RESUMEN
Oxidized protein adducts are formed under conditions of oxidative stress and may represent a valuable biomarker for a variety of diseases which share this common aetiology. A suitable candidate biomarker for oxidized proteins is protein-bound 3,4-dihydroxyl-l-phenylalanine (l-DOPA), which is formed on 3'-hydroxylation of tyrosine residues by hydroxyl radicals. Existing methodologies to measure protein-bound l-DOPA employ lengthy acid hydrolysis steps (ca. 16h) which may cause artifactual protein oxidation, followed by HPLC with detection based on the intrinsic fluorescence of l-DOPA. We report a novel method for the measurement of protein-bound l-DOPA which involves rapid hydrolysis followed by pre-column concentration of 6-aminoquinolyl-derivatives using cloud-point extraction. The derivatized material is resolved by reversed-phase HPLC in less than 30min and has derivatization chemistry compatible with both UV and fluorescent detection, providing detection down to the femtomole level. The method provides identical results to those found with highly specific ELISA-based techniques and requires only basic instrumentation. The stability of the 6-aminoquinolyl-derivatives together with the fast and sensitive nature of the assay will be appealing to those who require large sample throughput.
Asunto(s)
Aminoácidos/química , Proteínas Sanguíneas/química , Leucocitos Mononucleares/química , Levodopa/análisis , Albúmina Sérica Bovina/química , Amidinas/química , Cromatografía Líquida de Alta Presión , Humanos , Hidrólisis , Microextracción en Fase Líquida , Oxidación-Reducción , Unión ProteicaRESUMEN
Nanosized titanium dioxide (TiO2) is a common component of sunscreen preparations and cosmetics as it reflects UV and visible light in accordance to Rayleigh's law. However, in aqueous environments, TiO2 is an efficient photocatalyst, producing superoxide O2â»· and hydroxyl (HO·) radicals, which are highly damaging to biomolecules. We investigated the role of TiO2 in promoting the peroxidation of linoleic acid (LA) alone and in the presence of hypochlorous acid (HOCl). TiO2 significantly enhanced peroxidation of LA, which was further enhanced in the presence of HOCl. This latter finding involved the formation of singlet molecular oxygen in a Russell-type mechanism appearing to involve preformed lipid hydroperoxides (LOOH). In addition to lipid peroxidation, HOCl also mediated formation of 18:1 monochlorohydrins, which in the presence of TiO2 appeared to decompose to kinetic products which supplemented peroxidation of linoleic acid. We present a theoretical mechanism which fits the available experimental data and may partially explain the dichotomy associated with HOCls role in lipid modification.