* Supported by grants No. NB and MH from the United States Public Health Service and by a grant from the Epilepsy Foundation of America.

Transcription

1 JOURNAL OF NEUROBIOLOGY, VOL. 2, NO. 2, PP EFFECT OF THE CONVULSANT METHIONINE SULFOXIMINE ON THE IN VWO UPTAKE AND METABOLISM OF D-METHIONINE IN RAT BRAIN* NORA E. GHITTONIt and OTTO Z. SELLINGERS Mental Health Researh Institute, University of Mihigan Medial Center, Ann Arbor, Mihigan 4814 SUMMARY D-methionine was administered intraperitoneally to rats in traer (2-2.5 pmoles/kg) and large doses (4.7 mmoles/kg) and the brain levels of total (D + L) methionine, as well as of ysteine (+ystine) and glutathione were determined. The effet of o-administering the onvulsant agent, L-methionine-DL-sulfoximine (MSO) was also examined. The administration of traer doses of Cl2-D-methionine resulted in a doubling of total brain methionine within 4 hours post-injetion, but only in a moderate inrease of the levels of ysteine. When C14-D-methionine was used as traer, the peak inrease of the isotopi methionine pool and a peak aumulation of.35 "/o of the injeted radioativity were noted in the brain within 1.5 hours. When 4.7 mmoles/kg of D-methionine were administered, total brain methionine and ysteine inreased by 4- and 3-fold respetively, the former peaking at 1.5 hr and the latter at 2.5 hr post-injetion. The administration of MSO retarded the attainment of these peaks. It ould also be shown that while about 65% of the total brain methionine existed as the D-isomer 1.5 hr after its administration, only 46% was still present as the D-isomer.5 hr later. When MSO was administered simultaneously with D-methionine, the orresponding perentage values stood at 7% at 1.5 and at 2 hr. The results, therefore, suggest that even though D-methionine reahes the brain largely unhanged, its uptake and its onversion to the natural L-isomer may be inhibited by MSO under ertain onditions and hene its onversion to ysteine retarded. INTRODUCTION The disposition of in uiuo administered D-amino aids by brain tissue has been investigated by Lajtha and his assoiates (Lajtha and Toth, * Supported by grants No. NB-6294 and MH-7417 from the United States Publi Health Servie and by a grant from the Epilepsy Foundation of Ameria. t Postdotoral trainee, Present address: Fauldad de Farmaia y Bioquimia, Universidad de Buenos Aires, Buenos Aires, Argentina. $ To whom requests for reprints should be addressed by John Wiley & Sons, In.

2 154 GHITTONI AND SELLINGER 1962, 1963; Lajtha, 1968) who worked prinipally with the D-isomers of leuine, lysine and phenylalanine, and by Chirigos et al. (196) and Guroff and Udenfriend (1962) who examined the uptake of D-tyrosine and D- tryptophan. In all ases, little if any raemization of the administered D- amino aids to the natural L-isomers was noted. More reently Shah et al. (1968) reported that L-methionine markedly redued the in uiuo uptake by the brain of dihydroxyphenylalanine while D-methionine had no suh effet. In uitro, Nakamura (1963) noted that L- and D-methionine interfere equally effetively with the uptake of L-histidine by brain slies. Cerebral ell-free preparations have also been examined for their ability to reat with D-amino aids (Burh et al., 1956; Neims et al., 1966; Goldstein, 1966) and the presene of D-amino aid oxidase has been reported in the brain of several speies. More reently, de Marhi and Johnston (1969) asribed to D-amino aid oxidase the erebellar and spinal ord enzymati ativity whih they found to reat with glyine and with several other D-amino aids. Pursuing our long-standing interest in the mehanism of ation of the onvulsant methionine sulfoximine (Lamar and Sellinger, 1965; de Robertis et al., 1967; Sellinger et al., 1968; Ghittoni et al., 197; Ghittoni and Sellinger, 197) we reently tested the ability of several analogs and derivatives of methionine to protet rats against epilepti seizures indued by this drug and we noted that D-methionine was as effetive as the L-isomer (Sellinger et al., 1968). Subsequently, we desribed effets of L-methionine on the regional and intraellular disposition of ["I-MSO in rat brain (Ghittoni et al., 197) and hanges in the levels of erebral methionine and ysteine eliited by the administration of MSO (Ghittoni and Sellinger, 197). In the present report we desribe results of experiments whih demonstrate the ability of brain tissues to take up D-methionine, onvert it to the L-isomerand form ysteine therefrom. A study of the effets of MSO on the erebral disposition of D-methionme is also inluded. METHODS Materials. The following ompounds were obtained as indiated: L-methionine- DL-sulfoximine, D-methionine, and ninhydrin from Piere Chemial Co., Rokford, 111.; L-methionine from Nutritional Biohemials In., Cleveland, Ohio; CI4-(methyllabeled) D-methionine (s.a.: 6.2 mci/mmolr) from ICN Co., City of Industry, Calif; snake venom L-amino aid oxidase and hog kidney D-amino aid oxidase (3.6 units mg and 4.5 unitslmg, respetively) and beef liver atalase (53,65 units/mg) from Worthington Biohem. Co., Freehold, N. J.; CoA, aetyl phosphate and phosphotransaetylase (s.a.: 1,2 units/mg) from Boehringer Mannheim, New York, N. Y.; Dowex-AGP X mesh), hloride form, from Bio-Rad Laboratories, Rihmond, Calif; Dowex 5 W-X8 from Baker Chem. Co., Phillipsburg, N. J.; PPO and dimethyl POPOP from Pakard Instruments, Downers Grove, Ill.; Biosolv-3 from Bekman Instruments Co., Palo Alto, Calif.; 5,5'-dithiobis (2 nitrobenzoi aid) from K & K Laboratories, Plainview, N. Y.; 3-hydrazinoquinoline dihydrohloride from Eastman Organi Chem. Co., Rohester, N. Y.; dithiothreitol from Calbiohem.,

