Variability of standard clinical protein assays in the analysis of a model urine solution of fragmented albumin

Eppel, Gabriela Alejandra; Nagy, S.; Jenkins, Margaret A.; Tudball, Ronald; Daskalakis, Michael; Balazs, N.D.H.; Comper, Wayne D.

doi:10.1016/s0009-9120(00)00156-9

Cited by 43 publications

(34 citation statements)

References 15 publications

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“…The pyragallol red assay overestimates albumin by 7%, underestimates transferrin by 4%, and IgG by 59%. Albumin fragments lead to underestimation of urinary proteins by a variety of commercially available assays (9). Slight overestimation of albumin, underestimation of albumin fragments, and gross underestimation of IgG by this method are likely the explanation for finding an excellent correlation between total intact albumin and total urinary protein.…”

Section: Discussionmentioning

confidence: 97%

On the nature of proteinuria with acute renal injury in patients with chronic kidney disease

Agarwal¹

2005

American Journal of Physiology-Renal Physiology

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Agarwal, Rajiv. On the nature of proteinuria with acute renal injury in patients with chronic kidney disease. Am J Physiol Renal Physiol 288: F265-F271, 2005; doi:10.1152/ajprenal.00318.2004.-Albuminuria is an excellent marker of cardiovascular and renal prognosis. Commercially available tests of immunodetectable albumin in the urine may not identify posttranslationally modified albumin that makes it undetectable to antibodies. Also, it is unclear whether albumin is degraded to smaller fragments, such as through proteolysis, in the course of acute renal injury. In 20 men with chronic kidney disease, we measured excretion rates of urinary protein (pyragallol red), immundetectable urinary albumin (immunoturbidimetry), and urinary total intact albumin (HPLC) after a single dose of 100 mg intravenous iron sucrose administered over 5 min. Fragmentation of urinary albumin and carbonylation of urinary proteins were assessed by immunoblotting. Results showed that iron infusion increased carbonylation of plasma and urinary proteins in a time-dependent manner. A transient increase in urinary excretion rates of total protein, immunodetectable urinary albumin, and total intact albumin was seen. Fragmentation and loss of immunoreactivity of albumin paralleled the changes in total protein excretion. In conclusion, fragmentation, loss of immunoreactivity, and oxidation of albumin in a time-dependent manner may underestimate the extent of injury with the immunoreactive microalbumin assay. Measurement of total intact albumin may better quantify acute renal injury. oxidative stress; albuminuria PROTEINURIA IS A HALLMARK of chronic renal injury, the quantity and quality of which are excellent markers of both the cardiovascular and renal prognosis (13). Traditionally, the occurrence of albuminuria has been held as a marker of altered glomerular permselectivity (3), whereas the occurrence of small-molecular-weight proteinuria has been linked to renal tubular damage (2). While albuminuria has been well studied in models of chronic kidney disease (CKD), relatively little is known about the occurrence of albuminuria with acute renal injury in people with CKD (6). The processing of urinary albumin with acute renal injury is also poorly defined.Data are emerging that antibody-based tests of albuminuria may not identify posttranslationally modified albumin that makes it immunounreactive (11). If immunoreactive domains on urinary albumin were lost in the setting of acute renal injury, it would make the use of urinary total intact albumin, a test not based on immunodetection but molecular size, more desirable over tests of urinary albumin based on immunodetection. Whether acute renal injury makes albumin immunounreactive is not known.Acute renal failure is characterized by activation of intrarenal proteases, which can potentially degrade albumin to smaller fragments. Whether degradation of albumin occurs with acute renal injury is also unclear. If albumin were fragmented, then neither a total intact albumin test nor the conventional, commer...

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Section: Discussionmentioning

confidence: 97%

On the nature of proteinuria with acute renal injury in patients with chronic kidney disease

Agarwal¹

2005

American Journal of Physiology-Renal Physiology

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“…Proteolysis and chemical modification of albumin probably destroy some epitopes, but, apparently, polyclonal antibodies still react with enough epitopes to sustain turbidimetric reactions. A previous study of trypsin digestion of albumin (29 ), using a lower ratio of trypsin to albumin, concluded, "Many techniques used to assay patient urine samples are unable to detect fragmented albumin. .…”

Section: Discussionmentioning

confidence: 99%

Reactivity of Urinary Albumin (Microalbumin) Assays with Fragmented or Modified Albumin

Sviridov¹,

Drake

Hortin³

2008

Clinical Chemistry

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Background: Controversy exists regarding occurrence and measurement of structural variants of albumin in urine. In this study, we examined cross-reactivity of in vitro modified albumins in assays for urine albumin (microalbumin). Methods: We analyzed albumin modified by reagents, trypsin, or physical treatments or differing in primary sequence (animal albumins) with an immunoturbidimetric assay (Beckman LX20) using goat antiserum and a competitive immunoassay (Siemens Immulite) using a monoclonal antibody. We assessed occurrence of albumin fragments in urine by use of Western blotting of 24 specimens. Results: Chemical modification, modest sequence substitution (gorilla albumin), or cleavage of albumin by cyanogen bromide (CNBr) had little effect on reactivity in the LX20 assay. Albumin extensively cleaved with trypsin retained partial reactivity. The Immulite assay generally was affected more severely by albumin modifications and sequence changes. Western blots of fresh urine specimens or specimens stored at −80 °C showed little albumin fragmentation, but some specimens stored for 3 years at −20 °C had extensively fragmented albumin that was detected by the LX20 but not the Immulite assay. Conclusions: Nearly equivalent reactivity of intact albumin and CNBr fragments in the immunoturbidimetric assay indicates reactivity of antibodies with multiple epitopes throughout albumin. Therefore, it is difficult to abolish reactivity of albumin in this type of urine albumin assay. Differential sensitivity of 2 assays to albumin modification identifies a potential source of assay nonequivalence in measuring urinary albumin, particularly for specimens stored at −20 °C.

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“…Conventional antibodies generated against native serum albumin do not recognize modified intact urinary albumin (12)(13)(14). In fact, we have tested in excess of 30 different polyclonal and monoclonal albumin antibodies and have none that detect immunochemically nonreactive albumin.…”

Section: Discussionmentioning

confidence: 99%

“…These antibodies have been raised against serum albumin and not urinary albumin. It is clear that immunoassays measure an active epitope in the urine; however, this epitope may not be associated exclusively with intact albumin (12)(13)(14). The immunochemically nonreactive nature of this albumin molecule may be attributable to the fact that the epitope is altered by a conformational change as a result of incomplete processing by the lysosomal pathway, which is compromised in diabetes but not in healthy individuals (12,13 ), or to the attachment of ligands such as glucose or fatty acids, which are increased in diabetes.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Immunochemically Nonreactive Urinary Albumin

Osicka

Comper

2004

Clinical Chemistry

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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 (r 2 ‫؍‬ 0.83) than concentrations measured by native PAGE and RIA (r 2 ‫؍‬ 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 (r 2 ‫؍‬ 0.84) than concentrations measured by reducing SDS-PAGE and HPLC (r 2 ‫؍‬ 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

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Variability of standard clinical protein assays in the analysis of a model urine solution of fragmented albumin

Cited by 43 publications

References 15 publications

On the nature of proteinuria with acute renal injury in patients with chronic kidney disease

On the nature of proteinuria with acute renal injury in patients with chronic kidney disease

Reactivity of Urinary Albumin (Microalbumin) Assays with Fragmented or Modified Albumin

Characterization of Immunochemically Nonreactive Urinary Albumin

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