Detection of oxidative stress in heart by estimating the dinitrophenylhydrazine derivative of malonaldehyde

Accurate estimation of the oxidative stress in heart is necessary because the pathogenesis of many heart diseases are believed to be mediated at least in part from the development of oxidative stress resulting from the generation of oxygen free radicals and reduced antioxidant defense system. The most widely used method for this purpose has been the estimation of malonaldehyde (MDA), a lipid peroxidation product, by the thiobarbituric acid (TBA) reaction method. However, because of the nonspecificity of this method, the results are often erroneous. The present report describes a method using high-performance liquid chromatography (HPLC) to estimate MDA. To develop the oxidative stress, two different models were used: ischaemic-reperfused heart and perfusing the heart with a hydroxyl radical (OH•) generating system. The coronary effluents obtained from the isolated rat heart before ischaemia and during the reperfusion of ischaemic heart, as well as during the perfusion of the heart with the OH• generating system were collected, derivatized with 2,4-dinitrophenylhydrazine (DNPH) and extracted with pentane. Aliquots of 25 μl in acetonitrile were injected onto a Beckman Ultrasphere C18 (3 μm) column. The products were eluted isocratically with a mobile phase containing acetonitrile-water-acetic acid (40: 60:0.1, v/v/v), measured at 307 nm using a Waters M-490 multichannel UV detector and collected for gas chromatography-mass spectrometry (GC-MS). The peaks were identified by co-chromatography with DNPH derivatives of authentic standards, peak addition, and by GC-MS. The retention time for MDA-DNPH was 5.3 min. The increase in MDA peak in the post-ischemic heart and during the perfusion with OH• generating system, was reduced by the OH• scavengers, superoxide dismutase (SOD) plus catalase. The results of this study indicate that only a small amount of MDA is formed in the ischemic reperfused myocardium, and that the MDA-TBA method errorneously overestimates the MDA information. This method allows rapid and accurate estimation of MDA to monitor the oxidative stress in heart.