3 D-METHIONINE IN RAT BRAIN 155 Los Angeles, Calif.; FAD from Sigma Co., St. Louis, Mo.; N-aetyl-D-methionine from Cylo Chemial Co., Los Angeles, Calif.; and hloroplatini aid from Matheson, Coleman, and Bell, East Rutherford, N. J. Adult (2-25) male rats (Sprague-Dawley) were injeted i.p. with a onvulsant dose of MSO (.94 mmole/kg) and, unless stated otherwise, a 5-fold molar exess of D-methionine whih inluded, when desired, a traer dose of C 14-D-methionine. Traer C14-D-methionine was also administered alone but when MSO was given simultaneously, the molar ratio of MSO to D-methionine was about 1 :.25. Preparation of the free amino aid pool. The animals were killed by deapitation and the rapidly exised brain was weighed and homogenized in 1% (w/v) piri aid to make a 1% (w/v) tissue suspension whih was entrifuged at 12, X g for 1 min. The sediment was washed one with 1% piri aid and the pooled supernatants were passed through a olumn (1 X 2 m) of Dowex AG2 X 1 (1-2 mesh) in the hloride form. The effluent ontained the free amino aids. As reported previously (Lamar and Sellinger, 1965), MSO is quantitatively eluted from the Dowex olumn only after elution with.2 M HC1. Determination of erebral methionine. Total methionine. The following proedure was adopted for the quantitative determination of total methionine in the brain. The sulfur-ontaining amino aids in the Dowex effluent (see above) were determined by the hloroplatinate proedure of Awwad and Adelstein (1966) whih measures methionine, glutathione, and ysteine. The two latter ompounds were therefore determined independently by the proedures of Jelyn (1962) and Gaitonde (1967), respetively. After quantitative orretion for the olor ontributions of glutathione 2nd ysteine at Aroo in the proedure of Awwad and Adelstein 11966), methionine was alulated by applying the following formula: [Methionine] = [Sulfur amino aids] -.66 X (glutathione) + -~ X (ysteine).75 where.18,.66, and.75 are the Aeoo molar equivalent values for glutathione, ysteine, and methionine. L-methionine. The L-isomer of methionine was assayed by ondensing its keto analog, a-keto-?-methyl meraptobutyrate (KMB) obtained after the ation of L-amino aid oxidase, with 3-hydrazinoquinoline (Robins et al., 1956). The assay system ontained 2 pmoles of Tris buffer, ph 7.8, 1 Units of L-amino aid oxidase and 42 units of atalase in addition to L-methionine and was inubated for 2 hr at 3 C. Aliquots of the aidified inubation mixture were assayed for keto aids as desribed by Robins et al. (1956). The proedure was standardized with a-keto-glutarate. D-methionine. The speifi yeast D-amino aid N-aetyltransferase (Shmitt and Zenk, 1968) was used to N-aetylate the D-methionine present in the brain after its intraperituneal administration. The assay system was as desribed by Shmitt and Zenk (1968), exept that the onentration of aetyl phosphate (Lipmann and Tuttle, 1945) was 4 pmoles/tube. Inubations were at 3" for 1 hr. To onfirm the identity of the aetylated produt, aliquots of the inubation mixtures were hromatographed on Whatman 3MM paper asending in 1-butanol-gl. aeti aid-water 12: 1 : 4 by volume) for omparison with authenti N-aetyl-D-methionine (Rr:.87). Radioative measurements. ClJ-D-methionine was heked for stereospeifi purity as reeived from the supplier and also after treatment with L- and D-amino aid oxidase. Radioativity was determined in 15 ml of sintillation fluid ontaining 4 g of 2,5-dyphenyloxazole (PPO) and.1 g of 1,4 bis- (4 methyl-5-phenyloxazolyl) benzene [dimethyl POPOP] per liter of toluene or 1.9 g of PPO,.7 g of dimethyl POPOP and 6 g of naphthalene/liter of xylene, dioxane, and ethyl ellosolve (1:3:1 by volume). All radioativity measurements were performed in a Unilux I1 liquid sintillation spetrometer (Nulear Chiago, Des Plaines, Ill.) with an 8-85% effiieny. Quenhing orretions were applied using the hannels ratio method. 1

