'Oxygen Level in a Tissue' - What Do Available Measurements Really Report?

The aim of the paper is to discuss what currently is feasible clinically to measure the level of oxygen and how that measurement can be clinically useful. Because oxygen in tissues is quite heterogeneous and all methods of measurement can only provide an average across heterogeneities at some spatial and temporal resolution, the values that are obtained may have limitations on their clinical utility. However, even if such limitations are significant, if one utilizes repeated measurements and focuses on changes in the measured levels, rather than 'absolute levels', it may be possible to obtain very useful clinical information. While these considerations are especially pertinent in cancer, they also pertain to most other types of pathology.

Clinical and Statistical Considerations when Assessing Oxygen Levels in Tumors: Illustrative Results from Clinical EPR Oximetry Studies.

The success of treatment for malignancies, especially those undergoing radiation therapy or chemotherapy, has long been recognized to depend on the degree of hypoxia in the tumor. In addition to the prognostic value of knowing the tumor's initial level of hypoxia, assessing the tumor oxygenation during standard therapy or oxygen-related treatments (such as breathing oxygen-enriched gas mixtures or taking drugs that can increase oxygen supply to tissues) can provide valuable data to improve the efficacy of treatments. A series of early clinical studies of tumors in humans are ongoing at Dartmouth and Emory using electron paramagnetic resonance (EPR) oximetry to assess tumor oxygenation, initially and over time during either natural disease progression or treatment. This approach has the potential for reaching the long-sought goal of enhancing the effectiveness of cancer therapy. In order to effectively reach this goal, we consider the validity of the practical and statistical assumptions when interpreting the measurements made in vivo for patients undergoing treatment for cancer.

Skeletal Muscle Oxygenation Measured by EPR Oximetry Using a Highly Sensitive Polymer-Encapsulated Paramagnetic Sensor.

We have incorporated LiNc-BuO, an oxygen-sensing paramagnetic material, in polydimethylsiloxane (PDMS), which is an oxygen-permeable, biocompatible, and stable polymer. We fabricated implantable and retrievable oxygen-sensing chips (40 % LiNc-BuO in PDMS) using a 20-G Teflon tubing to mold the chips into variable shapes and sizes for in vivo studies in rats. In vitro EPR measurements were used to test the chip's oxygen response. Oxygen induced linear and reproducible line broadening with increasing partial pressure (pO2). The oxygen response was similar to that of bare (unencapsulated) crystals and did not change significantly on sterilization by autoclaving. The chips were implanted in rat femoris muscle and EPR oximetry was performed repeatedly (weekly) for 12 weeks post-implantation. The measurements showed good reliability and reproducibility over the period of testing. These results demonstrated that the new formulation of OxyChip with 40 % LiNc-BuO will enable the applicability of EPR oximetry for long-term measurement of oxygen concentration in tissues and has the potential for clinical applications.

Calculation of dose conversion factors for doses in the fingernails to organ doses at external gamma irradiation in air.

Absorbed doses to fingernails and organs were calculated for a set of homogenous external gamma-ray irradiation geometries in air. The doses were obtained by stochastic modeling of the ionizing particle transport (Monte Carlo method) for a mathematical human phantom with arms and hands placed loosely along the sides of the body. The resulting dose conversion factors for absorbed doses in fingernails can be used to assess the dose distribution and magnitude in practical dose reconstruction problems. For purposes of estimating dose in a large population exposed to radiation in order to triage people for treatment of acute radiation syndrome, the calculated data for a range of energies having a width of from 0.05 to 3.5 MeV were used to convert absorbed doses in fingernails to corresponding doses in organs and the whole body as well as the effective dose. Doses were assessed based on assumed rates of radioactive fallout at different time periods following a nuclear explosion.

Ion-exchange in melanin: an electron spin resonance study with lanthanide probes.

