Tardivel S, Médétognon J, Randoux C, Kébédé M, Drüeke T, Daudon M, Hennequin C, Lacour B
Laboratoire du Métabolisme Minéral des Mammiféres, EPHE, Physiologie, Faculté de Pharmacie, F-92296 Châtenay-Malabry Cedex, France.
Urol Res. 1999 Aug;27(4):243-9. doi: 10.1007/s002400050117.
In the past few years, alpha-1-microglobulin (alpha1m) has been copurified from human urine with bikunin, a potent inhibitor of calcium oxalate (CaOx) crystallization in vitro. In this study, we have purified alpha1m without bikunin contamination and investigated its possible role in CaOx crystallization by in vitro and in vivo studies. Alpha-1m was purified with an anti-alpha1m antibodies CNBr-activated sepharose column. Two molecular species of alpha1m of respectively 30 and 60 kDa were purified. For each protein, two blots of 30 and 60 kDa cross-reacted with anti-alpha1m antibodies, suggesting that these two forms were derived one from the other. Both protein species inhibited CaOx crystallization in a dose-dependent manner in two in vitro tests. In the first test, the presence of alpha1m of 30 kDa (8 microg/ml) in a medium containing 0. 76 mM CaCl(2) (with (45)Ca) and 0.76 mM Ox(NH(4))(2) inhibited CaOx crystallization by 38% as estimated by supernatant radioactivity after 1 h of agitation. In the second test, CaOx kinetics were examined for 3 to 10 min in a turbidimetric model at 620 nm. The presence of alpha1m of 30 kDa in a medium containing 4 mM CaCl(2) and 0.5 mM Na(2)Ox inhibited CaOx crystallization by 41.5%, as estimated by the slope modification of turbidimetric curve. Alpha-1m can be considered as another inhibitor of urinary CaOx crystal formation, as shown by the present in vitro studies. Using an ELISA assay, we found that urinary alpha1m concentration was significantly lower in 31 CaOx stone formers than in 18 healthy subjects (2.95 +/- 0.29 vs 5.34 +/- 1.08 mg/l respectively, P = 0.01). The decreased concentration of alpha1m in CaOx stone formers could be responsible in these patients, at least in part, for an increased risk of CaOx crystalluria.
在过去几年中，α1微球蛋白（α1m）已从人尿液中与比基尼宁一起被共纯化出来，比基尼宁是一种在体外对草酸钙（CaOx）结晶有强大抑制作用的物质。在本研究中，我们纯化了无比基尼宁污染的α1m，并通过体外和体内研究探讨了其在CaOx结晶中的可能作用。α1m用抗α1m抗体偶联的溴化氰活化琼脂糖柱进行纯化。纯化出了分子量分别为30 kDa和60 kDa的两种α1m分子形式。对于每种蛋白质，30 kDa和60 kDa的两条印迹带均与抗α1m抗体发生交叉反应，表明这两种形式是由一种衍生而来。在两项体外试验中，这两种蛋白质形式均以剂量依赖方式抑制CaOx结晶。在第一项试验中，在含有0.76 mM氯化钙（含45Ca）和0.76 mM草酸铵的培养基中加入30 kDa的α1m（8 μg/ml），搅拌1小时后，通过上清液放射性估计，CaOx结晶被抑制了38%。在第二项试验中，在620 nm的比浊法模型中对CaOx动力学进行了3至10分钟的检测。在含有4 mM氯化钙和0.5 mM草酸钠的培养基中加入30 kDa的α1m，通过比浊曲线斜率的改变估计，CaOx结晶被抑制了41.5%。如目前的体外研究所显示，α1m可被视为尿CaOx晶体形成的另一种抑制剂。通过酶联免疫吸附测定（ELISA），我们发现31例CaOx结石患者尿液中α1m浓度显著低于18例健康受试者（分别为2.95±0.29 vs 5.34±1.08 mg/l，P = 0.01）。CaOx结石患者中α1m浓度降低可能至少部分导致了这些患者CaOx结晶尿风险增加。
