高效液相色谱法测定精浆中过氧化脂质水平及其在男性不育症研究中的应用

Determination of Lipid Peroxidation in Human Seminal Plasma by High Performance Liquid Chromatography and Its Diagnostic Value of Male Infertility

采用高效液相色谱法测定精浆中过氧化脂质 (lipidperoxidation ,LPO)含量 ,研究了有正常生育能力的男子和不育症患者精浆中LPO含量水平差异及其对男子不育症的影响。精浆样品经酸化后 ,分解生成的丙二醛 (malon dialdehyde ,MDA)与硫代巴比妥酸 (thiobarbituricacid ,TBA)缩合反应形成紫红色产物 ,以LichrospherC18化学键合硅胶为固定相 ,0 0 2 5mol/LKH2 PO4(pH 6 2 ) 甲醇 (体积比为 5 8∶4 2 )为流动相进行色谱等度分离 ,在 5 32nm波长下检测 ,并对各组测定结果进行统计学分析。在所建立的分析条件下 ,MDA TBA缩合物的色谱保留时间约为 4min ,峰形清晰对称 ,与样品中其余内源性物质分离完全 ,定量准确。MDA浓度在 0 10～ 2 5 0 μmol/L内与峰面积之间的线性良好。MDA峰面积批间 (n =5 )、批内 (n =7)测定的相对标准偏差分别为 3 8%和 3 1% ,样品测定回收率为 90 0 %～ 98 8%。方法已应用于因脂质过氧化反应过高所致男性不育症的研究。除阻塞性无精子症组外 ,其余各不育组与正常生育组相比 ,精浆中MDA水平均有显著性差异 (P <0 0 1)。

A simple and reliable high performance liquid chromatographic (HPLC) method has been developed and validated for the analysis of malondialdehyde (MDA) in human seminal plasma. After human seminal plasma was hydrolyzed, MDA, one of the hydrolysis products, reacted with thiobarbituric acid (TBA) to form MDA(TBA)_2, a red-colored adduct with a maximum absorbance at 532 nm. HPLC separation of the adduct in human seminal plasma was performed on a Lichrospher C_(18) column. A mobile phase composed of 0.025 (mol/L) KH_2PO_4 (pH 6.2)-methanol in 58∶42 (v/v) was found to be the most suitable ratio for this separation at a flow rate of 1.0 (mL/min) and enabled the baseline separation of the adduct with isocratic elution. Under the chromatographic conditions described, the MDA-TBA adduct had a retention time of approximately 4 min, and good separation and detectability of MDA in human seminal plasma samples were obtained. The method proved to be linear in the range of MDA from 0.10 μmol/L to 2.50 (μmol/L). The relative standard deviations of MDA analysis within- and between-assay were 3.1% (n=7) and 3.8% (n=5), respectively. The average recoveries were 90.0%-98.8% for the human seminal plasma samples. The method has been successfully applied to the study of male infertility induced by overproduction of lipid peroxidation in male reproductive system. Exception of obstructive azoospermic group, MDA concentrations of seminal plasma in control group made very significant difference from those in other infertile groups (P<0.01).