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

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

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2871%2980319-8

Immunoassay using antigen—enzyme conjugates

/pdf/insulin-i131-metabolism-in-human-subjects-demonstration-of-2dqbkiapju.pdf

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

https://dm5migu4zj3pb.cloudfront.net/manuscripts/103000/103183/JCI55103183.pdf

Tissue distribution of I131 labeled human serum albumin following intravenous administration.

The role of serum albumin in the causation of cardiac edema has been disputed. Starling (1) postulated that circulatory stasis resulting from cardiac insufficiency is responsible for anoxia and increased permeability of the capillary membrane, the consequence of which is the passage of albumin and water into the extravascular spaces. Although Smirk (2) observed evidence of increased capillary permeability to water and crystalloids in congestive heart failure, Stead and Warren (3) demonstrated, by direct analysis, that the protein concentration of cardiac edema fluid was not elevated above that of the edema fluid produced by tourniquet stasis in normal subjects. Other studies have directed attention to the low serum albumin concentrations occasionally encountered in heart failure (4-7), and have suggested or implied (4, 8) a causal relationship to the pathogenesis of cardiac edema. Abnormalities in liver function tests and morphologic evidence of liver injury in severe congestive failure (9-14) have appeared to strengthen the possibility that hepatic synthesis of albumin may be impaired in this condition. Alternatively, low serum albumin concentrations have been attributed to inadequate dietary protein intake or poor absorption from a congested gastrointestinal tract (15, 16). Since the plasma concentration of albumin is not an adequate measure of the total quantity of circulating and extravascular albumin stores, it was of interest to measure the amount of total exchangeable albumin in subjects with heart failure employing I'8l labeled albumin as a tracer. The rate of metabolism of I18l labeled albumin was also studied with the aim of evaluating the ability of the subject in heart failure to synthesize albumin. Although there may yet remain some reservations regarding the validity of this tracer in the

https://dm5migu4zj3pb.cloudfront.net/manuscripts/103000/103184/JCI55103184.pdf

Distribution and metabolism of i131 labeled human serum albumin in congestive heart failure with and without proteinuria

/pdf/the-effect-of-large-doses-of-desiccated-thyroid-on-the-1pqzrdrb5t.pdf

The Effect of Large Doses of Desiccated Thyroid on the Distribution and Metabolism of Albumin-I131 in Euthyroid Subjects1

/pdf/blood-volume-alterations-in-congestive-heart-failure-27lw3f9tr0.pdf

Arthur Bauman

Papers

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

Tissue distribution of I131 labeled human serum albumin following intravenous administration.

Distribution and metabolism of i131 labeled human serum albumin in congestive heart failure with and without proteinuria

The Effect of Large Doses of Desiccated Thyroid on the Distribution and Metabolism of Albumin-I131 in Euthyroid Subjects1

Blood volume alterations in congestive heart failure