School of Pathology and Laboratory Medicine, The University of Western Australia, Nedlands.
Ann Clin Biochem. 2010 Mar;47(Pt 2):151-7. doi: 10.1258/acb.2009.009247. Epub 2010 Feb 11.
Previous studies have shown that albumin in stored urine samples degrades over time, and that albumin losses are greatest in samples with low pH conditions (pH < 5). Furthermore, the high-performance liquid chromatography (HPLC) assay for urinary albumin has been shown to be particularly susceptible to the effects of prolonged storage.
Frozen urine samples, stored for 12 months at -70 and -20 degrees C, were analysed for albumin fragmentation. Urinary protease activity was investigated in vitro in urine adjusted to pH 2.3-2.5. Albumin was measured by nephelometry, HPLC and sodium dodecyl sulphate-polyacrylamide gel electrophoresis.
In the unadjusted samples, albumin was degraded in 11 out of 40 samples stored at -20 degrees C. In the in vitro experiments, both endogenous albumin and exogenous albumin added to urine were rapidly degraded into large fragments within minutes after adjustment to low pH. The fragments produced were consistent with those produced during digestion with pepsin and urinary degradation was completely inhibited by pepstatin. Albumin concentration measured by HPLC was most dramatically affected, with near-complete loss of albumin-sized material within one hour of incubation at pH 2.3-2.5. Sample reactivity with antiserum in a nephelometry assay initially declined then increased, possibly due to exposure of internal epitopes during albumin digestion.
This study demonstrated that proteases are present and active in stored human urine samples. Urinary albumin digestion occurred in a manner consistent with activity of endogenous urinary proteases. Adjustment to neutral pH or addition of protease inhibitors may be useful techniques for sample preservation.
先前的研究表明，储存尿液样本中的白蛋白会随时间降解，而在 pH 值较低（pH < 5）的样本中，白蛋白损失最大。此外，已证明用于尿液白蛋白的高效液相色谱（HPLC）分析特别容易受到长时间储存的影响。
对储存在-70°C 和-20°C 下长达 12 个月的冷冻尿液样本进行白蛋白片段化分析。在 pH 值为 2.3-2.5 的尿液中体外研究尿蛋白酶活性。通过散射浊度法、HPLC 和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定白蛋白。
在未经调整的样本中，在 40 个储存在-20°C 的样本中有 11 个样本的白蛋白发生降解。在体外实验中，在调整至低 pH 值后，内源性白蛋白和添加到尿液中的外源性白蛋白在数分钟内迅速降解为大片段。产生的片段与胃蛋白酶消化产生的片段一致，尿液降解被胃蛋白酶抑制剂完全抑制。用 HPLC 测定的白蛋白浓度受影响最大，在 pH 2.3-2.5 孵育 1 小时内，白蛋白大小的物质几乎完全丢失。在散射浊度测定中，与抗血清的反应最初下降然后增加，这可能是由于白蛋白消化过程中暴露了内部表位。
本研究表明，蛋白酶存在于储存的人尿液样本中并具有活性。尿液白蛋白的消化方式与内源性尿蛋白酶的活性一致。调整至中性 pH 值或添加蛋白酶抑制剂可能是有用的样本保存技术。
