Compound-Specific and Intramolecular δ

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 δ15NHIS (δ15NHIS-Total) values, and there are no reported intramolecular δ15NHIS 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 δ15NHIS-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 δ15NHIS values of α-N (δ15NHIS-α) and side chain-N (δ15NHIS-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 (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 N isotope analysis.