4 156 GHITTONI AND SELLINGER RESULTS Effet of a traer dose of D-methionine on total brain methionine. The i.p. injetion of a traer dose of C14-D-methionine (between 2 and 2.5 pmoles/kg of body weight) resulted in about 4 hr in an elevation of total brain methionine (Fig. 1) to twie the endogenous levels of L- methionine (.59 Mmoles/g of brain) (Ghittoni and Sellinger, 197). The o-administration of MSO (Fig. 1, dashed line) appeared to stimulate this proess during the first half hour presumably by rapidly mobilizing the entry of extraerebral stores of L-methionine into the brain (Ghittoni and Sellinger, 197), or possibly, but less likely, by speifially failitating the entry into the brain of the administered D-isomer by virtue of a diret effet on the blood-brain barrier. It should be noted that when C14-Dmethionine was administered alone (Fig. 1, solid line) a lowering of the levels of total methionine in the brain ourred during the first half hour; this may have been due to differenes in the rates of flux of the administered D-methionine into, and of endogenous L-methionine out of, the brain with a onsequent, temporary net derement of the overall measurable methionine. Maximal radioativity, expressed as pm/pmole of total methionine (Fig. 2) was reahed within 3 min after C"-D-methionine alone and 1 hr later when MSO was o-administered. Subsequently, the radioativity present as methionine dereased to idential levels, irrespetive of whether MSO was administered. Maximal erebral as well O 7 P 12-. m / / 5 I - - I I I -I Hours after administration Fig. 1. Total brain methionine after the administration of a traer dose of D-methionine =k MSO. D-methionine ( pmoles/kg of body weight) was administered intraperitoneally and total brain methionine was determined. The y-interept represents the endogenous L-methionine onentration (.59 pmoleslg). (-) D-methionine; (---) D-methionine +.94 mmoles/kg of MSO.

5 D-METHIONINE IN RAT BRAIN Hours after administration Fig. 2. The time ourse of erebral labelling. C14-D-methionine (s.a.: 6.2 mci/- mmole, 4.5 X lo6 pm/rat) was administered and the radioativity of the aid-soluble materials determined, as indiated. (-) C14-D-methionine; (---) C14-D-methionine +.94 mmoles/kg of MSO. as renal aumulation of the radioativity, expressed as the per ent of the administered radioative dose, also ourred between 3 and 9 min. Its deay was faster in the brain than in the kidney, however, and was differentially influened by MSO, so that more radioativity appeared in the brain and less in the kidney when MSO was given than when it was omitted (Fig. 3, dashed lines). The administration of traer amounts of CI4-D-methionine also led to a slow, but signifiant trend toward higher than endogenous brain ysteine levels (Fig. 4); interestingly, this trend was initially opposed by the administration of MSO but later appeared to be stimulated by it (Fig. 4, dashed line). Effet of a seizure-protetive dose of D-methionine on total brain methionine. Figure 5 illustrates the hanges of erebral methionine after the injetion of 4.7 mmolesikg of D-methionine (solid line) +.94 mmoles/kg of MSO (dashed line). It should be noted that sine in Fig. 5 as in Fig. 1 the value at the y-axis interset represents the endogenous L-methionine (Ghittoni and Sellinger, 197), there was an apparent doubling of the endogenous methionine within 3 min, presumably as a result of a massive and largely unimpeded surge of the injeted D-isomer into the brain. Peak aumulation ourred 1.5 hr after D-methionine alone and.5 hr later when MSO was also given. Figure 5 also illustrates that although the administration of MSO had no apparent

