Folia Psychiatrica et Neurologica Japonica, Vol. 24, No. 4, 1970

L-[UJ4C] Tyrosine Metabolism of the Perfused Cat Brain with High Plasma Phenylalanine

or without Plasma Tyrosine

Shosuke WATANABE, Katsusuke MITSUNOBU, Takanori SANNOMIYA, and Saburo OTSUKI

Departriierit of Neiiro-Psychiatry, Okaynnia Wriivcrsity Medical School, Okayania

ABSTRACT Cat brain perfusion was carried out with

standard artificial blood containing 2 mg/dl of L-tyrosine and also with standard blood not containing tyrosine, for 70 minutes in each group. In addition, another group was perfused with the tyrosine-containing arti- ficial blood plus 50 mg/dl of L-phenyl- alanine. Each of these experiments was con- ducted to see how such blood would affect the tyrosine metabolism of the brain. The results are summarized briefly.

Tn the perfusion without addition of tyrosine, the tyrosine content of the brain decreased markedly. Furthcr, there was an outflow of the brain tyrosine into the venous blood. The free tyrosine of the brain is main- ly supplied from the blood tyrosine.

In the high plasma phenylalanine perfusion, the transport of the blood tyrosine into the brain was inhibited competitively by the blood phenylalanine, but output of tyro- sine from the brain to the blood was not disturbed.

( 3 ) Tn the high plasma phenylalanine perfusion, the incorporation of tyrosinc into the brain protein fraction from the brain acid soluble fraction was lower than that of

(1 )

(2 )

Received for publication Jan. 25, 1971.

the control group. During the high plasma phenylala-

nine perfusion, there was observed a marked increase of phenylalanine, and a decrease of tyrosine, threonine, isoleucine and leucine in the brain.

(4)

INTRODUCTORY STATEMENT It is known that tyrosine or the other

aromatic amino acids content of the brain rises in response to elevated blood levels of these amino acids4v8’.

The entrance of tyrosine into the brain tissue can be inhibited by the other aromatic amino acids and the long-chain nliphatics, probably by a competitive mechanism. How- ever, the direct demonstration of inhibition of tyrosine uptake by phenylalanine is diffi- cult because of the concomitant increase in blood tyrosine following intrapcritoneal ad- ministration of phenylalanineg’.

In the brain perfusion method, the com- position of artificial blood which is perfused through the brain can be prepared at will and hence it is easy to maintain the high con- centration of plasma phenylalanine or the deficiency of plasma tyrosine throughout the cxperimcnts.

In order to examine the transport of the blood tyrosine into the brain, the cat brain perfusions were conducted with blood con-

220 s. WA~AN.ABI!, K. bIITSUNOBU, T. SANNOhllYA and s. Ol-SUKI

tilining tyrosinc and also with blood not con- taining tyrosine. Next, in ordcr to examine the clfccts of high plasma phenylalanine on the metabolism of amino acids and protein of the brain. another group was perfused with blood containing tyrosine, phenyalanine and 1 U-14C]tyrosine.

MATERIALS AND METtIODS

The cat brain perfusion was con- ducted by ;I modification of Geiger's meth- 0 ~ 1 ~ ~ . This is an open perfusion system where blood once circulated through the brain does not re-cntcr the brain.

( I )

( 2 ) The standard artificial blood which con-

tains 2 mg/dl of L-tyrosine151 was used, but tyrosine was not added in the experiments w i t h blood not containing tyrosine. As for the high plasma phenylalanine the perfusion blood was prepared by adding SO mg/dl of L-phcnylnlanine to the standard blood. In addition, 10-30 of L-[U-14C]tyrosine (the product of the Radiochemical Centre, SA: 5.5 mCi/m mol) was added to 2,500 nil of the artificial blood. The purity of the [U-14C]tyrosinc sample used proved to be over 99% by two dimensional paper chro- matography. As for the control group the stanclard blood with [U-14C]tyrosine was used.

