India ink: A potential clinically applicable EPR oximetry probe

India Ink: A Potential Clinically Applicable EPR Oximetry Probe Harold M. Swartz, Ke Jian Liu, Fuminori Goda, Tadeusz Walczak Using a material that already is in widespread use in humans, India ink, the first EPR measurements in a human have been made, using the India ink in a pre-existing tattoo. The EPR spectra of India ink are very sensitive to the partial pressure of oxygen (PO,), thereby making it feasible to use this approach to measure pOz in tissues in patients. This potentially provides a means to measure this parameter directly with a sensitivity, accuracy, and repeatability that have not been available previously, and thereby to be able to individualize and guide treatment of diseases such as cancer and peripheral vascular insufficiency. Key words: oxygen, EPR, in vivo, clinical.

future, using EPR oximetry. EPR oximetry is based on the fact that molecular oxygen can interact with paramagnetic materials, changing their EPR spectra. The changes in the EPR spectrum can be calibrated with PO, and then used to measure PO, in various biological systems (6). Most EPR oximetry has been conducted using nitroxide-free radicals as the paramagnetic materials but recently a carbon-based material called fusinite, a derivative of coal, has been shown to have an EPR signal, which is much more sensitive to oxygen, with a sensitivity better than 1mmHg at low PO, (7,8). It is also very stable physicochemically resulting in little or no toxicity in vifro and in vivo. These properties, together with recent advances in low frequency EPR instrumentation, which have made in vivo EPR feasible, have made EPR oximetry capable of measuring PO, in tissues of living animals with the sensitivity, accuracy, and repeatability that is required for most purposes, but, as noted above, the persistence of these particles makes it problematical to adapt this to clinical use. India ink is made from carbon black of oil, coated with gelatin to obtain an aqueous suspension (9). It has been used in oriental countries for writing and painting for hundreds of years. In many parts of the world, it is also used for decoration as tattoos. In clinical medical practice India ink has already been used extensively in patients as a short-term and permanent marker in the skin, mucosal tissue, and tumors. Tattooing with India ink is a standard procedure for permanent marking for radiation therapy and endoscopic treatments (10-14). In an analysis of over 3500 patients who were administered India ink for medical purposes in published studies, no serious problems of toxicity from tattoos with India ink were reported (15).

INTRODUCTION

There is an increasing recognition that it would be very useful to have a means to be able to directly measure the partial pressure of oxygen (PO,) in tissues with sufficient sensitivity, accuracy, and ease (1).This would enable clinicians to relate many diseases (such as cancer and peripheral vascular insufficiency) and their treatment to perhaps the most pertinent variable affecting the status of the disease and/or its therapy: the actual pOz of the tissue. The existing methods for making these measurements in vivo, however, have very significant limitations (2, 3). Considerable effort is being devoted to solve this problem and some progress has been made. Electron paramagnetic resonance (EPR, or ESR) is one of the most promising techniques for accomplishing this goal but the most useful paramagnetic materials used with this technique (lithium phthalocyanine and fusinite coal) (4, 5) are particles that persist in tissues indefinitely. While the persistence of these particles offers a uniquely useful and sensitive means to make repeated noninvasive measurements of PO, over periods of months or years from the same site, this persistence makes the demonstration of their safety for human use a formidable task. We report here the discovery of the usefulness of another particulate substance, India ink, which shares many of the favorable properties of lithium phthalocyanine and fusinite coal, but which already is in widespread use in humans. This appears to make it feasible routinely to measure PO, in tissues in humans in the near

EXPERIMENTAL Animal Preparation

BALB/c mice weighing 20 to 25 g were obtained from Charles River Laboratories (Wilmington, MA). India ink was injected into the animal through a 26-gauge needle. Since anesthesia significantly affects PO,, the animal, which was restrained in a plastic holder, was conscious throughout of the experiment.

MRM 31:229-232 (1994) From the Department of Radiology, Dartmouth Medical School, Hanover, New Hamsphire. Address correspondence to: Dr. H. M. Swartz, Dartmouth Medical School, HB 7252, Hanover, NH 03755. Received September 3, 1993; revised November 10, 1993; accepted November 10, 1993. This research was supported by NIH grant GM 34250 and used the facilities of the IERC at Dartmouth supported by NIH grant RR-01811. 0740-3194/94 $3.00 Copyright 0 1994 by Williams & Wilkins All rights of reproduction in any form resewed.

EPR Measurements

The spectra in the human subject and in animals were obtained using an EPR spectrometer constructed in our laboratory with a low frequency (1.2 GHz, L-band) microwave bridge (16). Some calibrations and studies of physicochemical properties of India ink were performed 229

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using a Varian E-109E EPR spectrometer (9.5 GHz, Xband). Typical settings for the spectrometers were: magnetic field, 3210 gauss (X-band)/392 gauss (L-band);microwave power, 5 mW modulation frequency, 100 1612. Modulation amplitude was set at one third of the EPR line width. The EPR spectra on both spectrometers were collected, stored, and manipulated using the software EW (Scientific Software Inc., Normal, IL) installed on IBM compatible computers. Calibration of EPR Line Width with p 0 2

