AMERICAN JOURNAL OF PHYSIOLOGY Vol. 229, No. 3, September 1975. Printed in U.S.A.
Folate
transport
in the central
nervous
system
REYNOLD SPECTOR AND A. V. LORENZO (With the Technical Assistance of Peter Levy) Department of Neurology, Children’s Has-ital Medical Center, Department of Medicine, Peter Bent Brigham Hospital, and Departments of Medicine and Pharmacology, Harvard Medical School, Boston, Massachusetts 02111.5
SPECTOR, REYNOLD, AND A. V. LORENZO. Folate transport in the central nervous system. Am. J. Physiol. 229(3): 777-782. 1975.Methyltetrahydrofolic acid or folic acid was infused intravenously at a constant rate into conscious untreated or methotrexate-pretreated rabbits. After 150 min, at equivalent plasma concentrations, folic acid or methyltetrahydrofolic acid readily entered the choroid plexus, but only methyltetrahydrofolic acid readily entered the cerebrospinal fluid and probably brain by a saturable transport system. In contrast, after intraventricular injections, folic acid but not methyltetrahydrofolic acid was cleared from cerebrospinal fluid to blood by a saturable system. Intraventricular injection of folic acid at concentrations that saturated folic acid clearance from cerebrospinal fluid did not affect the transport of methyltetrahydrofolic acid from blood into cerebrospinal fluid. These results suggest that the transport system for methyltetrahydrofolic acid, which is about half-saturated at normal plasma concentrations, helps maintain the cerebrospinal fluid and probably brain methyltetrahydrofolic acid concentrations within relatively narrow limits. Moreover, folic acid, which the brain cannot utilize, is transported from cerebrospinal fluid. A possible locus for the systems that transport folic acid from and methyltetrahydrofolic acid into the cerebrospinal fluid is the choroid plexus. saturable;
cerebrospinal
fluid;
choroid
METHODS
AND
MATERIALS
The (&)-[14C]MeTHF (60 mCi/mmol) and [3H]FA (17, 23 Ci/mmol) were obtained from Amersham/Searle Corp., Arlington Heights, Ill. The (+)-[3H]MeTHF (160 mCi/mmol) was biosynthesized from [3H]FA in vivo in rat liver and isolated by DEAE-Sephadex and Sephadex G-15 chromatography (2, 16, 29). Crystalline FA was obtained from Sigma Chemical Co., St. Louis, MO. and methotrexate (Mtx) from Lederle Laboratories, Pearl River, N. Y. In vivo studies. In order to determine the entry of (+)MeTHF into CP, brain, and CSF from blood, unanesthetized, overnight-fasted New Zealand white rabbits, weighing 1.4-l .9 kg, were infused intravenously with 10 ml of chilled normal saline containing 5 mg sodium ascorbate, 2 mg thiourea and 4, 10, 40, or 230 lug (+)-[14C]MeTHF (26). Two milliliters of this solution were given over 10 min and the rest at a constant rate over 140 min. At the end of the infusion, the rabbit received a rapid intravenous injection of pentobarbital; and blood, 0.8-1.2 ml of cisternal CSF, CP, and brain were removed within 4 min (26). After weighing, CP were homogenized in 0.3 ml Hz0 containing 0.01 ml mercaptoethanol (ME). Whole brain, after weighing, was homogenized in 8.0 ml water. Duplicate aliquots of CSF (0.2 ml), plasma (0.2 ml), CP homogenate (0.02 ml), and brain homogenate were assayed for 14C in Aquasol (26). Th e nature of the 14C in the CSF, plasma, and CP homogenate was determined as described below (see Assays). To assess the stereospecificity of MeTHF entry into CSF, CP, and brain, two other rabbits were treated as above except that both 4 pug (+)-[14C] MeTHF and 2 pg (+)-[3H]MeTHF were placed in the infusion syringe. Folic acid entry into CP, brain, and CSF from blood was measured in rabbits infused as above, except that 10 pug FA and 50 PCi [3H]FA (rather than [14C]MeTHF) were placed in the infusion syringe and the rabbits were infused intravenously with either 0.3 mg/kg (body wt) or 3.0 mg/kg Mtx 30 min before beginning the FA infusion. The Mtx was employed to delay the metabolism of the FA (30). After sacrifice, the concentration of 3H in the tissues and the nature of the 3H in the CSF, plasma, and CP homogenate were determined (see Assays). To assess the possibility that Mtx might inhibit MeTHF entry into CSF, CP, and brain, two other rabbits were infused as above with 10 pg (+)[14CjMeTHF and no [3H]FA ‘or FA in the infusion syringe 30 min after 0.3 mg/kg iv Mtx. The concentration of i4C and the nature of the 14C in CSF, CP, and plasma were determined (see Assays). To measure the efflux of folates from CSF, either 0.1 &i
plexus
REDUCED FOLATES are present at higher concentrations in cerebrospinal fluid (CSF) and brain than plasma (1, 15). However, the enzyme that catalyzes the reduction of folic acid (FA) to tetrahydrofolic acid (THF), dihydrofolate reductase, is not present in mammalian brain (17). The high levels of reduced folates in CSF and brain are thought to be due to transport of reduced folates from blood, principally L(+)-5methyltetrahydrofolate ((+)-MeTHF) (16). This folate, (+)-MeTHF, is the major folate in plasma (2, 11). Levitt et al. (16) suggested that a transport mechanism for reduced folates from blood into CSF through the blood-CSF barrier might reside in the choroid plexus (CP). Subsequently, it was shown that the isolated CP contains a specific folate transport system. The characteristics of this system were compatible with the hypothesis that the choroid plexus is a locus of the transport of reduced folates from blood to CSF (5, 29). The purposes of this study are: I) to define the characteristics of the entry of MeTHF and FA into CSF, brain, and CP from blood; 2) to document the characteristics of the efflux of FA and MeTHF from CSF into blood, brain, and CP after intraventricular injections; and 3) to provide in vivo evidence that the CP is a locus for transport of MeTHF from blood into CSF as well as a locus for transport of FA from CSF to blood by an independent system.
777
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778
R.
of [3H]FA or [3H] inulin or 5.0 nCi of (&)-[14C]-MeTHF or (+)-(3H)MeTHF were injected into the left lateral ventricle of a rabbit anesthetized with pentothal (26). Each isotope was injected in a volume of 0.1 ml of chilled, artificial CSF which contained 50 pug sodium ascorbate, 20 pg thiourea, and, in some cases, 0.1 mg FA. After 2 h, the rabbit was reanesthetized with pentothal and sacrificed. Immediately, 0.8-1.2 ml of cisternal CSF, the left lateral and fourth ventricular CP (15 mg) and whole brain were obtained (26). The CSF, CP, and brain (after weighing) were assayed for radioactivity as above, and the nature of the radioactivity in the CSF and CP homogenate was determined (see Assays). The effect of saturating FA efflux from CSF on MeTHF influx into CSF from blood was studied by injecting 0.1 &i [3H]FA with or without 5.0 pug FA in 0.1 ml artificial CSF into the left lateral ventricle. After 30 min, 10 pg of ( &)-[14C]MeTHF in 10 ml chilled, normal saline with 5 mg sodium ascorbate and 2 mg thiourea were infused intravenously at a constant rate over 90 min into the conscious rabbit. At the end of the infusion, the animal was sacrificed and 0.8-1.2 ml of cisternal CSF, the left lateral and fourth ventricular CP, the right lateral CP, and brain were obtained. The CSF, CP, and brain (after weighing) were assayed for 3H and 14C and the nature of the 3H and 14C in CSF and the CP homogenates was determined (see Assays). The two lateral and fourth ventricular CP weighed about 20 mg. Assays. The methods for establishing the purity of the [3H]FA, ( &)-[14C]-MeTHF, ( +)-[3H]MeTHF, and [3H]inulin as determined by cellulose thin-layer (TLC) or paper chromatography have been described (3, 28, 29). In this study, for the folate chromatography, MN 300 UV 254 0.1 mm thin-layer cellulose plates (Brinkmann Instruments) were used exclusively and were chromatographed in 3 % NH&l (wt/vol), pH = 6.2 with 0.5 % ME. Except for the (+)-[3H]MeTHF, which was 90 % pure, all the other radiochemicals were greater than 95 % pure. The [3H]FA required repurification on Sephadex 25 periodically. The nature of the 14C or 3H in the CP after (a)-[14C]MeTHF, [3H]FA, or (+)-[3H]MeTHF injections was determined by the following procedures. CP homogenates (containing ME) were covered, heated at 75°C for 30 min, and centrifuged (2, 29). Duplicate aliquots (0.02 ml) of supernate and carrier (rt)MeTHF or FA were spotted and chromatographed on TLC plates (3, 29). The radioactivity corresponding to the FA or (&)MeTHF carrier spots was assayed and corrected for recovery. To determine the nature of the 14C or 3H in CSF and plasma after injections of (+)-[14C]MeTHF or (+)-[3H]MeTHF, the CSF and ultrafiltrates of plasma were chromatographed. The ultrafiltrates of plasma were obtained at 37°C by ultrafiltering 2.0 ml fresh plasma added to 0.5 ml artificial CSF through a Millipore ultrafilter (PSED 13 10) in a 2.5-ml stirred chamber at 5-lb pressure with 95 % N2: 5 % CO2 (25, 27). The first 0.2 ml of ultrafiltrate was discarded, and a total of 2 ml of ultrafiltrate from the 5-ml plasma solution (2 chambers) were collected in chilled tubes containing 5 mg sodium ascorbate and 2.0 mg thiourea. (A correction for loss through the filter which depended on
SPECTOR
AND
A.
