Magic Angle Spinning NMR and ¹H−³¹P Heteronuclear Statistical Total Correlation Spectroscopy of Intact Human Gut Biopsies

Abstract

Previously we have demonstrated the use of 1H magic angle spinning (MAS) NMR spectroscopy for the topographical variations in functional metabolic signatures of intact human intestinal biopsy samples. Here we have analyzed a series of MAS 1H NMR spectra (spin-echo, one-dimensional, and diffusion-edited) and 31P-{1H} spectra and focused on analyzing the enhancement of information recovery by use of the statistical total correlation spectroscopy (STOCSY) method. We have applied a heterospectroscopic cross-examination performed on the same samples and between 1H and 31P-{1H} spectra (heteronuclear STOCSY) to recover latent metabolic information. We show that heterospectroscopic correlation can give new information on the molecular compartmentation of metabolites in intact tissues, including the statistical "isolation" of a phospholipid/triglyceride vesicle pool in intact tissue. The application of 31P-1H HET-STOCSY allowed the cross-assignment of major 31P signals to their equivalent 1H NMR spectra, e.g., for phosphorylcholine and phosphorylethanolamine. We also show pathway correlations, e.g., the ascorbate-glutathione pathway, in the STOCSY analysis of intact tissue spectra. These 31P-1H HET-STOCSY spectra also showed different topographical regions, particular for minor signals in different tissue microenvironments. This approach could be extended to allow the detection of altered distributions within metabolic subcompartments as well as conventional metabonomics concentration-based diagnostics.

Analytic Properties of Statistical Total Correlation Spectroscopy Based Information Recovery in 1H NMR Metabolic Data Sets

“…There are now numerous applications of STOCSY and closely related covariance techniques for enhanced information recovery from low dimensional NMR data sets on multiple and single samples [3][4][5][6][7][8][9][10][11][12][13] . The STOCSY approach has been applied to the structural assignment problem in a variety of NMR metabolic profiling contexts, such as deconvolution of overlapped chromatographic peaks in LC-NMR 7 , delineation of drug metabolism in molecular epidemiology studies 5 and separation of different molecular signatures in diffusion edited spectroscopy 6 .…”

Use of 1H and 31P HRMAS to evaluate the relationship between quantitative alterations in metabolite concentrations and tissue features in human brain tumour biopsies

“…Moreover, in this study, the acquisition of 2D 1 H-1 H TOCSY and 1 H-31 P HSQC correlation spectra has enabled the identification of the spin systems for the main metabolites in 31 P spectra and has confirmed the assignments with the homonuclear and the heteronuclear correlations. Previously it had been demonstrated that the metabolic information could be improved by the combination of 1D 1 H and 31 P spectra, constructing afterwards a kind of virtual 2D heteronuclear correlation [18,39]. This 1D heteronuclear strategy has been applied in human to the study of gut biopsies to detect different tissue microenvironments [18] and in rat liver for the detection of druginduced hepatotoxicity [39].…”

“…Previously it had been demonstrated that the metabolic information could be improved by the combination of 1D 1 H and 31 P spectra, constructing afterwards a kind of virtual 2D heteronuclear correlation [18,39]. This 1D heteronuclear strategy has been applied in human to the study of gut biopsies to detect different tissue microenvironments [18] and in rat liver for the detection of druginduced hepatotoxicity [39]. The study here presented, has shown the feasibility of 1 H-31 P HSQC spectra acquisition on human brain tumour tissue with signal to noise ratio adequate to observe the signals from the main metabolites identified in the 31 Additionally, a remarkable advantage of the 31 P spectra is that allow an estimation of the pH inside the tissue samples, that relies in the change in the chemical shift of some phosphorylated metabolites present in the living tissues.…”

“…In humans, 1 H and 31 P 1D HRMAS experiments have been used to study the changes in cervical cancer and have showed a higher content of PC and PE in cancer tissue compared to normal tissue [17]. The combined use of 1 H and 31 P 1D HRMAS experiments has been also reported in human samples of gut biopsies [18] to reveal changes between different tissue microenvironment using 1D 1 H and 31 P spectra to construct virtual 2D heteronuclear correlation spectra that connect all protons on the molecule to the heteroatom [18]. It has been observed that in the ex vivo 31 P spectra of tissues there are no significant signals from the high-energy phosphates (ATP or PCr) due to the rapid metabolism either during the sample preparation or during the spinning [16,17].…”

Metabonomic investigations into the global biochemical sequelae of exposure to the pancreatic toxin 1‐cyano‐2‐hydroxy‐3‐butene in the rat

“…[22] We have previously shown that Statistical TOtal Correlation SpectroscopY (STOCSY) analysis of biofluid samples can be a highly informative structural assignment aid for drugs and endogenous metabolites, [23,24] and that STOCSY approaches can also be used to obtain structural information in ongoing chemical reactions, [25] in LC-NMR separations [26] and even to provide differential compartmental information in MAS-NMR studies on intact tissues. [27,28] Here, we show a new use for the STOCSY approach to investigate differential pathway correlations between xenobiotic and endogenous metabolites to evaluate drug metabolite toxicity and detoxification processes from biofluid profiles.…”