EPR Oximetry Research Articles

Critical oxygen tension in rat brain: a combined (31)P-NMR and EPR oximetry study.

The relationship between cerebral interstitial oxygen tension (Pt(O(2))) and cellular energetics was investigated in mechanically ventilated, anesthetized rats during progressive acute hypoxia to determine whether there is a "critical" brain Pt(O(2)) for maintaining steady-state aerobic metabolism. Cerebral Pt(O(2)), measured by electron paramagnetic resonance oximetry, decreased proportionately to inspired oxygen fraction. (31)P-nuclear magnetic resonance measurements revealed no changes in P(i), phosphocreatine (PCr)/P(i) ratio, or intracellular pH when arterial blood oxygen tension (Pa(O(2))) was reduced from 145.1 +/- 11.7 to 56.5 +/- 4.4 mmHg (means +/- SE). Intracellular acidosis, a sharp rise in P(i), and a decline in the PCr/P(i) ratio developed when Pa(O(2)) was reduced further to 40.7 +/- 2.3 mmHg. The corresponding Pt(O(2)) values were 15.1 +/- 1.8, 8.8 +/- 0.4, and 6.8 +/- 0.3 mmHg. We conclude that over a range of decreasing oxygen tensions, cerebral oxidative metabolism is not sensitive to oxygen concentration. Oxygen becomes a regulatory substrate, however, when Pt(O(2)) is decreased to a critical level.

Pharmacological modifications of the partial pressure of oxygen in murine tumors: evaluation using in vivo EPR oximetry.

EPR oximetry using an implantable paramagnetic probe was used to quantify the partial pressure of oxygen (pO(2)) in tissues in a transplantable mouse tumor model (TLT) after administration of 34 different vasodilators belonging to one of the following classes: angiotensin-converting enzyme inhibitors, calcium antagonists, alpha antagonists, potassium channel openers, beta-blockers, NO donors, and peripheral vasoactive agents. Twenty-four compounds were efficient in significantly increasing the local pO(2) in a majority of tumors. The increase of local pO(2) using pharmacological treatments was lower than that achieved by using oxygen or carbogen breathing. This technique offers an unprecedented tool for rapidly and accurately measuring treatment-induced modifications of pO(2) in tumors. Magn Reson Med 42:627-630, 1999.

Impact of the antimetastatic drug Batimastat on tumor growth and PO2 measured by EPR oximetry in a murine mammary adenocarcinoma.

Patients treated for early breast cancer have only a 10–15% probability of local relapse (Fowble, 1991), but the likelihood of eventual recurrence with distant metastases can be much higher depending on the original stage of the cancer. It is from this systemic disease that patients die (Willner et al., 1997). Treatment of malignant tumors has relied heavily on the use of cytotoxic agents, targeted at the increased metabolic and proliferative activity of the cancer cell. An alternative strategy has been to develop low toxicity therapies that can be administered continuously over a period of several years to stabilize malignant disease, and it is in this category that treatment with matrix metallopro-teinase inhibitors (MMPIs) lie.

Preparation and characterization of a highly sensitive EPR oximetry probe for biological application: [formula omitted] Jay L.Zweier and Periannan Kuppusamy

Preparation and characterization of a highly sensitive EPR oximetry probe for biological application: [formula omitted] Jay L.Zweier and Periannan Kuppusamy

EPR kinetic studies of oxygen release in thylakoids and PSII membranes: a kinetic intermediate in the S3 to S0 transition.

Time-resolved EPR oximetry has been used to determine the oxygen release kinetics in spinach thylakoids and PSII membranes. We observe release kinetics with half-times of approximately 0.85 and approximately 1.45 ms for thylakoids and PSII membranes, respectively, which are in close agreement with the EPR determined Yz decay kinetics for the S3 --> --> S0 transition in these systems. The results show conclusively that water-oxygen chemistry is not a rate-limiting step in the donor side of PSII under normal turnover conditions. By analyzing the oxygen release kinetics in thylakoids under nonphysiological, but still functionally competent conditions (low pH or high salt), we observed an initial delay in the O2 release of up to 200 microseconds following flash turnover from the S3 state. This is the first direct indication of a probable quasi-stable intermediate in the S3 --> --> S0 turnover of PSII, possibly representing the putative S4 state. Under conditions more closely approaching physiological, no such delay was resolved, indicating that the S4 --> O2 transition occurs within 50 microseconds under such circumstances. Two possible reaction sequences for O2 formation consistent with these and other data are discussed. It is suggested that the more probable form of "S4" is in fact the S3 + Yz* combination, which must undergo some molecular rearrangement on the tens to hundreds of microseconds time scale before O2 formation chemistry occurs.

