Separation of small metabolites and lipids in spectra from biopsies by diffusion-weighted HR-MAS NMR: a feasibility study

Abstract

High Resolution Magic Angle Spinning (HR-MAS) NMR allows metabolic characterization of biopsies. HR-MAS spectra from tissues of most organs show strong lipid contributions that are overlapping metabolite regions, which hamper metabolite estimation. Metabolite quantification and analysis would benefit from a separation of lipids and small metabolites. Generally, a relaxation filter is used to reduce lipid contributions. However, the strong relaxation filter required to eliminate most of the lipids also reduces the signals for small metabolites. The aim of our study was therefore to investigate different diffusion editing techniques in order to employ diffusion differences for separating lipid and small metabolite contributions in the spectra from different organs for unbiased metabonomic analysis. Thus, 1D and 2D diffusion measurements were performed, and pure lipid spectra that were obtained at strong diffusion weighting (DW) were subtracted from those obtained at low DW, which include both small metabolites and lipids. This subtraction yielded almost lipid free small metabolite spectra from muscle tissue. Further improved separation was obtained by combining a 1D diffusion sequence with a T2-filter, with the subtraction method eliminating residual lipids from the spectra. Similar results obtained for biopsies of different organs suggest that this method is applicable in various tissue types. The elimination of lipids from HR-MAS spectra and the resulting less biased assessment of small metabolites have potential to remove ambiguities in the interpretation of metabonomic results. This is demonstrated in a reproducibility study on biopsies from human muscle.

Metabolic profiling of listeria rhombencephalitis in small ruminants by 1H high‐resolution magic angle spinning NMR spectroscopy

“…High‐resolution magic angle spinning (HR‐MAS) nuclear magnetic resonance (NMR) spectroscopy is an established technique to study small‐molecule metabolites of tissue biopsies . The development of HR‐MAS has enabled the acquisition of well‐resolved spectra directly on intact tissues, such as brain biopsies obtained in vivo, almost reaching the quality of solution NMR spectra by attenuating the effects of line broadening mechanisms due to magic angle spinning .…”

Improvements in lipid suppression for 1H NMR‐based metabolomics: Applications to solution‐state and HR‐MAS NMR in natural and in vivo samples

“…When the relaxation filter is extended to 500 ms with the milk, artefacts appear in the CPMG spectra (Figure ), whereas the spectral line shape is preserved with the adiabatic spinlock. As per Diserens et al, a 400‐μs interpulse delay was used for CPMG, which required 625 × 180 o pulses to achieve a 500‐ms filter. Figure S1 compares increasing the CMPG train from 40 to 800 ms using a 400‐μs interpulse delay.…”

“…However, for samples that are naturally rich in lipids (tissues, various foods, living, and whole organisms), broad 1 H‐NMR signals from the lipids often dominate the aliphatic region of the spectrum. Although these signals contain important information themselves, they overlap with many of the smaller metabolite signals of interest and are problematic in solution‐state and HR‐MAS NMR . Particularly for environmental metabolomics, it would be highly beneficial if experiments can be performed in vivo, since under the current practice of buffer‐based extraction of metabolites, as valuable information could be lost in the sample preparation procedure.…”

“…It is well known that combinations of relaxation filters and diffusion filters can be used to select subcomponents (liquids, gels, and solids) in heterogeneous samples . Diserens et al extended this concept in a pioneering study that specifically targeted lipids in 1 H HR‐MAS of biological tissue . In tissue samples, because lipids are abundant, and many of them have restricted mobility (micelles, vesicles, and membranes), diffusion editing provides a relatively clean “lipid‐only” spectrum with other small metabolites such as carbohydrates and amino acids suppressed.…”

“…It would also be helpful if the lipid suppression technique can be used in standard solution‐state NMR spectroscopy, where the lack of narrowing by MAS can result in broader signals (especially for water due to a range of environments) in natural samples. In this paper, we build upon the pioneering lipid suppression technique proposed by Diserens and co‐workers . Specifically here, (a) the CMPG block is replaced with an adiabatic spinlock (which is more forgiving to pulse miscalibration and permits longer filtering times without J‐modulation); (b) a shaped presaturation W5 WATERGATE (SPR‐W5‐WATERGATE) is integrated for improved water suppression.…”