ENZYMIC METHOD FOR QUANTITATIVE DETERMINATION OF NANOGRAM AMOUNTS OF TOTAL AND OXIDIZED GLUTATHIONE - APPLICATIONS TO MAMMALIAN BLOOD AND OTHER TISSUES
ENZYMIC METHOD FOR QUANTITATIVE DETERMINATION OF NANOGRAM AMOUNTS OF TOTAL AND OXIDIZED GLUTATHIONE - APPLICATIONS TO MAMMALIAN BLOOD AND OTHER TISSUES
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DOI：
10.1016/0003-2697(69)90064-5
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发表时间：
1969-01-01
影响因子：
2.9
通讯作者：
TIETZE, F
中科院分区：
文献类型：
--
作者：
TIETZE, F
本文提出了一种测定纳克级谷胱甘肽的新方法，该方法是基于谷胱甘肽（GSH）或谷胱甘肽硫苷（GSSG）对TPNH和酵母谷胱甘肽还原酶（GSHR）混合物还原埃尔曼试剂（DTNB）的催化作用。与以前的分析方法不同，本文所述的程序可有效测量未知混合物的总谷胱甘肽（GSH + GSSG）含量，并且不会受到其他硫醇组分存在的明显干扰。有人建议，谷胱甘肽在这个系统中的催化作用驻留在GSH的连续酶促再生，本最初或酶促形成GSSG，其与巯基试剂的相互作用后。该方法的灵敏度足以测定细胞外组织液（如血浆、唾液和尿液）中的总谷胱甘肽，这些组织液通常含有极低水平的谷胱甘肽，基本上无需对样品进行预处理。全血谷胱甘肽测定也是如此，其中初步程序仅限于从10 μl样品中制备1：100的溶血产物。根据已发表的程序，用NEM预处理组织提取物以与游离GSH形成无酶活性的复合物，从而可以测定其中通常存在的低水平氧化型谷胱甘肽。使用上述分析方法测定大鼠血液、肾脏和肝脏的总谷胱甘肽和氧化型谷胱甘肽含量，所得值与先前研究者获得的值一致。
A method for the analysis of nanogram quantities of glutathione has been developed which is based on the catalytic action of GSH or GSSG in the reduction of Ellman reagent (DTNB) by a mixture of TPNH and yeast glutathione reductase. Unlike previous methods of analysis the procedure described here effectively measures the total glutathione (GSH + GSSG) content of unknown mixtures and is not subject to appreciable interference by the presence of other thiol components. It is suggested that the catalytic action of glutathione in this system resides in the continual enzymic regeneration of GSH, present initially or formed enzymically from GSSG, following its interaction with the sulfhydryl reagent. The sensitivity of the method is such as to permit the determination of total glutathione in extracellular tissue fluids such as plasma, saliva, and urine normally containing very low levels of this material, essentially without pretreatment of the sample. The same is true for glutathione determinations of whole blood, in which the preliminary procedure is confined to the preparation of a 1:100 hemolyzate from as little as 10 μl of sample. Following published procedures, the pretreatment of tissue extracts with NEM to form an enzymically inactive complex with free GSH allowed the determination of the low levels of oxidized glutathione normally present therein. The use of the foregoing analytical method in the determination of total and oxidized glutathione contents of rat blood, kidney, and liver gave values in good agreement with those obtained by previous investigators.