title: Recovery correction technique for NMR spectroscopy of perchloric acid extracts using DL-valine-2,3-d2: validation and application to 5-fluorouracil-induced brain damage.
abstract: OBJECTIVE
  We explored a recovery correction technique that can correct metabolite loss during perchloric acid (PCA) extraction and minimize inter-assay variance in quantitative (1)H nuclear magnetic resonance (NMR) spectroscopy of the brain and evaluated its efficacy in 5-fluorouracil (5-FU)- and saline-administered rats.
  
  
  METHODS
  We measured the recovery of creatine and dl-valine-2,3-d2 from PCA extract containing both compounds (0.5 to 8 mM). We intravenously administered either 5-FU for 4 days (total, 100 mg/kg body weight) or saline into 2 groups of 11 rats each. We subsequently performed PCA extraction of the whole brain on Day 9, externally adding 7 µmol of dl-valine-2,3-d2. We estimated metabolite concentrations using an NMR spectrometer with recovery correction, correcting metabolite concentrations based on the recovery factor of dl-valine-2,3-d2. For each metabolite concentration, we calculated the coefficient of variation (CEV) and compared differences between the 2 groups using unpaired t-test.
  
  
  RESULTS
  Equivalent recoveries of dl-valine-2,3-d2 (89.4 ± 3.9%) and creatine (89.7 ± 3.9%) in the PCA extract of the mixed solution indicated the suitability of dl-valine-2,3-d2 as an internal reference. In the rat study, recovery of dl-valine-2,3-d2 was 90.6 ± 9.2%. Nine major metabolite concentrations adjusted by recovery of dl-valine-2,3-d2 in saline-administered rats were comparable to data in the literature. CEVs of these metabolites were reduced from 10 to 17% before to 7 to 16% after correction. The significance of differences in alanine and taurine between the 5-FU- and saline-administered groups was determined only after recovery correction (0.75 ± 0.12 versus 0.86 ± 0.07 for alanine; 5.17 ± 0.59 versus 5.66 ± 0.42 for taurine [µmol/g brain tissue]; P < 0.05).
  
  
  CONCLUSIONS
  A new recovery correction technique corrected metabolite loss during PCA extraction, minimized inter-assay variance in quantitative (1)H NMR spectroscopy of brain tissue, and effectively detected inter-group differences in concentrations of brain metabolites between 5-FU- and saline-administered rats.
authors.affiliation: Division of Functional Imaging, Research Center for Innovative Oncology, National Cancer Center Hospital East Kashiwanoha.
authors.forename: Ryutaro
authors.initials: R
authors.lastname: Nakagami
authors.forename: Masayuki
authors.initials: M
authors.lastname: Yamaguchi
authors.forename: Kenji
authors.initials: K
authors.lastname: Ezawa
authors.forename: Sadaaki
authors.initials: S
authors.lastname: Kimura
authors.forename: Shusei
authors.initials: S
authors.lastname: Hamamichi
authors.forename: Norio
authors.initials: N
authors.lastname: Sekine
authors.forename: Akira
authors.initials: A
authors.lastname: Furukawa
authors.forename: Mamoru
authors.initials: M
authors.lastname: Niitsu
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Fujii
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism*; D001927:Brain Diseases / Q000378:metabolism*; D003401:Creatine / Q000378:metabolism; D005472:Fluorouracil / Q000008:administration & dosage*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D010472:Perchlorates; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012965:Sodium Chloride / Q000008:administration & dosage; D014633:Valine / Q000378:metabolism*
chemical_list: D010472:Perchlorates; D012965:Sodium Chloride; D014633:Valine; D003401:Creatine; D005472:Fluorouracil; C576518:Perchloric Acid