CRITIQUE ON THE K-PYROANTIMONATE METHOD FOR SEMIQUANTITATIVE ESTIMATION OF CATIONS IN CONJUNCTION WITH ELECTRON MICROSCOPY

The histochemical method employing potassium pyroantimonate in conjunction with electron microscopy has been investigated using carefully controlled preparation techniques and very sensitive atomic absorption analysis of cations. A critique on the reliability and limitations of the method based on test tube and in vitro experiments is given. The method is sensitive to Ca ++ , Mg ++ and Na + at the 10 –6 , 10 –5 and <10 –2 M levels, respectively. Under defined conditions a linear ~l:l ratio of cation present to cation precipitated occurs above these levels. Approximate solubility products have been estimated. Under the test conditions, K + does not precipitate as a pyroantimonate salt, and neither K + nor OsO 4 influences cation precipitaton at physiologic concentrations. Unbuffered, Tris-HCl-buffered and weakly buffered NaHCO 3 media at pH 7.2-7.8 give statistically similar results with Na + precipitation. The pyroantimonate ion can compete with chelators, ethylenedinitrilotetraacetic acid and ethylene glycol bis-N, N'-tetraacetic acid, for divalent cations when employed simultaneously. These chelators effectively remove Ca ++ but not Mg ++ from embryonic myocardium, and their effects on Na + and K + balance are not marked if employed for relatively short periods. Electron micrographic examples of cation precipitates are given in support of certain findings. A brief discussion of the significance of pyroantimonate grain size, the discrepancy between the ratio of intra- and extracellular precipitates and guidelines for the use of the method are included.