Successful Spin Trapping of the Hydroxyl Radical by Direct
In Vivo EPR
1Ke Jian Liu, 2Graham S. Timmins, 1Harold M. Swartz
1EPR Center, Dartmouth Medical School, Hanover, NH 03755, and
2University of Wales College of Medicine, Cardiff CF14 4XN, Wales, UK
INTRODUCTION: The detection of spin trapped radical adducts by EPR is
a particularly powerful
technique for the sensitive and specific detection, identification, and
relative quantitation of
short-lived free radicals. While the technique has been used widely and
successfully in
biological studies in model systems, there are some potential limitations
with “standard“
techniques that use conventional high frequency (X-band) EPR spectroscopy.
The conventional
approach is for wet samples such as cells or tissues to sizes of much less
than one ml. Therefore
extensive preparation and alteration is required to study tissue samples
from animal models,
which could result in problems such as artifactual radical adduct formation
and decay of adducts
during sample preparation.
METHODS: The development of low-frequency EPR spectrometers of
sufficient sensitivity
provides an attractive alternative detection technique for spin trapping
studies: the direct
detection of radical adducts in intact animals. In addition to avoiding
potential artifacts from the
preparation of the samples, it provides advantages such as real time
detection and determination
of the location in an organism.
RESULTS: Although there is a great deal of interest in studying the
formation and reactions of
HO. in biological systems, HO. has proven especially difficult to detect in
cells or in vivo by spin
trapping. We now have studied the feasibility of in vivo EPR detection of
spin trapped HO.
radical adducts of two related nitrones, 5,5-dimethyl-1-pyrroline-N-oxide
(DMPO) and 5-
diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). DEPMPO has been
reported to
have some advantages for spin trapping in biological systems. The
anesthetized mice pretreated
with DMPO or DEPMPO were injected with Fe (III) and 5-aminolevulinic acid
(ALA) and
inserted in the whole-body resonator of a 1.2 GHz EPR spectrometer. An EPR
signal was
observed which was consistent with the assignment as the HO. radical adduct.
The half-life of
this radical adduct in vivo was approximately one to two minutes. Injection
of dimethylsulfoxide
(DMSO) directly after the spin trap (DEPMPO) in the above protocol resulted
in the spectrum
which is consistent with methyl adduct of DEPMPO. No adduct signal was
observable under
same conditions using DMPO.
CONCLUSIONS: It is clear that DEPMPO can successfully form HO.
radical adducts in vivo that
are sufficiently stable that they can be observed by In Vivo EPR. In this
respect DEPMPO offers
significant advantages compared to DMPO, probably as a result of differences
in the stability of
the HO. radical adducts in vivo, because the rate of trapping of HO. is
approximately equivalent
for DMPO and DEPMPO. In conclusion, the use of DEPMPO has allowed the first
direct in vivo
EPR detection of radical adducts of the hydroxyl radical, and it has also
demonstrated that
oxidation of ALA can produce HO.in vivo. It is anticipated that the EPR
spin-trapping
techniques described here will allow further study in vivo of reactions
thought to involve HO..
This study was presented at the Oxygen Society annual meeting in 1998;
Manuscript was published in Free Radical Biology Medicine:
G.S. Timmins, K.J. Liu, E. J.H. Bechara, Y. Kotake, and H.M. Swartz,
“Trapping of Free Radicals
with Direct In Vivo EPR Detection: A Comparison of
5,5-Dimethyl-1-Pyrroline-N-oxide
(DMPO) and 5-Diethoxyphosphoryl-5-Methyl-1-Pyrroline-N-oxide (DEPMPO) as
Spin Traps
for HO• and SO4•-, Free Rad. Biol. Med., 27:329-333(1999).