Separation and Enrichment of the Active Component of Carbon Based Paramagnetic Materials for Use in EPR Oximetry

Carbon based paramagnetic materials are frequently used for EPR oximetry, especiallyin vivo,but the EPR spectra of these materials often have more than one paramagnetic center and/or relatively low signal intensity. To determine whether the multi-components of carbon based materials could be separated and enriched in the active component, we used density gradient centrifugation to separate the materials into several fractions. We studied two types of coals, gloxy and Pocahontas, and found these materials to have large density distribution. The separated density fractions had very different EPR spectra and intensities. The active component from the coal material had a more homogeneous EPR signal and significantly increased EPR signal intensity, whereas for India ink, only slight changes were observed. This result can be very useful in the development of better probes for EPR oximetry.

The measurement of oxygen in vivo using EPR techniques

The measurement of in vivo using EPR has some features which have already led to very useful applications and this approach is likely to have increasingly wide and effective use. It is based on the effect of oxygen on EPR spectra which provides a sensitive and accurate means to measure quantitatively. The development of oxygen-sensitive paramagnetic materials which are very stable, combined with instrumental developments, has been crucial to the in vivo applications of this technique. The physical basis and biological applications of in vivo EPR oximetry are reviewed, with particular emphasis on the use of EPR spectroscopy at 1 GHz using particulate paramagnetic materials for the repetitive and non-invasive measurement of in tissues. In vivo EPR has already produced some very useful results which have contributed significantly to solving important biological problems. The characteristics of EPR oximetry which appear to be especially useful are often complementary to existing techniques for measuring oxygen in tissues. These characteristics include the capability of making repeated measurements from the same site, high sensitivity to low levels of oxygen, and non-invasive options. The existing techniques are especially useful for studies in small animals, where the depth of measurements is not an overriding issue. In larger animals and potentially in human subjects, non-invasive techniques seem to be immediately applicable to study phenomena very near the surface (within 10 mm) while invasive techniques have some very promising uses. The clinical uses of EPR oximetry which seem especially promising and likely to be undertaken in the near future are long-term monitoring of the status and response to treatment of peripheral vascular disease and optimizing cancer therapy by enabling it to be modified on the basis of the measured in the tumour.

EPR Oximetry Mapping (EPROM) of cartilage formed in a hollow fiber bioreactor

EPR Oximetry Mapping (EPROM) of cartilage formed in a hollow fiber bioreactor

Electron paramagnetic resonance kinetic studies of the S states in spinach PSII membranes

The decay kinetics of Y ⋅ Z have been detected using time-resolved EPR to obtain the half-times of the S-state transitions in the oxygen-evolving complex of spinach PSII membranes. The half-times were determined by deconvoluting the signal II vf kinetics for the individual S-state contributions observed in a series of single turnover flashes. The Kok parameters required for deconvolution were estimated from the oxygen flash pattern measured in situ using nitroxide-based EPR oximetry. The half-times of the individual S-state transitions in PSII membranes were found to be: 70 μs for S 0→S 1, 110 μs for S l→S 2, 180 μs for S 2→S 3, and 1400 μs for S 3→S 0. Comparison of the S-state transition kinetics in PSII membranes with those of thylakoids shows that the S-state transitions are slowed in PSII membranes with the largest effect being observed for the S 3→S 0 transition. Suitable conditions for applying EPR oximetry to PSII membranes are described.

What does EPR oximetry with solid particles measure--and how does this relate to other measures of PO2?

