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 likely provides additional information on these pathways and associated N fluxes. Very few studies have reported molecular average δ15NHIS (δ15NHIS-Total) and there are no reported intramolecular δ15NHIS data for natural materials due to the technical limitations of available methods. Here, we present a novel analytical approach for compound-specific and intramolecular δ15N of poly-nitrogenous AAs using HIS as an example, such analytical scheme can be adapted to obtain position-specific δ15N of other key AAs with two N atoms 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 δ15NHIS-Total measurement, while the other undergoes NaClO oxidation, selectively converting α-N and a minor fraction of sidechain-N to NO₂⁻ at a known ratio. δ15NHIS of α-N (δ15NHIS-α) and sidechain-N (δ15NHIS-s) are then calculated from these two results. Our findings reveal that α-N is consistently enriched in ¹⁵N relative to sidechain-N in both commercial HIS powder (Δδ15Nα-s = ~ + 8‰) and biological samples (Δδ15Nα-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 δ15N.