A critical study of techniques for determining the cytological position of alkaline phosphatase.

The equilibrium lies well over on the side of free phosphate. Studies of the histochemical localization of alkaline phosphatase were made by Robison (1923), who showed that if a section of bone is incubated in a solution containing a phosphoric acid ester and calcium, the phosphate which is split off from the ester by phosphatase is precipitated as calcium phosphate in the vicinity of the enzyme. Considerable advances in our understanding of the mechanism of bone formation were made with the use of this technique. But it is not until comparatively recently that the technique has been developed to provide a close description of the histological distribution of phosphatase, or of its cytological distribution. Takamatsu (1939) and Gdmorri (1939) published, almost simultaneously, descriptions of a method for determining the localization of alkaline phosphatase in sections prepared by the usual histological methods. In their procedure a piece of tissue is fixed with 80 % alcohol, dehydrated with alcohol, cleared with benzene or cedarwood oil, and embedded in wax at a temperature not exceeding 6o° C. Sections are cut from this block with a microtome, mounted in the usual manner on microscope slides, the wax removed with xylol, after which the sections are taken down through alcohol-water mixtures into distilled water. These sections are incubated at 37 C. in a solution containing glycerophosphate and calciuml, at pH 9-3. Calcium phosphate is deposited in the sections as a result of the enzymatic splitting of glycerophosphate. The sections are treated with cobalt nitrate, washed with distilled water, treated with ammonium sulphide, washed in tap water and mounted in Canada balsam. Cobalt phosphate is less soluble than is calcium phosphate; consequently the treatment of a section with cobalt solution changes any calcium phosphate present to cobalt phosphate. Also, cobalt sulphide is less soluble than cobalt phosphate; consequently the treatment of a section with ammonium sulphide converts the cobalt phosphate into cobalt sulphide. Cobalt sulphide is black; consequently a black stain appears in the sections in the positions where initially calcium phosphate had been precipitated following enzymatic hydrolysis of glycerophosphate. A typical result is shown in PL 3, fig. 1. In the papers quoted above, and in numerous publications by other authors, it has been claimed that the histological distribution of the precipitated cobalt sulphide corresponds to the distribution of phosphatase in the sections, and that the site of the precipitate is even an exact guide to the cytological distribution of phosphatase. No proof of these claims has hitherto appeared, though in some cases by experiments on enzymatic activity of fragmented tissues, conducted by the ordinary biochemical techniques for estimating, phosphatase activity, it is possible to confirm the qualitative correctness of the results obtained (Fell & Danielli, 1943). If the cytological distribution of phosphatase is in fact revealed by this technique, invaluable information would thereby be made available. But from a cytological point of view no reliance whatever can be placed in the technique until answers can be provided to three questions. These are: