Improvements in the Preparation of Insulin. Alkaline Extraction of Pancreas.

THE original method for preparing insulin from normal fresh pancreas, worked out by Collip [1923], depended on the extraction of the minced tissue with plain alcohol. Doisy, Somogyi and Shaffer [1923] found that they could obtain considerably larger yields of the active principle by extraction with strongly acid alcohol. The fact that insulin in acid solution is readily adsorbed by a great variety of substances, while in faintly alkaline solutions this is not the case, [Dudley, 1923] suggested that possibly the yield of insulin might be increased by extracting the minced pancreas in an alkaline medium. The only danger which we anticipated was that in such a process we should have conditions favourable to the action of trypsin which is known rapidly to destroy insulin [Dudley, 1923]. Our fears in this respect proved to be groundless and by a very simple modification of the method of alcoholic extraction we have been able to obtain between four and five times the amount of insulin which we had been getting by the original method. We carry out the process as follows. One kilogram of fresh ox pancreas is minced into one litre of 95 % spirit; 85 g. sodium bicarbonate are stirred into the mixture, which is then poured into the mincer and reminced; this procedure is repeated a second time. The mixture is then allowed to stand with frequent stirring for two hours at room temperature, or if desired it may be kept in the cold room overnight. It is then poured on to a stout cloth, which is squeezed as thoroughly as possible in a suitable press. The turbid filtrate which has a PH of about 7*5 is treated with 14 times its volume of 95 % spirit and placed in the cold room (30) overnight. It is then filtered through a folded paper; 10 cc. of glacial acetic acid are added to the filtrate, which is then evaporated in vacuo, in a water-bath at 40450. When the volume of the residue has reached about 150 cc., the fat which has separated is removed by shaking out with light petroleum and the aqueous layer is freed from traces of this solvent by a few minutes' distillation under the conditions described above.