Variability of standard clinical protein assays in the analysis of a model urine solution of fragmented albumin.
OBJECTIVE
This study investigates the sensitivity of various standard clinical techniques in the detection of albumin fragments. The significance of this work is in the detection of urinary proteins, such as albumin, which has recently been discovered to be excreted as mainly peptide fragments as a result of filtered albumin undergoing degradation during renal passage. All filtered proteins undergo a similar degradation process.


METHODS
Albumin digested with trypsin was used as a model urine solution. The solution was assayed for albumin concentration by various methods including the biuret assay that is known to detect urinary albumin fragments. The digest solution was also analyzed by various clinically used chromagen assays, electrophoretic and chromatographic methods to determine whether they are able to detect the fragmented protein.


RESULTS
The benzethonium chloride, Coomassie blue, and pyrogallol red assays for urine protein, the immunoassay for human albumin and sodium dodecyl sulfate polyacrylamide gel electrophoresis with Coomassie blue staining were unable to detect the albumin fragments. Capillary electrophoresis was sensitive to the fragments but with low resolution. High-performance liquid chromatography gave the best results.


CONCLUSIONS
Many techniques utilized to assay patient urine samples are unable to detect fragmented albumin and, hence, will severely underestimate albumin and protein excretion.
Eppel
G A
GA
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
Nagy
S
S
Jenkins
M A
MA
Tudball
R N
RN
Daskalakis
M
M
Balazs
N D
ND
Comper
W D
WD
D000419:Albuminuria / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D016482:Urinalysis / Q000379:methods*
D010446:Peptide Fragments; D012710:Serum Albumin, Bovine