V.
LORENZO
concentration (5-22 %) was required (25, 27).) The ultrafiltrates of plasma and CSF were then passed through small DEAE-Sephadex columns (0.6 cm x 11 cm). The (+)C3H]MeTHF (3 n C i ) was added to the CSF or ultrafiltrate of plasma to identify the [14C]MeTHF peak. The column was eluted by means of a linear concentration gradient of phosphate buffer, pH = 6, constructed by placing 45 ml of 0.1 M phosphate buffer in the mixing chamber and 45 ml of 0.8 M phosphate buffer in the reservoir (16). The buffer contained 2 mg/ml Na ascorbate. Two-milliliter aliquots were collected from the column, and the radioactivity was determined in them. In the two groups of two rabbits that received 4 pg [14C]MeTHF intravenously, the CSF samples as well as the ultrafiltrates of plasma were pooled before chrcmatography to attain adequate numbers of disintegrations per minute. In the two groups of rabbits that received 10 rug [14C]MeTHF intravenously, the CSF samples in each group were pooled before chromatography. The nature of the 3H in CSF after [3H]inulin or [3H]FA injections was determined on 0.02-ml aliquots of unaltered or concentrated CSF by paper or thin-layer chromatography (3, 28, 29). I n some samples of CSF (and brain homogenates), the percentage of 3H that was not volatile was determined by evaporating to dryness. The nature of the 3H in plasma of rabbits infused with [3H]FA was determined by adding 0.1 ml plasma, 0.3 ml HzO, and 0.04 ml ME to 1.6 ml ethanol, mixing, and centrifuging. The supernate was assayed for 3H; 1.4 ml of supernate was taken to dryness and redissolved in 0.03 ml HZO. Ten microliters were spotted on TLC plates with carrier FA, and 0.01 ml was assayed for 3H. The 3H on the FA carrier spot was measured and corrected for recovery of a standard. The binding of [3H]FA to plasma could not be determined by ultrafiltration because of excessive nonspecific binding by the filter (Millipore PSED1310) (25, 27). However, less than 5 % of [3H]FA was bound to plasma proteins as determined by gel filtration of plasma from rabbits 2) which were injected with 0.3 mg/kg Mtx and sub( nsequently received over a 2.5-h period 10 pug FA plus 50 &i [3H]FA (25). Endogenous levels of folate in overnight-fasted rabbits were measured in CSF, plasma, and supernates of CP (after treatment of CP homogenates containing ME at 75°C for 30 min) by a competitive protein-binding assay using P-lactoglobulin (32). RESULTS
The total endogenous folate in serum, CSF, and CP is indicated in Table 1. The unbound plasma, CSF, and CP concentrations of [14C]MeTHF as well as the 14C in brain in rabbits infused with ( &)-[14C]MeTHF intravenously for 2.5 h are indicated in Table 2. Also shown in parentheses are the ratios of CSF and CP [14C]MeTHF (or brain 14C) to unbound plasma [14C]MeTHF. When the CSF of those rabbits in Table 2 was chromatographed on DEAESephadex columns, the 14C appeared as several peaks but greater than 73 % of the 14C in all samples (n = 7) was associated with MeTHF. When the ultrafiltrates of plasma from those rabbits that received 4 lug (=t)-[l*C]MeTHF were chromatographed on DEAE-Sephadex columns, 65 %
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FOLATE
TRANSPORT
TABLE
THE
779
CNS
Total endogenous folate in tissues
1.
Serum CSF Choroid
~-
IN
6.5 & 1.7 q/ml 32 rt 6 q/ml 3.6 If: 1.1 ,q/g
plexus
(4) (4) (5)
Values are means Ifi SE. The numbers of determinations parentheses. Total folate in overnight-fasted animals termined in the tissues by the competitive binding assay lactoglobulin (32). TABLE 2. Total [14C]MeTHF after intravenous [14C]Me THF --____-
in tissues at 2.5 h
Amount
of (&)-[l*C]MeTHF
Infused
40 fig; n
Unbound plasma, ml
CSF,
0.36 ng/
rig/ml
2.30 0.50 [45.00 * 4.001
12.10
0.31
0.65 0.07 (0.31 zt 0.08)
wvt rig/g
$
0.29 (0.80)
Choroid w/g
plexus,
30.24 (84.00)
0.86 rt 0.11 (0.41 It 0.12) 108.90 zt33.60 (56.60 GZ5.80)
23Opg; n = 3
99.30 13.40 [55.00 zf 6.001 &
[60 .OO]
1.82
*
Brain,
=2
* [19.oo]*
are in was deusing p-
5.60 0.60 (0.06 rfs: 0.00)
zk (0.15)
3.63 zk (0.30) * 337.50 (27.90)
19.20 2.80 (0.19 0.00)
900.40 *ML50 (9.10 * 1.50)
Values are means =t SE. Rabbits were infused intravenously with 4, 10, 40, or 230 pg (rt)-[14C]MeTHF over 2.5 h. At 2.5 hr, the animals were sacrificed, and choroid plexus, CSF, and ultrafiltrates of plasma were assayed for [i4C]MeTHF. Brain was assayed for 14C. * The values in brackets under each plasma value represent the percentage of plasma 14C at 2.5 h as unbound [14C]MeTHF. t In parentheses, under each value of CSF, brain and choroid plexus is the ratio of tissue [14C]MeTHF to unbound plasma [14C]MeTHF (except in brain as noted) in that group of animals at 2.5 h. STh e values given for brain were calculated on the assumption that the i4C in brain was [i4C]MeTHF.
of the ultrafilterable 14C was associated with MeTHF, whereas in those rabbits that received 230 pg (k)-[‘“ClMeTHF, 80 % of the ultrafilterable 14C was associated with MeTHF. Rabbit plasma appeared to bind a significant amount of MeTHF as does human serum (18, 19). The 14C in the CP appeared as a single peak on the TLC plates corresponding to the MeTHF carrier spot (n = 8), except in two CP where only 80 % of the 14C was associated with carrier MeTHF. For the purpose of calculation, all the 14C in the choroid plexuses was assumed to be [14C]MeTKF. However, because of the relatively low specific activity of the (&)-(14C]-MeTHF, the 0.02-ml aliquots of the supernates of the choroid plexuses (in those rabbits injected with 4 or 10 pug (=t)-[14C]MeTHF intravenously or 5 nCi intraventricularly) contained less than 100 dpm. Therefore, the quantitative assumption that all the 14C in the choroid plexus was [14C]MeTHF may not be entirely correct because of the difficulty in accurately detecting radiolabeled metabolites with the small amount of 14C applied to the TLC plate.
Table 2 shows that the ratio of [14C]MeTHF in CSF and CP to unbound [14C]MeTHF in plasma decreased with increasing plasma concentrations of unbound [14C]MeTHF. This was also true for the ratio of brain W to unbound plasma [14C]MeTHF. These results suggested that MeTHF entered CSF, CP, and possibly brain by a saturable transport system with an increase of approximately 5 rig/ml of unbound [14C]MeTHF decreasing the tissue-to-plasma ratios by approximately 50 %. However, since the total unbound MeTHF (endogenous MeTHF + [14C]MeTHF) was not measured in these rabbits, there existed the possibility that the endogenous unbound MeTHF in the plasma might significantly increase after injection of 10, 40, or particularly 230 pg of [14C]MeTHF (22). Second, the [14C]MeTHF may not mix completely with the endogenous MeTHF even in 2.5 h (31). Thus, at best, 5 rig/ml is a rough estimate of the half-saturation concentration of MeTHF entry into CSF and CP. In the two rabbits that received both (+)-[3H]MeTHF and (+)-[14C]MeTHF b y intravenous infusion over 2.5 h, the ratios of CSF to unbound plasma (+)-[3H]MeTHF in the two rabbits were 1.3 and 1.7 times the comparable ratios of CSF to unbound, plasma (+)-[14C]MeTHF. The appearance of FA in CSF, CP, and brain after intravenous FA over 2.5 h is indicated in Table 3. In the plasma of the two rabbits that received 0.3 mg/kg Mtx pretreatment, 63 % of the plasma 3H was [3H]FA. In the CSF, only 19 % of the 3H was nonvolatile. Volatile 3H in CSF was assumed not to be folic acid. Since the nonvolatile 3H was not chromatographed, the concentrations of nonvolatile 3H in CSF and brain are upper limits for the appearance of FA in CSF and brain. The minimal appearance of FA in CSF as opposed to CP differed significantly (P < .05) from the behavior of (&)-[14C]MeTHF, which entered CSF at least 15 times more readily (Table 3). Also, the concentration of 14C in brain was significantly higher (P < .05) than nonvolatile 3H. This difference in brain would be more marked if a correction for isotopes in residual blood in brain (~2 %) were made. The percentages of intraventricularly injected MeTHF, FA, and inulin in CSF after 2 h are indicated in Table 4. FA left CSF more rapidly than MeTHF and inulin. By adding 0.1 mg carrier FA to the injectate, the exit of FA from CSF was significantly slowed. Moreover, the large accumulation of MeTHF and FA in the CP was depressed by the addition of carrier FA to the injectate. The disappearance of pH]FA from CSF could not be accounted for by a change of 3H concentration in brain. Thus, FA (and possibly MeTHF), like certain weak acids and ions (7), appeared to be transported from CSF by a saturable mechanism. Further evidence for this conclusion is presented in Table 5 where 5 ,ug FA carrier injected into the lateral ventricle also significantly decreased [3H]FA efflux from CSF. Data presented in Table 5 suggest that depression of [3H]FA efflux from CSF and into CP, by the concurrent injection of 5 ,ug carrier FA into the lateral ventricle, did not affect [14C]MeTHF entry into CSF or 14C entry into brain from blood. However, the concentration of [14C]MeTHF in the CP did appear lower (P < .Ol) in those rabbits that received carrier intraventricular FA. Although the injec-
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780
R.
