Use of laser-excited fluorescence to measure mixed-function oxidase activity.

Abstract

The microsomal mixed-function oxidase (MFO) system is involved in the metabolism of various chemical compounds. Polycyclic aromatic hydrocarbons are metabolized by the cytochrome P-448 enzyme system, which contains MFOs. Induction of this MFO activity may be useful as an indicator of the toxicity of the inducer material. We have successfully used a portable centrifugal analyzer equipped with an argonion laser light source to quantify cytochrome P-448 activity induced in mouse-liver microsomes by exposing the animal's skin to different doses of liquids derived from a coal-liquefaction process. The MFO activity was determined kinetically by measuring the rate at which the highly fluorescent compound, resorufin, produced by the oxidation of 7-ethoxyresorufin substrate, was formed. The 514.5-nm laser excitation beam was directed with a fiber-optic bundle from the laser to the cuvets of a specially designed rotor; emitted fluorescence was monitored at 90 degrees to the incident beam through a 560-nm cut-on secondary filter. Use of the laser excitation source allowed very low MFO activities to be measured: picomole quantities of resorufin could be determined. We anticipate that the increased sensitivity of the method described here will allow MFO activities to be determined in body fluids and skin.