To Örebro University

Introduction: Ghrelin concentrations decline during puberty by an unclear mechanism. Acylated ghrelin (AG) is unstable in sampling tubes, but no standardized sampling protocol exists. We hypothesized that ghrelin levels decrease as a consequence of increased gonadotropin-releasing hormone (GnRH) signalling and that the addition of a protease inhibitor to sampling tubes preserves the AG levels.

Methods: In this randomized, placebo-controlled, cross-over study, 13 girls with suspected central precocious puberty were included. They performed an adjusted GnRH stimulation test twice and were given Relefact LHRH (R)(100 mu g/m(2)) or saline in a randomized order. Blood was sampled repeatedly for 150 min for the analysis of hormone concentrations. Oestradiol levels were only measured at baseline. The protease inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) was added to the sampling tubes. Specific ELISA kits were used for the analysis of AG and desacylated ghrelin (DAG) levels.

Results: Neither AG nor DAG levels changed after GnRH analogue injection in comparison to saline. The addition of AEBSF preserved AG levels (650.1 +/- 257.1 vs. 247.6 +/- 123.4 pg/mL, p < 0.001) and decreased DAG levels (51.9 [12.5-115.7] vs. 143.5 [71.4-285.7] pg/mL, p < 0.001). Both AG and DAG levels were inversely associated with insulin levels (r = -0.73, p = 0.005, and r = -0.78, p = 0.002, respectively). AG levels were inversely associated with oestradiol levels (rho = -0.57, p = 0.041).

Conclusion: Ghrelin levels do not decrease following a pharmacological dose of a GnRH analogue in the short term in girls. Addition of a protease inhibitor to the sampling tubes decreases AG degradation, resulting in preserved AG and decreased DAG levels. (C) 2022 The Author(s). Published by S. Karger AG, Basel