Determination of phthalate-induced rat liver cytochrome P-450 IVA1 by a fluorimetric enzymatic assay and by chemiluminescence detection on Western blots

An enzymatic assay for cytochrome P-450 IVA1 as determined by the formation of 12-hydroxylauric acid from lauric acid, using liquid chromatography with fluorescence detection has been compared with a commercial Western blot kit for this protein using enzyme enhanced chemiluminescence detection. The comparison was performed with samples of liver homogenates of rats exposed to the plasticizer di(ethylhexyl)phthalate (DEHP). Both methods showed sufficient sensitivity to detect the protein in control animals. The comparison resulted in a good correlation between both methods with a correlation coefficient of 0.96. The resulting no-effect levels for DEHP were also found to be the same. It is concluded that although the enzymatic method has a better reproducibility and dynamic range, both methods can be used for the determination of the cytochrome P-450 IVA1 in rat liver homogenates as an indicator of peroxisome proliferation.