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

Abstract

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.

Current Issues in Measurement and Reporting of Urinary Albumin Excretion

“…The proportions of fragments have been observed to increase with kidney disease (78 -81 ) and possibly with prolonged storage of specimens at Ϫ20°C (47 ). The sequences of several large fragments in urine have been identified, and some were detected in plasma, suggesting that some fragments in urine were from plasma (81 ).…”

“…Urine is enriched relative to plasma in low molecular weight peptide components and amino acid derivatives such as hippuric acid and phenylacetylglutamine (47,70,71 ). On a molar basis, the urinary concentrations of these compounds usually exceed that of albumin, so there may be substantial ligand binding to albumin (70,71 ).…”

Prevalence and Prognostic Value of Elevated Urinary Albumin Excretion in Patients With Chronic Heart Failure

“…Second, since timed urine collection is cumbersome in a multicenter setting, first morning spot collection was preferred for ease of implementation, better compliance, and to minimize the influence of circadian rhythm. Third, prolonged frozen storage of urine samples may lead to various changes in albumin, 33 underestimate the number of patients with abnormal excretion of albumin 34 and affect its prediction of outcome. 35 However, frozen urine samples are often used in large multicenter studies where albuminuria is determined by a central laboratory, 24,36 and our samples were kept at Ϫ70°C.…”

Indicazioni per la misura dell’albumina nelle urine per l’accertamento e il monitoraggio della nefropatia diabetica

“…Alcuni anni fa questo problema era apparso particolarmente importante in quanto era stato ipotizzato che l'albumina "modificata" non fosse riconosciuta dagli antisieri dei saggi immunochimici e che, quindi, i comuni metodi di laboratorio fornissero consistenti sottostime della concentrazione della proteina. In realtà, studi successivi più approfonditi hanno potuto dimostrare che gli antisieri policlonali sono in grado di riconoscere anche le forme modificate della proteina e quindi il problema è stato superato [9,10].…”