Marked discrepancies have previously been observed between insulin-like activities by rat fat pad assay (ILA) and insulin levels by radio-immunoassay (IRI) for the native sera. As miscellaneous factors in the serum may affect these assays, separation of serum insulin by means of salt-ethanol extraction, acid-ethanol extraction or gel filtration was carried out prior to assay.
Samples were dog sera obtained from the pancreatic and femoral veins, either at fasting or after glucose injection. The salt-ethanol procedure originally devised by Romans, was modified for the extraction of serum insulin. Recoveries of endogenous IRI by the salt-ethanol and the acidethanol procedures were 90% and 72% respectively, almost identical with recoveries of
131I-insulin added to sera. By gel filtration with Sephadex G 50,
131I-insulin was eluted separately from most of serum proteins. Serum was separated by this method into two fractions, one of which (Fr. A) contained most of serum proteins, and the other (Fr. B) was eluted following Fr. A corresponding to the peak of
131I-insulin.
In contrast to discrepancies of ILA and IRI in the native sera, ILA and IRI values of these extracts and fraction B were much closer to each other. These data demonstrate that IRI behaves like the pancreatic insulin in extraction and gel filtration procedures, and suggest that serum endogenous insulin assayed as IRI also possesses biological activity corresponding to its immunological reactivity.
Fraction A by gel filtration, despite the lack of any appreciable IRI, showed a marked ILA by fat pad assay. The ILA of a depancreatized dog was measured as 405μU/m
l, although its IRI was undetectable. This ILA was not recovered by either extraction procedures nor in Fr. B by gel flit ration, but a maked ILA was detected in Fr. A. Fraction A, as well as insulin and fraction B, stimulated various parameters of fat pad metabolism such as glucose uptake, net gas exchange,
14C incorporation from glucose-U-
14C into CO
2, total lipid and glycogen.
Cysteine treatment suppressed ILA of both Fr. A and Fr. B, while anti-insulin serum suppressed ILA of Fr. B but not that of Fr. A. Four point assay by fat pad revealed parallelism between the extracts or fraction B and crystalline insulin, but deviation from parallelism was significant between Fr. A and insulin.
These results suggest that the difference between ILA and IRI is mainly attributable to the ILA in fraction A. This ILA is probably due to factors entirely different from crystalline insulin, although it possesses many similar biological effects with insulin on adipose tissue metabolism in vitro.
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