Simultaneous Determination ofl-Ascorbic Acid, Glutathione, and Their Oxidized Forms in Ozone-Exposed Vascular Plants by Capillary Zone Electrophoresis

Abstract

Simultaneous determination of ascorbic acid (AA), glutathione (GSH), and oxidized glutathione (GSSG) in vascular plants has been achieved by capillary zone electrophoresis. A background electrolyte containing a phosphate buffer of pH 7 and hexadimethrin bromide for dynamic control of electroosmotic flow was used. Separation was performed in less than 5 min. The signal was simultaneously monitored at 265 and 200 nm. Dehydroascorbic acid (DHA) was determined in another aliquot of the same plant extract upon reduction with dl-homocysteine. Limits of detection of AA, GSH, and GSSG ranged from 0.2 to 0.8 μg mL-1, which corresponded from 0.04 to 0.16 mg per 100 g fresh weight of tomato, watermelon, and potato leaves. Plants exposed to a high ozone concentration (1600 μg m-3) in a laboratory fumigation system during 1, 2, 3, and 5 h showed progressive ozone-induced oxidative stress. This could be seen as a simultaneous decrease of the AA/(AA+DHA) and GSH/(GSH+GSSG) ratios; however, the decline rate was higher for AA/(AA+DHA) than for GSH/(GSH+GSSG) in plants cultivated in the field, in open-top chambers, during ca. 50 days with much lower ozone concentrations (mean concentrations, 10 and 80 μg m-3).

Simultaneous Determination of Glutathione and Glutathione Disulfide in an Acid Extract of Plant Shoot and Root by Capillary Electrophoresis

“…In this sense, our results confirm this tendency and are coincident with the results of several authors 5,8 . Likewise, the levels found in tomato root showed the same orders of magnitude as those reported by Rellan et al 8 in the root of Beta vulgaris, which they report as 92.1 and 46.1 nmol g -1 fr wt.…”

“…Similarly, linearity was good, with a correlation coefficient above 0.999 and a linear range up to 80 µM. Limit of detection values are comparable to those reported by Herrero et al 5 using CE with a diode array detector or those reported by Carru et al 12 using photometric detection. Values are also similar to those found through chromatographic techniques 8 , although not as low as those found through CE coupled to the use of more sensitive detectors such as laser-induced fluorescence 18 and mass spectrometry 19 .…”

“…Measurement reproducibility was established in relation to area under the curve and migration time, recording both parameters under the above mentioned experimental conditions five times in a day (intraday) and for five consecutive days (interday) ( Table 3). In most of the studies where capillary electrophoresis has been used for glutathione determination, the required separation time ranges from 3 to 7 min 5,8,11 . Studies where time is below this range are less frequent.…”

“…This function involves oxidation of the thiol group to form mainly glutathione disulfide (GSSG) 1,2 . Attempts have been made to relate changes in the levels of both peptides present in tissues or fluids to stressful situations resulting from various environmental conditions such as heavy metals, ozone, luminic radiation, among others [3][4][5] . In this respect, the GSH/GSSG ratio has been utilized rather than the individual levels of each peptide as an indicator of oxidative status in plants and animals 2 .…”

“…There are several studies dealing with glutathione determination in plant shoot 5,8 , with concentration values ranging from a few nmol g -1 to several hundreds of nmol g -1 . On the contrary, root has received less attention, even though it is the fundamental organ for nutrient absorption and also the main way of xenobiotic uptake by the plant.…”