In plasma obtained from normal fasting subjects insulin was precipitated by anti-insulin serum. Both the precipitate and the supernatant were extracted with acid-alcohol. Insulin or insulin-like activity was measured in the extracts by immunoassay, glucose uptake by the rat hemi-diaphragm and CO2 production by the rat epididymal fat pad. The amount of insulin found in plasma before extraction was quantitatively recovered from the insulin-anti-insulin precipitate extracted with acid-alcohol as measured by the immunoassay. No insulin was detected in the extracted supernatant. In close agreement were the results obtained with the diaphragm. Only the adipose tissue was able to demonstrate ILA in the extracted supernatant. This ILA was present even in control samples (buffer with known amounts of Crystalline Insulin added) in spite of the fact that all the added insulin had been recovered in the precipitate.

These results suggest that in normal human plasma all the insulin is readily available to react with insulin antibodies. Results with the epididymal fat pad method stroagly suggest the possibility that factors other than insulin stimulate the production of CO2 from glucose.

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