1 Department of Anesthesiology, Kyorin University, School of Med-icine, Mitaka, Japan.

2 Address reprint request to: Dr. Takehiko Iijima, Department ofAnesthesiology, Kyorin University, School of Medicine, Kyorin,Japan. Tel: 181-422-47-5511, ext 2410, 3544; Fax: 181-422-43-1504; E-mail: iijmt@kyorin-u.ac.jp

Relationship between Oxidation of Glutathione andReactive Nitrogen Species during the Early-ReperfusionPhase of Cerebral Ischemia

Takehiko Iijima, 1 Hideaki Sakamoto,1 Chikako Okada,1 and Yasuhide Iwao1

(Accepted May 8, 2002)

A timed profile of glutathione oxidation and reactive nitrogen species during reperfusion aftercerebral ischemia in rat was obtained. Dialysate was collected every 25 min from a microdial-ysis probe inserted into the cerebral cortex before and after cerebral ischemia. NO2

2, NO32, and

reduced and oxidized glutathione (GSH, GSSG) were detected by high-performance liquid chro-matography. GSH and GSSG increased and reached a peak: 3408 6 1710% (mean 6 SE) at 25min of reperfusion (P , 0.0001) and 329 6 104% at 50 min of reperfusion (P 5 0.06), respec-tively. Oxidation ratio decreased from 0.82 6 0.04 to 0.42 6 0.07 (P , 0.0001) at 25 min ofreperfusion. NO3

2 levels significantly decreased (68.3 6 9.1%) (P , 0.01) during ischemia andremained lower than the control value during reperfusion. NO2

2 levels did not significantlychange. These data suggest that GSH releases during early phase of reperfusion and that itsrapid oxidation contributes to prevent an increase in reactive nitrogen species.

KEY WORDS: Glutathione; nitrate; nitrite; ischemia; free radical; reactive nitrogen species.

Neurochemical Research, Vol. 27, No. 6, June 2002 (© 2002), pp. 497–500

4970364-3190/02/0600–0497/0 © 2002 Plenum Publishing Corporation

trite and nitrate is a good indicator of free radical for-mation. In contrast, analysis of the balance of glutathione,reduced-type (GSH) and oxidized-type (GSSG) may bea good indicator of free radical trapping. We examinedGSH and GSSG concentrations simultaneously with ni-trite and nitrate during the reperfusion phase and discusshere the involvement of these concentrations in thepathophysiology of neuronal death.

EXPERIMENTAL PROCEDURE

General Preparation.Fifteen Wistar rats were anesthetizedwith 1% of isoflurane and mechanically ventilated after tracheostomy.Both carotid arteries were exposed, and silk strings were put in placefor later ligation. The left femoral artery and vein were cannulatedfor the purpose of exsanguination (artery), blood pressure monitor-ing (artery), and drug infusion (vein), respectively. The animals(weight 280–360 g) were mounted on the stereotaxic frame to fix thecranium, and the left temporal bone was fenestrated for insertion of

INTRODUCTION

Glutamate is released during ischemic insult andmay have a harmful effect on neurons. In contrast tothe theory involving glutamate in neuronal death, glu-tamate becomes glutathione, which is a strong freeradical trapper (1). Free radicals may play a key rolein neuronal damage. The balance between free radicalsand their trappers seems to determine the destiny ofneuron. It is difficult to quantify free radical levels, asthey diminish rapidly. Nitrite (NO2

