Background: Preproghrelin is a complex prohormone, produced in the gastrointestinal tract, that produces several peptides with structural and functional heterogeneity. Preproghrelin is clived into a 28-AA peptide that can be acylated (AG) or not (DAG). Whereas AG is a powerful GH secretagogue and orexigenic peptide, the other variant DAG acts as a functional ghrelin antagonist. Ghrelin acylation depends on the activity of the enzyme Ghrelin-O-Acyl-Transferase (GOAT) and conversion of AG to DAG in blood samples is dependent on esterase activities. Thus adequate methods to collect and process blood samples are critical to accurately assay AG/DAG ratio in animal and human blood and understand their physiological relevance.

Methods: Plasma samples were collected from mouse, rat and human blood with or in the absence of protease inhibitors. PHMB 1 mM was used as a protease inhibitor in rodents. Aprotinin 250 KIU, AEBSF 0.2-2 mg/ml and P800 a cocktail of inhibitors were used in healthy humans or Anorexia Nervosa patients. In addition, plasma samples were supplemented or not with HCl 0.1N. Human samples were measured before and after storage at -20°C or -80°C. AG and DAG were assayed with selective immunoassays (Bertin Pharma).

Results: In all 3 species, AG was best recovered when plasma samples were acidified with HCl just before storage. AG/DAG ratio in acidified conditions was 0.3-0.5 in rodents and close to 1 in humans. Acidification post-freezing was inefficient. AG/DAG ratio was higher when samples were first stored at -20°C for 8 days and collected with AEBSF or aprotinin as a protease inhibitor than with P800.

Conclusion: These results demonstrate that 1) accurate measurement of AG/DAG ratio in blood samples requires adequate processing and storage conditions and 2) AG/DAG ratio varies according to the species and the pathophysiological conditions.