Ex vivo instability of glycated albumin: A role for autoxidative glycation

Ex vivo protein modifications occur within plasma and serum (P/S) samples due to prolonged exposure to the thawed state—which includes temperatures above −30 °C. Herein, the ex vivo glycation of human serum albumin from healthy and diabetic subjects was monitored in P/S samples stored for hours to months at −80 °C, −20 °C, and room temperature, as well as in samples subjected to multiple freeze-thaw cycles, incubated at different surface area-to-volume ratios or under different atmospheric compositions. A simple dilute-and-shoot method utilizing trap-and-elute LC-ESI-MS was employed to determine the relative abundances of the glycated forms of albumin—including forms of albumin bearing more than one glucose molecule. Significant increases in glycated albumin were found to occur within hours at room temperature, and within days at −20 °C. These increases continued over a period of 1–2 weeks at room temperature and over 200 days at −20 °C, ultimately resulting in a doubling of glycated albumin in both healthy and diabetic patients. It was also shown that samples stored at lower surface area-to-volume ratios or incubated under a nitrogen atmosphere experienced less rapid glucose adduction of albumin—suggesting a role for oxidative glycation in the ex vivo glycation of albumin.