The N-depleted results of threonine are linked to increased activity of threonine ammonia-lyase, and show potential as a possible biomarker for protein intake and/or gluconeogenesis. We hypothesize that the inverse nitrogen equilibrium isotope effects of Schiff base formation, between AAs and pyridoxal-5'-phosphate cofactor enzymes, play a key role in the bioaccumulation and depletion of N in the biomolecules of living organisms and contributes to the variability in the nitrogen trophic level effect. Copyright © 2017 John Wiley & Sons, Ltd.