Average pH values measured in tumor and mammary gland tissues in wild type C57Bl/6 mice using different techniques (Bobko et al., 2012).
Magnetic resonance spectroscopic and imaging techniques are methods of choice for
2. Nitroxides as functional EPR probes
2.1. EPR-based oximetric applications of the nitroxides
EPR oximetry is one of the most promising and rapidly developing techniques for measurement of oxygen in living tissues (Swartz, 2004). An advantage of EPR oximetry is that it is based on pure physical interaction, Heisenberg spin exchange, between paramagnetic molecules of probe and oxygen, and does not interfere with oxygen metabolism, therefore providing a basis for noninvasive oxygen measurements in biological systems. Initially, the paramagnetic probes for EPR oximetry were almost exclusively nitroxides (Backer et al., 1977; Lai et al., 1982; Froncisz et al., 1985; Bacic et al., 1989; Chan et al., 1989; Hyde & Subszynski, 1989). Both the longitudinal (T1) and transverse (T2) relaxation times of the NRs can be affected by collisions of the nitroxide with dissolved oxygen. The first observation of oxygen-induced NR broadening in various solvents was reported by Povich (Povich, 1975). Two years later the T2 oximetry method was proposed and applied to follow mitochondrial respiration in samples containing about 100 liver cells (Backer et al., 1977). T1-sensitive EPR oximetry was developed by Hyde et al (Froncisz et al., 1985; Hyde et al., 1990). T2 oximetric applications using NRs are preferred for biological applications in low viscosity solutions because T2 is close to the collision rate resulting in optimal EPR sensitivity. On the other hand, T1 oximetry may have an advantage for applications in highly viscous environments and for nitroxide-labeled macromolecules due to the fact that the nitroxide T1 >> T2. T1 oximetric methods include pulsed saturation-recovery, continuous wave saturation and rapid passage displays (Hyde & Subszynski, 1989).
The typical effect of oxygen on the low-field component of the EPR spectrum of NRs is shown in Figure 1 for the five-membered ring pyrroline CTPO nitroxide. In the absence of oxygen each spectral component of the triplet spectrum shows additional superhyperfine splittings with 12 protons of four methyl groups and proton at carbon C-4 of the heterocycle. An increase in oxygen concentration results first in the broadening of the lines of the superhyperfine structure followed by the increase of the enveloped EPR linewidth, Hpp.
Note that most six- and five-membered ring NRs with larger numbers of protons in the radical heterocycle do not reveal superhyperfine structure even in the absence of oxygen,
e.g. TEMPO radical of the piperidine type (see Fig. 2). The oxygen-induced line-broadening effects vary slightly with the NR structure and are about Hpp/[O2]500 mG/100% oxygen or 450 mG/mM of oxygen (see Fig. 1 and 2).
The well-developed chemistry of the NRs (Volodarsky et al., 1994; Hideg et al., 2005; Karoui et al., 2010) allows manipulation of their structure and properties, including charge, presence of hydrophilic or hydrophobic groups, and ability to be targeted. Small neutral NRs normally easily penetrate cellular membranes and are equally distributed throughout the intracellular and extracellular environments. Conversely, charged NRs will not cross the plasma membrane and thus can be used to measure oxygen concentrations in the extracellular compartment (Glockner et al., 1993; Baker et al., 1997). NRs encapsulated in liposomes or linked to carrier molecules can be used to achieve organ or tissue selectivity (Gallez et al., 1993). In general, NRs have low toxicity and can be administered to an animal by infusion or by intraperitoneal, intravenous or intratissue injection. To protect NRs against reduction and enhance their oxygen sensitivity Liu et al. (Liu et al., 1994) encapsulated NRs in proteinaceous microspheres filled with an organic liquid. The authors used encapsulated NR to measure the changes in oxygen concentration
Figure 3 demonstrates an example of the L-band EPR oximetry application using TEMPO nitroxide (see Fig.2 for the structure) for monitoring ischemia-induced oxygen depletion in isolated rat hearts (Zweier & Kuppusamy, 1988; Kuppusamy et al., 1994). The spectra showed a gradual decrease in the linewidth over the duration of ischemia approaching the linewidth observed in the absence of oxygen. Oxygen consumption observed in cardiopleged hearts subjected to global ischemia (Kuppusamy et al., 1994) was significantly slower than in noncardiopleged hearts (Zweier & Kuppusamy, 1988). This would be expected since contractile function of cardiopleged hearts is arrested. The oxygen consumption data for cardiopleged heart were obtained from spectral-spatial EPRI which allowed for spatially resolved oxygen mapping but required long acquisition times (16 min in Fig. 3a). PEDRI provides a faster alternative for oxygen mapping. Application of PEDRI for myocardium oxygen mapping was first demonstrated in perfused sheep heart using a high concentration, 4 mM, of Fremy’s salt nitroxide (Grucker & Chambron, 1993).
