High-performance liquid chromatography study of the pharmacokinetics of various spin traps for application to in vivo spin trapping

Free Radical Biology & Medicine, Vol. 27, Nos. 1/2, pp. 82– 89, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/99/$–see front matter

PII S0891-5849(99)00042-8

Original Contribution HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY STUDY OF THE PHARMACOKINETICS OF VARIOUS SPIN TRAPS FOR APPLICATION TO IN VIVO SPIN TRAPPING KE JIAN LIU,* YASHIGE KOTAKE,‡ MARGARET LEE,* MINORU MIYAKE,* KENT SUGDEN,† ZHENQIANG YU,‡ HAROLD M. SWARTZ*

and

*Departments of Radiology and †Chemistry, Dartmouth College, Hanover, NH and ‡Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA (Received 16 November 1998; Revised 15 February 1999; Accepted 15 February 1999)

Abstract—In vivo spin trapping is potentially a very useful tool to investigate the role of free radicals in physiologic processes and disease development. Unfortunately, knowledge on the stability and distribution of spin traps in living systems is limited. Therefore, in our study, we selected 11 acyclic and cyclic nitrone spin traps with diverse properties to determine their pharmacokinetics in mice. At varying times after intraperitoneal administration, we measured the concentration of the spin traps in the liver, heart, and blood. Our results showed that most spin traps were rapidly absorbed and were approximately evenly distributed throughout the mouse body. It was also found that most of the traps were relatively stable in vivo with more than half of the injected amount still available for spin trapping free radicals after an hour. Two of the 11 tested spin traps, however, decomposed after injection. These results indicate that for a successful in vivo spin trapping experiment, the stability of the spin trap is not of major concern, but the time course of distribution may be important. © 1999 Elsevier Science Inc. Keywords—Pharmacokinetics, Spin trapping, High-performance liquid chromatography, Electron paramagnetic resonance, Free radicals

INTRODUCTION

Due to their high reactivity, most biologically important free radicals are short lived and usually exist in extremely low steady state concentrations. This makes direct detection by EPR difficult. For many such short lived radicals, the spin trapping technique has often been used [2,6,7]. This technique converts the short-lived radicals into longer lived species (spin adducts) through reactions with a spin trap. It has already proven to be a useful tool in detecting free radicals, and has been widely used in chemical and biochemical studies [8]. Recently, with the development of low frequency EPR instrumentation, it has become possible to carry out spin trapping experiments in intact laboratory animals to trap free radicals in real time and at the site of formation [9 –17]. This approach complements the in vitro analysis of free radicals generated in vivo [18 –20]. Potentially, in vivo in situ spin trapping/EPR spectroscopy could provide direct information on biological processes that free radicals are involved. Therefore, in vivo spin trapping has several advantages, including: (i) noninvasively measuring the free radicals directly at the location where they evolve,

There is increasing evidence that suggests that free radicals play an important role in the development of many diseases, especially those associated with ischemiareperfusion injury and other types of uncontrolled oxidations. Free radicals are also believed to have a role in many physiologic processes, ranging from intermediates in enzyme reactions to effectors [1–5]. In addition, the toxicity of various drugs and chemicals are mediated through the generation of free radicals. Whereas the existence of free radicals in these biological processes can be inferred from end product analysis or from the effects of antioxidants or enzymes, electron paramagnetic resonance (EPR) spectroscopy allows for direct detection and extensive characterization of their generation and reactions. Address correspondence to: Dr. K. J. Liu, 7785 Vail Building, Department of Radiology, Dartmouth Medical School, Hanover, NH 03755, USA; Tel: (603) 650-1806; Fax: (603) 650-1717; E-Mail: [email protected]. 82

