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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS
Center of Biomedical Research Excellence, College of Pharmacy, University of New Mexico, Albuquerque, New Mexico (M.M., J.S., S.L., H.S., W.L., Z.Y., A.P., K.J.L.); School of Chinese Medicine, University of Hong Kong, Hong Kong (J.S.); Medical Biotechnology Center, University of Maryland Biotechnology Institute and Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland (J.P.Y.K.); and Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy and Center for Electron Paramagnetic Resonance Imaging for In Vivo Physiology and Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, Maryland (G.M.R.)
Received April 17, 2006; accepted June 2, 2006.
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Abstract |
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; Zauner et al., 2002). However, measurement of O2 levels in animal tissues using different imaging modalities is not a trivial task. For example, although O2 is paramagnetic, electron paramagnetic resonance (EPR) spectroscopy cannot directly detect this molecule at 37°C and requires the development of molecular probes that can report O2 concentrations in different experimental models. Once such family of stable free radical probes is trityls, whose EPR spectral lines are broadened in the presence of O2 due to the interaction of the two paramagnetic species (Elas et al., 2003). Not surprisingly, changes in the EPR spectral line widths of trityl radicals and other stable free radicals have been used to estimate O2 concentrations in homogenous solutions and isolated cell preparations (Elas et al., 2003; Khan et al., 2003; Shen et al., 2003; Kutala et al., 2004; Liu et al., 2004). With the development of low-frequency EPR spectroscopy with the capability to image free radicals (Halpern et al., 1989; Halpern and Bowman, 1991), reliable real-time estimates of O2 concentration in the vascular bed of a mouse tumor has recently been achieved using a polyanionic trityl radical (Elas et al., 2003).
As a result of their charge, however, trityl radicals are not particularly useful for measurement of tissue oxygenation in the brain, because delivery of these free radicals to brain tissue at sufficiently high concentration for imaging purposes is difficult, at best. Recently, we demonstrated that after intraperitoneal administration of 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (1), this nitroxide was able to cross the blood-brain barrier. Thereafter, upon esterase hydrolysis, 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (2) was entrapped in brain tissue (Shen et al., 2006). Through EPR spectroscopy, nitroxide 2 was found to accurately estimate O2 levels in homogeneous aqueous solutions (Shen et al., 2006). In light of this successful pilot study, we now describe results from a series of in vivo experiments designed to assess uptake of structurally disparate nitroxides that cross the blood-brain barrier after different routes of administration. From these experiments, nitroxide 1 and trans-3,4-di(acetoxymethoxycarbonyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (5), but not (2,2,5,5-tetramethylpyrrolidin-1-oxyl-3-ylmethyl)amine-N,N-diacetic acid diacetoxymethyl ester (3) (Fig. 1), exhibited favorable pharmacokinetic and pharmacodynamic profiles. Because nitroxide 1 is easier to synthesize and is more soluble in aqueous buffers than nitroxide 5, it seems that nitroxide 1 is the best of the current family of acetoxymethoxycarbonyl-containing nitroxides as an EPR proimaging agent for quantitating O2 levels in mouse brain.
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Materials and Methods |
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, p. 206; Kao and Rosen, 2004; Rosen et al., 2005). Nitroxides 1 and 3 were dissolved in 95% ethanol as 0.5 M stock solutions. For cellular and animal experiments, a stock solution was diluted 10-fold with PBS before use. The final concentration of ethanol was 0.0075 to 0.0126 mmol/kg body weight; no adverse effects were observed at these ethanol levels. Synthesis of nitroxide 5 is summarized schematically in Fig. 2 and is described in detail below. Because nitroxide 5 was poorly soluble in 95% ethanol, a stock solution of 0.2 M nitroxide 5 was made in N,N-dimethylacetamide/H2O [1:9 (v/v)] and then diluted 10-fold with PBS for animal experiments.
