A randomized trial of the effect of a GnRH analogue injection on ghrelin levels in girls.

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® (100 g/m2) 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.