title: Quantitative13C Traces of Glucose Fate in Hepatitis B Virus-Infected Hepatocytes.
abstract: Quantitative characterization of13C-labeled metabolites is an important part of the stable isotope tracing method widely used in metabolic flux analysis. Given the long relaxation time and low sensitivity of13C nuclei, direct measurement of13C-labeled metabolites using one-dimensional13C NMR often fails to meet the demand of metabolomics studies, especially with large numbers of samples and metabolites having low abundance. Although HSQC-based 2D NMR methods have improved sensitivity with inversion detection, they are time-consuming and thus unsuitable for high-throughput absolute quantification of13C-labeled metabolites. In this study, we developed a method for absolute quantification of13C-labeled metabolites using naturally abundant TSP as a reference with the first increment of the HMQC pulse sequence, taking polarization transfer efficiencies into consideration. We validated this method using a mixture of13C-labeled alanine, methionine, glucose, and formic acid together with a mixture of alanine, lactate, glycine, uridine, cytosine, and hypoxanthine, which have natural13C abundance with known concentrations. We subsequently applied this method to analyze the flux of glucose in HepG2 cells infected with hepatitis B virus (HBV). The results showed that HBV infection increased the cellular uptake of glucose, stimulated glycolysis, and enhanced the pentose phosphate and hexosamine pathways for biosynthesis of RNA and DNA and nucleotide sugars to facilitate HBV replication. This method saves experimental time and provides a possibility for absolute quantitative tracking of the13C-labeled metabolites for high-throughput studies.
authors.affiliation: State Key Laboratory of Genetic Engineering, Zhongshan Hospital and School of Life Sciences, Fudan University , Shanghai International Centre for Molecular Phenomics, Collaborative Innovation Center for Genetics and Development, Shanghai 200438, China.
authors.forename: Qianfen
authors.identifier: 0000-0003-2483-0087
authors.initials: Q
authors.lastname: Wan
authors.affiliation: CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences , Wuhan 430071, China.|Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310058, China.
authors.forename: Yulan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Genetic Engineering, Zhongshan Hospital and School of Life Sciences, Fudan University , Shanghai International Centre for Molecular Phenomics, Collaborative Innovation Center for Genetics and Development, Shanghai 200438, China.
authors.forename: Huiru
authors.initials: H
authors.lastname: Tang
mesh_terms: D002247:Carbon Isotopes; D005947:Glucose / Q000032:analysis* / Q000378:metabolism; D056945:Hep G2 Cells; D006515:Hepatitis B virus / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism* / Q000821:virology*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure
chemical_list: D002247:Carbon Isotopes; C000615229:Carbon-13; D005947:Glucose