Journal of Cerebral Blood Flow and Metabolism 12:603-612 © 1992 The International Society of Cerebral Blood Flow and Metabolism Published by Raven Press, Ltd., New York

Regional Cerebral L-e4C-Methyl]Methionine Incorporation into Proteins: Evidence for Methionine Recycling in the

Rat Brain

§Anna M. Planas, Christian Prenant, *tBernard M. Mazoyer, :j:Dominique Comar, and Luigi Di Giamberardino

INSERM U.334 and *CEA, Service Hospitalier Frederic loliot, Orsay, and tAntenne d'Informatique Medicale, Hopital Robert Debre, Paris, France; and :f:Centre de Recherches du Cyclotron, Universite de Liege, Liege, Belgium

and §Departmento de Farmacoiogia y Toxicoiogia, CSIC, Barcelona, Spain

Summary: The specific activity (SA) of free methionine was measured in plasma and in different regions of the rat brain at 15, 30, or 60 min after intravenous infusion of L-[I4C-methyl]methionine . Within these time periods, an apparent steady state of labeled free methionine in plasma and in brain was reached. However, the brain-to-plasma free methionine SA ratio was found to be �0.5, showing that an isotopic equilibrium between brain and plasma was not attained. This suggests the presence of an endog­enous source of brain free methionine (likely originating from protein breakdown), in addition to the plasma

Attempts to measure the regional rate of protein synthesis in the brain with positron emission tomog­raphy (PET) have been made using several llC_ labeled amino acids, principally L-[llC-methyl]­methionine (Bustany et ai., 1983, 1985; Ericson et ai., 1987) and L-[1_11C]leucine (Phelps et ai., 1984; Hawkins et ai., 1989). L-[llC-Methyl]Methionine is currently employed in PET for the evaluation of local amino acid uptake in various physiological and pathologic conditions of the human brain (Comar et ai., 1981; Bergstrom et ai., 1987a,b; Mosskin et ai., 1987; Hatazawa et ai., 1989; Mineura et ai., 1991; O'Tuama et aI., 1991). Yet for neither methionine nor leucine has a satisfactory model been so far

Received July 3, 1991; final revision received November 19, 1991; accepted February 10, 1992.

Address correspondence and reprint requests to Dr. A. M. Planas at INSERM U .334, SHFJ, 4, Place du General Leclerc, F-91401 Orsay Cedex, France.

Abbreviations used: ANOYA, analysis of variance; OPT, o-phthaldialdehyde; PCA, perchloric acid; PET, positron emis­sion tomography; SA, specific activity; TCA, trichloroacetic acid.

603

source. The contribution of this endogenous source to the content of free methionine varies significantly among the different brain regions. Our results indicate that the regional rates of protein synthesis measured with L­

[ " C-methyi]methionine using positron emission tomogra­phy would be underestimated, since the local fraction of brain methionine derived from protein degradation would not be considered. Key Words: Apparent steady state­Brain amino acid recyciing-L-[I4C-methyi]Methionine­Positron emission tomography-Protein turnover­Regional cerebral protein synthesis.

developed that would allow the estimation of local rates of cerebral protein synthesis (Smith et ai., 1988; Hargreaves-Wall et aI., 1990). This is mostly due to the fact that amino acids derived from pro­tein breakdown in the human brain might be recy­cled for protein synthesis. Recycling has been shown to occur in the rat brain by the presence of a pool of leucine (and other amino acids) (Seta et ai., 1973; Smith et ai., 1988; Hargreaves-Wall et ai., 1990) and a pool of leucyl-tRNA (Smith et ai., 1988; Hargreaves-Wall et ai., 1990) and valyl-tRNA (Smith et ai., 1991) that do not readily exchange with the plasma. No similar studies have been made with 14C-methyl- or IIC-methyl-Iabeled methionine, although the absence of amino acid recycling has been reported in a recent study using L-esS]methio­nine (Grange et ai., 1991a,b).

In the present work we examined the fate of L-e4C-methyl]methionine in the plasma and in sev­eral regions of the rat brain after systemic infusion. We measured the concentrations of the labeled frac­tions, i.e., free methionine, metabolites, and pro­teins, and the endogenous content of free methio-

604 A. M. PLANAS ET AL.

nine and proteins in plasma and in different brain regions. These measurements allowed for the eval­uation of the contribution of protein breakdown to the content of free methionine in different regions of the rat brain and for the estimation of the corre­sponding rates of methionine incorporation into proteins.

