Universidad de Alcalá, Departamento de Química Analítica, Química Física e Ingeniería Química, Ctra. Madrid-Barcelona Km. 33.600, 28871, Alcalá de Henares (Madrid), Spain.
Universidad Autónoma de Madrid, Departamento de Biología, Facultad de Ciencias, Campus de Cantoblanco, Calle Darwin, 2, 28049, Madrid, Spain.
Anal Chim Acta. 2024 Nov 15;1329:343190. doi: 10.1016/j.aca.2024.343190. Epub 2024 Sep 2.
Apoptotic bodies play an important role in the cellular communication as a consequence of the great variety of biomolecules they harbor. There is evidence that 1st generation apoptotic bodies from HK-2 cells induced by cisplatin or UV light trigger apoptosis in naïve HK-2 cells whereas 2nd generation apoptotic bodies activate cell proliferation showing an opposite effect. Thus, the development of new analytical strategies to quantify the changes in the involved metabolites is imperative to shed light on the biological mechanisms which trigger apoptosis and cell proliferation.
A LC-(Q-Orbitrap)MS method has been developed to quantify the metabolites unequivocally identified in the apoptotic body fluid from HK-2 cells in our previous works based on untargeted metabolomics. Thus, two different columns and gradients were tested and the HILIC column was selected taking into account the retention times and chromatographic separation. Also, different normal collision energies were tested for each metabolite and the parallel reaction monitoring was chosen to carry out the quantitative analysis. Once the method was optimized, it was evaluated in terms of linearity, limits of detection and quantification, matrix effects, accuracy, and precision, for each metabolite. Limits of detection ranged from 0.02 to 1.4 ng mL. A total of 9 relevant metabolites proposed as potential biomarkers to reveal metabolic differences among apoptotic bodies from HK-2 cells were quantified and some insights about the biological relevance were discussed.
The first targeted metabolomics methodology enabling the quantification of relevant metabolites in apoptotic bodies from HK-2 cells was developed using LC-(Q-Orbitrap)MS. Pyridoxine, kynurenine, and creatine concentrations were determined in apoptotic bodies from HK-2 cells treated with cisplatin and UV light. Phenylacetylglycine, hippuric acid, butyrylcarnitine, acetylcarnitine, carnitine, and phenylalanine were determined in 1st and 2nd generation apoptotic bodies from HK-2 cells treated with cisplatin. Concentrations determined were useful to establish their biological role in the metabolism.
凋亡小体作为细胞通讯的重要组成部分，由于其携带的大量生物分子而具有重要作用。有证据表明，顺铂或紫外线诱导的 HK-2 细胞的第一代凋亡小体可诱导未成熟的 HK-2 细胞发生细胞凋亡，而第二代凋亡小体则激活细胞增殖，表现出相反的效果。因此，开发新的分析策略来定量研究参与代谢物的变化对于揭示触发细胞凋亡和细胞增殖的生物学机制至关重要。
本研究基于之前的非靶向代谢组学工作，建立了一种 LC-(Q-Orbitrap)MS 方法来定量分析 HK-2 细胞凋亡小体液体中明确鉴定的代谢物。因此，测试了两种不同的色谱柱和梯度，考虑到保留时间和色谱分离，选择了亲水作用色谱柱。此外，还针对每种代谢物测试了不同的正常碰撞能量，并选择平行反应监测进行定量分析。方法优化后，对每种代谢物的线性、检测限和定量限、基质效应、准确度和精密度进行了评估。检测限范围为 0.02 至 1.4ng/mL。共定量了 9 种有潜力的生物标志物，这些生物标志物可能揭示 HK-2 细胞凋亡小体之间的代谢差异，并对其生物学相关性进行了讨论。
本研究首次建立了基于 LC-(Q-Orbitrap)MS 的靶向代谢组学方法，可定量分析 HK-2 细胞凋亡小体中的相关代谢物。采用该方法，测定了顺铂和紫外线处理的 HK-2 细胞凋亡小体中的吡哆醇、犬尿氨酸和肌酸浓度，还测定了顺铂处理的 HK-2 细胞第一代和第二代凋亡小体中的苯乙酰甘氨酸、马尿酸、丁酰肉碱、乙酰肉碱、肉碱和苯丙氨酸浓度。所测定的浓度有助于确定它们在代谢中的生物学作用。
