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

The overall goal of this procedure is to obtain high resolution metabolite profiles from rat brain extracts. This is accomplished by first harvesting rat brains and snap freezing these in liquid nitrogen. The second step is to extract brain metabolites by homogenizing tissue in methanol chloroform water, followed by removal of denatured proteins and organic solvents.

Next, the aqueous samples are lyophilized and all samples are resol in well-defined solvents. For optimal NMR spectroscopic analysis, the final step is to carefully set up and optimized metabolomic NMR experiments. Ultimately, NMR Spectra are required under well-defined experimental conditions and evaluated using optimized data processing protocols.

Though this method can provide insight into brain metabolism, it can also be applied to other soft tissues such as tumors, liver, kidney, and other organs. First, remove a previously prepared frozen brain tissue sample from the minus 80 degrees Celsius freezer. Then immediately transfer the tissue sample to a mortar partially filled with liquid nitrogen.

Use a liquid nitrogen cold pestle to break the frozen brain tissue into smaller pieces that easily fit into the test tubes used for tissue homogenization. Following this mix, the small pieces of frozen brain tissue thoroughly weigh out 250 to 350 milligrams of the tissue and transfer it to a test tube filled with a total of four milliliters of ice cold methanol. Every time pieces of frozen tissue are added to the test tube, homogenize these immediately with the tissue homogenizer.

After the last piece of frozen rat brain sample has been added to the test tube and homogenized, transfer the homogenate to a greater than or equal to 20 milliliter glass vial. Close the screw cap and place the homogenate on ice for 15 minutes. Next, add four milliliters of ice cold chloroform to the homogenate and vortex thoroughly.

When finished, set the homogenate on ice for 15 minutes. Add four milliliters of water to the homogenate after vortexing thoroughly let the homogenate stand at minus 20 degrees Celsius overnight. For complete phase separation, transfer the brain tissue homogenate solution to a greater than or equal tube 20 milliliter chloroform resistant centrifuge tube and centrifuge the sample at 13, 000 G and four degrees Celsius for 40 minutes.

Following centrifugation, use a paster pipette to transfer the upper phase to an appropriate 15 milliliter methanol resistant tube. Then use a fresh paster pipette to transfer the lower phase to an appropriate, greater than or equal tube 15 milliliter chloroform resistant tube, and place it on ice while keeping the tube with the water methanol phase on ice. Evaporate the methanol by directing a dry nitrogen stream into the extract solution.

Terminate the evaporation process when nitrogen bubbling. No longer causes volume reduction in the extract solution. After placing the tube with the methanol chloroform phase on ice, evaporate the methanol by directing a dry nitrogen stream onto the surface of the extract solution.

When all of the solvent is evaporated, close the tube and keep the sample at minus 80 degrees Celsius until ready for NMR analysis. To prepare the aqueous phase for lyophilization, transfer the aqueous sample to a thoroughly rinsed 50 milliliter centrifuge tube. Then freeze the extract solution by rotating the centrifuge tube in liquid nitrogen such that the inner surface of the tube is progressively covered by the frozen liquid.

When all the liquid is frozen, cover the tube with a punctured screw cap to allow the vapor to escape and place the tube in a wide neck vacuum filter bottle. After attaching the bottle to the freeze dryer, start the lyophilization, terminate the lyophilization process. When the sample is entirely dry, keep the sample at minus 80 degrees Celsius in a closed tube until used for NMR analysis.

For phosphorus NMR analysis of phospholipids, dissolve the dried lipids in 700 microliters of a five to four to one mixture of deuterated chloroform methanol, and a 200 millimolar aqueous CDTA solution. Transfer the sample to a micro centrifuge tube using a direct displacement micro pipette with a chloroform resistant tip. At this point, set the temperature regulation of the NMR probe to the desired target value, which is usually 25 degrees Celsius centrifuge.

The phospholipid extract solution at four degrees Celsius and 11, 000 G for 30 minutes to spin down the solid residues in the sample. When finished, transfer 600 microliters of the super natin to a high quality NMR tube with a five millimeter outer diameter. Prepare the appropriate coaxial insert stem filled with an aqueous 20 millimolar methylene diphosphate solution at pH 7.0 for chemical shift referencing and quantitation.

Place this insert in the NMR tube filled with the phospholipid extract solution. Next, fit the NMR tube with the appropriate spinner and transfer it to the NMR magnet. Spin the sample at 15 to 20 hertz after waiting until the sample has adjusted to the set temperature.

Carefully minimize the magnetic field in homogeneity across the sample by adjusting the on axis and off axis shim coil currents set the NMR spectrum acquisition parameters to the optimal values, which may vary as a function of magnet field strength. Once the spectrum acquisition has finished, process the free induction decay using optimized parameters to obtain the best results for the whole range of phospholipid resonances. Repeat processing using at least two different filtering procedures.

Finally filter very weak and broad signals without significant overlap by ization to increase the signal to noise ratio. In this experiment, extremely narrow spectral lines were obtained for all spectral regions analyzed even in very crowded regions. Peaks at a distance of less than 0.01 P PP M are almost completely separable at 400 megahertz.

As a consequence, qualitative and quantitative analysis of the numerous small, but currently unassigned peaks can be envisaged in the future line narrowing by choosing 1000 millimolar instead of 200.Millimolar. CDTA was desired but resulted in superposition of phospholipid signals line narrowing by choosing 277 Kelvin instead of 297. Kelvin was desired but also resulted in superposition of phospholipid signals.

A decrease in CDTA concentration from 200 millimolar to 50 millimolar only results in a modest increase in line width and in almost no change in chemical shifts. However, note that the tissue concentration in the 50 millimolar CDTA extract is half as high as it is in the 200 millimolar CDTA extract. Explaining the relatively narrow lines, a large number of quantifiable phospholipids were detected covering a considerable concentration range.

Spectral resolution is even sufficient to routinely detect partial splitting of certain peaks. As a consequence, qualitative and quantitative analysis of further phospholipid subgroups can be envisaged in the future. While attempting this procedure, it's important to remember to carry out each single step with extreme care to guarantee optimal metabolite analysis.