A colorimetric method for the standardization of heparin preparations.

IT is a fact well known to histologists that certain basic dyes undergo a change of tint when taken up by particular tissue elements, especially cartilage, mucus and the granules of the so-called 'mast cells'. The chemistry of this phenomenon, named 'metachromasia' by Ehrlich, has been studied in some detail by Lison [1935], This investigator concluded that the change of colour in the dye is due to the formation of the tautomeric imino-base, accompanied by an increased degree of molecular aggregation; and that all the substances which show the metachromatic staining contain organically combined sulphuric acid. Any sulphuric acid ester, in fact, provided that its molecular weight is not too low, will produce the characteristic colour change when added to an aqueous solution of a metachromatic dye. Now most of the substances known to be active in delaying the clotting of blood have large molecules containing esterified sulphuric acid; and, conversely, most substances having this structure appear to be anticoagulants [cf. Huggett & Rowe, 1933; Bergstrom, 1936; Chargaff et al. 1936]. Thus anticoagulant activity, and the capacity for giving the metachromatic reaction, are related to the same type of chemical structure. The effectiveness of any substance in producing the colour change appears, indeed, as will be shown, to be roughly proportional to its anticoagulant potency. Heparin co7ntains esterified sulphuric acid [Jorpes, 1935], and gives the metachromatic reaction with toluidine blue [Jorpes et al. 1937]. The reactionhas ,been used with success as a histochemical test for heparin [Jorpes et al. 1937; Wilander, 1939]. It has not, however, been applied-hitherto for the standardization of heparin preparations; and the purpose of this paperdis to specify conditions under which such an application is possible. The dye which has been chosen is toluidine blue.