Life Sciences Vol. 16, pp . 1571-1582 Printed in the U .S .A .
Pergamon Prese
FOLATE TRANSPORT IN THE CHOROID PLEXUS* Chi-Po Chent and Conrad Wagner Biochemistry Research Laboratory VA Hospital, Nashville, Tennessee 37203 and Department of Biochemistry Vanderbilt University School of Medicine Nashville, Tennessee 37203 (Received in final form May 1, 1975) SUMMARY The uptake of 5-methyltetrahydrofolic acid (5-MTHF) by the isolated choroid plexus of hog was studied and shown to be both tanperature and time dependent . Uptake of 5-MTHF by the isolated choroid plexus was a saturable process and exhibited a Kt of 0 .9 x 10 -6 M and Vmax of 1.39 nnwle/gm dry wt/min . The system did not require the presence of sodium ion nor was it ouabain sensitive. The presence of metabolic inhibitors, e .g ., 2,4-dinitrophenol, did not suppress the uptake rate . Deprivation of oxygen also did not affect the rate of 5-MTHF transport. Addition of folic acid to the incubating medium led to countertransport of intracellular 5-MTHF . Efflux studies also indicated that the majority of the intracellular 5-MTHF was rapidly exchangeable and therefore probably present in the cell water in a free state . Chromatographic analyses confirmed that 5-MTHF was not metabolically altered during the transport process. It is suggested that 5-methyltetrahydrofolic acid is transported in the isolated choroid plexus via a carriermediated process . The mechanism of folgte transport between blood and cerebrospinal fluid (CSF) has not been fully established . The observation of higher concentrations of folgte, mainly in the form of 5-methyltetrahydrofolic acid (5-MTHF), in the CSF than in the serum (1-7), the selective high conservation of folates in the central nervous system (CNS) (8) and the relatively constant ratio between CSF and serum folgte concentrations among various individuals (2,3,6,9) suggest the existence of a homeostatic mechanism for the maintenance of CSF folgte concentrations . Levitt and his associates (10) and Chanarin and his co-workers (11) found that 5-MTHF is transferred from the blood across the blood-brain barrier by a carrier-mediated mechanism. The transport of folgte from the CSF to the blood, however, has not been studied . It has been well established that the choroid plexus is one of the many possible sites in the central nervous systen which functions in maintaining *This work was supported in part from grant ßi469 from the Nutrition Foundation . tPresent Address : Section of Pharmacology and Toxicology, School of Pharmacy, University of Connecticut, Storrs, Conn . 06268
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the homeostasis in this region (12-15) . In this communication we report our studies on the mechanisms of 5-MTHF transport in the isolated choroid plexus of the hog . MATERIALS AND METHODS 1.
Tissues
The experiments were carried out using isolated choroid plexuses of hogs which were obtained from a local slaughterhouse . After stunning and decapitation, the hog skulls were opened by an electric chopper and the tissues were quickly removed from the lateral ventricles and stored in ice-cold Krebs-Ringer's (K-R) phosphate solution (pH 7 .2) before returning to the laboratory . The time interval between obtaining the tissues and the beginning of experiments was usually one to two hours . The viability of the choroid plexus was established by demonstratin the active transport of glucose which is known to take place in this tissue 16,17) . Choroid plexuses from the lateral ventricles weighing 75 to 125 mg were used in all experiments . The water content of choroid plexus was found to average 86% of the wet weight . 2.
Labeled and Non-labeled Compounds
The labeled compounds used in this study were obtained from the following sources : folic acid-3H (FA-3H) and glucose-14C from New England Nuclear . 5-methyltetrahydrofolic acid-3H (5-MTHF-3H) was prepared from FA-3H by Mr . W. T . Briggs in this laboratory by a combination of chemical and enzymological techniques with subsequent purification by DEAE-Sephadex column chromatography (18) . The non-labeled compounds used in this study were obtained from the following commercial sources : folic acid, ouabain, probenecid, dipher~ylhydantoin from Sigma, folinic acid from General Biochemicals, Phenobarbital sodium from Mallinckrodt, L-glutamic acid from Calbiochem, p-aminobenzoic acid from City Chemical, 2,4-dinitrophenol from Matheson, Coleman and Bell . Pteroic acid was prepared in this laboratory by the method of Levy and Golfiman (19) . Purified dihydrofolate reductase was a generous gift of Dr . Roy Kisliuk, Tufts University . 3.
Incubation Conditions
Uptake studies were done by placing two choroid plexuses in 20 ml glass vials containing 5 ml of K-R phosphate solution (pH 7 .2) . The K-R phosphate solution (pH 7 .2) was prepared as described in Manametric Techniques (20) . The incubation media contained a trace amount of 5-MTHF-3H (10 uCi/7 .45 x 10-8 ymoles/L) . The uncapped vials were shaken in a Gyrotory water bath shaker for one hour at 37oC, or as otherwise indicated . In some experiments using anaerobic conditions, the incubating media and vessels were gassed thoroughly with 100% N2 and incubated after capping (12) . In efflux studies, cho~oid plexuses were preincubated for one hour at 37oC in the presence of 5-MTHF- H. The tissues were then transferred to fresh media at the end of the preincubation period . At every 15-minute interval, the tissues were rapidly transferred into fresh media. After one hour, the tissues were removed and extracted with 2 ml of 1% sodium ascorbate solution (adjusted to pH 6 .0) . The sum of the radioactivity released into the media after each 15-minute period and that remaining in the tissue extract was set to 100% . The results were calculated as a cumulative percentage of total radioactivity released from the choroid plexus at each time interval .
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In the studies on countertransport, the choroid plexuses were preincubated for 60 minutes in order to introduce labeled 5-MTHF into the tissue . At the end of the preincubation period, unlabeled folic acid was added to the incubation media to achieve a final concentration of 10-6M . The radioactivity remaining in the choroid plexus was measured at different time intervals . 4.