MATERIALS AND METHODS

Materials L-e4C-MethyllMethionine (50 mCi/mmol) was pur­

chased from New England Nuclear (Dupont, Paris, France) as a solution containing 70% ethanol, which was evaporated under vacuum at 2Ye. Purity was checked by HPLC analysis using a precolumn derivatization tech­nique (see below). Prior to injection, L-[14 C-methyll­methionine was dissolved in saline containing 0.05% J3-mercaptoethanol. Reagents, unless otherwise noted, in­cluding o-phthaldialdehyde (OPT) and methionine, were from Sigma Chemicals (La Verpilliere, France). Buffers for HPLC were prepared using Milli-Q grade water (Mil­lipore S.A., Saint-Quentin-en-Yvelines, France) and methanol was from Merck (Nogent-sur-Marne, France). HPLC column eluants were filtered with O.5-fLm FH­Millipore filters and degassed by sonication. Scintillation liquid (Optifluor) was from Packard Instruments (Rungis, France), and disintegrations per minute was measured by the external standard method with a Packard J3-counter.

Animals Ten male Sprague-Dawley rats (243 ± 24 g body wt)

with ad libitum access to food and tap water were can­nulated under halothane anesthesia in a tail vein and an artery. Rats were allowed to recover from the anesthesia under mild restraint for � 2 h before the beginning of the experiment.

Experimental procedure [14C]Methionine infusion. Constant concentrations of

[14Clmethionine in plasma were attained by the infusion technique of Patlak and Pettigrew (1976) using a program­mable pump (Harvard Apparatus, Les Ulis, France). The infusion program was calculated from the plasma time course of [14Clmethionine, determined by independent experiments in a different set of rats after a single intra­venous bolus injection of e4C]methionine. The infusion technique can be summarized as follows: At zero time a [14Clmethionine bolus injection (36 fLCi) was given simul­taneously with the beginning of the infusion of a e4Clme­thionine solution (0.1 /-LCi//-L1) dispensed by the pro­grammed pump. The total amount of [14Clmethionine in­jected was � 100 fLCi at 15 min, 150 /-LCi at 30 min, and 200 fLCi at 60 min. This was equivalent to 1.75, 2.63, and 3.5 fLmol of methionine, respectively.

During the infusion period, arterial blood samples were withdrawn at different times and rats were decapitated at either 15 (n = 3), 30 (n = 4), or 60 (n = 3) min. Decap­itation was carried out immediately after the last blood sample was taken and without stopping the infusion. The brain was rapidly removed, kept on ice, and dissected into eight different regions: cerebellum, medulla, stria­tum, hippocampus, hypothalamus, thalamus, cortex, and white matter, the latter not corresponding to any partic­ular anatomical cerebral region but to a pool of white

J Cereb Blood Flow Metab, Vol. 12, No. 4, 1992

matter-enriched areas. Brain samples were rapidly weighed, immersed in liquid nitrogen, and then stored at - 80°C until further analysis.

Plasma was separated from arterial blood and plasma proteins were precipitated with 5% trichloroacetic acid (TCA; 30 fLI TCA/20 fLl plasma) and centrifuged at 11, 300 g for 5-10 min at 4°C. Pellets were washed twice with 30 fLl of TCA before being solubilized with 30 fLI of 1 N NaOH and kept overnight at 4°C prior to the measure­ment of protein content using the Lowry et al. method (1951) and to the determination of protein-associated ra­dioactivity. Total radioactivity from the supernatant was measured, and an HPLC analysis was carried out to sep­arate free [14C]methionine from its labeled metabolites and to measure the free endogenous methionine normally present in plasma.

Brain tissue was homogenized in 5 vol (wt/vol) of 0.33 M perchloric acid (PCA) by sonication on ice. Total tissue radioactivity was determined in aliquots of homogenized tissue, and the homogenate was then centrifuged at 25,000 g for 10 min at 4°C. The supernatant was filtered through a 0.45-fLm Millex-HV 4 filter (Millipore S.A.), ra­dioactivity was measured, and an HPLC analysis was performed, as will be described later. The cerebral pro­tein-containing precipitate was washed twice with 5 vols of 0.33 M PCA followed by 0.16 M PCA to eliminate remaining acid-soluble products. The final precipitate was solubilized with 1 N NaOH (same volume as that used for PCA precipitation) and kept overnight at 4°C prior to the measurement of radioactivity and protein content.