The technique of in vivo EPR oximetry has been more widely introduced only recently (Ferrari et al., 1994, Halpern et al., 1994, Swartz et al., 1994). Our laboratory has concentrated on spectroscopy at 1.2GHz, using particulate oxygen-sensitive paramagnetic materials placed at the site (or sites) of interest (Dunn et al., 1995, Gallez et al., 1996, Goda et al., 1996, Goda et al, 1995, Jiang et al., 1995, Liu et al., 1995, Liu et al., 1994a, Liu et al., 1994b, O’Hara et al., 1995a, O’Hara et al., 1995b, Swartz et al., 1992). This form of EPR oximetry has a number of features that seem potentially advantageous for making biologically useful measurements. These features include 1) the capability of making repeated non-invasive measurements from the same site without the need for anesthesia (after the placement of oxygen-sensitive paramagnetic materials at the site (or sites) of interest); 2) high accuracy and sensitivity, especially for lower levels of oxygen; 3) rapid response (seconds); 4) measurements which are not perturbed by factors such as pH, temperature, osmotic strength; 5) a high degree of stability and inertness in biological systems; 6) the availability of the oxygen-sensitive paramagnetic materials in a variety of forms ranging from a slurry of very small particles to a single macroscopic crystal; 7) the calibration is very stable; and 8) there is a very high degree of specificity of the measurements because there usually are no other EPR responsive materials present in sufficient concentrations to affect the measurements. EPR oximetry, of course, also has some potential limitations and uncertainties. These include 1) a certain level of invasiveness; for many uses the particulate oxygen-sensitive paramagnetic materials need to be physically placed at the site(s) of interest 2) availability; at the present time the instrumentation required for these measurements is not yet widely available 3) depth of penetration; the most sensitive approach, using an exciting frequency of I GHz, limits non-invasive measurements to a depth of about 10 mm; and 4) the nature of the parameter that is measured by the technique is not fully understood by many scientists. The purpose of this paper is to address the last question, doing so in a context that relates EPR oximetry to other methods.

Effect of lipopolysaccharide on intra-phagosomal oxygen concentration as measured by EPR oximetry.

Lipopolysaccharide (LPS) is the endotoxin from the outer membrane of Gram-negative bacteria that is associated with the high morbidity and mortality in patients with septic shock (Morrison, 1978). Our previous studies have shown that LPS can influence mitochondrial oxygen consumption in a variety of cell types (James and Jackson, 1995; Jackson, 1996) and alter the oxygen utilization of organs in experimental septic shock (James and Bacic, 1996). It is also suggested that LPS can augment the respiratory burst associated with phagocytosis in macrophages. We sought to investigate the effects of LPS on the respiratory burst and the distribution of oxygen within macrophages.

Potential for EPR oximetry to guide treatment planning for tumors.

A major contributing factor in the failure of solid tumors to be locally controlled by radiation therapy (RT) is the relative radiation resistance of hypoxic cells of tumors compared to well-oxygenated cells. Recently clinical studies have established the presence of hypoxic regions in human tumors (1,2). Further studies confirmed that relatively high tissue pO2 in human tumors correlated with positive outcomes of radiation therapy and that poor outcomes were associated with tumors with low pO2 (3–6). As a result of available studies, the conclusion has been drawn that the effective level of oxygenation in individuals could not be predicted based on tumor type, histology, staging, or even size, but had to be measured (7). With the development of methods to measure the pO2 in tumors (8), it now seems feasible to determine if this parameter, which can be of crucial importance in radiation therapy, can be used to improve treatment by permitting individualized optimization of therapy on the basis of the pO2 in the tumor.

Histological assessment of rodent CNS tissues to EPR oximetry probe material.

The effects of the paramagnetic oxygen sensing material, lithium phthalocyanine (LiPc) and fusinite were assessed in the brain of Mongolian gerbils and the spinal columns of rats respectively, to determine if there are histologically discernible changes in the tissue surrounding the probe material. This information is essential for the evaluation of the role of EPR oximetry in the measurements of pO2 in the CNS; the technique has great potential value for such measurements because it reports on the pO2 accurately and sensitively and, after the initial placement, measurements can be made repeatedly without invasive procedures or anesthesia. Histologic assessments demonstrated the inert nature of both the fusinite and LiPc EPR probes in rodent CNS tissue over relatively long (2 month) time periods. The fusinite suspensions and LiPc crystals (size range of approximately 100-200 microns) remained well localized to the point of injection and created mild acute tissue reaction on implantation (which appeared to resolve quickly) and virtually no tissue reaction at later times. The majority of the implanted fusinite and LiPc material was present extracellularly in the brain and spinal cord. MRI provided an accurate, noninvasive assessment of probe placement and was able to investigate pathologic effects (hemorrhage, edema, necrosis) associated with the probe placement and treatment effects.

