S.F. Kuku, A. Zeidler, D.S. Emmanouel, A.I. Katz, and A.H. Rubenstein, Department of Medicine, University of Chicago, Chicago, Illinois 60637, and N.W. Levin, and A. Tello, Department of Medicine, Henry Ford Hospital, Detroit, Michigan 48202. ABSTRACT: Immunoreactive plasma glucagon (IRG) in normal subjects and patients with chronic renal failure, diabetic ketoacidosis and diabetic hyperosmolar syndrome circulates in several forms. In the diabetic patients most IRG eluted coincidentally with the extracted, purified pancreatic hormone (MW3500), while in normal subjects a high molecular weight component predominated. In striking contrast, the major component of plasma IRG in patients with chronic renal failure was of intermediate size (MW+9000), consistent with proglucagon. The accumulation of this form of IRG suggests that the kidney plays an important role in its metabolism. If there are differences in the biological activity of the various circulating components of IRG, the significance of immunoreactive glucagon levels in some disease states will require reassessment. Plasma immunoreactive glucagon (IRG) concentrations are raised in patients with chronic renal failure (CRF) (1,2). This is mainly due to a reduction in the metabolic clearance rate of the hormone in this condition (3). The levels of IRG observed in uremia can be of the order associated with diabetic ketoacidosis, and glucagon has been incriminated as being at least partially responsible for the glucose intolerance which characterizes renal insufficiency (4,5). Although attractive, this hypothesis fails to explain the improvement of carbohydrate intolerance following dialysis (4), despite the fact that IRG concentrations are not lowered by this procedure (1). It has been recently shown that most of the IRG activity measured in the plasma of fasting normal and diabetic subjects is in a high molecular weight form (6,7). The biological activity of this component has not been established yet. Because of the possibility that the various forms of circulating IRG may have different biological activity, we have used gel filtration to characterize the plasma IRG in CRF patients and compared it with that of normal subjects and patients with other known hyperglucagonemic states such as diabetic Submitted October 1, 1975.

ketoacidosis (DKA) (8) and diabetic hyperosmolar syndrome (DHS) (9). PATIENTS AND METHODS. Blood samples obtained from 5 fasting normal subjects and 6 fasting chronic renal failure patients (CRF), 2 DKA and 2 DHS patients were collected in iced tubes containing Trasylol (500 units/ml.) and centrifuged at 4°C. Plasma was gel-filtered immediately or stored at -20°C until processed. Plasma (1.0 or 2.0 ml.) was gelfiltered on 50x1 cm Biogel P-30 columns. Elution was carried out under gravity at room temperature with 0.2 M glycine buffer (pH 8.8) containing 0.25% human serum albumin, 1% normal lamb serum and 50 units/ml Trasylol. One millilitre fractions were collected and the total volume was assayed. The columns were calibrated using 125j_ gamma globulin, porcine proinsulin (MW 9033) , insulin (MW 5777), glucagon (MW 3485), 125I-glucagon and Na 1 2 5 I . Plasma glucagon was measured by a double antibody radioimmunoassay using an antibody specific for pancreatic glucagon (30 K, obtained from Dr. R. Unger, Dallas). A beef-pork pancreatic glucagon standard was used (Eli Lilly Co., Indianapolis). The glucagon was labeled (10) with 125j an(j purified on QAE Sephadex (11) . Sheep anti-rabbit gamma globulin was used as the


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JCE & M , 1976 Vol 42 . No 1

precipitating antibody. Benzamidine [0.01 M final dilution], a proteolytic enzyme inhibitor, was added to the assay system. The assay sensitivity was 6 pg. per tube. RESULTS. Mean plasma IRG levels in the hyperglycemic diabetic syndromes (1525+578 pg/ml) and patients with renal failure (540+40 pg/ml) were significantly higher (p

Heterogeneity of plasma glucagon: patterns in patients with chronic renal failure and diabetes.

Immunoreactive plasma glucagon (IRG) in normal subjects and patients with chronic renal failure, diabetic ketoacidosis and diagetic hyperosmolar syndr...
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