6 158 GHITTONI AND SELLINGER KIDNEY *t Hours after administration Fig. 3. The total radioativity of brain and kidney after the administration of a traer dose of D-methionine. For details of injetion, see the aption for Fig. 1. The total tissue radioativity is expressed as % of the injeted dose. (-) D-methionine; (---) D-methionine +.94 mmoles/kg of MSO. effet on the initial doubling of total erebral methionine, it appeared to slow down the rate of its subsequent aumulation in the brain, possibly by ausing a preferred efflux of the L-isomer or, more diretly, by gradually impeding the otherwise free passage of the D-isomer through the blood-brain barrier. The plats of Fig. 5 also show that, within the 4-hr experimental period, there was no net depletion of methionine in the brain as a result of the administration of MSO. When a traer dose of CL4-D-methionine aompanied the 4.7 mmoles kg of C?-D-methionine, a sustained inrease of erebral radioativity, presumably present mostly as methionine, was noted (Fig. 6), in ontrast to the situation arising after the administration of traer doses of C14-Dmethionine alone (Fig. 2) when the radioativity rapidly deayed after 1.5 hr. Similarly, Fig. 7(a) illustrates the sustained retention of more than.5% of the injeted dose in 4 hr. Conversely, renal retention did not exeed 1.5% most probably beause the D-amino aid was effetively atabolized in this organ by the highly ative D-amino aid oxidase sys-

7 D-METHIONINE IN RAT BRAIN r al v) -.2 m I- I I I I ' I Hours after administration Fig. 4. Total brain ysteine after the administration of a traer dose of D-methionine. The onditions were as desribed in the aption for Fig. 1. Cysteine (+ ystine) was determined by the method of Gaitonde (1967). The y-interept indiates the endogenous onentration of ysteine:.52 pmoles/g. (-) D-methionine; (---) D- methionine +.94 mmoles/kg of MSO. r.24 ' I I I 1 I Hours after administration Fig. 5. Total brain methionine after the administration of large dose of D-methionine. 4.7 mmoles/kg of D-methionine were administered intraperitoneally. Otherwise, all onditions were as desribed in the aption for Fig. 1. (-) D-methionine +.94 mmoles/kg of MSO.

8 .I 16 GHITTONI AND SELLINGER X 3.r L g 25- / P.I v) + C 3 Hours after administration Fig. 6. The time ourse of erebral labelling. A large dose (4.7 mmoles/kg) of (2'2-Dmethionine whih also ontained 4.5 X 16 pm of C'4-D-methionine was injeted. See the aption for Figure 2 for details. [ -) D-methionine; (---) D-methionine +.94 mmoleslkg of MSO. tem. Cysteine values in the brain peaked at 2.5 hr and then began to return to ontrol levels (Fig. 8). The effet of MSO on the entry and the aumulation in the brain of the seizure-protetive amounts of D-methionine was negligible, as shown by the dashed line of Figs. 6 and 7. Yet, as shown by the dashed line of Fig. 8, a diminished onversion to ysteine was eliited and ould be sustained for about 3 hr. As doumented below (Table l), an inhibitory effet of MSO on the onversion of the D-methionine to the L-isomer may be invoked to explain these findings. Effet of lowering the molar ratio of D-methionine to MSO. Figures 1 and 5 showed that the administration of MSO with low or high D-methionine resulted in an inrement of total brain methionine over and above the endogenous value of.59 pmoles/g (Ghittoni and Sellinger, 197) whih was half-maximal at 2.5 hr. This effet of MSO is represented in Fig. 1 by.92 pmoles/g and in Fig. 5 by.145 bmoles 'g of total brain methionine. Sine these hanges in total brain methionine were eliited by traer vs. seizure-protetive amounts, it was of interest to examine the effet of intermediate molar ratios of D-methionine to MSO. Figure 9 shows the results of an experiment in whih groups of

9 D-METHIONINE IN RAT BRAIN 161-6r 6-5- v) L= BRAIN.o.- 5 V m D CY m t 3- KIDNEY <----- I LU I Hours after administration Fig. 7. The total radioativity of brain and kidney after the administration of a large dose of D-methionine. For details of injetion, see the aption for Fig. 6. For other details, see the aption for Fig. 3. (-) D-methionine; (---) D-methionine +.94 mmoles/kg of MSO. - m 1 I I I E ' I Hours after administration Fig. 8. Total brain ysteine after the administration of a large dose of D-methionine. See the aption for Fig. 6 for details of injetion and the aption for Fig. 4 for all other details. (-) D-methionine; (---) D-methionine +.94 mmoles/kg of MSO.

