Glucagon Degrading Activity

Abstract

To evaluate the effect of glucagon degrading activity (GDA) on radioimmunoassay (RIA) of glucagon, I measured GDAs in plasma, serum, lysed red blood cells (RBC), and suspension of mononuclear cells and granulocytes. Serum levels of GDA in patients with various diseases were also examined. 1. Serum GDA values in normal subjects (control) measured by TCA precipitation method were 4.6 +/- 2.2% (mean +/- S.D.). GDA values in plasma treated with citrate and those in serum treated with aprotinin were not different from those in nontreated serum. GDAs in plasma treated either with EDTA and aprotinin or with EDTA alone were significantly lower than those in control serum, but the values in plasma treated with heparin were markedly higher than those in control serum. 2. GDAs in RBC lysate and suspension of mononuclear cells and granulocytes remained low up to the concentration of 23 X 10(4) RBC/microliters and 5000 mononuclear cells or granulocytes/microliters, respectively. GDAs in RBC lysate and mononuclear cells were markedly suppressed by the treatment with EDTA, whereas GDAs in granulocytes were inhibited by the treatment with aprotinin. 3. Markedly high values of GDA were obtained in serum of patients with pancreas diseases, liver diseases, renal diseases and hyperthyroidism. However, in four patients these elevated levels were restored to normal value after recovery from these disorders. The elevated GDA values in serum were suppressed to normal value by the addition of EDTA and aprotinin. 4. On Bio-Gel P-6 column chromatography, 125I-glucagon incubated with patient serum containing high GDA values revealed several peaks eluted after 125I-glucagon. 5. In healthy subjects, immunoreactive glucagon (IRG) levels in nontreated serum were not different from those in plasma treated either with EDTA and aprotinin or with heparin. 6. In patients showing high serum GDA levels, serum GDA levels were not significantly related to IRG levels in plasma treated with EDTA and aprotinin. These results indicate that a series of treatment of blood samples before assay: addition of EDTA and aprotinin to the blood samples, immediate separation of plasma from blood cells, and storage at 4 degrees C is recommended to avoid breakdown of glucagon by GDAs.