4-hydroxy-2-nonenal and 1-methyl-2-phenylindole

The 1-methyl-2-phenylindole colorimetric assay is considered specific for malondialdehyde (MDA) and 4-hydroxynonenal (HNE) in mammalian systems, but its specificity in plant tissues is unknown. This study demonstrates that the assay produces a purple/blue chromophore with an absorbance peak at 586 nm for a malondialdehyde standard, while aqueous extractions from Ribes spp. Beta vulgaris, and Lycopersicon esculentum tissues produce an orange chromophore with an absorbance maximum at 450 nm and a large shoulder that extends to 700 nm. No distinctive MDA peak was discernable in plant samples at lambda=586 nm and absorbance was attributed to background interference. The reaction between sucrose and 1-methyl-2-phenylindole produced an orange chromophore with a spectrum similar to those obtained from plant extractions, suggesting that simple sugars are the likely source of background interference. This study demonstrates that the 1-methyl-2-phenylindole colorimetric assay is non-specific for detecting MDA and HNE in plants and its use is cautioned due to interference, particularly from sugars.

Topics: Aldehydes; Beta vulgaris; Biochemistry; Colorimetry; Evaluation Studies as Topic; Hydrochloric Acid; Indoles; Lipid Peroxidation; Malondialdehyde; Mesylates; Oxidative Stress; Plant Proteins; Ribes; Spectrophotometry

Under acidic and mild-temperature conditions, 1-methyl-2-phenylindole was found to react with malondialdehyde (MDA) and 4-hydroxyalkenals to yield a stable chromophore with intense maximal absorbance at 586 nm. The use of methanesulfonic acid results in optimal yields of chromophore produced from MDA as well as from 4-hydroxynonenal. By contrast, the use of hydrochloric acid results in an optimal yield of chromophore produced from MDA and a negligible reaction of 4-hydroxynonenal. Taking advantage of such chromogenic reactions, we developed a new colorimetric assay of lipid peroxidation. Using a methanesulfonic acid-based medium, MDA and 4-hydroxyalkenals can be measured at the 586 nm wavelength. However, the presence of endogenous inhibitors of the reaction with 4-hydroxyalkenals is common, and this means that the latter may be underestimated in some biological samples. The assay performed in a hydrochloric acid-based medium enables the specific measurement of MDA in the presence of 4-hydroxyalkenals. Upon hydrolysis of Schiff bases in hydrochloric acid (pH 1.5), either assay can be used to specifically measure the amount of total MDA in biological samples because 4-hydroxyalkenals undergo an irreversible cyclization reaction under the hydrochloric acid-based conditions of hydrolysis. The two assays were applied to the determination of the amount of MDA alone and of MDA and 4-hydroxyalkenals in an in vitro model of lipid peroxidation. This methodology was also used to clarify complex patterns of tissue-specific MDA production in vivo, following hydrolysis of Schiff bases, in rodents treated with doxorubicin.

Topics: Aldehydes; Animals; Chromogenic Compounds; Colorimetry; Female; Humans; Indoles; Lipid Peroxidation; Male; Malondialdehyde; Mice; Mice, Inbred BALB C; Rats; Rats, Sprague-Dawley; Sensitivity and Specificity