Pharmacokinetics of various spin traps

thereby reducing the possibility of artifacts due to sample handling and processing; (ii) monitoring free radical formation and decay in “real time;” (iii) determining if specific pathways demonstrated in vitro actually occur in vivo, where several competing free radical pathways may exist; and (iv) quantitating intermediate species. There are many factors and conditions that could make the application of in vivo spin trapping technique more successful. One of which would be the in vivo pharmacokinetics of spin traps. The knowledge of the stability and distribution of the spin trap in living systems would enable us to determine the optimal time to start or terminate the experiment, and the concentrations of spin trap needed in the tissue for sufficient detection. Despite the importance of such information, the reports in the literature regarding the stability and distribution of spin traps is limited [21–25], and no systematic approaches have been developed. The aim of the present study is to investigate the in vivo pharmacokinetics after intraperitoneal administration of several selected spin traps with particular emphasis on the kinetics of distribution and elimination. The spin traps chosen for this study were selected to represent a diverse range of physicochemical properties and selectivity toward specific free radicals, and were chosen from commercial sources or previously synthesized spin traps. If the promise of the in vivo EPR spin trapping are fulfilled, it should become possible to carry out a large number of studies bearing on significant biological problems in that the role of free radicals has been postulated. The results will help elucidate the pathogenesis of oxidative damage and help develop means to modify potentially damaging effects.

MATERIAL AND METHODS

Materials The pharmacokinetics of the following 11 spin traps were measured and their structures are shown in Fig. 1. The spin traps were selected from more than 50 nitrone compounds. Nitrones that were not selected had one or more apparently hydrophobic substituents such as halogens, alkyl groups, nitro groups, and cyano groups. PBN-type. Phenyl-N-tert-butylnitrone (PBN); (N-tbutylnitronyl)-N-tert-butylnitrone (BNBN); 2-sulfoxy phenyl-N-tert-butylnitrone sodium salt (2-SPBN); 4-methoxy phenyl-N-tert-butylnitrone (4-MOPBN); 4-(diethylamino) phenyl-N-tert-butylnitrone (4-NE2PBN), phenyl-N-(hydroxy-tert-butyl)nitrone (PBOHN). PyBN-type. 4-Pyridyl N-tert-butylnitrone (4-PyBN); 4-pyridyl N-oxide N-tert-butylnitrone (4-PyOBN).

83

DMPO-type. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO); 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO); 5-sodiumcarboxy-5-methyl pyrroline N-oxide (CMPO). PBN, DMPO, and 2-SPBN were purchased from Aldrich (Milwaukee, WI, USA), and DEPMPO was obtained from Oxis International (Portland, OR, USA). All other spin traps were originally synthesized by Dr. Edward G. Janzen and his colleagues in his published [26 –33] and unpublished studies and have been stored at the Oklahoma Medical Research Foundation. The spin trapping properties of many of the spin traps used in this study will be published separately elsewhere.

Treatment of animal and preparation of tissue samples BALB/c mice weighing 18 –20 g were obtained from Charles River Laboratories (Wilmington, MA, USA). A total of about 130 mice were used in this study. Mice were injected intraperitoneally with a spin trap that was dissolved either in saline or corn oil. At each specific time up to 120 min after injection, 0.5 ml blood was drawn into a heparinized syringe, by bleeding from the axillary vessels of the anesthetized mouse (ketamine/xylazine, 90/9 mg/kg, IP). Livers and hearts were removed, rinsed in saline, blotted, and weighed. The tissues were then homogenized with known amount of saline using a tissue grinder. Control mice were injected with saline. The homogenates or blood samples obtained from control mice were spiked with 5 ␮l of known concentration of the spin trap, and were subjected to the same extraction procedures. These extracts were used as concentration standards. Because the 11 spin traps were expected to have different water solubility, we developed three different extraction procedures. Based on the predicted physicochemical properties, we selected one of these methods to maximize the extraction efficiency. DMPO-type traps with high water solubility. The homogenate was transferred to an Eppendorf tube and centrifuged for 5 min. Fifty, twenty-five, and twenty-five microliters of 70% HClO4 was added to the supernatant from the liver, heart, and blood, respectively. After vortexing and centrifugation, the supernatant was filtered through a 0.2 ␮m syringe filter. This filtrate was injected to high-performance liquid chromatography (HPLC) to record the chromatogram. This procedure was used for DMPO, DEPMPO, and CMPO. PBN-type traps with intermediate water solubility. The homogenate was transferred to an Eppendorf tube and centrifuged for 5 min. Two hundred microliters of meth-

84

K. J. LIU et al.

Fig. 1. Chemical structure of the nitrone spin traps used in the present study.

