Oxidation of Glycerol to Formaldehyde by Microsomes: Are Glycerol Radicals Produced in the Reaction Pathway?

Microsomes and reconstituted systems containing cytochrome P450 can oxidize glycerol to formaldehyde in a reaction catalyzed by an oxidant produced from the interaction of nonheme iron with H₂O₂. To evaluate the mechanism for this oxidation, the generation of glycerol radicals by various systems was compared to rates of formaldehyde production from glycerol. Photolysis of H₂O₂, oxidation of xanthine by xanthine oxidase in the presence of iron catalysts, or NADPH-dependent microsomal electron transfer in the presence of ferric-EDTA produced hydroxyl radicals. In the presence of glycerol these reaction systems produced DMPO-glycerol radical adducts which were detected by ESR spectroscopy. Despite the production of ·OH and glycerol spin-trapped adducts by these reaction systems, very low amounts or nondetectable amounts of formaldehyde were produced from the glycerol. However, significant amounts of formaldehyde were observed when microsomes were incubated in the presence of ferric ammonium sulfate or ferric-ATP, although ·OH production was lower with these iron catalysts than with ferric-EDTA. These results fail to support correlation between ·OH production and oxidation of glycerol to formaldehyde. Under conditions in which glycerol was oxidized to formaldehyde, no glycerol radical species could be observed with DMPO as the spin-trapping agent. These results suggest the oxidant (not ·OH) derived from the interaction of H₂O₂ with iron apparently cleaves glycerol to formaldehyde without the formation of a radical intermediate. Alternatively, the radical intermediate may be produced at a too low concentration to be detected or the radical intermediate may not be formed as a free species and therefore cannot be spin-trapped. By comparing microsomal rates of formaldehyde production from glycerol and glycerol-d₅, a kinetic isotope effect of about three was observed. The kinetic isotope effect was due to changes in V_max for formaldehyde production and not K_m for glycerol, suggesting that breakage of a carbon-hydrogen bond was a rate-determining step in the overall pathway of glycerol oxidation to formaldehyde.