Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR

Summary

The sensitivity enhancement provided by dissolution dynamic nuclear polarization (DNP) enables following metabolic processes in real time by NMR and MRI. The characteristics and performances of a dedicated dissolution DNP setup designed for study enzymatic reactions are discussed.

Abstract

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of metabolic transformations. Hyperpolarization via dissolution DNP circumvents part of the sensitivity issues thanks to the large out-of-equilibrium nuclear magnetization stemming from the electron-to-nucleus spin polarization transfer. The high NMR signal obtained can be used to monitor chemical reactions in real time. The downside of hyperpolarized NMR resides in the limited time window available for signal acquisition, which is usually on the order of the nuclear spin longitudinal relaxation time constant, T₁, or, in favorable cases, on the order of the relaxation time constant associated with the singlet-state of coupled nuclei, T_LLS. Cellular uptake of endogenous molecules and metabolic rates can provide essential information on tumor development and drug response. Numerous previous hyperpolarized NMR studies have demonstrated the relevancy of pyruvate as a metabolic substrate for monitoring enzymatic activity in vivo. This work provides a detailed description of the experimental setup and methods required for the study of enzymatic reactions, in particular the pyruvate-to-lactate conversion rate in presence of lactate dehydrogenase (LDH), by hyperpolarized NMR.

Introduction

Dynamic nuclear polarization (DNP),^1,2 a technique designed to enhance the nuclear spin polarization, i.e., the imbalance between 'up' and 'down' spin populations (P = [N_↑ - N_↓] / [N_↑ + N_↓]), was first introduced in the 1950's. Nuclear spins such as ¹³C can be polarized up to P = 10^-1 in favorable conditions, typically at a temperature on the order of 1 K and in a magnetic field of 3.357 T.^3,4 A breakthrough for biological applications came in the early 2000's with the development of dissolution DNP which consists in di....

Protocol

NOTE: All data analysis was performed using commercial software.

1. Prepare the Polarizing Solution

1. Prepare 2 ml of a 1.12 M ¹³C-labeled sodium pyruvate (Na⁺[CH₃-CO-¹³COO]^-, substrate) solution doped with 33 mM of TEMPOL radical (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl, polarizing agent)⁴ in 2:1 D₂O/d₆-ethanol for ¹³C observations. CAUTION: Handling precautions must be taken due to the flammable nature of d₆-ethanol.
2. Verify that the radical concentration is optimal in terms of achieved polarization.
   1. Change slightl....

Results

NMR signal gains using dissolution DNP
The DNP effect consists in the transfer of the high polarization of unpaired electron spins, typically from stable radical molecules, to NMR-active nuclei, under microwave irradiation of the sample. The most often-used free radicals are TAM(OXO63) and TEMPOL.⁴ Polarization procedures using TEMPOL may be optimized by 'cross-polarization'.²⁵

Optimizing the concentrat.......

Discussion

The critical points of the dissolution DNP NMR experiment are: (i) the level of polarization attained for the substrate, which determines the lowest product concentration necessary for experiments as well as the number of signal acquisitions that can be performed and (ii) the lifetimes of magnetization, compared to the duration of the transfer between the polarization and the detection sites and to the rate of substrate transformation. The injection system of the dissolution DNP setup herein described allows for sample t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DNP polarizer	Vanderklink s.a.r.l (Switzerland)	///	Cryostat and electronic equipment for sample polarization
Vacuum system components	Edwards vacuum (France)	Various	- turbomolecular pumping setup - membrane pumping setup - high capacity roots pumping system - vacuum fittings and components
DNP 3.35T Magnet	Bruker (France)
500 MHz NMR Spectrometer	Bruker (France)
Origin 8.0	OriginLab (US)		Data analysis software
Chemicals
SODIUM PYRUVATE-1-13C, 99 ATOM % 13C	Sigma Aldrich (France)	490709
ETHANOL-D6, ANHYDROUS, 99.5 ATOM % D	Sigma Aldrich (France)	186414
4-Hydroxy-TEMPO 97%	Sigma Aldrich (France)	176141
Deuterium oxide	Sigma Aldrich (France)	151882
reduced nicotinamide adenine dinucleotide (NADH)	Sigma Aldrich (France)
ethylene-diaminetetraacetic acid (EDTA)	Sigma Aldrich (France)
dithiothreitol (DTT)	Sigma Aldrich (France)
phosphate buffer, pH = 7.0	Sigma Aldrich (France)
LDH enzyme in	Sigma Aldrich (France)	L-2500
bovine serum albumin, BSA	Sigma Aldrich (France)