Changes are induced in the electron spin resonance signal amplitude and microwave power saturation of the naturally occurring free radical in melanin by bound paramagnetic ions. The changes serve as experimental observables in competition experiments between diamagnetic and paramagnetic metal ions for melanin binding sites and between melanin and ethylenediaminetetraacetic acid for paramagnetic metal ions. Evidence is presented for the existence of several specific types of metal binding sites. The interaction of copper with free radicals leading to loss of electron spin resonance signal amplitude is magnetic in nature and not, as has been supposed, chemical.

Free radical increases in cancer: evidence that there is not a real increase.

The controversial finding of an increase in free radicals with the development of cancer was restudied with Walker 256 carcinosarcoma cells. It was confirmed that such an increase appears to occur, but it was also demonstrated that it is not a real increase. With growth of the tumor, the electron spin resonance lines for lyophilized samples became narrower, resulting in an increase in peak-to-peak height measurements, but there was no change in the total number of spins. The signals for lyophilized tumor samples were different from those for the same samples before lyophilization. Changes in line shape indicated that lyophilization. Changes in line shape indicated that lyophilized tumor samples contain a different mixture of radicals than lyophilized samples of normal tissue.

Quinoid radio-toxin (QRT) induced metabolic changes in mice: an ex vivo and in vivo EPR investigation.

Radio-toxins are toxic metabolites produced by ionizing irradiation and have toxic effects similar to those caused by direct irradiation. We have investigated the effect of a quinoid radio-toxin (QRT) obtained from gamma-irradiated potato tuber on various organs in mice using ex vivo and in vivo EPR spectroscopy. Results indicate a decrease in the activity of ribonucleotide reductase enzyme in spleen of mice treated with 0.2mg QRT. A dose of 2mg QRT was fatal to mice within 45-60 min of treatment. Nitrosyl hemoglobin complexes alpha-(Fe(2+)-NO)alpha-(Fe(2+))beta-(Fe(2+))(2) were detected from spleen, blood, liver, kidney, heart, and lung tissue samples of mice treated with lethal doses of QRT. A significant decrease of pO(2) in liver and brain was observed after administration of QRT at the lethal dose. The time of the appearance of the nitrosyl hemoglobin complex and its intensity varied with the dose of QRT and the type of tissue. These results indicate that the effect of the QRT is more prominent in spleen and to a lesser extent in liver and blood. The QRT action at the lethal doses resulted in an increased hypoxia over time with disruption of compensatory adaptive response. The results indicate similar outcome of QRT as observed with gamma-irradiation.

Radiation dose reconstruction from L-band in vivo EPR spectroscopy of intact teeth: Comparison of methods.

In vivo EPR tooth dosimetry is a more challenging problem than in vitro EPR dosimetry because of several potential additional sources of variation associated with measurements that are made in the mouth of a living subject. For in vivo measurement a lower RF frequency is used and, unlike in the in vitro studies, the tooth cannot be processed to optimize the amount and configuration of the enamel that is measured. Additional factors involved with in vivo measurements include the reproducibility of positioning the resonator on the surface of the tooth in the mouth, irregular tooth geometry, and the possible influence of environmental noise. Consequently, in addition to using the theoretical and empirical models developed for analyzing data from measurements of teeth in vitro, other unconventional and more robust methods of dose reconstruction may be needed. The experimental parameter of interest is the peak-to-peak amplitude of the spectrum, which is correlated to the radiation dose through a calibration curve to derive the reconstructed dose. In this study we describe and compare the results from seven types of computations to measure the peak-to-peak amplitude for estimation of the radiation induced signal. The data utilized were from three sets of in vivo measurements of irradiated teeth. Six different teeth with different doses were placed in the mouth of a volunteer in situ and measurements of each tooth were carried out on three different days. The standard error of dose prediction (SEP) is used as a figure of merit for quantifying precision of the reconstruction. We found that many of the methods gave fairly similar results, with the best error of prediction resulting from a computation based on a Lorentzian line model whose center field corresponds to the known parameter of the radiation-induced EPR spectra of teeth, with corrections from a standard sample that was measured as part of the data acquisition scheme. When the results from the three days of measurement were pooled, the SEP decreased dramatically, which suggests that one of the principal sources of variation in the data is the ability to precisely standardize the measurements conditions within the mouth. There are very plausible ways to accomplish improvements in the existing procedures.

