Context
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.
Materials
- Vacutainers BD cat# 367899; 6 ml K2 EDTA
- AEBSF (4-(2-Aminoethyl) benzenesulphonyl fluoride hydrochloride) stock solution at 200 mg/ml
- Acylated Ghrelin (human) Easy Sampling ELISA kit (cat# A05306)
- Unacylated Ghrelin (human) Easy Sampling ELISA kit (cat# A05319)
Protocol
Venous blood was collected into three separate EDTA vacutainers from eight individuals who fasted overnight. Treatment of the blood was performed immediately after collection. For EDTA + AEBSF samples, AEBSF stock solution was added at 1:100 to whole blood. After gentle mixing by inversion (x3) of the recapped tubes, the whole blood was centrifuged at 1500 g, 4 °C for 5 min. The plasma was then rapidly aliquoted and immediately frozen on dry ice. Samples were stored at either -20 °C or -80°C until the time of assay.
Plasma samples were thawed on ice, centrifuged for 1 min at 1500 g, 4 °C and kept on ice before transferring to the assay plates. Samples were measured in duplicate (50 µl per well) for AG and UAG using double-antibody sandwich ELISAs from Bertin Bioreagent.
Results
AG and UAG levels measured in samples collected in EDTA tubes without [E] or with the addition of AEBSF [A] after 1 day or 6 months of storage at either -20 °C or -80 °C.
Non-AEBSF plasma contained low AG and high UAG indicating rapid conversion of AG to UAG. However, AEBSF plasma, stored at -80 °C and measured at 0 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) and 1.8 (0.5) at each time point with no significant effect of storage period.
Customer
Delhanty PJ, Huisman M, Julien M, Mouchain K, Brune P, Themmen AP, Abribat T, van der Lely AJ.
The acylated (AG) to unacylated (UAG) ghrelin ratio in esterase inhibitor-treated blood is higher than previously described. Clin Endocrinol (Oxf). 2015 Jan;82(1):142-6. doi: 10.1111/cen.12489. Epub 2014 Jun 2. PubMed PMID: 24810977.