Low sensitivity of the NRs to oxygen tensions below 40 mmHg and concentration-induced line broadening complicate quantitative oximetric applications of the NRs. Typically, the self-broadening effect is about 100-200 mG/mM of NRs, and may interfere with accurate oxygen measurements at NR concentrations above 100 M. Application of deuterated NRs provides significant enhancement in sensitivity to low oxygen concentrations due to the narrowing linewidth, Hpp (Kuppusamy et al., 1994; Gallez et al., 1996a; Velan et al., 2000). Alternatively, measurement of “the depth of resolution” of the NRs superhyperfine structure (e.g. parameter a/A for the CTPO shown in the Fig.1) provides similar or even better sensitivity to oxygen than perdeuterated NRs, and has been used for oxygen measurements in cellular and enzymatic systems (Backer et al., 1977; Sarna et al., 1980; Lai et al., 1982). However, both approaches still suffer from concentration-induced line broadening which is difficult to disentangle from oxygen-induced broadening. To overcome this problem, Halpern et al. proposed selectively deuterated nitroxide with only one hydrogen hyperfine splitting, mHCTPO (see Scheme 1) (Halpern et al., 1994). Increasing NR concentration but not oxygen results in narrowing of hydrogen hyperfine splitting. This allows for discrimination between oxygen- and concentration-induced line broadening, and therefore, for quantitative oxygen detection in living tissues with sensitivity about 10 mmHg. The authors demonstrated an efficiency of low-field 250 MHz EPR oximetry in combination with a mHCTPO probe to report oxygen concentration in murine FSa and NFSa fibrosarcomas 7 cm deep in tissues of a living animal. An oximetric 2D (1spectral/1 spatial) spectral image of the tumor was also obtained allowing, in principle, direct assessment of tumor hypoxia to determine the usefulness of radiation and chemotherapy adjuvants directed to hypoxic cell compartments. In another application, cell-permeable perdeuterated NR, PDT (see Scheme 1), was applied to measure oxygenation level in radiation-induced fibrosarcoma (RIF-1) in mice (Kuppusamy et al., 1998). The data showed a 3-fold lower level of oxygenation of the tumor tissue compared with that of the normal muscle.
Chemical structures of the NRs referred to in the text: 4-protio-3-carbamoyl-2,2,5,5-tetraperdeuteromethyl-3-pyrrolinyl-1-15N-oxy (mHCTPO) and 4-oxo-2,2,6,6-tetramethylpiperidine-d16-1-15
Distribution of the NRs in living tissue can be measured using EPRI techniques as demonstrated in Fig. 3a. Moreover, spectral–spatial imaging can be performed which contains a complete spectral profile, as a function of field, at each spatial voxel element. Because the spatial and spectral dimensions are fully separable, information about local linewidth, and hence local oxygen concentration, can be derived independently from local spin density (Velan et al., 2000; Kuppusamy & Zweier, 2004). To date most NRs imaging applications have been performed using CW EPRI. Nitroxide imaging by time-domain pulsed EPR had not been attempted until recently because of the short spin-spin relaxation times, typically under 1 microsecond. Nevertheles, recent advances in RF electronics have enabled the pulses on the order of 10-50 ns (Murugesan et al., 1997) and improved spectrometer recovery times, therefore providing an opportunity for
EPR applications of other oxygen-sensitive paramagnetic materials include soluble trityl radicals (Krishna et al., 2002; Bobko et al., 2009) and particulate probes such as lithium phthalocyanine particles (Liu et al., 1993; Presley et al., 2006) and carbonaceous materials (chars, coals, carbon blacks) (Clarkson et al., 1998). It should be noted that particulate probes such as lithium phthalocyanine and synthetic char are suitable for measurements of oxygen partial pressure in place of implantation whereas soluble probes such as nitroxides and trityl compounds more suitable for imaging experiments.
