Compound-Specific and Intramolecular δ¹⁵N 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 δ¹⁵N values likely provide additional information on these pathways and associated N fluxes. Very few studies have reported molecular average δ¹⁵N_HIS (δ¹⁵N_HIS-Total) values, and there are no reported intramolecular δ¹⁵N_HIS data for natural materials due to technical limitations of available methods. Here, we present a novel analytical approach for compound-specific and intramolecular δ¹⁵N values of poly-nitrogenous AAs using HIS as an example. This scheme can be adapted to obtain position-specific δ¹⁵N 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 NO₃^- using UV-persulfate oxidation for δ¹⁵N_HIS-Total measurement, while the other undergoes NaClO oxidation, selectively converting α-N and a minor fraction of side chain-N to NO₂^- at a known ratio. The δ¹⁵N_HIS values of α-N (δ¹⁵N_HIS-α) and side chain-N (δ¹⁵N_HIS-s) are then calculated from these two results. Our findings reveal that α-N is consistently enriched in ¹⁵N relative to side chain-N in both commercial HIS powder (Δδ¹⁵N_α-s = ∼ +8‰) and biological samples (Δδ¹⁵N_α-s = ∼+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.