24. Measurement of Membrane Permeability of a Spin Trap, 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) by ESR Spectrometry

Kazunori Anzai, Yoshiko Furukawa^*, and Toshihiko Ozawa
(^*Kyoritsu College of Pharmacy)

Keywords: liposome, DMPO, ESR, spin-trapping, membrane permeability

Oxygen-derived radicals have been detected by using a spin-trapping technique with ESR spectrometry, mainly in solutions containing subcellular components. With this technique, very short-lived oxygen-derived radicals react with spin traps to yield long-lived radicals. On the other hand, few successful results have been reported using this technique with cellular systems. One reason fot the poor success with cellular systems is that a membrane-permeable spin trap must be used to detect oxygen-derived radicals generated inside the cells. No measurements have been reported on membrane permeability of various spin traps. In the present study, we tried to measure the membrane permeability of the most useful spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) with a liposomal system.

Liposomes were made with egg phosphatidylcholine by a reverse-phase evaporation method. Large unilamellar vesicles corresponding to the size of living cells can be prepared by this method. Hydroxyl radicals (*OH) were generated by the reaction of H₂O₂ with Cu complex (Cu(II)en₂) or homolysys of H₂O₂ upon UV-irradiation. The radical adduct between DMPO and *OH (DMPO-OH) was detected with an ESR spectrophotometer (FR-30, JEOL, Tokyo, Japan).

DMPO-OH was prepared and this was mixed with liposomal suspension. The ESR signal derived from outside the liposomes became invisible on using a membrane-impermeable spin-broadening reagent, potassium tris(oxalato) chromate(III). The time course of the ESR signal of DMPO-OH derived from inside the liposomes gave us information on the membrane permeability of DMPO-OH. Even at the first observed point in the time course (at 43 s), we observed a large signal intensity and this intensity slowly decreased. This meant that the permeation of DMPO-OH through the liposomal membrane was rapid and was completed within 43 s.

Next, we tried to measure the membrane permeability of intact DMPO. Liposomes containing Cu(II)en₂ inside were mixed with DMPO and an aliquot of the mixture was mixed with membrane-permeable H₂O₂. The time course of the intensity of the ESR signal derived from DMPO-OH formed inside the liposomes should give us information about the membrane-permeation rate of DMPO. We detected a significant signal intensity at 30 s after the mixing and the intensity remained almost constant during 26 min. We tried a different procedure to measure the DMPO-permeation through the liposomal membrane: *OH was produced by UV irradiation of H₂O₂ in the presence of the liposomes and *OH outside the liposomes was quenched with a high concentration of polyethylene glycol 4000. This experiment also gave the same result that the membrane-permeation rate of DMPO was fast. These results suggested the possibility of detecting radicals generated inside cells by using DMPO.