Metabolic Profiling of the Effects ofd-Galactosamine in Liver Spheroids Using1H NMR and MAS-NMR Spectroscopy

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Abstract
We report here the combined application of 1H magic angle spinning (MAS) and high-resolution NMR spectroscopy and pattern recognition methods to study the effects of a model toxin (d-galactosamine) in liver spheroid cultures. 1H NMR spectra of metabolic profiles of spheroids showed closer similarities to intact liver spectra than those of isolated hepatocytes, suggesting their superiority as an in vitro model system. Batches of spheroids were prepared from male Sprague Dawley rat livers and incubated in control hepatocyte medium or medium containing d-galactosamine (4 or 20 mM) for 4 or 24 h. Intact spheroids were packed into rotors and analyzed using MAS-NMR spectroscopy or homogenized and analyzed using conventional 1H NMR spectroscopy. Principal components analysis, (PCA), of the NMR data revealed separation of control and d-galactosamine-treated spheroids based on changes in the concentrations of the triglycerides and elevations in cholesterol and esters. The absence of cholesterol in hepatocytes and the relative under-representation of the lipid resonances offer an important advantage of spheroids over hepatocytes for the 1H NMR studies of fatty liver. Orthogonal signal correction (OSC) was used as a data filter to remove non-dose-dependent variation from the NMR spectra, improving the classification of treated spheroids and controls. This work shows that useful metabolic information can be obtained on drug toxicity by the use of combined MAS-NMR and high-resolution NMR of liver spheroids and that such studies may enhance the validation of in vitro techniques against in vivo models for metabolic profiling.

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