M. Hermeslima et al., QUANTIFICATION OF LIPID-PEROXIDATION IN TISSUE-EXTRACTS BASED ON FE(III)XYLENOL ORANGE COMPLEX-FORMATION, Free radical biology & medicine, 19(3), 1995, pp. 271-280
Commonly used spectrophotometric methods for determining the extent of
lipid peroxidation in animal tissue extracts, such as measurements of
diene conjugation and thiobarbituric acid reactive substances (TBARS)
, have been criticized for their lack of specificity. This study shows
that lipid hydroperoxides can be effectively quantified in animal tis
sue extracts using an assay based on the formation of a Fe(III)xylenol
orange complex. Addition of H2O2, cumene hydroperoxides, or methanoli
c tissue extracts to an acidic reaction mixture containing 0.25 mM Fe(
II) and 0.1 mM xylenol orange caused the formation of a broad Fe(III)x
ylenol orange complex absorbance peak at 560-580 nm with a correspondi
ng decrease in the xylenol orange peak at 440 nm. Complex formation me
asured at 580 nm was saturable with both xylenol orange and Fe (II) co
ncentration. Addition of ascorbic acid, GSH, and cysteine (0.3-5 mM) c
aused a saturable reduction of the Fe(III)xylenol orange complex. Form
ation of the Fe(III)xylenol orange complex was linear with the amount
of tissue extract added. A significant correlation (r = 0.88, p < 0.00
5) existed between the xylenol orange method of estimating lipid perox
idation and the conventional TBARS assay in a series of animal tissues
tested. The time course of increase in A580nm in tests using tissue e
xtracts was typical of a free radical reaction; a lag phase was follow
ed by a log phase. No increase in A580nm was observed up to 24 h when
highly peroxidizable arachidonic acid was assayed. These results indic
ate that the formation of the Fe(III)xylenol orange complex reflects a
chemical amplification of the original level of lipid hydroperoxides
present in tissue extracts and that peroxidizable lipids do not influe
nce the assay. The potential usefulness of the xylenol orange assay fo
r comparative biochemical and toxicological studies of oxidative stres
s is discussed.