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Detection and Isolation of Apoptotic Bodies to High Purity
Published on: August 12, 2018
Quantification of relevant metabolites in apoptotic bodies from HK-2 cells by targeted metabolomics based on liquid
Samuel Bernardo-Bermejo1, Ana B Fernández-Martínez2, Francisco Javier Lucio-Cazaña3
1Universidad de Alcalá, Departamento de Química Analítica, Química Física e Ingeniería Química, Ctra. Madrid-Barcelona Km. 33.600, 28871, Alcalá de Henares (Madrid), Spain.
Background:
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.
Results:
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-1. 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.
Significance:
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.
Insights
This study developed a new LC-MS method to quantify metabolites in apoptotic bodies, revealing key biomarkers that differentiate cell death and proliferation signals. This advances understanding of cellular communication and disease mechanisms.
Area of Science:
- Cell Biology
- Analytical Chemistry
- Metabolomics
Background:
- Apoptotic bodies are crucial for intercellular communication, carrying diverse biomolecules.
- First-generation apoptotic bodies can induce apoptosis, while second-generation bodies promote proliferation, highlighting distinct biological roles.
- Quantifying metabolite changes in apoptotic bodies is essential for understanding these opposing cellular effects.
Purpose of the Study:
- To develop and validate a targeted metabolomics method for quantifying specific metabolites in apoptotic bodies.
- To identify potential biomarkers indicative of metabolic differences in apoptotic bodies.
- To elucidate the biological roles of quantified metabolites in cellular processes.
Main Methods:
- Development of a liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry (LC-(Q-Orbitrap)MS) method.
- Optimization involved testing different columns (HILIC selected), gradients, and collision energies.
- Quantitative analysis utilized parallel reaction monitoring (PRM) and method validation included linearity, LOD/LOQ, matrix effects, accuracy, and precision.
Main Results:
- A robust LC-(Q-Orbitrap)MS method was established for the targeted quantification of metabolites in apoptotic bodies.
- Method validation demonstrated high sensitivity with limits of detection ranging from 0.02 to 1.4 ng/mL.
- Nine key metabolites were quantified, providing insights into metabolic differences and biological relevance.
Conclusions:
- The first targeted metabolomics methodology for quantifying metabolites in HK-2 cell-derived apoptotic bodies was successfully developed.
- Specific metabolite concentrations (e.g., pyridoxine, kynurenine, creatine, phenylacetylglycine) were determined in response to cisplatin and UV treatments.
- The quantified metabolite levels provide valuable data for understanding their specific biological roles in apoptosis and proliferation.