3,4-Dicyano-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (8). To a solution of 3-cyano-2,2,5,5-tetramethyl-1-pyrrolinyloxyl (7) (3.2 g; 14.4 mmol; prepared as described by Rozantsev, 1970, p. 207) in 95% ethanol (100 ml) was added 100 ml of an aqueous solution of potassium cyanide (3.05 g; 46.9 mmol) and ammonium chloride (2.6 g; 49.1 mmol). This mixture was heated at 70°C for 7 h, at which point the reaction was cooled to room temperature. The solution was then saturated with NaCl and extracted with ether (5 x 100 ml). The combined ether solutions were dried over anhydrous MgSO4, filtered, and reduced to dryness in a rotary evaporator. The resulting mixture of compounds was purified on silica gel. A small amount of starting material was removed by elution with hexane/ether (2:1). Subsequent elution with hexane/ether (1:1) afforded trans-3,4-dicyano-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (8a) (1.7 g; 46% yield), which was recrystallized from ether/hexane [mp = 141-142°C; IR (CHCl3): 2215 cm-1(CN)]. Increasing the polarity of the solvent to hexane/ether (1:2) eluted cis-3,4-dicyano-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (8b) (0.7 g; 18% yield), which was recrystallized from ether/hexane [mp = 84°C; IR (CHCl3): 2220 cm-1 (CN)] (Mathew and Dodd, 1985).
Nitroxide 5. A mixture of 8a (1 g; 5.2 mmol) and 2 M NaOH (60 ml) was warmed to 90°C for 3 days, at which point the reaction was cooled and the aqueous solution was extracted with ether. The remaining aqueous solution was cooled in an ice bath, acidified with 10% HCl, and extracted with ether. The organic solution was dried over anhydrous MgSO4, filtered, and rotary evaporated to dryness, leaving a light yellow solid. Recrystallization from acetone/benzene afforded trans-3,4-dicarboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (6) (1 g; 89% yield; mp = 222-224°C with decomposition) (Mathew and Dodd, 1985; Chatani et al., 2005).
To a mixture of nitroxide 6 (0.6 g; 2.6 mmol) and K2CO3 (1.08 g; 7.8 mmol) in Me2SO (2 ml) was added bromomethyl acetate (0.79 g; 0.51 ml; 5.2 mmol; Aldrich Chemical Co., Milwaukee, WI). The reaction was stirred at room temperature for 3 h, at which point methylene chloride (50 ml) was added. This mixture was washed with water (3 x 100 ml). The organic solution was dried over anhydrous Na2SO4, filtered, and reduced to dryness on a rotary evaporator. The residual Me2SO was removed under high vacuum. The resultant yellow oil was purified by silica gel chromatography (chloroform/ether; 49:1) to afford nitroxide 5 (0.77 g; 85%), which was recrystallized from hexane [mp = 89-90°C; IR (CHCl3): 1769 cm-1 (broad ester peak)]. Anal. (C16H24NO7): calculated, C = 51.34%; H = 6.46%; N = 3.74%; found, C = 51.35%; H = 6.41%; N = 3.75%.
Cell Culture. Sprague-Dawley rats were maintained and used in compliance with the principles set forth in the Guide for Care and Use of Laboratory Animals and approved by the University of New Mexico Animal Care and Use Committee. Primary cultured cortex neurons were prepared from embryonic day 15 Sprague-Dawley rats, as described previously with modification (Furuichi et al., 2005). In brief, dissociated cell suspensions were plated at a density of 2 x 106 cells/well on poly-L-lysine-coated six-well plates (BD Biosciences, San Diego, CA) with Neurobasal/2% B27 (Invitrogen, Carlsbad, CA) containing 0.5 mM glutamine (Sigma-Aldrich, St. Louis, MO), 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were maintained in a humidified incubator at 37°C, in 5% CO2, 95% air. On the 10th day of culture, the cells were used for experimentation.