2) and nitrate (NO32)

are the products that are formed from a strong cyto-toxic oxidant, peroxynitrate (2). The release of both ni-

the microdialysis probe. The dura was punctured with a needle, anda microdialysis probe (Eicom Co. Ltd., Kyoto, Japan) with a tipmade of a 2-mm permeable membrane was gently inserted into thecerebral cortex using a manipulator fixed on the bar of the stereo-taxic frame. The tip of a laser Doppler flowmeter (Advance Co. Ltd.,Tokyo, Japan) was also projected on the adjunct of the microdialy-sis probe. A tiny thermocouple (Unique Medical Co. Ltd., Tokyo,Japan) was inserted under the dura, and the temperature of the sur-face of the cortex was kept between 36.0° and 37.0°C by a radiationlamp (Nihon Kohden Co. Ltd., Tokyo, Japan). The microdialysisprobe was perfused with Ringer’s solution at a speed of 2 ml/min,and dialysate was collected into sampling tubing rinsed in advancewith sterilized distilled water. Sampling was started after 30 min ofstabilization after preparation. To collect 50 ml of dialysate sample,each condition was maintained for 25 min. Hypotension down to40 mm Hg was induced by exsanguination to a heparinized syringeafter the ligation of both carotid arteries. Exsanguination was con-tinued until the laser Doppler flowmetry values reached 20% of theinitial value, thus confirming ischemia. After 25 min of ischemia,exsanguinated blood was reperfused, and ligations of the carotid ar-teries were released. Reperfusion was then maintained for 125 min,with a total of seven samples being collected, including the controland ischemia samples.

Biochemical Analysis.Nitrate and nitrite levels (from 12 of15 samples) were measured using high-performance liquid chro-matography (HPLC) (ENO-200, Eicom Co. Ltd., Kyoto, Japan). Tenmicroliters of dialysate was injected into the HPLC machine, thesensitivity of which was 1028 M 3 10 ml (0.1 pM). The sample wasseparated in a reversed-phase analytical column and detected by aUV detector after the diazo reaction. A standard solution, 50 mM ofNO2

2 and NO32, was used for calibration.

The sample for glutathione measurement was chromatographi-cally separated by a precolumn (AC-ODS; Eicom Co. Ltd.) and a dif-ferential column (SC-50DS; Eicom Co. Ltd.). The mobile phase was99% 0.1 M phosphate buffer containing 1% methanol. Adding octa-sulfonic acid prolonged the retention time of GSH and contributed toan easy separation of GSH from other contamination. A gold elec-trode was used as an electrochemical detector because gold hasstrong specific reactivity with thiols (ECD-300; Eicom Co. Ltd.). Theelectrode was clamped with 1600 mV. GSSG was reduced by dithio-threitol (DTT, 100 mM) for 3 min, which was dissolved in 0.1 M bi-carbonate buffer (pH 9.5) to which EDTA2Na was added afterbubbling with nitrogen. As such, GSSG was measured as GSH. TheGSSG content was calculated by subtraction of the GSH (first meas-urement) content from the measured GSH after reduction by DTT,which contains GSSG. Standard solution (100 mM) was preparedfrom stock solution diluted 1000-fold for each experiment.

Statistical Analysis.Repeated ANOVA was used for statisticalanalysis of series of data. As a post hoc test, Fisher’s PLSD wasused. StatView version 5.0 software (Brain Power, Calabasas, CAwas used for the calculations. Values are crude values in dialysate.We did not try to convert the value to extracellular concentration be-cause efficiency of collection varies depending on the peameabilityof dialysis membrane. Values were expressed as mean 6 SE.

RESULTS

GSH and GSSG.GSH significantly increased justafter the start of ischemic conditions from 0.41 6 0.14to 2.63 6 0.91 mM (P , 0.05) and later greatly in-

creased during reoxygenation, reaching a peak of 6.4361.62 mM (3408 6 1710%) (P , 0.0001) at 25 min ofreperfusion (Fig. 1). This series of values was statisti-cally significant (F-value 8.896, P , 0.0001). GSSGdid not significantly increase during ischemia (1.82 60.83 mM control, 0.92 6 0.30 mM ischemia), later in-creased, and then reached a peak value of 4.14 61.71 mM (329 6 104%) at 50 min of reperfusion(Fig. 1). This series of changes was not statisticallysignificant (F-value 2.099, P 5 0.0617).

The oxidation ratio [2GSSG/(2GSSG 1 GSH)]significantly decreased from 0.82 6 0.04 to 0.42 60.07 (F-value 5.695, P , 0.0001) at 25 min of reper-fusion (Fig. 3).

