A fast and accurate method to measure both oxidative stress and vitality in a single organ slice

Abstract

Increased oxidative stress does not necessarily cause an organ to suffer from oxidative damage, since antioxidant systems to protect organs are present. However, when a decrease in the vitality of an organ coincides with an increase in oxidative stress, increased oxidative damage is likely. A sequential method for the measurement of both energy status and oxidative stress in the same sample has been developed. The novelty of this method lies in the combination of efficiency and accuracy. Nucleotides and malondialdehyde (MDA) of 80 different samples can be released in a perchloric environment with ultrasonic treatment instead of homogenization. Malondialdehyde concentration can be measured after complexing with 2,4-dinitrophenylhydrazine without any homogenization, solvent phase extraction, and centrifugation steps. Yields of both malondialdehyde and nucleotides were similar to those of the homogenization procedure. Detection limit was 141 fmol for MDA and 22.5 pmol for the nucleotides. Furthermore, the stability of the malondialdehyde-2,4-dinitrophenylhydrazine complex after 3 weeks at -20 degrees C is excellent 99.7% (+/-5.6). Nucleotides are stable for the same time period. Spiking of samples with MDA and nucleotides showed good recoveries (102.5% (+/-5.0) and 99.8% (+/-7.9), respectively). The present data show an accurate method to measure both the energy status and the oxidative stress in a single organ slice with a minimum of effort and time.