Abstract
Glucagon circulates in concentrations in the low picomolar range, which is demanding regarding the sensitivity of the methods for quantification applied. In addition, the differential and tissue specific proteolytic processing of the glucagon precursor and the presence in of several glucagon-like sequences, not only in the precursor of glucagon, but also in a number of other peptides of the glucagon-secretin family of peptides, put special demands on the specificity of the assays. Finally, experience has shown that unspecific interference of plasma components has presented additional problems. All of these problems have resulted in a lot of diverging results concerning measured and reported glucagon responses in both humans and experimental animals that have and still are causing considerable debate and controversy. There is very solid evidence that glucagon is an important hormone in human and mammalian metabolism, but its precise physiological role in glucose and lipid metabolism and in metabolic disease has been difficult to establish, not least because of these difficulties. It was our purpose with this review to discuss the methods of glucagon quantification and discuss pitfalls and sources of error. We also reviewed some of the dogmas regarding glucagon secretion in the light of the methodological difficulties.
Highlights
Because of the low concentrations of circulating glucagon, direct measurements in plasma had to await the development of sufficiently sensitive techniques, and the advent of the radio-immunoassays allowed Roger Unger in Dallas, Texas, who was in close contact with Berson and Yalow, to build on their experience from the development of the insulin assay [1], enabling him to publish the first glucagon assay, almost simultaneously with the insulin assay, in 1959 [2]
Proglucagon would be formed in both the intestinal mucosa and in the pancreas, but would undergo differential post-translational processing, leading to formation of glicentin and oxyntomodulin in the gut, and GRPP, glucagon, and a small C-terminal fragment in the pancreas
The recovery after extraction is not 100%, but is rather constant, and effectively removes the interference. Using this procedure and high affinity C-terminal antisera, the plasma concentration of glucagon typically falls from 7–10 mol/L in the fasting state down to around 1 pmol/L [42], consistent with the near complete cessation of glucagon secretion from isolated perfused pancreas preparation exposed to high glucose concentrations
Summary
Because of the low concentrations of circulating glucagon, direct measurements in plasma had to await the development of sufficiently sensitive techniques, and the advent of the radio-immunoassays allowed Roger Unger in Dallas, Texas, who was in close contact with Berson and Yalow, to build on their experience from the development of the insulin assay [1], enabling him (and Eisentraut) to publish the first glucagon assay, almost simultaneously with the insulin assay, in 1959 [2]. A full publication appeared in 1961 [3] and the assay was reported to have a detection limit of 50 pg/mL (16–17 pmol/L)- sensational for the time, but not quite enough to reliably measure physiological levels in humans. Assay improvements along the way made it possible to study meal response in both healthy and in individuals with type 2 diabetes, resulting in the citation classic by Müller et al from 1970 [9], showing suppression of glucagon by carbohydrates but lack of suppression and hypersecretion of glucagon in patients with T2DM. The group was subsequently fortunate enough to develop a sensitive and specific antiserum from rabbit 30 K with improved sensitivity and specificity [10], and the glucagon assay based on this antiserum, which was commercialized, dominated the field of glucagon research for decades. Other assays with similar or improved specificity and sensitivity were developed
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