Analyses of Radioactivity
At the end of the incubation period, the plexuses were removed, gently blotted, placed on a preweighed filter paper and weighed on an analytical balance . Tissues were then extracted with 2 ml of 1% sodium ascorbate solution (pH 6 .0) with a combination of freézing-thawing and sonication . The samples were centrifuged and aliquots of the supernatant were added to glass vials containing 10 ml of scintillation fluid (Scintisol-complete, Isolab, Akron, Ohio) . Radioactivity was measured with a liquid scintillation spectrometer (Tri-Carb, Model 3315, Packard Instrument Co ., Chicago, I11 .) with an efficiency of 35% . Adjustment for quenching was made by the channels ratio method (21) . The amounts of folate trapsported were expressed as rmoles/gm dry wt/hr ± S .E .M . The results were analyzed by subjecting the difference of mean values between control and other conditions to Student's t-test of significance (22) . 5.
Identification of Folate Compounds by Chromatography
The extent of macromolecule-folate binding was estimated by passing the sample solutions through a 0 .9 x 8 cm Bio-Gel P-6 column equilibrated with 0 .01 M phosphate buffer (pH 7 .0) containing 0.01 M mercaptoethanol and eluted with the same solvent . The column was calibrated with blue dextran. One-half ml fractions were collected and counted in 10 ml of scintillation fluid . The possible formation of folate metabolites during the transport process was tested by chromatography of the samples on a 0 .9 x 27 an column of A-25 DEAE-Sephadex equilibrated with 0.1 M phosphate buffer (pH 6.0) contain ing 20 mM mercaptoethanol . The sample solution was applied to the column and subsequently washed with 50 ml of 0 .1 M phosphate buffer (pH 6 .0) . Materials were eluted at 4°C by phosphate buffer (pH 6 .0) with a linear gradient from 0.1 M to 2 .0 M, also containing 20 mM mercaptoethanol . The final concentration of 2.0 M phosphate buffer was obtained after passage of 500 ml of eluting buffer (23 ). Non-radioactive 5-MTHF was added with the sample as a marker . Fractions of 3 .0 ml were collected and monitored by measurenent of radioactivity and ultraviolet absorbante at 290 nanometers . RESULTS 1.
Viability of the Choroid Plexus
The viability of the tissue was examined by incubating the choroid plexus in the K-R phosphate solution (pH 7 .2) containing either 100 uM or 1 uM glucose-14C . After 60 minutes, the T/M (tissue concentration/medium concentra tions) ratios were found to be 5 .76 ± 0.17 (n=4) and 12 .04 ± 1 .06 (n=4) for 100 uM and 1 uM glucose, respectively . The existence of an accumulative process in the choroid plexus is a strong indication that the tissues are still actively functional . 2.
The Uptake of 5-MTHF When 7 .45 x 10 -8 M 5-MTHF was incubated with the isolated choroid
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plexus, the initial uptake was very rapid . As shown in Fig . 1, an appreciable amount of 5-MTHF was already taken up by the choroid plexus after 5 minutes of incubation . The rate of uptake slowed after prolonged incubation and had essentially plateaued after 60 minutes . Only about 5% of the added substrate had been taken up at this time . Fig . 1 also shows that when more than a 10-fold higher concentration
3 .0 2.6 H
3
2 .0
m0 î U' w N W O i z
L~ L0 0 .5
10
20
30
40
60 TI M E
80
70
80
g0
(mln.)
FIG . 1 Time course of 5-methyltetrahydrofolic acid uptake and counter-transport phenomenon . At 60 min . unlabeled folic acid was added to the incubation medium to achieve a final concentration of 10 6 M and incubation was terminated at different time intervals (dotted line) . of folic acid was added to the uptake medium after 60 minutes of incubation, there was a rapid exit of labeled 5-MTHF from the tissues, indicating that counter-transport may be taking place. 3.
Identification of 5-MTHF in the Tissue Extract
Choroid plexuses which had been incubated with [3H] 5-MTHF for one hour were extracted as described in Materials and Methods. When the tissue extracts were chromatographed on small columns of Bio-Gel P-6 in order to separate free and bound folate derivatives, about 30% of the radioactivity was associated with the high molecular weight material (Fig . 2) . If the tissue extracts were boiled prior to chromatography on the Bio-Gel columns, no folate was found associated with the high molecular weight fraction . Ion-exchange chromatography of the boiled tissue extracts showed that more than 80% of the radioactivity taken up b the tissue co-chramatographed in a single peak with authentic 5-MTHF (Fig . 3~ .
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0
a
0 zw u w a
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20
15 10 5 0
FIG. 2 Radioactivity found Chromatographic identification of free and bound folates . in the aqueous extract of choroid plexus after one hour incubation with labeled folgte was chramatographed on 0.9 x 8 an Bio-Gel P-6 columns equilibrated with 0 .01 M phosphate buffer (pH 7.0) containing 0.01 M mercaptoethanol and eluted Fractions of 0.5 ml were collected . The first peak was with the same solvent. eluted in void volume . 4.
Variation of Incubation Conditions
As shown The uptake of 5-MTHF under different conditions was studied. in Fig . 4, the uptake of 5-MTHF was unaffected by the elimination of sodium Uptake was from the medium, or by the elimination of 02 during the incubation . Only also unaffected by the addition of ouabain or dinitrophenol (10-3 M) . incubation at 0°C resulted in a significant decrease in the uptake of 5-MTHF . The absence of arty requirement for sodium ions or oxygen together with the lack of ouabain sensitivity indicates that active transport of 5-MTHF is probably not operating in the choroid plexus . This is confirmed by the absence of inhibition in the presence of 2,4-dinitrophenol . 5.
Kinetics of 5-MTHF Transport
The rate of 5-MTHF transport increased at higher concentrations of the substrate . In these experiments, measurements of total uptake were made after Although it is not 5-minute incubation in order to study initial rates . certain that the initial rate of uptake is linear during the first five minutes, As shown in shorter incubation times are not practical with this method . Fig . 5, saturation is approached at higher concentration of 5-MTHF . The observed Kt for 5-MTHF obtained from best fitted regression analysis on the double reciprocal plot was 0 .9 x 10 -6 M . The apparent Vmax was 1 .39 rmoles/gm dry wt/min . 6.