HPLC analysis A precolumn fluorescent reaction was performed in

plasma and tissue acid-soluble fractions using OPT as the derivatizing agent, basically according to the method of Roth (1971). Briefly, at least I day before utilization, a stock of OPT -reduced solution consisting of 0.05 M OPT, 24.97% ethanol, 74.9% borax (2% sodium tetraborate, pH 10), and 0.13% J3-mercaptoethanol was prepared. This so­lution was diluted three times with the borax solution, at least 1 h before use. The acidity of the samples was neu­tralized by the OPT alkaline solution. Two volumes of this solution were added to 1 vol of the samples, allowing the reaction to take place in the dark during 1.5 min. After this time, samples were injected into the HPLC system. HPLC separation was carried out in a reverse-phase an­alytical column (/-LBondapak-CI8, 10 fLm x 30 cm; Wa­ters-Millipore) equipped with a Guard-Pak pre column (CI8; Waters) using an isocratic system with acetate buffer (pH 5.5, 40% 0.1 M potassium acetate with 0.01 M ethylenediaminetetraacetate and 60% methanol) running at I ml/min. The sample methionine content was quanti­fied with fluorimetric detection of the OPT-derivatized amino acid by using the external standard method. Frac­tions were collected sequentially every 0.5 min to mea­sure radioactivity.

THEORETICAL APPROACH TO EVALUATE

THE CONTRIBUTION OF PROTEIN

BREAKDOWN TO BRAIN FREE METHIONINE

AND METHIONINE INCORPORATION RATE

In steady-state conditions, the free methionine content of brain (MT, nmollg) equilibrates with

METHIONINE INCORPORATION INTO BRAIN PROTEINS 605

plasma free methionine (Mp, nmol/ml). A pro­grammed intravenous infusion of [14C]methionine allows the achievement of an apparent steady state between free [14C]methionine in the brain (MT *, nCi/g) and in the plasma (Mp *, nCi/mI). Let (J be the brain-to-plasma free methionine specific activity (SA) ratio measured during a programmed infusion of labeled methionine:

MT*/MT (J = Mp*/Mp

(1)

A value of (J < 1 indicates that, during the experi­mental time, there should be an endogenous source supplying brain free methionine (i.e. , protein break­down), since not all free methionine in the brain tissue derives directly from the plasma. The per­centage contribution of protein breakdown to brain free methionine would then be (I - (J) x 100.

An apparent rate of methionine incorporation into proteins (Ra, nmollmin/g of brain) can be cal­culated as follows:

P*(D Ra = -------

JOT[(Mp*/Mp)t]dt

(2)

where P* (nCi/g brain) is the measured amount of labeled methionine that has been incorporated into proteins during the infusion time T (min) and the denominator is the time integral of the plasma SA.

Assuming that the brain SA of methionyl-tRNA is identical to that of free methionine, the true rate of methionine incorporation into proteins (R) can be calculated as follows (Hargreaves-Wall et al. , 1990):

R-1

Statistical analyses

P*/T MT*/MT

(3)

Statistical analyses were carried out using the BMDP package (BMDP Statistical Software, Los Angeles, CA, U. S.A.). Two-way analyses of vari­ance (ANOY As) with a repeated-measures design were carried out to test differences between groups of time and between brain regions. By taking the regions as the within factor, these analyses ac­counted for the dependence between brain regions arising from the same rat. The grouping factor was the time (IS, 30, or 60 min). Differences between brain regions were tested using pooled pairwise Stu­dent t tests by taking the variance given by the ANOY A. Their degree of significance was evalu­ated using the Bonferroni probability, which cor­rects for the large number of tests made. Results are presented as mean values ± SD.

RESULTS

Plasma

[14C]Methionine infusion. Figure 1 shows typical results from IS-min and I-h infusions of [14C]me­thionine. Constant plasma levels of [14C]methionine (nCi/ml) were achieved as soon as 5-10 min after the beginning of the infusion. The average ± SD (n

= 10) constant plasma level of [14C]methionine was 92 ± 51 nCi/ml, corresponding to an increase of 3.9% of the mean endogenous methionine level, which was found to be 41 ± 6 nmol/ml. After 10--15 min, a 14C-Iabeled acid-precipitable fraction (i.e., protein fraction) appeared in the plasma and in­creased steadily. The plasma acid-soluble radioac­tive fraction contained not only free labeled methi­onine but also its metabolites, which exhibit a very moderate increase up to 60 min. At this time, 38% of the label in the acid-soluble fraction corre­sponded to free labeled metabolites in plasma.

Brain regions

[14C]Methionine time course. The accumulation of labeled methionine in different brain regions was

0.3,-------------------,

0.2

E 0.1 ;:;; (J

---- Methionine (!) ---fi--- Metabolites

:::1. - rs-----fr--_�-6--tJ, � o+-�---�--�--�----�

:: 0 5 10 15 > ;: Time (min) to,) as o

'C as �

as E (/j as

Il.

