Ghrelin is an acylated peptide with growth-hormonereleasing activity (1 ). It was first isolated from rat and human stomach during the search for an endogenous ligand to the "orphan" G-protein-coupled receptor, growth hormone secretagogue receptor (2 ). The peptide contains 28 amino acids, and n-octanoylation of the Ser-3 hydroxyl group is necessary for biological activity. Most studies have focused on the somatotropic and orexigenic roles of ghrelin; therefore, little is known about the kinetics of this peptide. Because the ester bond is both chemically and enzymatically unstable, elimination of the octanoyl modification of ghrelin can occur during storage, handling, and/or dissolution in culture medium (3 ). Because of increased interest in ghrelin measurements, a standardized method of sample collection is required.In the present study, which focused on the active form of ghrelin, we investigated the effects of anticoagulants and storage conditions on ghrelin stability. To distinguish the active form of ghrelin, we established two ghrelinspecific RIAs; N-RIA recognizes the N-terminal, octanoylmodified portion of the peptide, whereas C-RIA recognizes the C-terminal portion. Thus, the value determined by N-RIA specifically measures active ghrelin, whereas the value determined by C-RIA gives the total ghrelin immunoreactivity, including both active and desacyl ghrelin (4 -6 ). The minimum detectable quantities in the N-and C-RIAs were 5.0 and 50 pmol/L, respectively. The respective intra-and interassay CV were 3% and 6% for the N-RIA and 6% and 9% for the C-RIA (n ϭ 8 assays). Data are reported as the mean (SD). Comparisons of the time course of ghrelin concentrations between subgroups were made by two-way ANOVA for repeated measures, followed by the Scheffé test. P Ͻ0.05 was considered statistically significant.All blood samples were taken from three healthy male volunteers who gave written informed consent. Blood was taken from the forearm vein and immediately divided into tubes for serum and plasma preparation using (a) disodium EDTA (1 g/L) with aprotinin (500 000 kIU/ L), (b) disodium EDTA alone, (c) heparin sodium, or (d) no anticoagulant. Synthetic human ghrelin was added to each blood sample at a final concentration of 40 g/L; each sample was then sequentially divided into two aliquots for incubation at either 4 or 37°C. After incubation for 0, 30, and 60 min, blood samples were centrifuged, diluted 1:200 in RIA buffer, and subjected to ghrelin-specific RIAs. A comparison of the effects of different anticoagulants on the detected ghrelin concentrations is shown in Table 1A. Although the serum and three different plasma samples tested gave comparable results for total ghrelin by C-RIA, the N-RIA gave ghrelin concentrations that were significantly decreased at 37°C. When the ghrelin was measured by N-RIA, serum samples were highly affected by such treatment; samples stored for 60 min at 37°C lost ϳ35% of the ghrelin compared with the basal values at 0 min (P Ͻ0.05). The ghrelin concentrations in samples co...

Ghrelin is a novel growth hormone (GH)-releasing peptide, isolated from the stomach, which is identified as an endogenous ligand for GH secretagogues receptor. Although both ghrelin and its specific receptor are expressed in blood vessels, the cardiovascular effects of ghrelin remain unknown. To clarify whether ghrelin has a vasodilatory effect in humans, the response of forearm blood flow (FBF) to intra-arterial infusion of ghrelin was examined in eight healthy volunteers using a plethysmograph. In addition, hormonal responses to ghrelin were studied. Ghrelin increased FBF in a dose-dependent manner. Pretreatment with NG-monomethyl-l-arginine (l-NMMA), a nitric oxide synthase inhibitor, did not inhibit the FBF response to ghrelin, although l-NMMA significantly inhibited GH release. Serum insulin-like growth factor-I (IGF-I) level was not altered by ghrelin administration. Plasma cyclic guanosine 3', 5'-monophosphate level, a second messenger of nitric oxide, was also not altered. These results suggest that ghrelin has vasodilatory effects possibly through GH/IGF-I/nitric oxide-independent mechanisms.

Ghrelin is a novel growth hormone (GH)-releasing peptide, isolated from the stomach, which may also cause a positive energy balance by stimulating food intake and reducing fat utilization. However, whether glucose influences the release of ghrelin remains unknown. Accordingly, we examined circulating levels of ghrelin and GH in response to the intravenous or oral administration of 50 g of glucose in eight healthy humans. After the administration of intravenous glucose (50 g), the plasma ghrelin level decreased significantly from 127+/-9 to 98+/-9 fmol/ml (P<0.01), associated with an increase in plasma glucose from 85+/-3 to 357+/-19 mg/dl (P<0.01). Ingestion of 50 g of glucose decreased the plasma ghrelin level significantly from 134+/-12 to 97+/-15 fmol/ml (P<0.01), associated with an increase in plasma glucose from 93+/-3 to 166+/-10 mg/dl (P<0.01). The decrease in the plasma ghrelin level lasted for more than 30 min after recovery of the plasma glucose level. In conclusion, ghrelin secretion may be suppressed, at least in part, by an increased plasma glucose level in healthy humans.

Ghrelin is a novel GH (growth hormone)-releasing peptide isolated from the stomach. The cardiovascular and hormonal effects of the subcutaneous administration of ghrelin in humans remain unknown. Six healthy volunteers each received subcutaneous administration of three doses of ghrelin (1, 5 or 10 microg/kg) and placebo; the order of administration was randomized, and separate doses were given at least 24 h apart. The serum GH level dose-dependently increased from 0.5 +/- 0.4 to 3.6 +/- 2.1 ng/ml (1 microg/kg ghrelin; P=0.99 compared with baseline), 27.1 +/- 12.0 ng/ml (5 microg/kg; P<0.01 compared with baseline) and 45.4 +/- 12.8 ng/ml (10 microg/kg; P<0.01 compared with baseline) 30 min after ghrelin administration. Subcutaneous administration of ghrelin did not significantly alter circulating levels of corticotropin, cortisol, insulin-like growth factor-1, noradrenaline or adrenaline, although 10 microg/kg ghrelin slightly increased the prolactin level. No significant changes in heart rate or mean arterial pressure were observed. In contrast, the left ventricular ejection fraction, as assessed by echocardiography, increased dose-dependently from 63.5 +/- 0.6% to 65.1 +/- 0.9% (1 microg/kg ghrelin; P=0.97 compared with baseline), 69.6 +/- 1.3% (5 microg/kg; P<0.01 compared with baseline) and 71.5 +/- 0.9% (10 microg/kg; P<0.01 compared with baseline) 30 min after ghrelin administration. These haemodynamic and hormonal changes were still apparent 60 min after ghrelin injection. In conclusion, subcutaneous administration of ghrelin dose-dependently induced relatively specific GH release and enhanced cardiac performance in humans.