Cobalt speciation assay for human serum, Part II. Method validation in a study of human volunteers ingesting cobalt(II) chloride dietary supplement for 90 days

Abstract

A new analytical method for determining cobalt (Co) species in human serum by size exclusion chromatography with inductively coupled plasma mass spectrometry (SEC-ICP-MS) was applied to serum samples collected from 12 human volunteers who participated in a Co(II) chloride supplement study involving ingestion of 1 mg Co/day for up to 90 consecutive days. The study protocol included determination of serum total Co by acid digestion followed by ICP-MS. Co speciation assay measurements were conducted for up to 13 time points per individual spanning from one to two weeks before dosing began to two weeks after dosing ceased. The Co speciation assay showed good recovery >91% relative to total Co measurements. Undiluted serum demonstrated uniform fractions of large molecular Co defined as Co bound to albumin and other proteins >50 kDa at 96% and the residual as small molecular Co defined as free Co(II) and <1 kDa Co-complexes for individual serum Co concentrations up to 146 μg/L. There were no dose-related changes in Co distribution. Analysis of the same serum samples with tenfold dilution in 0.1 M acetic acid led to a lower fraction of large molecular Co at 87%, with the difference between diluted and undiluted measurements being 8.4%. The difference noted between undiluted and diluted large molecular Co may be attributed to Co release from albumin. Data demonstrated that large molecular Co was the predominant Co species in both undiluted and diluted human serum over a broad range of in vivo Co concentrations, reflecting high albumin–Co binding capacity. These data validate the Co speciation assay and may be employed in understanding further the toxicokinetics and dose-response relationships for Co species.

Keywords:

• cobalt
• cobalt supplement
• cobalt–chromium alloy implants
• occupational cobalt exposure

Acknowledgements

The authors would like to acknowledge Russell Gerads and Hakan Gurleyuk for their valuable contributions to refining the analytical methods and for peer review of this manuscript. All of the authors are employed by ChemRisk, a consulting firm that provides scientific advice to the government, corporations, law firms, and various scientific/professional organizations. ChemRisk has been engaged by DePuy Orthopedics, Inc., a manufacturer of prosthetic devices, some of which contain cobalt. This paper was prepared and written exclusively by the authors without review or comment by DePuy employees or counsel. It is likely that this work will be relied upon in medical research, nutrition research, and litigation. Some of the authors may be called upon to serve as expert witnesses. The funding for this paper and for the analytical work and method development was provided by DePuy, with additional funding provided by ChemRisk.