0.4

0.3

0.2

0.1

0 0

rr---

15

---- Methionine --0---· Metabolites ---0-- Proteins

30 45 Time (min)

@

60

FIG. 1. Time course of plasma 14C-label expressed as f.LCilml of plasma. Values correspond to [14C]methionine, 14C_ labeled proteins, or 14C-labeled metabolites of representative rats infused with [14C]methionine for 15 min (a) and 1 h (b). The programmed infusions were performed according to the method of Patlak and Pettigrew (1976).

J Cereb Blood Flow Metab, Vol. 12, No.4, 1992

606 A. M. PLANAS ET AL.

measured at 15, 30, and 60 min of infusion. Table 1 shows the mean cerebral distribution of total brain label label incorporated into brain proteins (i.e., the �cid-precipitable fraction), free [14C]methio­nine and labeled metabolites. Data shown in this tabl� were obtained by averaging the normalized measurements of eight brain regions for three to four rats per time point. Normalization was carried out for each brain region of each rat by dividing by the plasma e4C]methionine concentration. The to­tal brain radioactivity increased 125% from 15 to 60 min. The incorporation of label into proteins seemed to be linear between 15 and 60 min (r =

0.9). At 15 min, �35% of total brain label had al­ready been incorporated into proteins; at 30 min, label incorporation was 57%; and at 60 min, it Was 61 %. The remaining percentage of the total label corresponded to acid-soluble radioactivity (e4C]­methionine and its labeled metabolites). The per­centage of unmetabolized e4C]methionine was 42% at 15 min, 16% at 30 min, and 14% at 60 min. La­beled metabolites represented 23% at 15 min, 27% at 30 min, and 25% at 60 min. The level of free e4C]methionine was found to be constant between 15 and 60 min (see Table 1).

The apparent distribution ratios of [14C]methio­nine in different brain regions are presented in Ta­ble 2. No statistically significant differences be­tween 15, 30, and 60 min were found using a two­way ANOVA (F = 1.4; df = 2, 7; p = 0.3), or between brain regions (F = 1.2; df = 7, 72; p =

0.3). Label distribution in brain regions. The regional

distribution of the labeled proteins, the free e4C]­methionine, and the labeled metabolites was deter­mined for each time point and is presented here for the 60-min infusion (Fig. 2). At this time, the mean value for labeled proteins was 140 ± 84 nCi/g for the whole brain (mean of eight cerebral regions). The corresponding concentration of free [14C]methion­ine was 27 ± 12 nCi/g. Comparison between regions was carried out by a two-way ANOV A after nor-

TABLE 1. Brain 14C-label distribution

Brain 15 min 30 min 60 min fraction (n = 3) (n = 4) (n = 3)

Total 0.778 ± 0.20 1.854 ± 0.24 2.883 ± 0.47 Proteins 0.282 ± 0.07 1.057 ± 0.17 1.759 ± 0.30 Metabolites 0.247 ± 0.15 0.988 ± 0.19 1.349 ± 0.92 Methionine 0.292 ± 0.10 0.284 ± 0.04 0.341 ± 0.03

Values are the averages ± SD of eight brain regions per rat for (n) rats per time group, expressed as nCi/g .brain normalized by nCi/ml of plasma [14C]methionine. Companson between th

.e la­

beled brain methionine concentrations at the different times showed no statistically significant differences.

J Cereb Blood Flow Metab, Vol. 12, No.4, 1992

TABLE 2. Apparent distribution ratios for [14C]methionine

Region

Cerebellum Medulla Striatum Hippocampus Hypothalamus Thalamus Cortex White matter

15 min (n = 3)

0.304 ± 0.11 0.267 ± 0.06 0.331 ± 0.14 0.280 ± 0.12 0.291 ± 0.12 0.264 ± 0.10 0.338 ± 0.11 0.258 ± 0.05

30 min (n = 4)

0.263 ± 0.16 0.209 ± 0.05 0.271 ± 0.16 0.314 ± 0.13 0.339 ± 0.06 0.256 ± 0.03 0.350 ± 0.04 0.273 ± 0.02

60 min (n = 3)

0.318 ± 0.02 0.364 ± 0.11 0.406 ± 0.08 0.227 ± 0.06 0.500 ± 0.21 0.365 ± 0.02 0.446 ± 0.16 0.331 ± 0.05

Values are the means ± SD of three to four rats at each time and are presented as nCi/g brain [J4C]methionine normalized, for each rat by the corresponding nCi/ml plasma e4C]methionine. A two-wa; analysis of variance showed no significant differences between time groups.

malizing values for the corresponding plasma e4C]methionine concentration. At 60 min, the amount of label associated with tissue proteins was found to be significantly different between brain re­gions (F = 3.19; df = 7, 14; P < 0.03). However, no regional differences became apparent for the amount of free e4C]methionine and 14C-Iabeled me­tabolites. Therefore, the brain radioactive acid­soluble fraction containing free e4C]methionine and 14C-Iabeled metabolites was found to be relatively evenly distributed throughout the brain (see Fig. 2). On average, �65% of the acid-soluble label (i.e., not associated with proteins) corresponded to la­beled metabolites at 60 min, while the labeled me­tabolites accounted for <40% of free radioactivity in the plasma.

