Plasma biomarker proteins for detection of human growth hormone administration in athletes

Sock-Hwee Tan,Vita Birzniece,Ken Ho,Alamgir Khan,Natasha Care,Albert Lee,Dana Pascovici,Mark P Molloy

doi:10.1038/s41598-017-09968-7

Abstract

Human growth hormone (GH) is a naturally occurring hormone secreted by the pituitary gland with anabolic and growth-promoting activities. Since an increased availability of recombinant GH (rGH) for the treatment of GH-deficient patients, GH has been abused in sports and it is prohibited. “GH-isoform” and “biomarkers” tests are currently available for detection of GH abuse in sports, however both methods suffer from shortcomings. Here, we report on a proteomic approach to search for novel protein biomarkers associated with rGH administration in non-elite athletes. In this study, participants received either placebo or rGH for 8 weeks, and were followed over a 6-week washout period. We used 2-D DIGE and iTRAQ LC-MS/MS analyses to expose rGH-dependent marker proteins. Eight rGH-dependent plasma proteins namely apolipoproptein-L1, alpha-HS-glycoprotein, vitamin D-binding protein, afamin, insulin-like growth factor-binding protein-3, insulin-like growth factor-binding protein-ALS, lumican and extracellular matrix proteins 1 were identified. Apolipoprotein L1 and alpha-HS-glycoprotein were validated by Western blots to confirm their identities and expression patterns in rGH- and placebo-treated subject cohorts. Independent confirmation of these putative GH-responsive biomarkers would be of value for clinical practices and may have sports anti-doping utility.

Highlights

Human growth hormone (GH) is a naturally occurring, 191 amino acid peptide hormone secreted by the pituitary gland which consists of several isoforms, mainly 22 kDa and 20 kDa isoforms[1]
Plasma samples obtained from nonelite athletes whom were administrated with 2 mg/day recombinant GH (rGH) or placebo for 8 weeks[4] was analysed by 2-D DIGE following immunodepletion of the top seven most abundant plasma proteins (Fig. 1)
We applied an unbiased proteomic approach using two independent techniques in a search for protein biomarkers associated with GH administration in non-elite athletes

Summary

Introduction

Human growth hormone (GH) is a naturally occurring, 191 amino acid peptide hormone secreted by the pituitary gland which consists of several isoforms, mainly 22 kDa and 20 kDa isoforms[1]. The World Anti-Doping Agency (WADA) prohibits the use of any form of exogenous GH and growth factor products in sports[6]. The Biomarkers test is an indirect method which is based on measuring increased levels of GH-responsive proteins such as insulin-like growth factor 1 (IGF-1) and pro-collagen type III amino-terminal pro-peptide (P-III-NP)[10, 13, 14]. After cessation of GH administration, IGF-1 levels decrease rapidly within a week to homeostatic levels whereas P-III-NP decline more slowly, returning to baseline by six weeks[16]. This provides an opportunity to use the biomarkers test for both ‘in and out of competition’ stages

Methods

Results

Conclusion