Assciys mid measurement.s of brain specimens and of cerebrul arteriul and venoiis hlood sainples: ( i ) Froctionotion of ericli .srrl>stnnce. The brain was taken out after 70 min of perfusion, frozen-dried with dry ice-acetone mixture, and the cerebral cortex was homogenized in 10 volumes of S% TCA, and the supernatant thus obtain- cd was taken ils the acid soluble fraction. The 5% TCA sediment obtained in this in- stance was washed repeatedly 5 times with 10 volumes of 5 96 TCA and the extractions wcrc carried out with acetone, ethanol: ether mixture( 3: 1 , v/v), ch1oroform:methanol

Composition of perfusion blood

( 3 )

mixture ( 2 : 1 , v/v) and ether in the ordcr mentioned. Then after evaporating the or- ganis solvents it was again dissolved in cho1oform:methanol mixture and this solu- tion was taken as the lipid fraction. The insoluble component was added to the pro- tein fraction. The sediment remaining attcr the extraction of lipid wac dissolved i n 10 volumes of 5% TCA, heated at 90°C tor 15 minutes and the nucleic acid fr? ' c t' ion was obtainedl8). The residue was washed once with 5 % TCA, and further wnshcd with acetone, then with ether, and the wa\hcd sample was taken as the protein fraction.

Isolation arid qircitititative u n r i l j ~ s i s of jrre nmino cicitls of tlie bruin. The 5 % TCA supernatant was fractionated through a Dowex 50-x4 H' column by succc\sivc elution with water and with 4 N-ammonia solution, and the latter fraction was dried under low pressure. The amino acid fraction so prepared was analyzed with the Yanagi- moto amino acid analyzer (Model LC-4), using a Anibcrlitc CG- I20 column and elut- ing with sodium cilratc buffer\. As for the de t cr m i n at ion of brain tyros i ne r ad ioac t i v i t y , the cffluent of the column containing only tyrosinc wa4 separated, desalted and the radioactivity was measured by the method to be mentioned later. The content of brain tyrosine was determined also by Udenfricnd and Cooper's methoci20'. The isolation and CI u an t i t ;I t ive an ;i I y si s of glut am ate , asp ar t ;I t c and glutaminc was done by the moelific a t ' ion of Kurahasi'c method"' alreacly clc~cribedli' .

Qitnntitcitive nnulysis of tottrl jree amino acids of the bruit?. Total free amino acids was determined with the acid solublc fraction of the brain by a modification of ninhydrin method"].

(iv) Determination of blood tyrmitie. The blood sample (whole blood) was dc- proteinized with 20% TCA and the amount of the whole blood tyrosine was determined by Udenfriend and Cooper's method2"'.

( i i )

(iii)

Tyrosine Metabolism of Perfuwd Brain 22 1

Thcn from thc tyrosinc value so obtained the amount of blood cell tyrosine was sub- tracted.

( v ) Meti.sitremerits of rritlioiictivity. To 0.5 ml of cach snniplc 12 ml of thc solution of 1 g 2.5-diplicnyloxazolc (PPO) and 100 nig I .4-bis-2-(4-mcthyl-S-phcnyloxazolyl)- benzcnc (dimcthyl POPOP) containcd in 1 liter of toluene-ethanol ( 7 : 5 , v/v) was added, and the radioactivity WIS measured with thc Packard liquid scintillation spcc- tromctcr. With a portion of spccimens ;I

given quantity was placcd in ;I counting vial, dried to solid by evaporation. then this solid substance was dissolved in 0.3 ml of 1 M- hyamin solution in methanol, and to this solution 15 ml cach of the solution of P P O and dimcthyl POPOP containcd in 1 liter of toluene was :~dtlcd. and thc ratlionctivity W;IS

measured by the identical mcthod. As for measuring the radioactivity of protcin frnc- tion thc protein fraction of a given dry weight was put into a counting vial and the radioactivity was mcnsurcd by thc abovc- mentioned hyaminc-tolucne mcthnnol.

The corrections were madc by thc extcrnal and internal stanclardization nnd the radio- activities were expressed as c1.p.m.

