Direct dynamic measurement of intracellular and extracellular lactate in small‐volume cell suspensions with ¹³C 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.

Metabolic Studies of Tumor Cells Using [1‐13C] Pyruvate Hyperpolarized by Means of PHIP‐Side Arm Hydrogenation

“…The rate constant k PL reflects the LDH activity and the membrane transport of lactate and pyruvate, while the colorimetric assay is carried out on cells lysate. Several studies have been carried out using HP metabolites that allowed to discriminate between the intra and extracellular metabolites (pyruvate and lactate), and to gain information about the transport speed through the cells membrane, in cells cultures and in‐vivo …”

Correlation of hyperpolarized 13C‐MRI data with tissue extract measurements

“…This is different from previous results in cells using a different tumour model, 26 as well as a number of other studies demonstrating that 13 C-lactate production is dominated by the exchange of label (i.e., with the endogenous lactate pool) rather than the net conversion of pyruvate into lactate. 26,28,38,41,42 Based on the present results, the exchange-based mechanism appears to be largely determined FIGURE 6 Examples of high-resolution NMR spectra are shown. Note that, despite differences in the signal amplitude, satelliting [3-13 C]Lac peaks correspond to approximately 10% 13 C fractional enrichment of lactate.…”

Pyruvate cellular uptake and enzymatic conversion probed by dissolution DNP‐NMR: the impact of overexpressed membrane transporters

“…Similar enhancement values were obtained in other recent studies of in-cell pyruvate metabolic transformation. [15] These experiments used D 2 O for sample dissolution, which does not impact on the final value of polarization enhancement obtained at the exit from the polarizer but slightly favors the relaxation times of pyruvate carbon C-1 during transport, compared to the use of water. Pyruvate C-1 relaxation time constants have values ranging between 45 and 65 s [26] depending on the main magnetic field.…”

“…[11] These kinetics are driven by (i) the cell membrane crossing rate of pyruvate and (ii) the enzymatic conversion rate of pyruvate to lactate. [12,13,15] Previous studies drew comparisons between lactate buildup rates in cells with membrane pyruvate transporters inhibited or not. [12][13][14] The purpose of the present study is to contribute to the clarification of the role of membrane transporters in the observed kinetics, revealing the impact of their expression level on the observed pyruvate-to-lactate transformation kinetics.…”