Immunoassay Of Endogenous Plasma Insulin In

tion of hemolysis of insulin-sensitized red blood cells; however, the lower limit of detectability by this technique was approximately 0.1 fig (2.8 mU) making it unsuitable for determination of plasma insulin. Loveless (32) has used certain normal human subjects, in whom the skin can be locally sensitized to insulin (by the intracutaneous injection of human anti-insulin serum) to assay insulin by the whealing response obtained. Aside from the inconvenience associated with this method, the lower limit of detectability was 200 fiU beef insulin per ml and human plasma insulin was not detectable, a result attributed in part to the lesser reactivity of human insulin (32).

Reported estimates of plasma insulin concentrations, derived from the various biological assay procedures, have varied widely. Thus, the in vitro diaphragm assay has yielded values ranging from 40 to 80 ^U per ml (33) to as high as 4,600 ^U per ml (34) in fasting plasmas and from about 130 to 800 /xU per ml (33) to 9,000 to 22,000 fxU per ml (35) after glucose in normal subjects. Measuring the increase in oxidation of glucose-1-C14 to C1402 by rat epididymal adipose tissue in vitro, Martin, Renold and Dagenais (3) found that the insulin-like activity of fasting normal plasma in this preparation corresponded to 50 to 350 /iU of insulin per ml. Pfeiffer, Pfeiffer, Ditschu-neit and Ahn (36), using the same assay, found that plasma diluted 1:2 gave higher and more consistent insulin concentrations and reported normal fasting levels of 135 to 680 per ml in 15 normal human subjects, with concentrations frequently exceeding 2,000 to 4,000 /*U per ml after tolbutamide and metahexamide. Employing the immunoassay method we have observed generally much smaller increases in peripheral insulin concentration after large doses of sodium tolbutamide, administered intravenously or by mouth, than after glucose given by the same routes to normal or diabetic men (37).

It is generally agreed (5, 34) that dilution of plasma or serum increases markedly the estimated insulin concentration in the diaphragm assay and similar observations have been made in the rat epididymal fat pad assay (36). This phenomenon has been either attributed to the presence of inhibitory substances in the plasma (5, 34) or interpreted as indicating that insulin-like activity of serum as measured by the isolated rat diaphragm

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