anol was added to the 200 ␮l portion of the supernatant from the liver and heart. The solution was transferred to a Millipore Ultrafree-MC centrifugal filtration unit (Biomax-10 with 10,000 MW cutoff), and centrifuged for 30 min. The filtrate was injected to HPLC to record the chromatogram. This procedure was used for 4-PyOBN, PBOHN, 4-PyBN, and PBOHN. PBN-type traps with poor water solubility. The homogenate or blood was added with 15 ml chloroform, shaken vigorously for 5 min in a separator funnel, and allowed to stand for 5 min to separate the two phases. The chloro-

form layer was collected and the solvent was removed by evaporation to dryness under vacuum and nitrogen stream. Two milliliters of methanol/water (70:30) was added to the residue to collect the spin trap. The solution was filtered through a 0.2 ␮m syringe filter and injected to HPLC. This procedure was used for PBN, 4-MOPBN, 4-NE2PBN, and BNBN. Measurement of spin trap concentration by HPLC The spin trap tissue concentration was measured using a Hewlett-Packard (Palo Alto, CA, USA) 1090 HPLC

Pharmacokinetics of various spin traps

85

Table 1. Spin Trap Solutions Administered to Mice and HPLC Conditions for Analysis of the Concentration of the Spin Traps in Tissue

Spin traps PBN-type PBN BNBN 2-SPBN 4-MOPBN 4-NE2PBN PBOHN PyBN-type 4-PyBN 4-PyOBN DMPO-type DMPO DEPMPO CMPO

Retention time (min)

Detection wavelength (nm)

Solution administered to mouse

CH3OH/H2O ratio, flow rate (ml/min)

0.1 ml 20 mg/ml saline 0.5 ml 4 mg/ml saline 0.1 ml 20 mg/ml saline 0.1 ml 20 mg/ml saline 0.1 ml 20 mg/ml corn oil 0.15 ml 20 mg/ml saline

70:30%, 0.75 60:40%, 0.8 (decomposed in vivo) 70:30%, 0.75 75:25%, 0.8 65:35%, 0.75

4.33 4.55

295 330

4.34 5.22 5.68

305 350 290

0.1 ml 20 mg/ml saline 0.1 ml 50 mg/ml saline

45:55%, 0.75 30:70%, 0.75

6.45 6.25

290 330

0.1 ml 100 mg/ml saline 0.1 ml 100 mg/ml saline 0.1 ml 100 mg/ml saline

20:80%, 1.0 40:60%, 0.8 (decomposed in vivo)

3.46 6.07

230 235

system equipped with a photo diode array detector. The column was a Microsorb 5-mm C-18 (15 cm ⫻ 4.6 mm) column, and the injection volume was 20 ␮l. Other conditions listed in Table 1, were adjusted for each specific spin trap so that the retention time was between 4 – 8 min. For example, the conditions for DEPMPO were: mobile phase ⫽ methanol/water (40:60); flow rate ⫽ 0.8 ml/min; detection wavelength ⫽ 235 nm. Calculation of the spin trap concentration in tissue Quantification of the concentration of spin traps in tissue was based on the chromatographic peak height, that was confirmed to be linear in the range measured in this study. Blood and tissue homogenates from the control (saline treated) mice were spiked with each authentic spin trap, and then treated with the same extraction procedures. These spiked samples were used as standards for calculating the spin trap concentration in the extract, by comparing the peak heights obtained from the chromatogram. The spin trap concentration in the liver, heart, and blood was then back-calculated, by normalizing with the weight of the tissue, dilution factor, and HPLC injection volume. The concentration was expressed as microgram spin trap per gram tissue weight (␮g/g). For blood, we used the same unit (␮g/g) by assuming that 1 ml blood weighs about 1 g. The calculated concentration in the liver and heart probably contains some spin traps from the residual blood because the liver and heart was not perfused. But this error is expected to be small because the animal has been bled before the organs were removed. RESULTS

Each concentration value presented here was an average from two mice, and usually the two values were

within 20%. The small sample size is justifiable because the purpose of the study was not to measure the exact metabolic rate of each spin trap in vivo, but rather to obtain potential candidates for in vivo spin trapping applications by screening relatively large number of spin traps. By semi-quantitatively determining the trend of decay within a certain limit of error, it is hoped that this information will serve as a guideline for designing future in vivo spin trapping experiments. Figure 2 shows a typical chromatogram of the extract from homogenized mouse liver tissue. To achieve a signal from the spin trap that was well separated from the rest of the peaks, the ratio of methanol/water for the

Fig. 2. A typical HPLC chromatogram of extract of homogenized liver tissue (trace A, solid line) and a standard solution of the spin trap in saline (trace B, dash line) under the same HPLC conditions. The liver was removed, homogenized, and extracted 30 min after intraperitoneal injection of 0.15 ml of 20 mg/ml PBOHN aqueous solution into a 20-g mouse.