Nitrate and Nitrite.Nitrite levels did not changeafter ischemia (127 6 47 pM control, 105 6 511 pMduring ischemia) (F-value 0.850, P 5 0.5369) (Fig. 2).

Nitrate levels significantly decreased after thestart of ischemia from 1011 6 388 pM to 486 6 131 pM(68.3 6 9.1%) and remained lower than the controlvalue even at 125 min of reperfusion. This decreasewas statistically significant different from the controlvalue (F-value 2.630, P 5 0.0249) (Fig. 2).

DISCUSSION

Glutamate levels have been widely shown to in-crease during ischemia and are assumed to have aharmful effect on neurons. This theory has been sup-ported by an in vitro challenge study. However, gluta-mate can be converted to glutathione, which is a potent

498 Iijima, Sakamoto, Okada, and Iwao

Fig. 1. Timed profile of GSH and GSSG during the reperfusionphase after 25 min of global ischemia (n 5 15). GSSG increasedafter the start of reperfusion, while GSH began to increase duringischemia. The increase in GSH was approximately 10 times greaterthan that of GSSG. 25 min indicates 0–25 min of reperfusion afterischemia. *P , 0.05, **P , 0.01, ***P , 0.0001, significantlydifferent from the control value.

radical scavenger and is recognized to exert protectiveeffects against oxygen stress.

As we demonstrated in the present study, levels ofglutathione, which is synthesized by released gluta-mate and cystein, rapidly increase during reperfusionafter ischemia. Reduced glutathione is synthesized asfollows:

L-Glutamic acid1 L-cysteine1ATPj l-g-glutamyl-L-cysteine 1 ADP 1 Pi

L-g-Glutamyl-L-cysteine1ATP1glycinejReducedglutathione 1 ADP 1 Pi

Synthesis of GSH requires ATP. Therefore, largeincreases in GSH during reoxygenation can be ex-plained. GSH increased during ischemia and dramati-

cally increased up to approximately 3000% during earlyphase of reperfusion. This increase in GSH may ex-plain the rapid reduction of glutamate during reperfu-sion in previous studies (3,4). GSSG did not increaseduring ischemia and increased after the beginning ofreperfusion. This delay in the increase may be reason-able because oxidant is produced only when oxygen isavailable after ischemia. Such a delay in GSSG pro-duction has also been reported in a renal ischemicmodel (5).

The oxidation ratio decreased after ischemia; ourdata are in the opposite direction of the data presentedby Baek et al. using a gerbil ischemic model (6). In thebrain, substrate for glutathione, i.e., glutamate, isabundant during reperfusion; therefore, it is reasonablethat GSH increases rapidly during reperfusion, andthen the oxidation ratio should be reduced during theearly phase of reperfusion. In the heart, glutamate isnot released as much as in the brain during reperfu-sion. Even in ischemic heart, GSH release precedesGSSG release and the oxidation ratio decreases duringearly phase of reperfusion (7). Our data are in line withthe data from ischemic heart. Decrease in the oxidationratio during reperfusion may be supported by the factthat reactive oxygen species does not increase duringthe early phase of reperfusion.

In the present study, the increase in GSH levelswas approximately 10 times greater than that ofGSSG. McCoy et al. reported that GSSG is only ap-proximately 1% of GSH in the ischemic kidney (5).Under the circumstances of oxygen deficiency, mostof the GSH seems to be catabolized to glutamate andcystein, again to produce ATP, with only a small por-tion of GSH apparently being oxidized to GSSG (8).