3. [3H]FA or nonvolatile 3H in tissues or [14C]MeTHF or W in tissues after intravenous C3H]FA or (&-[14C]Me THF, respectively
TABLE
I
Rabbits Infused with 10 pg [sH]FA* After 3 mg/kg Mtx
Tissue
(93 = 2)
Unbound plasma [3H]FA (I, ZZ) or [i4C]MeTHF (III), nghl CSF nonvolatile 3H (I, II) or [‘“ClMeTHF (ZZZ), ng/
II
Rabbits Infused with 10 pg [3H]FA After 0.3 mg/kg Mtx (?z = 2)
III
Rabbits Infused with 10 pg [l*C]gM3eTJJJrk’fter
3.10
1.80
0.04 @*Ol>$
0.04 (0.01)
0.278 (0.15)
Injected
Radiochemical
Amount
74.9 *5 .o* (9) 60.1 rfi3.9 (5) 52.3 sfrl.5 (2)
Values are means & SE. The number of rabbits are in parentheses below each value. Rabbits, anesthesized with pentothal, were injected in the left lateral ventricle with 0.1 ml of artificial CSF containing the radiochemical. Two hours after the injection, the animal was sacrificed and the amount of the original radiochemical in CSF and the left lateral and fourth ventricular choroid plexuses was determined. The 3H or 14C content (and not the original radiochemical) was determined in whole brain. The values reported are the percentage of the injected radiochemical (or of the injected radioactivity in the case of brain) recovered in an assumed 3 ml CSF, 15 mg choroid plexus, and 7.5 g brain or the sum of these. * Values differed from the respective value in that column after the injection of (&)-[14C]MeTHF with P < .Ol (Dunnett’s test (9)) or P < .05 (Scheffe’s method for multiple comparisons in the Gaussion analysis of variance (6)). t Value differed with P < .05 (Dunnett’s test).
ionized at pH = 7.4, to diffuse appreciably across the blood-CSF and blood-brain barriers (16, 26) and by the presence of a saturable carrier acting to transport reduced folates from blood into CSF and brain. Moreover, at norma1 plasma concentrations, this carrier would have to be predominantly saturated. Levitt et al. (16) showed that, after the intravenous injection of MeTHF and FA, MeTHF appeared in CSF, but FA did not. However, Levitt et al. did not analyze the 3H activity in plasma after the injection of C3H]FA. In the present study, we have shown the [14C]MeTHF entered rabbit CP and CSF by a saturable mechanism with a one-half saturation concentration of about 5 rig/ml (Table 2). This is approximately the normal rabbit serum concentration. Similarly, in humans, the half-saturation concentration for folate transport from blood into human CSF is about 5 rig/ml (13). I\/roreover, by pretreating rabbits with Mtx to depress FA metabolism (30), we have been able to show that FA appearance in CSF was more than an order of magnitude lower at comparable radiochemical concentrations in plasma than MeTHF appearance (Table 3). However, firm conclusions about entry of FA into CSF (and brain) are not possible, because FA appeared to disappear from CSF into blood by a saturable mechanism (Tables 4 and 5). The [3H]FA uptake by brain could not explain this result (Table 4). Consequently, the minimal appearance
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FOLATE
TRANSPORT
IN THE
781
CNS
TABLE 5. Percent of L3H]FA and [14C]Me THF in tissues 2 h after intraventricular injection of 13H]FA and subsequent intravenous infusion of (&>-[14C)Ue THF Rabbits Injected With 0.1 LcCi [3H]FA (n = 4) [3H]FA
CSF (3 Choroid
ml)
16.7k6.8
plexus
6.6rt0.6
[14C]MeTHF 10-S
Sum
(7.5
g)
21.8~4.0 45.6zt4.3
X
2.3zkO.2
2.4zto.
(20 mg> Brain
Rabbits [3H]FA
8.OzkO.8
12.7ztO.6
Injected With 0.1 pCi and 5 pg FA (n = 3) [ldC]MeTHF 10-Z
[3H]FA
52.6zfr4.3 (P < .02) 1
2.0*0.2 (P > 0.1) l.lztO.1
o.o=fro.o (P < .Ol)
(P
.l)
(P >
0.1)
9.6&l (P >
.6 0.1)
70.9&l (P -c
X
.Ol)
6.5zk1.2 .l
.05j
Values are means & SE. Number of rabbits(n) in parentheses. Rabbits were injected in the left lateral ventricle with 0.1 &i [3H]FA in 0.1 ml artificial CSF with or without 5 pg carrier FA. Thirty minutes later, each rabbit was infused intravenously with 10 pg [l*C]MeTHF over 90 min at a constant rate. At the end of the infusion, the rabbit was sacrificed and the amount of [3H]FA and [‘“ClMeTHF in CSF and choroid plexus was measured. The content of l*C and 3H in brain were also measured. The values reported are the percentage of the injected radiochemical (or of the injected radioactivity in the case of brain) in an assumed 3 ml CSF, 20 mg choroid plexus, and 7.5 g brain or the sum of these. The P values (in parentheses) were calculated for each percentage in the rabbits injected with, versus those without, carrier FA by using the Student t test, two tailed (6).
of FA in CSF (and brain) may be due to saturable efflux transport from CSF as well as poor penetration into CSF. This finding of MeTHF appearing in CSF more readily than FA may explain why MeTHF, which is about as potent an inducer of seizures as FA when injected intracisternally or intracortically (2 1), is a much stronger convulsant when injected intravenously ( 12). Another explanation for these data would be that Mtx inhibits the entry of FA more than MeTHF into CSF (and brain) from blood. Although unlikely in our view, this remains possible. A second alternative explanation would be that tracer concentrations of [3H]FA (without carrier) were labile when injected into CSF in vivo; that is, the disappearance of C3H]FA was due not to transport of [3H]FA from CSF but rather to alteration of [3H]FA as by 3H exchange with HzO. However, we believe this unlikely because, when 0.01 &i of [3H]FA p er milliliter of fresh rabbit CSF was incubated at 37°C under 95 % 02: 5 % CO2 for 2 h, no destruction of the [3H]FA occurred (unpublished observations). Enzymes that employ MeTHF (20) as well as the transport system of the gut (33) are stereospecific and require or favor (+)-MeTHF. In this study, we observed that (-I-)MeTHF achieved CSF to unbound plasma levels 1.5 times those of (&)-MeTHF in two rabbits. Second, the transport of [14C]MeTHF into CSF and 14C into brain after intravenous (&)-[14C]MeTHF was not depressed by saturation of FA efflux (Table 5). Thus, the transport of (+)MeTHF into CSF appeared specific (16) and independent of the CSF concentration of FA. Evidence that the isolated CP may be a locus for trans-
port of MeTHF into CSF has been presented (5, 29). The Kt- for (+)-[14C]MeTHF into the CP in vitro was 8 rig/ml. In vivo, the CP concentrated MeTHF (Tables 2 and 3) with a half-saturation concentration of about 5 rig/ml in plasma. The concentrating ability (over 2.5 h) of the CP on a weight basis for (It)-[14C]MeTHF was greater than liver and kidney (unpublished observations), and the levels of folate in CP were comparable to liver and kidney (Table 1) (2, 4). However, a previously unexplained finding was the greater affinity of FA than MeTHF for the isolated CP uptake system (29), even though FA does not readily appear in CSF in vivo (16; this study). W,e postulate that this in vitro phenomenon is due to the exposure of both the CSF and blood sides of the isolated CP epithelial cells to the medium. In vivo, there are tight junctions between the CP epithelial cells (8), and one side of the CP, the ciliated side, is bathed by CSF where the other side faces the blood (8). Our data, showing that FA was transported from CSF more readily than MeTHF, would be consistent with the hypothesis that the ciliated side of the CP preferentially extracted and transported folic acid from the CSF. On the other hand, MeTHF appeared to be more readily transported than FA from blood to CSF -presumably via the CP. Thus, in vivo, if this analysis is correct, FA would be preferentially transported through the CP and out of CSF, and MeTHF would be preferentially transported from blood through the CP and into CSF. In vitro, where both sides of the CP are exposed to the medium, this selectivity of the CP cannot be observed since only CP uptake is measured (29). However, the finding that intraventricularly injected FA depressed the concentration of MeTHF in CP after an intravenous infusion of MeTHF (Table 5) suggests that the preference of the blood side of the CP for MeTHF was not absolute. In vitro, in the isolated CP, carrier FA depressed [14C]MeTHF uptake, although less readily than carrier MeTHF (29). Alternative explanations of this CP data are possible. In vitro, and presumably in vivo, a large part of the MeTHF and FA is bound inside the CP (29; unpublished observations). Therefore, the unbound concentration differential between CP and CSF and blood could be much smaller than indicated in Tables 1-5, and conclusions based on total tissue to unbound plasma or CSF levels could be misleading. It is also possible that FA is less readily released from the CP into the CSF when entering from the blood side. Second, it is possible that a saturable system for MeTHF from CSF to ,blood (via the choroid plexus) exists that was not demonstrated in this study. This might be because the choroid plexus system was saturated in vivo by the endogenous level of 32 rig/ml MeTHF in the CSF (Table 1) plus the 40 ng MeTHF injected with the intraventricular injection (Table 4). In vitro, the choroid plexus transport system for MeTHF is one-half saturated at 8 rig/ml (& = 18 mM) MeTHF in the medium (29). Although we believe bidirectional, saturable transport of MeTHF between CSF and plasma is unlikely because of the large concentration gradient between CSF and plasma (Table l), the data from this study cannot exclude this possibility. This study does not provide direct evidence that the CP is the sole or even primary mechanism for MeTHF trans-
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782
R. SPECTOR
port into CSF and provides no evidence for the route of MeTHF entry into brain. However, the saturable uptake system of the CP for MeTHF, like that for folate in liver, kidney, and gut (10, 14, 22, 23, 33; unpublished observations), suggests that the CP may perform a transport role in the CNS similar to its role in ascorbic acid transport from blood into CSF (26). Wh erever the exact locus (loci) of .MeTHF transport into the CNS, MeTHF readily appeared in CSF by a mechanism that was one-half saturated at normal plasma concentrations of MeTHF. Thus, at low the mechanism for transporting plasma concentrations, MeTHF from blood to CSF would be less than one-half saturated and would pump proportionately more MeTHF into CSF. At high plasma concentrations, the transport mechanism is predominately saturated, and MeTHF would enter CSF predominately by diffusion. The saturable entry of MeTHF into the CNS complements the regulation of plasma MeTHF levels by the gut, liver, and kidney (10,
AND A. V. LORENZO
14, 22, 23, 33, 34; unpublished observations), thus offering the CNS further protection against plasma fluctuations. Moreover, folic acid, which cannot be employed by the brain (17), is pumped from the CSF by a saturable mechanism. In conclusion, MeTHF and FA levels in the CNS would appear to be homeostatically regulated by these systems even in the face of gross alterations in either dietary or blood levels. The authors thank Drs. Norman Uretsky and Robert Snodgrass for their counsel and support and Dr. David E. Drum for his measurements of the endogenous folate contents of the tissues. This study was supported in part by National Institutes of Health Grants NS 05172 and NS-HD 09704 and The Children’s Hospital Medical Center Mental Retardation and Human Developmental Research Program Grant HD 03773. A. V. Lorenzo is a recipient of a National Institutes of Health Career Development Award HD 18519. Received
for
publication
20 August
1974.
