Effect of a GnRH Injection on Ghrelin Concentrations in Girls With Suspected Premature Puberty

The purposes of the study is to investigate whether circulating levels of ghrelin decreases after an iv injection of GnRH in girls with suspected central premature puberty and to investigate the effects of different post sampling handling on the preservation of acylated ghrelin.

Patients with clinically suspected central premature puberty undergo a GnRH stimulation test to have an appropriate diagnosis. 20 such girls will be recruited to this study and a modified GnRH stimulation test will be done. The modification includes prolonged blood sampling time (0, 30, 60, 90, 120, and 150 min after the GnRH injection instead of the usual 0, 30, 60, and 90 min). The girls will also be investigated twice. In randomised order they will be given either GnRH or placebo (isotone saline), on different days. One week to one month will separate the different investigation days. The proteinase inhibitor 4-(2-aminoethyl) benzene-sulfonyl fluoride hydrochlorid (AEBSF) will be added to some of the blood tubes, and hydrochloric acid will be added to some of the plasma. All plasma samples will be frozen and all analyses will be done at the same time. Acylated ghrelin will be analyzed by Millipore's specific ELISA kit and deacylated ghrelin will be analyzed by Cayman's specific ELISA kit. Anthropometric data as well as pubertal state will be collected. P-glucose, S-insulin, S-follicle stimulating hormone (FSH), S-luteinizing hormone (LH), and S-estradiol will also be analyzed.

A single intravenous injection of gonadotropin releasing hormone GnRH (Relefact LHRH 0,1 mg/mL). Dose: 100 micrograms per square meter body surface. Maximal dose: 100 micrograms.

A single intravenous injection of sodium chlorid (9 mg/mL). Dose: the same volume as the volume given/to be given with GnRH, that is maximal dose = 1 mL = 9 mg sodium chlorid.

Gonadotropin releasing hormone GnRH (Relefact LHRH) iv injection, single dose, 100 micrograms/square meter body surface, maximum dose: 100 microgram.

Sodium chlorid 9 mg/mL will be given as a single iv injection with the same volume as the volume used for the active substance injection.

Paired comparison will be done between the acylated ghrelin concentration after GnRH injection and the acylated ghrelin concentration after saline injection. Paired comparison will also be done between deacylated ghrelin concentration after GnRH injection and deacylated ghrelin concentration after saline injection.

Deacylated ghrelin concentrations in samples treated with the inhibitor AEBSF, in samples treated with both AEBSF and hydrochloric acid, and in samples not treated with either AEBSF or hydrochloric acid

Deacylated ghrelin concentrations in samples treated with the inhibitor AEBSF, in samples treated with both AEBSF and hydrochloric acid, and in samples not treated with either AEBSF or hydrochloric acid

Deacylated ghrelin concentrations will be compared at 150 min after iv injections between different handling procedures.

Inclusion Criteria: Female gender. Clinical suspicion of central precocious puberty. A GnRH stimulation test is to be performed to make appropriate diagnosis. Exclusion Criteria: Age < 1 year. Weight < 10 kg. Untreated hypo- or hyperthyroidism. Diabetes of any kind. BMI > 3 z-score. Growth hormone treatment. Syndrome or suspected syndrome. Tumour or congenital malformation of the hypothalamus or the pituitary gland. (Neuroradiology does not have to be performed only because of the study.) On going ulcus ventriculi or the patient has gone through surgery that removed part of the ventricle or changed it markedly in any other way.