Nitrogen isotopic discrimination in dietary amino acids: The threonine anomaly

RATIONALE: The Threonine Anomaly relates to an observation made 25 years ago on the change in Thr nitrogen isotopic ratio in mammalian metabolism. Unlike all other amino acids, Thr in body protein is found to be depleted (rather than enriched) in N-15 relative to dietary Thr. Interpreting isotopic discrimination has become a useful source of ecological and palaeodietary information and it is desirable that the underlying processes are understood. METHODS: The principal enzyme of threonine catabolism, suggested to be responsible for the anomaly, threonine dehydratase, was prepared from rat liver. A time course of incubation of the enzyme with pure threonine was followed, and samples of residual threonine prepared for isotopic analysis by combustion in an automated carbon and nitrogen analyser coupled to a continuous flow isotope ratio mass spectrometer. RESULTS: We show experimentally, in vitro, that the enzymic reaction catabolising Thr cannot be responsible for its N-15 depletion. Plots of delta N-15 against both reaction time course and percentage completion show in fact an accelerating enrichment. CONCLUSIONS: A previously advanced suggestion that the unique catabolic mechanism for threonine was responsible for the anomalous depletion in (15)Nis clearly not the case. We therefore offer alternative explanations, based on threonine's role at an organismal rather than cellular level. Copyright (C) 2016 John Wiley & Sons, Ltd.