REFERENCES 1. 2. 3. . 4.
5.
10. 11.
12. 13.
14. 15.
ALPERIN, J. B., AND M. E. HAGGARD. Cerebrospinal fluid folate (CFA) and the blood-brain barrier. Clin. Res. 18 : 40, 1970. BIRD, 0. D., V. M. MCGLOHON, AND J. N. VAITKUS. Naturally occurring folates in the blood and liver of the rat. Anal. Biochem. 12 : 18-35, 1965. BROWN, J. P., G. E. DAVIDSON, AND J. M. SCOTT. Thin-layer chroatography of pteroylglutamates and related compounds. J. Chromatog. 79 : 195-207, 1973. BROWN, J. P., G. E. DAVIDSON, AND J. M. SCOTT. The identification of the forms of folate found in the liver, kidney and intestine of the monkey and their biosynthesis from exogenous pteroylglutamate (folic acid). Biochim. Biophys. Acta 343 : 78-88, 1974. CHEN, C. P., AND C. WAGNER. Transport of folic acid (FA) and 5methyltetrahydrofolic acid (5-MTHF) in the isolated choroid plexus. Federation Proc. 33 : 7 16, 1974. COLQUHOUN, D. Lectures on Biostatistics. Oxford : Clarendon, 197 1, p. 148, 211. CSERR, H. F. Physiology of the choroid plexus. Pharmacol. Rev. 51: 273-311, 1971. DAVSON, H. T. The Physiology of the Cerebrosflinal Fluid. Boston : Little, Brown, 1967. DUNNETT, C. W. A multiple comparison procedure for comparing several tratments with a control. J. Am. Sot. Agron. 50: 1096-l 121, 1955. GORESKY, C. A., H. WATANAKE, AND D. G. JOHNS. The renal excretion of folic acid. J. Clin. Invest. 42 : 1841-1849, 1963. HERBERT, V., A. R. LARRABEE, AND ,J. M. BUCHANAN. Studies on the identification of a folate compound of human serum. J. CZin. Invest. 41: 1134-l 138, 1962. HOMMES, 0. R., AND E. A. M. T. OBBENS. Liver function and folate epilepsy in the rat. J. Neural. Sci. 20: 269-272, 1973. JENSEN, 0. N., AND 0. V. OLESEN. Folic acid concentrations in cerebrospinal fluid in relation to anticonvulsant drugs and cerebral atrophy. Acta Psych. Stand. 47 : 206-2 12, 1971. JOHNS, D. G., S. SPERTI, AND A. S. V. BURGEN. The metabolism of tritiated folic acid in man. J. CZin. Invest. 40 : 1684-1695, 1961. KOREVAAR, W. C., M. A. GEYER, S. KNAPP, L. L. Hsu, AND A. J. MANDELL. Regional distribution of 5-methyltetrahydrofolic acid in brain. Nature New BioZ. 245 : 244-245, 1973.
16. LEVITT, M., P. F. NIXON, J. H. PINCUS, AND J. R. BERTINO. Transport characteristics of folates in cerebrospinal fluid; a study utilizing doubly hydrofolate. 17.
labelled 5-methyltetrahydrofolate J. CZin. Invest. 50: 1301-l 308,
and
5-formyltetra-
1971.
MAKULU, D. R., E. F. SMITH, AND J. R. BERTINO. Lack of dihydrofolate reductase activity in brain tissue of mammlian species : possible implications . J. Neurochem. 21 : 241-245,1973.
18. MARKKANEN, T., AND 0. PELTOLA. Binding of folic acid activity by body fluids. Acta HaematoZ. 43 : 272-279, 1970. 19. MARKKANEN, T., P. HIMANEN, R.-L. PAJULA, AND G. MOLNAR. Binding of folic acid to serum proteins. Acta Haematol. 50 : 284-292, 1973. 20. NIXON, P. F., AND J. R. BERTINO. Enzymic preparations of radiolabelled (+)-L-5-methyltetrahydrofolate and (+)-L-5-formyltetrahydrofolate. Anal. Biochem. 43 : 162- 172, 197 1. 21. OBBENS, E. A. M. T., AND 0. R. HOMMES. The epileptogenic effects of folate derivatives in the rat. J. Neural. Sci. 20: 223-229, 1973. 22. PRATT, R. F., AND B. A. COOPER. Folates in plasma and bile of man after feeding folic acid-3H and 5-formyltetrahydrofolate (folinit acid). J. CZin. Invest. 50 : 455-462, 1971. 23. SELHUB, J., H. BRIN, AND N. GROSSOWICZ. Uptake and reduction of radioactive folate by everted sacs of rat small intestine. European J. Biochem. 33 : 433-438, 1973. 24. SPAANS, F. No effect of folic acid supplement on CSF folate and serum vitamin B1:! in patients on anticonvulsants. Epilepsia 11 : 40341 1, 1970. 25. SPECTOR, R., D. T. KORKIN, AND A. V. LORENZO. A rapid method for the determination of salicylate binding by the use of ultrafilters. J. Pharm. Pharmacol. 24: 786-789, 1972. 26. SPECTOR, R., AND A. V. LORENZO. Ascorbic acid homeostasis in the central nervous system. Am. J. Physiol. 225 : 757-763, 1973. 27, SPECTOR, R., R. VERNICK, AND A. V. LORENZO. Effects of pressure on the plasma binding of digoxin and ouabain in an ultrafiltration apparatus. Biochem. Pharmacol. 22 : 2485-2487, 1973. 28. SPECTOR, R., AND A. V. LORENZO. Inhibition of penicillin transport from the cerebrospinal fluid after intracisternal inoculation of bacteria. J. CZin. Invest. 54: 3 16-325, 1974. 29. SPECTOR, R., AND A. V. LORENZO. Folate transport by the choroid plexus in vitro. Science 187 : 54-O-542, 1975. 30. SPECTOR, R. G. Folic acid and convulsions in the rat. Biochem. Pharmacol. 20 :’ 1730- 1732, 197 1. 31. WATERMAN, N., L. SCHARFENBERGER, AND M. J. RAFF. Rate of binding of antibiotics to canine serum protein. Antimicrob. Agents Chemotherapy 5 : 294-295, 1974. 32. WAXMAN, S., AND C. SCHREIBER. Measurement of serum folate levels and serum folic acid-binding protein by 2H-PGA radioassay. Blood 42: 281-290, 1973. 33. WEIR, D. G., J. P. BROWN, D. S. FREEDMAN, AND J. M. SCOTT. The absorption of the diastereoisomers of 5-methyltetrahydropteroylglutamate in man: a carrier-mediated process. CZin. Sci. Mol. Med. 45: 625-631, 1973. 34. WHITEHEAD, V. M., R. PRATT, A. VIALLET, AND B. A. COOPER. Intestinal conversion of folinic acid to 5-methyltetrahydrofolate in man. Brit. J. Haematol. 22 : 63-72, 1972.