Efflux of 5-MTHF
Most of the 5-MTHF taken up by the choroid plexus was freely exchangeable with the medium . This is shown on Table I . The tissue was first preloaded with labeled 5-MTHF by incubation in 7 .45 x 10-8 M substrate for 60 minutes. The tissue was then transferred to fresh medium at 15-min . intervals. After 60 minutes, over 85X of the 5-MTHF taken up in the first period of
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I z
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O
H u a
a
w u z
a u
0 m
u a 0
am
H
m a
ô
a
FIG. 3 Chromatographic identification of folates on DEAE-Sephadex column . Choroid plexuses were extracted after incubation with labeled folate as described in the text and chramatographed on a 0.9 x 27 cm column of A-25 DEAE-Sephadex with an increasing gradient from 0.1 M to 2 .0 M . Unlabeled 5-MTHF was added with authentic 5-MTHF which was measured by its absorbante at 290 nm . The ultraviolet absorbing peak which eluted between fractions 30 and 40 was due to the ascorbate which had been added to the tissue extract as an antioxidant . incubation was released to the median . The remaining 20% of the radioactivity is probably due to degradation products of 5-MTHF since the authentic unlabeled material also exhibited absorbante at 290 rm in fractions which eluted before the main peak . TABLE I Tissues were preincubated for 60 min. at 37°C in the presence of 7 .45 x 10-8 M 5-MTHF . The tissues were then transferred to fresh media every 15 min . (see Ina bation Conditions) . Time (min .) 15 30 45 60 7.
Percent of Total (Mean t SEM) 57 .19 73 .76 81 .52 86 .39
± f ± ±
1 .30 1 .68 1 .71 1 .68
(n=4) (n-4 (n=4 ; (n=4)
Effect of Various Drugs on the Uptake of 5-MTHF Several drugs were tested for their effect on the transport of 5-MTHF .
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CONTROL
TRIS
NZ
OUABAIN ION
DNP
IÔ3M
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FIG . 4 The uptake of 5-MTHF by the isolated choroid plexus was determined under different conditions . DNP, 2,4-dinitrophenol .** P < 0.01 These are shown in Table II . Neither probenecid, which is transported by a mechanism involving organic anions in the choroid plexus, nor dipher~ylhydantoin, which has been reported to affect the intestinal transport of folgte derivatives, was inhibitory . On the other hand, Phenobarbital, at a concentration of 10 -3 M, caused significant inhibition . TABLE II Effect of Various Drugs on the Uptake of 5-Methyltetrahydrofolate in the Isolated Choroid Plexus Addition Control Probenecid Dipher~ylhydantoin Phenobarbital *N .S, 8.
Conc . 10 -3 M 10 -3 M 10 -3 M
Percent of Control 100 93 .02 121 .84 54 .13
(n-4 (n~4 ; n-4 (n=4~
P Value N.S .* N.S . 0 .0025
non-significance
Effect of Folgte Analogues on the Transport of 5-MTHF
The uptake of 5-MTHF by the choroid plexus was inhibited by a number of folgte analogues . This is shown in Table III . Folic acid, folinic acid, methotrexate and pteroic acid, all were potent inhibitors of 5-MTHF transport when tested at 10- 6 M. Para-aminobenzoic acid and L-glutamic acid, which make up portions of the folic acid molecule, were also inhibitory when tested at
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1/S (PM) 1 FIG . 5 A double reciprocal plot of the rate of 5-MTHF uptake . plotted by the method of regression analysis .
The linear graph was
higher concentrations . TABLE III Effect of Folate Analogues on the Uptake of
5-Met~ltetrahydrofolate in the Isolated_ Çhoroid Plexus Uptake in the absence of addition was taken as the control value . Addition None Folic acid Folinic acid Methotrexate Pteroic acid p-Aminobenzoic acid L-Glutamic acid
Conc . 10-6 10 -6 10 -6 10 -6 10 -3 10 -3
M M M M M M
Percent of Control
P Value
100 41 .31 22 .25 49 .89 27 .27 43 .11 69 .18
-0.001 0.001 0.001 0.001 0.05 0.05
DISCUSSION Levitt and co-workers (10) and Chanarin et al . (11) have demonstrated that the transport of 5-MTHF from the blood to the CSFin both the dog and man is probably a carrier-mediated process across the blood-brain barrier . Allen and Klipstein (8) found that the central nervous system has a selective conservation mechanism for maintaining high folate activity in spite of subnormal
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concentrations of this vitamin which may occur in the blood or other tissues . Several workers (2,3,6,9) have also reported that the ratio of folate concentrations between the CSF and serum ranains rather constant in a variety of abnormal conditions . Such observations would suggest a mechanism in the choroid plexus and/or elsewhere in the central nervous system which would operate to regulate the exit of folate from the CSF to the blood and thus maintain the homeostasis in this region . The 5-MTHF in the CSF may be derived from turnover of 5-MTHF in the brain tissue which then passes from the interstitial fluid of the brain into the CSF . The presence of a so-called blood-CSF barrier between the CSF and the blood could then contribute to the observed concentration difference of 5-MTHF between the CSF and the serum . In the past decade, many researchers have stressed the importance of transport mechanisms in the choroid plexus or elsewhere in the CNS in the regulation of the concentration of various substances in the CNS (14,24) . The participation of the choroid plexus in the efflux transport processes in vivo (15,24) and the ability of the choroid plexus incubated in vitro to accunu~e substances against a concentration gradient are generallyaccepted as factors which help to maintain the relatively stable mileu in the central nervous systan (12,14) . The results of the present studies suggest the existence of a carriermediated transport system for 5-MTHF in the choroid plexus . In addition to the saturation kinetics exhibited by the uptake of 5-MTHF (Fig . 5), the choroid plexus also demonstrates countertransport by folic acid (Fig . 1) . Specificity of uptake is also danonstrated by the inhibition produced in the presence of folate analogues . Table III shows that folic acid, folinic acid, methotrexate and pteroic acid all decrease the uptake of 5-MTHF . If the concentration of 5-MTHF in the tissue after 60 minutes uptake is calculated from a number of experiments and compared to the medium concentration, the ratio (T/M) usually exceeded 1 .0 suggesting the existence of same active process to maintain this gradient . The absence, however, of any inhibition by ouabain or 2,4-dinitrophenol, as well as lack of aqy dependence on sodium together with the absence of inhibition under anaerobic conditions all tend to rule out the operation of an active transport process in the uptake of 5-MTHF by the choroid plexus . It Beans possible, therefore, that the concentration gradient is maintained by the binding of 5-MTHF to tissue components . Such binding may take place with several macromolecular species with varying degrees of affinity . Fig . 2 shows that at least 30X of the 5-MTHF taken up after 60 min. i s bound strongly enough to a macromolecule to ranain associated during chromatography on a Bio-Gel column . This does not exclude the possibility that additional 5-MTHF is bound to other species with a lower affinity that would dissociate during the chromatography . The binding of folate derivatives to protein species in various tissues has been daranstrated recently in our own laboratory (25) as well as others (26,27) . This binding has been shown to be non-covalent, probably involving electrostatic interactions between the folate derivative and the binder . This is also consistent with the demonstration that the majority of the transported 5-MTHF in the choroid plexus was freely exchangeable with the medium (Table I) and was unchanged 5-MTHF (Fig . 3) . This, in addition to the reversibility of the direction of flux by the addition to the incubation medium of a substance which competitively inhibits 5-MTHF influx, is indicative of the countertransport phenomenon (28) and implies the operation of a mobile carrier . Moreover, the danonstration of countertransport of 5-MTHF by folic acid indicates that this substance not only competes with 5-MTHF for sites on the carrier molecule but also utilizes this same carrier, at least in part, for its own transport .(29) .
