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

采用高效液相色谱法测定精浆中过氧化脂质(lipid peroxidation,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(malondialdehyde,MDA)与硫代巴比妥酸(thiobarbituric acid,TBA)缩合反应形成紫红色产物,以Lichrospher C18化学键合硅胶为固定相,0.025 mol/L KH2PO4 (pH 6.2)-甲醇(体积比为58∶42)为流动相进行色谱等度分离

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

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 C18 column. A mobile phase composed of 0.025 mol/L KH2PO4 (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 micromol/L to 2.50 micromol/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).