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). Therefore, the in vitro cellular studies here were conducted at 21°C. Neuronal cells (2.5 x 106 cells/ml) were incubated with 50 µM nitroxide 1 or 3 at 21°C with gentle shaking for 60 min, at which point the cells were centrifuged, and the cell pellet was washed with PBS. This washing procedure was repeated three times. The neurons were resuspended in HBSS at 2.5 x 106 cells/ml. One aliquot of the cell sample was transferred into a Teflon tube, placed into the cavity of the EPR spectrometer, and the spectrum was recorded at room temperature. This time point was considered to be t = 0 in Fig. 3. The remaining cell suspension was gently agitated at 21°C, and at 30 and 60 min, aliquots of the cells were removed, centrifuged, washed with PBS, and resuspended in HBSS. EPR spectra of these aliquots were then acquired at room temperature. An X-band EleXsys 540 EPR spectrometer (Bruker, Newark, DE) was used with the following parameters: microwave power, 20 mW; center field, 3340 G; modulation frequency, 100 kHz; and modulation amplitude, 0.3 G. Pharmacokinetics of Uptake of Nitroxides 1, 3, and 5 in Mouse Brain. We determined the preferable route of administration that will achieve the maximum retention of the nitroxide in the brain by measuring the pharmacokinetics of nitroxides 1, 3, and 5. Mice (C57 strain, weighing 18-20 g) were obtained from Charles River Laboratories, Inc. (Wilmington, MA). Animal housing, care, and application of experimental procedures were in accordance with the institutional guidelines and approved by University of New Mexico Animal Care and Use Committee. Mice were maintained under appropriate lighting conditions for 4 days with free access to food and water before experimentation. On the day of experimentation, mice were anesthetized by inhalation of 4% isoflurane in N2O/O2 (70%: 30%) and were maintained under anesthesia by 1% isoflurane in N2O/O2 (70%:30%); the mouse core temperature was maintained at 37°C by using a heating pad. In a typical experiment (as shown in Fig. 4), after a mouse was anesthetized, nitroxide 1 or 3, at the dose of 0.39 or 0.23 mmol/kg body weight, respectively, was injected i.a., i.v., or i.p. Nitroxide administration caused no obvious changes in animal behavior or any signs of acute toxicity. For intra-arterial and intravenous injections, Tygon microtubing (0.010-in. i.d., 0.030-in. o.d.; Saint-Gobain PPL Corp., Bridgewater, NJ) was cannulated before administration of nitroxide. For the arterial route, the right carotid artery was exposed surgically, and the external carotid artery was ligated with a 6-0 silk suture and then the tube was inserted into the right common carotid artery and was fixed with a 6-0 silk suture. For the intravenous route, the tube was cannulated into the right femoral vein. In experiments depicted in Fig. 6, mice received nitroxide 1 or 5 intravenously at a dose of 6 µl/g body weight of a 20 mM stock solution, prepared by diluting a 0.2 M stock solution in N,N-dimethylacetamide/H2O [1:9 (v/v)] 10-fold with PBS.
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Pharmacodynamics of Nitroxides 1, 3, and 5 in Mouse Brain. After the mice (C57 strain, weighing 18-20 g) were anesthetized, either nitroxide 1 or 3 was injected intra-arterially, intravenously, or intraperitoneally at a dose 0.39 or 0.23 mmol/kg body weight, respectively. EPR spectra were recorded from the mouse head using the head resonator, immediately after tuning the spectrometer and then every 2 min for 10 min. The animals were then removed from the magnet, and the thorax was surgically opened. A 23-gauge butterfly needle was inserted immediately into left ventricle, and a part of the right atrium was excised to allow the blood to drain. Normal saline (2.0-2.5 ml/min) was infused via an infusion pump through the butterfly needle until clear saline emerged from the right atrium. The animals were then returned to the spectrometer, and EPR spectra were again recorded. The perfusion procedure took approximately 8 min on average. Pharmacodynamic experiments were also conducted to compare nitroxides 1 and 5 after intravenous administration of each compound (6 µl/g body weight of a 20 mM stock solution). EPR spectra were recorded using the following instrumentation settings: microwave frequency, 1.12 GHz; microwave power, 18 mW; center field, 412 G; modulation frequency, 100 kHz; and modulation amplitude, 1.0 G.