In vivoevaluation of redox state using nitroxide probes
Regulation of tissue redox status is important for maintenance of normal physiological conditions in the living body. Disruption of redox homoeostasis may lead to oxidative stress and can induce many pathological conditions such as cancer, neurological disorders and ageing. The intracellular thiols, and particularly the redox couple of glutathione, GSH, and its disulfide form, GSSG, are considered the major regulators of the intracellular redox state (Schafer & Buettner, 2001). Therefore, noninvasive spectroscopic evaluation and imaging of tissue redox status and, in particularly, GSH redox status, could have clinical applications (Kuppusamy & Krishna, 2002; He et al., 2004; Swartz et al., 2007; Hyodo et al., 2008; Ojha et al., 2008; Roshchupkina et al., 2008; Bobko et al., 2012).
2.2.1. Nitroxides as redox-sensitive paramagnetic probes
NRs introduced into biologically relevant systems are predominantly observed in the radical and hydroxylamine forms and exist in redox equilibrium as shown in Scheme 2. The reduction of NRs to EPR-silent hydroxylamines in many cases is an unfavorable factor that limits their applications. On the other hand, the EPR-measured rates of NR reduction depend on overall tissue redox status allowing for the differentiation of normal and pathological states (Kuppusamy et al., 2002; Ojha et al., 2008; Bobko et al., 2012). The reduction of cell-permeable NRs to hydroxylamines is primarily intracellular and, therefore to a great extent is determined by intracellular redox status. On the other hand, cell-impermeable nitroxides allow for detection of reducing capacity of extracellular microenvironment.
Illustration of the nitroxide/hydroxylamine redox couple. In general, for most biologically relevant samples one-electron reduction of the nitroxides prevails and the equilibrium is strongly shifted towards the hydroxylamine (Kocherginsky & Swartz, 1995).
Cancer and ischemic heart disease, two leading causes of mortality in the United States, represent pathologic conditions with compromised redox state. Figure 5 demonstrates the application of cell-permeable CP nitroxide (see Scheme 3) for EPR
Tumor cells are known to generate significant alterations in the redox status. This status is an important determinant in the response of the tumor to certain chemotherapeutic agents, radiation, and bioreductive hypoxic cell cytotoxins (Cook et al., 2004). Figure 6 illustrates the application of cell-impermeable RSG nitroxide (see Scheme 3) designed to access both extracellular tumor redox and pH (Bobko et al., 2012), latter measurements being discussed in the Section 2.3. The extracellular reducing capacity of tumor tissue was found to be about four-fold higher compared with that of the normal mammary gland (Fig. 6). Inhibition of tumor angiogenesis with granulocyte-macrophage colony-stimulating factor (GM-CSF) resulted in significant “normalization” of the tumor redox status (Fig. 6b) as well as correlating with a decrease in tumor growth and metastases (Eubank et al., 2009).
Representative structures of the NRs used for
EPR studies using cell-permeable nitroxides support significantly higher reducing capacity of the tumor tissues. Figure 7 demonstrates L-band EPRI application for redox mapping of the tumor in living mice (Kuppusamy & Krishna, 2002; Kuppusamy et al., 2002) using CMP nitroxide. A significant decrease in CMP reduction rates after treatment with a GSH depleting agent clearly demonstrates a central role of GSH in tissue redox homeostasis. Note that in general, appreciable chemical reduction of the NRs by GSH is not observed (Finkelstein et al., 1984; Glebska et al., 2003; Bobko et al., 2007b). However GSH significantly contributes to the reduction of the NRs indirectly by acting as a secondary source of reducing equivalents (Takeshita et al., 1999; Kuppusamy et al., 2002; Bobko et al., 2007b).