RESULTS Correliitiori betbt'een the blood ty-

rosine concentration rind tlie rrcid solirhle tj3rosirie conc'eritrtifiori of tlie brriiri (Tablc

( 1 )

1 ) . Using two kinds of the artificial blood to be pcrfused. one without any tyrosinc and the othcr supplcmented with 0.1 12 Ifmol 'ml of L-tyrosinc. thc brain perfusion w a s con- ducted for 70 min on 5 cats per cach arti- licial blood. In thc blood containing tyrosinc the tlin'crcncc after deducting thc amount of blood cell tyrosinc from that of wholc blood tyrosinc proved to bc 0.1 1 ~ ~ m o l / m l , which coinciclctl wcll with the amount of tyrosinc added.

The amount of thc acid solublc tyrosinc of thc brain pcrfused with blood not con- taining tyrosinc decreased mnrkcdly ; namely. two out of thc 5 cases the brain tyrosine complctcly disappeared and in thc remain- ing thrce i t became only a trace amount or 0.008 ;mol 'g brain. In addition, the avcr- agc artcriovcnous difference of tyrosine contents w a s -0.0056 pmol/g brain/min, showing acid solublc tyrosine of the brain flowing out into thc venous blood.

O n thc othcr hand, when thc plusma tyrosine concentration was maintained at the normal level of 0.1 1 ,umol/nil, thc brain tyrosinc amount w a s 0. I36 //mol/g brain. The arteriovcnous difTcrcncc of tyrosine con- tents was 0.0048 pmol,:g brain 'min, indi- cating the uptake of blood tyrosine by the br ;i i n .

( 2 ) Brrriri nietiibolism in tlie perfitsion M W i Iiigli plLi.smrr ~~heriylcilotiine. Aftcr add- ing L-[U-14C]tyrosine to artificial perfusion

Table 1 .-- Correlation Between Blood Tyrosine Concentration and Acid Soluble Tyrosine Concentration of Perfused Cat Brain

With tyrosine With tyrosine No. of exp. 5 5 Blood tyrosine concentration 0 0.11*0.02

~~- (pmol/ml)

(pmol/g brain)

of tyrosine contents -0. 0056fO.oO14 0.0048f 0.0oO4 (pmol/g brain/min)

Values are meansfS.D.

Brain tyrosine concentration 0-0.008 0.136f0.012

Arterio-venous differences

222 S. WATANAHE, K. MITSUNOBLJ, T. SANNOMWA and S. O T S U K I

Table 2.-Tyrosine Transport into Brain and Acid Soluble Tyrosine Concentration of Perfused Cat Brain

No. of exp. High plasma

phenylalalanine'h 3

Control 5

SA of tyrosine in cerebral

SA of tyrosine in cerebral

Arterio-venous difference3

venous blood

arterial blood

of tyrosine contents ( p i o l / g brain/min)

x loo 91.4-1-6.7 95.3-1-2.6

Uptake of tyrosine into blain from arterial blood 1 0.036 k 0.01 2 0.023-1-0.009 (pmol/g brainlmin)

into venous bloodt 0.031 k0.011 0.03010.008 (,umol/g brain/min)

Output of tyrosinc from brain

Tyrosine concentration of brain (pmol/g brain) 0.136+0.012 0.066tO. 008

Values arc means t S.D. The brain was pcrfused with the standard blood plus 50mg/dl of L-phenyla- lanine.

+ Calculated values. See text.

blood, thc standard brain perfusion was done on 5 cats and high plasma phenylalanine pcrfusion also on 5 cats.

( i ) The tyrosine transport into the brain m t l t i le ucid solrrble tyrosine coriceritrcitian of the brain (Table 2) . In either experi- ment the specitic activity (SA) of the venous blood was found to be 90-95% of the SA of the arterial blood, showing dilution of blood 14C-tyrosinc due to the output of tyrosine from the brain. However, there was no signilicunt difference between the control

group and high plasma phenylalanine group. On the other hand, the average arterio-

venous difference of tyrosine contents in the standard blood perfusion was 0.0048 /miol/ g brain/min, indicating the uptake of blood tyrosine by the brain, but in the high plasma phenylalanine perfusion it was, on the con- trary, -0.007 1 pmol/g brain/min, showing tyrosine tlowing out into the venous blood.

