# Compound-Specific and Intramolecular δ15N Analysis of a Poly-Nitrogenous Amino Acid: Histidine

## TL;DR

This paper introduces a new method to analyze nitrogen isotope ratios within histidine, revealing differences in nitrogen atoms from different parts of the molecule.

## Contribution

A novel analytical approach for compound-specific and intramolecular δ15N analysis of poly-nitrogenous amino acids is developed.

## Key findings

- α-N in histidine is consistently enriched in 15N compared to side chain-N in commercial and biological samples.
- The method can be adapted for other poly-nitrogenous amino acids like glutamine.
- The technique uses ion-exchange chromatography and selective oxidation to measure position-specific δ15N values.

## Abstract

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.