Thermospray mass spectrometry of inositol mono- and polyphosphates, separated by ion-exchange chromatography, was evaluated for its potential as a general method for quantitative analysis of these substances. The only ions of significant abundance that are produced by the thermospray ionization process result from the total loss of phosphate from inositol. Thus inositol mono-, tris- and hexakisphosphate each gave mass spectra consisting solely of [MH]+ and [MNH4]+ of inositol. When the chromatographic eluates are passed through a heated reactor prior to the thermospray source maximal yields of these ions are obtained. The sensitivity of the technique falls short of that needed for a general method for biological applications, because the lower limit of detection is about 100 pmol microliter-1. Inositol phosphates peracetylated on C-hydroxyls were also studied, with separation by ion-exchange chromatography. Again, thermospray ionization produces totally dephosphorylated species, with the highest-mass ions retaining all of the acetyl groups, even when using the thermal reactor. Losses of acetate were also observed. Sensitivity with the acetyl derivative was comparable to that with the underivatized inositol phosphates.

Pretreatment of cryostat sections of dog kidney with sodium salts of various anions in acetate buffer at pH 4.0 for periods of time ranging up to 6 hours produces differential inhibition of non-specific alkaline phosphatase activity as determined by the Gomori method. The degree of anionic effect is dependent on the nature of the anion and modified by the cation present. The effect also varies directly with the anionic concentration, duration of exposure and temperature of the pretreatment buffer and inversely with the pH. On the basis of the differential suppression of enzymatic activity by the various anions, and the conditions under which these effects were evident, distinctive inhibition profiles were established for the several phosphomonoesterases tested, including the renal and intestinal alkaline phosphatases and the smooth muscle 5'-nucleotidases of several species. The similarity of the results obtained in various biochemical studies on other enzymes reported in the literature and those obtained histochemically in these experiments suggests a common mechanism of action; this is presumed to be a differential effect of anions on the dissociation of apoenzyme and coenzyme. The method described provides a simple and effective means of differentiating, under histochemical conditions, closely related and even apparently identical enzymes.

Methods for the permethylation of inositol phosphates (i.e. the formation of the completely substituted C-O-methyl/P-O-methyl derivatives) have been studied as a precursor to preparing C-O-methyl inositols where the remaining inositol hydroxyl groups are at the positions originally occupied by the phosphomonoesters. Classical sodium-driven methylations, diazomethane methylations and methylation with methyl trifluoromethanesulfonate were studied and only the latter was found to produce completely alkylated inositol phosphates. Treatment of the permethylated substrates with methanolic HCl removed the dimethylphosphate groups to produce C-O-methyl inositols which are candidates for negative ion chemical ionization gas chromatographic/mass spectrometric analysis as heptafluorobutyryl C-O-methyl inositols. As an example, gas chromatographic/mass spectrometric analysis of myo-inositol 1,2,6-trisphosphate was carried out by methylation, dephosphorylation and conversion to the tris(heptafluorobutyryl) derivative. Detection at the low-femtomole level was achieved by this means. A limitation of the method may be that the methylation procedure appears to produce a variable degree of phosphate positional isomerization, with resulting loss of specificity. If stable isotope internal standards were available for the inositol polyphosphates of interest, this limitation could be compensated for.