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The uptake of 5-MTHF by the hog choroid plexus is in contrast to studies carried out by Rubin and his collaborators (30) using methotrexate, a folate analogue . These investigators found that T/M ratios of 6 to 7 were attained and transport of methotrexate by rabbit choroid plexus was significantly inhibited by incubation under N2 or by the addition of 2,4-dinitrophenol at 10-4 M concentration. Ouabain (10-5 M) did not inhibit . The uptake of methotrexate by the rabbit choroid plexus was not inhibited by folic acid or folinic acid and, therefore, arty active transport mechanism which may be responsible for methotrexate accumulation is probably distinct from the systen responsible for the transport of 5-MTHF and other natural forms of folate . The intestinal transport of 5-MTHF has been examined in considerable detail by several laboratories (23,31-33) . The consensus of all these studies using a variety of techniques is that passage of 5-MTHF across the rat intes tine is a passive process . There was no evidence for accumulation of 5-MTHF nor was there any indication of competition by analogues or energy requirement . The data presented here tend to indicate that an energy independent carriermediated transport system for 5-MTHF is present in the choroid plexus . Other factors which are involved in the maintenance of high folate concentration in the brain tissue and CSF are the mechanisms of transport from the blood to the brain tissue and from the tissue to the CSF. Since this present work was completed, a report has appeared which describes studies on the transport of folic acid and 5-MTHF by the isolated rabbit choroid plexus (34) . This latter study confirms the presence of a saturable, specific, uptake system for folates in this tissue . In contrast to the data presented here, however, Spector and Lorenzo claim that uptake of folates is an energy dependent process since they obtained high T/M ratios after 15 minutes incubation and showed inhibition of folate uptake by a combination of dinitrophenol and iodoacetate in the absence of glucose . They were unable to show any inhibition under anaerobic conditions in the absence of glucose. Since iodoacetate may inactivate a protein carrier in addition to serving as an inhibitor of glycolysis and the high T/M ratios may be explained by tissue binding of transported folate, the claim of an energy dependent process being involved should be viewed with caution . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 .
J . B. ALPERIN and M. E . HAGGARD, Clin . Res. 18 40 (1970) . M. B. BOWERS, JR . and E. H. REYNOLDS, Lancette 1376 (1972) . M. E . BOYKIN and H. HOOSHMAND, Neurolog~4~3 (1970) . V . HERBERT and R. ZALUSKY, Fed. Proc . 20 453 (1961) . T. MARKKANEN and P. HIMANEN,Înt. _J . Vitam. Nutr . Res . 41 79-85 (1971) . E . H. REYNOLDS, B. B . GALLAGHER, R H. MATTSON, M .BOWERS, and A. L. JOHNSON, Nature 240 155-157 (1972) . D . G. WELLS and H . J . CÂSEY, Brit . Med. J . 3 834-836 (1967) . C. D. ALLEN and F . A . KLIPSTEIN, ~Neuro~l~ 20 403 (1970) . E . H. REYNOLDS, R . H . MATTSON, and B . B . GALLAGHER, Neurology _22 841844 (1972) . M. LEVITT, P . F. NIXON, J . H . PINCUS, and J . R . BERTINO, _J . Clin . Invest . 50 1301-1308 (1971) . Î . CHANARIN, J. PERKY, and E . H. REYNOLDS, Clin . Sci . _Mol . _Med . _46 369373 (1974) . C . P . CHEN, Active transport of myoinositol in the isolated choroid plexus . Ph .D . dissertation, University of Kentucky (1973) . T. Z . CSAKY, Choroid plexus . In : Handbook of Neurochemistry , Vol . 2 . A. Lajtha (ed .), Plenum Press, New or 1 6~. H. F. CSERR, Physiol . Rev . 51 273-311 (1971) .
