Growth hormone secretagogues: out of competition

Abstract

Growth hormone secretagogues (GHS) constitute a new GH deficiency treatment increasing exponentially in number and improved potency and bioavailability over the last decade. The growth hormone releasing activity makes these compounds attractive for the artificial improvement of the human sports skills, now that recombinant human growth hormone (rhGH) administration is effectively detected. The GHS family is extremely diverse both in number and chemical heterogeneity and keeps growing continuously. In this paper, a general screening test is proposed. To develop a universal method, the single common property of growth hormone secretagogues has been targeted: their capacity to bind to the GHS receptor 1a (GHS-R1a). Pretreated urine samples have been tested in a competition assay where eventually the GHS presence detached a radiolabelled ligand from the receptor in a dose-dependent manner. Blank urine samples were processed to determine potential age, gender and exercise effects, and to define a threshold beyond which a specimen is considered positive. Samples from a growth hormone releasing peptide 2 (GHRP-2) excretion study corroborated the screening assay applicability with a detection window of approximately 4.5 h, and results were confirmed by comparison with a dedicated LC-MS quantification of the intact compound.

Detection of Growth Hormone Releasing Peptides in Serum by a Competitive Receptor Binding Assay

“…The basal state was subsequently recovered and remained unaltered until at least 24 h post-administration. The detection time window appeared shorter in the serum as the drugs can be detected for 2 hours after administration versus 6 hours in urine samples [29], although these data are similar to that obtained previously from GHRP-2 and GHRP-6 in serum samples with other methods [26,27].…”

“…There were several important reasons for our interest in the Here we present an experimental study which was built on the previous screening method described by Pinyot et al and Ferro et al in urine samples [22,28,29] to develop an alternative method that uses serum as a new matrix. The serum is a valuable matrix used for analysis of some doping substances e.g., CERA [30], human chorionic gonadotrophin [31], and growth hormone [32,33].…”

“…Urine: Urine samples were processed following the protocol established by Pinyot et al [29]. Briefly, urine samples were thawed and centrifuged for 15 min at 3500g at 4°C.…”

“…The previously described radio competition assay methodology to detect GHRPs in urine [22,28,29] was modified and simplified for the detection of these compounds in serum samples. As was expected, however, the direct application of the competitive assay on "unprocessed" serum samples (without any denaturing or immunocapture pretreatment) from 10 non-treated subjects showed an interfering effect.…”

Fit-for-Purpose Radio Receptor Assay for the Determination of Growth Hormone Secretagogues in Urine

“…After optimizing the runtime protocol of the screening method, five additional variables were studied to assess the sensitivity and robustness of the method developed by Pinyot et al 18,19 : incubation time conditions of binding assay (40 vs 70 min), number of washes at different pH values, food intake effect, interference with concomitant doping substances potentially present in real samples, and limit of decision.…”