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Aromatic hydroxylation from the reaction between hydroxyl radical and salicylate or its related compounds has been often utilized as a marker for the generation of hydroxyl radicals. We have investigated several technical aspects of applying this method to study hydroxyl radical production during cerebral ischemia and reperfusion using the hydroxylation of 4-hydroxybenzoic acid (4-HBA) to form 3,4-dihydroxybenzoic acid (3,4-DHBA). 4-HBA was administered to rats either through intravenous infusion, or by way of an in vivo microdialysis probe implanted in the brain. Dialysate containing 3,4-DHBA was collected and analyzed by HPLC with electrochemical detection. An endogenous compound was found to co-elute with 3,4 -DHBA but could be separated by varying the chromatographic conditions. Because of interrupted blood flow during cerebral ischemia and reperfusion, delivery of 4-HBA through the microdialysis probe is a preferred method to systemic administration such as intravenous infusion. It is concluded that the oxidation of 4-HBA to 3,4-DHBA can be a reliable and accurate indicator for the formation of hydroxyl radical in vivo if the experiments are well designed to avoid potential pitfalls associated with technical difficulties of the method.

Chromium (VI) compounds are widely recognized as human carcinogens. Extensive studiesin vitroand in model systems indicate that the reactive intermediate, Cr (V), generated by cellular reduction of Cr (VI), is likely the candidate for the ultimate carcinogenic form of chromium compounds. Here we review our current understanding of the in vivo reduction of Cr (VI) and its related free radical generation. Our results demonstrate that Cr (V) is indeed generated from the reduction of Cr (VI)in vivoand that Cr (V) thus formed can mediate the generation of free radicals. Cr (V) and its related free radicals are very likely to be involved in the mechanism of Cr (Vl)-induced toxicity and carcinogenesis. These studies also illustrate thatin vivoEPR spectroscopy and magnetic resonance imaging can be very useful and powerful tools for studying paramagnetic metal ions in chemical and biochemical reactions occurring in intact animals. (Mol Cell Biochem 222: 41-47, 2001)

General anesthesia is needed in many animal experiments in order to carry the studies out in an effective and appropriate manner. The concentration of oxygen in tissues is often a critical variable in many of these studies. Anesthesia, however, can affect tissue oxygenation because it can alter many physiological parameters, especially cardiopulmonary and metabolic parameters. While these physiological parameters can be measured, the relationship between them and tissue oxygenation may not be straightforward and further, the effects of anesthetics on these parameters vary with the type of anesthetic, the route of administration, and the method of administration. Therefore, it seems very desirable to directly measure the effects of anesthesia on tissue oxygenation.

Although nitric oxide (NO) is a central mediator during endotoxin-induced sepsis, direct detection of tissue NO in vivo, has until recently been difficult, and techniques have relied on indirect measurement of bi-products in blood or invasive technology. We have utilized electron paramagnetic resonance (EPR) in conjunction with the spin-trapping technique to detect NO directly, and non-invasively, from the tissue of septic mice. Relative signal intensity arising from NO complexed with iron and diethyldithiocarbamate (DETC) measured directly from the liver and kidney of mice given endotoxin was maximal at 6 hours post endotoxin. We failed to detect an EPR signal from mice given pyrogen-free saline. The quality of the EPR signal obtained (high signal to noise ratio of 15:1) using our experimental set-up and L-band EPR hardware was such that we were able to establish a time course of NO production in tissue following endotoxin, and measurement of NO from other organs (kidney and spleen). Our EPR results probably reflected NO arising from inducible NO-synthase enzymes as a result of endotoxin stimulation. This technique was extended to experiments in which we first implanted an oxygen sensitive material (gloxy) into the liver of mice, and then monitored NO production folowing endotoxin. Due to the fact that the EPR spectrum from gloxy and that of NO-Fe-(DETC)2 do not overlap, we were able to monitor NO production and pO2 simultaneously in tissue, in real time.

Oxygenation (pO2) is a major variable in many types of cancer treatment. This includes radiation therapy1–6, photodynamic therapy7–9 and certain chemotherapeutic agents10–17. The pO2 level can affect the response of tumors to therapy, especially in the critical range below 10 mmHg, where increasing hypoxia results in increased resistance to radiotherapy18. If it were possible to measure pO2 in tumors, then it might be possible to optimize the type and timing of treatment.

General anesthesia is known to alter cardiopulmonary and hematological parameters which affect tissue oxygenation. However, the effect of various types of anesthetics on the relationship between brain tissue pO2 and these physiologic parameters is still largely unknown.

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 measurement 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 µm) 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.

Polarographic micro-electrode measurements are very useful for measuring pO2 in vivo, especially for measurements of the variation of pO2 within a tumor (1,2,8). This method has several advantages, including: it is the only direct method currently in extended use in the clinical setting; it can provide data on microscopic heterogeneity; and it is fairly widely available. While the micro-electrode method has become a type of “gold standard” for measurement of pO2 in tissues, it has some limitations and disadvantages: it can be technically difficult; it has limited resolution at the very low levels of pO2 that are important for many clinically relevant processes; and it can perturb the tissues significantly, especially when used in repeated studies to monitor pO2 in tissues over time. Repeated measurements are especially desirable to follow the effect on tissue pO2 after treatment with some drugs (e.g. anti-cancer drugs and anesthetics) and radiation, the effects of acute and chronic ischemia, and changes in respiratory factors. Electron paramagnetic resonance (EPR) oximetry appears to offer some complimentary advantages for such studies: it can monitor pO2 continuously and/or repeatedly at the exactly the same localized area in tissue in vivo without the need for anesthesia; it can resolve small differences in pO2 even at the very low levels that occur pathophysiologically; and it can be used in a variety of settings.

For many years membrane science was essentially subdivided into two major fields: artificial membranes used mainly in separation/purification technology and biological membranes. During the last ten years however it has become evident that artificial biofunctional and biomimetic membranes, containing immobilized biological components and/or are created on the basis of principles of native biomembranes1,2 can have very similar properties. This provides an excellent avenue to use the properties of biomembranes to develop more effective artificial membranes for separation/purification technology and to use artificial membranes to elucidate the structure and function of native membranes.

The purpose of this paper is to provide an illustrative description of the current state of development of the use of electron paramagnetic resonance (EPR, or completely equivalently, electron spin resonance or ESR) to measure the partial pressure of oxygen (pO2) in tissues in vivo under physiological conditions. This summary is based on published and unpublished results from our laboratory (1–7) and does not attempt to describe the results of other laboratories which also are working along related lines (8–10). The pertinent features of our technique are illustrated. We also consider the current limitations of the technique and likely developments in the near future. Our evaluation is that: this technique now is suitable for immediate use in small animals; within a short period of time instruments will be available facilitating its use in larger animals; and preliminary studies are imminent in human subjects (7).