Electron paramagnetic resonance kinetic studies of the S states in spinach thylakoids.

The Tyrz+ decay kinetics have been analyzed by using time-resolved EPR to determine the half-time of each Si-->S(i + 1) transition in the O2-evolving complex of spinach thylakoids under physiological conditions. Using dark-adapted thylakoids and appropriate single-turnover flash sequences, we were able to detect the signal IIvf kinetics of the Tyrz+ S0-->Tyrz S1, Tyrz+ S1-->Tyrz S2, Tyrz+ S2-->Tyrz S3, and Tyrz+ S3-->(S4)-->Tyrz S0 transitions. To correct for damping of the S state synchronization during the flash sequence, the Kok parameters were estimated by measuring the oxygen flash pattern in situ using nitroxide-based EPR oximetry. Following deconvolution of the individual S state contributions, the signal IIvf decay kinetics yield the following half-times for the S state transitions: S0-->S1 in 40-60 microseconds, S1-->S2 in 85 microseconds, S2-->S3 in 140 microseconds, and S3-->(S4)-->S0 in 750 microseconds. Preliminary results with detergent-solubilized PSII membranes suggest that the S3-->S0 transition at least is slowed by a factor of approximately 2 in this system. Ramifications of these half-times in terms of electron transfer events on the donor site of PSII are discussed.

EPR Linewidth (T2) Method to Measure Oxygen Permeability of Phospholipid Bilayers and Its Use to Study the Effect of Low Ethanol Concentrations

It is well known that continuous-wave EPR spectra of nitroxide probes (labels) introduced into phospholipid bilayers are sensitive to molecular oxygen. However, accurate determination of oxygen broadening from these experiments is complicated by the complex shapes of EPR spectra, which are strongly influenced by anisotropic restricted motion of the probe molecules. An accurate method is presented to extract the oxygen broadening from the spectra measured with and without oxygen and at the same temperature. The method is based on a fast convolution algorithm with Levenberg–Marquardt optimization. This method was previously applied to EPR oximetry with nitroxides exhibiting rotational motion in the fast limit. It is shown that for several membrane spin probes, the oxygen broadening can be described as homogeneous; thus, a one-linewidth-parameter fitting model is appropriate. The method is applied to measure permeability profiles of model membranes composed from 1,2-dimyristoyl-sn-glycero-3-phosphocholine above and below the main phase transition. For both membrane phases, the broadening of doxyl- and sterol-type labels is found to be homogeneous, a finding consistent with the model of Heisenberg exchange between molecular oxygen and spin probes. As an example, the method is applied to study the ethanol effect on local oxygen permeability of a phospholipid bilayer. It is shown that ethanol concentrations as low as 1% (v/v) increase oxygen permeability of the bilayer. The effect is larger at the surface of the membrane than at its center, indicating that ethanol molecules interact primarily within the polar head region of the bilayer.

Effect of repetitive ischemia on myocardial oxygen tension in isolated perfused and hypoperfused rat hearts.

The objective of this study was to determine the effects of repetitive ischemia on myocardial oxygen tension (pO2), consumption, and delivery in crystalloid normoperfused (perfusion pressure>70 mmHg) and hypoperfused (perfusion pressure approximately 50 mmHg) constant flow isolated rat hearts. EPR oximetry with lithium phthalocyanine was used to measure myocardial pO2. Baseline myocardial pO2 (means +/- SE) was 185 +/- 13 mmHg (normoperfused) and 162 +/- 14 mmHg (hypoperfused). Myocardial pO2 fell to < 1 mmHg during no-flow ischemia. After recovery from repetitive ischemia, myocardial pO2 and coronary resistance increased significantly in all hearts; oxygen consumption and left ventricle work decreased in normoperfused hearts, although not significantly compared with controls, and did not change significantly in hypoperfused hearts. Increased myocardial pO2 in the normoperfused group may be due to decreased oxygen consumption and/or increased local delivery, while increased myocardial pO2 in the hypoperfused hearts is due to increased local oxygen delivery.