Regional endogenous methionine and protein contents. Results of the content of free endogenous methionine in the brain from rats studied at 15, 30, and 60 min were pooled together, since no signifi­cant differences between times were found by ANOVA (F = 0.58; df = 2, 7; p = 0.58). The average of the mean brain regional methionine con­tent was 26 ± 8 nmol/g. Mean regional results are shown in Table 3. Differences between regions were found by a two-way ANOVA (F = 5.20; df =

7, 63; p < 0.001). Comparison between regions showed that the white matter (p < 0.01), medulla (p < 0.005), and cerebellum (p < 0.001) all had a smaller methionine content than the striatum.

Results of the measurement of regional protein content are also shown in Table 3. No correlation was observed between the protein content and the amount of free methionine in the different brain re­gions.

Brain-to-plasma SA ratio

[14C]methionine SA was determined for each brain region and plasma at 15, 30, and 60 min. No

METHIONINE INCORPORATION INTO BRAIN PROTEINS 607

FIG. 2. Tissue label distribution of 14C after 60-min infusion of [14C]methionine. Values are f1Cilg of brain normalized by the concentration of [14C]methionine in plasma and are the means ± SD of three rats. Results are presented for eight different regions (cerebellum, CE; me­dulla, ME; striatum, STR; hippocampus, HIP; hy­pothalamus, HY; thalamus, TH; cortex, CTX; white matter, WM).

>- 3 -> -(,,) CO o 't:I CO ...

c: CO ...

.a " CI) N CO E ... o Z

2

1

o

statistically significant differences between time points (F = 1; df = 2, 7; p = 0.4) were found. The brain-to-plasma SA ratios were calculated for the different brain regions and are presented in Table 4 for each time. The mean ratio of brain to plasma was 0.50 ± 0.16. The ANOV A showed that glo­bally, within the brain, there were significant differ­ences between regions (F = 3; df = 7, 63; p < 0.01). However, the comparison between particular regions did not show any specific differences.

Sources of brain free methionine

The fraction of brain free methionine supplied by the plasma and that originated from protein degra­dation were calculated (see Materials and Methods) from the e4C]methionine results presented above. The estimations of these fractions for different brain regions are presented in Fig. 3.

The amount of brain free methionine supplied by protein breakdown varied significantly between brain regions (F = 4.5; df = 7, 63; p < 0.001) and was higher in striatum than in white matter (p <

TABLE 3. Brain regional methionine and protein contents

Brain Methionine Protein region (nmol/g tissue) (mg/g tissue)

Cerebellum 18.95 ± 5.69 98.8 ± 8 Medulla 19.97 ± 5.81 112.4 ± 5 Striatum 33.06 ± 7.08 104.7 ± 15 Hippocampus 28.58 ± 8.93 89.9 ± 17 Hypothalamus 28.43 ± 8.21 75.5 ± 15 Thalamus 26.24 ± 7.24 107.8 ± 20 Cortex 28.23 ± 7.82 113.1 ± 9 White matter 21.11 ± 5.01 \03.9 ± 19

Values are the means ± SO of \0 rats. The regional methionine and protein contents were found to be not significantly different between rats infused with [14C]methionine for different periods of time, so results were pooled.

• Methionine

Brain Regions

� Metabolites o Proteins

0.02), cerebellum (p < 0.001), and medulla (p < 0.001). The average of the different brain regions was 14 ± 8 nmollg. In contrast, the amount of me­thionine supplied by the plasma was found to be very similar between brain regions (F = 1.2; df =

7, 63; p = 0.3), with an average of 13 ± 4 nmol/g (see Fig. 3).

Estimation of rates of methionine incorporation

into proteins

The average rate of label incorporation into pro­teins (P*/D was 2.5 ± 0.7 nei/min/g. The ANOV A test showed global significant differences between brain regions (F = 7.2; df = 7, 49; p < 0.001). Nevertheless, specific differences between particu­lar regions did not became apparent.

