The importance of sample preparation and storage in glutathione analysis.
The importance of sample preparation and storage in glutathione analysis.
复制标题
样品制备和储存在谷胱甘肽分析中的重要性。
DOI：
10.1006/abio.1993.1254
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发表时间：
1993
影响因子：
2.9
通讯作者：
Francetic,DJ
中科院分区：
文献类型：
--
作者：
Roberts,JC;Francetic,DJ
关于生物组织中谷胱甘肽（GSH）测定所用的样品制备程序，文献中几乎没有一致性。大多数程序使用酸使组织样品均质化，以从混合物中沉淀蛋白质并使氧化变化最小化。其他人在均质化中使用5，5 ′-二硫代-双（2-硝基苯甲酸）（DTNB），其仅用于封闭硫醇基团。目前的研究进行批判性地比较两种方法的样品制备产生的GSH值确定的Tietze方法在许多器官的小鼠。结果发现，当使用DTNB代替酸制备组织样品时，肾脏、肝脏和胰腺GSH水平被严重低估。当用AT-125预处理动物时，这种差异被消除，证实了γ-谷氨酰转肽酶的参与，该酶负责GSH降解的第一步。GSH加入到DTNB中的肾匀浆中，以时间依赖性方式快速且连续地降解。与此相反，在大多数情况下，GSH加入到酸性匀浆产生稳定的GSH值长达8小时后，样品制备。酸匀浆在−70°C下储存12个月后，10份样品中有9份的结果与原始测量结果相同，误差在10%以内。
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′-dithio-bis(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 γ-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°C for 12 months gave results identical to original measurements, within 10% error, for 9 of 10 samples tested.