10 - 162 GHITTONI AND SELLINGER Table 1 Effet of MSO on the Conversion of D- to L-Methionine* ~ - _~ Time after administration of D-methionme f MSO 1.5 hr 2 hr ~.- - ~~- - D-methionine D-methionine D-methionine + MSO D-methionine + MSO Methionine - ~ in brain pmoles/g % p/moles % pmoles/g % pmoles/g % Totalt D-isomer L-isomer Ratio: D,L Perent L-hange due to MSO * D-methionine (4.7 mmoles/kg) was administered alone or simultaneously with.94 mmoles/kg of MSO to groups of rats, one half of whih were killed 1.5 hr and the other 2 hr post-injetion. Total and D-methionine levels were determined (see Methods) ~ t Determined analytially by the proedure of Awwad and Adelstein (1966), see Methods. 1 Determined enzymatially by the proedure of Shmitt and Zenk (1968). 5 Calulated by differene between the total and the D-isomer values. rats were injeted with.94 mmolesjkg of MSO alone or simultaneously with.23 (traer), 1.15, 3.3, and 4.7 mmolesjkg of D-methionine and erebral methionine and ysteine were determined 2.5 hr later. In onfirmation of some of our previous results (Ghittoni and Sellinger, 197), the injetion of MSO alone resulted in a fall of erebral methionine from an endogenous level of.59 pmoles/ g to.42 pmoles/g and an elevation of erebral ysteine from an endogenous level of.52 pmoles/g to.93 pmoles,/g. However, as expeted, the joint administration of MSO or 2.3 mmoles/kg of D-methionine resulted in maximal levels of total brain methionine of about.1 pmoles/g; yet interestingly, the levels of ysteine remained onstant at about.1 pmolelg irrespetive of the molar ratio of D-methionine to MSO. Brain glutathione. The erebral levels of glutathione were not affeted by the administration of MSO and D-methionine alone or together and its tissue levels, determined in 4 animals under a number of different experimental onditions, average 1.1 =t.12 pmoleslg of brain (Kandera et al., 1968; Himwih and Agrawal, 1969). Conversion of D- to L-methionine. In their study of the efflux of amino aids from the mouse brain, Lajtha and Toth (1962) noted no signifiant metabolism of intraerebrally injeted D-amino aids and onluded that the D-isomers of leuine, lysine and phenylalanine left the brain unhanged, both as a result of net outflow and of replaement by the natural L-isomers. Subsequently, Lajtha and Toth (1963) suggested

11 D-METHIONINE IN RAT BRAIN L ru n m W- bp.- m D- Methionine administered with MSO, m moles/kg Fig. 9. The effet of lowering the D-methionine-MSO ratio on total brain methionine and ysteine..94 mmoles/kg of MSO were administered simultaneously with D- methionine as indiated, and all the animals were killed 2.5 hr later. The y-interept represents erebral levels of methionine (---) and ysteine (.---a) 2.5 hr after the administration of.94 mmoles/kg of MSO alone [taken from Ghittoni and Sellinger 197) 1. that an ineffiient inward transport was operative for the D-, but not for the L-amino aids. Sine the results of the massive D-methionine administration [Figs. 6 and 7(a)] indiated a steady inrease of the radioativity in the brain and, more speifially, of the radioativity present as methionine, it beame of interest to disriminate between the D- and the L-methionine isomers and, if possible, determine the proportions of eah isomer at seleted times after the administration of the unnatural isomer. To this effet, 2 groups of rats were injeted with D-methionine and 2 groups with D-methionine and MSO and half of the animals in eah group were killed 1.5 and 2 hr later. These partiular time points were hosen beause of the findings depited in Fig. 5 whih showed total brain methionine to be maximal 1.5 hr after the injetion of D-methionine alone and 2 hr after the joint injetion of D-methionine and MSO. Thus, the experimental design made it possible to assess the effet of MSO on the D- to L-ratio at times when total brain methionine was either dereasing or inreasing. Table 1 shows that when D-methionine was administered alone, the value of the ratio of D- to L-methionine fell by more than 1% in 3 min, i.e., from 1.77 at 1.5 hr to.87 at 2 hr; whereas, when, MSO was also administered, it dereased by less than 5%, i.e., from 2.4 to 1.7. Another indiation of the quantitative extent of the onversion of D- to L-methionine and of the effet of MSO on this proess was obtained