A suspension of India ink in phosphate buffered solution (80 mg/ml) was drawn into a gas permeable Teflon tube (Zeus Industrial Products, Raritan, NJ; 0.623 mm inner diameter; wall thickness, 0.038 mm). This Teflon tube was folded twice and inserted into a quartz EPR tube open at both ends. PO, in the perfusing gas was monitored and measured by an oxygen analyzer (Sensor Medics, Model OM-11, Anaheim, CA) calibrated with air and nitrogen. The quantitative dependence on PO, of the EPR spectrum was obtained by measuring line width (LW) as a function of PO, in the perfusing gas, with LW defined as the difference in magnetic field between the maximum and minimum of the first derivative recording of the signal. Measurement of p02 in Human and Mice in Vivo

Adult mice were injected in the gastrocnemius muscle with 10 pl of a suspension of India ink and then placed in the gap of the magnet with the L-band loop gap detector positioned over the injection area. When required, blood flow was restricted by a ligature around the upper leg. For the EPR measurements in a human subject, a volunteer with an extensive tattoo on his lower arm positioned the black area of the tattoo under the loop gap detector. Results and Discussion

India ink has an EPR spectrum that is very sensitive to PO,. The LW of the EPR spectrum was reversibly broadened by oxygen (Fig. 1).The effect of oxygen on the line

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width is particularly sensitive in the region of biological interest (1-40 mmHg), with a capability of determining differences of less than 1.0 mmHg. The stability and sensitivity of the response of India ink to PO, was studied in the gastrocnemius muscles of mice in vivo by making repeated measurements of the EPR spectra over an extended period of time. The response to decreases of pOz was indicated by a narrowed LW and increased line height when the blood flow to the leg was restricted by the tightening of a tourniquet. The corresponding values of PO, before and after the constriction of the blood flow were 14.2 ? 0.5 and 1.2 ? 0.2 mmHg, respectively (Fig. 2). The response of India ink was sufficiently rapid to follow the process of deoxygenation, which was completed in less than 15 s. The response of India ink in muscle to oxygen was maintained during the 39 days of our observations. We obtained EPR spectra from a human volunteer with an extensive tattoo on his arm when the blood flow to the skin was constricted with a tourniquet (Fig. 3). The EPR spectrum changed accordingly, with the LW decreasing from 4050 to 3400 mgauss, reflecting the decrease of oxygenation of the skin. This confirms that even under these very suboptimal conditions, in which the paramagnetic substance was unselected and in which India ink had been in tissue for a prolonged period of time, this approach can detect changes in pOz. Our observation, we believe, is the first in vivo EPR measurement in a human subject. This approach appears to have potential for immediate useful clinical applications. With appropriate calibration it could be used repeatedly to monitor PO, in tumors for individualizing therapy (particularly, radiation therapy). It also could be used for the characterization of the status of oxygenation in tissues in patients with peripheral vascular disease and then used to evaluate the effectiveness of therapy. The guidance of therapy would be done by providing, for the first time, a means to measure directly the impact of therapeutic drugs and surgical procedures on the actual PO, of the tissues which are at risk from impaired blood supplies.

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FIG. 1. Calibration of the EPR LW of India ink with pop in various media. The calibration curve with pop was independent of the media which were: oleic acid (A), serum (O),and phosphate buffered solution (0).

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FIG. 2. Measurement of pop

in vivo in the gastrocnemius muscles of unanesthetized mice. The corresponding PO, before (open symbols) and after (solid symbols) the constriction of the blood flow were 14.2 2 0.5 and 1.2 ? 0.2 mmHg, respectively. Different symbols represent individual animals.

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Time (day) The detailed mechanism for the interaction of India ink with oxygen is currently under investigation, but even in the absence of such knowledge, with proper calibration, it should report PO, accurately and reliably in clinical use, as well as in experimental animals. The calibrations can be made readily because the response to p 0 2 is independent of the medium (Fig. 1).India ink is widely available as a commercial material; although, presumably because of variations in the materials that are used andlor in the process of making the product, we have

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FIG. 3. In vivo EPR measurements of

found that India ink from different manufacturers has different oxygen dependent changes in the line width and intensity of the EPR signal. Therefore, each batch needs to be calibrated. The EPR signal appears to consist of more than one type of paramagnetic center, which sometimes complicates the analysis of the oxygen-dependent changes in the EPR spectra. Our preliminary studies suggest that this is due to differences in some minor components. The understanding of these variations would not only solve the immediate problem, but also could serve as a clue for finding andlor making formulations of India ink which are optimized for in vivo EPR oximetry. The ability of existing low frequency EPR instrumentation to make measurements of pOz in vivo is clearly illustrated in this report, although it would be useful (and it is quite feasible) to improve the instrumentation further for clinical use. With our current setup of the instrument, i.e., working at a microwave frequency of 1.2 GHz with an extended loop detector, we are able to obtain reasonable signal-to-noise ratio with a maximum penetration depth of 10 mm. While there are many useful applications within that depth, it may be desirable to go to lower frequency EPR spectrometers to expand the depths which can be studied (17), although the use of lower frequencies does lead to increased difficulties with sensitivity.

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popfrom a human tattoo.

Spectra were obtained before and after constriction of the blood flow by means of a rubber tourniquet around the arm. The LW of the EPR spectra changed from 4050 mgauss to 3400 mgauss. The correspondingvalues of pop were unknown because a calibration of the response to pop for the material used for the tattoo was not available.

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