86

K. J. LIU et al.

mobile phase was varied. The assignment of the signal was confirmed by spiking the samples with authentic spin trap having the same retention time. To further confirm the assignment, the ultraviolet-vis absorption spectrum at the peak of the signal, obtained simultaneously by the photo diode array detection system during chromatogram data acquisition, was compared to that of a pure aqueous solution of the spin trap. The peak height of the spin trap signal was a linear function of the concentration of the spin trap under the present conditions, providing the basis for the quantitative measurement from in vivo samples. Table 1 summarizes the experimental conditions used to make these measurements. The difference in injection volume and concentration indicates the adjustments that were made to accommodate the differences in solubility and absorption/extinction coefficients of the various spin traps. Because perchloric acid is a strong oxidant that was used to deproteinize the tissue samples of DMPO-type traps, we investigated its effect on the peak height. By comparing the chromatograms of the standard aqueous solution of the spin trap with and without perchloric acid, we found no difference in the peak height. Because perchloric acid could oxidize PBN-type spin traps, however, a different extraction procedure was used, that utilizes a centrifugal filtering device retaining the concentrated protein and other molecules greater than the molecular cutoff of 10,000. To determine the stability and distribution of the spin traps in vivo, the concentration was followed in the liver, heart, and blood of mice for up to 120 min after the intraperitoneal injection. Figure 3A–3I shows the time dependence of the concentration of 9 spin traps in the heart, liver, and blood of mice. With the exception of 4-PyBN, and to some extent for BNBN and 4-PyOBN, the concentrations in the three investigated organs were very similar for most of the traps. Most spin traps tested were rapidly absorbed after intraperitoneal injection. After 15 min, that was the earliest time point we measured, the maximum concentration had already been reached for almost all acyclic and cyclic nitrones. The maximum concentration was usually close to the average concentration for the whole body, as calculated by the total dose divided by body volume, suggesting that the spin traps were relatively evenly distributed. Further, there was no evidence of rapid metabolism before the first measurement at 15 min. The results in Fig. 3 indicate that most of the spin traps were relatively stable in vivo; 60 min after injection, about 50% of the nitrones was still available in the organs to spin trap free radicals. 4-MOPBN, 4NE2PBN, and PBOHN showed greater clearance rate. Among the 11 acyclic and cyclic nitrones studied, the extracted tissue samples from 2-SPBN and CMPO did

not produce any HPLC peak at the expected retention time, whereas a new signal appeared at a different retention time. The ultraviolet-vis absorption spectrum of this new signal was also different from that of the authentic sample of 2-SPBN or CMPO. To investigate the cause of the observation, and distinguish between the two possibilities (traps being preferentially distributed in organs not measured in the study, or traps being destroyed upon in vivo administration), we mixed authentic 2-SPBN or CMPO with homogenized tissue from non-treated mice and followed the same extraction procedure for HPLC analysis. The chromatogram obtained was the same as the one for the in vivo injection, indicating that both 2-SPBN and CMPO are not stable in vivo, and are rapidly destroyed upon injection. The mechanism of this instability was not investigated. DISCUSSION

The results from the present study indicate that most of the spin traps were rapidly taken up by the animal, and were stable in vivo after intraperitoneal administration. Apart from a few exceptions, notably 4-PyBN, 4-PyOBN, and BNBN, the spin traps investigated in this study distribute relatively evenly in the liver, heart, and blood. Also, most spin traps had a significant amount still available in the living tissue 1 h after injection, and for some spin traps, such as BNBN, 4-PyBN, and DMPO, significant amounts remained after 2 h. The mechanism of the decrease of concentration over time was not investigated in this study, but it is likely to be due to excretion. The results indicate that the optimal timing for spin trapping free radicals is likely to be within 30 min after administration. It can also be concluded that for successful trapping and detection of free radicals, the bottleneck is the rapid decay of the spin adducts, and not the stability of the spin traps. Increasing the lifetime of the spin adduct, or maintaining the concentration of the spin adducts at detectable levels, will be the key factors in future development of spin trapping in intact animals. The 11 acyclic and cyclic nitrones we selected for this study had very different water solubility and chemical stability in vivo. To obtain a higher extraction efficiency and to recover the traps in the tissue without damaging them, we developed three different extraction procedures to accommodate the diverse properties of the traps. One of the important procedures in detection is deproteinizing that reduces the interference in HPLC analysis. Perchloric acid often is used to deproteinize biological samples, but it is a strong oxidizing agent that could oxidize the spin traps. DMPO-type spin traps were found to be resistant to perchloric acid treatment and it was therefore appropriate to use perchloric acid when extracting

