Liu S, Ansari N H, Wang C, Wang L, Srivastava S K
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX,77555-0647 USA.
Curr Eye Res. 1996 Jul;15(7):726-32. doi: 10.3109/02713689609003455.
Purpose. To develop a rapid and accurate method for the quantification of reduced glutathione (GSH) and oxidized glutathione (GSSG) using micro-quantities of ocular lens. Methods. The epithelium, cortex and nucleus of the lens were separated and also the whole lens was homogenized in 3% metaphosphoric acid. The homogenate was ultrafiltered by centrifugation at 10,000 g in an Amicon microconcentrator, molecular weight cut off 3,000 g. The method does not require prior derivatization of the glutathiones. The filtrate was analyzed on a Microsorb-MV by a high performance liquid chromatography (HPLC) column using an isocratic solvent system (3% methanol and 10 mM potassium phosphate, pH 3.0) and detection at 200 nm.
The GSH and GSSG were eluted from the HPLC column at retention times 5 and 10 min, respectively. The detection limit was 100 pmoles applied to the column. The recovery of GSH and GSSG added to the tissue samples was 97-100%.
A fast and sensitive HPLC-method for the quantification of picomole quantities of GSH and GSSG in ocular lens, which does not require prior derivatization, has been developed.
目的。开发一种使用微量晶状体快速准确地定量还原型谷胱甘肽（GSH）和氧化型谷胱甘肽（GSSG）的方法。方法。将晶状体的上皮、皮质和核分离，并且将整个晶状体在3%偏磷酸中匀浆。匀浆在Amicon微浓缩器中以10,000 g离心进行超滤，截留分子量为3,000 g。该方法不需要谷胱甘肽预先衍生化。滤液在Microsorb-MV上通过高效液相色谱（HPLC）柱进行分析，使用等度溶剂系统（3%甲醇和10 mM磷酸钾，pH 3.0）并在200 nm处检测。
GSH和GSSG分别在保留时间5分钟和10分钟时从HPLC柱上洗脱。检测限为施加到柱上100皮摩尔。添加到组织样品中的GSH和GSSG的回收率为97 - 100%。
已开发出一种快速灵敏的HPLC方法，用于定量晶状体中皮摩尔量的GSH和GSSG，该方法不需要预先衍生化。