The apparent rates of methionine incorporation (Ra) were calculated as described in Materials and Methods, and mean regional rates are shown in Ta­ble 5. The two-way ANOVA showed that rates

TABLE 4. Regional ratios of brain to plasma methionine specific activity (SA)

Brain SA/plasma SA

Brain 15 min 30 min 60 min region (n = 3) (n = 4) (n = 3)

Cerebellum 0.50 ± 0.20 0.24 ± 0.11 0.70 ± 0.02 Medulla 0.60 ± 0.24 0.68 ± om 0.60 ± 0.13 Striatum 0.37 ± 0.12 0.29 ± 0.12 0.50 ± 0.05 Hippocampus 0.34 ± 0.12 0.38 ± 0.14 0.57 ± 0.03 Hypothalamus 0.42 ± 0.20 0.54 ± 0.13 0.58 ± 0.09 Thalamus 0.47 ± 0.23 0.47 ± 0.05 0.52 ± 0.\3 Cortex 0.35 ± 0.15 0.60 ± 0.16 0.68 ± 0.25 White matter 0.47 ± 0.08 0.56 ± 0.16 0.60 ± 0.17

Values are the means ± SO of (n) rats. The regional ratios of brain to plasma SA were found to be not significantly different between rats infused with [14C]methionine for different periods of time.

J Cereb Blood Flow Metab, Vol. 12, No. 4, 1992

608 A. M. PLANAS ET AL.

-C)

� o E t: -

CI) t: t: o .-

s::. -CI) E t: as � a::I

40 • from plasma

� from protein-breakdown

30

20

10

o CE ME STR HIP HY

Brain regions

were not significantly different between the studied times. However, regional Ra values were globally different (F = 17.3; df = 7, 49; p < 0.001). Regional comparisons carried out using pairwise Student t tests showed that Ra was higher in cerebellum than in striatum and in white matter (p < 0.05). The brain average Ra was 0.87 ± 0.3 nmol/min/g brain tissue.

The rate of methionine incorporation into brain proteins (R) was estimated at 2.3 ± 1 nmollmin/g tissue. Estimations for the different brain regions are shown in Table 5. Although no specific signifi­cant differences between particular regions were found, the ANOV A test showed global differences between the brain regions (F = 2.7; df = 7, 49; p < 0.02). Nevertheless, this test showed no significant differences (F = 1.18; df = 6, 42; p = 0.3) if only gray matter regions were considered. The average rate of total incorporation for the gray matter was

TABLE 5. Regional rates of methionine incorporation into brain proteins

Brain region

Cerebellum Medulla Striatum Hippocampus Hypothalamus Thalamus Cortex White matter

Apparent incorporation

rate per g of brain tissue (nmollmin/g)

Ra 1.10 ± 0.4 0.96 ± 0.3 0.69 ± 0.2 0.79 ± 0.4 0.79 ± 0.3 0.90 ± 0.3 1.02 ± 0.3 0.65 ± 0.2

Incorporation rate per g

of brain tissue (nmol/min/g)

Ri 2.05 ± 0.9 2.08 ± 0.5 2.58 ± 1.5 2.99 ± 2.2 2.42 ± 1.1 2.61 ± 0.8 2.67 ± 0.9 1.58 ± 0.7

Values are the means ::t SD of \0 rats. Ra is the apparent rate of methionine incorporation into proteins; Ri is the rate of me­thionine incorporation into proteins.

J Cereb Blood Flow Me/ab, Vol. 12, No.4, 1992

TH CTX WM

FIG. 3. Free methionine content in brain originated either di­rectly from plasma or from pro­tein degradation. Values were estimated from the measured brain-to-plasma specific activity ratio (0') and the tissue methio­nine content (MT) (see Materials and Methods). Results show the means ± SO of 10 rats (nmol/g brain) and are presented for eight cerebral regions (cerebel­lum, CE; medulla, ME; striatum, STR; hippocampus, HIP; hypo­thalamus, HY; thalamus, TH; cortex, CTX; white matter, WM).

2.6 ± 1.3 nmollmin/g, significantly different (p < 0.001) from that of the white matter, which was 1.6 ± 0.7 nmollmin/g.

DISCUSSION

The use of methyl-labeled methionine for PET studies (Comar et aI., 1981; Bustany et aI., 1983, 1985; Ericson et aI., 1987) has been regarded as more problematic than that of carboxyl-labeled leu­cine (see, e.g., Smith et aI., 1980; Phelps et aI., 1984; Hawkins et aI., 1989) owing to an active de­methylation pathway leading to many different la­beled metabolic compounds in both plasma and brain (Jones et aI., 1985; Lundqvist et aI., 1985; Buonomo et aI., 1988; Ishiwata et aI., 1988, 1989, 1991). This may explain why no satisfactory com­partmental model has been so far developed to accurately describe the kinetic behavior of L­e'C-methyl]methionine in humans (Mazoyer et aI., 1989). Our study indicates that relatively more la­beled metabolites were produced in the brain than in the plasma and underscores the need for further studies to develop a kinetic model accounting for brain methionine metabolism.

