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Received on July 7, 2004
Accepted on August 26, 2004
Proteomics and Protein Markers |
1 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia, and AusAm Biotechnologies Inc., New York, NY
* To whom correspondence should be addressed. E-mail: wayne.comper{at}med.monash.edu.au.
Background: Conventional immunoassays underestimate the urinary albumin concentration because intact albumin in urine exists in two forms, immunoreactive and immunochemically nonreactive.
Methods: Urinary albumin concentration measured by HPLC (which measures total albumin, i.e., the sum of immunoreactive albumin + immunochemically nonreactive albumin) or RIA was compared with densitometric analysis of albumin bands in diabetic urine samples separated by either native polyacrylamide gel electrophoresis (PAGE) or reducing sodium dodecyl sulfate (SDS)-PAGE. Immunochemically nonreactive albumin was also isolated from diabetic urine (relative amount detected, 70-80% of the expected) and was tested for contamination by common urinary proteins by native PAGE, ELISA, and capillary electrophoresis.
Results: Urinary albumin concentrations measured by native PAGE and HPLC were better correlated (r2 = 0.83) than concentrations measured by native PAGE and RIA (r2 = 0.62) because under native conditions both native PAGE and HPLC detect total albumin and not only the immunoreactive albumin alone that is measured by RIA. Urinary albumin concentrations measured by reducing SDS-PAGE and RIA were better correlated (r2 = 0.84) than concentrations measured by reducing SDS-PAGE and HPLC (r2 = 0.65) because under reducing conditions immunochemically nonreactive albumin is unstable and fragments into many smaller peptides. The partially purified preparation was found to contain <1% contamination by common urinary proteins and is stable to freezing and frequent freeze/thaw cycles.
Conclusions: The results are consistent with the interpretation that immunochemically nonreactive albumin has a limited number of polypeptide chain scissions and is held together by noncovalent intrachain bonding and disulfide bonds. Detection of this molecule is likely to be of clinical importance in diagnosing kidney disease as well as cardiovascular disease.
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