Tissue targeted metabonomics: metabolic profiling by microdialysis sampling and microcoil NMR

Abstract

The concentration of low molecular weight compounds in tissues can yield valuable information about the metabolic state of an organism. Studies of changes in the metabolic state or metabonomics can reflect disease pathways, drug action, or toxicity. This research aims to develop a new approach, tissue targeted metabonomics. Microdialysis sampling and microcoil NMR analysis are employed to compare basal and ischemic metabolic states of various tissues (blood, brain, and heart) of Sprague-Dawley rats. Microdialysis sampling is localized, making the metabolic profile tissue specific. Coupling to NMR analysis is highly advantageous, because a complete metabolic profile is obtained in a single spectrum. However, small sample volumes and low analyte concentrations make analysis of microdialysis samples challenging. Microcoil NMR uses low sample volumes and has improved mass sensitivity, relative to standard 5 mm probes. The coupling of these techniques is a potentially powerful tool for metabonomics analysis.

Fig. 1

Spectral comparison of whole plasma and plasma dialysate from the same rat analyzed by NMR. (A) Plasma dialysate (50% D₂O with TSP) analyzed by microcoil NMR. (B) Whole plasma (20% D₂O) analyzed with a triple-axis gradient probe.

Fig. 2

Expansion of the heart dialysate spectra before and during myocardial ischemia to show the aliphatic (A) and aromatic (B) spectral regions. For each set of spectra, the upper spectrum was measured before ischemia and the bottom spectrum was measured using dialysis samples collected during ischemia. The depletion of glucose occurs during ischemia as the heart rapidly utilizes its tissue stores to meet the heart’s large energy demand. Shifts were observed in the aromatic region during ischemia, suggesting that pH fluctuations may have occurred.

Fig. 3

Plasma dialysate before (A) and during (B) myocardial ischemia. Changes in the levels of lactate (1.40 ppm), acetate (2.05 ppm), and acetone (2.15 ppm) are observed during ischemia. The asterisk (*) indicates internal standard resonances. Arrows mark key metabolic changes observed during ischemia.

Fig. 4

Brain dialysate before (A) and during (B) myocardial ischemia. Note the appearance of glycerol (3.4–3.8 ppm) and succinate (2.2 ppm) during ischemia and changes in propionate (1.2 ppm) and lactate (1.3 ppm) levels, demonstrating that brain tissue metabolism is affected by myocardial ischemia. Arrows mark key metabolic changes observed during ischemia.