Aliquots of plasma were analyzed for total myostatin, free myostatin (both DGDF80, R&D Systems, UK), FLRG (DFLRG0, R&D Systems, UK), activin A (DAC00B, R&D Systems, UK), FLRG (DFLRG0, R&D Systems, UK), and GDF11 (DY1958, R&D Systems, UK) were analyzed according to manufactures instructions. Total myostatin was measured by the acidification release method. Briefly, 100 μL plasma was treated with 50 μL 1N HCl, vortexed and incubated at room temperature for 10 min then neutralized with 50 μL 1.2 N NaOH and further diluted in 200 μL manufacturer provided diluent for a final dilution of 1:4, similar to previously validated methods (Lakshman et al. 2009 ). Plasma samples for free myostatin were diluted 1:4 in identical diluent, without acidification or neutralized treatment. All samples and standard were analyzed in triplicate. Of the 88 participants, 56 GDF11 results were either below limit of detection of our assay (31.3 pg/mL) or otherwise undetectable, and are thus not reported here.
As GDF11 has been previously reported to have a high structural similarity to myostatin/GDF8 (Poggioli et al.2016 ), we also directly compared the GDF11 ELISA used here against recombinant myostatin in a physiological (0–2000 pg/mL) and supra‐physiological (20,000 pg/mL) range, with no cross reactivity reported (Fig. 1 ).
As GDF11 has been previously reported to have a high structural similarity to myostatin/GDF8 (Poggioli et al.