NMR-Based Identification of Metabolites in Polar and Non-Polar Extracts of Avian Liver

Abstract

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 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 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.

Quantitative 1H NMR Metabolomics Reveal Distinct Metabolic Adaptations in Human Macrophages Following Differential Activation

“…All NMR spectra were collected at 298 K (25 °C) using a Bruker 600 MHz ( 1 H Larmor frequency) AVANCE III solution NMR spectrometer, equipped with a SampleJet automatic sample loading system, a 5-mm triple resonance ( 1 H, 15 N, 13 C), liquid-helium-cooled three-channel inverse (TCI) detection NMR cryoprobe, and Topspin software (Bruker version 3.2). Then, 1D 1 H NMR spectra acquisition was performed using the Bruker-supplied excitation sculpting (ES)-based ‘zgesgp’ pulse sequence [71,72], and NMR spectra were recorded with 256 scans and a 1 H spectral window of 9615.38 Hz. Free induction decays (FIDs) were collected with 32K data points and a dwell time interval of 52 µsec, amounting to a data acquisition time of 1.7 s. Recovery delay (D1) times between acquisitions were set to 1 s, resulting in an overall 2.7 s relaxation recovery delay between scans [73,74].…”

An Optimization of Liquid–Liquid Extraction of Urinary Volatile and Semi-Volatile Compounds and Its Application for Gas Chromatography-Mass Spectrometry and Proton Nuclear Magnetic Resonance Spectroscopy

“…The DCM (LLE) can extract metabolites with a range of polarities (particularly relatively non polar compounds), however it would not be expected to be that efficient in extracting polar compounds, such as ionic compounds [38]. The literature data related to non-polar compound analysis from biological fluids and tissues is very limited, usually including on the 1 H NMR spectra a general description of the groups of compounds [39]. In another report, the urine extracts were dissolved in different deuterated solvents (MeOD, DMSO, DMF, MeCN, Acetone, CDCl 3 and DCM), with subsequent monitoring of the levels of five metabolites: hippurate, creatinine, lactate, histidine and alanine [38].…”

Plasma metabolomic profiling of healthy pregnant mares and mares with experimentally induced placentitis

“…Immediately prior to analysis, plasma samples were thawed at room temperature. Two hundred µL of plasma was added to 400 µL of 10% deuterium oxide (Sigma Aldrich) (D 2 O) and 90% water containing 0.1-mmol/L trimethylsilyl propionate (Sigma Aldrich) (TSP), centrifuged at 3500 g for 10 minutes and the supernatant transferred 13,[23][24][25] Preliminary identification based on those sources was then confirmed with 2D spectral analysis. To perform quantitative comparisons between mares, individual time points and groups, a second method of integration was performed in which metabolitespecific peaks were manually integrated and weighted to the TSP integral.…”