Pharmacokinetics of various spin traps

Fig. 3. Stability and distribution of various spin traps in vivo. At each specific time after intraperitoneal injection, the animals were killed, and organs were removed and homogenized. The concentrations of the spin traps in the liver, heart, and blood of mice were measured using HPLC. Each data point was the average of two mice.

87

88

K. J. LIU et al.

DMPO spin traps. Perchloric acid was not used for PBN-type traps because it oxidized and decomposed them. Consequently, a centrifugal filtering device was employed to reduce the protein content in these aqueous extract solutions. For lipophilic PBN-type traps, extraction with chloroform was efficient, achieving an extraction efficiency better than 95%. This number is based on the calculation from the distribution of the traps in the organic phase using the data on the octanol-water partition coefficients [34]. Because of their diverse water solubility, the mobile phase for the HPLC analysis had to be adjusted so that retention times for each spin trap fell in a convenient range when we used a single C-18 column. Two of the 11 nitrones investigated in the study, 2-SPBN and CMPO, decomposed after injection into the mouse. The HPLC chromatogram of the tissue extract had a peak at a different retention time than the authentic spin trap. Both 2-SPBN and CMPO were found to have been destroyed when they were mixed with homogenized liver and heart samples (data not shown), indicating that they are readily metabolized or decomposed by the tissue. This indicates that the synthesized spin trap CMPO may not be useful in vivo, and that the selection of spin traps for in vivo applications should not be purely based on results from chemical studies [35–37]. This could be particularly important when the chemical study indicates a good stability of its spin adducts. A case in point is CMPO, that has been shown to produce a CMPO/•OH adduct with a life time longer than DMPO/ • OH in aqueous solution (our unpublished results). An alternative explanation for the disappearance of the expected peaks in the chromatogram for 2-SPBN and CMPO is the possible conjugation of the spin traps, causing the change of the retention time in the chromatogram. This change of retention time, however, was only observed in the extraction from animal tissue, but not from standard samples in saline. Whether the “altered” spin traps, either decomposed or conjugated, would maintain their function of trapping free radicals is unknown, and was not investigated. The spin traps with relatively poor water solubility (such as 4-NE2PBN) were efficiently taken up after injection, suggesting that hydrophobic spin traps could be useful if appropriate vehicles are used. Therefore, we plan to test hydrophobic spin traps that were omitted from the present study. In conclusion, the present study demonstrates that most of the spin traps that were selected are reasonably stable in vivo, and are evenly distributed in the liver, heart, and blood. Therefore in vivo spin trapping experiments in biological systems can be designed with confidence of the availability of adequate levels of these spin traps.

Acknowledgement — We are grateful to Dr. Edward G. Janzen for allowing us to use spin traps that were synthesized by his group at the University of Georgia, the University of Guelph, and the Oklahoma Medical Research Foundation. This research is supported in part by a grant from National Institutes of Health (R21 HL60623) and the Animal Gift Program of Charles River Laboratory. This research also used the facilities of the EPR Center for the Study of Viable Systems at Dartmouth that is supported by the National Institutes of Health Grant P41 RR11602. The authors thank Dr. G. M. Rosen for many helpful discussion and comments of the manuscript.