Application of the NRs as redox-sensitive contrast agent for EPRI, MRI and PEDRI (also termed as Overhauser magnetic resonance imaging, OMRI) was recently discussed (Hyodo et al., 2008). In summary, NRs provide a useful tool for quantitative assessment and mapping of the redox environment in living tissues.
2.2.2. Nitroxides as glutathione-sensitive paramagnetic probes
The redox couple of glutathione, GSH, and its disulfide form, GSSG, is considered the major regulator of the intracellular redox state (Schafer & Buettner, 2001). Therefore, GSH redox status
EPR spectroscopy in combination with thiol-specific nitroxides allows for determination of the accessible thiol groups in various biological macromolecules, such as human plasma low-density lipoproteins (Kveder et al., 2003) and erythrocyte membranes (Soszynski & Bartosz, 1997). This approach normally requires purification of the sample from the unbound label and can not be used
Structures of the disulfide nitroxyl biradicals, R1SSR1, R2SSR2 and 15N-R2SSR2.
EPR spectra of the RSSR labels are significantly affected by intra-molecular spin exchange between two radical fragments resulting in appearance of “biradical” spectral components in addition to the conventional triplet spectral pattern of the mononitroxide. Figure 8a demonstrates the typical changes of the EPR spectra of R2SSR2 upon GSH addition consistent with splitting of the biradical disulfide bond and formation of two monoradicals due to the reaction of thiol-disulfide exchange, RSSR + GSH → RSSG + RSH. Figure 8b shows the corresponding kinetics of the monoradical component increase allowing for the calculation of the rate constant. The RSSR labels being lipophilic compounds diffuse easily across cellular membranes where they reacts with intracellular GSH providing a reliable EPR approach for determination of GSH
The convenient time window of the reaction of the imidazolidine RSSR labels, R2SSR2 (Khramtsov et al., 1997) and 15N-R2SSR2 (Roshchupkina et al., 2008) allows for quantitative measurement of GSH content by the analysis of their EPR spectral change kinetics. The kinetics approach looses the attractive simplicity of the static EPR measurements performed
Figure 9a shows typical
Intracellular GSH has been shown to be one of the major factors modulating tumor response to a variety of commonly used anti-neoplastic agents, such as cisplatin (Rabik & Dolan, 2007). The 15N-R2SSR2 probe has been used to compare
As can be concluded from figures 9b and 10, the EPR signal changes of the R2SSR2 probe show biphasic character: (i) comparatively fast monoradical signal increase due to the reaction with GSH, and (ii) slow signal decay due to bioreduction. A comparatively long EPRI acquisition time of about 5 min did not allow us to resolve the first phase of the kinetics from images given in Figure 10. With the development of faster imaging techniques, e.g. functional PEDRI discussed in the section 2.3.3, paramagnetic disulfide RSSR nitroxides can be used as dual function intracellular GSH and redox imaging probes.
In summary, it seems likely that EPR and EPR-based imaging approaches to visualize redox processes in living tissues, and in particular to assess intracellular GSH, will become increasingly utilized and valuable tools.
In vivospectroscopy and imaging of pH using nitroxide probes
Spatially and temporarily addressed pH measurements
2.3.1. pH effect on the EPR spectra of stable nitroxides
The first pH effect on EPR spectra of stable NRs was observed in strong acids and was related to protonation of the nitroxyl fragment itself (Hoffman & Eames, 1969; Malatesta & Ingold, 1973). Imino nitroxyl radicals (Ullman & Osiecki, 1970) were apparently the first reported stable NRs with spectral sensitivity in physiological range but did not find applications as pH probes due to the complexity of their EPR spectra and rapid reduction to EPR silent products in biological fluids (Woldman et al., 1994; Haseloff et al., 1997; Bobko et al., 2004). The observed pH effects on the NRs of piperidine and pyrrolidine types with ionizable functional groups were impractically small (Hsia & Boggs, 1972; Quintanilha & Mehlhorn, 1978; Nakaie et al., 1981; Mathew & Dodd, 1985; Khramtsov & Weiner, 1988; Saracino et al., 2002) due to the long distance between radical center and ionizable group.
