The cobalt–albumin binding assay: Insights into its mode of action

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. 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. 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. 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.