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Folate
transport
in the central
nervous
system
REYNOLD SPECTOR AND A. V. LORENZO (With the Technical Assistance of Peter Levy) Department of Neurology, Children’s Has-ital Medical Center, Department of Medicine, Peter Bent Brigham Hospital, and Departments of Medicine and Pharmacology, Harvard Medical School, Boston, Massachusetts 02111.5
SPECTOR, REYNOLD, AND A. V. LORENZO. Folate transport in the central nervous system. Am. J. Physiol. 229(3): 777-782. 1975.Methyltetrahydrofolic acid or folic acid was infused intravenously at a constant rate into conscious untreated or methotrexate-pretreated rabbits. After 150 min, at equivalent plasma concentrations, folic acid or methyltetrahydrofolic acid readily entered the choroid plexus, but only methyltetrahydrofolic acid readily entered the cerebrospinal fluid and probably brain by a saturable transport system. In contrast, after intraventricular injections, folic acid but not methyltetrahydrofolic acid was cleared from cerebrospinal fluid to blood by a saturable system. Intraventricular injection of folic acid at concentrations that saturated folic acid clearance from cerebrospinal fluid did not affect the transport of methyltetrahydrofolic acid from blood into cerebrospinal fluid. These results suggest that the transport system for methyltetrahydrofolic acid, which is about half-saturated at normal plasma concentrations, helps maintain the cerebrospinal fluid and probably brain methyltetrahydrofolic acid concentrations within relatively narrow limits. Moreover, folic acid, which the brain cannot utilize, is transported from cerebrospinal fluid. A possible locus for the systems that transport folic acid from and methyltetrahydrofolic acid into the cerebrospinal fluid is the choroid plexus. saturable;
cerebrospinal
fluid;
choroid
METHODS
AND
MATERIALS
The (&)-[14C]MeTHF (60 mCi/mmol) and [3H]FA (17, 23 Ci/mmol) were obtained from Amersham/Searle Corp., Arlington Heights, Ill. The (+)-[3H]MeTHF (160 mCi/mmol) was biosynthesized from [3H]FA in vivo in rat liver and isolated by DEAE-Sephadex and Sephadex G-15 chromatography (2, 16, 29). Crystalline FA was obtained from Sigma Chemical Co., St. Louis, MO. and methotrexate (Mtx) from Lederle Laboratories, Pearl River, N. Y. In vivo studies. In order to determine the entry of (+)MeTHF into CP, brain, and CSF from blood, unanesthetized, overnight-fasted New Zealand white rabbits, weighing 1.4-l .9 kg, were infused intravenously with 10 ml of chilled normal saline containing 5 mg sodium ascorbate, 2 mg thiourea and 4, 10, 40, or 230 lug (+)-[14C]MeTHF (26). Two milliliters of this solution were given over 10 min and the rest at a constant rate over 140 min. At the end of the infusion, the rabbit received a rapid intravenous injection of pentobarbital; and blood, 0.8-1.2 ml of cisternal CSF, CP, and brain were removed within 4 min (26). After weighing, CP were homogenized in 0.3 ml Hz0 containing 0.01 ml mercaptoethanol (ME). Whole brain, after weighing, was homogenized in 8.0 ml water. Duplicate aliquots of CSF (0.2 ml), plasma (0.2 ml), CP homogenate (0.02 ml), and brain homogenate were assayed for 14C in Aquasol (26). Th e nature of the 14C in the CSF, plasma, and CP homogenate was determined as described below (see Assays). To assess the stereospecificity of MeTHF entry into CSF, CP, and brain, two other rabbits were treated as above except that both 4 pug (+)-[14C] MeTHF and 2 pg (+)-[3H]MeTHF were placed in the infusion syringe. Folic acid entry into CP, brain, and CSF from blood was measured in rabbits infused as above, except that 10 pug FA and 50 PCi [3H]FA (rather than [14C]MeTHF) were placed in the infusion syringe and the rabbits were infused intravenously with either 0.3 mg/kg (body wt) or 3.0 mg/kg Mtx 30 min before beginning the FA infusion. The Mtx was employed to delay the metabolism of the FA (30). After sacrifice, the concentration of 3H in the tissues and the nature of the 3H in the CSF, plasma, and CP homogenate were determined (see Assays). To assess the possibility that Mtx might inhibit MeTHF entry into CSF, CP, and brain, two other rabbits were infused as above with 10 pg (+)[14CjMeTHF and no [3H]FA ‘or FA in the infusion syringe 30 min after 0.3 mg/kg iv Mtx. The concentration of i4C and the nature of the 14C in CSF, CP, and plasma were determined (see Assays). To measure the efflux of folates from CSF, either 0.1 &i
plexus
REDUCED FOLATES are present at higher concentrations in cerebrospinal fluid (CSF) and brain than plasma (1, 15). However, the enzyme that catalyzes the reduction of folic acid (FA) to tetrahydrofolic acid (THF), dihydrofolate reductase, is not present in mammalian brain (17). The high levels of reduced folates in CSF and brain are thought to be due to transport of reduced folates from blood, principally L(+)-5methyltetrahydrofolate ((+)-MeTHF) (16). This folate, (+)-MeTHF, is the major folate in plasma (2, 11). Levitt et al. (16) suggested that a transport mechanism for reduced folates from blood into CSF through the blood-CSF barrier might reside in the choroid plexus (CP). Subsequently, it was shown that the isolated CP contains a specific folate transport system. The characteristics of this system were compatible with the hypothesis that the choroid plexus is a locus of the transport of reduced folates from blood to CSF (5, 29). The purposes of this study are: I) to define the characteristics of the entry of MeTHF and FA into CSF, brain, and CP from blood; 2) to document the characteristics of the efflux of FA and MeTHF from CSF into blood, brain, and CP after intraventricular injections; and 3) to provide in vivo evidence that the CP is a locus for transport of MeTHF from blood into CSF as well as a locus for transport of FA from CSF to blood by an independent system.
777
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778
R.
of [3H]FA or [3H] inulin or 5.0 nCi of (&)-[14C]-MeTHF or (+)-(3H)MeTHF were injected into the left lateral ventricle of a rabbit anesthetized with pentothal (26). Each isotope was injected in a volume of 0.1 ml of chilled, artificial CSF which contained 50 pug sodium ascorbate, 20 pg thiourea, and, in some cases, 0.1 mg FA. After 2 h, the rabbit was reanesthetized with pentothal and sacrificed. Immediately, 0.8-1.2 ml of cisternal CSF, the left lateral and fourth ventricular CP (15 mg) and whole brain were obtained (26). The CSF, CP, and brain (after weighing) were assayed for radioactivity as above, and the nature of the radioactivity in the CSF and CP homogenate was determined (see Assays). The effect of saturating FA efflux from CSF on MeTHF influx into CSF from blood was studied by injecting 0.1 &i [3H]FA with or without 5.0 pug FA in 0.1 ml artificial CSF into the left lateral ventricle. After 30 min, 10 pg of ( &)-[14C]MeTHF in 10 ml chilled, normal saline with 5 mg sodium ascorbate and 2 mg thiourea were infused intravenously at a constant rate over 90 min into the conscious rabbit. At the end of the infusion, the animal was sacrificed and 0.8-1.2 ml of cisternal CSF, the left lateral and fourth ventricular CP, the right lateral CP, and brain were obtained. The CSF, CP, and brain (after weighing) were assayed for 3H and 14C and the nature of the 3H and 14C in CSF and the CP homogenates was determined (see Assays). The two lateral and fourth ventricular CP weighed about 20 mg. Assays. The methods for establishing the purity of the [3H]FA, ( &)-[14C]-MeTHF, ( +)-[3H]MeTHF, and [3H]inulin as determined by cellulose thin-layer (TLC) or paper chromatography have been described (3, 28, 29). In this study, for the folate chromatography, MN 300 UV 254 0.1 mm thin-layer cellulose plates (Brinkmann Instruments) were used exclusively and were chromatographed in 3 % NH&l (wt/vol), pH = 6.2 with 0.5 % ME. Except for the (+)-[3H]MeTHF, which was 90 % pure, all the other radiochemicals were greater than 95 % pure. The [3H]FA required repurification on Sephadex 25 periodically. The nature of the 14C or 3H in the CP after (a)-[14C]MeTHF, [3H]FA, or (+)-[3H]MeTHF injections was determined by the following procedures. CP homogenates (containing ME) were covered, heated at 75°C for 30 min, and centrifuged (2, 29). Duplicate aliquots (0.02 ml) of supernate and carrier (rt)MeTHF or FA were spotted and chromatographed on TLC plates (3, 29). The radioactivity corresponding to the FA or (&)MeTHF carrier spots was assayed and corrected for recovery. To determine the nature of the 14C or 3H in CSF and plasma after injections of (+)-[14C]MeTHF or (+)-[3H]MeTHF, the CSF and ultrafiltrates of plasma were chromatographed. The ultrafiltrates of plasma were obtained at 37°C by ultrafiltering 2.0 ml fresh plasma added to 0.5 ml artificial CSF through a Millipore ultrafilter (PSED 13 10) in a 2.5-ml stirred chamber at 5-lb pressure with 95 % N2: 5 % CO2 (25, 27). The first 0.2 ml of ultrafiltrate was discarded, and a total of 2 ml of ultrafiltrate from the 5-ml plasma solution (2 chambers) were collected in chilled tubes containing 5 mg sodium ascorbate and 2.0 mg thiourea. (A correction for loss through the filter which depended on
SPECTOR
AND
A.
