Breukels, V., et al., Direct dynamic measurement of intracellular and extracellular lactate in small-volume cell suspensions with (13)C hyperpolarised NMR. NMR Biomed, 2015. 28(8): p. 1040-8.
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 alpha-cyano-4-hydroxycinnamic acid.
[NMR paper] Probing the intracellular glutathione redox potential by in-cell NMR spectroscopy.
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Angew Chem Int Ed Engl. 2014 Jan 7;53(2):447-50
Authors: Rhieu SY, Urbas AA, Bearden DW, Marino JP, Lippa KA, Reipa V
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Non-invasive and real-time analysis of cellular redox processes has been...
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A detailed characterization of an NMR flow probe for use in direct-injection sample analysis is presented. A 600-MHz, indirect detection NMR flow probe with a 120-microl active volume is evaluated in two configurations: first as a stand-alone small volume probe for the...
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[NMR paper] Direct measurement of angles between bond vectors in high-resolution NMR.
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Angles between two interatomic vectors are measured for structure elucidation in solution nuclear magnetic resonance (NMR). The angles can be determined directly by using...
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[NMR paper] Direct measurement of angles between bond vectors in high-resolution NMR.
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Biochemistry. 1990 Mar 13;29(10):2617-22
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Direct dynamic measurement of intracellular and extracellular lactate in small-volume cell suspensions with (13)C hyperpolarised NMR
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