An automated assay of urinary alanine aminopeptidase activity
An automated assay of urinary alanine aminopeptidase activity
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DOI：
10.1515/cclm.2008.103
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发表时间：
2008-01-01
影响因子：
6.8
通讯作者：
Steiner, Michael
中科院分区：
文献类型：
--
作者：
Holdt, Birgit;Peters, Eva;Steiner, Michael
背景：尿酶，包括刷状边界酶丙氨酸氨基肽酶（AAP），是肾毒性的可靠生物标志物，是肾小管损伤的早期指标。本研究的目的是开发和验证自动尿AAP活性测定。方法和结果：为此，我们采用了l -丙氨酸-4-硝基苯胺为底物的光度测试，用于专用的临床化学分析仪。由于没有确定AAP活性的校准器，我们使用光度测试评估了市售对照材料的AAP活性。该材料随后被用作自动测定的校准器。质量保证由独立的控制材料完成。在AAP分析之前，使用Sephadex G50对尿样进行快速凝胶过滤步骤是必要的，以避免氨和氨基酸对酶活性的低估。我们将自动光度法与人工光度法进行了比较，发现两种方法之间具有良好的相关性（r(2) = 0.946, n = 50）。结论：我们得出的结论是，新开发的自动化检测是一种用户友好的分析工具，可以在中高通量实验室中检测尿液AAP活性。潜在的应用包括临床诊断工作和毒理学研究。
Background: Urinary enzymes, including the brush border enzyme alanine aminopeptidase (AAP), are reliable biomarkers of nephrotoxicity and represent early indicators of tubular damage. The aim of the present study was to develop and validate an automated urinary AAP activity assay.Methods and results: For this purpose, we adapted a photometric test using the substrate L-alanine-4-nitroanilide to a dedicated clinical chemistry analyser. Due to the unavailability of calibrators with established AAP activity, we assessed the AAP activity of a commercially available control material using the photometric test. This material was subsequently employed as a calibrator for the automated assay. Quality assurance was accomplished with independent control materials. A fast gel filtration step of urine samples using Sephadex G50 was demonstrated to be necessary before AAP analysis to avoid underestimation of enzyme activity due to ammonia and amino acids. We compared the automated assay with a manual photometric assay and obtained excellent correlation between both methods (r(2) = 0.946, n = 50).Conclusions: We conclude that the newly developed automated assay represents a user-friendly and analytical tool to determine urinary AAP activity in a medium-to high-throughput laboratory. Potential applications include clinical diagnostic work and toxicological studies.