Analysis of Inositol Phosphates and Phospholipids

The inositol ring contains six hydroxyl residues, with positions 1,4, and 5 phosphorylated in IP3 (Fig. 1). A scheme for the metabolism of phosphoinositides is shown on Fig. 2 (Dean and Beaven, 1989). 

In case of smooth muscle tissue cultures, LiCl (1-10 mM) is added to the medium just prior to the addition 

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FIGURE 1 D-myo-lnositol (planar and chair configuration) and D-myo-inositol 1,4,5-trisphosphate [I(1,4,5)P3]. The arrow indicates the site of the ester link with diacylglycerol in phosphatidylinositol. The negative charge of the phosphate groups is not indicated. From Dean and Beaven (1989, Fig. 1, p. 200). 

The frozen muscle is pulverized, and the powder is extracted with a trichloroacetic acid (TCA) or perchloric acid (PCA) solution. After removal of the precipitated proteins, the supernatant extracts are freed of excess acid. TCA is removed by extraction with diethyl ether, and PCA is removed by precipitation with KOH at neutral pH. The inositol phosphates in the watery extract are separated by chromatography. 

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