Stable imidazoline and imidazolidine NRs have been proven to be the most useful spin probes for EPR spectroscopy and imaging of pH (Khramtsov et al., 2000, Khramtsov, 2005) due to the large effect of pH on their EPR spectra and large number of structures synthesized. Scheme 5 illustrates the chemical origin of the pH effect on EPR spectra of these types of NR. Protonation results in an EPR-detected difference in hyperfine splitting, aN, and g-factor (aN≈ 1 G and g≈0.0002) between R and RH+ forms (Khramtsov et al., 1982). For NR with an equilibrium constant
Reversible protonation of the nitrogen atom N-3 of the imidazoline and imidazolidine NRs, and chemical structures of the IR1-IR5 referred in the Figure 11. Two main resonance structures are shown illustrating higher unpaired electron density on nitrogen atom N-1 in the unprotonated form.
Up to the present a wide variety of pH-sensitive NRs have been developed with different ranges of pH sensitivity, labeling groups, lipophilicity and stability towards bioreduction (Khramtsov & Weiner, 1988; Khramtsov & Volodarsky, 1998; Kirilyuk et al., 2004; 2005; Voinov et al., 2005; Bobko et al., 2012). These spin pH probes, together with low-field EPR-based techniques, offer unique opportunities for non-invasive pH assessments in living animals in compartments with widely varying pH ranges. Figure 11 exemplifies a set of nitroxide pH probes that cover all pH ranges from acidic values observed in stomach and ischemic tissues to alkaline values characteristic of mitochondria. The potential applications are enormous, as tumors and ischemic areas may have acidic pH values compared to surrounding tissues, local areas of infection or inflammation can exhibit specific localized pH reductions allowing infection to be imaged and localized.
2.3.2. In vivo pH monitoring using pH-sensitive nitroxides
Low-field EPR spectroscopy using spin pH probes has been shown to be a valuable tool for
The acidic extracellular pH (pHe) in tumors has a number of important consequences, playing a role in tumor initiation, progression, and therapy (Gillies et al., 2004). To be used for monitoring of tumor extracellular tissue acidosis, the nitroxide pH probe has to (i) possess pH-sensitive spectral properties in the pH range from about 6.5 to 7.2, (ii) have enhanced stability to survive a reducing tumor microenvironment, and (iii) not penetrate cellular membranes to ensure probe targeting to extracellular space. These properties were achieved by synthesizing RSG nitroxide (Scheme 3) with pKa≈6.6 (Fig. 11) being ideally fitted for detection of extracellular tumor tissue acidity. The bulky ethyl substitutes at positions 2 and 5 around the NO fragment of the RSG are introduced to enhance its stability towards bioreduction (Kirilyuk et al., 2004). The binding of the radical to hydrophilic tripeptide, GSH, prevents probe diffusion across the plasma membrane, and therefore, enforces probe localization to extracellular aqueous volumes (Woldman et al., 2009). Recently the RSG probe has been used to monitor tissue pHe in mice bearing breast cancer tumors for assessment of therapeutic effectiveness of various treatments (Bobko et al., 2012). It was observed that tumor pHe is about 0.4 pH units lower than in normal mammary gland tissue in agreement with microelectrode data (see Table 1). Note that RSG nitroxide can be used as a dual function pH and redox probe allowing for concurrent
|pH values measured in tumors and mammary glands|
|L-band EPR||pH microelectrode||PEDRI|
|Tumor||6.60±0.07 (n=3)||6.70±0.05 (n=3)||6.7±0.1|
|Gland||6.98±0.05 (n=3)||7.01±0.05 (n=3)||7.1±0.1|
The enhanced stability of the RSG probe allowed for development of an EPR approach for monitoring ischemia-induced acidosis in isolated perfused rat hearts (Komarov et al., 2012). RSG probe demonstrated excellent EPR signal stability in the heart while 90% of the previously used IR2 probe (Khramtsov, 2005) was reduced in myocardial tissue within 5 minutes. As seen in Figure 13 ischemic preconditioning improved pH homeostasis during the global no-flow ischemia. Similar kinetics of myocardial acidification observed by the cell-impermeable RSG and cell-permeable IR2 probes support fast pH equilibration between intracellular and extracellular spaces in agreement with data obtained previously using glass microelectrodes and 31P-NMR (Asimakis et al., 1992; Lundmark et al., 1999).
