Saponification

SAPONIFICATION in the strict sense of the word means the decomposition of fats by an alkali with the formation of glycerin and soaps, but this term is used to denote in general any kind of hydrolysis of esters, and frequently other hydrolysis reactions of organic compounds as well, for example: the conversion of nitriles into acids, the decomposition of acid amides into an acid and ammonia, the decomposition of acetals into an alcohol and an aldehyde. Saponification can be brought about by heating with water (more easily by the action of superheated steam), heating with an acid, heating with an alkali, or by the action of sodium alcoholate. The reaction of the saponification of an ester into an acid and an alcohol is reversible: CH₃.CO.O.C₂H₅ + H₂O ⇌ CH₃.CO.OH + C₂H₅.OH. If, however, saponification is carried out by the action of an alkali or alcoholate, it goes to completion, because the acid is obtained in the form of a salt and leaves the reaction sphere. Similarly to the cited reaction of the saponification of ethyl acetate, the saponification of fats also proceeds; for example, triolein breaks down into oleic acid and glycerin: C₃H₅(O.CO.C₁₇H₃₃)₃ + 3H₂O = C₃H₅(OH)₃ + 3C₁₇H₃₃.CO.OH. Saponification proceeds stepwise, passing through the stages of formation of diolein and monolein. Such saponification also occurs under the action of lipases (see). (See also Digestion.) Saponification in histological practice. During conventional fixation of tissue in formalin, the dissolution of potassium and sodium salts of fatty acids occurs, as a result of which these types of soaps disappear under given conditions for observation; only calcium soaps are not subject to dissolution in formalin. In order to observe all types of soaps histologically, i.e., completed saponification as a whole, one strives during fixation to convert all soaps into insoluble fatty-acid calcium. For this purpose, fixation of pieces of the studied tissue is performed according to Fischler in 10% formalin saturated with calcium salicylate. Sections obtained on a freezing microtome are subjected to mordanting in a concentrated solution of copper acetate for 2-24 hours in a thermostat; after washing the sections in distilled water, they are stained with hematoxylin and then differentiated according to Weigert's method for myelin fibers (see Weigert staining methods) with the difference that the sections are kept in hematoxylin for a shorter time (not less than 20 minutes) and differentiated until the red blood cells are decolorized. Soaps with this method are stained blue-black. According to Fischler's instructions, simultaneously with processing a part of the material by the method just described, another part of the same material must be subjected to the same processing, but with fixation in formalin without the addition of calcium salicylate, which gives a blue-black coloration only to fatty acids; a comparison between sections processed by these two methods makes it possible to judge the amount of fatty acids and soaps in the tissue (see Lipoids).