Exp. Brain Res. 30, 65-74 (1977)
Experimental Brain Resea"ch 9
Springer-u
1977
Penetration of 14C-Antipyrine and 14C-Barbital into the Choroid Plexus and Cerebrospinal Fluid of t h e Rat in Vivo C.E. Johanson and D.M. Woodbury Department of Pharmacology,Universityof Utah Collegeof Medicine, Salt Lake City, Utah 84132, USA
Summary. The nature of the transport and distribution of two organic compounds, antipyrine and barbital, in the in vivo choroid plexus was studied in the adult rat. The rate of penetration of *4C-barbital into the lateral ventricular plexus (LVP) was approximately one fourth that of 14C-antipyrine; :a similar quantitative relationship was found with respect to the penetration of these two drugs into the cerebrospinal fluid (CSF). The 14C-antipyrine and 14C-barbital spaces for the choroid plexus (as well as for skeletal~muscle for comparison), together with radioisotope data for the extracellular fluid volume (3H-inulin) and residual erythrocyte volume ( ~ r , e r y t h r o c y t e space~, were used to analyze the partitioning of these drugs between the extracellular and intracellular fluids. The steady-state distribution o f ~4C-antipyrine was characterized by its penetration into virtually the entire water of all the tissue compartments investigated; there was no concentration gradient for radioantipyrine from plasma to choroidal epithelium to CSF. On the other hand, the steady-state concentration of [~4C]barbital in the epithelial cells of the LVP was about 20% less than that in plasma H20, but about 10% greater than that in the CSF. Additional data suggest that the steady-state level of radiobarbital in the epithelium of the LVP is determined primarily by the H § gradient across the cell membrane and perhaps also by sieving of barbital by the CSF secretory process. Although carrier barbital (20 mg/kg) did not significantly change the [~4C]barbital space in LVP, it is not possible to rule out carrier-mediated transport of barbital in this tissue. Key words: Cerebrospinal fluid transport - Rat choroid plexus - Drug distribution- Antipyrine - Barbital
Introduction The penetration of organic compounds into the cerebrospinal fluid (CSF) has been explained on the basis that the barrier between blood and CSF behaves as
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C.E. Johanson and D.M. Woodbury
a l i p o i d l a y e r ( M a y e r et al., 1959; B r o d i e et al., 1960). H o w e v e r , t h e r e has b e e n a p a u c i t y o f in vivo studies d e s c r i b i n g t h e u p t a k e o f o r g a n i c c o m p o u n d s b y t h e c h o r o i d plexus, i.e., t h e m e m b r a n o u s i n t e r f a c e s e p a r a t i n g b l o o d f r o m C S F . A c o n s i d e r a b l e b o d y o f e v i d e n c e i n d i c a t e s t h a t t h e c h o r o i d p l e x u s is an i n t e g r a l p a r t o f t h e b l o o d - C S F b a r r i e r (see r e v i e w b y Cserr, 1971); thus, with r e s p e c t to those organic compounds which are neither transported by a carrier mechanism n o r t r a p p e d i n t r a c e l l u l a r l y , it is c o n c e i v a b l e t h a t t h e r e l a t i v e r a t e o f u p t a k e o f such c o m p o u n d s b y t h e c h o r o i d p l e x u s a r e similar to t h e i r r e l a t i v e r a t e s o f p e n e t r a t i o n into t h e CSF. Thus, o n e a s p e c t o f this i n v e s t i g a t i o n c o n c e r n s a c o m p a r a t i v e analysis o f t h e r a t e s o f u p t a k e o f two r a d i o l a b e l l e d drugs o f d i f f e r e n t lipophilicity, a n t i p y r i n e a n d b a r b i t a l , b y c h o r o i d p l e x u s a n d CSF. It is also o f i n t e r e s t to a n a l y s e t h e e x t e n t o f the s t e a d y - s t a t e u p t a k e o f p h a r m a c o l o g i c a l agents b y t h e c h o r o i d p l e x u s in o r d e r to a s c e r t a i n m e c h a n i s m s o f t r a n s p o r t in this r e g u l a t o r y tissue. Thus, a n o t h e r a s p e c t o f this i n v e s t i g a t i o n has i n v o l v e d t h e c o m p a r t m e n t a l analysis o f t h e i n t r a - t i s s u e d i s t r i b u t i o n o f 14C-antipyrine a n d 14C-barbital b o t h in t h e c h o r o i d p l e x u s ( a n e p i t h e l i a l tissue) and, for c o m p a r i s o n , in s k e l e t a l m u s c l e (a n o n - e p i t h e l i a l tissue). S u c h a n analysis o f t h e p a r t i t i o n i n g o f t h e s e drugs b e t w e e n e x t r a c e l l u l a r a n d i n t r a c e l l u l a r fluids at t h e s t e a d y s t a t e p r o v i d e s d e d u c t i v e i n f o r m a t i o n c o n c e r n i n g m e c h a n i s m s o f t r a n s p o r t in t h e tissues of i n t e r e s t .
