Mitton K P, Trevithick J R
Department of Biochemistry, University of Western Ontario, London, Canada.
Methods Enzymol. 1994;233:523-39. doi: 10.1016/s0076-6879(94)33058-1.
The HPLC-EC method has good specificity for the analysis of glutathione, tocopherol, and ascorbate. The same HPLC system can be used for all three analysis with changes of mobile phase and the electrode cell to match the procedure required. The same C18 reversed-phase column has been used with a refillable guard column for 3 years with no noticeable loss of resolving power. The main advantage of the glutathione procedure was the ability to monitor both GSH and GSSG, which allowed us to confirm that loss of GSH in the diabetic rat lens does not result in the appearance of GSSG. The main benefit of the tocopherol procedure was the ability to measure the tocopherol content of a single rat lens. Our previous experience with UV or fluorescence detection showed those methods to be not sensitive enough for a single lens determination. The mammalian lens has the lowest tocopherol content of the tissues of the eye, 10 to 40 times less than most body tissues as measured by gas chromatography-mass spectrometry (GC-MS). The better sensitivity of electrochemical detection has allowed for a single lens determination, keeping the number of experimental animals to a minimum. An advantage of the ASC analysis procedure was the extra specificity imparted by both the chromatography and the detector as well as the ability to estimate the total ascorbate (ASC plus DHAA) and DHAA content. Other reducing agents such as GSH and uric acid can interfere in colorimetric methods that rely on the reducing action of ASC. The very high GSH content of the mammalian lens was a concern when choosing a procedure. GSH levels exceeding 10 times the level of lens samples were found to yield no response using the HPLC-EC procedure for ASC. The only disadvantage with electrochemical detection was that the electrode response could drift with time, requiring more frequent calibration with standards. We continue to utilize these methods to examine the prevacuole loss of ASC and GSH in the diabetic rat lens model of cataract.
高效液相色谱 - 电化学检测法（HPLC - EC）在分析谷胱甘肽、生育酚和抗坏血酸盐方面具有良好的特异性。通过改变流动相和电极池以匹配所需程序，同一高效液相色谱系统可用于这三种分析。同一根C18反相柱搭配可再填充的保护柱已使用3年，分离能力未见明显下降。谷胱甘肽分析方法的主要优点是能够同时监测还原型谷胱甘肽（GSH）和氧化型谷胱甘肽（GSSG），这使我们能够确认糖尿病大鼠晶状体中GSH的减少并不会导致GSSG的出现。生育酚分析方法的主要优势在于能够测量单个大鼠晶状体中的生育酚含量。我们之前使用紫外或荧光检测的经验表明，这些方法对于单个晶状体的测定不够灵敏。通过气相色谱 - 质谱联用（GC - MS）测量，哺乳动物晶状体的生育酚含量在眼部组织中是最低的，比大多数身体组织低10至40倍。电化学检测的更高灵敏度使得能够对单个晶状体进行测定，从而将实验动物数量降至最低。抗坏血酸盐（ASC）分析程序的一个优点是色谱法和检测器都赋予了额外的特异性，以及能够估算总抗坏血酸盐（ASC加脱氢抗坏血酸（DHAA））和DHAA含量。其他还原剂如GSH和尿酸会干扰依赖ASC还原作用的比色法。在选择分析程序时，哺乳动物晶状体中极高的GSH含量是一个需要考虑的问题。发现当GSH水平超过晶状体样品水平10倍时，使用HPLC - EC法分析ASC时无响应。电化学检测的唯一缺点是电极响应会随时间漂移，需要更频繁地用标准品进行校准。我们继续使用这些方法来研究糖尿病大鼠白内障晶状体模型中ASC和GSH的液泡前体损失情况。
