66 DETECTION OF NITRIC OXIDE [6]

chelating agent. Also, although it is widely believed that only NO can react with MGD and DETC, reports suggest that several NO metabolites (such as nitroxyl anion) can react with MGD. 44-46

C o n c l u s i o n s

With proper precautions in the use of the technique, combined with the selection of appropriate methods, EPR spectroscopy provides a very effective method for the simultaneous measurement of oxygen and nitric oxide in functioning biological systems, both in vitro and in vivo. This approach is especially valuable when repeated measurements are needed.

A c k n o w l e d g e m e n t

The authors acknowledge the Biomedical Technology Research Center at the EPR Center for Viable Systems at Dartmouth Medical School, supported by the National Center for Research Resources, NIH Grant P41 RR11602, Hanover, New Hampshire, and NIH Grant P01 GM51630. The Department of Cardiology, W.H.R.I., is supported by the British Heart Foundation.

44 K. Tsuchiya, M. Yoshizumi, H. Houchi, and R. Mason, J. Biol. Chem. 275, 1551 (2000). 45 y. Xia, A. J. Cardounel, A. E Vanin, and J. L. Zweier, Free Radic. Biol. Med. 29, 793 (2000). 46 A. M. Komarov, D. A. Wink, M. Feelish, and H. H. Schmidt, Free Radic. Biol. Med. 28, 739 (2000).

[6] Electron Paramagnetic Resonance Studies of Nitric Oxide in Living Mice

By ANDREI M. KOMAROV

I n t r o d u c t i o n

Electron paramagnetic resonance (EPR) observation in small living animals is feasible only for stable paramagnetic products, and the problem of free radical stabilization does not have a generic solution. Sulfur ligands are known to stabilize nitrosyliron complexes.l,2 Vanin and co-workers 3 introduced diethyldithiocarba- mate (DETC) as a precursor forming the lipophilic DETC-Fe-nitric oxide (NO) complex in animal tissues. However, DETC forms insoluble complexes with iron

1 j. F. Gibson, Nature (Lond.) 196, 64 (1962). 2 C. C. McDonald, W. D. Phillips, and H. E Mower, J. Am. Chem. Soc. 87, 3319 (1965). 3 L. N. Kubrina, W. S. Caldwell, P. I. Mordvintcev, I. V. Malenkova, and A. E Vanin, Biochim. Biophys.

Acta 1099, 233 (1992).

Copyright 2002, Elsevier Science (USA). All rights reserved.

METHODS IN ENZYMOLOGY, VOL. 359 0076-6879/02 $35.00

[61 In Vivo EPR OF NITRIC OXIDE 67

and therefore requires a separate iron injection to produce in vivo EPR-visible nitrosyliron complex. This problem has been solved using a water-soluble com- plex of N-methyl-D-glucamine dithiocarbamate (MGD) and iron (MGD-Fe) for EPR detection of endogenous nitric oxide in the circulating tail blood of live con- scious m i c e . 4-6 Dithiocarbamate NO traps yield an intense three-line EPR signal at ambient temperatures, thus allowing the study of in situ and real-time nitric oxide generation and spatial distribution of NO in small living animals and isolated organs. 7-11 This article describes a method for tracing NO metabolism and distri- bution in endotoxin-treated mice using in vivo low-frequency EPR spectroscopy in combination with an extracellular MGD-Fe nitric oxide trapping complex.

P r o p e r t i e s of D i t h l o c a r b a m a t e - F e C o m p l e x Re l ev an t to in Vivo NO D e t e c t i o n

1. Bis(dithiocarbamato)nitrosyliron(II) complexes12 are tetrasulfur complexes of iron(II) coordinated to a nitrogen atom of nitrogen monoxide. This square pyra- midal complex contains a single unpaired electron and a low-spin iron (S = 1/2) in the formal oxidation state Fe(I), d 7.13 The MGD-Fe-NO complex displays EPR spectrum consisting of three lines (g = 2.04 and aN = 12.5 G) at room tempera- ture due to the interaction of an unpaired electron with the 14N nucleus (nuclear spin ---- 1). 4 The MGD-Fe-15NO complex yields a two-line EPR pattern (g = 2.04 and aN = 17.6 G) characteristic for the 15N isotope (nuclear spin = 1/2). 14

