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Proteomics and Protein Markers |
1 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
2 AusAm Biotechnologies Inc., New York, NY.
aAddress correspondence to this author at: Department of Biochemistry and Molecular Biology, Monash University, Wellington Rd., Clayton, Victoria, Australia 3800. Fax 61-3-9903-1117; 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.
The following articles in journals at HighWire Press have cited this article:
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  K. Kania, E. A Byrnes, J. P Beilby, S. A R Webb, and K. J Strong Urinary proteases degrade albumin: implications for measurement of albuminuria in stored samples Ann Clin Biochem, March 1, 2010; 47(2): 151 - 157. [Abstract] [Full Text] [PDF]
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E. J Lamb, F. MacKenzie, and P. E Stevens How should proteinuria be detected and measured? Ann Clin Biochem, May 1, 2009; 46(3): 205 - 217. [Abstract] [Full Text] [PDF]
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 R. T. Gansevoort and E. Ritz Hermann Senator and albuminuria--forgotten pioneering work in the 19th century Nephrol. Dial. Transplant., March 1, 2009; 24(3): 1057 - 1062. [Abstract] [Full Text] [PDF]
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W. G. Miller, D. E. Bruns, G. L. Hortin, S. Sandberg, K. M. Aakre, M. J. McQueen, Y. Itoh, J. C. Lieske, D. W. Seccombe, G. Jones, et al. Current Issues in Measurement and Reporting of Urinary Albumin Excretion Clin. Chem., January 1, 2009; 55(1): 24 - 38. [Abstract] [Full Text] [PDF]
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A. Shaikh, J. C. Seegmiller, T. M. Borland, B. E. Burns, P. M. Ladwig, R. J. Singh, R. Kumar, T. S. Larson, and J. C. Lieske Comparison between Immunoturbidimetry, Size-Exclusion Chromatography, and LC-MS to Quantify Urinary Albumin Clin. Chem., September 1, 2008; 54(9): 1504 - 1510. [Abstract] [Full Text] [PDF]
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 B. Haraldsson, J. Nystrom, and W. M. Deen Properties of the Glomerular Barrier and Mechanisms of Proteinuria Physiol Rev, April 1, 2008; 88(2): 451 - 487. [Abstract] [Full Text] [PDF]
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C. Granier, K. Makni, L. Molina, B. Jardin-Watelet, H. Ayadi, and F. Jarraya Gene and protein markers of diabetic nephropathy Nephrol. Dial. Transplant., March 1, 2008; 23(3): 792 - 799. [Full Text] [PDF]
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D. Sviridov, S. K. Drake, and G. L. Hortin Reactivity of Urinary Albumin (Microalbumin) Assays with Fragmented or Modified Albumin Clin. Chem., January 1, 2008; 54(1): 61 - 68. [Abstract] [Full Text] [PDF]
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 D. J. Magliano, K. R. Polkinghorne, E. L.M. Barr, Q. Su, S. J. Chadban, P. Z. Zimmet, J. E. Shaw, and R. C. Atkins HPLC-Detected Albuminuria Predicts Mortality J. Am. Soc. Nephrol., December 1, 2007; 18(12): 3171 - 3176. [Abstract] [Full Text] [PDF]
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J. W. Brinkman, D. de Zeeuw, H. J. Lambers Heerspink, R. T. Gansevoort, I. P. Kema, P. E. de Jong, and S. J.L. Bakker Apparent Loss of Urinary Albumin during Long-term Frozen Storage: HPLC vs Immunonephelometry Clin. Chem., August 1, 2007; 53(8): 1520 - 1526. [Abstract] [Full Text] [PDF]
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 H. H. Otu, H. Can, D. Spentzos, R. G. Nelson, R. L. Hanson, H. C. Looker, W. C. Knowler, M. Monroy, T. A. Libermann, S. A. Karumanchi, et al. Prediction of Diabetic Nephropathy Using Urine Proteomic Profiling 10 Years Prior to Development of Nephropathy Diabetes Care, March 1, 2007; 30(3): 638 - 643. [Abstract] [Full Text] [PDF]
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N. Babic, T.S. Larson, S.K. Grebe, S.T. Turner, R. Kumar, and R.J. Singh Application of liquid chromatography-mass spectrometry technology for early detection of microalbuminuria in patients with kidney disease. Clin. Chem., November 1, 2006; 52(11): 2155 - 2157. [Full Text] [PDF]
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O. T.M. Chan and D. A. Herold Chip Electrophoresis as a Method for Quantifying Total Microalbuminuria Clin. Chem., November 1, 2006; 52(11): 2141 - 2146. [Abstract] [Full Text] [PDF]
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T. Peters Jr How should we measure the albumin in urine? Clin. Chem., April 1, 2006; 52(4): 555 - 556. [Full Text] [PDF]
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D. Sviridov, B. Meilinger, S. K. Drake, G. T. Hoehn, and G. L. Hortin Coelution of Other Proteins with Albumin during Size-Exclusion HPLC: Implications for Analysis of Urinary Albumin Clin. Chem., March 1, 2006; 52(3): 389 - 397. [Abstract] [Full Text] [PDF]
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