The methionine SA was found to be two times lower in the brain than in plasma (mean brain-to­plasma SA ratio = 0.5). This suggests that there is a fraction of methionine in the brain that is not di­rectly supplied by plasma. Evidence shows that a similar phenomenon does exist in the brain for var­ious amino acids (including valine, lysine, tyrosine, arginine, leucine, and glycine), since full equilib­rium between the SA of free amino acids in plasma and brain cannot be reached, sometimes even after 6 h of infusion (Seta et aI., 1973; Smith et aI., 1988;

METHIONINE INCORPORATION INTO BRAIN PROTEINS 609

Hargreaves-Wall et aI. , 1990). In contrast, Grange et ai. (1991a,b) reported the brain-to-plasma SA ra­tio to be unity after the infusion of e5S]methionine, indicating that no methionine recycling would be taking place. We have no explanation for this dis­crepancy.

The actual immediate precursor pool for protein synthesis is the pool of aminoacyl-tRNA. During steady-state conditions, the brain SA of aminoacyl­tRNA has been found to be similar to the SA of the corresponding free amino acid in studies carried out using labeled leucine (Smith et al. , 1988; Har­greaves-Wall et aI. , 1990) and also using [35S]me_ thionine (Grange et aI. , 1991a,b).

Images of the brain have been obtained after the administration of labeled amino acids to animals us­ing autoradiography (e.g. , Dwyer et aI. , 1982; Ing­var et aI. , 1985; Lestage et aI. , 1987; Bodsch et aI. , 1988; Kirikae et aI. , 1988) and to humans using PET (e.g., Bustany et aI. , 1983; Ericson et aI. , 1987; Hawkins et al. , 1989). These images showed marked differences in the total accumulation of la­bel in the different brain regions, which were attrib­uted to differences in the rate of amino acid incor­poration into proteins. Moreover, autoradiographic images showing real regional differences in the ac­cumulation of label into proteins have been ob­tained by washing the free label out of the tissue sections (Y oshimine et aI. , 1987; Widmann et aI. , 1991). In agreement with these observations, we showed here that while the concentrations of la­beled methionine and labeled metabolites presented little variation among the different brain regions studied at a given time, there was significant re­gional variation in the incorporation of label into proteins (see Fig. 2) and in the apparent rate of methionine incorporation (Ra) (see Table 5). Within the regions with high content of gray matter (all the regions studied but the white matter), we found a correlation (r = 0.78, p < 0.05) between the appar­ent incorporation rate (Ra) and the brain-to-plasma SA ratio (rr) . This indicates that the brain SA is related to the incorporation of label into proteins in each region. The relationship between these two independent measurements may be relevant to techniques that do not allow for the direct determi­nation of brain SA (i.e. , PET and autoradiography).

Our results indicate that error might arise, when measuring cerebral rates of amino acid incorpora­tion into proteins, if the fraction of brain methionine derived from amino acid recycling is not taken into account (not only for the calculation of the absolute rate of amino acid incorporation into brain proteins, but also for the study of the relative rates in the different brain regions). The estimation of amino

acid incorporation into proteins 1 or 2 h after the intravenous injection of labeled tracers would ac­count for only � 50% of the total incorporation in brain tissue due to the dilution effect of amino acid recycling. In addition, a simple twofold global cor­rection of the measured rate of protein synthesis (Ra) might not be valid since we found no correla­tion between the apparent rate of incorporation (Ra) and the rate of incorporation (R) for the different brain regions. This indicates that the apparent rates of methionine incorporation (the only rates that can be determined in PET studies) may not reflect the real rates of methionine incorporation into proteins. Therefore, care must be taken in interpreting any alterations in the apparent rates found under differ­ent pathologic conditions.

The white matter-enriched region showed consis­tently low values for all the parameters studied, and the total incorporation rate accounted for only 60% of that of the averaged gray matter. This suggests that protein synthesis is lower in the white than in the gray matter. Our results are in agreement with those of Smith et ai. (1980), who reported leucine incorporation rates 50% lower in white matter than in gray matter.