Peroxynitrite is produced from superoxide (O22)

and possesses strong oxidative properties. It peroxi-dates lipids and interferes with enzymes in the TCAcycle (9). NO2

2 and NO32 are located downstream

from the peroxynitrite cascade (2). Therefore, NO22 and

NO32 can be considered representative parameters of

oxygen-reactive species (10). We observed no increasein these products during the early phase of reperfusion,instead observing a decrease in NO2

2 and NO32 levels,

although these levels have been reported to increasesoon after ischemic insult in an electrospin resonancestudy (11). During oxygen depletion, such oxidantsmay not be produced because of limited oxygen avail-ability. Pearl et al. reported a reduction in exhalednitric oxide after 90 min of hypoxia in piglets (12).Clark has also reported that NO2

2 and NO32 levels in-

crease only after 30–48 h in head-injured patients (13).And in a myocardial infarction model, NO2

2 and NO32

Glutathione and Reactive Nitrogen Species during Reperfusion after Cerebral Ischemia 499

Fig. 2. Timed profile of NO22 and NO3

2 during the -phase after25 min of global ischemia (n 5 12). NO2

2 did not significantlychange under the different experimental conditions, while NO3

2

significantly decreased (P , 0.05) during ischemia and reperfusion.25 min indicates 0–25 min of reperfusion after ischemia. *P , 0.05,**P , 0.01, significantly different from the control value.

Fig. 3. Oxidation ratio of glutathione during ischemia andreperfusion. Note that oxidation ratio decreased and reached bottomwhen GSH release reached peak during 0–25 min of reperfusionafter ischemia. *P , 0.05, **P , 0.01, ***P , 0.0001, significantlydifferent from the control value.

levels also increased 3 days after experimental is-chemia (14). As such, increases in NO2

2 and NO32

seems to be late phenomena occurring a certain intervalafter ischemia. We observed that only NO3

2 levels aresignificantly reduced, implying an increase in NO2

2 andNO3

2 ratio. Rizzo et al. also reported, based on a micro-dialysis study in the brain, that NO2

2 NO32 increases

during reoxygenation (15). NO• reacts with ferrous oxy-hemoglobin to yield NO3

2; as such, the increase inNO2

2/NO3 indicates some interference with NO• pro-duction. Our results also show that NO2

2 NO32 in-

creased during rapid production and conversion of GSHand GSSG simultaneously. Therefore, it is conceivablethat NO• was at least partly trapped by GSH.

The therapeutic window of ischemic neuronaldeath is reported to be 45–60 min during the reperfu-sion phase (16). The period for the appearance and re-duction of this buffer for reactive oxygen speciesseems to be in line with this therapeutic window. Thedynamic change of thiols and reactive oxygen species,as demonstrated in this study, may be involved in themechanism of neuronal death after ischemia.

In the ischemic skin model, GSH has beendemonstrated to play a protective role against ischemiccell injury (17). The trapping of reactive oxygen (ni-trogen) species exerted by GSH may also play an im-portant role in ischemic neuronal injury. Furtherstudies elucidating the relationship between GSH pro-duction and neuronal death after ischemic insult re-main to be carried out.

In conclusion, we have found that the conversionof GSH to GSSG rapidly commences during the reper-fusion phase and terminates within 100 min. Simulta-neous decrease in NO3

2 suggests that GSH is consumedby free-radical trapping.

ACKNOWLEDGMENTS

This study was supported by a Grant-in-Aid for Scientific Re-search from the Ministry of Education and Science No. 11671521.

REFERENCES

1. Chen, Y., Varitiainen, N. E., Ying, W., Chan, P. H., Koistinaho,J., and Swanson, R. A. 2001. Astrocytes protect neurons fromnitric oxide toxicity by a glutathione-dependent mechanism.J. Neurochem. 77:1601–1610.

2. Cuzzocrea, S., Zingarelli, B., and Caputi, A. P. 1998. Role ofconstitutive nitric oxide synthase and peroxynitrite productionin a rat model of splanchnic artery occlusion shock. Life Sci. 63:789–799.

3. Iijima, T., Iijima, C., Iwao, Y., and Sankawa, H. 2000. Differ-ence in glutamate release between retina and cerebral cortex fol-lowing ischemia. Neurochem. Int. 36:221–224.

4. Benveniste, H., Drejer, J., Shousboe, A., and Diemer, N. H.1984. Elevation of the extracellular concentration of glutamateand asparate in rat hippocampus during transient cerebral isch-emia. J. Neurochem. 43:1369–1374.