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R . SPECTOR and A . V . LORENZO, _J . Pharmacol . Exptl . Thera . _185 642648 (1973) . T . Z . CSAKY and B . M . RIGOR, Life Sci . 3 931-936 (1964) . T . Z . CSAKY and B . M . RIGOR, Pro . Brain Res . 29 147-154 (1968) . P . F . NIXON and J . R . BERTINO~aI .iochc~n . ~ 162-173 (1971) . C . C . LEVY and P . GOLDMAN, J . BiôT. CFem.~2~2 1533-2938 (1967) . W . W . UMBREIT, R . H . BURRIS, agi . F . STAUFFER, Manametric Techniques , 3rd Ed . Burgess, Minneapolis (1957) . _in Biolo ical C . H . WANG and D . L . WILLIS, Radiotracer Methodol Science . Prentice-Hall, Englewo~CT~i~ts 1 6 G . W . SNEDECOR and W . G . COCHRAN, Statistical methods . The Iowa State University Press, Ames, Iowa (1968) . W . STRUM, P . F . NIXON, J . R . BERTINO, and H . J . BINDER, _J . Clin . Invest . 50 1910-1916 (1971) . J. R . PAPPENHEIMER, S . R . HEISEY, and E . F . JORDAN, _Am . _J . Physiol . _200 1-10 (1961) . M . ZAMIEROWSKI and C . WAGNER, Biochen . Bio s . _Res . Cammun ., in press (1974) . R . CORROCHER, G . DESANDRE, M . L . PACOR, and A . V . HOFFBRAND, Clin . _Sci . Mol . Med . 46 551-554 (1974) . . B A . KAMENand J . D . CASTON, J . Lab . Clin . Med . 8 1 164-174 (1974) . .13109-183 (1961) . W . WILBRANDT and T . ROSENBERG,Pharmacy -Rev . ~716~197~ . C . P . CHEN and C . WAGNER, Fed . Proc R . RUBIN, E . OWENS, and D . BALL, _Cancer Res . 28 689-694 (1968) . MATTY, _J . Physiol . _236 653J . A. BLAIR, I . T . JOHNSON, and A J. 661 (1974) . L . ELSBORG, Dan . Med . Bull . 21 1-11 (1974) . S . MOMTAZI and -V . HERBERT, Am. J . Clin . Nutr . 2 6 23-29 (1973) . Science 187 540-542 (1975) . R . SPECTOR and A . V . LORENZ
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FOLATE TRANSPORT IN THE CHOROID PLEXUS* Chi-Po Chent and Conrad Wagner Biochemistry Research Laboratory VA Hospital, Nashville, Tennessee 37203 and Department of Biochemistry Vanderbilt University School of Medicine Nashville, Tennessee 37203 (Received in final form May 1, 1975) SUMMARY The uptake of 5-methyltetrahydrofolic acid (5-MTHF) by the isolated choroid plexus of hog was studied and shown to be both tanperature and time dependent . Uptake of 5-MTHF by the isolated choroid plexus was a saturable process and exhibited a Kt of 0 .9 x 10 -6 M and Vmax of 1.39 nnwle/gm dry wt/min . The system did not require the presence of sodium ion nor was it ouabain sensitive. The presence of metabolic inhibitors, e .g ., 2,4-dinitrophenol, did not suppress the uptake rate . Deprivation of oxygen also did not affect the rate of 5-MTHF transport. Addition of folic acid to the incubating medium led to countertransport of intracellular 5-MTHF . Efflux studies also indicated that the majority of the intracellular 5-MTHF was rapidly exchangeable and therefore probably present in the cell water in a free state . Chromatographic analyses confirmed that 5-MTHF was not metabolically altered during the transport process. It is suggested that 5-methyltetrahydrofolic acid is transported in the isolated choroid plexus via a carriermediated process . The mechanism of folgte transport between blood and cerebrospinal fluid (CSF) has not been fully established . The observation of higher concentrations of folgte, mainly in the form of 5-methyltetrahydrofolic acid (5-MTHF), in the CSF than in the serum (1-7), the selective high conservation of folates in the central nervous system (CNS) (8) and the relatively constant ratio between CSF and serum folgte concentrations among various individuals (2,3,6,9) suggest the existence of a homeostatic mechanism for the maintenance of CSF folgte concentrations . Levitt and his associates (10) and Chanarin and his co-workers (11) found that 5-MTHF is transferred from the blood across the blood-brain barrier by a carrier-mediated mechanism. The transport of folgte from the CSF to the blood, however, has not been studied . It has been well established that the choroid plexus is one of the many possible sites in the central nervous systen which functions in maintaining *This work was supported in part from grant ßi469 from the Nutrition Foundation . tPresent Address : Section of Pharmacology and Toxicology, School of Pharmacy, University of Connecticut, Storrs, Conn . 06268
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the homeostasis in this region (12-15) . In this communication we report our studies on the mechanisms of 5-MTHF transport in the isolated choroid plexus of the hog . MATERIALS AND METHODS 1.
Tissues
The experiments were carried out using isolated choroid plexuses of hogs which were obtained from a local slaughterhouse . After stunning and decapitation, the hog skulls were opened by an electric chopper and the tissues were quickly removed from the lateral ventricles and stored in ice-cold Krebs-Ringer's (K-R) phosphate solution (pH 7 .2) before returning to the laboratory . The time interval between obtaining the tissues and the beginning of experiments was usually one to two hours . The viability of the choroid plexus was established by demonstratin the active transport of glucose which is known to take place in this tissue 16,17) . Choroid plexuses from the lateral ventricles weighing 75 to 125 mg were used in all experiments . The water content of choroid plexus was found to average 86% of the wet weight . 2.
Labeled and Non-labeled Compounds
The labeled compounds used in this study were obtained from the following sources : folic acid-3H (FA-3H) and glucose-14C from New England Nuclear . 5-methyltetrahydrofolic acid-3H (5-MTHF-3H) was prepared from FA-3H by Mr . W. T . Briggs in this laboratory by a combination of chemical and enzymological techniques with subsequent purification by DEAE-Sephadex column chromatography (18) . The non-labeled compounds used in this study were obtained from the following commercial sources : folic acid, ouabain, probenecid, dipher~ylhydantoin from Sigma, folinic acid from General Biochemicals, Phenobarbital sodium from Mallinckrodt, L-glutamic acid from Calbiochem, p-aminobenzoic acid from City Chemical, 2,4-dinitrophenol from Matheson, Coleman and Bell . Pteroic acid was prepared in this laboratory by the method of Levy and Golfiman (19) . Purified dihydrofolate reductase was a generous gift of Dr . Roy Kisliuk, Tufts University . 3.
Incubation Conditions
Uptake studies were done by placing two choroid plexuses in 20 ml glass vials containing 5 ml of K-R phosphate solution (pH 7 .2) . The K-R phosphate solution (pH 7 .2) was prepared as described in Manametric Techniques (20) . The incubation media contained a trace amount of 5-MTHF-3H (10 uCi/7 .45 x 10-8 ymoles/L) . The uncapped vials were shaken in a Gyrotory water bath shaker for one hour at 37oC, or as otherwise indicated . In some experiments using anaerobic conditions, the incubating media and vessels were gassed thoroughly with 100% N2 and incubated after capping (12) . In efflux studies, cho~oid plexuses were preincubated for one hour at 37oC in the presence of 5-MTHF- H. The tissues were then transferred to fresh media at the end of the preincubation period . At every 15-minute interval, the tissues were rapidly transferred into fresh media. After one hour, the tissues were removed and extracted with 2 ml of 1% sodium ascorbate solution (adjusted to pH 6 .0) . The sum of the radioactivity released into the media after each 15-minute period and that remaining in the tissue extract was set to 100% . The results were calculated as a cumulative percentage of total radioactivity released from the choroid plexus at each time interval .
