Coelution of Other Proteins with Albumin during Size-Exclusion HPLC: Implications for Analysis of Urinary Albumin

Abstract

AbstractBackground: Size-exclusion HPLC has been used as an alternative to immunoassays for quantifying urinary albumin (microalbumin). Systematically higher values for the HPLC method have been proposed to result from nonimmunoreactive albumin.Methods: We evaluated separation of purified proteins and urinary components by size-exclusion HPLC using a Zorbax Bio Series GF-250 column eluted with phosphate-buffered saline. Urinary components eluting in the “albumin” peak were analyzed by mass spectrometry and reversed-phase HPLC.Results: Several proteins, such as transferrin, α1-proteinase inhibitor, α1-acid glycoprotein, and α2-HS glycoprotein, analyzed as purified components, were not resolved from albumin by size-exclusion HPLC. Peaks for other proteins, such as IgG and urinary components identified as dimers of α1-microglobulin and immunoglobulin light chains, overlapped with the albumin peak. Profiles of urine specimens showed variable amounts of components overlapping with albumin. Furthermore, the albumin peak obtained by size-exclusion HPLC was found by mass spectrometry and reversed-phase HPLC to contain multiple components in addition to albumin.Conclusions: Size-exclusion HPLC does not resolve albumin from several other proteins in urine. The albumin peak resolved by this technique, although predominantly composed of albumin, contains several coeluting globulins that would contribute to overestimation of albumin concentration by size-exclusion HPLC.