The average Concentration of acid soluble tyrosine of the brain in the standard blood perfusion was 0.136 ,umol/g brain, but i t

Table 3.--Incorporation of Blood [U-I4C]Tyrosine into Acid Soluble Fraction of Pcrfused Cat Brain

Control High plasma phenylalanine Exp. No.

I 2 3 4 5 Mean-1-S.D. 6 7 8 9 10 M e a n i S . D .

A : Total radioactivity of acid soluble 20400 5280 5320 3190 6270 21 10 fraction of brain 15800 4600 4440 1910 (d.p.m/g brain)

B : Ihlioactivity of blood tyrosine 23700 5s50 6730 13800 26200 6580 (ci. p. m . /id) 25600 6130 17800 7970

A Exloo ( 5 % ) 86.3 61.8 95.3 73.9 79.1 79.5k11.2 23.1 25.0 23.9 24.0 32.1 25.6k3.3

?Same as in the Table 2.

Tyrosine Metabolism of Perfused Brain 223

decreased to 0.066 pmol/g brain in the high plasma phenylalaninc perfu4on.

(ii) [ U-14C]tyrosine metabolism of the brain: The incorporcition of blood [U-14C] tyrosine into the acid .sohble frrrction of the bmin. In the control group the total radio- activity of the acid soluble fraction proved to be 79.5*1 1.2% of that of the blood, but in the group perfused with high plasma phenylalaninc it was 26.6L3.496, proving to be about one third of the control (Table 3 ) .

The radioactivity distribution in the amino acids of the brain was calculated, taking the radioactivity of the acid soluble fraction as 100 (Table 4). The radioactivity of gluta- mate, aspartate and glutamine was very minimal and SO-90% of the radioactivity was contained in tyrosine, which proved that the distribution of radioactivity both in the control group and high plasma phenylala-

Table 4.-Radioactivity Distribution in Amino Acids of Perfused Cat Brain

High plasma Control phenylalanine

3 3 No. of exp.

Glutamate 1 .2k0 .3 (96) 1 .6k0 .7 (%) Aspartate 0 . 3 i 0 . I 0 . 4 k 0 . 2 Glutamine 0 .3k0 . 1 0.9kO. 3 Tyrosine 87.8k3.3 80.5+3.4

Total acid soluble

Values are means_tS.D. %me as in the Table 2.

- ~~~ ~ ~ ~

~ ~ ~~ -~

100 100

nine perfusion tended to be similar, indicat- ing that in the high plasma phenylalanine perfusion the entrance of tyrosine into the brain is markedly inhibited.

The RSA of the acid solirble tyrosine of the brcrin (Table 5 ) . The RSA of the brain tyrosine obtained by dividing the SA of the brain tyrosine by the SA of the blood tyro- sine times 100 was 52.0k5.6 in the control group and it was 30.2kS.7 in the high plasma phenylalanine perfusion, which showed a relative decrease.

The incorporation of [ U-14C]tyrosine into the brain protein (Table 6 ) . In order to compare the incorporation of [U-14C]tyrosine to the brain protein in both groups, the rate of incorporation was represented by the frac- tion obtained by dividing the radioactivity of the brain protein fraction by the radio- activity of the acid solublc fraction times 100. By this representation the control group showed the value of 54.9k17.2 while the high plasma phenylalanine group 3 9 . 2 t 14.5, showing relatively low values and suggesting that there occurs disturbance in the brain protein synthesis from the amino acid pool in the brain.

Cliunges in the brain anzino acids level (Table 7 ) . After the brain perfusion with high plasma phenylalanine for 70min the amount of the brain phenylalanine was markedly increased, reaching as high a level as about 5.5 times the normal level. In con-

(iii)

Table S.-RSA of Acid Soluble Tyrosine in Perfused Cat Brain

Control High plasma phenylalanine" ~~ Exp. No.

1 2 Mean 6 7 8 9 Mean+S.D.

A : SA of acid soluble tyrosine in brain 1170(X) (d.p.m./pmol) 1110oo

tyrosine 202000 B : SA 0.f blood

(d.p.rn. /pol) 239000

41900 60500 47300 15000

98400 245000 141000 75900

57.6 46.3 52.0 42.6 33.5 24.1 19.8 30.21k8.7 A BX100 (%I

"Same as in the Table 2.

224 S. WATANABE, I(. MITSUNOBU, T. SANNOMIYA and S. O-ISUKI

Table 6.--lncorporation of [U-14C]Tyrosine into Protein Fraction of Perfused Cat Brain

Control High plasma phenylnlanine*: Exp. No.

A : Radioactivity of protein fraction 9560 1860 3900 1.510 3020 639 of brain I0300 1930 1330 5xx (d.p.ni./g brain)

B: Radioactivity of acid soluble 20400 52x0 5300 3190 6170 71 10 fraction of brain I5800 3600 44-10 1910 (d.p.m./g brain)

1 2 3 4 5 MeanI rSD. 6 7 8 9 10 hlcan 1-S.D. ~~ ~ ~~~ ~ ~

