Methods for liquid- and solid-state CP-MAS NMR spectroscopy of untreated tissue biopsies.
We describe a method for NMR analysis of rapidly cooled or frozen biopsies and report its use on rat liver. Since the metabolic state of the biopsies can be expected to reflect the in vivo state, the method may be used as an alternative to the traditional examination of a perchloric acid extract of the biopsy. Perfusion-cooling of rat liver was applied as an efficient means of preserving the metabolic state. This method ensures very rapid cooling without interruption of the oxygen supply to the liver, and the results show that biopsies taken subsequently maintain energy metabolites near in vivo concentrations for at least 40-60 min. High resolution, natural abundance 13C liquid-state NMR spectroscopy could be carried out within this time frame on the untreated biopsy. In addition, the biopsy was frozen for 13C cross polarization-magic angle spinning solid-state spectroscopic examination, which was carried out at -40, -100, and -150 degrees C. The solid-state spectra allowed analysis of the relative glycogen content of the intact liver tissue, which showed good correlation with chemically measured glycogen on the same samples. Furthermore it was observed that the C1-carbon of glycogen in all liver samples splits into two resonances (5.4 ppm apart) in the solid state but not in the liquid state. This suggests that two conformational states of glycogen are populated, with rapid equilibration in the liquid state but no equilibration in the frozen state.
Quistorff
B
B
NMR Center, Panum Institute, University of Copenhagen, Denmark.
Frye
J S
JS
Bock
K
K
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001706:Biopsy; D002247:Carbon Isotopes; D003080:Cold Temperature; D005947:Glucose / Q000378:metabolism; D007511:Ischemia / Q000378:metabolism; D008099:Liver / Q000098:blood supply / Q000737:chemistry* / Q000378:metabolism; D008112:Liver Glycogen / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008297:Male; D010477:Perfusion; D051381:Rats; D017208:Rats, Wistar
D002247:Carbon Isotopes; D008112:Liver Glycogen; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D005947:Glucose