Zhao Libo, Liu Zhini, Wang Sihan, Xiong Xin, Zhao Hongzhi, Cai Zongwei, Li Xiaona
Department of Pharmacy, Peking University Third Hospital, Beijing 100191, China; Therapeutic Drug Monitoring and Clinical Toxicology Center of Peking University, Beijing 100191, China.
Department of Pharmacy, Banan Hospital of Chongqing Medical University, Chongqing 401320, China.
J Pharm Biomed Anal. 2025 Nov 15;265:117026. doi: 10.1016/j.jpba.2025.117026. Epub 2025 Jun 17.
Pyrimidine intermediates, essential components of DNA/RNA, serve as energy transducers and signaling mediators in cellular communication pathways. However, research on pyrimidine metabolism in acute kidney injury (AKI) is hindered by the limited variety of metabolites and poorly understood underlying mechanisms. To date, no liquid chromatography coupled to mass spectrometry (LC-MS) method has achieved comprehensive coverage of pyrimidine metabolism. In this study, we developed an ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous detection of 10 pyrimidine metabolites. The method demonstrated a linear range of 1.2-2400 ng/mL, with reproducible recovery rates and consistent matrix effect. Both intra-day and inter-day accuracy and precision were within acceptable limits. Most metabolites exhibited stability under various conditions, including room temperature, autosampler, -40°C preservation, and after three freeze-thaw cycles. The method enabled extensive detection of pyrimidine metabolites, ensuring effective separation of all target compounds and achieving satisfactory peak shapes, particularly for the challenging monophosphate nucleotides. The method was successfully applied to matched plasma-urine biofluids from critically ill patients. In AKI inpatients, plasma levels of uracil, thymidine, and 2'-deoxyuridine were significantly reduced, while cytosine and cytidine levels were elevated. Additionally, a decrease in urinary cytidine concentration was observed in AKI patients. These findings demonstrate disturbances in the pyrimidine metabolic pathway in AKI patients, suggesting that the onset of AKI in critically ill patients may be closely associated with the accumulation of uremic solutes. The reliable quantification provided novel insights into the disease pathogenesis of AKI.
嘧啶中间体作为DNA/RNA的重要组成部分，在细胞通讯途径中充当能量转换器和信号介质。然而，急性肾损伤（AKI）中嘧啶代谢的研究受到代谢物种类有限以及潜在机制了解不足的阻碍。迄今为止，尚无液相色谱-质谱联用（LC-MS）方法能够全面覆盖嘧啶代谢。在本研究中，我们开发了一种超高效液相色谱-串联质谱联用（UHPLC-MS/MS）方法，用于同时检测10种嘧啶代谢物。该方法的线性范围为1.2-2400 ng/mL，回收率具有可重复性，基质效应一致。日内和日间的准确度和精密度均在可接受范围内。大多数代谢物在各种条件下都表现出稳定性，包括室温、自动进样器、-40°C保存以及经过三次冻融循环后。该方法能够广泛检测嘧啶代谢物，确保所有目标化合物有效分离，并获得令人满意的峰形，特别是对于具有挑战性的单磷酸核苷酸。该方法已成功应用于重症患者匹配的血浆-尿液生物流体。在AKI住院患者中，尿嘧啶、胸苷和2'-脱氧尿苷的血浆水平显著降低，而胞嘧啶和胞苷水平升高。此外，在AKI患者中观察到尿胞苷浓度降低。这些发现表明AKI患者嘧啶代谢途径存在紊乱，提示重症患者AKI的发生可能与尿毒症溶质的积累密切相关。可靠的定量分析为AKI的疾病发病机制提供了新的见解。
