Studies on Alum-Precipitated Insulin

Abstract

The precipitation of active biological principles, such as diphtheria toxoid, by alums suggested to us the possibility of preparing an alum-precipitated insulin. After trial of several preliminary procedures, we adopted the following technic:

To 100 cc. of ordinary insulin U40 is added 10 cc. of a 50% (by weight) solution of NaAl(SO₄)_2.12 H₂O in sterile distilled water. A heavy turbidity forms instantly and, upon mixing, a copious precipitation occurs. After standing for 24 hours in the refrigerator at 5°-10°C, the mixture is centrifuged at 3500 r.p.m. for 30 minutes. The clear supernatant fluid is now decanted from the sediment. Bio-assay of this fluid, performed in the usual manner on rabbits, shows that it contains only 5-10% of the original insulin unitage.

To this supernatant fluid is now added a further portion of 10 cc. of 50% soda-alum solution. After remaining for one week in the refrigerator at 5°C, a second crop of precipitate is obtained. This crop is separated by centrifuging from the supernatant fluid which now proves insulin-free by bioassay.

The precipitate obtained by the action of alum on insulin solutions, hereinafter referred to as alum precipitated insulin, is a white amorphous powder, insoluble in alcohol, ether and petroleum ether and slightly soluble in neutral (pH 7) distilled water. It is readily soluble in undiluted serum, but only slightly soluble in serum diluted 1:5 with saline. In solutions of acidity greater than pH 2.5, it dissolves completely. In solutions of pH 2.5 to 7, it forms insoluble suspensions, the minimum solubility being obtained in solutions of pH 6.

On dissolving separately the 2 precipitates in solutions of pH 1 to pH 2 and assaying the resultant clear solutions on rabbits, it is found that the first precipitate contained 90-95% of the total original insulin unitage. The second precipitate contained the remaining 5-10%. Thus, we find that a quantitative recovery of the active principle from insulin solution may be effected by fractional alum precipitation.

Chemical analysis of the precipitate showed a concentration of 0.10 to 0.12 γ of aluminum and 4.0 to 7.0 γ of non-protein nitrogen per unit of insulin.

For experimental and clinical trial, the precipitate of 100 cc. of insulin U-40 was suspended in 50 cc. of sterile distilled water containing 0.1 cc. N/10 HC1 per liter, to which has been added 02% of phenol. This suspension now assays 80 units of alum-precipitated insulin per cc.

Hypoglycemic activity of the suspension was studied in the following manner: A series of 44 rabbits weighing between 1.72 and 2.28 kg., which had been starved for 24 hours, were injected sub-cutaneously with a suspension of 3 units of the alum-precipitated insulin. In view of the relative uniformity of our results, Table I is limited to the blood sugar levels observed in series of 6 rabbits.

These results show that 3 units of alum-precipitated insulin cause a blood glucose depression in rabbits of 62 to 110 mg. % in 7 to 11½ hours, with recovery to the preprandial glycemia in 15 to 24 hours. Hypoglycemic tetany (insulin shock) was evident in no case.

Three days after recovery from the alum-precipitated insulin, each of the animals in the above series received injections of 3 units of ordinary insulin. The maximum depression of blood sugar was 74 to 122 mg. % in 1 to 2 hours, with insulin shock in all 6 cases.