气／质联用法检测反式花生四烯酸对糖尿病大鼠视网膜微血管的硝化应激损伤评价

Evaluation of nitrative stress on retinal capillary by detection of trans-arachidonic acid with GC/MS in diabetic rats

背景反式花生四烯酸（TAAs）是NO2介导的花生四烯酸异构化的主要产物，是硝化应激的特异性脂质标志物。NO2介导的损伤是缺血性视网膜病变可能的发病机制之一，先前的研究证实高糖可以引发硝化应激。目的应用气／质联用（GC／MS）法检测TAAs水平以评价高糖引发的硝化应激对视网膜微血管的损害程度。方法2周龄雄性SD大鼠100只随机分为模型组和正常对照组，应用链脲佐菌素（STZ）60mg／kg一次性腹腔注射诱导糖尿病动物模型。应用改良合成法，花生四烯酸（AA）经环氧化及去氧化制得TAAs的标准品14E—AA。GC／MS检测造模后2、4、8、12、16周时血清中TAAs及AA的含量，选择离子79，计算峰面积之比。结果50只大鼠成功建立糖尿病模型，其高血糖状态持续在实验期。14E—AA纯度达到97．3％，可作为标准品使用。糖尿病大鼠造模成功50只，每组10只，造模后2周、4周模型组大鼠血清中TAAs／AA（反式与）顺式之比与正常对照组相比差异无统计学意义（t=-0．376、t=-0．642，P〉0．05）；造模后8、12、16周糖尿病大鼠血清中TAAs／AA值为0．1042、0．1568和0．1895，明显高于对照组的0．0832、0．0910和0．1100，差异均有统计学意义（t=-36．409、t=-166．714、t=-148．212，P〈0．05）。结论糖尿病大鼠血清中TAAs／AA水平随病程的延长而增加，提示硝化应激对视网膜微血管的损伤程度与病程的长短有关。建立GC／MS检测TAAs体系可为缺血性微血管病变的研究提供新的评价指标。

Background Trans-arachidonic acids （TAAs） the major products of NO2＇ -mediated isomerization of arachidonic are specific lipid markers of nitrative stress and acid （AA）, and they represent a possible new mechanism of NO2＂ -induced toxicity in ischemic retinopathy. Our previous study demonstrated that high glucose induce nitrative stress response. Objective This study aimed to evaluate the effect of nitrative stress on retinal capillary in diabetic rats by detecting the TAAs with gas chromatography and mass spectrometry （GC/MS）. Methods One hundred 2-week-old clean male SD rats were randomly divided into control group and diabetic group. Diabetic rat models were induced by intraperitoneal injection of streptozotiein of 60 mg/kg,and the successful models were identified as the blood glucose level 〉 16.7 mmol/L. The synthesis method was modified in this study and arachidonic acid was utilized as starting material. 14E-AA was obtained as a standard sample via expoxidation and deoxidation in a safe and practical pattern. Serum TAAs and AA in diabetic rats were detected by GC/MS in 2,4,8, 12 and 16 weeks after injection of streptozoticin. The ratio of peak area of TAAs to AA with selected ion of 79 was estimated and compared with normal rats. Results Fifty rats had been in Diabetic models were established in 50 rats with the successful rate 100%, and the high blood glucose level in model rats retained throughout the experimental duration. GC/MS results showed that the 14E-AA was obtained with the purity of 97.3% , which provided a standard sample to nitrative stress-related research as a premise. No significant differences were found in serum TAAs/AA values at 2 weeks and 4 weeks between model group and control group （t =-0. 376, t =-0. 642, P〉0.05）. However, serum TAAs/AA values in at 8,12 and 16 weeks after streptozoticin injection were considerably elevated in comparison with those of control group （0.0832 vs 0. 1042,0.0910 vs0. 1568,0. 1100 vs 0. 1895; t=-36. 409 ,t=-166. 714,t=-148. 212 ,P〈0.05）. Conclusion Elevation of serum TAAs/AA is associated with diabetic duration,implying that damage of nitrative stress to retinal capillary is a parallel procedure to diabetic course. Establishment of GC/MS detection system offers a new evaluating indicator in the research on microvascular ischemic disease and could be implemented in clinical testing.