Factors affecting the ascorbate- and phenolic-dependent generation of hydrogen peroxide in Dulbecco's Modified Eagles Medium.
Ascorbate and several polyphenolic compounds have been reported to undergo oxidation in cell culture media to generate hydrogen peroxide (H2O2), but the mechanism underlying this has not been established. We therefore investigated the parameters affecting H2O2 production. H2O2 generation from ascorbate, gallic acid and other phenolic compounds in Dulbecco's Modified Eagles' Medium (DMEM) at 37 degrees C under 95% air - 5% CO2 was not significantly inhibited by high (5-10 mM) concentration of EGTA, o-phenanthroline or desferriox-amine, but partial inhibition by EDTA and diethylene-triaminepentaacetic acid (DTPA) was observed. Incubation of DMEM alone at 37 degrees C led to an upward drift of pH, even under an atmosphere of 95% air - 5% CO2. Prevention of this pH rise by increasing the concentration of N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (Hepes) buffer lowered the levels of H2O2 generated by ascorbate and phenolic compounds, but there was still substantial H2O2 generated at pH 7.4. Mixtures of ascorbate and phenolic compounds led to less H2O2 generation than would be expected from the rates observed with ascorbate or phenolic compounds alone. Ascorbate prevented the loss of gallic acid incubated in DMEM. The role of metal ions and other constituents of the culture medium in promoting H2O2 generation is discussed.
Wee
Liang Meng
LM
Department of Biochemistry, Faculty of Medicine, National University of Singapore MD 7 #03-08, 8 Medical Drive, Singapore 117597, Singapore.
Long
Lee Hua
LH
Whiteman
Matthew
M
Halliwell
Barry
B
D001205:Ascorbic Acid / Q000378:metabolism*; D002374:Catalase / Q000378:metabolism; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D005707:Gallic Acid / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007477:Ions; D004369:Pentetic Acid / Q000494:pharmacology; D013054:Spectrophotometry, Atomic; D013696:Temperature; D013997:Time Factors
D002614:Chelating Agents; D003470:Culture Media; D007477:Ions; D004533:Egtazic Acid; D005707:Gallic Acid; D004369:Pentetic Acid; D004492:Edetic Acid; D006861:Hydrogen Peroxide; D002374:Catalase; D001205:Ascorbic Acid