Department of Clinical Medicine, Aarhus University, Denmark; Research Unit for Molecular Medicine, Aarhus University and Aarhus University Hospital, Denmark; Hammel Neurorehabilitation Centre and University Research Clinic, Denmark.
Department of Forensic Medicine, Aarhus University, Denmark.
J Pharm Biomed Anal. 2023 Apr 1;227:115304. doi: 10.1016/j.jpba.2023.115304. Epub 2023 Feb 16.
Kynurenine metabolites are emerging as promising clinical biomarkers in several diseases, especially within psychiatry. Unfortunately, they are difficult to detect, particularly the challenging neurotoxic metabolite quinolinic acid (QUIN). The aim of this study was twofold: First, to develop a liquid chromatography-mass spectrometry method (LC-MS) for simultaneous targeted quantification of key kynurenine metabolites together with untargeted metabolomics, and second, to demonstrate the feasibility of the method by exploring serum/plasma and gender differences in 120 healthy young adults between 18 and 30 years of age. A range of analytical columns (C18 and biphenyl columns) and mobile phases (acidic and alkaline) were systematically evaluated. The optimized LC-MS method was based on a biphenyl column, a water-methanol gradient with 0.2% formic acid, and authentic isotope-labeled standards for each kynurenine metabolite. Precision and accuracy of targeted quantification of the key kynurenine metabolites tryptophan (TRP), kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), and QUIN were excellent, far exceeding the acceptance criteria specified by international guidelines. Median inter- and intra-day precision were < 6% in serum and plasma; the median accuracy was 2.4% in serum and 8% in plasma. Serum concentrations were ≤ 10% different from the corresponding concentrations in plasma for all kynurenine metabolites in healthy young adults. Men had higher levels (8-18%) of TRP, KYN, and KYNA than women (p ≤ 0.009), while no differences were observed for 3-HK and QUIN (p > 0.70). Incurred sample reanalysis of 10% of the samples yielded a median difference < 5% from the initial measurement, demonstrating the robustness of the method. Besides the targeted quantification of key kynurenine metabolites, our method was found to be suitable for simultaneous untargeted metabolomics analyses of hundreds of metabolites. A range of compound classes could be detected including amino acids, nucleic acids, dipeptides, antioxidants, and acylcarnitines, making explorative studies highly feasible. For example, we identified an additional kynurenine metabolite, 2-Quinolinecarboxylic acid, which was 47% higher in males than females (adjusted p-value = 0.001). In conclusion, in this study, we present a reliable and robust LC-MS method for simultaneous targeted and untargeted metabolomics ready for both research and clinical use. We show that both serum and plasma can be used for kynurenine studies, and the reported gender differences are in accordance with the literature. Future studies should consider using biphenyl-based LC-MS columns to successfully detect QUIN.
犬尿氨酸代谢物在几种疾病中作为有前途的临床生物标志物而出现，尤其是在精神病学中。不幸的是，它们很难被检测到，特别是具有挑战性的神经毒性代谢物喹啉酸（QUIN）。本研究的目的有两个：首先，开发一种液相色谱-质谱法（LC-MS），用于同时对关键犬尿氨酸代谢物进行靶向定量分析，并进行非靶向代谢组学分析，其次，通过探索 18 至 30 岁之间的 120 名健康年轻成年人的血清/血浆和性别差异，证明该方法的可行性。我们系统地评估了一系列分析柱（C18 和联苯柱）和流动相（酸性和碱性）。优化的 LC-MS 方法基于联苯柱、含 0.2%甲酸的水-甲醇梯度以及每个犬尿氨酸代谢物的真实同位素标记标准品。关键犬尿氨酸代谢物色氨酸（TRP）、犬尿氨酸（KYN）、犬尿氨酸酸（KYNA）、3-羟基犬尿氨酸（3-HK）和 QUIN 的靶向定量的精密度和准确度都非常好，远远超过国际指南规定的可接受标准。血清和血浆中，关键犬尿氨酸代谢物的日内和日间精密度中位数<6%；血清中的准确度中位数为 2.4%，而血浆中的准确度中位数为 8%。在健康年轻成年人中，所有犬尿氨酸代谢物的血清浓度与相应的血浆浓度相差≤10%。男性的 TRP、KYN 和 KYNA 水平比女性高（8-18%）（p≤0.009），而 3-HK 和 QUIN 则无差异（p>0.70）。对 10%的样本进行重新分析，结果表明与初始测量相比，中位数差异<5%，证明了该方法的稳健性。除了对关键犬尿氨酸代谢物进行靶向定量分析外，我们的方法还适用于同时对数百种代谢物进行非靶向代谢组学分析。可以检测到包括氨基酸、核酸、二肽、抗氧化剂和酰基肉碱在内的多种化合物类别，使探索性研究变得非常可行。例如，我们鉴定了一种额外的犬尿氨酸代谢物，2-喹啉羧酸，其在男性中的含量比女性高 47%（调整后的 p 值=0.001）。总之，在这项研究中，我们提出了一种可靠且稳健的 LC-MS 方法，可同时进行靶向和非靶向代谢组学分析，既适用于研究也适用于临床应用。我们表明，血清和血浆都可用于犬尿氨酸研究，并且报告的性别差异与文献一致。未来的研究应考虑使用联苯基 LC-MS 柱来成功检测 QUIN。
