Petyaev I M, Hunt J V
University of Cambridge, Department of Pathology, UK.
Biochim Biophys Acta. 1997 Apr 21;1345(3):293-305. doi: 10.1016/s0005-2760(97)00005-2.
The reaction rate between superoxide and nitro-blue tetrazolium (NBT) is known to be accelerated/catalysed by micellar systems. Previous reports suggest that an accelerated rate of NBT reduction by micellar systems may be the result of either the binding of organic substrates such as NBT to the micellar phase giving a more favourable environment for superoxide reduction (an orientation effect), or the electrostatic interaction between micelles and superoxide. Here we show, using three different superoxide generating systems, that micelles composed of a number of different lipids or human low density lipoprotein (LDL) accelerates the apparent reaction between superoxide and NBT. Evidence in favour of an accelerated production of superoxide as opposed to the accelerated reduction of NBT is provided and we propose that the accelerated production of superoxide is a consequence of increased oxygen solubility in the lipid, rather than aqueous, phase. This is supported by: 1. The absence of any spectrophotometric changes due to interaction between lipid or LDL and reagents used. 2. The ability of micelles composed of a number of different fatty substances, including LDL, to accelerate superoxide generation, assessed by NBT reduction. 3. The behaviour of micelles, which appears to be one of substrate rather than catalyst, during the acceleration of NBT reduction. This is confirmed by the use of a known micellar catalyst, Triton-X100. This suggests that lipids contribute to the reaction as a substrate rather than a catalyst. 4. The inability of LDL to accelerate NBT reduction by potassium superoxide, a reaction which is independent of bimolecular oxygen. 5. The inability of LDL to accelerate NBT reduction when added after superoxide generation. 6. Studies that show LDL can enhance an NBT-independent monitor of oxidation, namely the transition metal-catalysed oxidation of vitamin C. 7. Estimations of the solubility of oxygen in LDL which appear to be consistent with reported physical measurements. Furthermore, we show that LDL modification can alter LDL-mediated micellar acceleration of superoxide generation. Extensive oxidation of LDL decreases micellar acceleration and minimal oxidation enhances it. We suggest that LDL micellar acceleration might serve as a novel approach to studying human LDL.