Q-band EPR biodosimetry in tooth enamel microsamples: feasibility test and comparison with x-band.

A comparative study of electron paramagnetic resonance dosimetry in Q- and X-bands has shown that Q-band is able to provide accurate measurements of radiation doses even below 0.5 Gy with tooth enamel samples as small as 2 mg. The optimal amount of tooth enamel for dose measurements in Q-band was found to be 4 mg. This is less than 1% of the total amount of tooth enamel in one molar tooth. Such a small amount of tooth enamel can be harmlessly obtained in an emergency requiring after-the-fact radiation dose measurement. The other important advantage of Q-band is full resolution of the radiation-induced EPR signal from the native, background signal. This separation makes dose response measurements much easier in comparison to conventional X-band measurements in which these overlapping signals necessitate special methods for doses below 0.5 Gy. The main disadvantages of Q-band measurements are a higher level of noise and lower spectral reproducibility than in X-band. The effect of these negative factors on the precision of dose measurements in Q-band could probably be reduced by improvement of sample fixation in the resonance cavity and better optimization of signal filtration to reduce high-frequency noise.

Protocol for emergency EPR dosimetry in fingernails.

There is an increased need for after-the-fact dosimetry because of the high risk of radiation exposures due to terrorism or accidents. In case of such an event, a method is needed to make measurements of dose in a large number of individuals rapidly and with sufficient accuracy to facilitate effective medical triage. Dosimetry based on EPR measurements of fingernails potentially could be an effective tool for this purpose. This paper presents the first operational protocols for EPR fingernail dosimetry, including guidelines for collection and storage of samples, parameters for EPR measurements, and the method of dose assessment. In a blinded test of this protocol application was carried out on nails freshly sampled and irradiated to 4 and 20 Gy; this protocol gave dose estimates with an error of less than 30%.

EPR dosimetry in chemically treated fingernails.

By using EPR measurements of radiation-induced radicals it is possible to utilize human fingernails to estimate radiation dose after-the-fact. One of the potentially limiting factors in this approach is the presence of artifacts due to mechanically induced EPR signals (MIS) caused by mechanical stress during the collection and preparation of the samples and the so-called background (non-radiation) signal (BKS). The MIS and BKS have spectral parameters (shape, g-factor and linewidth) that overlap with the radiation-induced signal (RIS) and therefore, if not taken into account properly, could result in a considerable overestimation of the dose. We have investigated the use of different treatments of fingernails with chemical reagents to reduce the MIS and BKS. The most promising chemical treatment (20 min with 0.1 M dithiothreitol aqueous solution) reduced the contribution of MIS and BKS to the total intensity of EPR signal of irradiated fingernails by a factor of 10. This makes it potentially feasible to measure doses as low as 1 Gy almost immediately after irradiation. However, the chemical treatment reduces the intensity of the RIS and modifies dose dependence. This can be compensated by use of an appropriate calibration curve for assessment of dose. On the basis of obtained results it appears feasible to develop a field-deployable protocol that could use EPR measurements of samples of fingernails to assist in the triage of individuals with potential exposure to clinically significant doses of radiation.

Development and evaluation of biocompatible films of polytetrafluoroethylene polymers holding lithium phthalocyanine crystals for their use in EPR oximetry.