Statistical Analysis. All data are expressed as mean ± S.E. The Student's unpaired t test was used to assess the statistical significance of differences.
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10 to 20% of the initial intracellular concentration of the nitroxides was lost from the cells. These data suggest that nitroxides 1 and 3 may be excellent EPR proimaging agents for estimating O2 levels in mouse brain.
Pharmacokinetics of Nitroxides 1 and 3 in Mouse Brain via Three Different Routes of Administration. Although isolated cells do not exhibit the same dynamic properties of an intact animal, based on data depicted in Fig. 3, we expected the concentrations of nitroxide 1 and nitroxide 3 in the mouse head to be similar, and, perhaps nitroxide 3 might achieve even higher levels in this organ. Thereafter, diffusion across the blood-brain barrier followed by in situ hydrolysis would liberate the corresponding nitroxides 2 and 4, which, being predominantly charged at physiological pH, should be retained in brain tissue. After intraperitoneal administration of nitroxides 1 and 3, the peak EPR signal intensity and the t
of these nitroxides in the mouse head (i.e., brain tissue with the associated vasculature) were essentially the same (Fig. 4). In contrast, intravenous or intra-arterial administration led to a different scenario (Fig. 4, B and C). After either intravenous or intra-arterial administration, the peak EPR signal intensity of nitroxide 1 was always greater than that found for nitroxide 3. The disparity between nitroxides 1 and 3 is approximately 2-fold at the maximum (Fig. 4, B and C). With nitroxide 1, the t was determined to be 35, 19, and 21 min after intraperitoneal, intravenous, and intra-arterial administration, respectively (Fig. 4, A-C). In contrast, the t values for nitroxide 3 were 34, 13, and 10 min, following intraperitoneal, intravenous, or intra-arterial administration, respectively (Fig. 4, A-C).
Pharmacodynamics of the Nitroxides 1 and 3 in Mouse Brain. The pharmacokinetic studies presented in Fig. 4 cannot differentiate between nitroxides that have crossed the blood-brain barrier and become trapped in brain tissue from nitroxides that are merely passively retained in the brain vasculature. Therefore, we conducted experiments designed to measure the distribution of nitroxides in these two compartments after administration through different routes. In a typical study, a group of mice was injected intraperitoneally with nitroxide 1. EPR spectra were recorded from the heads of mice before and after blood was completely displaced from the vasculature by saline perfusion. Differences in the EPR spectral peak height before and after emptying of the vasculature gave an estimate of the amount of nitroxide that was in the vascular bed as well as the amount that had been transported into and entrapped in the brain tissue. The findings of these studies are summarized in Fig. 5.
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), whereas after intraperitoneal injection, additional metabolism of these nitroxides and the charged nature of nitroxides 3 and 4 must be contributing factors along with bioreduction for diminished concentration of these nitroxides in the mouse head. In addition, the peak bolus concentration attained in the brain was undoubtedly much higher for i.v. and i.a. administration than for i.p. injection.
After blood was completely displaced from the vasculature by perfusion with normal saline, the fraction of nitroxides 1 and 3 that crossed the blood-brain barrier and was entrapped in brain tissue (as nitroxides 2 and 4) was virtually the same, independent of the route of administration (Fig. 5, A and B). This finding was surprising given the highly charged nature of nitroxide 4. For nitroxide 1, the fractional retention values in brain tissue following i.p., i.v., and i.a. administration were 0.49 ± 0.12, 0.50 ± 0.11, and 0.62 ± 0.06, respectively. Corresponding values for nitroxide 3 were 0.61 ± 0.10, 0.56 ± 0.15, and 0.52 ± 0.06 for i.p., i.v., and i.a. administration, respectively.
