Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Summary

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

Abstract

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

Introduction

Murine models have been utilized extensively in brain research¹. Genotype-phenotype correlations have been investigated in mouse and rat brains by studying gene expression at the RNA and/or protein levels on the one hand, and morphological, functional, electrophysiological and/or behavioral phenotypes on the other^2-6. However, to completely understand the mechanisms linking phenotype to genotype, it is imperative to investigate the molecular events downstream of protein expression, i.e. the metabolism of the biochemical substrates upon which enzymes act⁷. This requirement led, over the past 10 to 15 years, to a renaissance of ....

Protocol

NOTE: ANIMAL ETHICS STATEMENT
Animal studies on rats followed the guidelines valid in France, and were approved by the local Ethics Committee (#40.04, University of Aix-Marseille Medical School, Marseille, France).

1. Harvesting and Freezing Rat Brain

1. Prepare items required: liquid nitrogen (N_2liq.) in Dewar that is large enough to keep a freezing clamp (at least 2-3 L volume); anesthetic (e.g., isoflurane, or ketamine/xylazine); anesthesia chamber; sterile dissection tools: surgical scissors, scalpel, forceps; tissue wipes and bottle with cleaning alcohol (ethanol); needles (25 G); 1 ml and 10 ml ....

Results

To obtain best resolution in metabolic NMR spectra of brain and other tissue extracts, it has long been common practice to remove or mask metal ions (most importantly: paramagnetic ions) present in extract solutions. This has been achieved either by adding a chelating agent such as EDTA or CDTA to the extract¹⁹, or by passing the extract through an ion exchange resin such as Chelex-100²⁰. The results presented in Figure 1 demonstrate that this step is not necessary for ¹H.......

Discussion

NMR spectroscopy is an efficient method for measuring concentrations of chemical compounds in solution in a very reproducible and accurate manner. However, to obtain high-quality data it is necessary to adhere to certain rules concerning sample preparation and analysis. In the determination of metabolite concentrations by NMR spectroscopy, neither the generation nor the reception of the NMR signal dominates the quantitation error, unless the intensity of an observed signal approaches the detection threshold (particularly.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Isoflurane	Virbac	Vetflurane	Anesthetic for animals
Isoflurane vaporizer	Ohmeda	Isotec 3	Newer model available: Isotec 4
Scalpel, scissors, forceps, clamps	Harvard Apparatus Fisher Scientific	various various	Surgical equipment for animals
Freeze-clamp tool	homebuilt	n/a	Tong with aluminium plates, to be inserted in liquid nitrogen for cooling
Dewar	Nalgene	4150-4000
Liquid nitrogen	Air Liquide	n/a
Nitrogen gas	Air Liquide	n/a
Nitrogen evaporator	Organomation Associates	N-EVAP 111	Can be replaced by homebuilt device
Mortar	Sigma-Aldrich	Z247472
Pestle	Sigma-Aldrich	Z247510
Tissue homogenizer	Kinematica	Polytron	With test tubes fitting homogenizer shaft
Electronic scale	Sartorius	n/a
Methanol	Sigma-Aldrich	M3641
Chloroform	Sigma-Aldrich	366910
Glass centrifuge tubes	Kimble	45500-15, 45500-30	Kimax 15 ml, 30 ml tube
Microcentrifuge tubes	Kimble	45150-2	Kimax 2 ml tube; should replace "Eppendorf" tube if compatible with centrifuge rotor
Polystyrene pipettes	Costar Corning	Stripettes	5 and 10 ml volumes
Deuterochloroform	Sigma-Aldrich	431915	99.96% deuterated
Deuterium oxide	Sigma-Aldrich	423459	99.96% deuterated
Deuterium chloride	Alpha Aesar	42406	20% in deuterium oxide
Sodium deuteroxide	Sigma-Aldrich	164488	30% in deuterium oxide
Lyophilizer	Christ	Alpha 1-2
Cold centrifuge	Heraeus	Megafuge 16R
pH meter	Eutech Cybernetics	Cyberscan
CDTA	Sigma-Aldrich	D0922
Cesium hydroxide	Sigma-Aldrich	516988
NMR tubes	Wilmad	528-PP
NMR stem coaxial insert	Sigma-Aldrich	Z278513	By Wilmad
NMR pipettes	Sigma-Aldrich	Z255688
Pipettes	Eppendorf	Research	With tips for volumes from 0.5 to 1,000 μl
Pipet-Aid	Drummond	XP
NMR spectrometer	Bruker	AVANCE 400	including probe and other accessories
NMR software	Bruker	TopSpin 1.3	newer version available: Topspin 3.2
Water-soluble standard compounds	Sigma-Aldrich	various
Phospholipid standard compounds	Avanti Polar Lipids Doosan Serdary Sigma-Aldrich	various various various	Source for plasmalogens, but may be <70 - 80% purity
Methylenediphosphonate	Sigma-Aldrich	M9508
TSP-d4	Sigma-Aldrich	269913