An assessment of the effects of sample ischaemia and spinning time on the metabolic profile of brain tumour biopsy specimens as determined by high-resolution magic angle spinning (1)H NMR.
High-resolution magic angle spinning (HRMAS) (1)H NMR of biopsy tissue provides a biochemical profile that has potential diagnostic and prognostic value, and can aid interpretation of the lower-resolution (1)H-NMR spectra obtained in vivo. However, the biochemical profile obtained may be affected by experimental factors such as a period of ischaemia before snap-freezing of the biopsy tissue for subsequent analysis and the mechanical stress of the spinning procedure of HRMAS itself. We have used normal rat brain cortex as a 'gold standard', either funnel-frozen or deliberately allowed to become ischaemic for set periods of time before snap-freezing, to quantitatively investigate these two effects. In addition, we have compared biochemical changes that occur in normal rat brain during HRMAS (spun continuously at 5 kHz for 4 h at 4 degrees C as could be required for a two-dimensional acquisition) with those that occur in biopsy samples from low-grade and high-grade adult human astrocytomas, during the same HRMAS procedure. Significant changes due to delayed initial sample freezing were noted in metabolites associated with glycolysis (alanine, glucose and lactate), as expected. However, for the funnel-frozen rat tissue at 4 degrees C, there were even more significant changes, which appear to be the result of extended spinning at 5 kHz. In particular, the 18% total creatine increase observed is unlikely to be de novo synthesis of creatine. More likely, the asymptotic exponential increase in creatine suggests an exponential release of an NMR-invisible bound creatine store as a result of tissue damage from mechanical stress of sample spinning. Overall, it appears that tissue ischaemia during biopsy excision and delays in snap-freezing may have less significant effects on metabolite profile than the prolonged spinning times required for two-dimensional HRMAS, and this must be accounted for when results are being interpreted.
Opstad
Kirstie S
KS
Division of Basic Medical Sciences, St George's, University of London, UK. kopstad@sgul.ac.uk
Bell
B Anthony
BA
Griffiths
John R
JR
Howe
Franklyn A
FA
D000818:Animals; D014408:Biomarkers, Tumor / Q000032:analysis*; D001706:Biopsy / Q000379:methods; D002545:Brain Ischemia / Q000378:metabolism* / Q000473:pathology*; D001932:Brain Neoplasms / Q000378:metabolism* / Q000473:pathology*; D045744:Cell Line, Tumor; D005260:Female; D005910:Glioma / Q000378:metabolism / Q000473:pathology; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D011522:Protons; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*; D013113:Spin Labels
D014408:Biomarkers, Tumor; D011522:Protons; D013113:Spin Labels