~~~

A -- x 100 (%) 46.8 6 5 . 1 35.2 41.9 73.2 51.4+14.3 47.4 29.9 67.6 30.9 30.3 3 V . 3 1 1 . 3 B

"Same a s in the Table 2.

Table 7.-Aniino Acids Level of Perfused Cat Bruin DISCUSSION

No. of exp.

~~

Glutanxite \- Aspartate 1 Glutarninet Thrconine W i n e Glycine Alaninc Valine Methioninc Isoleucine Lcucine Tyrosinel f Phenylalanine GABA Total amino

acid W

Control 4

7.31 k 1.68 1 .30 t0 .02 3.61-L-0.10 0.751k0.01 0.71 1 0 . 09 1 .33 t0 .23 0.51 + O . 21 0.25rt0.03 O.52*0. 10 0 . I5i:0.04 0.2410.05

0 . 136k0.012 0.22k0.03 1.291.0.12

39.17-kO.80

High plasnia phenylalanine'!-

4

7 . ~ 1 i l . h S l.h9+0. I 6 3 . 61 +0. 35 0.4010.11 0.76t-0.38 1.1610.21 I . 18-1-0.48 0. 37k0. 04 0.49k0.17 0. I0i-0.02 0.1310.02

0.066k0.008 I . lhF0 .02 1.17i0. 16

38.76t3.34

Expressed as pmol/g brain. Values are means

:I: Same as in the Table 2. .(_ Determined by the niodificatiod of Kura-

hasi's method. 1 (-Determined by Udcnfriend and Cooper's

met hod. S Determined by the modification of nin-

hydi-in niethod (Kosen). The others wcrc determined by amino acid

analyzer iising a Aniberlite CQ-120 column.

:t~ S. D.

trast to this, thc brain tyrosine was decreased by about 50%. Threonine, isolcucine and leucine tended to show a slight decreased.

Whcn the brain perfusion is conducted with the artiticial blood kept ai-ound tlic normal tyrosinc level of 0.1 I !cniol/nil. the level of the br:iin tyrosine can be main- taincd at around the normal value of 0.136 pmol/g brain. At that stage the nrtcrio- venous dill'erencc of tyrosine contents is 0.0048 pmol/g brain lniin, showing ;I con- stant uptake of tyrosine into thc brain. Chirigos et d."' administcrcd a lnrgc dose of L-tyrosine intraperitoneally to rats and f o u n d 1-2 hours later thc ratio of the brain tyrosine to thc plasma tyrosine to bc 1.04-1.32, showing a dynamic txilancc of tyrosinc bc- tween the brain and the plasma. Wc Ii:lve also obtained the brnin to plnsnin ratio of 1.24 by our brain pcrf'usion, ivhich pr;ic- tically coincides with their result.

On the contrary, when the brain pcrfucinn is carried out lor 70 min with tyrosinc-frcc blood. the brain tyrosine can he obscrvcd only in a trace amount but its outflow into the venous blood can be rccognized. This means that the phenyl:ilnnine hydroxylasc activity in the brain tissue is very low and the brain tyrosine is mainly supplied from the blood tyrosine. Nagatsu, Levitt and Udenfriend13' have found the presence of tyrosine hydroxylase in the brain and Ikeda.

