Eppel G A, Nagy S, Jenkins M A, Tudball R N, Daskalakis M, Balazs N D, Comper W D
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
Clin Biochem. 2000 Aug;33(6):487-94. doi: 10.1016/s0009-9120(00)00156-9.
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.
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.
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.
Many techniques utilized to assay patient urine samples are unable to detect fragmented albumin and, hence, will severely underestimate albumin and protein excretion.
本研究调查了各种标准临床技术在检测白蛋白片段方面的敏感性。这项工作的意义在于检测尿蛋白，如白蛋白，最近发现由于滤过的白蛋白在肾脏通过过程中发生降解，其主要以肽片段的形式排泄。所有滤过的蛋白质都经历类似的降解过程。
用胰蛋白酶消化的白蛋白用作模型尿液溶液。通过各种方法测定该溶液中的白蛋白浓度，包括已知可检测尿白蛋白片段的双缩脲法。还用各种临床使用的显色测定法、电泳和色谱法分析消化液，以确定它们是否能够检测到片段化的蛋白质。
用于尿蛋白检测的苯扎氯铵法、考马斯亮蓝法和邻苯三酚红法、人白蛋白免疫测定法以及考马斯亮蓝染色的十二烷基硫酸钠聚丙烯酰胺凝胶电泳均无法检测到白蛋白片段。毛细管电泳对片段敏感，但分辨率较低。高效液相色谱法给出了最佳结果。
许多用于检测患者尿液样本的技术无法检测到片段化的白蛋白，因此会严重低估白蛋白和蛋白质的排泄量。