2. NO and the water-soluble dithiocarbamate-Fe complex react with a rate constant 15-17 from 106 to 108 M -1 s -1 (the rate constant of NO scavenging by

4 A. Komarov, D. Mattson, M. M. Jones, P. K. Singh, and C.-S. Lai, Biochem. Biophys. Res. Commun. 195, 1191 (1993).

5 C.-S. Lai and A. M. Komarov, FEBSLett. 345, 120 (1994). 6 C.-S. Lai and A. M. Komarov, in "Bioradicals Detected by ESR Spectroscopy" (H. Ohya-Nishiguchi

and L. Packer, eds.), p. 163. Birkhauser Verlag, Basel, 1995. 7 V. Quaresima, H. Takehara, K. Tsushima, M. Ferrari, and H. Utsumi, Biochem. Biophys. Res.

Commun. 221, 729 (1996). 8 H. Fujii, J. Koscielniak, and L. J. Berliner, Magn. Reson. Med. 38, 565 (1997). 9 Z. Yohimura, H. Yokoyama, S. Fujii, E Takayama, K. Oikawa, and H. Kamada, Nature Biotechnol.

14, 992 (1996). lo A. M. Komarov, Cell. Mol. Biol. 46, 1329 (2000). ll p. Kuppusamy, E Wang, A. Samoilov, and J. L. Zweier, Magn. Reson. Med. 36, 212 (1996). 12 G. A. Brewer, R. J. Butcher, B. Letafat, and E. Sinn, lnorg. Chem. 22, 371 (1983). 13 R. J. Butcher and E. Sinn, lnorg. Chem. 19, 3622 (1980). 14 y. Kotake, T. Tanigawa, M. Tanigawa, and I. Ueno, Free Radic. Res. 23, 287 (1995). 15 S. Pou, E Tsai, S. Porasuphatana, H. Halpern, G. V. R. Chandramouli, E. D. Barth, and G. M. Rosen,

Biochim. Biophys. Actu 1427, 216 (1999). 16 S. V. Paschenko, V. V. Khramtsov, M. E Skatchkov, V. E Plysnin, and E. Bassenge, Biochem.

Biophys. Res. Commun. 225, 577 (1996). 17 V. Misik and E Riesz, J. Phys. Chem. 1@t), 17986 (1996).

68 DETECTION OF NITRIC OXIDE [61

oxyhemoglobin is 3 x 107 M -1 s -1, and rapid reactions of NO with other radical species are in the range of 109-10 l° M-is- l) . 18 In vivo NO trapping efficiency of the MGD-Fe complex is 50% or higher based on the attenuation of the plasma nitrate/nitrite level following MGD-Fe complex injection in endotoxin-treated mice. 19 The partition coefficients of MGD-Fe and MGD-Fe-NO complexes in octanol/water mixtures are 0.01 and 0.001, respectively. 11 Therefore, based on their physical properties, these complexes should be confined to intravascular and extracellular aqueous compartments. Note that extracellular MGD-Fe and intra- cellular DETC-Fe complexes bind NO equally in isolated ischemic myocardium when they are applied at the same dose. 2° Such a comparison in vivo is complicated by their potentially different clearance rate and distribution.

3. MGD cannot use tissue iron and should be supplemented with exogenous iron. 21 DETC chelates intracellular-free iron in most tissues to yield lipophilic DETC-Fe "traps" However, in brain tissue, 22'23 isolated myocardium, 2° or for in vivo EPR studies, 7 DETC requires a separate subcutaneous injection of the Fe- citrate complex (135/zmol/kg of FeSO4 plus 0.64 mmol/kg of sodium citrate) 24 to produce the EPR-visible DETC-Fe-NO complex. Note that iron supplementation enhances the in vivo NO trapping efficiency of dithiocarbamate, thus increasing the EPR signal of NO complexes, but it does not change nitric oxide production in tissue when it is given after iNOS activation [i.e., 6 hr after lipopolysaccharide (LPS)]. 21

E x p e r i m e n t a l P r o c e d u r e s

Materials

MGD and N6-monomethyl-L-arginine (NMMA) can be obtained from Calbiochem (San Diego, CA). The powder form of MGD should be sealed under ni- trogen gas and stored desiccated at 4°. 25 15N2-guanidino-L-arginine (15N-arginine) is purchased from Cambridge Isotope Laboratories (Wobum, MA). Sources of other chemicals are specified in the procedure.

