represented by microvessels. Although the mass of PtdIns, PtdIns(4)P and PtdIns(4,5)P₂ was comparable in synaptomsomes and microvessels, the incorporation of [³H]myo-inositol into phosphorylated PtdIns in microvessels was less than that in synaptosomes.