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

Abstract

The everlasting pharmacological development is continuously producing new substances with potential doping abuse. Among these, secretagogues are very prone to misuse by athletes for their properties to release growth hormone (GH) and some limitations in the actual analytical methods to detect them. In this paper, an in-depth study on the key variables of the radio receptor method previously developed by our group is performed and a fit-for-purpose protocol is established. Thus, this sensitive and robust screening method is proposed as an intelligent and preventive antidoping method to detect new growth hormone secretagogues (GHSs) in exceptional suspicious urine samples obtained from athletes and will support the current detection methods based on liquid chromatography-mass spectrometry (LC-MS).

Structure‐activity relationship for peptídic growth hormone secretagogues

“…Cell culture, membrane preparations and competition binding assay parameters have previously been published by Pinyot et al and Ferro et al . Briefly, HEK293 cells stably expressing GHSR1a was obtained from Dr R. Smith (Baylor College of Medicine, Houston, TX, USA) through Dr F. Casanueva (University of Santiago de Compostela, Spain).…”

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

“…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].…”

“…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.…”

“…Cell culture, membrane preparations and competition binding assay parameters have previously been published by Pinyot et al [22] and Ferro et al [28] Data were analysed and plotted with GraphPad Prism 5 (San Diego, CA, USA) or Excel software. Statistical analysis was performed using Excel and SPSS programs.…”

Evaluation of fibronectin 1 in one dried blood spot and in urine after rhGH treatment

“…These were a daily subcutaneous injection of 0.067 mg À1 kg À1 day À1 person -1 s.c. of 22 kDa rhGH daily for 14 days as described by Ferro et al [36] FN1 ELISA FN1 levels in one DBS and urine were measured with enzymatic methods using the fibronectin ELISA kit (Abcam, Cambridge, MA, USA) described for serum, plasma, and urine by the manufacturer. These were a daily subcutaneous injection of 0.067 mg À1 kg À1 day À1 person -1 s.c. of 22 kDa rhGH daily for 14 days as described by Ferro et al [36] FN1 ELISA FN1 levels in one DBS and urine were measured with enzymatic methods using the fibronectin ELISA kit (Abcam, Cambridge, MA, USA) described for serum, plasma, and urine by the manufacturer.…”