No abstract

The principal source of hydrogen peroxide in mitochondria is thought to be from the dismutation of superoxide via the enzyme manganese superoxide dismutase (MnSOD). However, the nature of the effect of SOD on the cellular production of H(2)O(2) is not widely appreciated. The current paradigm is that the presence of SOD results in a lower level of H(2)O(2) because it would prevent the non-enzymatic reactions of superoxide that form H(2)O(2). The goal of this work was to: a) demonstrate that SOD can increase the flux of H(2)O(2), and b) use kinetic modelling to determine what kinetic and thermodynamic conditions result in SOD increasing the flux of H(2)O(2). We examined two biological sources of superoxide production (xanthine oxidase and coenzyme Q semiquinone, CoQ(*-) that have different thermodynamic and kinetic properties. We found that SOD could change the rate of formation of H(2)O(2) in cases where equilibrium-specific reactions form superoxide with an equilibrium constant (K) less than 1. An example is the formation of superoxide in the electron transport chain (ETC) of the mitochondria by the reaction of ubisemiquinone radical with dioxygen. We measured the rate of release of H(2)O(2) into culture medium from cells with differing levels of MnSOD. We found that the higher the level of SOD, the greater the rate of accumulation of H(2)O(2). Results with kinetic modelling were consistent with this observation; the steady-state level of H(2)O(2) increases if K<1, for example CoQ(*-)+O(2)-->CoQ+O(2)(*-). However, when K>1, e.g. xanthine oxidase forming O(2)(*-), SOD does not affect the steady state-level of H(2)O(2). Thus, the current paradigm that SOD will lower the flux of H(2)O(2) does not hold for the ETC. These observations indicate that MnSOD contributes to the flux of H(2)O(2) in cells and thereby is involved in establishing the cellular redox environment and thus the biological state of the cell.

No abstract

We have developed a rapid, inexpensive, and reliable assay for the determination of ascorbate using a plate reader. In this assay, ascorbic acid is oxidized to dehydroascorbic acid using Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy) and then reacted with o-phenylenediamine to form the condensation product, 3-(dihydroxyethyl)furo[3,4-b]quinoxaline-1-one. The rate of appearance of this product is monitored over time using fluorescence. With this method, it is possible to analyze 96 wells in less than 10min. This permits the analysis of 20 samples with a full set of standards and blanks, all in triplicate. The assay is robust for a variety of samples, including orange juice, swine plasma, dog plasma, and cultured cells. To demonstrate the usefulness of the assay for the rapid determination of experimental parameters, we investigated the uptake of ascorbate and two different ascorbate derivatives in U937 cells. We found similar plateau levels of intracellular ascorbate at 24h for ascorbate and ascorbate phosphate. However, the intracellular accumulation of ascorbate via the phosphate ester had an initial rate that was three to five times slower than that via the palmitate ester. Only lower concentrations of the palmitate ester could be examined because the ethanol needed as solvent decreased cell viability; it behaved similarly to the other two compounds at lower concentrations. To come to these conclusions, only nine plates needed to be analyzed to provide us with the end result after only 7h of analysis. This clearly demonstrates the strength of the plate reader assay, which allows the analysis of large-sample sets in a fraction of the time required for the methods that are most commonly used today. The assay is quick, is very economical, and provides results with uncertainties on the order of only 5%.

Although its concentration is generally not known, glutathione peroxidase-1 (GPx-1) is a key enzyme in the removal of hydrogen peroxide (H 2 O 2 ) in biological systems. Extrapolating from kinetic results obtained in vitro using dilute, homogenous buffered solutions, it is generally accepted that the rate of elimination of H 2 O 2 in vivo by GPx is independent of glutathione concentration (GSH). To examine this doctrine, a mathematical analysis of a kinetic model for the removal of H 2 O 2 by GPx was undertaken to determine how the reaction species (H 2 O 2 , GSH, and GPx-1) influence the rate of removal of H 2 O 2 . Using both the traditional kinetic rate law approximation (classical model) and the generalized kinetic expression, the results show that the rate of removal of H 2 O 2 increases with initial GPx r , as expected , but is a function of both GPx r and GSH when the initial GPx r is less than H 2 O 2 . This simulation is supported by the biological observations of Li et al.. Using genetically altered human glioma cells in in vitro cell culture and in an in vivo tumour model, they inferred that the rate of removal of H 2 O 2 was a direct function of GPx activity)GSH (effective GPx activity). The predicted cellular average GPx r and H 2 O 2 for their study are approximately GPx r 51 mm and H 2 O 2 :5 mm based on available rate constants and an estimation of GSH. It was also found that results from the accepted kinetic rate law approximation significantly deviated from those obtained from the more generalized model in many cases that may be of physiological importance.

Singlet oxygen initiates lipid peroxidation via a nonfree radical mechanism by reacting directly with unsaturated lipids to form lipid hydroperoxides (LOOHs). These LOOHs can initiate free radical chain reactions leading to membrane leakage and cell death. Here we compare the ability and mechanism by which three small-molecule membrane antioxidants (beta-carotene, alpha-tocopherol and nitric oxide) inhibit lipid peroxidation in membranes. We demonstrate that beta-carotene provides protection against singlet oxygen-mediated lipid peroxidation, but does not slow free radical-mediated lipid peroxidation. Alpha-Tocopherol does not protect cells from singlet oxygen, but does inhibit free radical formation in cell membranes. Nitric oxide provides no direct protection against singlet oxygen exposure, but is an exceptional chain-breaking antioxidant as evident from its ability to blunt oxygen consumption during free radical-mediated lipid peroxidation. These three small-molecule antioxidants appear to have complementary mechanisms for the protection of cell membranes from detrimental oxidations.

No abstract

Nitric oxide (NO • ) is an effective chain-breaking antioxidant in free radical-mediated lipid oxidation (LPO). It reacts rapidly with peroxyl radicals as a sacrificial chain-terminating antioxidant. The goal of this work was to determine the minimum threshold concentration of NO • required to inhibit Fe 2+ -induced cellular lipid peroxidation. Using oxygen consumption as a measure of LPO, we simultaneously measured nitric oxide and oxygen concentrations with NO • -and O 2 -electrodes. Ferrous iron and dioxygen were used to initiate LPO in docosahexaenoic acid-enriched HL-60 and U937 cells. Bolus addition of NO • (1.5 μM) inhibited LPO when the NO • concentration was greater than 50 nM. Similarly, using (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate (PAPA/NO) as a NO • donor we found that an average steady-state NO • concentration of at least 72 ± 9 nM was required to blunt LPO. As long as the concentration of NO • was above 13 ± 8 nM the inhibition was sustained. Once the concentration of NO • fell below this value, the rate of lipid oxidation accelerated as measured by the rate of oxygen consumption. Our model suggests that the continuous production of NO • that would yield a steady-state concentration of only 10 -20 nM is capable of inhibiting Fe 2+ -induced LPO.