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In the studies on countertransport, the choroid plexuses were preincubated for 60 minutes in order to introduce labeled 5-MTHF into the tissue . At the end of the preincubation period, unlabeled folic acid was added to the incubation media to achieve a final concentration of 10-6M . The radioactivity remaining in the choroid plexus was measured at different time intervals . 4.
Analyses of Radioactivity
At the end of the incubation period, the plexuses were removed, gently blotted, placed on a preweighed filter paper and weighed on an analytical balance . Tissues were then extracted with 2 ml of 1% sodium ascorbate solution (pH 6 .0) with a combination of freézing-thawing and sonication . The samples were centrifuged and aliquots of the supernatant were added to glass vials containing 10 ml of scintillation fluid (Scintisol-complete, Isolab, Akron, Ohio) . Radioactivity was measured with a liquid scintillation spectrometer (Tri-Carb, Model 3315, Packard Instrument Co ., Chicago, I11 .) with an efficiency of 35% . Adjustment for quenching was made by the channels ratio method (21) . The amounts of folate trapsported were expressed as rmoles/gm dry wt/hr ± S .E .M . The results were analyzed by subjecting the difference of mean values between control and other conditions to Student's t-test of significance (22) . 5.
Identification of Folate Compounds by Chromatography
The extent of macromolecule-folate binding was estimated by passing the sample solutions through a 0 .9 x 8 cm Bio-Gel P-6 column equilibrated with 0 .01 M phosphate buffer (pH 7 .0) containing 0.01 M mercaptoethanol and eluted with the same solvent . The column was calibrated with blue dextran. One-half ml fractions were collected and counted in 10 ml of scintillation fluid . The possible formation of folate metabolites during the transport process was tested by chromatography of the samples on a 0 .9 x 27 an column of A-25 DEAE-Sephadex equilibrated with 0.1 M phosphate buffer (pH 6.0) contain ing 20 mM mercaptoethanol . The sample solution was applied to the column and subsequently washed with 50 ml of 0 .1 M phosphate buffer (pH 6 .0) . Materials were eluted at 4°C by phosphate buffer (pH 6 .0) with a linear gradient from 0.1 M to 2 .0 M, also containing 20 mM mercaptoethanol . The final concentration of 2.0 M phosphate buffer was obtained after passage of 500 ml of eluting buffer (23 ). Non-radioactive 5-MTHF was added with the sample as a marker . Fractions of 3 .0 ml were collected and monitored by measurenent of radioactivity and ultraviolet absorbante at 290 nanometers . RESULTS 1.
Viability of the Choroid Plexus
The viability of the tissue was examined by incubating the choroid plexus in the K-R phosphate solution (pH 7 .2) containing either 100 uM or 1 uM glucose-14C . After 60 minutes, the T/M (tissue concentration/medium concentra tions) ratios were found to be 5 .76 ± 0.17 (n=4) and 12 .04 ± 1 .06 (n=4) for 100 uM and 1 uM glucose, respectively . The existence of an accumulative process in the choroid plexus is a strong indication that the tissues are still actively functional . 2.
The Uptake of 5-MTHF When 7 .45 x 10 -8 M 5-MTHF was incubated with the isolated choroid
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plexus, the initial uptake was very rapid . As shown in Fig . 1, an appreciable amount of 5-MTHF was already taken up by the choroid plexus after 5 minutes of incubation . The rate of uptake slowed after prolonged incubation and had essentially plateaued after 60 minutes . Only about 5% of the added substrate had been taken up at this time . Fig . 1 also shows that when more than a 10-fold higher concentration
3 .0 2.6 H
3
2 .0
m0 î U' w N W O i z
L~ L0 0 .5
10
20
30
40
60 TI M E
80
70
80
g0
(mln.)
FIG . 1 Time course of 5-methyltetrahydrofolic acid uptake and counter-transport phenomenon . At 60 min . unlabeled folic acid was added to the incubation medium to achieve a final concentration of 10 6 M and incubation was terminated at different time intervals (dotted line) . of folic acid was added to the uptake medium after 60 minutes of incubation, there was a rapid exit of labeled 5-MTHF from the tissues, indicating that counter-transport may be taking place. 3.
Identification of 5-MTHF in the Tissue Extract
Choroid plexuses which had been incubated with [3H] 5-MTHF for one hour were extracted as described in Materials and Methods. When the tissue extracts were chromatographed on small columns of Bio-Gel P-6 in order to separate free and bound folate derivatives, about 30% of the radioactivity was associated with the high molecular weight material (Fig . 2) . If the tissue extracts were boiled prior to chromatography on the Bio-Gel columns, no folate was found associated with the high molecular weight fraction . Ion-exchange chromatography of the boiled tissue extracts showed that more than 80% of the radioactivity taken up b the tissue co-chramatographed in a single peak with authentic 5-MTHF (Fig . 3~ .
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H u a
0
a
0 zw u w a
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20
15 10 5 0
FIG. 2 Radioactivity found Chromatographic identification of free and bound folates . in the aqueous extract of choroid plexus after one hour incubation with labeled folgte was chramatographed on 0.9 x 8 an Bio-Gel P-6 columns equilibrated with 0 .01 M phosphate buffer (pH 7.0) containing 0.01 M mercaptoethanol and eluted Fractions of 0.5 ml were collected . The first peak was with the same solvent. eluted in void volume . 4.
Variation of Incubation Conditions
As shown The uptake of 5-MTHF under different conditions was studied. in Fig . 4, the uptake of 5-MTHF was unaffected by the elimination of sodium Uptake was from the medium, or by the elimination of 02 during the incubation . Only also unaffected by the addition of ouabain or dinitrophenol (10-3 M) . incubation at 0°C resulted in a significant decrease in the uptake of 5-MTHF . The absence of arty requirement for sodium ions or oxygen together with the lack of ouabain sensitivity indicates that active transport of 5-MTHF is probably not operating in the choroid plexus . This is confirmed by the absence of inhibition in the presence of 2,4-dinitrophenol . 5.
