A practical guide for the stabilization of acylghrelin in human blood collections

Abstract

Objective and methods: To better understand acylghrelin plasma stability, human synthetic acylghrelin was spiked into plasma and tracked by liquid chromatography tandem mass spectrometry. To investigate the best method for quantifying clinical plasma acylghrelin levels, pre- and postprandial human blood was collected from healthy volunteers (n=6) using various sample collections and treatments. Plasma ghrelin levels from human blood collections were analysed by enzyme-linked immunosorbant assay (ELISA).

Results: Acylghrelin's half-life in plasma was approximately 45 min with the formation of des-acylghrelin approaching 50% before the end of the 60-min incubation. Loss of acylghrelin inversely correlated with an increase in des-acylghrelin (P<0.008; r(2) =0.870). Plasma pretreated with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) or protease inhibitor cocktail without acidification resulted in no detectible acylghrelin losses. Acylghrelin measurements with AEBSF-treated blood were minimally 40% higher than sodium citrate/citric acid, K(2) EDTA, aprotinin/HCl and P800 collections. HCl addition to AEBSF-treated plasma did not provide enhanced acylghrelin stability and induced deacylation at and above the 100 mM final concentration. Pre- and postprandial ghrelin attenuation was investigated using aprotinin/HCl, AEBSF, protease inhibitor cocktail and no treatment for blood and plasma preparations. Fasting samples treated with AEBSF and protease inhibitor cocktail were approximately threefold higher than aprotinin/HCl and control treatments (P<0.03). Pre- and postprandial ghrelin attenuation was approximately twofold different (P<0.04) with significant counterintuitive trends in aprotinin/HCl and K(2) EDTA groups.

Conclusions: Our data suggest that AEBSF addition to K(2) EDTA blood immediately after collection without plasma acidification, processing on ice and 14-day 70 °C storage is the best treatment for accurately quantifying acylghrelin in human plasma.