V.
LORENZO
concentration (5-22 %) was required (25, 27).) The ultrafiltrates of plasma and CSF were then passed through small DEAE-Sephadex columns (0.6 cm x 11 cm). The (+)C3H]MeTHF (3 n C i ) was added to the CSF or ultrafiltrate of plasma to identify the [14C]MeTHF peak. The column was eluted by means of a linear concentration gradient of phosphate buffer, pH = 6, constructed by placing 45 ml of 0.1 M phosphate buffer in the mixing chamber and 45 ml of 0.8 M phosphate buffer in the reservoir (16). The buffer contained 2 mg/ml Na ascorbate. Two-milliliter aliquots were collected from the column, and the radioactivity was determined in them. In the two groups of two rabbits that received 4 pg [14C]MeTHF intravenously, the CSF samples as well as the ultrafiltrates of plasma were pooled before chrcmatography to attain adequate numbers of disintegrations per minute. In the two groups of rabbits that received 10 rug [14C]MeTHF intravenously, the CSF samples in each group were pooled before chromatography. The nature of the 3H in CSF after [3H]inulin or [3H]FA injections was determined on 0.02-ml aliquots of unaltered or concentrated CSF by paper or thin-layer chromatography (3, 28, 29). I n some samples of CSF (and brain homogenates), the percentage of 3H that was not volatile was determined by evaporating to dryness. The nature of the 3H in plasma of rabbits infused with [3H]FA was determined by adding 0.1 ml plasma, 0.3 ml HzO, and 0.04 ml ME to 1.6 ml ethanol, mixing, and centrifuging. The supernate was assayed for 3H; 1.4 ml of supernate was taken to dryness and redissolved in 0.03 ml HZO. Ten microliters were spotted on TLC plates with carrier FA, and 0.01 ml was assayed for 3H. The 3H on the FA carrier spot was measured and corrected for recovery of a standard. The binding of [3H]FA to plasma could not be determined by ultrafiltration because of excessive nonspecific binding by the filter (Millipore PSED1310) (25, 27). However, less than 5 % of [3H]FA was bound to plasma proteins as determined by gel filtration of plasma from rabbits 2) which were injected with 0.3 mg/kg Mtx and sub( nsequently received over a 2.5-h period 10 pug FA plus 50 &i [3H]FA (25). Endogenous levels of folate in overnight-fasted rabbits were measured in CSF, plasma, and supernates of CP (after treatment of CP homogenates containing ME at 75°C for 30 min) by a competitive protein-binding assay using P-lactoglobulin (32). RESULTS
The total endogenous folate in serum, CSF, and CP is indicated in Table 1. The unbound plasma, CSF, and CP concentrations of [14C]MeTHF as well as the 14C in brain in rabbits infused with ( &)-[14C]MeTHF intravenously for 2.5 h are indicated in Table 2. Also shown in parentheses are the ratios of CSF and CP [14C]MeTHF (or brain 14C) to unbound plasma [14C]MeTHF. When the CSF of those rabbits in Table 2 was chromatographed on DEAESephadex columns, the 14C appeared as several peaks but greater than 73 % of the 14C in all samples (n = 7) was associated with MeTHF. When the ultrafiltrates of plasma from those rabbits that received 4 lug (=t)-[l*C]MeTHF were chromatographed on DEAE-Sephadex columns, 65 %
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FOLATE
TRANSPORT
TABLE
THE
779
CNS
Total endogenous folate in tissues
1.
Serum CSF Choroid
~-
IN
6.5 & 1.7 q/ml 32 rt 6 q/ml 3.6 If: 1.1 ,q/g
plexus
(4) (4) (5)
Values are means Ifi SE. The numbers of determinations parentheses. Total folate in overnight-fasted animals termined in the tissues by the competitive binding assay lactoglobulin (32). TABLE 2. Total [14C]MeTHF after intravenous [14C]Me THF --____-
in tissues at 2.5 h
Amount
of (&)-[l*C]MeTHF
Infused
40 fig; n
Unbound plasma, ml
CSF,
0.36 ng/
rig/ml
2.30 0.50 [45.00 * 4.001
12.10
0.31
0.65 0.07 (0.31 zt 0.08)
wvt rig/g
$
0.29 (0.80)
Choroid w/g
plexus,
30.24 (84.00)
0.86 rt 0.11 (0.41 It 0.12) 108.90 zt33.60 (56.60 GZ5.80)
23Opg; n = 3
99.30 13.40 [55.00 zf 6.001 &
[60 .OO]
1.82
*
Brain,
=2
* [19.oo]*
are in was deusing p-
5.60 0.60 (0.06 rfs: 0.00)
zk (0.15)
3.63 zk (0.30) * 337.50 (27.90)
19.20 2.80 (0.19 0.00)
900.40 *ML50 (9.10 * 1.50)
Values are means =t SE. Rabbits were infused intravenously with 4, 10, 40, or 230 pg (rt)-[14C]MeTHF over 2.5 h. At 2.5 hr, the animals were sacrificed, and choroid plexus, CSF, and ultrafiltrates of plasma were assayed for [i4C]MeTHF. Brain was assayed for 14C. * The values in brackets under each plasma value represent the percentage of plasma 14C at 2.5 h as unbound [14C]MeTHF. t In parentheses, under each value of CSF, brain and choroid plexus is the ratio of tissue [14C]MeTHF to unbound plasma [14C]MeTHF (except in brain as noted) in that group of animals at 2.5 h. STh e values given for brain were calculated on the assumption that the i4C in brain was [i4C]MeTHF.
of the ultrafilterable 14C was associated with MeTHF, whereas in those rabbits that received 230 pg (k)-[‘“ClMeTHF, 80 % of the ultrafilterable 14C was associated with MeTHF. Rabbit plasma appeared to bind a significant amount of MeTHF as does human serum (18, 19). The 14C in the CP appeared as a single peak on the TLC plates corresponding to the MeTHF carrier spot (n = 8), except in two CP where only 80 % of the 14C was associated with carrier MeTHF. For the purpose of calculation, all the 14C in the choroid plexuses was assumed to be [14C]MeTKF. However, because of the relatively low specific activity of the (&)-(14C]-MeTHF, the 0.02-ml aliquots of the supernates of the choroid plexuses (in those rabbits injected with 4 or 10 pug (=t)-[14C]MeTHF intravenously or 5 nCi intraventricularly) contained less than 100 dpm. Therefore, the quantitative assumption that all the 14C in the choroid plexus was [14C]MeTHF may not be entirely correct because of the difficulty in accurately detecting radiolabeled metabolites with the small amount of 14C applied to the TLC plate.
Table 2 shows that the ratio of [14C]MeTHF in CSF and CP to unbound [14C]MeTHF in plasma decreased with increasing plasma concentrations of unbound [14C]MeTHF. This was also true for the ratio of brain W to unbound plasma [14C]MeTHF. These results suggested that MeTHF entered CSF, CP, and possibly brain by a saturable transport system with an increase of approximately 5 rig/ml of unbound [14C]MeTHF decreasing the tissue-to-plasma ratios by approximately 50 %. However, since the total unbound MeTHF (endogenous MeTHF + [14C]MeTHF) was not measured in these rabbits, there existed the possibility that the endogenous unbound MeTHF in the plasma might significantly increase after injection of 10, 40, or particularly 230 pg of [14C]MeTHF (22). Second, the [14C]MeTHF may not mix completely with the endogenous MeTHF even in 2.5 h (31). Thus, at best, 5 rig/ml is a rough estimate of the half-saturation concentration of MeTHF entry into CSF and CP. In the two rabbits that received both (+)-[3H]MeTHF and (+)-[14C]MeTHF b y intravenous infusion over 2.5 h, the ratios of CSF to unbound plasma (+)-[3H]MeTHF in the two rabbits were 1.3 and 1.7 times the comparable ratios of CSF to unbound, plasma (+)-[14C]MeTHF. The appearance of FA in CSF, CP, and brain after intravenous FA over 2.5 h is indicated in Table 3. In the plasma of the two rabbits that received 0.3 mg/kg Mtx pretreatment, 63 % of the plasma 3H was [3H]FA. In the CSF, only 19 % of the 3H was nonvolatile. Volatile 3H in CSF was assumed not to be folic acid. Since the nonvolatile 3H was not chromatographed, the concentrations of nonvolatile 3H in CSF and brain are upper limits for the appearance of FA in CSF and brain. The minimal appearance of FA in CSF as opposed to CP differed significantly (P < .05) from the behavior of (&)-[14C]MeTHF, which entered CSF at least 15 times more readily (Table 3). Also, the concentration of 14C in brain was significantly higher (P < .05) than nonvolatile 3H. This difference in brain would be more marked if a correction for isotopes in residual blood in brain (~2 %) were made. The percentages of intraventricularly injected MeTHF, FA, and inulin in CSF after 2 h are indicated in Table 4. FA left CSF more rapidly than MeTHF and inulin. By adding 0.1 mg carrier FA to the injectate, the exit of FA from CSF was significantly slowed. Moreover, the large accumulation of MeTHF and FA in the CP was depressed by the addition of carrier FA to the injectate. The disappearance of pH]FA from CSF could not be accounted for by a change of 3H concentration in brain. Thus, FA (and possibly MeTHF), like certain weak acids and ions (7), appeared to be transported from CSF by a saturable mechanism. Further evidence for this conclusion is presented in Table 5 where 5 ,ug FA carrier injected into the lateral ventricle also significantly decreased [3H]FA efflux from CSF. Data presented in Table 5 suggest that depression of [3H]FA efflux from CSF and into CP, by the concurrent injection of 5 ,ug carrier FA into the lateral ventricle, did not affect [14C]MeTHF entry into CSF or 14C entry into brain from blood. However, the concentration of [14C]MeTHF in the CP did appear lower (P < .Ol) in those rabbits that received carrier intraventricular FA. Although the injec-
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780
R.
