The development of new methods for the assessment of oxygen radical-mediated oxidative damage to biomolecules with special reference to lipids
The involvement of chemically-reactive oxygen radical species (RORS) in the pathogenesis of inflammatory joint diseases has been well documented. Much of the toxicity produced by increased superoxide ion (O2.-) and hydrogen peroxide (H2O2) generation has been attributed to the production of the highly reactive hydroxyl radical (.OH) which is mediated by low-molecular-mass iron chelates such as iron-citrate complexes. The .OH radical is extremely reactive and proving its formation in vivo is very difficult. Hence, assays for the assessment of .OH radical activity in the inflamed rheumatoid joint have involved identification and/or quantification of ‘unnatural’ chemical species produced by the attack of OH radical on a range of endogenous or, alternatively, therapeutically-administered exogenous 'target' molecules. This study involves: (1) An investigation of the precise chemical nature of intermediates in and so called 'endproducts' of the process of lipid peroxidation. Second-derivative (2D) electronic absorption spectrophotometry has been applied to the analysis of isomeric conjugated hydroperoxydienes, hydroxydienes and ketodienes and also to thiobarbituric acidreactive material in synovial fluid (SF) obtained from patients with inflammatory joint diseases. (2) A method for determining the extent of RORS-dependent peroxidation of polyunsaturated fatty acids (PUFAs) has been further developed for application to biological samples. This technique involves the conversion of conjugated hydroperoxydienes and compounds derived therefrom, i.e. oxodiene and hydroxydienes, to strongly chromophoric conjugated triene and tetraene species. The chromophore(s) produced are measured by their absorbance in the ultra-violet or near ultra-violet regions of spectra using 2D spectrophotometry. (3) An evaluation of the chemical nature of non-transferrin-bound iron in inflammatory synovial fluid is investigated by high-resolution, high-field proton (1H) nuclear magnetic resonance (NMR) spectroscopy, combined with the use of powerful iron(III) chelators (desferrioxamine and nitrilotriacetate), and the iron(lII)-binding protein apotransferrin in order to 'speciate' catalytic, low-molecular-mass iron complexes present in inflammatory SF samples. (4) The application of high field proton NMR spectroscopy to evaluate the abilities of the antioxidant thiol drug N-acetylcysteine and exogenous cysteine to protect metabolites present in intact inflammatory SF samples against oxidative damage arising from reactive oxygen radical species generated via gamma-radiolysis (5.00 kGy) in the presence of atmospheric oxygen.
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