Electron paramagnetic resonance (EPR) oximetry is a powerful technology that allows the monitoring of oxygenation in tissues. The measurement of tissue oxygenation can be achieved using lithium phthalocyanine (LiPc) crystals as oxygen reporters. In order to have biocompatibility for the sensing system and to assure long-term stability in the responsiveness of the system, we developed films of Teflon AF 2400 with embedded LiPc crystals. These systems can be used as retrievable inserts or parts of an implantable resonator or catheter. Atomic force microscopy studies revealed that the surface of the films was regular and planar. The response to oxygen of the sensor (EPR linewidth as a function of pO(2)) remained unchanged after implantation in mice, and was not affected by sterilization or irradiation. The use of resonators, holding LiPc embedded in Teflon AF 2400, implanted in the gastrocnemius muscle of rabbits allowed the monitoring of oxygen during several weeks. Several assays also demonstrated the biocompatibility of the system: (1) no hemolytic effect was noted; (2) no toxicity was found using the systemic injection test of extracts; (3) histological analysis in rabbit muscle in which the films were implanted for 1 week or 3 months was similar to standard polyethylene biocompatible devices. These advanced oxygen sensors are promising tools for future pre-clinical and clinical developments of EPR oximetry. These developments can be applied for other applications of biosensors where there is a need for oxygen permeable membranes.

The relationship between partial pressure of oxygen and perfusion in two murine tumors after X-ray irradiation: a combined gadopentetate dimeglumine dynamic magnetic resonance imaging and in vivo electron paramagnetic resonance oximetry study.

Changes of partial pressure of oxygen (pO2) and blood perfusion were studied in MTG-B and RIF-1 tumors (n = 5 each) before and after a single 20-Gy dose of X-ray irradiation. Using electron paramagnetic resonance oximetry, we have observed an initial fast decrease of pO2 after irradiation, followed by a slow increase. The time course of these changes was faster in the MTG-B tumors than in the RIF-1 tumors. Gadopentetate dimeglumine (Gd-DTPA) dynamic magnetic resonance imaging studies showed a reduction in uptake of Gd-DTPA at the time of minimum pO2 and a recovery at the time of maximum pO2 in each tumor. Previous work indicates that there is microscopic heterogeneity in tumors, with well-vascularized "capillary regions" being closer to capillaries than poorly vascularized "noncapillary regions." We propose a two-component (slow and fast) model of Gd-DTPA uptake that is designed to quantify the kinetics of these two compartments by analyzing the total tumor uptake kinetics without having to identify specific regions of interest. Total perfusion in the tumors was greatly reduced at the time of minimum oxygenation, and the volume of the slow component increased after irradiation. We conclude that a decrease in blood perfusion is one of the main causes of the decline in pO2 observed after irradiation.

Reduced tumor oxygenation by treatment with vinblastine.

Vinblastine (VLB) previously has been shown to perturb tumor blood flow, but the effect of these perturbations on tissue oxygenation is not known. The recent development of electron paramagnetic resonance (EPR) oximetry now has made it feasible to measure the effects of changes of perfusion on the pO(2) in tumors and normal tissues as a function of time and dose. We measured changes in tumor perfusion by Patent blue staining, tumor blood volume and microvascular permeability by contrast-enhanced magnetic resonance imaging, and tumor oxygenation by EPR in s.c. SA-1 murine tumors. We found that treatment with VLB induced dose-dependent reduction in tumor perfusion. One hour after i.p. treatment of mice with 2.5 mg/kg VLB, tumor perfusion was reduced to 20% of the pretreatment value and returned to close to original values within 48 h. A transient tumor blood flow-modifying effect of VLB was demonstrated also by contrast-enhanced magnetic resonance imaging; reduction of tumor blood volume and microvascular permeability was found. Reduced tumor oxygenation was found as measured by EPR oximetry, with the same time course of changes in tumor blood flow. Tumor oxygenation was reduced to 50% of pretreatment value 1 h after the treatment with 2.5 mg/kg VLB and returned to pretreatment levels within 24 h after the treatment. Although the directions of the changes in perfusion and oxygenation were similar, they were quantitatively different. Reduction in oxygenation of normal tissues, muscle, and subcutis also occurred but was smaller and returned to pretreatment values more quickly compared to the changes induced in the tumors. In conclusion, the present study demonstrates that VLB causes a profound reduction in tumor blood flow and oxygenation, which may have implications in controlling side effects of therapy and the planning of combined treatment with VLB, either with other chemotherapeutic drugs or with radiotherapy.

