用荧光分光光度法测量冠心病患者血浆谷胱甘肽

Plasma Glutathione of Patients with Coronary Heart Disease Measured by Fluorospectrophotometer

利用荧光分光光度计对还原型谷胱甘肽(GSH)及氧化型谷胱甘肽(GSSG)标准品进行全波长扫描,确定激发(λex)波长为334·4nm,发射(λem)波长为424nm以及5nm的谱带宽度,以pH8·3的磷酸盐缓冲液(PBS)作为缓冲液,用100μL10mmol·L-1邻苯二甲醛(OPA)甲醇溶液对GSH进行衍生化30min后测量荧光值(FU)。以标准品浓度为自变量,FU为应变量,求直线方程,YGSH=6·9+8·6X(r2=0·994),YGSSG=6·2+17·2X(r2=0·999),并制作标准曲线。据此测定三组研究对象血浆GSH及GSSG,并计算GSH/GSSG的氧化还原电位。研究发现,从正常对照组到冠心病前期组,再到冠心病组,GSSG逐渐升高,GSH和GSH/GSSG逐渐减少,氧化还原电位逐渐升高,向氧化方向偏移(P均<0·05)。该方法操作简单,精密度好,准确度高。

The standard samples of reduction form glutathione （GSH） and oxidization form glutathione （GSSG） were scanned with full-wavelength range to determine the excitation wavelength λex334.4 nm, the emission wavelength λem 424 nm, and the spectral bandwidth 5 nm respectively. Phosphate buffer saline （PBS） of pH 8. 3 served as buffer solution. GSH was incubated for 30 min with 100 μL o-phthaldehyde （OPA） of 10 mmol· L^-1 methyl alcohol solution for derivatization, and then fluorescence intensities were measured. With standard glutathione concentration being independent variable and fluorescence intensity being dependent variable, the linear equations for GSH and GSSG were deduced： YGSH =6. 9＋8. 6X （r^2 =0. 994） and YGSSG=6. 2＋17.2X （r^2 =0. 999）. Standard curves were done hereby. The plasma glutathione of three groups was then measured, and GSH/ GSSC redox potential was calculated according to Nernst equation. The results indicated that, from normal control group to prophase coronary heart disease group, then to coronary heart disease group, the GSH and GSH/GSSG ratio gradually reduced, GSSG and GSH/GSSG redox potential gradually increased （more positive） （all P〈0. 05）, and the redox potential shifted to oxidization direction along with the development of coronary heart disease. This fluorospectrophotometry method showed simple operation, and fine precision and accuracy.