Pharmacokinetics of Nitroxides 1 and 5 in Mouse Brain after Intravenous Administration. Because regardless of the route of administration of nitroxide 1 in brain tissue, its retention was substantially greater than that of nitroxide 3, we hypothesized that a more lipid-soluble acetoxymethoxycarbonyl analog of nitroxide 1 might further increase nitroxide concentration in the brain. Toward this goal, we synthesized nitroxide 5, which can be hydrolyzed to the doubly anionic nitroxide 6 (Fig. 1). We determined the pharmacokinetics of nitroxides 1 and 5, after intravenous administration of each compound (Fig. 6). The pharmacokinetic curves were essentially the same, with t values being 16 and 13 min for nitroxides 1 and 5, respectively. These results indicated that an additional acetoxymethoxycarbonyl group at position 4 on the nitroxide ring neither increased peak concentration of the nitroxide in the mouse head nor enhanced the kinetic profile.
Pharmacodynamics of the Nitroxides 1 and 5 in Mouse Brain. Because the pharmacokinetics of nitroxides 1 and 5 were essentially identical, we expected that there would not be a significant difference in the concentration of nitroxides 2 and 6 in brain tissue. The results shown in Fig. 7 confirm this hypothesis, even though nitroxide 5 is the more lipophilic of the two compounds. Although at physiological pH nitroxide 6 is doubly anionic and nitroxide 2 is singly anionic, there was no significant difference in the fraction of each nitroxide that was entrapped in brain tissue (Fig. 7). The fractional retention values were 0.55 ± 0.08 and 0.49 ± 0.09 for nitroxides 1 and 5, respectively.
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), we demonstrated that after intraperitoneal administration of nitroxide 1, this proimaging agent crossed the blood-brain barrier, where it was hydrolyzed to nitroxide 2. This metabolism allowed the paramagnetic compound to be entrapped in brain tissue. Based on this previous study, we prepared two nitroxides whose molecular differences would give us insight into structural features that might promote even higher concentrations of nitroxide in brain tissue. Because all the nitroxides have the same core structure, they are expected to be equally efficacious for O2 sensing. By altering the functional groups attached to the core nitroxide structure, we aimed to examine how substituents affected the pharmacokinetic and pharmacodynamic profile. We first synthesized nitroxide 3. Once this paramagnetic compound crossed the blood-brain barrier, hydrolysis would lead to nitroxide 4. At physiological pH, nitroxide 4 bears two negative charges and one positive charge, resulting in net anionic character (Fig. 1). Based on a previous report (Keana et al., 1987), we surmised that this compound would be more resistant to bioreduction than nitroxide 2. Moreover, the highly charged nature of nitroxide 4 would further retard its diffusion across the blood-brain barrier from brain tissue into the vasculature.