REFERENCE [1] Stohs, S. J. The role of free radicals in toxicity and disease. J. Basic Clin. Physiol. Pharmacol. 6:205–228; 1995. [2] Aust, S. D.; Chignell, C. F.; Bray, T. M.; Kalyanaraman, B.; Mason, R. P. Free radicals in toxicology. Toxicol. Appl. Pharmacol. 120:168 –178; 1993. [3] Halliwell, B. Mechanisms involved in the generation of free radicals. Pathol. Biol. 44:6 –13; 1996. [4] Knight, J. A. Diseases related to oxygen-derived free radicals. Ann. Clin. Lab. Sci. 25:111–121; 1995. [5] Gutteridge, J. M. Free radicals in disease processes: a compilation of cause and consequence. Free Radic. Res. Commun. 19:141– 158; 1993. [6] Mason, R. P.; Knecht, K. T. In vivo detection of radical adducts by electron spin resonance. Methods Enzymol. 233:112–117; 1994. [7] Janzen, E. G. Spin trapping. Acc. Chem. Res. 4:31– 40; 1971. [8] Janzen, E. G.; Haire, D. L. Two decades of spin trapping. In: Tanner. D. D., ed. Advances in free radical chemistry. Greenwich, CT: JAI Press Inc., 1990:253–295. [9] Jiang, J.; Liu, K. J.; Shi, X.; Swartz, H. M. Detection of shortlived free radicals by low-frequency electron paramagnetic resonance spin trapping in whole living animals. Arch. Biochem. Biophys. 319:570 –573; 1995. [10] Jiang, J.; Liu, K. J.; Joran, S. J.; Swartz, H. M.; Mason, R. P.; Detection of free radical metabolite formation using in vivo EPR spectroscopy: evidence of rat hemoglobin thiyl radical formation following phenylhydrazine administration. Arch. Biochem. Biophys. 330:266 –270; 1996. [11] Liu, K. J.; Jiang, J.; Swartz, H. M.; Shi, X. Low-frequency EPR detection of chromium(V) formation by chromium(VI) reduction in whole live mice. Arch. Biochem. Biophys. 313:248 –252; 1994. [12] Liu, K. J.; Shi, X.; Jiang, J. J.; Goda, F.; Dalal, N.; Swartz, H. M. Chromate-induced chromium(V) formation in live mice and its control by cellular antioxidants: an L-band electron paramagnetic resonance study. Arch. Biochem. Biophys. 323:33–39; 1995. [13] Halpern, H. J.; Yu, C.; Barth, E.; Peric, M.; Rosen, G. M. In situ detection, by spin trapping, of hydroxyl radical markers produced from ionizing radiation in the tumor of a living mouse. Proc. Natl. Acad. Sci. USA 92:796 – 800; 1995. [14] Fujii, H.; Zhao, B.; Koscielniak, J. Berliner, L. J. In vivo EPR studies of the metabolic fate of nitrosobenzene in the mouse. Magn. Reson. Med. 31:77– 80; 1994. [15] Mader, K.; Bacic, G.; Swartz, H. M. In vivo detection of anthralin-derived free radicals in the skin of hairless mice by lowfrequency electron paramagnetic resonance spectroscopy. J. Invest. Dermatol. 104:514 –517; 1995. [16] Xia, Y.; Khatchikian, G.; Zweier, J. L. Adenosine deaminase inhibition prevents free radical-mediated injury in the postischemic heart. J. Biol. Chem. 271:10096 –10102; 1996. [17] Yoshimura, T.; Yokoyama, H.; Fujii, S.; Takayama, F.; Oikawa, K.; Kamada, H. In vivo EPR detection and imaging of endogenous nitric oxide in lipopolysaccharide-treated mice. Nature Biotechnol. 14:992–994; 1996. [18] Burkitt, M. J.; Mason, R. P. Direct evidence for in vivo hydroxylradical generation in experimental iron overload: an ESR spintrapping investigation. Proc. Natl. Acad. Sci. USA 88:8440 – 8444; 1991. [19] Reinke, L. A.; Towner, R. A.; Janzen, E. G. Spin trapping of free radical metabolites of carbon tetrachloride in vitro and in vivo:

Pharmacokinetics of various spin traps

[20] [21]

[22] [23]

[24] [25]

[26]

