Maslamani S, Glenton P A, Khan S R
Department of Pathology, College of Medicine, University of Florida, Gainesville, Florida 32610, USA.
J Urol. 2000 Jul;164(1):230-6.
To determine the urinary crystallization inhibitory activity, urine is generally centrifuged and/or filtered. These preparative procedures may result in a total or partial removal of many macromolecular constituents implicated in crystallization. The main purpose of this study was to investigate the changes in urinary macromolecular composition following centrifugation and filtration.
Twenty-four hour urine samples were collected from human volunteers. Each was divided into 4 aliquots; one was filtered, the other was centrifuged, another was centrifuged and filtered. The control sample was neither filtered nor centrifuged. Total protein and lipid contents of each sample were determined. Proteins were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analysis was performed using antibodies against osteopontin (OPN), prothrombin (PT) related proteins, inter-alpha-inhibitor (IalphaI) related proteins, Tamm-Horsfall protein (THP), and albumin (ALB). The effect of processing on incorporation of urinary proteins in crystal matrices was also examined. Calcium oxalate crystals were produced in processed and unprocessed urine samples by the addition of sodium oxalate. Crystals were harvested, de-mineralized and their proteins analyzed by SDS-PAGE and Western blotting.
Processing reduced the amounts of both proteins and lipids in the urine. Previously we identified phospholipids in the matrix of calcium oxalate crystals as well as the filtrate and retentate removed during filtration and centrifugation. Phospholipids have a high affinity for calcium-containing crystals. In the case of proteins, those with high molecular weights appeared to be clearly affected by filtration and centrifugation. Processing also appeared to influence the incorporation of proteins in the crystals. The matrix of crystals produced in processed urine contained less THP than those made in unprocessed urine, apparently a result of the loss of this higher molecular weight protein during processing. Incorporation of PT-related proteins, particularly fragment 1, was increased.
We propose that selective inclusion of macromolecules is a result of an increase in available binding sites on crystal surfaces because of the removal of certain calcium binding substances such as phospholipids and proteins. Removal of larger macromolecules from the milieu may also provide a better access to the crystal surfaces.
为了测定尿液的结晶抑制活性，通常会对尿液进行离心和/或过滤。这些预处理程序可能会导致许多与结晶有关的大分子成分全部或部分被去除。本研究的主要目的是调查离心和过滤后尿液大分子组成的变化。
从人类志愿者收集24小时尿液样本。每个样本分成4份；一份进行过滤，另一份进行离心，第三份先离心再过滤。对照样本既不进行过滤也不进行离心。测定每个样本的总蛋白和脂质含量。使用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分析蛋白质。使用针对骨桥蛋白（OPN）、凝血酶原（PT）相关蛋白、α-抑制物（IαI）相关蛋白、Tamm-Horsfall蛋白（THP）和白蛋白（ALB）的抗体进行蛋白质印迹分析。还检查了处理对尿液蛋白质掺入晶体基质的影响。通过添加草酸钠在处理过和未处理过的尿液样本中生成草酸钙晶体。收集晶体，脱矿，并通过SDS-PAGE和蛋白质印迹分析其蛋白质。
处理降低了尿液中蛋白质和脂质的含量。此前我们在草酸钙晶体基质以及过滤和离心过程中去除的滤液和截留物中鉴定出了磷脂。磷脂对含钙晶体具有高亲和力。就蛋白质而言，高分子量的蛋白质似乎明显受到过滤和离心的影响。处理似乎也会影响蛋白质掺入晶体。处理过的尿液中产生的晶体基质所含的THP比未处理尿液中产生的晶体少，这显然是由于该高分子量蛋白质在处理过程中损失所致。PT相关蛋白，特别是片段1的掺入增加。
我们认为，由于去除了某些钙结合物质，如磷脂和蛋白质，晶体表面上可利用的结合位点增加，从而导致大分子的选择性掺入。从环境中去除较大的大分子也可能为晶体表面提供更好的接触途径。