Methods Drug Uptake Studies Forty-five male rats (Sprague-Dawley, 250-4 I0 g) were utilized in this aspect of the investigation. Each animal was anaesthetized with ether via cone, bilaterally nephrectomized (to minimize fluctuations in plasma drug concentration), and injected intraperitoneaUy with 10 ~Ci of either [a4C]antipyrine (11 mCi/mM, New England Nuclear) or [14C]barbital (8 mCi/mM, International Chemical & Nuclear). Rats injected with [14C]antipyrine were killed at 3, 6, 9, 12 and 15 rain post injection. Those animals which received radiobarbital were killed at 9, 12, 15 and 120 min after injection; in the study of uptake of 14C-barbital at 120 min, one group of rats received barbital (0.6 mg/kg) in the injection of t4C-barbital (10 ~tCi)while another group of rats received a dose of carrier barbital (20 mg/kg) administered concurrently with the injection of radioisotope (10 ~Ci). To terminate a given experiment, each rat was reanaesthetized with ether; immediately therafter, a 5 ml sample of blood was withdrawn from the abdominal aorta prior to exsanguination via this artery. (Each animal was etherized for a brief period, i.e., for a few minutes, at the start of an experiment when the renal pedicles were ligated and again at the termination of that same experiment when the rat was killed; this procedure was adopted so as not to introduce into the long-term (2 h) experiments complicating variables arising from continuous exposure to ether.) The pH of anaerobically-collectedblood was analysed on an Instrumentation Laboratory blood-gas apparatus (Model 213). CSF and brain were sampled from the cisterna magna and cerebral cortex, respectively. (Fluid withdrawn from the cisterna magna undoubtedly represents subarachnoid as well as ventricular fluid; however, that the concentration of solutes (e.g., urea) in cisternal fluid is similar to that in ventricular fluid is indicated from data by Kleeman et al., 1962 and by Bradbury et al., 1963.) With the aid of a microscope and fine forceps, the entire plexus from each lateral ventricle (LVP) was removed without fragmentation. Finally, a piece of skeletal muscle was excised from the thigh. A thorough description of the sampling techniques has been given previously (Johanson et al., 1974). Plasma samples, as well as those of various tissues, were digested in 1 M piperidine and added to scintillation cocktail (PPO, ethoxyethanol and toluene) for counting of a4C activity in a programmed Isocap 300. The tissue uptake of a given radioisotope was expressed as a space (Table 1) according to the formula:
Penetration of Drugs into In Vivo Choroid Plexus
space =
67
dpm/mg tissue water (or ~tl CSF water) dpm/~l plasma water
Thus, the finding of space equal to unity would be consistent with the interpretation that the radioisotope distributes into the entire water of a tissue.
Table 1. Distribution of 14C-antipyrine and 14C-barbital between plasma and various tissues in the rat a Time (min) after intraperitoneal injection of radioisotope
A. a4C-antipyrine spaces Choroid plexus Cerebral cortex Cerebrospinal fluid Skeletal muscle
B. a4C-barbital spaces Choroid plexus Cerebral cortex Cerebrospinal fluid Skeletal muscle
3 0.82 0.87 0.57 0.25
6 0.95 0.97 0.77 0.58
9 1.02 0.99 0.90 0.81
12 1.00 0.97 0.95 0.93
15 0.99 0.98 0.96 0.95
9 0.44 0.52 0.28 0.68
12 0.67 0.59 0.35 0.70
15 0.65 0.68 0.44 0.69
120 0.80 0.82 0.65 0.84
120 b 0.82 0.78 0.68 0.87
a Each entry in the table is a mean value for 3-5 animals. Spaces for both radioisotopes were determined by the formula given in Methods. Standard errors are generally 2-8 % of the respective means. For each tissue (and CSF) listed in part A there are no statistically significant differences between a4C-antipyrine spaces at 12 rain and those at 15 rain (P > 0.05, multiple range test); with respect to part B, the 14C-barbital spaces at 120 rain (without carrier barbital) are not significantly different from those corresponding spaces at 120 rain (with carrier barbital) b Results for rats receiving carrier barbital (20 mg/kg) in addition to radiobarbital
Compartmentation Studies Eighteen rats were used in the compartmentation studies, the purpose of which was to quantify the volume of the various water compartments in the tissues of interest (Table 2) for use in the calculation of intracellular and extracellular concentrations of [14C]antipyrine and [14C]barbital. For both LVP and muscle, water content was determined by the difference between wet and dry tissue weight. Because of the problem of evaporation associated with the small size of the LVP (i.e., < 1 rag), it was necessary to determine the true wet weight indirectly by a technique involving electrobalance weighing and graphical analysis (Johanson et al., 1976). With technique previously described (Johanson et al., 1974), measurements of extracellular fluid volume (by the steady-state distribution of [all]inulin, i.e., 60 rain uptake) and residual erythrocyte volume (with 5~Cr-tagged erythrocytes) were carried out in separate animals because of the limited amount of plexus tissue for analysis. In general, the materials and procedures (i.e. tissue sampling, analytical techniques and calculations) utilized in the compartmentation studies were similar to those used in the drug uptake studies. To calculate the concentration of a drug in a given tissue compartment, we applied the general equation: concentration = amount/volume (1). For example, the concentration of radiolabeled drug in parenchymal cell water is calculated by the formula: dpm(wt) - dpm(~0 - dpm(r~) V(wt) - V(e 0 - V(re)
