Lipid peroxidation measurement by thiobarbituric acid assay in rat cerebellar slices.
Lipid peroxidation by reactive oxygen species (ROS) is known to be involved in the damaging mechanism of several acute and chronic brain disorders. The most prominent and currently used assay as an index for lipid peroxidation products is the thiobarbituric acid assay (TBA test). It is based on the reactivity of an end product of lipid peroxidation, malondialdehyde (MDA) with TBA to produce a red adduct. However, it is known that the MDA levels are frequently overestimated, that the reaction lacks specificity and mainly reflects the susceptibility of brain tissue to the generation and degradation of newly formed lipid hydroperoxides under the TBA test conditions. The present paper shows that artifactual lipid peroxidation by TBA test conditions can be prevented and that the MDA level overestimation can be minimized in cerebellar slices. This can be done by incubating the slices in a continuous tissue perfusion system, by adding butylated hydroxytoluene (BHT) to the homogenization solutions and by carrying out the assay anaerobically on deproteinizated supernatants of cerebellar slice homogenates. The present research also showed that lipid peroxidation products generated during incubation of the slices by hydrogen peroxide (H2O2) could be measured without artifactual interference by the TBA test conditions.
Garcia
Yngo J
YJ
Departamento de Bioquímica, Facultad de Medicina, Universidad de Los Andes, Mérida 5101, Venezuela. garciayngo@intercable.net.ve
Rodríguez-Malaver
Antonio J
AJ
Peñaloza
Nancy
N
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002084:Butylated Hydroxytoluene / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006861:Hydrogen Peroxide / Q000494:pharmacology; D066298:In Vitro Techniques; D015227:Lipid Peroxidation / Q000187:drug effects / Q000502:physiology*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D013057:Spectrum Analysis; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D013858:Thiobarbiturates / Q000032:analysis*; D013997:Time Factors
D000975:Antioxidants; D013858:Thiobarbiturates; D002084:Butylated Hydroxytoluene; D006861:Hydrogen Peroxide; C029684:thiobarbituric acid