Detection of Follistatin-doping in urine and blood
Chapter S4 of WADA’s Prohibited List 2018 (“Hormone and metabolic modulators”) lists myostatin inhibitors under sub-chapter 4 (“Agents modifying myostatin function(s)”). Follistatin (FST) suppresses signaling of myostatin and subsequently leads to an increase in muscle mass and loss of body fat. FST is a secreted glycoprotein, which can be found in many tissues and organs (e.g. pituitary, bone marrow, ovary, kidney, liver, blood vessels). Due to alternative splicing, three FST-isoforms exist (FS-288, FS-300, and FS-315). The isoform with 315 amino acids (FS-315) is the dominant one. FS-315 can also be detected in blood. Typical concentrations in serum and plasma are in the range of 2-3 ng/mL.
So far, no approved follistatin pharmaceuticals are available. Nevertheless, follistatins can be bought from many internet providers for “research purposes”. Their products are labelled either “Follistatin”, “Follistatin 344”, or “Follistatin 315”. Most of these proteins are expressed in E. coli and hence lack the characteristic glycosylation of human endogenous follistatins. This fact will be exploited in order to detect doping with follistatins. After immunoaffinity purification (serum/plasma, urine), FST will be separated by electrophoresis (SDS-, SAR-, or IEF-PAGE) and detected by Western blotting. Due to the missing glycosylation, “black market” FSTs will not only differ in molecular mass but also isoelectric point (pI) from the endogenous FSTs.
Follistatin (FS), a myostatin-inhibiting protein, is prohibited according to chapter S4 of the "WADA Prohibited List 2022". While currently no approved pharmaceutical formulations of Follistatin are available, Follistatin can be bought on the black market. Most of the products are labelled "Follistatin 344" (FS344), few "Follistatin 315". A study on FS344 black market products was performed and an electrophoretic detection method for serum and urine developed. While only 9 of the 17 tested products actually contained Follistatin, in some of the others growth promoting peptides were found (e.g. MGF, GHRP-2). Suprisingly, all nine products contained His-tagged FS344 and a high degree of its oligomoers. The detection method is based on immunomagnetic purification followed by SDS-PAGE and Western bloting with a monoclonal anti-His antibody. Alternatively, a monoclonal anti-FS antibody can be used. For immunoprecipitation (IP), a polyclonal anti-Follistatin antibody is applied. An evaluation of suitable antibodies for IP and immunoblotting is also presented. Furthermore, practicall all currently available Follistatin standards were investigated. The detection limit of the method for back market FS344 in urine is ca 0.1 ng/mL for 10 mL. For a sample volume of 100 μL, an LOD of 5 ng/mL could be achieved for serum. Due to the presence of His-tags an unambiguous differentiation from endogenous Follistatin is possible.