Kinetics of 5-MTHF Transport
The rate of 5-MTHF transport increased at higher concentrations of the substrate . In these experiments, measurements of total uptake were made after Although it is not 5-minute incubation in order to study initial rates . certain that the initial rate of uptake is linear during the first five minutes, As shown in shorter incubation times are not practical with this method . Fig . 5, saturation is approached at higher concentration of 5-MTHF . The observed Kt for 5-MTHF obtained from best fitted regression analysis on the double reciprocal plot was 0 .9 x 10 -6 M . The apparent Vmax was 1 .39 rmoles/gm dry wt/min . 6.
Efflux of 5-MTHF
Most of the 5-MTHF taken up by the choroid plexus was freely exchangeable with the medium . This is shown on Table I . The tissue was first preloaded with labeled 5-MTHF by incubation in 7 .45 x 10-8 M substrate for 60 minutes. The tissue was then transferred to fresh medium at 15-min . intervals. After 60 minutes, over 85X of the 5-MTHF taken up in the first period of
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I z
E c O o~ N
O
H u a
a
w u z
a u
0 m
u a 0
am
H
m a
ô
a
FIG. 3 Chromatographic identification of folates on DEAE-Sephadex column . Choroid plexuses were extracted after incubation with labeled folate as described in the text and chramatographed on a 0.9 x 27 cm column of A-25 DEAE-Sephadex with an increasing gradient from 0.1 M to 2 .0 M . Unlabeled 5-MTHF was added with authentic 5-MTHF which was measured by its absorbante at 290 nm . The ultraviolet absorbing peak which eluted between fractions 30 and 40 was due to the ascorbate which had been added to the tissue extract as an antioxidant . incubation was released to the median . The remaining 20% of the radioactivity is probably due to degradation products of 5-MTHF since the authentic unlabeled material also exhibited absorbante at 290 rm in fractions which eluted before the main peak . TABLE I Tissues were preincubated for 60 min. at 37°C in the presence of 7 .45 x 10-8 M 5-MTHF . The tissues were then transferred to fresh media every 15 min . (see Ina bation Conditions) . Time (min .) 15 30 45 60 7.
Percent of Total (Mean t SEM) 57 .19 73 .76 81 .52 86 .39
± f ± ±
1 .30 1 .68 1 .71 1 .68
(n=4) (n-4 (n=4 ; (n=4)
Effect of Various Drugs on the Uptake of 5-MTHF Several drugs were tested for their effect on the transport of 5-MTHF .
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Choroidal Transport of Folgte
CONTROL
TRIS
NZ
OUABAIN ION
DNP
IÔ3M
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OT C
FIG . 4 The uptake of 5-MTHF by the isolated choroid plexus was determined under different conditions . DNP, 2,4-dinitrophenol .** P < 0.01 These are shown in Table II . Neither probenecid, which is transported by a mechanism involving organic anions in the choroid plexus, nor dipher~ylhydantoin, which has been reported to affect the intestinal transport of folgte derivatives, was inhibitory . On the other hand, Phenobarbital, at a concentration of 10 -3 M, caused significant inhibition . TABLE II Effect of Various Drugs on the Uptake of 5-Methyltetrahydrofolate in the Isolated Choroid Plexus Addition Control Probenecid Dipher~ylhydantoin Phenobarbital *N .S, 8.
Conc . 10 -3 M 10 -3 M 10 -3 M
Percent of Control 100 93 .02 121 .84 54 .13
(n-4 (n~4 ; n-4 (n=4~
P Value N.S .* N.S . 0 .0025
non-significance
Effect of Folgte Analogues on the Transport of 5-MTHF
The uptake of 5-MTHF by the choroid plexus was inhibited by a number of folgte analogues . This is shown in Table III . Folic acid, folinic acid, methotrexate and pteroic acid, all were potent inhibitors of 5-MTHF transport when tested at 10- 6 M. Para-aminobenzoic acid and L-glutamic acid, which make up portions of the folic acid molecule, were also inhibitory when tested at
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1/S (PM) 1 FIG . 5 A double reciprocal plot of the rate of 5-MTHF uptake . plotted by the method of regression analysis .
