title: The cobalt-albumin binding assay: insights into its mode of action.
abstract: BACKGROUND
  We previously hypothesized that the N-terminus of human serum albumin (HSA) is altered during ischemic events, thus establishing the foundation for the cobalt-HSA binding assay phenomenon. In this investigation, we attempt to clarify the mode of action of the cobalt-HSA binding assay by direct observations of cobalt binding to HSA.
  
  
  METHODS
  High pressure liquid chromatography coupled to positive electrospray ionization mass spectrometry (HPLC/MS) was used to study cobalt binding to HSA in the plasma of patients with and without evidence of myocardial ischemia.
  
  
  RESULTS
  Strong binding of cobalt to the N-terminus of HSA occurs at pH>7.0. No differences in cobalt binding to the N-terminus of HSA are observed in ischemic versus non-ischemic patients' plasma despite differences in the cobalt-HSA binding assay. Plasma free cysteine/cystine ratio appears to play a role in the quantitative response of the cobalt-HSA binding assay.
  
  
  CONCLUSIONS
  The main determinants of the cobalt-HSA binding assay mechanism of action include but are not limited to: the proportion of intact N-terminus of HSA, HSA concentration, plasma cysteine/cystine ratio, plasma pH, and the state of oxidation of cys34 of HSA. Assay improvements that consider and take these factors into account could lead to an improved cobalt-HSA binding assay with greater clinical utility.
authors.affiliation: Swedish Medical Center, Trauma Research Laboratory, Englewood, CO, USA; Swedish Medical Center, Emergency Department, Englewood, CO 80113, USA. dbaror@dmibio.com
authors.forename: David
authors.initials: D
authors.lastname: Bar-Or
authors.forename: Leonard T
authors.initials: LT
authors.lastname: Rael
authors.forename: Raphael
authors.initials: R
authors.lastname: Bar-Or
authors.forename: Denetta S
authors.initials: DS
authors.lastname: Slone
authors.forename: Charles W
authors.initials: CW
authors.lastname: Mains
authors.forename: Nagaraja K R
authors.initials: NK
authors.lastname: Rao
authors.forename: C Gerald
authors.initials: CG
authors.lastname: Curtis
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003035:Cobalt / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D012709:Serum Albumin / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D012709:Serum Albumin; D003035:Cobalt