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Age Trends in Growth and Differentiation Factor-11 and Myostatin Levels in Healthy Men, Measured Using Liquid Chromatography Tandem Mass Spectrometry: Differential Response to Testosterone
Liming Peng 1 2, Thiago Gagliano-Jucá 2, Karol M Pencina 2, Srinivasan Krishnan 3, Zhuoying Li 2, Russell P Tracy 4, Ravi Jasuja 2, Shalender Bhasin 1 2
Abstract:
Background: Growth and differentiation factor (GDF)-11 controls embryonic development and has been proposed as an anti-aging factor. GDF-8 (myostatin) inhibits skeletal muscle growth. Difficulties in accurately measuring circulating GDF-11 and GDF-8 have generated controversy.
Methods: We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous measurement of circulating GDF-8 and GDF-11 that employs denaturation, reduction and alkylation; cation-exchange solid-phase extraction; tryptic digestion; followed by separation and quantification using two signature peptides for MRM and C-terminal [ 13C6 15N4]-Arg peptides as internal standards. We evaluated age trends in serum GDF-11 and GDF-8 concentrations in community-dwelling healthy men, 19 years or older and determined the effects of graded testosterone doses on GDF-8 and GDF-11 concentrations in healthy men in a randomized trial.
Results: The assay demonstrated linearity over a wide range, lower limit of quantitation 0.5 ng/mL for both proteins, and excellent precision, accuracy, and specificity (no detectable cross-reactivity of GDF-8 in GDF-11 assay or of GDF-11 in GDF-8 assay). Mean±SD (median±1QR) GDF-8 and GDF-11 levels in healthy community-dwelling men, 19 years and older, were 7.2±1.9 (6.8±1.4) ng/mL. Neither GDF-8 nor GDF-11 levels were related to age or body composition. Testosterone treatment significantly increased serum GDF-8 but not GDF-11 levels.
Conclusions: The LC-MS/MS method for the simultaneous measurement of circulating total GDF-8 and GDF-11 demonstrates the characteristics of a valid assay. Testosterone treatment increased GDF-8 levels, but not GDF-11. Increase in GDF-8 levels by testosterone treatment which increased muscle mass, suggests that GDF-8 acts as a chalone to restrain muscle growth.
Methods: We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous measurement of circulating GDF-8 and GDF-11 that employs denaturation, reduction and alkylation; cation-exchange solid-phase extraction; tryptic digestion; followed by separation and quantification using two signature peptides for MRM and C-terminal [ 13C6 15N4]-Arg peptides as internal standards. We evaluated age trends in serum GDF-11 and GDF-8 concentrations in community-dwelling healthy men, 19 years or older and determined the effects of graded testosterone doses on GDF-8 and GDF-11 concentrations in healthy men in a randomized trial.
Results: The assay demonstrated linearity over a wide range, lower limit of quantitation 0.5 ng/mL for both proteins, and excellent precision, accuracy, and specificity (no detectable cross-reactivity of GDF-8 in GDF-11 assay or of GDF-11 in GDF-8 assay). Mean±SD (median±1QR) GDF-8 and GDF-11 levels in healthy community-dwelling men, 19 years and older, were 7.2±1.9 (6.8±1.4) ng/mL. Neither GDF-8 nor GDF-11 levels were related to age or body composition. Testosterone treatment significantly increased serum GDF-8 but not GDF-11 levels.
Conclusions: The LC-MS/MS method for the simultaneous measurement of circulating total GDF-8 and GDF-11 demonstrates the characteristics of a valid assay. Testosterone treatment increased GDF-8 levels, but not GDF-11. Increase in GDF-8 levels by testosterone treatment which increased muscle mass, suggests that GDF-8 acts as a chalone to restrain muscle growth.
