Plasma Levels Of Ghrelin During Different GH Provocative Tests In Boys With Short Stature

BackgroundGhrelin, a novel peptide isolated from rat stomach and subsequently found also in humans, was recently identified as the endogenous ligand for growth hormone (GH) secretogogue (GHS) receptor (GHSR). Like other GHSs, activation of the receptor stimulates GH secretion from the pituitary gland. However, the accurate regulating mechanism and effect of ghrelin were still unclear. Some studies suggested ghrelin-GHSR increased GH secretion by stimulated growth hormone relating hormone (GHRH) secretion in hypothalamus and GH secretion directly in pituitary gland, but the accurate mechanism of ghrelin in regulating GH synthesis and/or secretion was not full understood yet. We had known that arginine and insulin induced hypoglycemia stimulated GH secretion, but to our knowledge there are no data on ghrelin levels during insulin tolerance test (ITT) and the arginine stimulation test or GHRH test and the relationship among ghrelin, insulin induced hypoglycemia, arginine and GHRH-GH axis.ObjectiveIn order to investigate the relationship among arginine, insulin induced hypoglycemia, ghrelin, and GHRH-GH axis, we studied the plasma ghrelin levels at fasting and each time-point during GH provocative tests and GHRH test in growth hormone deficiency (GHD) and idiopathic short stature (ISS) children.Subjects and Methods PatientA total of 30 short stature boys were enrolled in the Children's Hospital of Zhejiang University School of Medicine from June 2005 to September 2005. The criteria for the diagnosis of short stature boys were as following: the heights of all these children were at least 2SD lower than the mean height of the same gender and age (from national statistics for Chinese school children in 1995). Children with other endocrinal, nutritional, metabolic, chromosomal, mental disorder, or family factor might cause short stature were excluded in the study. They were all prepuberty and included 10 cases of complete GHD (CGHD) with a mean age of 10.50 ± 2.35 years and 10 cases of ISS with a mean age of 8.24 ±1.81 years. They were all diagnosed by two GH provocative tests (arginine and insulin) after an overnight fast in 2 different days. Moreover, GHRH test were done for different diagnosis the abnormality of hypothalamus or pituitary gland in other 10 cases of GHD with a mean age of 10.55 ± 2.99 years and blood samples at every time point were collected for ghrelin measurement as well.. Control groups consisted of 10 healthy normal height boys with a mean age of 9.75 ± 2.51 years and the fasting blood samples were collected.Consent was obtained from the parents and the ethical committee of the Children Hospital of Zhejiang University School of Medicine. GH provocative testsITT (0.1 U-kg"1) and arginine stimulation test (0.5 mg-kg"1) was performed initially to differential diagnosis. Insulin (low peak of blood glucose <2.5 mmol/L) or arginine were administered by intravenous injection after dilution and blood samples were performed at 0, 15, 30, 60 and 90 min for GH measurements and ghrelin measurement. Normal GH secretion was defined by at least one test above 10 ug/L. GHD were defined as the stimulated GH peak of two tests all low than 10 ug/L. CGHD were defined as stimulated GH peak of two test less than 5 ug/L. GHRH testSermoreline acetate 1 ug-kg"1 (HaiNan Zhonghe Pannaceuticals Co., Ltd, China,) was administered by intravenous injection at the first day, followed by 1 ug-kgr1 bysubcutaneous injection for 3 days and then 1 ug-kg"1 was administered by intravenous injection in fifth day. Blood samples were obtained at 0, 30, 60, and 90 min for GH and ghrelin measurements in the last day. Plasma ghrelin measurementTwo milliliter venous bloods was obtained by venipuncture and were placed in a tube with 30 ul 10% ethylenediaminetetra acetic acid and 20 ul aprotinin. Plasma all were isolated and stored at -70°C until analysis. Plasma ghrelin levels, including Ser3-octanoyl and Ser3-des-octanoyl ghrelins, were measured by radioimmunoassay according to the manufacturer's protocol. The commercially available kits were purchased from the Phoenix Pharmaceuticals Inc (USA) with a sensitivity of 10 ng/L. The intra-assay and inter-assay CVs were 6% and 12% respectively. StatisticsStatistical analyses were conducted by using SPSS software (11.5 version). Quantitative data with normal distribution were presented as mean ± S.D. As plasma ghrelin levels of some samples were lower than 10 ng/L, they were treated as 10 ng/L. All the plasma levels were transformed into logarithms [ln(ghrelin)] before analysis because the distribution was skewed. The statistical significance between means was estimated by one-way ANOVA followed by LSD multiple comparisons or Student's t test when appropriate. Repeated measure general liner model analysis of the difference of plasma ghrelin levels during different stimulated test in GHD group and ISS group. The relation between stimulated ghrelin peak and stimulated GH peak was analyzed by bivariate correlation. Differences were considered statistically significant atP<0.05Results1. The fasting plasma ghrelin levels in controls, GHD groups (included 10 cases of GH provocative tests and 10 cases of GHRH test) and ISS group were 6.80±0.45, 5.39±1.75 and 6.19±0.60 respectively with a significant difference (F=4.126, P=0.024). LSD multiple comparisons showed that fasting In(ghrelin) levels in controls were higher than GHD group (i*=0.008), but similar with ISS group (P=0.229).2. The plasma ghrelin levels at every time points in ISS groups were higher than GHD groups. Moreover, we found the plasma ghrelin levels in 2 cases with GHD were very low. One case of CGHD were lower than 10 ng/L at fasting baseline and did not increased during two GH provocative tests, and the plasma ghrelin levels of the another one case were 10 ng/L except at 90 min during arginine stimulation test.3. Although the plasma ghrelin levels in ISS group at different time point during GH provocative tests were higher than GHD group, these were not significant difference between two groups (P>0.05 respectively).4. Repeated measure general liner model analysis was done for analysis the difference of ghrelin between two GH provocative tests, and age was treated as a covariate because the mean age between the two groups were different. We found the plasma ghrelin levels were not significant associated with age during both two GH provocative tests (P=0.659, P=0.125, respectively). During arginine stimulation test, the plasma ghrelin levels were significantly different at different time points (P=0.003), but no significant difference between two groups (P=0.100). During ITT, the plasma ghrelin levels were different between GHD and ISS group (P=0.029), but no significant difference among different time point (P=Q.216). The ghrelin peak did not significantly correlated with stimulated GH peak both during arginine stimulation test and ITT 0=0.161, P=0.498;r=0.207, P=0.382 respectively).5. Among 10 GHD children done with GHRH test, 6 cases were due to hypothalamus abnormality (H-GHD, GHRH stimulated peak >5 ug/L) and 4 cases were due to adenohypophyseal abnormality (GHRH stimulated peak < 5 ug/L). We found that the plasma ghrelin levels of one H-GHD case were lower than 10 ng/L as well. The mean ln(ghrelin) levels of different time points were 6.01±1.39, 6.06±1.44 and 5.86±1.83 respectively without a significant difference (F=0.043, i>=0.958). However, ghrelin peak were inversely correlated with the stimulated GH peak during GHRH test (r=-O.O35, P=0.036).Conclusion1. Ghrelin has an important role on GH secretion and abnormal secretion of ghrelin might be a reason of GHD.2. GH secretion stimulated by arginine might partly by activation ghrelin-GHSR signaling.