Metabolites present in liver provide important clues regarding the physiological state of an organism.The aim of this work was to evaluate a protocol for high-throughput NMR-based analysis of polar and non-polar metabolites from a small quantity of liver tissue.We extracted the tissue with a methanol/chloroform/water mixture and isolated the polar metabolites from the methanol/water layer and the non-polar metabolites from the chloroform layer.Following drying, we re-solubilized the fractions for analysis with a 600 MHz NMR spectrometer equipped with a 1.
7 mm cryogenic probe.In order to evaluate the feasibility of this protocol for metabolomics studies, we FENUGREEK - BLESSED THISTLE analyzed the metabolic profile of livers from house sparrow (Passer domesticus) nestlings raised on two different diets: livers from 10 nestlings raised on a high protein diet (HP) for 4 d and livers from 12 nestlings raised on the HP diet for 3 d and then switched to a high carbohydrate Sweatpants diet (HC) for 1 d.The protocol enabled the detection of 52 polar and nine non-polar metabolites in 1H NMR spectra of the extracts.We analyzed the lipophilic metabolites by one-way ANOVA to assess statistically significant concentration differences between the two groups.
The results of our studies demonstrate that the protocol described here can be exploited for high-throughput screening of small quantities of liver tissue (approx.100 mg wet mass) obtainable from small animals.