Black Magic and EPR Oximetry: From Lab to Initial Clinical Trials
Nadeem
Khan 1,
Huagang Hou1, Patrick Hein2, Richard J. Comi2,
Jay C. Buckey2,
Oleg Grinberg1, Ildar Salikhov1,
Shi Y. Lu1, Hermine Wallach2, and Harold M. Swartz1
1EPR Center for the Study
of Viable Systems, Dartmouth Medical School, Hanover, NH 03755, USA;
2Department
of Medicine, Dartmouth-Hitchcock Medical Center, Lebanon, NH 03756, USA
INTRODUCTION
It would be very useful
to have a method to directly and repetitively measure the partial pressure of
oxygen (pO2) in tissues.
This would be especially desirable for
planning and evaluating therapy for tumors and vascular insufficiencies. A
variety of techniques
are available for measuring pO2 in tissues;
however, none of these techniques has been shown to have the properties needed
for
optimal experimental and clinical use (i.e. sensitivity, accuracy, ease,
and ability to make measurements repeatedly). EPR oximetry is
one of the most
promising techniques for accomplishing this goal. It is based on the fact that
molecular oxygen can interact with
paramagnetic materials such as nitroxides,
lithium phthalocyanine, coals such as fusinite, chars, and India ink, affecting
their EPR
spectra in a reproducible manner that is proportionate to the amount
of oxygen.
In order to extend the
application of EPR oximetry to humans, India ink is the probe of choice because
appropriate India inks
have EPR signals whose line widths are sensitive to
changes in oxygen concentrations; most importantly, India ink already has been
used extensively in humans (as a marker for the skin, lymphatics, mucosa, and
tumors, and for decoration as tattoos).To facilitate
the application of EPR
oximetry in human subjects, we have developed an India ink that has good
sensitivity to oxygen, high stability in tissues,
good signal intensity, and
minimal toxicity. Here, we report the various properties of this India ink,
results obtained from our animal
experiments, and our first preliminary
clinical results, which are part of the first systematic clinical use of EPR
oximetry.
MATERIALS AND METHODS
India ink
We identified an
effective India ink, Higgins black magic waterproof ink (No. 4465), through a
search of local commercial sources of inks.
This ink has an excellent signal to noise ratio. To increase the signal
intensity, the ink was concentrated to 20% of its original volume
by heating at 90°C for six to seven hours.
Prior to injection, the ink was autoclaved for one hour at 121°C.
EPR Measurements
In vivo EPR measurements were
carried out on 1.2 GHz (L - band) EPR spectrometers. Typical
settings for the spectrometer were:
incident microwave power, 80 mW; magnetic
field center, 400 gauss; scan range, 120 gauss; and scan time 12-15 seconds.
Modulation amplitude was set at less than one-third of the EPR line width.
RESULTS
The non-dialyzed
concentrated Higgins black magic ink was used in our first clinical
experiments. The measurements in volunteers
were carried out in the first
specifically developed clinical L-band EPR spectrometer at the Dartmouth-Hitchcock Medical Center.
We injected the ink in
the feet of healthy volunteers at the sites of greatest risk for patients with
diabetic peripheral vascular diseases
(under the first metatarsal head) and
also at the site where transcutaneous oxygen measurements often are made (first
interosseous dorsal space)
(Figure 1a). The typical EPR spectrum obtained from
these injections (7-10µl of ink) is shown in Figure 1b.
The tissue pO2
has been measured over several weeks (Figure 2).
1a 1b
Figure 1:
a: The cosmetic result of non-dialyzed concentrated Higgins black magic ink
injection in
the first interosseous dorsal space (between the first and second toes).
b: EPR signals obtained from only 7-10µl of concentrated ink before (upper
tracing) and
after (lower tracing) temporary muscle compression.
Figure 2:
pO2 measured by EPR using concentrated Higgins black ink (~ 5 µl) under the
plantar first
metatarsal head of a healthy volunteer. Measurements of baseline tissue pO2, pO2
after
muscle compression, and pO2 after compression was released, i.e. recovery, were
done on
Dec. 11, 2002. Tissue pO2 was measured again on April 30, 2003. The EPR
measurements for
baseline and recovery were done for a period of ~15 minutes and for ~ 5 minutes
during
muscle compression (Mean ± SD).
DISCUSSION
The Black Magic India
ink was reduced to 20% of its original volume to achieve better signal
intensity with minimal volume.
This ink has an approximately three-to-four
times better signal to noise ratio as compared with the earlier India ink.
The
line width is sensitive to changes in oxygen concentrations, and no power
saturation was observed at 1.2 GHz.
The line width of the EPR signals was not
significantly affected by changes in pH, mild broadening, mild oxidizing, or
reducing agents;
however, significant changes were observed with change in
temperature. The in vivo results in animals indicate that a stable pO2
was observed by day 14. At all time points, the line width of the EPR signal
observed after muscle compression was similar to that
obtained with 0% perfused
oxygen in the calibration. These results indicate that there is no change in
the calibration of this
ink and the suitable time for initiating EPR
measurements is 7-14 days after injection of the ink.
We carried out our first
preliminary clinical experiments using EPR oximetry. Only 7-10 µl of the
concentrated ink was enough
to obtain an EPR signal, which is shown in Figure
1B. The signal to noise ratio observed was nearly 18-20 in single EPR scans
with
a scan time of 15 seconds. The results shown in Figure 2 indicate that it
is possible to do repeated measurements over at least several
months after the
ink injection, indicating that long-term follow-up studies are feasible. We are
very encouraged with these results
and are confident that the EPR technique
using India ink will be a non-invasive, fast, and reliable technique for pO2
measurements
in clinical studies. At present, we plan to carry out a systematic
study of these injected sites over longer periods and also to extend
the study
to more volunteers to obtain more data with the aim of developing EPR oximetry
as a valuable tool for clinical use.
ACKNOWLEDGMENTS
This work was supported by a NIH
(NIBIB) grant PO1 EB002180, “Measurement of pO2 in Tissues In
Vivo and In Vitro,”
and used the facilities of the
EPR Center for the Study of Viable
Systems supported by NIH (NIBIB) grant P41 EB002032.