Coelution of other proteins with albumin during size-exclusion HPLC: Implications for analysis of urinary albumin.
BACKGROUND
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, alpha1-proteinase inhibitor, alpha1-acid glycoprotein, and alpha2-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 alpha1-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.
Sviridov
Denis
D
Department of Laboratory Medicine, Warren Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892-1508, USA.
Meilinger
Bonnie
B
Drake
Steven K
SK
Hoehn
Gerard T
GT
Hortin
Glen L
GL
D000419:Albuminuria / Q000175:diagnosis / Q000652:urine; D001798:Blood Proteins / Q000652:urine*; D002851:Chromatography, High Pressure Liquid; D005189:False Positive Reactions; D006801:Humans; D012709:Serum Albumin / Q000032:analysis*
D001798:Blood Proteins; D012709:Serum Albumin