title: The acylated (AG) to unacylated (UAG) ghrelin ratio in esterase inhibitor-treated blood is higher than previously described.
abstract: BACKGROUND
  The acylated/unacylated ghrelin (AG/UAG) ratio has been reported to range from 0·02 to 0·3, 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.
  
  
  METHODS
  A 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 0-6 months at -20 or -80 °C.
  
  
  METHODS
  Healthy, non-obese, adults (n = 8; 4 women), age 26-42 yrs, after an overnight fast.
  
  
  METHODS
  AG and UAG stability following different methods of sample treatment and storage.
  
  
  RESULTS
  Non-AEBSF plasma contained low AG and high UAG (>270 pg/ml) indicating rapid conversion of AG to UAG. However, AEBSF plasma, stored at -80 °C and measured at 0, 1, 3 and 6 months contained AG and UAG ranges of 12-350 and 17-170 pg/ml, respectively. Mean (SEM) AG/UAG ratios were 1·7(0·3), 1·2(0·2), 1·5(0·3) and 1·8(0·5) at each time point with no significant effect of storage period.
  
  
  CONCLUSIONS
  AG 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.
authors.affiliation: Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Patric J D
authors.initials: PJ
authors.lastname: Delhanty
authors.forename: Martin
authors.initials: M
authors.lastname: Huisman
authors.forename: Michel
authors.initials: M
authors.lastname: Julien
authors.forename: Karine
authors.initials: K
authors.lastname: Mouchain
authors.forename: Patrick
authors.initials: P
authors.lastname: Brune
authors.forename: Axel P N
authors.initials: AP
authors.lastname: Themmen
authors.forename: Thierry
authors.initials: T
authors.lastname: Abribat
authors.forename: Aart J
authors.initials: AJ
authors.lastname: van der Lely
mesh_terms: D000215:Acylation; D000328:Adult; D004791:Enzyme Inhibitors; D004950:Esterases / Q000037:antagonists & inhibitors; D005260:Female; D054439:Ghrelin / Q000097:blood*; D006403:Hematologic Tests / Q000592:standards*; D006801:Humans; D008297:Male
chemical_list: D004791:Enzyme Inhibitors; D054439:Ghrelin; D004950:Esterases