18 D. A. Wink, M. B. Grisham, J. B. Mitchell, and E C. Ford, Methods Enzymol. 268, 12 (1996). 19 A. M. Komarov and C.-S. Lai, Biochim. Biophys. Acta 1272, 29 (1995). 20 A. M. Komarov, J. H. Kramer, I. T. Mak, and W. B. Weglicki, Mol. Cell. Biochem. 175, 91 (1997). 21 A. M. Komarov, I. T. Mak, and W. B. Weglicki, Biochim. Biophys. Acta 1361, 229 (1997). 22 V. D. Mikoyan, N. V. Voevodskaya, L. N. Kubrina, I. V. Malenkova, and A. E Vanin, Biochim.

Biophys. Acta 1269, 19 (1995). 23 V. D. Mikoyan, L. N. Kubrina, and A. E Vanin, Biofizika 39, 915 (1994). 24 L. N. Kubrina, V. D. Mikoyan, P. I. Mordvintcev, and A. E Vanin, Biochim. Biophys. Acta 1176,

240 (1993). 25 L. A. Shinobu, S. C. Jones, and M. M. Jones, Acta PharmacoL Toxicol. 54, 189 (1984).

[6] In Vivo EPR OF NITRIC OXIDE 69

Animal Procedures

The L-band (1.1 GHz) EPR experiment is performed with 15- to 20-g female ICR mice (Harlan Sprague-Dawley, Indianapolis, IN). The size of animals is re- stricted by the requirements of L-band EPR measurement (lowering the microwave frequency will allow the use of animals up to 30 g). Mice weighing 25-30 g are suitable for the S-band (3.5 GHz) EPR experiment and are used in our measure- ments taken from the murine tall. Each mouse is given 4 mg of LPS (Escherichia coli 026:B6 from Sigma, St. Louis, MO) via the lateral tail vein (note that none of the LPS-treated mice survive over 24 hr). At 6 hr, VEA mice are anesthetized with methoxyflurane (Pittman-Moore, Mundelein, IL) and then injected subcuta- neously with 0.4 ml of the MGD-Fe in water (326 mg/kg MGD and 34 mg/kg of FeSO4). 5 Over time, MGD, like other dithiocarbamate derivatives, decomposes in aqueous solution, yielding toxic carbon disulfide. 26 In addition, the iron incor- porated in the MGD-Fe complex can oxidize and precipitate in the air-saturated solution. Thus, it is important to prepare fresh MGD-Fe complex in deoxygenated water (yellow color of solution) maintaining a 5 : 1 MGD-to-iron ratio and aerate it just before injection (brown-colored solution). Measuring absorption at 340, 385, and 520 nm (extinction coefficients 20,000, 15,000, and 3000 M -l, respectively) could discover the presence of Fe(III) in the MGD-Fe complex. This could also be done by EPR of frozen aqueous solutions (77K) at g = 4.3 (characteristic EPR signal of ferric iron.) 27 Note that the administration of anaerobic MGD-Fe solu- tion to animals 28 is less desirable, as the exposure to oxygen (and generation of reactive species) will occur in vivo. Using mixtures of MGD-Fe and ascorbate to keep the iron in a reduced state is also prone to artifacts, as ascorbate reacts with nitrite to release NO. Note that EPR-visible MGD-Fe-NO complexes can be prepared using Fe(III) salts as an iron source. 29 This may be due to reduction of the EPR-silent Fe(III)-NO complex to a paramagnetic Fe(II)-No derivative as is the case with other nitrosyliron complexes. 27 Therefore, in vivo application of Fe(III) NO traps is possible, but it will lead to the loss of reductants such as ascorbate and thiols. Normal mice will tolerate four separate aliquots (0.4 ml each) of MGD-Fe solution prepared as described earlier and injected subcutaneously over 6 hr. 6 For measurement of 15NO production, animals receive a subcutaneous injection of 15N-arginine (10 mg per mouse in saline) and the MGD-Fe injection. For inhibi- tion experiments, at 6 hr after LPS treatment, mice are injected intraperitoneally with an aliquot of NMMA (50 mg/kg in saline).