12 164 GHITTONI AND SELLINGER in the following experiment,: 1.5 and 2 hr after the administration of a seizure-protetive dose of C *-D-methionine (+ a traer dose of CI4-Dmethionine) alone or jointly with a onvulsant dose of MSO, Dowex eluates of brain aid-soluble filtrates (see Methods) were onentrated and hromatographed before and after inubation with L-amino aid oxidase, and the amount of radioative KMB whih was derived exlusively from the CI4-L-methionine formed in vivo was determined. The results of this experiment revealed a 14% inrement in t.he L-isomer after D-methionine alone but a zero inrement after D-methionine and MSO, a finding in agreement with the results shown in Table 1 and whih provides additional doumentation of the blok by MSO of the onversion of D- to L-methionine. DISCUSSION The uninumbered ability of L-me thionine to penetrate the blood-brain barrier and to exhange with the L-methionine of mammalian brain was first demonstrated by Appel et al. (196) and later again by Shah et al. (1968). Daniel and Waisman (1969) reently determined a 3-fold inrease of methionine in the brain of weanling rats 2 hr after giving 11 mmoles,/kg of the L-isomer, while Hardwik et al. (197) noted that the administration of 1 mmoles/kg of L-methionine to guinea pigs led to profound metaboli alterations and death within 6 hr. In the present experiments the maximal injeted dose of 4.7 mmoles/kg of D-methionine eliited no toxi reations within the short experimental period studied. On the other hand, we are aware of no reports dealing with the in uiuo uptake of D-methionine by the brain. The experiments desribed in the first part of this report relating to traer amounts of C14-D-methionine injeted alone or with MSO, revealed an early drop of total brain methionine from the endogenous level of.59 pmoles g to.5 pmoles /g after the amino aid was given alone and an inrease to.8 pmoles /g after MSO was given with it (Fig. 1). In our opinion, it is unlikely that the initial derement was due to an exhange replaement by the ingoing D-isomer of more than equal amounts of outgoing 1,-methionine; rather, a more likely explanation is that D-methionine was initially rapidly onverted to ysteine, via raemization and transsulfuration. Indeed, as shown by the results of Fig. 4, total brain ysteine inreased from an endogenous value of.52 pmoles/g to.63 pmoles / g during the first 3 min post-administration. The inrease in total brain methionine noted when MSO was o-administered (Fig. 1, dashed line) did not lead to hanges in total brain ysteine; hene it must be onluded that MSO interfered with the onversion of D-methionine to ysteine. Evidene for this onlusion is disussed below. A more likely explanation of the inrease is then, in our opinion, a failitatory effet of MSO on the inward transport of D-methionine. We opt for this explanation beause of our reent observation of a similar, albeit

13 D-METHIONINE IN RAT BRAIN 165 reverse, failitatory effet of L-methionine on the initial uptake of [H3]- MSO in three different regions of the rat brain (Ghittoni et al., 197). Mehanistially, the failitation may take the form of an MSO-indued inrease in affinity of the D-methionine for a speifi arrier system, or for the arrier system whih mediates the transport of L-methionine and for whih D-methionine may also have a high degree of affinity; or, perhaps, it may involve no suh hange in affinity but a transient aeleration of the flux of the arrier-linked D-methionine into the brain instead. The dashed line of Fig. 2 suggests that the putative MSO-failitated inward transport brought proportionately more C l2 (of unknown isomeri distribution) than C -D-methionine into the brain, with the result that the values of ountslminlpmole of total brain methionine were appreiably lower under these onditions than when CI4-D-methionine was given alone (Fig. 2, solid line). Most likely, a large proportion of the inrease in total brain methionine was the result of the mobilization of peripheral L-mehhionine stores whih appeared to be able to enter the brain at a high rate for at least 4 hr (Fig. 1). Net efflux of radioativity from the brain began at about 1.5 hr [Figs. 2 and 3(a) ]; and there appeared to be no preferred retention of C14-D-methionine in the brain, sine the desending slopes of the urves of Figs. 2 and 3(a) were virtually idential. An analysis of the inward transport of the seizure-protetive doses of D-methionine (Sellinger et al., 1968), and of the effets of MSO thereon, reveals no partiular features whih would aid in the distintion between speifi mehanisms governing the inward transport of traer vs. massive doses. Obviously, as a result of the administration of the latter and of the apparently uninumbered passage of D-methionine through the blood-brain barrier, total brain methionine rose by about 4% above endogenous levels in about 1.5 hr. Yet unlike when traer amounts were given (Fig. l), it also sharply delined soon thereafter, reahing lose to endogenous values in 4 hr. A orrespondingly high onversion to ysteine appeared to aount for at least part of this deline, for the levels of the latter amino aid exeeded the endogenous values by about 3% in 2.5 hr (Fig. 8). The effet of MSO was to delay the appearane of the total brain methionine peak by 3 min and to blok the overall onversion of D-methionine to ysteine. There appeared to be no failitation by MSO of the initial inward transport of D-methionine into the brain as was supposedly the ase when traer amounts of D- methionine were administered (see above). Most likely, failitation by MSO ould not our due to the immediate sat.uration of all arrier sites with D-methionine. As shown previously (Ghittoni and Sellinger, 197), the i.p. administration of a onvulsant dose of MSO results in a transient elevation of L- methionine in the brain with a peak of about.1 pmole/g at 2 hr. Subsequently (i.e., as the animal nears the time of seizure) erebral L-methionine falls to below ontrol values of about.4 pmoles/g. Following the