Changes of oxygen tension in experimental tumors after a single dose of X-ray irradiation.

Electron paramagnetic resonance oximetry was used to measure the partial pressure of oxygen (pO2) in two types of tumor in vivo in C3H/HeJ mice. The pO2 in MTG-B (high hypoxic fraction) and RIF-1 (low hypoxic fraction) tumors was monitored prior to and at several time points after a single dose of X-ray irradiation (up to 7 days after treatment). Initial values of pO2 in RIF-1 (8.7 +/- 1.1 mm Hg; n = 14) were higher than that of pO2 in MTG-B (3.3 +/- 0.5 mm Hg; n = 19). The pO2 in both types of unirradiated tumors decreased slowly with tumor growth. Irradiation of tumors had a two-phase effect on pO2: an initial sharp decrease in pO2, followed by slow reoxygenation. After a 20-Gy radiation dose, the pO2 was 2.2 +/- 0.5 mm Hg at 6 h [significantly lower (P < 0.0001) than in control] and 3.2 +/- 0.5 mm Hg at 48 h [significantly higher (P < 0.02) than in control] in MTG-B, and 5.4 +/- 1.2 mm Hg at 24 h and 8.2 +/- 1.0 mm Hg at 72 h in RIF-1. The time course for these changes in pO2 was found to be independent of the doses in use in this study (10, 20, and 40 Gy). The occurrence of radiation-induced changes in pO2 and the different time courses of these changes suggest that repeated monitoring of pO2 in tumors during treatment could be used to enhance the efficacy of clinical treatments.

Oxidation of hydroxylamines to nitroxide spin labels in living cells.

In the presence of oxygen, cells can oxidize hydroxylamines, which are the products of the reduction of nitroxides in cells, back to nitroxides. Lipid-soluble hydroxylamines are oxidized much more rapidly than water-soluble ones, and most of this oxidation is inactivated by heat or trichloroacetic acid, indicating that the principal mechanism is enzyme-linked. The rates of oxidation of some lipophilic hydroxylamines are comparable to the rates of reduction of the corresponding nitroxides. Hydroxylamines formed by reduction of aqueous soluble nitroxides are not oxidized by cells, except for slight oxidation of some pyrrolidine derivatives. The latter is due to autoxidation. The kinetics of oxidation of reduced lipid-soluble nitroxides are all first-order with respect to hydroxylamines, regardless of the position of the nitroxide group along the carbon backbone, indicating that the oxidation occurs within the membrane. The oxidation of hydroxylamines in cells in inhibited by cyanide but not by antimycin A or SKF-525A. We also describe an effective method to oxidize hydroxylamines and follow this reaction; the method is based on the use of perdeuterated [15N]Tempone.

The products of the reduction of doxyl stearates in cells are hydroxylamines as shown by oxidation by 15N-perdeuterated Tempone.

The use of nitroxides in functional biological systems has increased greatly as it has become evident that such studies can provide valuable biophysical and metabolic data. This has led to a need to understand the nature of the metabolism of nitroxides and their products. This paper presents data indicating the value of 15N-perdeuterated Tempone specifically to indicate the amount of hydroxylamines that are present in a cellular system. Using this technique, we found that in the mammalian cells that we studied the principal or only products of reduction of doxyl stearates were the corresponding hydroxylamines.

Cellular metabolism of water-soluble nitroxides: effect on rate of reduction of cell/nitroxide ratio, oxygen concentrations and permeability of nitroxides.

