Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds

Abstract

The occurrence of malondialdehyde (MDA), a secondary end product of the oxidation of polyunsaturated fatty acids, is considered a useful index of general lipid peroxidation. A common method for measuring MDA, referred to as the thiobarbituric acid-reactivesubstances (TBARS) assay, is to react it with thiobarbituric acid (TBA) and record the absorbance at 532 nm. However, many plants contain interfering compounds that also absorb at 532 nm, leading to overestimation of MDA values. Extracts of plant tissues including purple eggplant (Solanum melongena L.) fruit, carrot (Daucus carota L.) roots, and spinach (Spinacia oleracea L.) leaves were assessed for the presence of MDA and other non-MDA compounds absorbing at 532 nm. A method described herein corrects for these interferences by subtracting the absorbance at 532 nm of a solution containing plant extract incubated without TBA from an identical solution containing TBA. The reliability and eciency of this spectrophotometric method was assessed by altering the relative ratios of exogenous MDA additions and/or extracts of red cabbage (Brassica oleracea L.) leaves containing interfering compounds and then measuring MDA recovery. Reliability was also validated through high-performance liquid chromatography and high-performance liquid chromatography-mass spectrometry techniques. Results indicated that over 90% of exogenously added MDA could be recovered through the improved protocol. If there were no corrections for interfering compounds, MDA equivalents were overestimated by up to 96.5%. Interfering compounds were not detected in vegetables such as lettuce (Lactuca sativa L.) and spinach which had low or negligible concentrations of anthocyanidin derivatives. Comparisons between the TBARS method presented here and two currently accepted protocols indicated that the new modi®ed method exhibits greater accuracy for quantifying TBA-MDA levels in tissues containing anthocyanins and/or other interfering compounds. This modi®ed protocol represents a facile and rapid method for assessment of lipid peroxidation in virtually all plant species that contain interfering compounds.

Oxidative stress and antioxidant systems in Guava (Psidium guajava L.) fruits during ripening

“…The samples were cooled in an ice bath, centrifuged at 8,000 x g for 15 min and the absorbance of the supernatant was recorded at 532 nm. Non specific absorbance of the sample at 660 nm was subtracted from the absorbance at 532 nm (Hodges et al, 1999). The concentration of MDA was calculated using the molar extinction coefficient of 155 mM -1 cm -1 .…”

Arsenic-induced oxidative stress in the common bean legume, Phaseolus vulgaris L. seedlings and its amelioration by exogenous nitric oxide

“…H 2 O 2 content was measured from the absorbance at 410 nm using a standard curve. Lipid peroxidation rates were determined by measuring the malondialdehyde (MDA) equivalents following the method of Hodges et al (1999). About 0.5 g of fresh tissue was homogenized in a mortar with 80 % ethanol.…”

“…Absorbance was measured at 450, 532 and 600 nm. Level of lipid peroxides was calculated following Hodges et al (1999) and expressed as nmol MDA g −1 fresh weight.…”

Effects of Aluminum on Light Energy Utilization and Photoprotective Systems in Citrus Leaves

“…Lipid peroxidation was determined by measuring the malondialdehyde (MDA) concentration (Hodges et al, 1999). There were five replicates per treatment (one leaf per replicate, one leaf per plant).…”