Roberts J C, Francetic D J
Department of Medicinal Chemistry, University of Utah, Salt Lake City 84112.
Anal Biochem. 1993 Jun;211(2):183-7. doi: 10.1006/abio.1993.1254.
There is little consistency in the literature regarding the procedures for sample preparation employed for the measurement of glutathione (GSH) in biological tissues. Most procedures use an acid to homogenize tissue samples to precipitate proteins from the mixture and to minimize oxidative changes. Others employ 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the homogenization, which serves only to block thiol groups. The present studies were undertaken to critically compare the two methods of sample preparation on resulting GSH values determined by the Tietze method in numerous organs of mice. It was found that kidney, liver, and pancreas GSH levels were seriously underestimated when DTNB was used instead of acid to prepare the tissue samples. This discrepancy was eliminated when the animals were pretreated with AT-125, confirming the participation of gamma-glutamyltranspeptidase, the enzyme responsible for the first step in the degradation of GSH. GSH added to kidney homogenates in DTNB was degraded rapidly and continuously in a time-dependent fashion. In contrast, GSH added to acid homogenates produced stable GSH values up to 8 h after sample preparation in most cases. Storage of acid homogenates at -70 degrees C for 12 months gave results identical to original measurements, within 10% error, for 9 of 10 samples tested.
关于生物组织中谷胱甘肽（GSH）测量所采用的样品制备程序，文献中几乎没有一致性。大多数程序使用酸来匀浆组织样品，以从混合物中沉淀蛋白质并尽量减少氧化变化。其他方法在匀浆过程中使用5,5'-二硫代双（2-硝基苯甲酸）（DTNB），其仅用于封闭巯基。本研究旨在严格比较两种样品制备方法对通过Tietze方法在小鼠多个器官中测定的GSH值的影响。结果发现，当使用DTNB而不是酸来制备组织样品时，肾脏、肝脏和胰腺的GSH水平被严重低估。当动物用AT-125预处理时，这种差异被消除，证实了γ-谷氨酰转肽酶的参与，该酶是GSH降解第一步的负责酶。添加到DTNB中的肾脏匀浆中的GSH以时间依赖性方式迅速且持续降解。相比之下，在大多数情况下，添加到酸匀浆中的GSH在样品制备后长达8小时产生稳定的GSH值。在-70℃下储存酸匀浆12个月，对于10个测试样品中的9个，结果与原始测量值相同，误差在10%以内。
