title: Phenotyping hepatocellular metabolism using uniformly labeled carbon-13 molecular probes and LC-HRMS stable isotope tracing.
abstract: Metabolite stable isotope tracing is a powerful bioanalytical strategy that has the potential to unravel phenotypic markers of early pharmaceutical efficacy by monitoring enzymatic incorporation of carbon-13 atoms into targeted pathways over time. The practice of probing biological systems with carbon-13 labeled molecules using broad MS-based screens has been utilized for many years in academic laboratories but has had limited application in the pharmaceutical R&D environment. The goal of this work was to establish a LCMS analytical workflow that was capable of monitoring carbon-13 isotope changes in glycolysis, the TCA and urea cycles, and non-essential amino acid metabolism. This work applies a standardized protein precipitation with 80% cold methanol and two distinct reverse-phase ion-pair liquid chromatography methods coupled to either a positive- or negative-ion mode high-resolution accurate mass spectrometry screening method. The data herein combines thousands of single-point peak integrations into a novel metabolite network map as a visualization aid to probe and monitor stable isotope incorporation in murine hepatocytes using uniformly labeled (13)C6 glucose, (13)C3 lactate, and (13)C5 glutamine. This work also demonstrates that nitrogen metabolism may have a large influence on the TCA cycle and gluconeogenic carbon fluxes in hepatocyte cell culture.
authors.affiliation: Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer, Inc., 558 Eastern Point Road, Groton, CT 06340, USA.
authors.forename: John K
authors.initials: JK
authors.lastname: Meissen
authors.affiliation: Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer, Inc., 558 Eastern Point Road, Groton, CT 06340, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Pirman
authors.affiliation: Cardiovascular and Metabolic Diseases Research Unit, Pfizer, Inc., 610 Main Street, Cambridge, MA 02139, USA.
authors.forename: Min
authors.initials: M
authors.lastname: Wan
authors.affiliation: Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer, Inc., 558 Eastern Point Road, Groton, CT 06340, USA.
authors.forename: Emily
authors.initials: E
authors.lastname: Miller
authors.affiliation: Cardiovascular and Metabolic Diseases Research Unit, Pfizer, Inc., 610 Main Street, Cambridge, MA 02139, USA.
authors.forename: Aditi
authors.initials: A
authors.lastname: Jatkar
authors.affiliation: Cardiovascular and Metabolic Diseases Research Unit, Pfizer, Inc., 610 Main Street, Cambridge, MA 02139, USA.
authors.forename: Russell
authors.initials: R
authors.lastname: Miller
authors.affiliation: Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer, Inc., 558 Eastern Point Road, Groton, CT 06340, USA.
authors.forename: Rick C
authors.initials: RC
authors.lastname: Steenwyk
authors.affiliation: Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer, Inc., 558 Eastern Point Road, Groton, CT 06340, USA. Electronic address: matthew.blatnik@pfizer.com.
authors.forename: Matthew
authors.initials: M
authors.lastname: Blatnik
mesh_terms: D000818:Animals; D002247:Carbon Isotopes / Q000032:analysis / Q000737:chemistry*; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D006019:Glycolysis; D022781:Hepatocytes / Q000378:metabolism*; D013058:Mass Spectrometry; D055442:Metabolome / Q000502:physiology; D015335:Molecular Probes / Q000737:chemistry*; D051381:Rats
chemical_list: D002247:Carbon Isotopes; D015335:Molecular Probes
keywords: Central carbon metabolism; Cytoscape; High resolution mass spectrometry (HRMS); Metabolic Flux; Metabolomics; Nitrogen metabolism