In order to interpret more accurately studies that have used nitroxides and to improve the efficacy of the use of nitroxides in both basic studies of cells and as contrast agents for in vivo NMR, we have initiated a systematic study of the distribution and metabolism of nitroxides in biological systems. Overall, the results provide a reasonably coherent picture of some aspects of the interactions between nitroxides and cells. Reduction of the nitroxides appears to be an intracellular process, so that one of the principal variables that affects the rate of reduction is the ability of a nitroxide to enter cells. The entrance of nitroxides into cells shows considerable variability and ranges from essentially no penetration (e.g., 2,2,6,6-tetramethylpiperidine-N-oxyl-4-trimethylamine), through rates that are comparable to rates of reduction (e.g., 2,2,5,5-tetramethyl-pyrrolidine-N-oxyl-3-carboxylic acid), to rates that are so fast that there is complete equilibrium between intracellular and extracellular compartments (e.g., Tempone). The presence of a charged group on the nitroxide appears to be the important variable that affects their ability to enter cells. Once a nitroxides enters the cell, the structure of the nitroxide, e.g., piperidine vs. pyrrolidine ring, is major factor that affects the rate of reduction. The rates of reduction increase with increasing concentrations of nitroxides. This indicates that the principal mechanism(s) of reduction do not saturate in the concentration range we studied. We observed no abrupt changes in the rates of reduction over the entire concentration range of cells and nitroxides that we studied, which suggests that the mechanism(s) of nitroxide reduction did not change. The presence of oxygen decreased the observed rate of reduction of many of the nitroxides and this effect was independent of the concentration of nitroxide.

Kinetics of enzyme-mediated reduction of lipid soluble nitroxide spin labels by living cells.

Nitroxide spin labels can be reduced to the corresponding hydroxylamines in cells. The selective action of inhibitors, and thermal and chemical inactivation demonstrate that the reduction of nitroxides in cells is an enzymatic or enzyme-mediated process. The kinetics of reduction of doxylstearates are affected by the position of the doxyl moiety along the stearic acid chain. The doxyl moiety of 5-doxylstearate is close to the membrane surface, and its reduction is first order with respect to the nitroxide, whereas the doxyl moieties of 10- and 12-doxylstearate are in the membrane hydrocarbon region and their reduction is a zero-order process. The reduction of 16-doxylstearate which usually has a mixture of first- and zero-order kinetics becomes zero order with addition of an extracellular broadening agent, potassium trioxalatochromiate(III). These results suggest that the rate of reduction of doxyl moieties is controlled by their accessibility to reducing equivalents, i.e., the rate-limiting step for the reduction of the doxyl moiety deep in the membrane is the diffusion of reducing equivalents within or into the membrane. The reduction of doxylstearates in cells is inhibited by rotenone but not antimycin A, cyanide, propyl gallate or SKF-525A. It appears that the reduction of doxylstearates takes place at the level of the ubiquinone in the respiratory chain in mitochondria in these cells.

Simultaneous measurements of the intra- and extra-cellular oxygen concentration in viable cells.

An EPR method that can measure the intra- and extra-cellular oxygen concentration [O2] simultaneously in vitro has been developed using specially designed nitroxides. In the presence of Fe(CN)6(3-) in the medium, intracellular [O2] is measured by a neutral 15N-nitroxide and extracellular [O2] is measured by a negatively charged 14N-nitroxide, since charged species do not enter cells and the EPR spectrum of a 15N-nitroxide does not overlap with that of a 14N-nitroxide. The method is based in part on the minimal broadening of negatively charged nitroxides by Fe(CN)6(3-) and the very effective broadening of neutral nitroxides by the same paramagnetic ions. Results with this method confirm the existence of gradients in [O2] between the extracellular and intracellular compartments in CHO cells and M5076 tumor cells, even without stimulation of cellular respiration by CCCP. The nature of the barrier that needs to be involved to account for the experimental results raises some significant questions.