It has been demonstrated that insulin induces the production of specific nonprecipitating antibodies, both in experimental animals and in humans.\" Lysis of sensitized red blood cells, paper electrophoresis,\" and salt precipitation have been used in methods of immunoassay of insulin. In a preliminary report the authors described a two antibody system of insulin assay. The present paper describes the method in greater detail and presents results of assays of rat plasma.

Immunoassay of Insulin: Two Antibody System: Plasma Insulin Levels of Normal, Subdiabetic and Diabetic Rats

Prediction of blood volume in normal human adults.

For years investigators have sought an assay for insulin which would combine virtually absolute specificity with a high degree of sensitivity, sufficiently exquisite for measurement of the minute insulin concentrations usually present in the circulation. Methods in use recently depend on the ability of insulin to exert an effect on the metabolism of glucose in vivo or in excised muscle or adipose tissue. Thus, the insulin concentration in plasma has been estimated: a) from the degree of hypoglycemia produced in hypophysectomized, adrenalectomized, alloxan-diabetic rats (1); b) from the augmentation of glucose uptake by isolated rat hemidiaphragm (2); or c) from the increased oxidation of glucose-1-C14 by the rat epididymal fat pad (3). Since there have been reports indicating the presence, in plasma, of inhibitors of insulin action (4) and of noninsulin substances capable of inducing an insulin-like effect (5,6), these procedures, while yielding interesting information regarding the effects of various plasmas on glucose metabolism in tissues, are of doubtful specificity for the measurement of insulin per se (5).

Immunoassay of endogenous plasma insulin in man

Over 50 years ago, Benedict (2) published his extensive monograph on the metabolism of fasting in man, in which he demonstrated that carbohydrate stores provide a small but significant component of the body's fuel for only the first few days. Thereafter, protein and fat are the sole sources of fuel, the former contributing 15% of the calories and the latter the balance. The primary role of fat as fuel was apparent to Benedict and his contemporaries; it is plentiful and expendable. The significance of the protein requirement, however, was less clear; in fact, it was not fully understood until nearly 20 years later when the obligatory dependence of the central nervous system on glucose was firmly established (3). Since glycogen stores in man were known to approximate only 200 g, it was readily apparent that glucose has to be derived from protein in order to maintain cerebral metabolism during a prolonged fast. More recently, our understanding of the fasted state has been further clarified by the demonstration that free fatty acid is both the major transport form of lipid leaving adipose tissue (4, 5) and a substrate that is

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Hormone-fuel interrelationships during fasting.

WE have previously reported on the immuno-assay of beef insulin and certain other animal insulins, employing antiserums from human subjects treated with commercial mixtures of beef and pork insulin1. The insulin-binding antibodies present in these anti-serums do not form precipitable complexes with insulin, but with the use of insulin labelled with iodine-131 the complexes are readily demonstrable by paper chromatography and electrophoresis2. Beef, pork, sheep and horse insulins can be assayed quantitatively by measurement of the degree of competitive inhibition of binding of any insulin labelled with iodine-1311–3. As might have been anticipated, however, human insulin competes too weakly in systems employing human antiserum to be measurable at concentrations which obtain in vivo. Furthermore, the lack of availability of significant quantities of pure human insulin precludes its use as an antigen for animal immunization. However, in the present work, it has been found that human insulin cross-reacts strongly with insulin-binding antibodies in guinea pigs immunized with crystalline beef insulin, and that guinea pig anti-beef insulin serum has characteristics suitable for the detection and measurement of human insulin at concentrations which exist in the plasma of normal fasting subjects.

Assay of Plasma Insulin in Human Subjects by Immunological Methods

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Insulin-i131 metabolism in human subjects: demonstration of insulin binding globulin in the circulation of insulin treated subjects

SummarySerum albumin, serum γ globulin, crystalline glucagon and crystalline insulin were observed to bind to diaphragm in vitro and to be eluted slowly on continuous washing with buffered solutions. A lack of reproducibility of binding of insulin at low concentrations is attributable in part to variable adsorption to glassware. A definite saturation of binding sites on diaphragm was not observed at the highest concentrations employed. Competitive inhibition was observed in the presence of heterologous proteins. Binding capacity was not altered by treatment of diaphragms with formaldehyde. Binding of insulin by isolated rat diaphragm in vitro is not demonstrably of biologic significance but is attributable to nonspecific surface adsorption of proteins.

Lack of Specificity of Insulin-I131-Binding by Isolated Rat Diaphragm:

Determination of trapped plasma in the centrifuged erythrocyte volume of normal human blood with radioiodinated (I131) human serum albumin and radiosodium (Na24).

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Katharina Newerly

Papers

Insulin-i131 metabolism in human subjects: demonstration of insulin binding globulin in the circulation of insulin treated subjects

Lack of Specificity of Insulin-I131-Binding by Isolated Rat Diaphragm:

Determination of trapped plasma in the centrifuged erythrocyte volume of normal human blood with radioiodinated (I131) human serum albumin and radiosodium (Na24).

Distribution and fate of intravenously administered modified human globin and its effect on blood volume. studies utilizing i131 tagged globin

Estimation of trapped plasma with I131 albumin; critique of methods.