Figure 4. HPLC separation of ³H-glutathione from lens homogenate compared to the standard ³H-glutathione injected into the rat

Lenses were removed after sacrifice of the rat at 30 min after injection of ³H-glutathione, weighed and homogenized in 1.0 ml PEDTA. 0.1 ml of 69% HClO₄ was then added to deproteinize the homogenate. An aliquot of the ³H-glutathione injected was also taken for HPLC using a C18 Bondapak column as described in the text. The volumes used for injection were 20 µl of deproteinized lens homogenate (total volume 1.09 ml) and 5 µl glutathione (diluted with 0.1 volumes of 60% HClO₄). The total radioactivity in the aliquots used was 6432 dpm/ 100 µl homogenate injected, and 1.029 x 107 dpm for 200 µl of standard glutathione which was injected. Fractions (0.5 ml) were collected at a flow rate of 1 ml/min into scintillation vials and counted to establish the elution profile. As can be seen, the radioactive glutathione appears to elute in fraction 9.