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Quantitative characterization of glutaminolysis in human plasma using liquid chromatography-tandem mass spectrometry

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Abstract

Glutaminolysis is the metabolic pathway that lyses glutamine to glutamate, alanine, citrate, aspartate, and so on. As partially recruiting reaction steps from the tricarboxylic acid (TCA) cycle and the malate-aspartate shuttle, glutaminolysis takes essential place in physiological and pathological situations. We herein developed a sensitive, rapid, and reproducible liquid chromatography-tandem mass spectrometry method to determine the perturbation of glutaminolysis in human plasma by quantifying 13 involved metabolites in a single 20-min run. A pHILIC column with a gradient elution system consisting of acetonitrile-5 mM ammonium acetate was used for separation, while an electrospray ionization source (ESI) operated in negative mode with multiple reaction monitoring was employed for detection. The method was fully validated according to FDA’s guidelines, and it generally provided good results in terms of linearity (the correlation coefficient no less than 0.9911 within the range of 0.05–800 μg/mL), intra- and inter-day precision (less than 18.38%) and accuracy (relative standard deviation between 89.24 and 113.4%), with lower limits of quantification between 0.05 and 10 μg/mL. The new analytical approach was successfully applied to analyze the plasma samples from 38 healthy volunteers and 34 patients with type 2 diabetes (T2D). Based on the great sensitivity and comprehensive capacity, the targeted analysis revealed the imperceptible abnormalities in the concentrations of key intermediates, such as iso-citrate and cis-aconitate, thus allowing us to obtain a thorough understanding of glutaminolysis disorder during T2D.

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Acknowledgments

The authors would like to thank the subjects for their participation.

Funding

This study was financially supported by the National Natural Science Foundation of China (No. 81403181), the Natural Science Foundation of Heilongjiang Province of China (No. QC2016109), and the Open Project Program of the MOE Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical University) (No. DQCP2017MS02).

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Author notes

  1. Yunfei Hua and Xuping Yang contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, Ministry of Education, Tongjiaxiang No. 24, Nanjing, 210009, Jiangsu, China

    Yunfei Hua, Xuping Yang, Ruiting Li, Linrui Li, Xia Yuan, Yuan Tian, Zunjian Zhang & Yin Huang

  2. Department of Neurology, the Second Affiliated Hospital, Harbin Medical University, Harbin, 150001, Heilongjiang, China

    Peifang Liu & Peijia Liu

  3. Key Laboratory of Myocardial Ischemia, Harbin Medical University, Ministry of Education, Harbin, 150001, Heilongjiang, China

    Peifang Liu & Yin Huang

  4. Wuxi Center for Drug Safety Control, Wuxi, 214028, Jiangsu, China

    Xiaoyi Hua

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  1. Yunfei Hua
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Correspondence to Zunjian Zhang or Yin Huang.

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All procedures performed in studies involving human participants were following the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

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Hua, Y., Yang, X., Li, R. et al. Quantitative characterization of glutaminolysis in human plasma using liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem 411, 2045–2055 (2019). https://doi.org/10.1007/s00216-019-01626-3

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  • DOI: https://doi.org/10.1007/s00216-019-01626-3

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