Date of Award
Master of Science (MS)
The oxidized and reduced forms of ascorbic acid (AA) are reactive and unstable compounds making their measurement difficult. Detection of small amounts of AA and dehydroascorbic acid (DHAA) is essential for determining the biochemical function of the vitamin in cells and subcellular organelles. While a variety of techniques exist which detect millimolar levels of AA, accurate assessment of pmol levels of AA and DHAA in a variety of tissues has not been perfected. In the present study, a method was developed for quantitation of AA and DHAA that combines HPLC separation with the advantages of increased sensitivity and selectivity available with coulometric electrochemical detection.
Tissues were homogenized in 0.3% meta-phosphoric acid containing 1 mM thiourea and 0.1 mM EDTA. Portions of the homogenates were either placed in 10% meta-phosphoric for AA analysis or in a 120 mM sodium phosphate buffer (pH 7.2) containing 0.1 mM EDTA and 1 mM thiourea, for total AA (AA +DHAA) measurement. Total AA was determined by reducing the endogenous DHAA in a sample with 10 mM p-mercaptoethanol and allowing the reduction to occur for 10 minutes at room temperature. The reaction was stopped with the addition of 10% meta-phosphoric acid.
The developed method was extended to assess the levels of AA and DHAA in nondiabetic and diabetic rat liver, kidney, brain and adrenal tissues. Results indicated that the AA levels in diabetic liver and kidney tissues were significantly less than control levels but there was no difference in AA levels in diabetic and nondiabetic brain and adrenal tissues. When compared to controls, DHAA concentration was greater in diabetic rat liver, kidney, and adrenal giands but not in brain tissue.
Fry, Debra A., "Measurement of Ascorbic Acid and Dehydroascorbic Acid in Mammalian Tissues using HPLC with Electrochemical Detection" (1991). Theses and Dissertations. 1014.