The linear graph was
higher concentrations . TABLE III Effect of Folate Analogues on the Uptake of
5-Met~ltetrahydrofolate in the Isolated_ Çhoroid Plexus Uptake in the absence of addition was taken as the control value . Addition None Folic acid Folinic acid Methotrexate Pteroic acid p-Aminobenzoic acid L-Glutamic acid
Conc . 10-6 10 -6 10 -6 10 -6 10 -3 10 -3
M M M M M M
Percent of Control
P Value
100 41 .31 22 .25 49 .89 27 .27 43 .11 69 .18
-0.001 0.001 0.001 0.001 0.05 0.05
DISCUSSION Levitt and co-workers (10) and Chanarin et al . (11) have demonstrated that the transport of 5-MTHF from the blood to the CSFin both the dog and man is probably a carrier-mediated process across the blood-brain barrier . Allen and Klipstein (8) found that the central nervous system has a selective conservation mechanism for maintaining high folate activity in spite of subnormal
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concentrations of this vitamin which may occur in the blood or other tissues . Several workers (2,3,6,9) have also reported that the ratio of folate concentrations between the CSF and serum ranains rather constant in a variety of abnormal conditions . Such observations would suggest a mechanism in the choroid plexus and/or elsewhere in the central nervous system which would operate to regulate the exit of folate from the CSF to the blood and thus maintain the homeostasis in this region . The 5-MTHF in the CSF may be derived from turnover of 5-MTHF in the brain tissue which then passes from the interstitial fluid of the brain into the CSF . The presence of a so-called blood-CSF barrier between the CSF and the blood could then contribute to the observed concentration difference of 5-MTHF between the CSF and the serum . In the past decade, many researchers have stressed the importance of transport mechanisms in the choroid plexus or elsewhere in the CNS in the regulation of the concentration of various substances in the CNS (14,24) . The participation of the choroid plexus in the efflux transport processes in vivo (15,24) and the ability of the choroid plexus incubated in vitro to accunu~e substances against a concentration gradient are generallyaccepted as factors which help to maintain the relatively stable mileu in the central nervous systan (12,14) . The results of the present studies suggest the existence of a carriermediated transport system for 5-MTHF in the choroid plexus . In addition to the saturation kinetics exhibited by the uptake of 5-MTHF (Fig . 5), the choroid plexus also demonstrates countertransport by folic acid (Fig . 1) . Specificity of uptake is also danonstrated by the inhibition produced in the presence of folate analogues . Table III shows that folic acid, folinic acid, methotrexate and pteroic acid all decrease the uptake of 5-MTHF . If the concentration of 5-MTHF in the tissue after 60 minutes uptake is calculated from a number of experiments and compared to the medium concentration, the ratio (T/M) usually exceeded 1 .0 suggesting the existence of same active process to maintain this gradient . The absence, however, of any inhibition by ouabain or 2,4-dinitrophenol, as well as lack of aqy dependence on sodium together with the absence of inhibition under anaerobic conditions all tend to rule out the operation of an active transport process in the uptake of 5-MTHF by the choroid plexus . It Beans possible, therefore, that the concentration gradient is maintained by the binding of 5-MTHF to tissue components . Such binding may take place with several macromolecular species with varying degrees of affinity . Fig . 2 shows that at least 30X of the 5-MTHF taken up after 60 min. i s bound strongly enough to a macromolecule to ranain associated during chromatography on a Bio-Gel column . This does not exclude the possibility that additional 5-MTHF is bound to other species with a lower affinity that would dissociate during the chromatography . The binding of folate derivatives to protein species in various tissues has been daranstrated recently in our own laboratory (25) as well as others (26,27) . This binding has been shown to be non-covalent, probably involving electrostatic interactions between the folate derivative and the binder . This is also consistent with the demonstration that the majority of the transported 5-MTHF in the choroid plexus was freely exchangeable with the medium (Table I) and was unchanged 5-MTHF (Fig . 3) . This, in addition to the reversibility of the direction of flux by the addition to the incubation medium of a substance which competitively inhibits 5-MTHF influx, is indicative of the countertransport phenomenon (28) and implies the operation of a mobile carrier . Moreover, the danonstration of countertransport of 5-MTHF by folic acid indicates that this substance not only competes with 5-MTHF for sites on the carrier molecule but also utilizes this same carrier, at least in part, for its own transport .(29) .
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The uptake of 5-MTHF by the hog choroid plexus is in contrast to studies carried out by Rubin and his collaborators (30) using methotrexate, a folate analogue . These investigators found that T/M ratios of 6 to 7 were attained and transport of methotrexate by rabbit choroid plexus was significantly inhibited by incubation under N2 or by the addition of 2,4-dinitrophenol at 10-4 M concentration. Ouabain (10-5 M) did not inhibit . The uptake of methotrexate by the rabbit choroid plexus was not inhibited by folic acid or folinic acid and, therefore, arty active transport mechanism which may be responsible for methotrexate accumulation is probably distinct from the systen responsible for the transport of 5-MTHF and other natural forms of folate . The intestinal transport of 5-MTHF has been examined in considerable detail by several laboratories (23,31-33) . The consensus of all these studies using a variety of techniques is that passage of 5-MTHF across the rat intes tine is a passive process . There was no evidence for accumulation of 5-MTHF nor was there any indication of competition by analogues or energy requirement . The data presented here tend to indicate that an energy independent carriermediated transport system for 5-MTHF is present in the choroid plexus . Other factors which are involved in the maintenance of high folate concentration in the brain tissue and CSF are the mechanisms of transport from the blood to the brain tissue and from the tissue to the CSF. Since this present work was completed, a report has appeared which describes studies on the transport of folic acid and 5-MTHF by the isolated rabbit choroid plexus (34) . This latter study confirms the presence of a saturable, specific, uptake system for folates in this tissue . In contrast to the data presented here, however, Spector and Lorenzo claim that uptake of folates is an energy dependent process since they obtained high T/M ratios after 15 minutes incubation and showed inhibition of folate uptake by a combination of dinitrophenol and iodoacetate in the absence of glucose . They were unable to show any inhibition under anaerobic conditions in the absence of glucose. Since iodoacetate may inactivate a protein carrier in addition to serving as an inhibitor of glycolysis and the high T/M ratios may be explained by tissue binding of transported folate, the claim of an energy dependent process being involved should be viewed with caution . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 .
J . B. ALPERIN and M. E . HAGGARD, Clin . Res. 18 40 (1970) . M. B. BOWERS, JR . and E. H. REYNOLDS, Lancette 1376 (1972) . M. E . BOYKIN and H. HOOSHMAND, Neurolog~4~3 (1970) . V . HERBERT and R. ZALUSKY, Fed. Proc . 20 453 (1961) . T. MARKKANEN and P. HIMANEN,Înt. _J . Vitam. Nutr . Res . 41 79-85 (1971) . E . H. REYNOLDS, B. B . GALLAGHER, R H. MATTSON, M .BOWERS, and A. L. JOHNSON, Nature 240 155-157 (1972) . D . G. WELLS and H . J . CÂSEY, Brit . Med. J . 3 834-836 (1967) . C. D. ALLEN and F . A . KLIPSTEIN, ~Neuro~l~ 20 403 (1970) . E . H. REYNOLDS, R . H . MATTSON, and B . B . GALLAGHER, Neurology _22 841844 (1972) . M. LEVITT, P . F. NIXON, J . H . PINCUS, and J . R . BERTINO, _J . Clin . Invest . 50 1301-1308 (1971) . Î . CHANARIN, J. PERKY, and E . H. REYNOLDS, Clin . Sci . _Mol . _Med . _46 369373 (1974) . C . P . CHEN, Active transport of myoinositol in the isolated choroid plexus . Ph .D . dissertation, University of Kentucky (1973) . T. Z . CSAKY, Choroid plexus . In : Handbook of Neurochemistry , Vol . 2 . A. Lajtha (ed .), Plenum Press, New or 1 6~. H. F. CSERR, Physiol . Rev . 51 273-311 (1971) .
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