In primary cultures of neuronal cells that had been incubated with nitroxide 1 or 3, intracellular concentration of nitroxide 4 was considerably greater than that found for nitroxide 2 (Fig. 3). The loss of nitroxide 4 from the cells, which is through an organic anion transport mechanism (Rosen et al., 2005), paralleled our previous study (Shen et al., 2006). In mice, pharmacokinetics of nitroxides 1 and 3 after intraperitoneal injection were essentially the same, with a t of 35 min for both compounds (Fig. 4A). In contrast, after intravenous and intra-arterial administration of nitroxide 1, peak concentration of this nitroxide was twice that observed when the identical experiments were conducted with nitroxide 3 (Fig. 4, B and C). These results suggest that intravenous or intra-arterial injection of nitroxide 1 is the best route of administration for achieving the highest concentration of nitroxide 2 in the brain for imaging O2. Interestingly, the results from the in vivo pharmacokinetics and pharmacodynamics experiments demonstrate that higher concentrations of nitroxide 2 compared with nitroxide 4 were observed in brain tissue, which is contrary to expectations based on in vitro cellular studies. This difference is probably the result of the disparity between the static system, i.e., cultured neurons, and the dynamic system, i.e., the functional and complex blood-brain barrier. The findings from the present study again underscore the importance of animal experiments when investigating compounds for targeted delivery to the brain. In the present study, we inferred that the form of the nitroxide retained in brain tissue is principally the carboxylate resulting from in situ enzymatic hydrolysis. This inference is founded on two published studies. First, in vitro biochemical experiments unequivocally demonstrated that carboxyl esterase rapidly hydrolyzes the acetoxymethyl ester (i.e., nitroxide 1), but not the methyl ester, of nitroxide 2 to the corresponding carboxylate (as shown in Fig. 1) (Sano et al., 2000). Second, we showed (Shen et al., 2006) that whereas the acetoxymethyl ester 1 was very effective for loading nitroxide 2 into brain tissue, the methyl ester was not, despite the greater lipophilicity of the methyl ester. These two studies led us to attribute the EPR signal in brain primarily to the carboxylate form of the nitroxide.
We also designed and synthesized nitroxide 5 based on the reasoning that inclusion of two acetoxymethoxycarbonyl groups at positions 3 and 4 would enhance the lipophilicity of the proimaging nitroxide. Furthermore, after hydrolysis to nitroxide 6, the doubly anionic nature of this molecule would further retard diffusion from brain tissue back into the vasculature. Even though the increased hydrophobicity of nitroxide 5 compared with nitroxide 1 dictated an 3-fold decrease in the concentration of nitroxides used in the pharmacokinetic studies shown in Fig. 6, still we achieved brain levels of nitroxide that should allow, based on previous studies (Shen et al., 2006), accurate estimates of O2 levels in vivo. Given that nitroxide 6 is a dianion at physiological pH, we were surprised that brain tissue levels of nitroxide 6 were not substantially higher than that found for the monoanionic nitroxide 2 (Fig. 7). The reason for this remains uncertain. Future studies are designed to prepare the [15N]perdeuterio-nitroxide 1 to determine whether this acetoxymethoxycarbonyl-containing nitroxide is an even better EPR proimaging agent than is nitroxide 1 for quantitating O2 levels in mouse brain.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: EPR, electron paramagnetic resonance; PBS, phosphate-buffered saline; mp, melting point; HBSS, Hanks' balanced salt solution; i.v., intravenous; i.a., intra-arterial; i.p., intraperitoneal.
Address correspondence to: Dr. Gerald M. Rosen, Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 725 West Lombard St., Baltimore, MD 21201. E-mail: grosen{at}umaryland.edu
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Chatani S, Nakamura M, Akahane H, Kohyama N, Taki M, Arata T, and Yamamoto Y (2005) Synthesis of C2-chiral bifunctionalised spin labels and their application to troponin C. Chem Commun 1880-1882.
Elas M, Williams BB, Parasca A, Mailer C, Pelizzari CA, Lewis MA, River JN, Karczmar GS, Barth ED, and Halpern HJ (2003) Quantitative tumor oxymetric images from 4D electron paramagnetic resonance imaging (EPRI): methodology and comparison with blood oxygen level-dependent (BOLD) MRI. Magn Reson Med 49: 682-691.[CrossRef][Medline]
Furuichi T, Liu W, Shi H, Miyake M, and Liu KJ (2005) Generation of hydrogen peroxide during brief oxygen-glucose deprivation induces preconditioning neuronal protection in primary cultured neurons. J Neurosci Res 79: 816-824.[CrossRef][Medline]
Griffeth LK, Rosen GM, Rauckman EJ, and Drayer BP (1984) Pharmacokinetics of nitroxide NMR contrast agents. Investig Radiol 19: 553-562.[CrossRef][Medline]
Halpern HJ and Bowman MK (1991) Low-frequency EPR spectrometers: MHz range, in EPR Imaging and In Vivo EPR (Eaton GR, Eaton SS, and Ohno K eds) pp 45-63, CRC Press, Inc., Boca Raton, FL.
