HISTOCHEMICAL DEMONSTRATION OF PHOSPHOLIPASE B (LYSOLECITHINASE) ACTIVITY IN RAT TISSUES

Abstract

A method has been developed for the histochemical demonstration of phospholipase B (lysolecithinase) of rat tissues. The enzyme attacks lysolecithin with liberation of 1 mole of glycerylphosphorylcholine and 1 mole of fatty acid. The recommended procedure involves use of 6-10 µ frozen sections, fixed in cold calcium-formol and incubated at 37°C in Tris buffered medium at pH 6.6 containing 2.2 x 10–3 M lysolecithin and 1% cobalt acetate. The fatty acid liberated by enzymatic hydrolysis is trapped as a cobalt precipitate and is then converted to a blackbrown precipitate by treatment with dilute ammonium sulfide in cold isotonic saline. Equivalent amounts of fatty acid and glycerylphosphorylcholine are recovered by extraction and analysis of the incubated sections and of the incubation medium, thus proving that lysolecithin hydrolysis occurs under the proposed reaction conditions. Staining is reduced by treating the sections with copper ions, mercury compounds, alcohols, acetone and by heating at 60°C prior to incubation with substrate. Lowering of the pH of the incubation medium has similar effect. These findings are interpreted as evidence of the enzymatic nature of the reaction. Cells exhibiting a positive staining are found in the lamina propria of the intestinal villi and crypts, in the red pulp of the spleen and in the interstitial tissue of lung, liver and thymus. Similar elements are present in bone marrow smears and in leukocyte preparations obtained by peritoneal lavage. The morphologic and staining characteristics of these cells correspond to those of the eosinophilic leukocytes. Physical and chemical agents (x-irradiation corticosteroids) which sharply decrease the number of eosinophils also reduce the number of cells shown histochemically to hydrolyze lysolecithin. A correspondent. diminution of phospholipase B activity of homogenates of the same tissues can be shown in vitro. Differences in tissue distribution and chemical properties distinguish the phospholipase B from less specific esterases and lipases.