26 To Martens, D. Langevin-Bermond, and M. B. Fleury, J. Pharmaceut. Sci. 82, 379 (1993). 27 A. E Vanin, X. Liu, A. Samouilov, R. A. Stukan, and J. L. Zweier, Biochim. Biophys. Acta 1474,

365 (2000). z8 T. Miyajima and Y. Kotake, Biochem. Biophys. Res. Commun. 215, 114 (1995). 29 S. Fujii, T. Yoshimura, and H. Kamada, Chem. Lett. 9, 785 (1996).

70 DETECTION OF NITRIC OXIDE [6]

Measurement of NO in Circulating Blood

Immediately after MGD-Fe complex injection, the mouse still resting in the plexiglass-restraining tube (24-mm diameter, with ventilation holes in the animal chamber) is transferred to a specially built platform on the S-band EPR spectrome- ter. No anesthetic agent is used and the animal remains conscious without apparent discomfort throughout the entire in vivo S-band EPR measurement. The tail of the animal is immobilized by taping it to a thin plexiglass rod and is then placed in- side a horizontally oriented loop-gap resonator. A 4-mm loop loop-gap resonator operating at 3.5 GHz 3° is recommended for this in vivo experiment because the tip of the mouse tail (2-3 mm) fits well into the diameter of the resonator. The measured unloaded Q of the empty resonator is 3000 and the loaded Q is 400 (with the presence of the mouse tail). 19 EPR spectra are recorded at room temperature using an EPR spectrometer equipped with an S-band microwave bridge. The field modulation amplitude is calibrated using Fremy salt as a standard. Instrument settings for this experiment are as follows: 100 G field scan, 30 sec acquisition time, 0.1 sec time constant, 2.5 G modulation amplitude, 100 KHz modulation fre- quency, and 25 mW microwave power (each of the spectra recorded is an average of nine individual 30-sec scans).

Measurement of NO in the Murine Body

Immediately after MGD-Fe complex injection, mice are put into a plexiglass- restraining tube for 1 hr, anesthetized with an intraperitoneal injection of Inactin, and placed in a disposable plastic holder inside the loop-gap L-band (1.1 GHz) resonator with a diameter of 25 mm (Fig. 1). 31 Note that anesthetized endotoxin- treated mice are more likely to die during EPR experiments than conscious animals. EPR measurements are carried out at room temperature using an L-band 1- to 2-GHz microwave bridge interfaced to an EPR spectrometer. Instrument settings for this experiment include 84 G field scan, 120 sec acquisition time, 1 sec time constant, 8 G modulation amplitude, 100 KHz modulation frequency, and incident power up to 500 mW (each spectrum is obtained from an average of four individual 120-sec scans). The modulation amplitude suggested for this experiment (8 G) is close to optimal and allows in vivo EPR signal measurement from head to tail. It should be noted that the peak-to-peak line width of the EPR signal for M G D - F e - NO is close to 4.0 G in solution, 14 but in tissue it can increase 50% or more. 2° We recommend reducing modulation amplitude to 4.0 G to observe a clear separation of hyperfine components for 14N and 15N isotopes in vivo.

3o W. Froncisz and J. S. Hyde, J. Magn. Reson. 47, 515 (1982). 31 W. Froncisz, T. Oles, and J. S. Hyde, J. Magn. Reson. 82, 109 (1989).

[6] In Vivo EPR OF NITRIC OXn3E 71

SMA microwave Coupling Coupling connector gears Modulation

~io just / Rotating I coils o I--1 / J coupling , . I k f--I / • ,~t= I rm _ _ / dipole ~ \p , , I I ,,=. I

brass __~"1 . . . . . . . . . . . . . . . . ~1 resonator ~ F ~ ~ I I

( - - I I I ~ [ ] [ ] [ ] I~:i . .-f i" I / protective I ~ / " % , \ 11\\\ I Exch'ange i l I -FIRR r- i J tube i ,' , elll

coil connector fiberglass " shield

FIG. 1. Murine L-band EPR loop-gap resonator for in vivo studies. Reproduced with permission from W. Froncisz, T. Oles, and J. S. Hyde, J. Magn. Reson. 82, 109 (1982).

