Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

Summary

Dynamic nuclear polarization with subsequent sample dissolution has enabled real-time studies of metabolism in biological systems. Hyperpolarized [1-¹³C]pyruvate was used to study lactate dehydrogenase activity in a prostate carcinoma cell line in vitro.

Abstract

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have emerged in conjunction with the state-of-the-art positron emission tomography with ¹⁸F-fluorodeoxyglucose ([¹⁸F]-FDG PET). ¹³C magnetic resonance spectroscopic imaging (¹³CMRSI) is a minimally invasive imaging method that enables the monitoring of metabolism in vivo and in real time. As with any other method based on ¹³C nuclear magnetic resonance (NMR), it faces the challenge of low thermal polarization and a subsequent low signal-to-noise ratio due to the relatively low gyromagnetic ratio of ¹³C and its low natural abundance in biological samples. By overcoming these limitations, dynamic nuclear polarization (DNP) with subsequent sample dissolution has recently enabled commonly used NMR and magnetic resonance imaging (MRI) systems to measure, study, and image key metabolic pathways in various biological systems. A particularly interesting and promising molecule used in ¹³CMRSI is [1-¹³C]pyruvate, which, in the last ten years, has been widely used for in vitro, preclinical, and, more recently, clinical studies to investigate the cellular energy metabolism in cancer and other diseases. In this article, we outline the technique of dissolution DNP using a 3.35 T preclinical DNP hyperpolarizer and demonstrate its usage in in vitro studies. A similar protocol for hyperpolarization may be applied for the most part in in vivo studies as well. To do so, we used lactate dehydrogenase (LDH) and catalyzed the metabolic reaction of [1-¹³C]pyruvate to [1-¹³C]lactate in a prostate carcinoma cell line, PC3, in vitro using ¹³CMRSI.

Introduction

Presently, the most widely used clinical method for tumor staging, restaging, treatment response monitoring, and recurrence detection of a wide variety of cancers is [¹⁸F]-FDG PET.¹ However, recently, several novel and alternative approaches have emerged. One of those methods is ¹³CMRSI. This technique involves the introduction of the ¹³C-molecule into a biological sample, followed by minimally invasive MRI to assess the metabolism in vitro or in vivo in real time. Nevertheless, the biggest challenge of ¹³CMRSI, compared to the other methods such as [¹⁸F]-FDG PET or computed tomography....

Protocol

1. Sample Stock Solution Preparation

1. Add gadoterate meglumine (GadM, 0.5 mol/L) to concentrated [1-¹³C]pyruvic acid to give a final concentration of 1-mmol/L GadM. Add trityl radical tris (8-carboxy-2,2,6,6-tetra-(hydroxyethyl)-benzo-[1,2-4,5]-bis-(1,3)-dithiole-4-yl)-methyl sodium salt (OX063) to this mixture to give a final concentration of 15 mmol/L. Vortex until complete dissolution.
   NOTE: This stock solution preparation is designed for usage with a 3.35-T preclinical DNP hyperpolarizer. When a 7-T clinical hyperpolarizer is used, the gadoterate meglumine is not required because, at a higher magnetic field, its benefits are negligible. ....

Results

The results of the “microwave sweep” are illustrated in Figure 3. It shows that the optimal microwave frequency for the [1-¹³C]pyruvate sample is at 94.156 GHz for the local 3.35-T hyperpolarizer. All following hyperpolarization experiment (n = 14) were performed using this microwave frequency with a power of 100 mW. The microwave irradiation was applied for 60 to 80 min, leading to a solid-state hyperpolarization higher than 90%. The results are presented in Figure 4. The hyp.......

Discussion

¹³CMRSI with hyperpolarized probes is a promising method to monitor metabolism in real time in vitro and in vivo. One very important aspect when employing this experimental process is the proper standardization, especially regarding in vitro experiments. First, the preparation of the sample needs to be done properly and consistently to achieve the same concentration of hyperpolarized material in each experiment. This requires a precise weighing of both the sample to be hyperpolarized.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HyperSense DNP Polariser	Oxford Instruments		3.35 T preclinical DNP hyperpolarizer
GE/Agilent MR901	GE Healthcare/Agilent Technologies		7 T preclinical MRI scanner, with small bore designed for experiments onrodent
Spinsolve Carbon	Magritek		1 T NMR spectrometer with permanent magnet
Deuterium Oxide	Sigma Aldrich	7789-20-0
Sodium phosphate dibasic	Sigma Aldrich	7558-79-4
Sodium phosphate monbasic	Sigma Aldrich	7558-80-7
Sodium hydroxide	Sigma Aldrich	1310-73-2
Disodium edetate	Sigma Aldrich	6381-92-6
Pyruvic acid - 13C1	Cambridge Isotopes Laboratories	CLM-8077-1
Dotarem (0.5 mmol/L)	Guerbet		gadoterate meglumine
tris (8-carboxy-2,2,6,6-tetra-(hydroxyethyl)-benzo-[1,2–4,5]-bis-(1,3)-dithiole-4-yl)-methyl sodium salt (OX063)	GE Healthcare		trityl radical used as a sourse of free electron
PC3 cell line	ATCC	CRL1435
F-12K medium	ATCC	30-2004
Fetal Bovine Serum	ATCC	SCRR-30-2020
Trypsine-EDTA Solution, 1x	ATCC	30-2101
Sample plastic cup	Oxford Instruments
Trypan blue	Bio-Rad	145-0013-MSDS