Objective: Considerable uncertainty remains regarding the veracity of measuring myokine irisin more than seven years after its original description. Unresolved issues include the nature of transcription of the irisin precursor fibronectin type III domain containing 5 (FNDC5) gene across species, the reliability of irisin levels measured with commercial enzyme-linked immunosorbent assays (ELISAs), and the overall validity of the recently published reference values for human serum measured with quantitative mass spectrometry. We utilized multiple species and measures to evaluate the robustness of commonly used reagents and methods for reporting irisin.
Methods: Amplification of cDNA was used to assess the FNDC5 transcript patterns in humans and mice. The specificity and sensitivity of different irisin antibodies were examined via western blotting. Quantification of circulating native irisin was conducted with mass spectrometry using an absolute quantification peptide for irisin.
Results: We show that there is a greater transcript diversity of human FNDC5 than currently annotated, but no indication of the expression of transcripts leading to a truncated form of irisin. Available irisin antibodies still bind to patterns of unspecific serum proteins, which compromise reliable measurements of irisin with ELISAs. Absolute quantification of irisin with labeled peptides by mass spectrometry is an advanced method but requires a multi-step sample preparation introducing uncontrollable variations in the measurement.
Conclusion: Our data represent an explicit warning against measuring circulating irisin using available methods. Measuring irisin is akin to chasing shadows.