Halpern HJ, Spencer DP, van Polen J, Bowman MK, Masssoth RJ, Nelson AC, Dowey EM, and Teicher BA (1989) An imaging radiofrequency electron spin resonance spectrometer with high resolution and sensitivity for in vivo measurements. Rev Sci Instrum 60: 1040-1050.[CrossRef]
Kao JPY and Rosen GM (2004) Esterase-assisted accumulation of 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinloxyl into lymphocytes. Org Biomol Chem 2: 99-102.[CrossRef][Medline]
Keana JFW, Pou S, and Rosen GM (1987) Nitroxides as potential contrast enhancing agents for MRI application: influence of structure on the rate of reduction by rat hepatocytes, whole liver homogenate, subcellular fractions and ascorbate. Magn Reson Med 5: 525-536.[Medline]
Khan N, Shen J, Chang TY, Chan CC, Fung PC, Grinberg O, Demidenko E, and Swartz H (2003) Plasma membrane cholesterol: a possible barrier to intracellular oxygen in normal and mutant CHO cells defective in cholesterol metabolism. Biochemistry 42: 23-29.[CrossRef][Medline]
Kutala VK, Parinandi NL, Pandian RP, and Kuppusamy P (2004) Simultaneous measurement of oxygenation in intracellular and extracellular compartments of lung microvascular endothelial cells. Antioxid Redox Signal 6: 597-603.[CrossRef][Medline]
Liu S, Timmins GS, Shi H, Gasparovic CM, and Liu KJ (2004) Application of in vivo EPR in brain research: monitoring tissue oxygenation, blood flow, and oxidative stress. NMR Biomed 17: 327-334.[CrossRef][Medline]
Mathew AE and Dodd JR (1985) Synthesis of substitute 2,2,5,5-tetramethylpyrrolidin-1-oxyl spin labels - pH sensitivity studies. J Heterocyclic Chem 22: 225-228.
Rosen GM, Burks SR, Kohr MJ, and Kao JPY (2005) Synthesis and biological testing of aminoxyls designed for long-term retention by living cells. Org Biomol Chem 3: 645-648.[CrossRef][Medline]
Rozantsev EG (1970) Free Nitroxyl Radicals, Plenum Press, New York.
Sano H, Naruse M, Matsumoto K, Oi T, and Utsumi H (2000) A new nitroxyl-probe with high retention in the brain and its application for brain imaging. Free Radic Biol Med 28: 959-969.[CrossRef][Medline]
Shen J, Khan N, Lewis LD, Armand R, Grinberg O, Demidenko E, and Swartz H (2003) Oxygen consumption rates and oxygen concentration in molt-4 cells and their mtDNA depleted (rho0) mutants. Biophys J 84: 1291-1298.[Medline]
Shen J, Liu S, Miyake M, Liu W, Pritchard A, Kao JPY, Rosen GM, Tong Y, and Liu KJ (2006) Use of 3-acetoxymethoxcarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl as an EPR oximetry probe: potential for in vivo measurement of tissue oxygenation in mouse brain. Magn Reson Med 55: 1433-1440.[CrossRef][Medline]
Tsuzuki Y, Fukumura D, Ossthuyse B. Koike C, Carmeliet P, and Jain RK (2000) Vascular endothelial growth factor (VEGF) modulation by targeting hypoxia-inducible factor-1alpha-hypoxia response element-VEGF cascade differentially regulates vascular response and growth rate in tumors. Cancer Res 60: 6248-6252.
Zauner A, Daugherty WP, Bullock MR, and Warner DS (2002) Brain oxygenation and energy metabolism: part I-biological function and pathophysiology. Neurosurgery 51: 289-301.[CrossRef][Medline]
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