Evaluation of DEPMPO as a Spin Trapping Agent in Biological
Systems
Ke Jian Liu, Minoru Miyake, Tomasz Panz, Harold Swartz
EPR Center for the Study of Viable Biological Systems, Department of
Radiology, Dartmouth
Medical School, Hanover, NH 03755 - USA
Free radicals have been implicated to play an important role in the
development of many
diseases, as well as having a role in many normal physiological processes,
ranging from
intermediates in enzyme reactions to roles as effectors. While the existence
of free radicals in
these biological processes can be inferred from end product analysis and/or
from the effects of
antioxidants or enzymes, the technique of EPR spectroscopy allows for their
direct detection.
Free radicals as a rule are very short lived and/or exist in extremely low
concentrations.
For many such short lived radicals, the technique of spin trapping can often
be used. However,
the application of the spin trapping technique in biological systems,
especiallyin vivo , has been
difficult because of the lack of stability of the spin adducts in the viable
systems where an array
of reducing systems can convert the paramagnetic adducts into EPR silent
products. Recently
there has increased activity in the design and synthesis of spin traps which
could produce adducts
more resistant to bioreduction. One of the newly synthesized traps, DEPMPO,
a phosphorylated
derivative of the widely used DMPO, has been reported to produce spin
adducts with longer
lifetime, particularly for the adduct of superoxide. The spin adducts of
DEPMPO usually have
characteristic EPR spectra, making it possible to identify the trapped
radical unambiguously. The
present study was undertaken to evaluate several aspects of DEPMPO as a spin
trapping agent in
biology systems with particular emphasis on its application in vivo.
We investigated cellular toxicity of DEPMPO using an assay for colony
formation in
CHO cells. The results indicate that there was no significant cytotoxicity
of DEPMPO up to a
concentration of 25 mM. At higher concentrations, above 50 mM, plating
efficiency decreased
drastically. The mechanism of this phenomenon was not investigated in
detail, but the
experiments performed ruled out the changes in ionic strength or pH, due to
addition of the spin
trap, as the potential cause.
The spin trap DEPMPO itself was reasonably stable in vivo. After IP
injection into a
mouse, DEPMPO was taken up rapidly (< 15 min), and distributed evenly in the
liver, heart, and
blood of the mice. 60 min after injection, about 50% of the injected DEPMPO
was still available
in the tissue.
In contrast to spin traps, spin adducts usually usually short lived in vivo.
We used SO3
adducts as our model system to evaluate and compare the spin trap DEPMPO and
DMPO. Under
in vivo conditions, the spin adduct DEPMPO/SO3 was 2-4 times more stable
than
DMPO/SO3 depending on the route of administration of the adducts. Using a
low frequency
EPR spectrometer, we were able to observe the spin trapped SO3 radical both
with DMPO and
DEPMPO directly in the intact mouse. DEPMPO had a detectable spin adduct
signal at a
concentration as low as 1 mM, while 5 mM of DMPO was required for a similar
intensity.
We conclude that DEPMPO is a good candidate as an in vivo spin trapping
agent. It
represents an improvement over the widely used DMPO in terms of the
stability of the spin
adducts. The observed lack of toxicity at concentrations of at least 25mm
should allow sufficient
concentrations to be employed in vivo.The results also suggest that with the
continuous
development of new spin traps, in addition to the improvement of the low
frequency EPR
instrumentation, more and more radicals will potentially become detectable
directly in intact
animals, which will greatly contribute to the study of the role of free
radicals in biology and
medicine.
This study was presented at the Rocky Mountain Conference on Analytical
Chemistry,
Denver, CO in July, 1998. A manuscript was submitted to Free Radical Biology
Medicine
and published in 1999:
K.J. Liu, M. Miyake, T. Panz, and H.M. Swartz, “Evaluation of DEPMPO as a
Spin Trapping Agent in
Biological Systems,” Free Rad. Biol Med. 26:714-721 (1999)