title: A high precision method for quantitative measurements of reactive oxygen species in frozen biopsies.
abstract: OBJECTIVE
  An electron paramagnetic resonance (EPR) technique using the spin probe cyclic hydroxylamine 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) was introduced as a versatile method for high precision quantification of reactive oxygen species, including the superoxide radical in frozen biological samples such as cell suspensions, blood or biopsies.
  
  
  METHODS
  Loss of measurement precision and accuracy due to variations in sample size and shape were minimized by assembling the sample in a well-defined volume. Measurement was carried out at low temperature (150 K) using a nitrogen flow Dewar. The signal intensity was measured from the EPR 1st derivative amplitude, and related to a sample, 3-carboxy-proxyl (CP•) with known spin concentration.
  
  
  RESULTS
  The absolute spin concentration could be quantified with a precision and accuracy better than ±10 µM (k = 1). The spin concentration of samples stored at -80°C could be reproduced after 6 months of storage well within the same error estimate.
  
  
  CONCLUSIONS
  The absolute spin concentration in wet biological samples such as biopsies, water solutions and cell cultures could be quantified with higher precision and accuracy than normally achievable using common techniques such as flat cells, tissue cells and various capillary tubes. In addition; biological samples could be collected and stored for future incubation with spin probe, and also further stored up to at least six months before EPR analysis, without loss of signal intensity. This opens for the possibility to store and transport incubated biological samples with known accuracy of the spin concentration over time.
authors.affiliation: Dept. of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway.
authors.forename: Kirsti
authors.initials: K
authors.lastname: Berg
authors.affiliation: Dept. of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway; Dept. of Medical Biosciences, Physiological Chemistry, Umeå University, Umeå, Sweden.
authors.forename: Madelene
authors.initials: M
authors.lastname: Ericsson
authors.affiliation: Dept. of Physics, Norwegian University of Science and Technology, Trondheim, Norway; Dept. of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden.
authors.forename: Mikael
authors.initials: M
authors.lastname: Lindgren
authors.affiliation: Dept. of Biomedical Engineering (MTÖ), County Council of Östergötland, Radiation Physics, Dept. of Medicine and Health Sciences, Linköping University, Linköping, Sweden.
authors.forename: Håkan
authors.initials: H
authors.lastname: Gustafsson
mesh_terms: D000818:Animals; D001706:Biopsy; D002138:Calibration; D003080:Cold Temperature; D003497:Cyclic N-Oxides; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods / Q000592:standards*; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000378:metabolism; D005615:Freezing; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009206:Myocardium / Q000737:chemistry* / Q000378:metabolism; D011759:Pyrrolidines; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000032:analysis*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013113:Spin Labels
chemical_list: C492269:1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine; D003497:Cyclic N-Oxides; D011759:Pyrrolidines; D017382:Reactive Oxygen Species; D013113:Spin Labels; C032289:2,2,5,5-tetramethyl-1-pyrrolidinyloxy-3-carboxylic acid