Quantification of Inositol and Phosphate

Inositol derived by dephosphorylation of purified inositol phosphates may be quantified. Phosphates are removed from the inositol ring, either by acid hydrolysis or by treatment with alkaline phosphatase, and the free inositol is determined by enzymatic conversion to wyo-inose with myo-inositol dehydrogenase and NAD+. The NADH thus formed is oxidized back to NAD+ with oxoloacetate to form malate, which is measured fluorometrically. This coupled reaction is stoichiometric and is sufficiently sensitive to assay 0.2 to 8 nmol of inositol per sample (Dean and Beaven, 1989 Palmer and Wakelam, 1989). 

The Pj liberated by acid or enzymatic hydrolysis of the inositol phosphates may also be assayed. An anion-exchange HPLC column, which separates the inositol phosphates, is coupled to a second column that contains immobilized alkaline phosphatase. The Pj that is released by the enzyme is measured color-imetrically. The system can detect 1 nmol of inositol phosphates in a single sample to indicate levels of IP3 of 13 to 40 nmol/g of tissue. The sensitivity of the phosphate assay is increased to the picomolar level with malachite green as a reagent (Dean and Beaven, 1989 Palmer and Wakelam, 1989). 

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