A method to reduce interference by sucrose in the detection of thiobarbituric acid-reactive substances

A thiobarbituric acid (TBA) reaction for measuring lipid peroxidation products was evaluated for interference by several ingredients commonly used in solutions to prepare or analyze tissue homogenates or subcellular organelles. These included sucrose (up to 100 mM final concentration in the assay medium), Tris-maleate (up to 40 mM), imidazole (up to 20 mM), inorganic phosphate (up to 10 mM), and 4- morpholinepropanesulfonic acid (up to 20 mM). When the samples were heated at 95 degrees C as recommended in some procedures, only sucrose significantly affected color development. Sucrose concentrations as low as 10 mM significantly increased absorbance at 532 nm of aqueous tetramethoxypropane (TMP) standards, and so the assay could not be applied reliably to tissue samples prepared in sucrose. Sucrose interference was only partially reduced by subsequent organic extraction (n-butanol plus pyridine), with measured absorbances remaining significantly greater (50-100%) than sucrose-free controls at sucrose concentrations of 20 mM or more. Modifying the assay to include sucrose in blanks and TMP standards failed to adequately correct for interference when the absorbance of unextracted (aqueous) solutions was measured. Further modification by adding sucrose to blanks and TMP standards, followed by butanol-pyridine extraction, gave standard curves that were linear, through the origin, and had slopes equivalent to those of sucrose-free standards. This modification enabled almost complete recovery (average 2% error) of known amounts of TMP added to aliquots of tissue homogenates containing amounts of sucrose that otherwise significantly interfered. Also, with the modified method the content of TBA-reactive substances in tissues homogenized in sucrose was found to be not significantly different from that measured in tissues homogenized in a noninterfering substance, KCl.