title: Plasma protein binding of luciferase substrates influences sensitivity and accuracy of bioluminescence imaging.
abstract: BACKGROUND
  Potential confounding factors in bioluminescence imaging (BLI)-quantification need to be identified to improve its accuracy and repeatability.
  
  
  OBJECTIVE
  The aim of this study was to study the degree to which plasma protein binding of BLI substrates influences signal intensity and quantification, both in vitro and in vivo.
  
  
  METHODS
  Protein binding was assessed using ultrafiltration and spectrophotometry. Effects of increasing serum protein concentrations were studied in vitro, in intact cells expressing Firefly or Renilla luciferase, and in vivo, in a doxorubicin-induced hypoalbuminemia mouse model.
  
  
  RESULTS
  Increasing concentrations of serum proteins showed an important negative effect on BLI signal intensity, both for D-luciferin and coelenterazine-dependent reactions. This was due to a decrease in the free fraction of the substrate in the presence of serum proteins of up to 88%. In vivo, hypoalbuminemia was associated with higher BLI signal intensity, although this was only significant in animals with a major decrease in albumin (<2 g/dL). Important kinetic differences, including earlier peak luminescence and a more rapid decline in luminescence, were observed for coelenterazine-dependent BLI under hypoalbuminemic conditions. Changes in protein concentrations can significantly influence BLI quantification and its presence decreases sensitivity in vitro. In vivo, effects on signal intensity and kinetics are only noted at severe hypoalbuminemia and can be neglected in most experimental designs. These findings again emphasize the need for careful interpretation of BLI quantification data.
authors.affiliation: In Vivo Cellular and Molecular Imaging Laboratory, Vrije Universiteit Brussel, Brussels, Belgium. mkeyaerts@gmail.com
authors.forename: Marleen
authors.initials: M
authors.lastname: Keyaerts
authors.forename: Carola
authors.initials: C
authors.lastname: Heneweer
authors.forename: Lea O Tchouate
authors.initials: LO
authors.lastname: Gainkam
authors.forename: Vicky
authors.initials: V
authors.lastname: Caveliers
authors.forename: Bradley J
authors.initials: BJ
authors.lastname: Beattie
authors.forename: Geert A
authors.initials: GA
authors.lastname: Martens
authors.forename: Christian
authors.initials: C
authors.lastname: Vanhove
authors.forename: Axel
authors.initials: A
authors.lastname: Bossuyt
authors.forename: Ronald G
authors.initials: RG
authors.lastname: Blasberg
authors.forename: Tony
authors.initials: T
authors.lastname: Lahoutte
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000378:metabolism*; D002460:Cell Line; D008156:Luciferases / Q000378:metabolism*; D049449:Luminescence; D008297:Male; D051379:Mice; D008819:Mice, Nude; D013379:Substrate Specificity
chemical_list: D001798:Blood Proteins; D008156:Luciferases
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