2型糖尿病患者血浆中抗氧化状态的测定

Determination of antioxidant status in the plasma of patients with type 2 diabetic mellitus

目的报道一种新的简单的过3-(4,5-二甲基噻唑)-2,5-二苯四唑溴(MTT)染料法来检测血浆抗氧化状态的高低。方法 MTT[50μL;5.0mg/mL磷酸盐缓冲液(PBS)]与100μL血浆在PBS中于37℃共同孵育30、60和120min,反应终点加入1.0mL的0.04mmol/L盐酸异丙醇,吸收峰在570nm。血浆中活性氧介质(ROS)通过二丁酸佛波醇酯(PDB)刺激粒细胞而产生,通过鲁米诺化学发光反应来测定。结果 15名健康成人血浆中抗氧化水平明显高于27例糖尿病患者(P<0.05)。血小板对MTT的减少影响较小,与人血清等量白蛋白浓度的小牛血清中MTT的被还原量较少。糖尿病患者具有较低的抗氧化能力。结论糖尿病患者血浆的抗氧化状态较正常人低,细胞外MTT的减少可能通过酶或非酶反应。

Objective To establish a simple technique to determine quantitatively the antioxidant status with 3- （4, 5-dimethyhhiazol-2-yl）-2, 5-diphenyhetrazolium bromide （MTF） staining method. Methods MTF （50μL; 5.0 mg/mL in PBS） was incubated with plasma （100 μL） in PBS for 30, 60 or 120 min at 37 ℃, the reaction terminated by adding 1.0mL of 0.04 mmoi/L hydrochloric acid in isopropanol （the absorption peaks was at 570 nm）. The content of reactive oxygen species （ROS） in plasma, generated in 12, 13-phorbol dibutyrate （PDB）-stimulated granulocytes, was determined with luminol-dependent ehemilmnineseence assay. Results The content of antioxidant status in plasma of healthy subjects （n=15） significantly higher （P〈0.05） than that in diabetic patients （n=27） over all time periods studied. The impact of platelets on the reduction of MTT was slight. Moreover, the reduction of MTF in bovine serum albumin was much lower than that in the human serum albumin （equivalent concentration to bovine serum albumin）. The antioxidant activity in plasma of patients with type 2 diabetic mellitus was lower. Conclusion The antioxidant activity in plasma of patients with type 2 diabetic mellitus was lower than that in normal persons, and the extracellular reduction of MTT by plasma may occur via enzymatic and non-enzymatic processes.