Comparison of protein carbonyl and antioxidant levels in brain tissue from intracerebral haemorrhage and control cases

Abstract

Background: In an attempt to develop a clearer understanding of the pathological mechanisms underlying intracerebral haemorrhage (ICH), the objective of this investigation was to obtain evidence for free radical-induced oxidative damage in brain tissue following intracerebral haemorrhage. Methods: Brain cortex samples were obtained from the ischaemic penumbra (overlying the haematoma) from 10 patients with spontaneous ICH and from six control cases (normal tissue obtained during tumour removal or aneurysm repair). Following extraction via homogenization and subsequent derivatization with dinitrophenylhydrazine (DNPH), tissue samples were analysed for the presence of protein carbonyl moieties (a hallmark of tissue protein oxidation). This procedure involved SDS-polyacrylamide gel electrophoresis and Western immunoblotting using a commercially available primary monoclonal antibody to DNPH, with final visualization of oxidized protein bands via enhanced chemiluminescence. Results: Samples from ICH cases showed a number (10–15) of well-defined bands of medium to strong staining intensity (not present in nonderivatized samples), corresponding to proteins of molecular mass 25–200 kDa, indicating the presence of oxidatively damaged proteins in these samples. However, tissue samples from control cases also showed the presence of oxidized protein bands, with fractionation patterns for individual ICH or control samples being qualitatively and quantitatively similar. In addition, there was no significant difference in the levels of the following antioxidants (as additional indirect markers of free radical activity) in ICH or control brain tissue: glutathione, glutathione peroxidase, glutathione reductase, catalase, superoxide dismutase, and total antioxidant status. Conclusions: It is concluded from the above data that (i) evidence for free radical involvement in ICH based on protein carbonyl analysis should be interpreted with caution, since normal brain tissue contains a surprisingly high proportion of oxidized proteins; (ii) since there is no evidence for increased protein oxidative damage or decreased tissue antioxidant levels in ICH, therapeutic strategies aimed at salvage of potentially viable tissue would not benefit from inclusion of protein protecting antioxidants.