In a previous report, serum insulin was extracted with efficient recovery by salt-ethanol extraction.

As the second method of separation of serum insulin gel filtration was adopted. Dog sera obtained from the pancreatic and the femoral veins were separated into two fractions (A and B) with Sephadex G-50. Fractions A and B corresponded respectively to the major peak of serum proteins and the portion where I-131-insulin added to serum was eluted. Insulin-like activity (ILA) was assayed by rat fat pad and immunoreactive insulin (IRI) by a double antibody method. Significant ILA was demonstrated in both fractions. IRI in serum appeared mostly in fraction B. ILA/IRI ratio was closer to 1.0 in fraction B than in serum. Fraction A tended to have fairly uniform ILA irrespectively of origin of serum, often exceeding the ILA of serum. Fraction A from a depancreatized dog serum had similar degree of ILA. Serum and both fractions stimulated various phases of fat pad metabolism similarly to insulin, including glycogen synthesis. Assays based on four different metabolic parameters gave essentially the same ILA values for both serum and fractions. ILA of fraction A differed from insulin in its nonneutralizability with anti-insulin serum, its poor suppressibility by cysteine, and the lack of parallelism of its regression line with that of insulin. Simple dilution and reconcentration of serum resulted in an increase in its ILA. Presumably, fraction B is primarily due to serum insulin with similar immunological and biological activities to pancreatic insulin. Whether or not ILA of fraction A is related to insulin remains unknown.

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