2.3.3. pH mapping of living tissues
Figure 14 demonstrates
Recently we made significant progress towards pH mapping of living tissue using functional PEDRI approaches (Khramtsov et al., 2010; Efimova et al., 2011). Fig. 15 demonstrates PEDRI pH mapping of a tumor-bearing mouse after injection of deuterated analog of the RSG radical in tumor and mammary gland. The deuterated probe (see Fig. 15) has a narrower linewidth (Hpp=1.2 G) than RSG (Hpp=2.1 G), and therefore higher spectral intensity. The EPR signal of the deuterated RSG probe is easily saturated by RF irradiation which is of critical importance for PEDRI experiments. In total, more than one order decrease in acquisition time was achieved for PEDRI pH mapping (24.8 s in Fig. 15) compared with CW EPRI. Two areas of probe localization correspond to tumor (Fig. 15b left) and mammary gland (Fig. 15b right) with higher pH heterogeneity in tumor. Mean pHe values in tumor and mammary gland were found to be in agreement with EPR spectroscopy and microelectrode data measured in the same mice (see Table 1). The PEDRI functional approach may find applications for pH mapping of other living tissues and has potential for applications to humans.
In vivoassessment of nitric oxide using nitronyl nitroxides
The measurement of nitric oxide
The physiological effects of NNR cannot be entirely explained by their specific radical-radical reactions with nitric oxide due to the rapid reduction of NNR to its corresponding hydroxylamines. The performed mechanistic studies of the NNR reaction with NO in a reducing environment (Bobko et al., 2004) demonstrate an ability of the NNR to react with NO in the presence of the reducing agent, ascorbate, as shown in Figure 16b. It has been shown that equilibrium between radical, NNR, and its hydroxylamine is normally strongly shifted towards diamagnetic hydroxylamine, therefore no initial EPR spectra were observed in the presence of ascorbate (Fig. 16b). However NO generation and its consequent reaction with NNR results in accumulation of paramagnetic NNR and appearance of the corresponding EPR spectra (Fig. 16b, 3min). During the reaction, NNR is transformed to INR followed by characteristic changes of EPR spectral pattern (Fig. 16b, 6 and 9 min). After the reaction was complete, INR was reduced in its diamagnetic form by ascorbate (Fig. 16b, 20 min). The presence of fluorine atoms in the structure of fNNR allowed us to monitor accumulation of diamagnetic hydroxylamine product by 19F NMR spectroscopy providing additional proof of the redox-sensitive mechanism of the NNR reaction with NO. The corresponding scheme of the reactions provides a plausible mechanism explaining the antagonistic action of NNR against NO in a reducing environment, a phenomenon well documented
The application of NNR for NO detection by EPR in biological systems with physiologically low rates of NO generation is limited due to the very rapid reduction of NNR, and particularly of INR, into diamagnetic EPR-silent product (Woldman et al., 1994; Haseloff et al., 1997; Bobko et al., 2004), so equilibrium radical concentration is lower than threshold of EPR detection. An alternative approach for NO detection using NNR is based on application of fluorinated NNRs, such as fNNR, in combination with 19F NMR spectroscopy. An
In recent decades functional EPR spectroscopy and imaging applications have moved closer to biomedical applicability. The bottleneck of
This work was partly supported by NIH Grant EB014542-01A1. VVK thanks Dr. Andrey Bobko for technical assistance and helpful discussion.