14 166 GHITTONI AND SELLINGER administration of MSO + D-methionine (4.7 mmoles/kg) (Fig. 5, dashed line), total brain methionine rose to peak levels of about 35% above the endogenous levels. However, in order to assess the true effet of MSO on the erebral uptake of D-methionine, we believe that L-methionine values existing after MSO alone (Ghittoni and Sellinger, 197) must be subtrated from the total methionirie values obtaining after MSO + D- methionine. We also assume that MSO does not seletively ativate any presumed mehanism governing the movement of D-methionine from the plasma into the peripheral tissues [a reasonable assumption in view of the results shown in Fig. 7(b)] so as to lead to lower plasma and brain methionine without involving "inward transport" mehanisms. On this basis, it may be alulated that MSO markedly impairs the idux of D-methionine into the brain. Expressed as per ent of the values shown by the full line of Fig. 5, the interferene ranged from 56% at 3 min to 28% at 2.5 hr. It may be noted that the administration of the onvulsant pentylenetetrazole did not inhibit the uptake of L-lysine by mouse brain (Lajtha and Toth, 1965). Sturman et al. (197) have reently shown a slight retardation of the erebral uptake of S",]-Lmethionine by vitamin BG-defiient rats whih was aompanied by a pronouned inrease of brain ystathionine. Comparison of the in uiuo uptakes of D-methionine by rat brain and of D-leuine, D-phenylalanine and D-lysine by mouse brain (Lajtha and Toth, 1963) reveals that of D-methionine to be the slowest. Thus (as estimated from the data of Fig. 5) 45 min after the injetion of D-methionine, total brain methionine rose by only 9 pmoles/1 g over the endogenous level, ompared to inrements of 2 and 9 pmoles/g. respetively, for phenylalanine and leuine (Lajtha and Toth, 1963). The alterations of the serebral levels of L-methionine aused by D- methionine and MSO are shown in Table 1. The data show a more rapid fall of the D-L ratio in the absene of MSO, yet the numerial value of this ratio was higher after the administration of MSO + D-methionine. These findings suggest that under the two different onditions the exhange of D-methionine for, and/or its onversion to, the L-isomer is regulated by different time onstants. An alternate alulation of the data of Table 1 reveals that while the levels of L-methionine were only.1 pmoles/g higher than the endogenous levels 1.5 hr after the injetion of D-methionine, they exeeded these levels by.29 prnoles/g at 2 hr. This finding points to the operation of an effiient erebral mehanism for the exhange of the D-isomer for, or its onversion to, the L-isomer. Conversely, the same alulation reveals that, provided the endogenous values subtrated were those of L-methionine at 1.5 and 2 hr after MSO alone (Ghittoni and Sellinger, 197), brain L-methionine levels were lower by,3 and.36 pmoles, g when MSO was administered than when it was not. Therefore, these results show a net positive effet of the administration of a D-amino aid on the erebral levels of the orresponding L-isomer and interferene with this onversion by a pharmaologially