Use of EPR oximetry with India ink to measure the pO2 in the liver in vivo in mice.

The partial pressure of oxygen (pO2) of the liver in vivo in unanesthetized mice was determined using electron paramagnetic resonance (EPR) oximetry with India ink. The EPR spectra were obtained using a low-frequency (1.2 GHz) EPR spectrometer with a loop gap cavity resonator. The line width of the India ink used in this experiment was reversibly broadened by oxygen and was particularly sensitive to pO2 below 30 torr. After the administration of India ink into the tail vein, the India ink particles were taken up mainly by Kupffer cells in the liver and in part by phagocytes in the spleen. The pO2 measured in the normal liver was about 14 torr and was constant for the 2-week experimental period. The pO2 decreased when measured at 1, 2, and 6 days after treatment with hepatotoxin (carbon tetrachloride (CCl4)); within 2 weeks, it returned almost to the initial level. Measurements by EPR at sacrifice of controls and CCl4-treated mice indicated that more than 90% of the India ink went to the liver; the spleen contained 4.7% of total amount in control mice and 8.8% in CCl4-treated mice when measured 2 weeks after the treatment. These data indicate the usefulness of India ink for measuring the pO2 of the liver in vivo and that the pO2 in the Kupffer cells is decreased when the liver is damaged by CCl4.

Accuracy of Oxygen Measurements in T2 (Line Width) EPR Oximetry

EPR oximetry is used for in vivo and in vitro measurements of oxygen in biological systems, including experimental animals. The accuracy of oxygen measurements in T2 (line width) EPR oximetry is significantly improved if least-squares simulation is used to extract the line width parameters. The oxygen effect on the EPR spectra of nitroxide solutions and aqueous suspensions of fusinite can be described as an additional homogeneous broadening that modifies the EPR spectrum of the oxygen-free probe. This allows one to use a one-parameter line width model in most cases. The simulations were carried out with the use of a fast-convolution algorithm followed by Levenberg-Marquardt optimization. The validity of error estimates provided by this method was tested on sets of experimental spectra taken under common conditions. It is shown that the accuracy of oxygen measurements in line width (T2) oximetry is determined not only by the probe sensitivity (rate of line width change versus oxygen concentration), but also by the signal-to-noise ratio, inhomogeneous contribution to the line shape (e.g., unresolved proton superhyperfine structure), and the spectral window. The accuracy of oxygen measurements is compared for aqueous solutions of two nitroxide radicals with different superhyperfine structure and for aqueous suspensions of fusinite.

The effects of endotoxin on oxygen consumption of various cell types in vitro: An EPR oximetry study

We have studied the effects of bacterial endotoxin on the oxygen consumption of a variety of target cells, and found that the rate of utilization of oxygen by treated cells was decreased in a time- and dose-dependent manner. Precise EPR measurement of oxygen concentrations enabled us to demonstrate that this effect was linked to mitochondrial dysfunction and was particular to each cell type. Such detailed knowledge on oxygen utilization by viable whole cells and the varied effects of endotoxin are as yet undocumented. Oxygen consumption was shown to decrease quite markedly in CHO cells and kidney cells from the cortex region. Cells from the kidney medulla region had lower baseline consumption and were stimulated to increased levels of oxygen consumption on addition of similar doses of endotoxin. Macrophages exhibited a dual response in that in addition to inhibiting mitochondrial oxygen consumption, endotoxin pretreatment primed these cells to exhibit an enhanced oxidative burst on stimulation with Zymosan. These results show that endotoxin has a direct effect on normal cellular oxygen consumption and is an important parameter that must be considered when following the early effects on cells and tissues during the septic syndrome.

India ink: A potential clinically applicable EPR oximetry probe

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 (pO2), thereby making it feasible to use this approach to measure pO2 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.