Lee Charlotte Wing Man, Altabet Mark A, Baca Jesus, Barrera Jason, Zhang Lin
Texas A&M University Corpus Christi, Corpus Christi, Texas 78412, United States.
School for Marine Science and Technology, University of Massachusetts Dartmouth, New Bedford, Massachusetts 02744, United States.
Anal Chem. 2025 Aug 5;97(30):16288-16295. doi: 10.1021/acs.analchem.5c01711. Epub 2025 Jul 24.
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.
组氨酸（HIS）是一种必需氨基酸（AA），在金属螯合和质子缓冲中具有关键的生理作用。它的三个氮（N）原子——一个α-氨基氮和咪唑侧链中的两个氮——通过不同的生物合成途径整合，并经历不同的分解代谢过程。因此，其分子内的δN值可能为这些途径和相关的氮通量提供额外信息。很少有研究报道分子平均δN（δN）值，由于现有方法的技术限制，尚无天然材料的分子内δN数据报道。在此，我们以组氨酸为例，提出了一种针对多氮氨基酸的化合物特异性和分子内δN值的新型分析方法。该方案可用于获取其他多氮氨基酸（如谷氨酰胺）的位置特异性δN值。未衍生化的组氨酸通过离子交换色谱（IC）分离，并分成两份。一份通过紫外过硫酸盐氧化完全氧化为NO用于δN测量，而另一份进行次氯酸钠氧化，以已知比例选择性地将α-N和一小部分侧链-N转化为NO。然后根据这两个结果计算α-N（δN）和侧链-N（δN）的δN值。我们的研究结果表明，在商业组氨酸粉末（ΔδN = ∼ +8‰）和生物样品（ΔδN = ∼ +3至25‰）中，α-N相对于侧链-N在氮含量上始终富集，这可能是由于通过脱氨基作用优先进行α-N分解代谢。这一发现支持了利用分子内氮同位素分析研究多氮氨基酸不同生物合成和分解代谢过程的潜力。