Ex Vivo Measurement o f Nitric Oxide Levels

Some animals are sacrificed 2 hr after M G D - F e complex injection. Wet tis- sue samples, including whole blood, liver, and kidney tissue, are transferred into a quartz tube (i.d., 2 mm) for EPR measurement at room temperature. Urine samples are collected from the urinary bladder and are transferred immediately to a quartz fiat cell for X-band EPR measurement. The urine sample is dark brown in color, characteristic of the presence of the M G D - F e complex. 19 The M G D - F e - N O com- plex in the urine is found to be stable at 4 ° for several hours. Reducing agents such as dithionite and ascorbate have been used ex vivo to convert diamagnetic EPR- silent derivatives of the dithiocarbamate-nitrosyliron complex into paramagnetic NO complexes. 32 Note, however, that the same procedure may generate NO from nitrite present in the sample. 33 Spectra are recorded at 22 ° with an X-band EPR spectrometer operating at 9.5 GHz. Instrument settings for this experiment include 100 G field scan, 4 rain acquisition time, 0.5 sec time constant, 2.5 G modulation amplitude, 100 KHz modulation frequency, and 100 mW microwave power. We recommend reducing the microwave power to 30 mW to observe clear separation

32 V. D. Mikoyan, L. N. Kubrina, V. A. Serezhenkov, R. A. Stukan, and A. E Vanin, Biochim. Biophys. Acta 1336, 225 (1997).

33 K. Tsuchiya, M. Takasugi, K. Minakuchi, and K. Fukuzawa, Free Radic. Biol. Med. 21, 733 (1996).

72 DETECTION OF NITRIC OXIDE [6]

lOG I I

FIG. 2. In vivo 1.1-GHz EPR spectra of the MGD-Fe-NO complex in LPS-treated mice. Mice are injected with 0.4 ml of the MGD-Fe complex (a) or the MGD-Fe complex plus NMMA (b) 6 hr after LPS administration. The instrumental settings are as described in the text. Each spectrum presented here is an average of four 120-see scans. The MGD-Fe-NO complex is formed in the liver by NO produced from endogenous L-arginine. Reproduced with permission from A. M. Komarov, Cell. Mol. Biol. 46, 1329 (2000).

of hyperfine components for I4N and 15N isotopes in the urine sample. 19 Note that the "background signal" of dithiocarbamate-Cu(II) chelates may complicate X-band EPR spectra of NO complexes in tissues and liquid samples. 19,34 MGD forms water-soluble complexes with Cu(II), t9 but it does not form complexes with tissue Cu(II), unlike DETC. 2°,21

E x a m p l e s of NO D e t e c t i o n in Living Mice

In Vivo EPR Detection of NO Generated from Endogenous L-Arginine

An intensive three-line EPR signal from the MGD-Fe-NO complex in the upper abdomen (liver region) of endotoxin-treated mice is detected by in vivo L-band EPR spectroscopy (Fig. 2a). The EPR spectral parameters of the sig- nal are identical to those of the MGD-Fe-NO complex in an aqueous solution of NO (g=2.04 and aN= 12.5 G). 4'10 Note that the in vivo EPR signal in the upper abdomen of endotoxin-treated mice is mostly due to the MGD-Fe-NO complex formed in situ in the liver tissue of live animals (except the contribu- tion from the blood signal ~7%). It is likely that the MGD-Fe-NO complex formed in the murine liver is excreted and concentrated in the bile, as is the case in rat. 35

34 y. Suzuki, S. Fujii, T. Torninaga, T. Yoshimoto, T. Yoshimura, and H. Kamada, Biochim. Biophys. Acta 1335, 242 (1997).

35 L. A. Reinke, D. R. Moore, and Y. Kotake, Anal. Biochem. 243, 8 (1996).

[6] In Vivo EPR OF NITRIC OXIDE 73

Administrat ion of N M M A (50 mg/kg) in septic shock mice decreases the level of the M G D - F e - N O complex in the upper abdomen below the L-band EPR detectabili ty limit (Fig. 2b). Ex vivo examination of isolated tissues and urine shows, however, that NO complex formation is not completely abolished by N M M A administration. 19 This may point to the possibil i ty of the secondary

Cnonenzymatic") NO formation from nitrite.