We have found the average total brain rate of methionine incorporation into proteins to be 2.6 nmollmin/g brain for the gray matter and 1.6 nmoll min/g for the white matter. Hargreaves-Wall et ai. (1990) have calculated the rates of leucine incorpo­ration into brain proteins from the label incorpora­tion rate over the SA of the brain leucyl-tRNA or the SA of the total brain free leucine. They have reported rates for leucine incorporation (0.6 nmol/ min/g) much lower than the rates we are presenting here for methionine. However, their experiments were carried out in anesthetized rats, and anesthe­sia may decrease the levels of brain amino acid up­take and protein synthesis (Hawkins et aI. , 1982; Miller et aI. , 1985; Lestage et aI. , 1987). In awake freely moving rats, Lestage et ai. (1987) found rates for methionine incorporation ranging from 0.2 to 3 nmol/g/min in �50 brain structures using autoradi­ography. Rates have also been reported for other amino acids: leucine, 3.2 nmol/min/g (Keen et aI. , 1989); phenylalanine, 5-12 nmollmin/g (Hawkins et aI. , 1982); and valine, 0.5-1.9 nmol/min/g (Kirikae et aI. , 1988).

Considering that the mean brain protein content was 100.6 mg/g tissue for global brain, the rate of methionine incorporation into proteins was 0.024 nmol/mg/min. Taking the methionine content of brain proteins to be 130 nmol/mg protein (Banos et aI. , 1973), 1% of total brain protein is synthesized (and presumably, in steady-state conditions, de-

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610 A. M. PLANAS ET AL.

graded) each hour. This corresponds to a global half-life of 2.5 days and to a turnover time of 3.6 days. However, slower turnover rates and longer half-lives are obtained when the estimations are made from the apparent rate of methionine incor­poration into proteins (Ra), i.e., based on the radio­active tracer incorporation only. In this case, we found that 0.4% brain protein is synthesized each hour. This corresponds to a half-life of 7 days and a turnover rate of 10 days, similar to those reported previously (for reviews see Dunlop, 1978; Waterlow et aI., 1978; Zak et aI., 1979, Shahbazian et aI., 1989). For example, Shahbazian et ai. (1987), using e4C]valine, reported the cerebral protein synthesis rate to be 0.6%/h, while Seta et al. (1973) have found that the turnover per hour varied from 0.3 to 0.7% depending on the amino acid considered, with half-lives ranging between 4.5 and 9 days.

Early studies (Kipnis et aI., 1961; Hod and Hershko, 1976; Vidrich et aI., 1977) suggested that the process of amino acid incorporation into pro­teins is coupled to the transport of amino acids across the cell membrane. This hypothesis would explain several in vitro observations, such as those of Gainer et ai. (1975), which showed that extracel­lular labeled leucine is incorporated into proteins preferentially to labeled leucine injected directly into neurons. An alternative hypothesis is that the radioactive amino acids incorporated into proteins derive from a precursor pool that is in equilibrium with the intracellular amino acids (Jones and McIl­wain, 1971; Mortimore et aI., 1972). In agreement with this hypothesis, the aminoacyl-tRNA synthe­tase responsible for the formation of the direct pre­cursor pool (i.e., the aminoacyl-tRNA) is associ­ated mainly with ribosomes rather than with the cell membrane (Ussery et aI., 1977).

To date, the in vivo studies have not resolved the issue of whether a membrane factor is implicated in the activation of the precursor pool for amino acid incorporation into proteins. However, in vivo ex­periments provided strong evidence supporting the hypothesis that amino acids originating from brain protein breakdown significantly contribute to brain protein synthesis (Garlick and Marshall, 1972; Seta et ai., 1973; Smith et aI., 1988; Hargreaves-Wall et aI., 1990). If recycling were a common feature for all the amino acids in the brain, methods based on the injection of radioactively labeled large neutral amino acids into the blood would underestimate the rate of cerebral protein synthesis.

In conclusion, this work presents evidence indi­cating that (a) there is an endogenous cerebral source of brain free methionine, which is likely to be the degradation of proteins; (b) the amount of

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free methionine liberated from protein breakdown varies depending on the brain region, contrarily to the amount of free methionine directly supplied by the plasma; (c) the rate of methionine incorporation appears to be homogeneous within the gray matter; (d) the total radioactivity of a particular brain region is directly related to the rate of incorporation of labeled methionine into proteins; and (e) the brain SA of the free methionine should be taken into ac­count to estimate the rate of protein synthesis.

Acknowledgment: Dr. A. M. Planas was supported by the Mercure Program of the French Foreign Office (Ser­vice pour la Science et la Technologie) and the Spanish Ministry of Education and Science (Direcci6n General de Investigaci6n Cientifica y Tecnica). The authors would like to thank Ms. R. Hassig, Ms. I. Doignon, and Ms. M. Clavier for technical assistance.

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