A sensitive procedure for the determination of protein bound 3,4-dihydroxyphenyl-alanine as a marker for posttranslational protein hydroxylation in human frontal cortex, liver, and red blood cells.

Abstract

Oxidative stress has been implicated as an initiating or at least aggravating factor in neurodegenerative diseases, atherosclerosis, stroke, and inflammatory diseases. Since reactive oxygen species (ROS) are short lived reactive species, the involvement of ROS in biological tissues has to be determined by the evaluation of ROS-modified marker molecules. In principle, ROS react unspecifically with nearly any kind of biomolecule depending on the site of their production. One of these relatively stable markers of oxidative stress is supposed to be protein-bound 3,4-dihydroxyphenylalanine (PB-DOPA; Gieseg et al., 1993). Current fluorimetric assays for the detection of PB-DOPA (Armstrong and Dean, 1995) turned out to be less sensitive than the electrochemical detection of PBDOPA. The former studies mainly used model proteins to investigate the biochemistry of in vitro oxidation of tyrosine induced by the iron/EDTA/ascorbate system in proteins. We now tried to further improve the sensitivity of the available methods in order to be able to determine the basal levels of PB-DOPA in various human tissues with high oxygen turnover rates (frontal cortex, liver and red blood cells) from healthy controls. In addition, we determined tyrosine levels in order to be able to establish a tissue specific ratio of PBDOPA to tyrosine in analogy to the well known ratio of 8-hydroxy-2‘deoxyguanosine/2‘deoxyguanosine for the determination of oxidative stress related DNA damage different organs and could be of value if ROS-mediated actions following trhe exposure to toxicants that cause free radical production shall be investigated.