3. [3H]FA or nonvolatile 3H in tissues or [14C]MeTHF or W in tissues after intravenous C3H]FA or (&-[14C]Me THF, respectively
TABLE
I
Rabbits Infused with 10 pg [sH]FA* After 3 mg/kg Mtx
Tissue
(93 = 2)
Unbound plasma [3H]FA (I, ZZ) or [i4C]MeTHF (III), nghl CSF nonvolatile 3H (I, II) or [‘“ClMeTHF (ZZZ), ng/
II
Rabbits Infused with 10 pg [3H]FA After 0.3 mg/kg Mtx (?z = 2)
III
Rabbits Infused with 10 pg [l*C]gM3eTJJJrk’fter
3.10
1.80
0.04 @*Ol>$
0.04 (0.01)
0.278 (0.15)
Injected
Radiochemical
Amount
74.9 *5 .o* (9) 60.1 rfi3.9 (5) 52.3 sfrl.5 (2)
Values are means & SE. The number of rabbits are in parentheses below each value. Rabbits, anesthesized with pentothal, were injected in the left lateral ventricle with 0.1 ml of artificial CSF containing the radiochemical. Two hours after the injection, the animal was sacrificed and the amount of the original radiochemical in CSF and the left lateral and fourth ventricular choroid plexuses was determined. The 3H or 14C content (and not the original radiochemical) was determined in whole brain. The values reported are the percentage of the injected radiochemical (or of the injected radioactivity in the case of brain) recovered in an assumed 3 ml CSF, 15 mg choroid plexus, and 7.5 g brain or the sum of these. * Values differed from the respective value in that column after the injection of (&)-[14C]MeTHF with P < .Ol (Dunnett’s test (9)) or P < .05 (Scheffe’s method for multiple comparisons in the Gaussion analysis of variance (6)). t Value differed with P < .05 (Dunnett’s test).
ionized at pH = 7.4, to diffuse appreciably across the blood-CSF and blood-brain barriers (16, 26) and by the presence of a saturable carrier acting to transport reduced folates from blood into CSF and brain. Moreover, at norma1 plasma concentrations, this carrier would have to be predominantly saturated. Levitt et al. (16) showed that, after the intravenous injection of MeTHF and FA, MeTHF appeared in CSF, but FA did not. However, Levitt et al. did not analyze the 3H activity in plasma after the injection of C3H]FA. In the present study, we have shown the [14C]MeTHF entered rabbit CP and CSF by a saturable mechanism with a one-half saturation concentration of about 5 rig/ml (Table 2). This is approximately the normal rabbit serum concentration. Similarly, in humans, the half-saturation concentration for folate transport from blood into human CSF is about 5 rig/ml (13). I\/roreover, by pretreating rabbits with Mtx to depress FA metabolism (30), we have been able to show that FA appearance in CSF was more than an order of magnitude lower at comparable radiochemical concentrations in plasma than MeTHF appearance (Table 3). However, firm conclusions about entry of FA into CSF (and brain) are not possible, because FA appeared to disappear from CSF into blood by a saturable mechanism (Tables 4 and 5). The [3H]FA uptake by brain could not explain this result (Table 4). Consequently, the minimal appearance
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FOLATE
TRANSPORT
IN THE
781
CNS
TABLE 5. Percent of L3H]FA and [14C]Me THF in tissues 2 h after intraventricular injection of 13H]FA and subsequent intravenous infusion of (&>-[14C)Ue THF Rabbits Injected With 0.1 LcCi [3H]FA (n = 4) [3H]FA
CSF (3 Choroid
ml)
16.7k6.8
plexus
6.6rt0.6
[14C]MeTHF 10-S
Sum
(7.5
g)
21.8~4.0 45.6zt4.3
X
2.3zkO.2
2.4zto.
(20 mg> Brain
Rabbits [3H]FA
8.OzkO.8
12.7ztO.6
Injected With 0.1 pCi and 5 pg FA (n = 3) [ldC]MeTHF 10-Z
[3H]FA
52.6zfr4.3 (P < .02) 1
2.0*0.2 (P > 0.1) l.lztO.1
o.o=fro.o (P < .Ol)
(P
.l)
(P >
0.1)
9.6&l (P >
.6 0.1)
70.9&l (P -c
X
.Ol)
6.5zk1.2 .l
.05j
Values are means & SE. Number of rabbits(n) in parentheses. Rabbits were injected in the left lateral ventricle with 0.1 &i [3H]FA in 0.1 ml artificial CSF with or without 5 pg carrier FA. Thirty minutes later, each rabbit was infused intravenously with 10 pg [l*C]MeTHF over 90 min at a constant rate. At the end of the infusion, the rabbit was sacrificed and the amount of [3H]FA and [‘“ClMeTHF in CSF and choroid plexus was measured. The content of l*C and 3H in brain were also measured. The values reported are the percentage of the injected radiochemical (or of the injected radioactivity in the case of brain) in an assumed 3 ml CSF, 20 mg choroid plexus, and 7.5 g brain or the sum of these. The P values (in parentheses) were calculated for each percentage in the rabbits injected with, versus those without, carrier FA by using the Student t test, two tailed (6).
of FA in CSF (and brain) may be due to saturable efflux transport from CSF as well as poor penetration into CSF. This finding of MeTHF appearing in CSF more readily than FA may explain why MeTHF, which is about as potent an inducer of seizures as FA when injected intracisternally or intracortically (2 1), is a much stronger convulsant when injected intravenously ( 12). Another explanation for these data would be that Mtx inhibits the entry of FA more than MeTHF into CSF (and brain) from blood. Although unlikely in our view, this remains possible. A second alternative explanation would be that tracer concentrations of [3H]FA (without carrier) were labile when injected into CSF in vivo; that is, the disappearance of C3H]FA was due not to transport of [3H]FA from CSF but rather to alteration of [3H]FA as by 3H exchange with HzO. However, we believe this unlikely because, when 0.01 &i of [3H]FA p er milliliter of fresh rabbit CSF was incubated at 37°C under 95 % 02: 5 % CO2 for 2 h, no destruction of the [3H]FA occurred (unpublished observations). Enzymes that employ MeTHF (20) as well as the transport system of the gut (33) are stereospecific and require or favor (+)-MeTHF. In this study, we observed that (-I-)MeTHF achieved CSF to unbound plasma levels 1.5 times those of (&)-MeTHF in two rabbits. Second, the transport of [14C]MeTHF into CSF and 14C into brain after intravenous (&)-[14C]MeTHF was not depressed by saturation of FA efflux (Table 5). Thus, the transport of (+)MeTHF into CSF appeared specific (16) and independent of the CSF concentration of FA. Evidence that the isolated CP may be a locus for trans-
port of MeTHF into CSF has been presented (5, 29). The Kt- for (+)-[14C]MeTHF into the CP in vitro was 8 rig/ml. In vivo, the CP concentrated MeTHF (Tables 2 and 3) with a half-saturation concentration of about 5 rig/ml in plasma. The concentrating ability (over 2.5 h) of the CP on a weight basis for (It)-[14C]MeTHF was greater than liver and kidney (unpublished observations), and the levels of folate in CP were comparable to liver and kidney (Table 1) (2, 4). However, a previously unexplained finding was the greater affinity of FA than MeTHF for the isolated CP uptake system (29), even though FA does not readily appear in CSF in vivo (16; this study). W,e postulate that this in vitro phenomenon is due to the exposure of both the CSF and blood sides of the isolated CP epithelial cells to the medium. In vivo, there are tight junctions between the CP epithelial cells (8), and one side of the CP, the ciliated side, is bathed by CSF where the other side faces the blood (8). Our data, showing that FA was transported from CSF more readily than MeTHF, would be consistent with the hypothesis that the ciliated side of the CP preferentially extracted and transported folic acid from the CSF. On the other hand, MeTHF appeared to be more readily transported than FA from blood to CSF -presumably via the CP. Thus, in vivo, if this analysis is correct, FA would be preferentially transported through the CP and out of CSF, and MeTHF would be preferentially transported from blood through the CP and into CSF. In vitro, where both sides of the CP are exposed to the medium, this selectivity of the CP cannot be observed since only CP uptake is measured (29). However, the finding that intraventricularly injected FA depressed the concentration of MeTHF in CP after an intravenous infusion of MeTHF (Table 5) suggests that the preference of the blood side of the CP for MeTHF was not absolute. In vitro, in the isolated CP, carrier FA depressed [14C]MeTHF uptake, although less readily than carrier MeTHF (29). Alternative explanations of this CP data are possible. In vitro, and presumably in vivo, a large part of the MeTHF and FA is bound inside the CP (29; unpublished observations). Therefore, the unbound concentration differential between CP and CSF and blood could be much smaller than indicated in Tables 1-5, and conclusions based on total tissue to unbound plasma or CSF levels could be misleading. It is also possible that FA is less readily released from the CP into the CSF when entering from the blood side. Second, it is possible that a saturable system for MeTHF from CSF to ,blood (via the choroid plexus) exists that was not demonstrated in this study. This might be because the choroid plexus system was saturated in vivo by the endogenous level of 32 rig/ml MeTHF in the CSF (Table 1) plus the 40 ng MeTHF injected with the intraventricular injection (Table 4). In vitro, the choroid plexus transport system for MeTHF is one-half saturated at 8 rig/ml (& = 18 mM) MeTHF in the medium (29). Although we believe bidirectional, saturable transport of MeTHF between CSF and plasma is unlikely because of the large concentration gradient between CSF and plasma (Table l), the data from this study cannot exclude this possibility. This study does not provide direct evidence that the CP is the sole or even primary mechanism for MeTHF trans-
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782
R. SPECTOR
port into CSF and provides no evidence for the route of MeTHF entry into brain. However, the saturable uptake system of the CP for MeTHF, like that for folate in liver, kidney, and gut (10, 14, 22, 23, 33; unpublished observations), suggests that the CP may perform a transport role in the CNS similar to its role in ascorbic acid transport from blood into CSF (26). Wh erever the exact locus (loci) of .MeTHF transport into the CNS, MeTHF readily appeared in CSF by a mechanism that was one-half saturated at normal plasma concentrations of MeTHF. Thus, at low the mechanism for transporting plasma concentrations, MeTHF from blood to CSF would be less than one-half saturated and would pump proportionately more MeTHF into CSF. At high plasma concentrations, the transport mechanism is predominately saturated, and MeTHF would enter CSF predominately by diffusion. The saturable entry of MeTHF into the CNS complements the regulation of plasma MeTHF levels by the gut, liver, and kidney (10,
AND A. V. LORENZO
14, 22, 23, 33, 34; unpublished observations), thus offering the CNS further protection against plasma fluctuations. Moreover, folic acid, which cannot be employed by the brain (17), is pumped from the CSF by a saturable mechanism. In conclusion, MeTHF and FA levels in the CNS would appear to be homeostatically regulated by these systems even in the face of gross alterations in either dietary or blood levels. The authors thank Drs. Norman Uretsky and Robert Snodgrass for their counsel and support and Dr. David E. Drum for his measurements of the endogenous folate contents of the tissues. This study was supported in part by National Institutes of Health Grants NS 05172 and NS-HD 09704 and The Children’s Hospital Medical Center Mental Retardation and Human Developmental Research Program Grant HD 03773. A. V. Lorenzo is a recipient of a National Institutes of Health Career Development Award HD 18519. Received
for
publication
20 August
1974.
