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

Abstract

Proton nuclear magnetic resonance (NMR) spectra of intact biological samples often show strong contributions from lipids, which overlap with signals of interest from small metabolites. Pioneering work by Diserens et al. demonstrated that the relative differences in diffusivity and relaxation of lipids versus small metabolites could be exploited to suppress lipid signals, in high‐resolution magic angle spinning (HR‐MAS) NMR spectroscopy. In solution‐state NMR, suspended samples can exhibit very broad water signals, which are challenging to suppress. Here, improved water suppression is incorporated into the sequence, and the Carr‐Purcell‐Meiboom‐Gill sequence (CPMG) train is replaced with a low‐power adiabatic spinlock that reduces heating and spectral artefacts seen with longer CPMG filters. The result is a robust sequence that works well in both HR‐MAS as well as static solution‐state samples. Applications are also extended to include in vivo organisms. For solution‐state NMR, samples containing significant amount of fats such as milk and hemp hearts seeds are used to demonstrate the technique. For HR‐MAS, living earthworms (Eisenia fetida) and freshwater shrimp (Hyalella azteca) are used for in vivo applications. Lipid suppression techniques are essential for non‐invasive NMR‐based analysis of biological samples with a high‐lipid content and adds to the suite of experiments advantageous for in vivo environmental metabolomics.

High-Resolution Magic Angle Spinning (HR-MAS) NMR-Based Fingerprints Determination in the Medicinal Plant Berberis laurina

“…Additionally, in order to improve spectral resolution [ 18 ], the HR-MAS technique is to be applied to the swollen sample in a suitable NMR solvent that provides some molecular motions [ 19 , 22 ]. In addition to the liquid state, HR-MAS NMR has been used in metabolic analyses in human, plant, and food stuff quality managements, genotype, phenotype, and organism cataloging, with interindividual comparisons, environmental toxicity, and pollution [ 24 , 25 , 26 , 27 , 28 , 29 , 30 ]. Moreover, crowded spectral overlaps and chemical structure elucidations can be facilitated by mapping all homo- and heteronuclear correlations through multidimensional (nD) NMR approaches [ 31 ].…”

Ex vivo Comprehensive Multiphase NMR of whole organisms: A complementary tool to in vivo NMR

“…However, the only drawback of using the F 0 generation is the over emphasized lipid signal. While this is not ideal, as lipids often mask other interesting signals [ 67 ], all components are labelled during 3 weeks of growth [ 14 ] and F 0 generations are commonly used successfully in vivo response studies [ 36 , 65 , 66 ]. As such, the use of F 0 vs. F 1 will likely come down to the cost of raising multiple generations of organisms, which will be driven by the reproduction rate, and food consumption rate of the organisms.…”

Understanding the Fate of Environmental Chemicals Inside Living Organisms: NMR-Based 13C Isotopic Suppression Selects Only the Molecule of Interest within 13C-Enriched Organisms

“…In this case, a DIPSI block is applied that acts as a T1ρ filter, during which signals from large molecules such as proteins preferentially dephase because of relaxation. The additional advantage of using a spin-lock such as DIPSI over the more common CPMG (Car–Purcell–Meiboom–Gill) approach is reduced RF heating and reduced J -modulation . In addition, if the Δ* delays (Figure A) are incremented, the experiment becomes a 1 H– 1 H 2D TOCSY experiment that has been 13 C filtered.…”

“…For 13 C isotopic suppression, an X filter and a double testing for adjacent nuclei with a gyration operator (TANGO) were used. For relaxation filtering a decoupling in the presence of scalar interactions (DIPSI) block was employed. , In addition, the sequence was also concatenated with 1 H shaped presaturation SPR-W5 WATER suppression by GrAdient Tailored Excitation (WATERGATE) to facilitate suppression of the wide water profile that is associated with in vivo biological specimens. ,,, The W5-WATERGATE segment was made from two delays alternating with nutations for tailored excitation (DANTE) blocks using 125 μs binomial delays (sidebands at 12 and −2 ppm). Furthermore, presaturation was applied via a continuous train of 2 ms rectangular pulses applied to the water during the relaxation delay .…”

“…The additional advantage of using a spin-lock such as DIPSI over the more common CPMG (Car−Purcell− Meiboom−Gill) approach is reduced RF heating and reduced J-modulation. 31 In addition, if the Δ* delays (Figure 1A) are incremented, the experiment becomes a 1 H− 1 H 2D TOCSY experiment that has been 13 C filtered. While not explicitly required in this study, such an implementation could be very useful if additional 1 H− 1 H correlations are required for spectral assignment of 1 H− 12 C species remaining after 13 C filtering.…”