High-Resolution Diffusion and Relaxation Edited One- and Two-Dimensional ¹H NMR Spectroscopy of Biological Fluids

Abstract

A new approach to the characterization of biomolecules in whole biological fluids is presented based on simplification of 1H NMR spectra by utilizing differences in molecular diffusion coefficients alone and combinations of relaxation and diffusion parameters. New NMR pulse sequences incorporating both spectral editing features together with solvent water resonance elimination are presented. The methods are exemplified using whole human blood plasma, and it is shown that it is possible to obtain NMR spectra of the slowly diffusing species (generally large molecules) by diffusion editing, the slowly relaxing species (generally small molecules) by spin relaxation editing, or spectra showing any range of molecular mobility using a combination of the two methods. The diffusion-based editing methods are also applicable to the selection of resonances in two-dimensional NMR spectroscopy of biofluids, and we show this for the first time by the production of 1H-1H diffusion-edited TOCSY spectra of human blood plasma where the resonance intensities are weighted according to the molecular diffusion coefficient. In this case, by measuring a diffusion-edited 1H-1H TOCSY NMR spectrum of plasma, it is possible to obtain signals from only the macromolecular components, and this may be of benefit in the analysis of blood lipoproteins. In complex biofluids, the combination of diffusion and relaxation editing brings about considerable spectral simplification leading to an easier resonance assignment process. We also demonstrate the production of 1H NMR spectra with intensities corresponding to diffusion coefficient rather than number of protons, and this opens up new possibilities for pattern recognition classification of samples based on altered molecular mobility features of biofluid components.

NMR spectroscopy – a modern analytical tool for serum analytics of lipoproteins and metabolites

“…By suppressing small metabolites during the measurement, it was possible for the first time to generate spectra of lipoproteins that did not have to be isolated from the serum first. Rather, any number of spectra were created based on the different mobility of the molecules by way of a suitable combination of relaxation and diffusion-edited NMR spectroscopy [7] . Furthermore, the diffusion coefficients of some metabolites were determined simultaneously in a single serum sample using two-dimensional homonuclear TOCSY (total correlated spectroscopy) [10] .…”

HRMAS NMR spectroscopy applications in agriculture

“…1), which may not only further improve spectral resolution, but also select coherence pathways and permit diffusion experiments to differentiate molecular species according to their degree of mobility in hydrated matrices. Moreover, edited (namely "filtered") pulse sequences, such as T 2 -and diffusion-based sequences, can be used in HRMAS systems [10][11][12]. This allows HRMAS spectroscopy to provide information on both polar and non-polar components of biological tissues or agrofood products without preliminary separations though extractions.…”

Analysis of biofluids and chemical mixtures in non-deuterated solvents with1H diffusion-weighted PFG phase-sensitive double-quantum NMR spectroscopy

“…3 Recently this methodology has undergone a revival. [4][5][6][7][8][9][10][11][12][13][14][15][16][17] This has been made possible by the introduction in high-resolution NMR of actively shielded gradient coils and by the development of 2D and 3D diffusion-ordered spectroscopy (DOSY) experiments. Actively shielded gradient coils greatly reduce the magnitude of eddy currents which can be induced by the rapid switching of magnetic field gradients.…”

“…The extension to three dimensions 10 has the advantage of removing overlap which is present in the 1D spectra. Typically a COSY, 10 NOESY 12 or TOCSY 13,15,17 sequence is appended to a stimulated-echo scheme. A series of 2D experiments are recorded with incrementation of the amplitude of the two diffusion gradients (G d 3 .…”