Insulin-like Activity by Fat Pad Assay and Immunoreactive Insulin: I. Their Correlation in Native Serum, Acid-ethanol Extract and Salt-ethanol Extract

In view of controversies on the relationship of ILA (insulin-like activity) and IRI (immunoreactive insulin) in blood, removal of extra factors other than insulin was attempted by means of salt-ethanol (acid-ethanol-NaCl) or acid-ethanol extraction methods prior to assay. The saltethanol procedure, a modification of Romans' method for the extraction of pancreatic insulin, proved to be more efficient in extraction. ILA was assayed by rat fat pad method using C-14-O₂ production from glucose-U-C-14 as index. IRI was assayed by a double antibody radioimmunoassay. Dog sera obtained from the pancreatic and femoral veins were chiefly used. Recoveries of endogenous IRI by saltethanol and acid-ethanol methods were 90 per cent and 72 per cent respectively, which were almost identical with recoveries of I-131-insulin added to sera. The ILA of the extracts differed considerably from the ILA of the original sera; the recovery scattered widely beween 5 and 116 per cent. In contrast to wide discrepancies between ILA and IRI in the native sera, ILA and IRI in the extracts were much closer to each other. There was a good parallelism between regression lines of ILA in the extracts and of standard insulin, but deviation was significant between the regression lines of insulin and untreated sera. The ILA in the extracts of dog and human serum was mostly suppressed with antiinsulin serum. Thus, endogenous IRI behaves like pancreatic insulin during extraction procedure and appears to possess biological activity corresponding to its immunoreactivity.