ContextThe acylated/unacylated ghrelin (AG/UAG) ratio has been reported to range from 002 to 03, suggesting biologically relevant independent regulation of each ghrelin isoform. However, AG is deacylated to UAG by esterases in blood samples, and esterase inhibition is critical for their accurate measurement. Our hypothesis is that at least part of the variation in reported AG and UAG values is due to inconsistent sample preparation. DesignA non-interventional study. Quantification with two different, commercially available, ELISA formats of AG and UAG in venous plasma stabilized or not with 4-(2-aminoethyl) benzenesulphonyl fluoride (AEBSF) and stored for 0-6months at -20 or -80 degrees C. ParticipantsHealthy, non-obese, adults (n=8; 4 women), age 26-42yrs, after an overnight fast. MeasurementsAG and UAG stability following different methods of sample treatment and storage. ResultsNon-AEBSF plasma contained low AG and high UAG (>270pg/ml) indicating rapid conversion of AG to UAG. However, AEBSF plasma, stored at -80 degrees C and measured at 0, 1, 3 and 6months contained AG and UAG ranges of 12-350 and 17-170pg/ml, respectively. Mean (SEM) AG/UAG ratios were 17(03), 12(02), 15(03) and 18(05) at each time point with no significant effect of storage period. ConclusionsAG and UAG levels measured in AEBSF-stabilized plasma indicate that the AG/UAG ratio is markedly higher than previously described and that UAG is a physiological component of the circulation. This highlights the importance of immediately stabilizing blood samples on collection for determination of both AG and UAG concentrations and provides a valuable tool for their measurement in physiological and interventional studies.