Determination of nonsuppressible insulin-like activity in human serum by a sensitive protein-binding assay

Abstract

We describe a sensitive protein-binding assay for non-suppressible insulin-like activity in human serum. It can detect as little as 0.2 microunits (corresponding to 0.5 ng) of the activity in 0.4 ml of the assay mixture. It is measured in a low-molecular-weight fraction (termed "biological material") obtained by chromatography of serum on Sephadex G-50 in 1 mol/liter acetic acid. This fraction has been shown earlier to contain nearly all this billogically active material that is present in serum. A partially purified carrier protein from human serum is used as the binding protein; different concentrations of a partially purified preparation of material with the activity serve as standards, which compete with 125l-labeled tracer for binding. Biological material dilutes more or less in parallel with the standard over a 10-fold concentration range. In the chromatographed serum fractions, displacing activity appears between 50 and 80% bed volume, with the peak at 60%, and coincides with the distribution and the peak of radioactivity obtained by chromatography of tracer. A good correlation (r = 0.88) is observed between the values determined for this activity in the rat fat-pad assay and the protein-binding assay, although the latter yields about two fold higher results (190 +/- 37 milliunits/liter vs. 345+- 65 milliunits/liter, mean values for 18 normal sera). Values determined in the protein-binding assay are decreased in hypopituitary patients (183 +/- 27 milliunits/liter) and increased in acromegalics (486 +/- 88 milliunits/liter), in accord with the results of the bioassay (68 +/- 21 milliunits/liter for hypopituitary patients, 293 +/- 53 for acromegalics).