In Vivo EPR Detection of l5NO Generated from 15N-Arginine

In endotoxin-treated mice, 15N-labeled L-arginine competes with the natural analog, producing a mixture of nitric oxide species (15NO and IaNO). Figure 3a exhibits an in vivo EPR signal (Fig. 3a) obtained from the upper abdomen of the septic shock mouse after administrating 15N-arginine. The EPR doublet (charac- teristic of 15N) of the MGD-Fe-15NO complex indicates the origin of nitric oxide from one of the guanidino nitrogens of 15N-labeled L-arginine. The EPR triplet of

a . /

lOG I I

FIG. 3. In vivo 1.1-GHz EPR spectra of MGD-Fe-15NO and MGD-Fe-14NO complexes detected in various regions of the body in LPS-treated mice. Mice are injected with 0.4 ml of the MGD-Fe complex and 10 mg of 15N-arginine, and EPR spectra are recorded from the (a) liver region, (b) head region, and (c) lower abdomen region of the body. The instrumental settings are as described in the text. Each spectrum presented is an average of four 120-sec scans, and the receiver gain is the same as for Fig. 2. Note that the doublet EPR pattern is arising from the MGD-Fe-15NO complex, whereas the MGD-Fe-14NO complex is contributing to the signal external "shoulders" and small center field peak. Reproduced with permission from A. M. Komarov, Ceil Mol. Biol. 46, 1329 (2000).

74 DETECTION OF NITRIC OXIDE [61

the MGD-Fe-14NO complex (i.e., the complex formed by nitric oxide originated from endogenous L-arginine) contributes external "shoulders" and a small center- field peak in the EPR spectrum (Fig. 3a). In vivo, the most intense EPR signal is found in the upper abdomen, which is consistent with the strongest signal having been seen in liver ex vivo. Figures 3b and 3c show EPR signals from the head and lower abdomen regions of live septic shock mice that received 15N-arginine; the two-line pattern of the MGD-Fe-15NO complex is evident. Note that there is a pronounced in vivo EPR signal in the head region ('-~60% of the signal intensity in the upper abdomen area). Because the water-soluble MGD-Fe complex is not likely to cross the blood-brain barrier, 36 the circulating MGD-Fe-NO complex may be contributing to the signal found in the head region (Fig. 3b). Nonetheless, the source of NO in the circulating MGD-Fe-NO complexes may be brain tissue where nitric oxide is generated locally by iNOS. 37

EPR signals from the kidney and urinary bladder containing urine are the most likely contributors to the in vivo EPR spectrum in the lower abdomen area (Fig. 3c).

C o n c l u s i o n

In vivo EPR spectroscopy and the isotopic tracing experiment with 15N-arginine show widespread activation of NO generation in septic shock mice. It is important to emphasize that the NO level detected in the mouse body by the EPR technique may not represent the true "free" NO level existing at any given moment, but rather that NO is trapped and stabilized by the NO-trapping reagent in m i c e . 19 Note that all redox forms of NO (NO +, NO, NO-) in vivo may contribute to MGD-Fe-NO complex, as in aerobic conditions the trapping agent is not selective for redox species of N O . 38'39 Note also that the direct reaction of the MGD-Fe trap with nitrite 4° is not likely the source of the MGD-Fe-NO complex in vivo, in view of the fact that nitrite levels in tissues and body fluids are low. Complex redox chem- istry of dithiocarbamate-iron NO traps is well compensated by their unique and important in vivo applications. In brief, recently developed dithiocarbamate-iron nitric oxide-complexing agents allow real-time detection of nitric oxide generation and distribution in small living animals by low-frequency EPR spectroscopy. This new technique significantly extends the capabilities of the traditional spin trapping approach.

36 T. Yoshimura, S. Fujii, H. Yokoyama, and H. Kamada, Chem. Lett. 4, 309 (1995). 37 M.-L. Wong, V. Rettori, A. A1-Shekhlee, P. B. Bongiorno, G. Canteros, S. M. McCann, P. W. Gold,

and J. Licinio, Nature Med. 2, 581 (1996). 38 A. M. Komarov, D. A. Wink, M. Feelisch, and H. H. H. W. Schmidt, Free Radic. Biol. Med. 28, 739

(2000). 39 A. M. Komarov, A. Reif, and H. H. H. W. Schmidt, Methods Enzymol. 359, [2], 2002 (this volume). 40 K. Tsuchiya and R. P. Mason, J. Biol. Chem. 275, 1551 (2000).