Quantitative ¹³C Traces of Glucose Fate in Hepatitis B Virus-Infected Hepatocytes

Abstract

Quantitative characterization of C-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 ofC nuclei, direct measurement of C-labeled metabolites using one-dimensionalC 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 of C-labeled metabolites. In this study, we developed a method for absolute quantification ofC-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 of C-labeled alanine, methionine, glucose, and formic acid together with a mixture of alanine, lactate, glycine, uridine, cytosine, and hypoxanthine, which have naturalC 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 the C-labeled metabolites for high-throughput studies.

A framework for tracer-based metabolism in mammalian cells by NMR

“…HSQC spectra offer at least two options for the measurement of label incorporation: (i) Measuring intensities from metabolite spin systems, which rise with the amount of label incorporation. This approach requires an unlabeled reference spectrum, and to derive absolute concentrations, it is also necessary to calibrate for different metabolites owing to variable coupling constants (see 18 and Materials and Methods ). (ii) Analysis of the complex multiplet patterns in the indirect 13 C-dimension arising from scalar couplings ( 1 J CC couplings) between adjacent 13 C-atoms.…”

Quantitative Isotopomer Rates in Real‐Time Metabolism of Cells Determined by NMR Methods

“…The maximum filter leakage is 2% when the filter periods are mis-set by +/-50Hz from the ideal values for a particular 1 H{ 13 C} resonance. When coupling constants are known, editing becomes a more realistic option for the quantification of label incorporations as the overall transfer function can be calculated based on the 1 JCH and T2 transverse relaxation times, as recently shown by Wan et al for 1D-HMQC [19] . However, this requires precise knowledge of these parameters.…”

“…The two most common analytical technologies in this context are mass spectrometry (MS) [1] and Nuclear Magnetic Resonance (NMR) spectroscopy. NMR is becoming an increasingly important method in this context [2] as it has the ability to quantify site-specific label incorporation and is suitable for detecting metabolism in primary patient cells placed in an NMR tube t measure metabolism in real-time [2][3][4] and to analyze labelled cell extracts in reasonable highthroughput [5][6][7][8][9][10] . NMR offers several options to measure 13 C or 15 N isotope incorporation.…”

“…However it is not clear what level of accuracy can be achieved owing to overlap with 13 C-coupled 1 H-signals in the undecoupled spectrum. Alternatively, Wan et al [19] suggested to calculate accurate concentrations from 1D-1 H-13 C-HSQC spectra using observed intensities and the pulse sequence specific transfer function. This is only possible if the 1 JCH coupling constant and relaxation rates are known which is unfortunately not the case for most metabolites.…”

Metabolomic Approaches to Study Chemical Exposure-Related Metabolism Alterations in Mammalian Cell Cultures

“…As life is built upon carbon chemistry, carbon-13 is a stable isotope tracer frequently used in NMR and MS metabolomics studies [ 69 , 70 , 71 ]. In NMR, individual 13C atoms can be tracked through isotopomers, i.e., isomers that differ only by the position of 13C, and followed over chemical transformations of metabolic pathways.…”