title: Direct dynamic measurement of intracellular and extracellular lactate in small-volume cell suspensions with (13)C hyperpolarised NMR.
abstract: Hyperpolarised (HP) (13)C NMR allows enzymatic activity to be probed in real time in live biological systems. The use of in vitro models gives excellent control of the cellular environment, crucial in the understanding of enzyme kinetics. The increased conversion of pyruvate to lactate in cancer cells has been well studied with HP (13)C NMR. Unfortunately, the equally important metabolic step of lactate transport out of the cell remains undetected, because intracellular and extracellular lactate are measured as a single resonance. Furthermore, typical experiments must be performed using tens of millions of cells, a large amount which can lead to a costly and sometimes highly challenging growing procedure. We present a relatively simple set-up that requires as little as two million cells with the spectral resolution to separate the intracellular and extracellular lactate resonances. The set-up is tested with suspensions of prostate cancer carcinoma cells (PC3) in combination with HP [1-(13)C]pyruvate. We obtained reproducible pyruvate to lactate label fluxes of 1.2 and 1.7 nmol/s per million cells at 2.5 and 5.0 mM pyruvate concentrations. The existence of a 3-Hz chemical shift difference between intracellular and extracellular lactate enabled us to determine the lactate transport rates in PC3. We deduced a lactate export rate of 0.3 s(-1) and observed a decrease in lactate transport on addition of the lactate transport inhibitor α-cyano-4-hydroxycinnamic acid.
authors.affiliation: Department of Radiology and Nuclear Medicine, Radboud University Medical Centre, Nijmegen, the Netherlands.
authors.forename: Vincent
authors.initials: V
authors.lastname: Breukels
authors.affiliation: Department of Urology, Radboud University Medical Centre, Nijmegen, the Netherlands.
authors.forename: Kees C F J
authors.initials: KC
authors.lastname: Jansen
authors.affiliation: Department of Radiology and Nuclear Medicine, Radboud University Medical Centre, Nijmegen, the Netherlands.
authors.forename: Frits H A
authors.initials: FH
authors.lastname: van Heijster
authors.affiliation: Institute of Physics of Biological Systems, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Andrea
authors.initials: A
authors.lastname: Capozzi
authors.affiliation: Institute for Molecules and Materials, Radboud University, Nijmegen, the Netherlands.
authors.forename: P Jan M
authors.initials: PJ
authors.lastname: van Bentum
authors.affiliation: Department of Urology, Radboud University Medical Centre, Nijmegen, the Netherlands.
authors.forename: Jack A
authors.initials: JA
authors.lastname: Schalken
authors.affiliation: Institute of Physics of Biological Systems, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Comment
authors.affiliation: Department of Radiology and Nuclear Medicine, Radboud University Medical Centre, Nijmegen, the Netherlands.
authors.forename: Tom W J
authors.initials: TW
authors.lastname: Scheenen
mesh_terms: D001692:Biological Transport; D066241:Carbon-13 Magnetic Resonance Spectroscopy / Q000379:methods*; D002452:Cell Count; D045744:Cell Line, Tumor; D045604:Extracellular Fluid / Q000378:metabolism*; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism*; D019344:Lactic Acid / Q000378:metabolism*; D008297:Male; D011471:Prostatic Neoplasms / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D019344:Lactic Acid; D019289:Pyruvic Acid
keywords: PC3; dynamic nuclear polarization; enzyme kinetics; hyperpolarized carbon-13; lactate; monocarboxylate transporter