Tyrosine Metabolism of Pcrfused Brain 225

Levitt and Udenfriend’O’ have reported that in the brain this tyrosine hydrox) lase makes phenylalanine undergo hydroxylation only to a small extent.

Now, supposing the brain tyrosine to be in a fixcd concentration at 7 0 min perfusion time, among the amount of radioactivity of artcrial blood ( R a ) , and that of vetiou< blood (RL) , SA of arterial blood tyrosinc (SA.), and that of brain tyrosine (SA b ) , and urterio- venous difference of tyrosine contents (C,-,), thc follouing two formulas can be formed. In which, A represents the amount of tyrosine uptake into the blain and y rep- resents the amount of tyrosinc output from the brain.

R, - (X i< SA a ) -t ( y >: SA I r ) = R X=C,, t-k.Y

when the amount ot the ritterial blood tyrosinc that hci\ entered to the brain and the amount of tyrosine i1ouir.g out of the brain into the vcnou\ blood arc calculated by these two formulas, the rejults come to what are shown in Table 2. Namely, in the pcrfusion of high plasma phenylalaninc, the rate of tyrosinc uptake by the brain comes to be 0.023’0.009 pmol/g brain/niin, which is 36% lcss than that nf the control ( 0 . 0 3 6 ~ 0 . 0 1 2 ,tlmol,’g brain/min), and the arterio-venous difference of tyrojinc contents is - 0.0071 2 0.0052 pmol/g b ra idmin , showing that the rate of tyrosinc output from the brain into the venous blood surpasses tlie rate of uptakc by thc brain. The exit rate of tyrosine into the venous blood is about the same in thc control and tlie high plasma phenylalanine group. These results indicate that in thc brain perfused with high plasma phenylalaninc the uptake of tyrosinc by the brain is clearly diminished and is inhibited competitively by phenylalaninc.

The amount of 14C-tyrosine incorporatcd into the acid soluble fraction of the brain in the high plasma phenylalanine perfusion

is about one third that of the control, and the tyrosine content of the brain decreases in the high plasma phenylalanine perfusion. Curoil‘ and Udenfriend*’ have demonstrated that, whcn phcnylalanine and tyrosine are injected siniultancously into the rat perito- neal cavity, the ratio of brain tyrosine con- tent to plasma tyrosinc is markedly de- creased as compared with the same in the singlc tyrosinc injection, and phcnylalanine clearly inhibits the uptake of tyrosine by the brain. With the brain sliccs of rat similar findings have been rcportcd7J4’. In the prcs- cnt experinicnts of high plasma phenylala- nine perfusion, the fact that the transport of tyrosine into the brain is inhibited coincides with the rcports of these workers, but it is an interesting finding that the amount of tyrosine outllowing from the brain into the venous blood is in the same with the control gro i t p.

Next, looking at the influence of high plasma phenylalanine on thc concentration of brain amino acids othcr than tyrosine, phcnylalanine as expeetcd increases to 5.5 times that in the control, and threonine, iso- leucine and leucine show a dcereasing tendency in the eonccntration. It is widcly recognized by many investigators”,G,8,1”’ that high circulating levels of phenylalanine raises the phenylalanine levcl in the brain. High concentrations of phenylalaninc, whether ad- ministered chronically in the dict o r acutcly by p a r e n t e d injection, have produced sig- nificant reductions of cerebral concentrations of threonine. valinc, mcthioninc, isoleucine, leucine, histidine and tyrosine2J2’.

I n the high plasma phenylalanine pcr- fusion, the incorporation of tyrosine into the brain protein is also decrcascd. It has been suggestcd that the protein synthesis of the brain is inhibitcd by the high levels of p h ~ n y l a l a n i n c ’ ~ ~ ~ ) . Therc is also the report that thc imbalance of the brain amino acids seems to be responsible for the inhibition of

226 S. W A ~ I A N ~ B I , I<. MITSUNOIIU, T. SANNOMIYA and S. O I S U K I

thc brain protcin synthesis16'

Ac I( NOW L c ffi1: hi E N T

Thc authors wish to acknowledge grate- fully the kind coopcration of Dr. Shigehiko Ihara, dircctor. and our colleagucs in the Research Institute of Psychiatry, Okayama Jikci Hospital.

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