Compound-Specific and Intramolecular δN Analysis of a Poly-Nitrogenous Amino Acid: Histidine.

Histidine (HIS) is an essential amino acid (AA) with key physiological roles in metal chelation and proton buffering. Its three nitrogen (N) atoms─one α-amino and two in the imidazole side chain─are incorporated through distinct biosynthetic pathways and undergo different catabolic processes. Thus, its intramolecular δN values likely provide additional information on these pathways and associated N fluxes. Very few studies have reported molecular average δN (δN) values, and there are no reported intramolecular δN data for natural materials due to technical limitations of available methods. Here, we present a novel analytical approach for compound-specific and intramolecular δN values of poly-nitrogenous AAs using HIS as an example. This scheme can be adapted to obtain position-specific δN values of other poly-nitrogenous AAs such as glutamine. Underivatized HIS is separated by ion-exchange chromatography (IC) and divided into two aliquots. One fraction is fully oxidized to NO using UV-persulfate oxidation for δN measurement, while the other undergoes NaClO oxidation, selectively converting α-N and a minor fraction of side chain-N to NO at a known ratio. The δN values of α-N (δN) and side chain-N (δN) are then calculated from these two results. Our findings reveal that α-N is consistently enriched in N relative to side chain-N in both commercial HIS powder (ΔδN = ∼ +8‰) and biological samples (ΔδN = ∼+3 to 25‰), likely due to preferential α-N catabolism via deamination. This finding supports the potential of studying diverse biosynthetic and catabolic processes of poly-nitrogenous AAs using intramolecular N isotope analysis.