三种尿毒素诱导高级氧化蛋白产物的生成

Formation of advanced oxidative protein products induced by three uremic toxins

目的探讨尿毒症患者体内长期积蓄的高水平尿毒素是否会通过参与高级氧化蛋白产物（AOPP）的生成而介导蛋白质的氧化损伤。方法以丙二醛（10mmol／L）、马尿酸（20mmol／L）和对甲酚（10mmol／L）为尿毒素代表。人血清白蛋白（HSA）、健康人和尿毒症患者血浆用20mmol／L的PBS（pH7．4）统一调整到质量浓度为50g／L，随后分别加入尿毒素至指定浓度。对照组内不加任何尿毒素。37℃分别温育0．5h和24h后，分别测定各组的AOPPs、蛋白巯基及二酪氨酸浓度。高效液相色谱（HPLC）法用于观察蛋白质的交联聚集情况。结果与对照组相比，尿毒素组的AOPP浓度平均增加了121．5％（P〈0．05）。尿毒素尚可降低蛋白巯基浓度，降低幅度平均达14．7％（P〈0．05）；同时蛋白二酪氨酸水平增至119．2％（P〈0．05）。HPLC结果表明尿毒素可以时间依赖性方式显著诱导高分子量AOPP（HMW—AOPPs）的生成，平均增幅为148．4％～333．3％（P〈0．05）。结论小分子类尿毒素可通过时间依赖性方式诱导HMW．AOPPs的生成而介导蛋白质的氧化损伤。除了髓过氧化物酶（MPO）-H2O2-Cl-通路的激活之外，尿毒素的介导损伤也是较为重要的体内AOPP生成机制之一。

Objective To study whether the uremic toxins accumulated long-term in uremia patients may be involved in oxidation of protein by forming advanced oxidative protein products （AOPPs）. Methods Malonylaldehyde （MDA）, hippuric acid （HA） and p-cresol were used as the representatives of uremic toxins. Human albumin serum （HSA）, plasma specimens from normal or uremia patients were incubated respectively with MDA （10 mmol/L）, HA （20 mmol/L） and p-cresol （10 mmol/L） or PBS （20 mmol/L, pH 7.4, as control groups） at 37℃ for 30 minutes or 24 hours, respectively. Those indices such as AOPPs, protein thiol groups （Pt-SH） and dityrosine were used as biomarkers of protein injury. High performance liquid chromatography （HPLC） was employed to identify the aggregation and cross-links of modified proteins. Results AOPPs levels in all groups containing poison compounds were significantly increased by 121.5%（P〈0.05） compared to that in control groups. Uremic toxins also resulted in over 14.7% loss in Pt-SH （P〈 0.05） and 119.2% increment in dityrosine, respectively （P〈0.05）. Meanwhile, the formation of HMW-AOPPs in a time-dependent manner was observed by HPLC and cross-linked protein levels were significantly increased by 148.45%-333.3% in comparison with control groups. Conclusion Uremic toxins can directly mediate the damage of proteins by inducing the formation of HMW- AOPPs in a time-dependent manner, which is also one of the mechanism of AOPPs production in vivo besides the activation of the myeloperoxidase-H2O2-Cl- pathway.