Abstract
Background: We recently reported that proper blood processing using the RAPID (reduced temperatures, acidification, protease inhibition, isotopic exogenous controls, dilution) method is of key importance before measuring plasma concentration and molecular forms of labile hormones such as acyl ghrelin, cholecystokinin-58, gastrin-releasing peptide and somatostatin-28 (Endocrinology 150:5113-8; 2009). Active glucagon-like peptide-1 (GLP1(7-36)amide and GLP-1(7-37)) can be produced in blood ex-vivo by an endopeptidase cleaving GLP-1(1-36) and GLP-1(1-37) or destroyed by the action of dipeptidyl aminopeptidase IV (DPP-IV) to produce GLP-1(9-36)amide or GLP-1(9-37). Aim: To investigate whether different blood processing influences the detection of active and total GLP-1 plasma levels. Methods: Blood was obtained between 8-9 AM from male Sprague-Dawley rats (300-350g) either fed ad libitum or 24 h fasted (n=5-6/group) and processed in three ways: 1. collected in ice cold tubes containing EDTA (7.5%, 10μl/1ml) followed by centrifugation, 2. same as 1. plus DPP-IV inhibitor (diprotin A, 50μM) or 3. RAPID method without isotopic standards (RAPD). Samples were stored at -80°C until radioimmunoassays for total and active GLP1. Results: Active GLP-1 levels did not change among metabolic conditions irrespective of the blood processing (P 0.05). Plasma formation and DPP-IV inhibition gave comparable results for circulating total GLP-1 plasma levels of ad libitum fed rats while RAPD processing resulted in lower levels (P 0.05). In a preliminary experiment ~50% of iodinated GLP-1(7-36) was lost in Falcon tubes as used in the RAPD method. Conclusion: Active GLP-1 does not change under the metabolic conditions tested. The two plasma formation methods yield similar results for the measurement of circulating active and total GLP-1, whereas RAPD processing unexpectedly results in lower plasma levels of total and active GLP-1. The use of isotopic exogenous standards will be important to determine recovery and peptide structure alteration ex-vivo possibly leading to changed blood levels of active or total GLP-1. NIDDK 41301/70851/73152
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