Dynamic UltraFast 2D EXchange SpectroscopY (UF-EXSY) of hyperpolarized substrates
Graphical abstract
Introduction
In the fields of chemistry and biology, multidimensional Nuclear Magnetic Resonance (NMR) acquisitions, which differentiate and correlate the resonances arising from individual sites onto multiple frequency axes, are commonly used to study structure, dynamics, reaction states, proteins, the chemical environment of molecules, or any other sample that contains nuclei possessing spin [1], [2], [3]. These experiments are intrinsically longer than their conventional one-dimensional (1D) counterparts. In general, 2D NMR techniques are limited by the inherent low sensitivity, resulting in acquisition times on the order of minutes to hours [1]. In carbon-13 NMR, this is particularly pronounced where less than 1% of carbon atoms possess the NMR detectable 13C isotope. Moreover, SNR suffers from an intrinsically lower gyromagnetic ratio of the 13C isotope.
Not surprisingly, there has been an increased interest in using nuclei in the ‘hyperpolarized’ state, whose spin population differences depart significantly from the ≈10−5 Boltzmann distribution. Dynamic Nuclear Polarization (DNP) yields over a 10,000-fold increase in SNR [2], which is far greater than what can be achieved by multiscan signal averaging. Hyperpolarization with its dramatic increase in sensitivity provides a unique opportunity to probe previously undetectable phenomena via NMR.
Signal detection of hyperpolarized substrates, however, is challenging due to nonrenewable longitudinal magnetization and short-lived signals. These challenges make conventional 2D NMR acquisition strategies incompatible with hyperpolarized substrates. Specifically, conventional acquisitions schemes for multidimensional NMR require an array of scans that are identical to one another aside from the serial incrementing of evolution delays. Given the non-renewable polarization and the shortened acquisition times due to signal decay by T1, 2D NMR acquisitions with hyperpolarized substrates requires sequence modifications.
Shapiro and Frydman proposed a method for thermally polarized samples, where the serial indirect domain t1 encoding of 2D NMR is replaced by a parallelized procedure allowing for different positions within a sample for inequivalent evolution times [4]. Then Frydman and Blazina extended this method to hyperpolarized substrates [1]. We propose a similar method utilizing their parallelized approach as the foundation for this work. The method presented here differs in several aspects: (1) it uses a symmetric slice selective excitation rather than a gradient acting in combination with a frequency-swept excitation for preparation and (2) it uses dephasing and rephasing gradients rather than an oscillating field gradient. The acquisition and reconstruction presented here relies on principles of phase accrual, which expands on our recently described 1D method Metabolic Activity Decomposition with Simulated Echo Acquisition Mode (MAD-STEAM) [5].
The key advancement presented in this work is its application to dynamic 2D EXchange SpectroscopY (EXSY) of hyperpolarized carbon-13 substrates, which we show can be used to detect bidirectional exchange. Exchange is particularly important, where both preclinical cell and animal studies of hyperpolarized substrates [6], [9], as well as the first-in-man clinical trial [10], have focused heavily on the exchange of hyperpolarized metabolites as markers of disease.
Magnetic Resonance Spectroscopy of hyperpolarized substrates provides a new tool for investigating tissue metabolism and kinetics in vivo [11], [12]. Previously experiments using MAD-STEAM showed that in addition to increased conversion of pyruvate-to-lactate in tumors, the less studied conversion of lactate-to-pyruvate was significantly smaller in tumors compared to normal tissue with a transgenic model of prostate cancer [13], consistent with a decreased LDH-B expression and increased monocarboxylate transporter 4 (MCT4) and LDH-A expression. However, the rate as measured can be corrupted by alanine-to-pyruvate and hydrate-to-pyruvate conversion, warranting a method to separate these signals to reveal the origin of this change.
Investigation of bidirectional flux and exchange has a number of applications to the metabolism field such as reductive carboxylation [14], [15], lipogenesis and its regulation of citrate and α-ketoglutarate [16], glutamine addiction [17], [18], [19], [20], gluconeogenesis, and the isoenzyme composition of LDH. Detection of these pathways has diagnostic and biomedical research potential. For instance, the directionality of reactions within the citric acid cycle has become an area of increased interest as reductive carboxylation has been shown to support tumor growth [14]. However, the signal from hyperpolarized experiments reports only on the bulk spin-exchange and cannot differentiate concomitant spin-exchange. 2D NMR techniques for hyperpolarized substrates could be further used to probe directionality of metabolic pathways. Moreover, 2D NMR could provide improved specificity to cancer metabolism and shed light on exchange and flux of hyperpolarized substrates.
Section snippets
Acquisition
Conventional dynamic EXSY acquisition schemes necessitate renewable longitudinal magnetization not available in hyperpolarized substrates. Additionally, conventional EXSY acquisition schemes require many repetitions to obtain the entire indirect spectral dimension (Fig. 1a). The dynamic UF (ultra fast)-EXSY pulse sequence is rapid and does not require renewable longitudinal magnetization making it ideal for hyperpolarized substrates (Fig. 1b). Key features include the symmetric slice selection
NMR experiments
These studies were conducted on a 14.1T wide-bore microimaging spectrometer equipped with 100 G/cm gradients and a 10 mm broadband probe (Agilent Technologies). The sequence shown in Fig. 1b was acquired with Δτ = 8.575 ms, tphase = 52 μs, Gphase = 5 G/cm, tcrush = 10 ms, Gcrush = 15 G/cm, Necho = 3, TM = 1–2 s temporal resolution, NTM = 5–20 repetitions, Δz = 3 mm, 20° flip, 64 spectral points, and 4006 Hz bandwidth. Noise was subtracted to remove cross peak artifacts. T2 signal loss between echoes was small and
Validation
The method was validated with a Bloch simulator (SpinBench, Heartvista, Palo Alto, CA) and with hyperpolarized phantom experiments where the hydration of pyruvate and LDH enzymatic activity were observed dynamically (Fig. 3a). To further validate the technique, pyruvate was saturated after allowing hyperpolarized pyruvate to be converted to hyperpolarized lactate via the LDH enzyme and cofactor NADH (Fig. 3a). As expected, only the conversion of lactate-to-pyruvate was observed.
In Fig. 3b, we
Conclusions
In this work, we present a new UltraFast method for acquiring dynamic 2D EXchange SpectroscopY (UF-EXSY) within a single acquisition using phase accrual. The presented dynamic UF-EXSY pulse sequence is rapid and does not require renewable longitudinal magnetization making it ideal for hyperpolarized substrates. This method overcomes the three main challenges associated with 2D NMR of hyperpolarized substrates: (1) 2D NMR experiments are time intensive, (2) longitudinal magnetization is not
Acknowledgments
The authors acknowledge Dr. Kayvan Keshari and Mark Van Criekinge for their development of the bioreactor system as well as Dr. Michael Lustig and Peter Shin for discussions on optimization, Drs. John Pauly and Adam Kerr for discussions on stimulated echoes, and Dr. Christian Frezza for discussions of abnormal cancer metabolism in renal cell carcinomas. The DNP polarizers and related infrastructure were supported by an NIH center Grant (P41EB013598). This study was also supported by NIH Grants
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