15 D-METHIONINE IN RAT BRAIN 167 ative methionine derivative in ontrast to the report by Lajtha and Toth (1963) who noted no signifiant alteration of the erebral L-leuine in the brain of Swiss mie reeiving D-leuine. REFERENCES APPEL, K. R., APPEL, E., and MAURER, W. (196). Konzentration und Austaushrate des freien Methionins im Gehirn der Ratte. Biohem : AWWAD, H. K. and ADELSTEIN, S. J. (1966). A quantitative method for the determination of the speifi radioativity of sulfur-ontaining amino aids separated by paper hromatography. Anal. Biohem. 16: BURCH, H. B., LOWRY,. H., PADILLA, A. M., and COMBS, A. M. (1956). Effets of riboflavin defiieny and realimentation on flavin enzymes of tissues. J. Biol. Chem. 223: CHIRIGOS, M., GREENGARD, P., and UDENFRIEND, S. (196). Uptake of tyrosine by rat brain in viuo. J. Biol. Chem. 235: DANIEL, R. G. and WAISMAN, H. A. (1969). The influene of exess methionine on the free amino aids of brain and liver of the weanling rat. J. Neurohem. 16: DE MARCHI, W. J. and JOHNSTON, G. A. R. (1969). The oxidation of glyine by D- amino aid oxidase in extrats of mammalian entral nervous tissue. J. Neurohem. 16: DE ROBERTIS, E., SELLINGER,. Z., RODRIGUEZ DE LORES ARNAIZ, G., ALBERICI, M., and ZIEHER, L. M. (1967). Nerve endings in methionine sulfoximine onvulsant rats: a neurohemial and ultrastrutural study. J. Neurohem. 14: GAITONDE, M. K. (1967). A spetrophotometri method for the diret determination of ysteine in the presene of other naturally ourring amino aids. Biohem. J. 14: GHITMNI, N. R., OHLSSON, W. G., and SELLINGER,. Z. (197). The effet of methionine on the regional and intraellular disposition of 3H-methionine sulfoximine in rat brain. J. Neurohem. 17: GHITTONI, N. E. and SELLINGER,. Z. (197). Cerebral methionine and ysteine levels after the injetion of the onvulsant methionine sulfoximine. Pharmaol. Res. Comms. 2: GOLDSTEIN, D. B. (1966). D-amino aid oxidase in brain: distribution in several speies and inhibition by pentobarbitone. J. Neurohem. 13: GUROFF, G. and UDENFRIEND, S. (1962). Studies on aromati amino aid uptake by rat brain in vivo. J. Biol. Chem. 237: HARDWICK, D. F., APPLEGARTH, D. A., COCKROFT, D. M., ROSS, P. M., and CALDER, R. J., (197). Pathogenesis of methionine-indued toxiity. Metabolism 19: HIMWICH, W. A. and AGRAWAL, H. C. (1969). Amino aids. In: Handbook of Neurohemistry, Lajtha, A. (Ed.), Plenum Press, New York, Vol. I, JOCELYN, P. C. (1962). The effet of glutathione on protein sulphydryl groups in rat liver homogenates. Biohem. J. 85: KANDERA, J., LEVI, G., and LAJTHA, A. (1968). Control of erebral metabolite levels. 11. Amino aid uptake and levels in various areas of the rat brain. Arh. Biohem. Biophys. 126: LAJTHA, A. (1968). Transport as ontrol mehanism of erebral metabolite levels. In: Progress in Brain Researh, Lajtha, A. and Ford, D. (Eds.). Elsevier Press, Amsterdam, Vol. 29, LAJTHA, A. and TOTH, J. (1962). The brain barrier system The efflux of intraerebrally administered amino aids from the brain. J. Neurohern. 9:

16 GHITTONI AND SELLINGER LAJTHA, A. and TOTH, J. (1963). The brain barrier system. V. Stereospeifiity of amino aid uptake, exhange and efflux. J. Neurohem. 1: LAJTHA, A. and TOTH, J. (1965). The effets of drugs on uptake and exit of erebral amino aids. J. Neurohem. 14: LAMAR, C., JR., and SELLINGER,. Z. (1965). The inhibition in uiuo of erebral glutamine synthetase and glutamine transferase by the onvulsant methionine sulfoximine. Biohem. Pharmaol. 14: LIPMANN, F. and TUTTLE, L. C. (1945). A speifi miromethod for the determination of ayl phosphates. J. Biol. Chem. 159: NAKAMURA, R. (1963). The transport of histidine and methionine in rat brain slies. J. Biohem. (Tokyo) 53: NEIMS, A. H., ZIEVERINK, w. D., and SMILACK, J. D. Distribution of D-amino aid oxidase in bovine and human nervous tissue. J. Neurohem. 13: ROBINS, E., ROBERTS, N. R., EYDT, K. M., LOWRY,. H., and SMITH, D. E. (1956). Mirodetermination of a-keto aids with speial referene to mali, lati, and glutami dehydrogenases in brain. J. Biol. Chem. 218: SCHMITT, J. H. and ZENK, M. H. (1968). Determination of D-amino aids by stereospeifi enzymi aetylation. Anal. Biohem. 23: SELLINGER,. z., AZCURRA, J. M., and OWLSSON, W. G. (1968). Methionine sulfoximine seizures. VIII. The dissoiation of the onvulsant and glutamine synthetase inhibitory effets. J. Pharmmol. Exptl. Therp. 164: SHAH, N. S., KAMANO, A., GLISSON, S., and CALLISON, D. (1968). Studies on the uptake of radiolabeled Dopa, 5-HTP and tryptophan in rat tissues in vivo: effet of methionine, tryptophan and some keto aids. Int. J. Neuropharmaol. 7: STURMAN, J. A., COHEN, P. A., and GAULL, G. E. (197). Metabolism on L-j5S-methionine in vitamin Bg defiieny: observations on ystathionuria. Biohem. Med. 3: 51Cb-523. YANG, S. F., Ku, H. S., and PRATT, H. K. (1967). Photohemial prodution of ethylene from methionine and its analogues in the presene of flavin mononuleotide. J. Bid. Chem. 242: