Special modifications of the fluorescent screening method for glucose-6-phosphate dehydrogenase deficiency.
Special modifications of the fluorescent screening method for glucose-6-phosphate dehydrogenase deficiency.
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作者：
E. Beutler;M. Mitchell
葡萄糖-6-磷酸脱氢酶（G-6-PD）缺乏该方法依赖于还原三磷酸吡啶核苷酸（TPNH）的荧光作为G-6-PD活性的指标。100毫升加10jd全血。含有葡萄糖-6-磷酸、TPN、缓冲液和皂苷的筛选溶液。在室温下培养5分钟后，在普通滤纸上形成一个斑点。让斑点干燥，然后在长波紫外线下检查荧光。我们现在对程序进行修改，这可能会在某些情况下增强其效用。已经发现，当使用最初描述的测试试剂时，滤纸上的荧光褪色相对较快。超过24小时后很难区分正常和缺乏酶的样品。虽然这不会对普通临床实验室造成任何问题，但它可能会限制该技术在实地调查中的实用性。在这类研究中，最好在较远的中心实验室用紫外光检查测试结果，可能要延迟几天。研究发现，用合适的三氢氯化物缓冲液代替磷酸盐缓冲液，可以大大提高荧光的稳定性和初始强度。第二种修饰使几乎完全消除轻微荧光成为可能，这种荧光有时在检查患有最轻微形式的葡萄糖-6-磷酸脱氢酶缺乏症的个体的血液时出现，特别是使用用tris缓冲的反应混合物。这种荧光可以通过在反应混合物中加入氧化谷胱甘肽（GSSG）来消除。当GSSG存在时，TPNH通过谷胱甘肽还原酶（GSSG- r）的作用被氧化。但是谷胱甘肽还原酶的活性通常只有葡萄糖-6-磷酸脱氢酶-磷酸葡萄糖醛酸脱氢酶活性的大约四分之一
for glucose-6-phosphate dehydrogenase (G-6-PD) deficiency.1 This method depends upon the fluorescence of reduced triphosphopyridine nucleotide (TPNH ) as an indicator of G-6-PD activity. Ten j.d. of whole blood are added to 100 jil. of a screening solution containing glucose-6-phosphate, TPN, buffer, and saponin. A spot is made on ordinary filter paper after 5 minutes incubation at room temperature. The spot is permitted to dry, and is then examined for fluorescence under long-wave UV light. We now introduce modifications of the procedure which may enhance its usefulness under certain circumstances. It has been found that when the test reagent originally described is used the fluorescence on filter paper fades relatively rapidly. It is difficult to differentiate normal from enzyme-deficient samples after more than 24 hours. While this does not pose any problem for the ordinary clinical laboratory, it may limit the usefulness of the technic for field surveys. In such studies it may be desirable to examine the results of tests under ultraviolet light in a distant central laboratory, possibly after several days’ delay. It has been found that the stability, and indeed the initial intensity, of fluorescence can be greatly enhanced by substituting an appropriate trishydrochloride buffer for phosphate buffer. A second modification makes it possible to eliminate almost entirely the slight fluorescence which sometimes appears when blood from individuals with the mildest forms of glucose-6-phosphate dehydrogenase deficiency are examined, especially using a reaction mixture buffered with tris. This fluorescence can be eliminated by adding oxidized glutathione (GSSG) to the reaction mixture. When GSSG is present, TPNH is oxidized through the action of glutathione reductase (GSSG-R). But the activity of glutathione reductase is normally only approximately one-quarter of that of the activity of the glucose-6-phosphate dehydrogenase-phosphogluconic dehydrogenase sys-