INTRODUCTION

Chemical Exchange Saturation Transfer (CEST) imaging presents the potential to in vivo probing of metabolic pathways. However, clinical implementation of the CEST method for metabolic imaging requires overcoming several technical obstacles, including: (1) the direct saturation of water that occurs for saturation of exchangeable protons with very small chemical shifts with respect to water; (2) the possible unintended saturation of other labile protons at the same resonance frequency; and (3) the inability to measure those labile protons in the compound that undergo very fast chemical exchange with water protons. To address these challenges, we propose to advance the development of CEST by promoting the analysis of the effect of J-couplings of homonuclear Hartmann–Hahn through-bond polarization transfer between aliphatic protons that are one to two bonds away from the target labile proton, rather than directly saturating the labile one. This novel J-coupling mediated CEST method would permit the specific targeting of metabolites with labile protons that are not accessible by traditional CEST methods. We tested this concept with desmosine (DES), a unique, pyridinium-containing amino acid that is generated from allysine and lysine by lysyl oxidases during the cross-linking and formation of mature elastin within the extracellular matrix in the lung. During elastolytic processes, DES can be released into the extracellular milieu and taken up by the circulation. When sampled in the blood and urine, DES can serve as an important biomarker for certain lung diseases.

METHODS

NMR spectra of 19mM desmosine (EMD Millipore Corp, USA) solution in PBS was measured on a Bruker 600MHz AVANCE HD III system at 5°C. Ultra-fast CEST Z-spectra (UFZ) were measured to confirm magnetization transfers between -NH in DES and water. Homonuclear J-coupling through-bond polarization transfers between -CH and -NH protons were measured by 2D total correlation spectroscopy (TOCSY). The CEST effects on the water signal intensities (WI) due to magnetization transfers between -NH and water protons were measured by using 1D selective TOCSY with irradiation frequencies centered respectively on the resonances of -CH protons, which are upfield from water resonance (WIup), and on the spectral points that are equal distances but downfield from the water resonance (WIdown), with mixing time between 10 and 300 ms. The value (WIdown-WIup)/WIdown was calculated to represent the J-coupling polarization transfer initiated CEST effect.

RESULTS

The regular CEST effects due to magnetization transfers between -NH protons in DES and water protons can be seen in Fig 1, where the z-spectra are compared with a spectrum of DES to indicate the resonance frequencies. The J-coupling induced through-bond polarization transfer between aliphatic -CH protons and the labile -NH protons can be observed in the 2D TOCSY in Fig 2. The above defined water resonance intensity values (WIdown-WIup)/WIdown measured with a 50 ms mixing time, clearly indicate 10-30% signal reductions when -CH protons (at 3.91, 3.84 and 3.76 ppm) that are one bond away from their corresponding -NH protons are excited compared with either control excitation frequencies (4.24, 3.42, and 2.49 ppm) or resonance frequencies of -CH protons that are more than one bond away (3.03 and 2.19 ppm). Representative (WIdown-WIup)/WIdown values as functions of mixing times are presented in Fig 3.

DISCUSSION

Our data presented here suggest the potential of testing changes in water resonance intensities due to a CEST effect from labile protons that are polarized through J-couplings following the irradiation of their neighboring aliphatic protons. The observed different effects on the changes of water intensities, seen when comparing irradiation of immediate neighboring aliphatic protons with those separated by multiple bonds, suggest that the observed TOCSY-CEST effect cannot be simply interpreted as due to a NOE effect. While our data demonstrate the potential of this new concept for CEST, more investigations are needed to evaluate the feasibility and challenges of this approach for in vivo imaging. Currently, we are employing this new method to quantify pathologic processes in models of lung disease.

CONCLUSION

Our proposal of developing J-coupling mediated CEST has the potential to overcome the obstacles currently experienced by traditional CEST in its clinical implementations. Our data presented here support further developments and investigations in this direction.

Acknowledgements

We gratefully acknowledge the support of the Massachusetts General Hospital Athinoula A. Martinos Center for Biomedical Imaging.

References

No reference found.