[27] [28]

effect of acute ethanol administration. Toxicol. Appl. Pharmacol. 112:17–23; 1992. Knecht, K. T.; Mason, R. P. In vivo spin trapping of xenobiotic free radical metabolites. Arch. Biochem. Biophys. 303:185–194; 1993. Cheng, H. Y.; Liu, T.; Feuerstein, G.; Barone, F. C. Distribution of spin-trapping compounds in rat blood and brain: in vivo microdialysis determination. Free Radic. Biol. Med. 14:243–250; 1993. Knecht, K. T.; Mason, R. P. The detection of halocarbon-derived radical adducts in bile and liver of rats. Drug. Metab. Dispos. 19:325–331; 1991. Chen, G.; Griffin, M.; Poyer, J. L.; McCay, P. B. HPLC procedure for the pharmacokinetic study of the spin-trapping agent, alphaphenyl-N-tert-butyl nitrone (PBN). Free Radic. Biol. Med. 9:93– 98; 1990. Liu, K. J.; Jiang, J.; Ji, L. L.; Shi, X.; Swartz, H. M. An HPLC and EPR investigation on the stability of DMPO and DMPO spin adducts in vivo. Res. Chem. Intermediates 22:499 –509; 1996. Rosen, G. M.; Halpern, H. J.; Brunsting, L. A.; Spencer, D. P.; Strauss, K. E.; Bowman, M. K.; Wechsler, A. S. Direct measurement of nitroxide pharmacokinetics in isolated hearts situated in a low-frequency electron spin resonance spectrometer: implications for spin trapping and in vivo oxymetry. Proc. Natl. Acad. Sci. USA 85:7772–7776; 1988. Janzen, E. G.; Blackburn, B. J. Detection and identification of short-lived free-radicals by electron spin resonance trapping techniques (spin trapping). Photolysis of organolead, tin and mercury compounds. J. Am. Chem. Soc. 91:4481– 4490; 1969. Janzen, E. G.; Liu, J-P. Radical addition reactions of 5,5-dimethyl-1-pyrroline-1-oxide. ESR spin trapping with a cyclic nitrone. J. Magn. Reson. 9:510 –512; 1973. Janzen, E. G.; Zawalski, R. C. Synthesis of nitronyl alcohols and their benzoate esters. J. Org. Chem. 43:1900; 1978.

89

[29] Janzen, E. G.; Wang, Y. Y.; Shetty, R.V. Spin trapping with a-pyridyl-N-oxide N-t-butyl nitrones in aqueous solutions. A unique ESR spectrum for the hydroxyl radical adduct. J. Am. Chem. Soc. 100:2923; 1978. [30] Janzen, E. G.; Dudley, R. L.; Shetty, R. V. Synthesis and electron spin resonance chemistry of nitronyl labels for spin trapping. ␣-Phenyl N-[5-(5-methyl-2,2-dialkyl-1,3-dioxanyl)] nitrones and ␣-(N-alkylpyridinium) N-(tert)-butyl) nitrones. J. Am. Chem. Soc. 101:243; 1979. [31] Janzen, E. G.; Shetty, R. V. Spin trapping chemistry of sodium 2-sulfonatophenyl t-butyl nitrone (Na⫹ 2-SPBN). A negatively charged water-soluble spin trap. Tetrahedron Lett. 35:3229 – 3232; 1979. [32] Janzen, E. G.; Shetty, R. V.; Kunanec, S. M. Spin trapping chemistry of 3,3,5,5-tetramethylpyrroline-N-oxide. An improved cyclic spin trap. Can. J. Chem. 59:756 –758; 1981. [33] Hinton, R. D.; Janzen, E. G. Synthesis and characterization of phenyl substituted C-phenyl-N-tert-butylnitrones and some of their radical adducts. EPR parameters for hydroxyl and hydroperoxyl spin adducts. J. Org. Chem. 57:2646 –2651; 1992. [34] Janzen, E. G.; West, M. S.; Kotake, Y.; DuBose, C. M. Biological spin trapping methodology. III. Octanol-water partition coefficients of spin-trapping compounds. J. Biochem. Biophys. Methods 32:183–190; 1996. [35] Kotake, Y.; Janzen, E. G. Decay and fate of the hydroxyl radical adduct of a-phenyl-N-tert-butylnitrone in aqueous media. J. Am. Chem. Soc. 113:9503–9506; 1991. [36] Janzen, E. G.; Kotake, Y.; Hinton, R. D. Stabilities of hydroxyl radical spin adducts of PBN-type spin traps. Free Radic. Biol. Med. 12:169 –173; 1992. [37] Janzen, E. G.; Hinton, R. D.; Kotake, Y. Substituent effect on the stability of the hydroxyl adduct of a-phenyl N-butyl nitrone (PBN). Tetrahedron Lett. 33:1257–1260; 1992.