The provision of J-coupling information along the J-coupling dimension aids metabolite identifications because J-couplings are insensitive to physiological factors (such as temperature or pH value) versus chemical shifts 16. A proton-decoupled 1D spectrum can be obtained from the skyline projection along the chemical shift dimension, facilitating metabolite assignments and quantifications. This approach separates chemical shifts and J-couplings into two different spectral dimensions. The 2D J-resolved ( JRES) 1H NMR spectroscopy 15 can yield a 2D spectrum with efficient acquisitions for complex metabolite mixtures. However, the applications of most 2D NMR methods are generally limited by their longer acquisition time. Moving from 1D to 2D 1H NMR spectroscopy is a natural solution for spectral congestion because 2D 1H NMR provides more molecular information and more accurate metabolite specificities. This limitation poses a considerable challenge for unique identification and quantification of metabolites. However, due to the limited range of proton chemical shifts and a large number of resonances from various metabolites, spectral congestion, even severe overlapping of spectral peaks, is generally encountered in 1D NMR spectra of tissue extracts 13 and biological fluids 14. Advantages of 1D 1H NMR approaches include (a) relatively rapid spectral acquisition and (b) direct measurement of metabolite concentrations using a single internal standard 12. To date, 1D 1H NMR spectroscopy is one of the most popular techniques for metabolite studies. For example, proton ( 1H) NMR spectra of biological fluids (such as blood, plasma or urine) are rich in metabolic information and thus are useful for studying endogenous metabolic changes caused by drug toxicities or diseases 10, 11. Benefiting from the high-throughput information relevant to biochemical and biological processes and from the intrinsic noninvasiveness involved in its measurements, NMR spectroscopy has been successfully applied in various fields, including functional toxicology 5, 6, environmental science 7, 8 and nutrition studies 9. NMR spectroscopy has been proven as a powerful tool for metabolite analyses of biological samples 1, 2, 3, 4. Furthermore, this method also can be applied to measurements of semisolid and viscous samples. It provides a significant contribution to metabolite analyses of biological samples and may be potentially applicable to in vivo samples. This method is a previously-unreported high-resolution 2D J-resolved spectroscopy for biological applications without specialised hardware requirements or complicated sample pretreatments. Metabolite analyses for a postmortem fish from fresh to decayed statuses are presented to further reveal the capability of the proposed method. A dramatic improvement in spectral resolution is evident in our contrastive demonstrations on a sample of pig brain tissue. In this study, we propose an NMR method to achieve high-resolution J-resolved information for metabolite analyses directly from intact biological samples. Due to the observed macroscopic magnetic susceptibility in biological tissues, current NMR acquisitions in measurements of biological tissues are generally performed on tissue extracts using liquid NMR or on tissues using magic-angle spinning techniques. NMR spectroscopy is a commonly used technique for metabolite analyses.
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