Introduction

One of the most promising, yet least delivering MRI approach is maybe an in vivo magnetic resonance spectroscopy (MRS). MRS provides a non-invasive window into an in vivo biochemistry – basically a virtual biopsy. Still, its application in medical diagnostics is restricted: routine spectroscopy is typically limited to spatial, chemical and temporal resolutions of cubic centimetres, mM and minutes.
There is a need for a new and simple MRS method that will provide full spectroscopic information about metabolites or other molecules in vivo.
Hyperpolarization techniques boost the signal of isotopically labelled metabolites and can provide requested spectroscopic information, however, it also requires a polarizer, administration of hyperpolarized substrate, specialized imaging sequences and an x-nuclei channel for the MRI system.
Here, we present a novel method to selectively prepare and detect the signal of endogenous metabolites.

Methods

We use singlet-state encoded magnetic resonance (SISTEM) spectroscopy (fig. 1A and 3A) to selectively prepare the signal of metabolites for background free MRS. The pulse sequence utilizes the unique properties of long-lived singlet spin states (LLSs): (i) long live-time; (ii) two-spin quantum state; (iii) preservation of LLS under the action of broad-band decoupling. The double spin states of LLS are not destroyed by saturation pulses (fig. 1A) as other spin-orders are and can be isolated using double quantum filters (fig. 3A). The metabolite N-acetylalanine (NAA) was chosen as a test system.
The method was tested on a 600 MHz spectrometer (Bruker Avance II) and 7 T MRI with a 30 cm bore (Bruker 7/30 ClinScan), equipped with a ¹H transmit-receive volume coil with an inner diameter of 8 cm for excitation and a single-loop pick-up coil with an inner diameter of 0.9 cm for acquisition.
As a test system a solution of 10 mM NAA in 600 μl D₂O (MS1) or in 300 μl D₂O mixed with 300 μl dairy cream (30% fat concentration, MS2) was used for experiments with NMR and a solution of 100 mM NAA in 1500 μl H₂O (MS3) was used for MRI studies.

Results

Two MRS sequences SISTEM-1,2 were successfully implemented on a high resolution 600 MHz NMR and preclinical 7 T MRI systems (fig. 1-3). The optimal value for SISTEM-1 (or SARKAR-II)¹ pulse sequence (fig. 1A) for NAA-CH₂ protons was found to be ca. 10 ms; sequence does not require precise settings (fig. 1B). The LLS was successfully populated and a lifetime of 6.5 s at 14.1 T and 2.5 kHz WALTZ-16 decoupling was obtained, that is almost 6 times longer than T₁ ≈ 1 s (fig. 1C). This long lifetime of LLS and preservation under the action of hard RF-pulses allows saturating all other spins with a broad-band decoupling. This is a more efficient spectrum editing method than T₁-weighted/differential approach.³
Without any dedicated fat or water suppression, only a very little NAA signal can be detected by a pulse-acquisition experiment (fig. 2B). Using SISTEM-1, however, strong suppression of fat and H₂O signal was achieved. This enabled an increase of the linear receiver gain, rg, by a factor of 320 (fig. 2C). It was realised in an inhomogeneous magnetic field (here: FWHM ca. 27 Hz).
Due to limited allowed power deposition in the tissue, SISTEM-2 instead of SISTEM-1 sequence was used in MRI experiments. In SISTEM-2, the broadband decoupling and singlet-to-magnetization transfer stages are replaced by an OPSYd (fig. 3A)². When applied, both water and NAA-CH₃ signals were strongly suppressed, while the strong NAA-CH₂ signal was observed (fig. 3B, C).
Zero-quantum coherences excited in SISTEM experiments evolve during the time period t₂ with the frequency equal to chemical shift difference (fig. 3A,D). By acquisition of a series of SISTEM-2 spectra with different t₂ and a consequent Fourier transformation, we were able to precisely determine the NAA-CH₂ chemical shift difference of ca. 52.7+-0.4 Hz (fig. 3E). Due to severe magnetic field inhomogeneity of MRI this is impossible in a simple pulse-acquisition experiment.
pH dependence of NAA spectrum was revealed by a high-resolution NMR (fig. 4). NAA-CH and NAA-CH₂ protons are much more sensitive to pH variation than usually observed singlet of NAA-CH₃.

Discussion

SISTEM allows separation of targeted long-lived signals by suppression of all the rest signals; the application may be to image the biodistribution of metabolites and drugs such as ethosuximide (Zarontin).
pH measurement may be another application. The frequency of SISTEM modulated signal is equal to the chemical shift difference (fig. 3). The value of the chemical shift difference is a function of pH and for NAA-CH₂ it changes from 0.2 ppm at pH 6 and above to 0.02 ppm at pH 3 (fig. 4). Hence, without a need for an external reference and correction of a magnetic field homogeneity and susceptibility, we were able to measure pH of model solutions.

Conclusion

We showed that LLS can be used to selectively prepare and measure the signals of coupled protons of chosen metabolites or drugs in the presence of water, inhomogeneous field and highly concentrated fatty solutions; before T₁ contrast was used predominantly to improve spectral resolution and therefore quickly relaxing spins were not accessible. Non-invasive measurement of pH value, imaging of drug distribution are some of the possible applications.

Acknowledgements

We acknowledge support by the Emmy Noether Program “metabolic and molecular MR” (HO 4604/2-2), the research training circle “materials for brain” (GRK 2154/1-2019), DFG - RFBR grant (HO 4604/3-1, № 19-53-12013), the Cluster of Excellence “Inflammation at Interfaces” (EXC306), BMBF grant e:Med Systems Medicine Initiative “Try-IBD” (01ZX1915C), the Cluster of Excellence “precision medicine in inflammation” (PMI 1267). Kiel University and the Medical Faculty are acknowledged for supporting the Molecular Imaging North Competence Center (MOIN CC) as a core facility for imaging in vivo. MOIN CC was founded by a grant from the European Regional Development Fund (ERDF) and the Zukunftsprogramm Wirtschaft of Schleswig-Holstein (Project no. 122-09-053).