The cobalt-albumin binding assay: insights into its mode of action.

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.