5. McCoy, R. N., Hill, K. E., Ayon, M. A., Stein, J. H., and Burk,R. F. 1988. Oxidant stress following renal ischemia: Changes inthe glutathione redox ratio. Kidney Int. 33:812–817.

6. Baek, S. H., Kim, J. Y., Choi, J. H., Park, E. M., Han, M. Y.,Kim, C. H., Ahn, Y. S., and Park, Y. M. 2000. Reduced glu-tathione oxidation ratio and 8 ohdG accumulation by mild isch-emic pretreatment. Brain Res. 856:28–36.

7. Tritto, I., Dulio, C., Santoro, G., Elia, P. P., Cirllo, P., Simone,C. D., Chiariello, M., and Ambrosio, G. 1998. A short burst ofoxygen radicals at reflow induces sustained release of oxidizedglutathione from post ischemic hearts. Free Radic. Biol. Med.24:290–297.

8. Juurlink, B. H. J., Schultke, E., and Hertz, L. 1996. Glutathionerelease and catabolism during energy substrate restriction in as-trocytes. Brain Res. 710:229–233.

9. Radi, R., Beckman, J. S., Bush, K. M., and Freeman, B. A. 1991.Peroxynitrite-induced membrane lipid peroxidation: The cyto-toxic potential of superoxide and nitric oxide. Arch Biochem.Biophys. 288:481–487.

10. Stuehr, D. J., Gross, S. S., Sakuma, I., Levi, R., and Nathan,C. F. 1989. Activated murine macrophages secrete a metaboliteof arginine with the bioactivity of endothelium-derived relaxingfactor and the chemical reactivity of nitric oxide. J. Exp. Med.169:1011–1020.

11. Kumura, E., Yoshimine, T., Iwatsuki, K., Yamanaka, K., Tanaka,S., Hayakawa, T., Shiga, T., and Kosaka, H. 1996. Generation ofnitric oxide and superoxide during reperfusion after focal cerebralischemia in rats. Am. J. Physiol. (Cell Physiol. 39)270:C748–C752.

12. Pearl, J. M., Nelson, D. P., Wellmann, S. A., Raake, J. L., Wag-ner, C. J., McNamara, J. L., and Duffy, J. Y. 2000. Acute hypoxiaand reoxygenation impairs exhaled nitric oxide release and pul-monary mechanics. J. Thorac. Cardiovasc. Surg. 119:931–938.

13. Clark, R. B., Kochanek, P. M., Obrist, W. D., Wong, H. R., Bil-liar, T. R., Wisniewski, S. R., and Marion, D. W. 1996. Cere-brospinal fluid and plasma nitrite and nitrate concentrationsafter head injury in humans. Crit. Care. Med. 24:1243–1251.

14. Akiyama, K., Suzuki, H., Grant, P., and Bing, R. J. 1997. Oxy-dation products of nitric oxide, NO2 and NO3, in plasma after ex-perimental myocardial infarction. J. Mol. Cell. Cardiol. 29:1–9.

15. Rizzo, V., Montalbetti, L., Rozza, A. L., Bolzani, W., Porta, C.,Balduzzi, G., Scoglio, E., and Moralli, R. 1998. Nitrite/nitratebalance during photoinduced cerebral ischemia in the rat deter-mined by high-performance liquid chromatography with UVand electrochemical detection. J. Chromatogr. 798:103–108.

16. Kuroiwa, T., Bonnekoh, P., and Hossmann, K. A. 1991. Thera-peutic window of halothane anesthesia for reversal of delayedneuronal injury in gerbils: Relationship to postischemic motorhyperactivity. Brain Res. 563:33–38.

17. Rees, R. S., Smith, D. J., Adamson, B., Im, M., and Hinshaw, D.1995. Oxidant stress: The role of the glutathione redox cycle inskin preconditioning. J. Surg. Res. 58:395–400.

500 Iijima, Sakamoto, Okada, and Iwao