A microplate adaptation of the thiobarbituric acid reactive substances assay to determine lipid peroxidation fluorometrically in small sample volumes

Résumé

A simple, fast, reproducible and low-cost assay for thiobarbituric acid reactive substances (TBARS) has been adapted for use with a microplate spectrofluorometer. The technique allows rapid analysis of multiple samples and requires a very small sample volume (50 µl of red cell homogenates from passerine birds at protein concentrations of 3.4-8.9 mg/ml in this study), what is of special interest for biomonitoring studies working with small-sized animals from which a limited amount of sample can be obtained. The TBARS test involves the reaction of thiobarbituric acid (TBA) with malondialdehyde (MDA) under heating (90°C), leading to the formation of products that can be measured fluorometrically using black 384-well plates at excitation/emission wavelength of 532/553 nm. The concentrations of peroxidized lipids in samples were determined by extrapolation from a MDA standard curve. Two different excitation/emission combinations (532/553 and 530/550 nm) were used and both pairs were suitable for this technique. Intraand inter-plate variability was < 20% and a good linearity of the standard curve was observed (R2 > 0.99). The research use of this microplate adaptation of the TBARS assay will provide further data and understanding of lipid peroxidation reducing the limitation of small sample volume.