REFERENCES 1. 2. 3. . 4.
5.
10. 11.
12. 13.
14. 15.
ALPERIN, J. B., AND M. E. HAGGARD. Cerebrospinal fluid folate (CFA) and the blood-brain barrier. Clin. Res. 18 : 40, 1970. BIRD, 0. D., V. M. MCGLOHON, AND J. N. VAITKUS. Naturally occurring folates in the blood and liver of the rat. Anal. Biochem. 12 : 18-35, 1965. BROWN, J. P., G. E. DAVIDSON, AND J. M. SCOTT. Thin-layer chroatography of pteroylglutamates and related compounds. J. Chromatog. 79 : 195-207, 1973. BROWN, J. P., G. E. DAVIDSON, AND J. M. SCOTT. The identification of the forms of folate found in the liver, kidney and intestine of the monkey and their biosynthesis from exogenous pteroylglutamate (folic acid). Biochim. Biophys. Acta 343 : 78-88, 1974. CHEN, C. P., AND C. WAGNER. Transport of folic acid (FA) and 5methyltetrahydrofolic acid (5-MTHF) in the isolated choroid plexus. Federation Proc. 33 : 7 16, 1974. COLQUHOUN, D. Lectures on Biostatistics. Oxford : Clarendon, 197 1, p. 148, 211. CSERR, H. F. Physiology of the choroid plexus. Pharmacol. Rev. 51: 273-311, 1971. DAVSON, H. T. The Physiology of the Cerebrosflinal Fluid. Boston : Little, Brown, 1967. DUNNETT, C. W. A multiple comparison procedure for comparing several tratments with a control. J. Am. Sot. Agron. 50: 1096-l 121, 1955. GORESKY, C. A., H. WATANAKE, AND D. G. JOHNS. The renal excretion of folic acid. J. Clin. Invest. 42 : 1841-1849, 1963. HERBERT, V., A. R. LARRABEE, AND ,J. M. BUCHANAN. Studies on the identification of a folate compound of human serum. J. CZin. Invest. 41: 1134-l 138, 1962. HOMMES, 0. R., AND E. A. M. T. OBBENS. Liver function and folate epilepsy in the rat. J. Neural. Sci. 20: 269-272, 1973. JENSEN, 0. N., AND 0. V. OLESEN. Folic acid concentrations in cerebrospinal fluid in relation to anticonvulsant drugs and cerebral atrophy. Acta Psych. Stand. 47 : 206-2 12, 1971. JOHNS, D. G., S. SPERTI, AND A. S. V. BURGEN. The metabolism of tritiated folic acid in man. J. CZin. Invest. 40 : 1684-1695, 1961. KOREVAAR, W. C., M. A. GEYER, S. KNAPP, L. L. Hsu, AND A. J. MANDELL. Regional distribution of 5-methyltetrahydrofolic acid in brain. Nature New BioZ. 245 : 244-245, 1973.
16. LEVITT, M., P. F. NIXON, J. H. PINCUS, AND J. R. BERTINO. Transport characteristics of folates in cerebrospinal fluid; a study utilizing doubly hydrofolate. 17.
labelled 5-methyltetrahydrofolate J. CZin. Invest. 50: 1301-l 308,
and
5-formyltetra-
1971.
MAKULU, D. R., E. F. SMITH, AND J. R. BERTINO. Lack of dihydrofolate reductase activity in brain tissue of mammlian species : possible implications . J. Neurochem. 21 : 241-245,1973.
18. MARKKANEN, T., AND 0. PELTOLA. Binding of folic acid activity by body fluids. Acta HaematoZ. 43 : 272-279, 1970. 19. MARKKANEN, T., P. HIMANEN, R.-L. PAJULA, AND G. MOLNAR. Binding of folic acid to serum proteins. Acta Haematol. 50 : 284-292, 1973. 20. NIXON, P. F., AND J. R. BERTINO. Enzymic preparations of radiolabelled (+)-L-5-methyltetrahydrofolate and (+)-L-5-formyltetrahydrofolate. Anal. Biochem. 43 : 162- 172, 197 1. 21. OBBENS, E. A. M. T., AND 0. R. HOMMES. The epileptogenic effects of folate derivatives in the rat. J. Neural. Sci. 20: 223-229, 1973. 22. PRATT, R. F., AND B. A. COOPER. Folates in plasma and bile of man after feeding folic acid-3H and 5-formyltetrahydrofolate (folinit acid). J. CZin. Invest. 50 : 455-462, 1971. 23. SELHUB, J., H. BRIN, AND N. GROSSOWICZ. Uptake and reduction of radioactive folate by everted sacs of rat small intestine. European J. Biochem. 33 : 433-438, 1973. 24. SPAANS, F. No effect of folic acid supplement on CSF folate and serum vitamin B1:! in patients on anticonvulsants. Epilepsia 11 : 40341 1, 1970. 25. SPECTOR, R., D. T. KORKIN, AND A. V. LORENZO. A rapid method for the determination of salicylate binding by the use of ultrafilters. J. Pharm. Pharmacol. 24: 786-789, 1972. 26. SPECTOR, R., AND A. V. LORENZO. Ascorbic acid homeostasis in the central nervous system. Am. J. Physiol. 225 : 757-763, 1973. 27, SPECTOR, R., R. VERNICK, AND A. V. LORENZO. Effects of pressure on the plasma binding of digoxin and ouabain in an ultrafiltration apparatus. Biochem. Pharmacol. 22 : 2485-2487, 1973. 28. SPECTOR, R., AND A. V. LORENZO. Inhibition of penicillin transport from the cerebrospinal fluid after intracisternal inoculation of bacteria. J. CZin. Invest. 54: 3 16-325, 1974. 29. SPECTOR, R., AND A. V. LORENZO. Folate transport by the choroid plexus in vitro. Science 187 : 54-O-542, 1975. 30. SPECTOR, R. G. Folic acid and convulsions in the rat. Biochem. Pharmacol. 20 :’ 1730- 1732, 197 1. 31. WATERMAN, N., L. SCHARFENBERGER, AND M. J. RAFF. Rate of binding of antibiotics to canine serum protein. Antimicrob. Agents Chemotherapy 5 : 294-295, 1974. 32. WAXMAN, S., AND C. SCHREIBER. Measurement of serum folate levels and serum folic acid-binding protein by 2H-PGA radioassay. Blood 42: 281-290, 1973. 33. WEIR, D. G., J. P. BROWN, D. S. FREEDMAN, AND J. M. SCOTT. The absorption of the diastereoisomers of 5-methyltetrahydropteroylglutamate in man: a carrier-mediated process. CZin. Sci. Mol. Med. 45: 625-631, 1973. 34. WHITEHEAD, V. M., R. PRATT, A. VIALLET, AND B. A. COOPER. Intestinal conversion of folinic acid to 5-methyltetrahydrofolate in man. Brit. J. Haematol. 22 : 63-72, 1972.
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