High-performance liquid chromatography-electrochemical detection of antioxidants in vertebrate lens: glutathione, tocopherol, and ascorbate.

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.