(2)
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C.E. Johanson and D.M. Woodbury
where dpm = disintegrations/min of radioactive drug, wt = wet tissue, ef = extracellular fluid, re = residual erythrocytes in sampled tissue, and V = fractional volume of water in a given compartment. The experimental values substituted into the terms of formula 2 were pooled means of steady-state values. Values of V for the various compartments are listed in Table 2; the dpm data for the respective compartments, which are expressed on a unit weight (of tissue) basis, were derived from the steady-state spaces listed in Table 1 and from untabulated data for erythrocytes. The calculation of drug concentration in the parenchymal cell compartment is based on observations that the volume comprised by endothelial ceils and connective tissue is negligible compared with parenchymal cell volume; see diagram drawn to scale of a choroidal villus by Dohrmann (1970). The pH gradient across the cell membrane may be the main factor affecting the distribution of a weak organic acid (such as barbital) between the intracellular fluid (icf) and extracellular fluid (ecf) of a given tissue. To test for this possibility, a modified form of the Henderson-Hasselbalch equation [base]/[acid] = antilog (pH-pKa) (3) was used to calculate the relative concentrations of ionized and unionized forms of the drug both in the icf and ecf; see footnote a in Table 3.
Table 2. Compartmentalization data for choroid plexus and skeletal musclea. Each entry represents a mean for 6 rats. Limits are standard errors Fractional volume (or "space") of water (mg/mg wet wt. of tissue) Choroid plexus Skeletal muscle
Compartment Total tissue ExtraceUular fluid Erythrocytesb (residual) Parenchymal cells
0.793-+0.010 a 0.202 + 0.007 0.045+-0.006 r 0.546 a
0.760+-_0.003 ~ 0.111 + 0.005 0.004-+0.001 r 0.645 a
" Determined by difference between wet and dry weights b Residual erythrocytes in sampled tissue constitute 6.6 and 0.6% of the wet weight of choroid plexus and skeletal muscle, respectively c Calculated from data for tissue content of erythrocytes (see footnote b) and an erythrocyte water content of 67.4 % d Calculated by difference (using pooled mean results for other compartments)
Table 3. Steady-state partitioning of tracers between intracellular and extracellular fluidsa. Each entry represents a mean concentration of radioisotope which has been calculated from average steady-state values in Tables 1 and 2; because of data pooling for the calculations, statistical parameters are not available Relative concentration (as % of plasma) of tracers in Cerebrospinal Choroid cell Skeletal muscle fluid H20 HzO cell H20 ~4C-antipyrine 14C-barbital
96 67 (90)"
99 78 (77) ~
94 81 (78) a
a The numbers in parentheses represent those concentrations expected if the unionized (but not the ionized) form of barbital penetrates the cell membrane to distribute between intracellular and extracellular fluids according to the pH gradient. A value of 7.8 was used for the pKa of barbital (Hogben et al., 1959); if a pK~ of 7.5 (Brodie et al., 1960) is used, the expected concentrations of barbital are approximately 10 % less than those values in parentheses
Penetration of Drugs into In Vivo Choroid Plexus
69
Results
Uptake of [14C]Antipyrine and [a4C]Barbital by Choroid Plexus and Other Tissues The uptake of [14C]antipyrine by both choroid plexus and cerebral cortex was extremely rapid; at 6 min post injection, this tracer had distributed into virtually all of the water of these richly-perfused tissues (Table 1, part A). The CSF, a large-cavity fluid, required several minutes longer to reach a steady state; thus, at 12-15 rain radioantipyrine had penetrated into virtually all of the water of the sampled CSF. The uptake of [14C]antipyrine by skeletal muscle trailed that of the various compartments in the central nervous system (CNS). The rate of uptake of [a4C]barbital by tissues within the CNS was markedly slower than the rate of uptake of [a4C]antipyrine (Table 1, part B). At 15 min post injection, a time at which [*4C]antipyrine had nearly achieved an equilibrium distribution in all tissues investigated, [~4C]barbital had distributed into only 0.65 of the water in choroid plexus and into 0.68 of that in cerebral cortex; with respect to the build-up in concentration of radiobarbital, the sampled CSF lagged considerably behind that of the plexus and cortex. The steady-state distribution of radiobarbital in all tissues (and CSF) was not significantly affected by carrier barbital (20 mg/kg IP); see 2 h data. In contrast to the findings for [~4C]antipyrine, the uptake of [~4C]barbital by the LVP was slower than that of muscle. That the influx of radiolabeled antipyrine and barbital into LVP is mainly from the blood, rather than from the CSF, is suggested by the finding that the CSF concentration of both drugs is always lower than that in the LVP (Table 1). Moreover, the movement of solute by diffusion from CSF to blood via the choroid plexus would be thwarted by an opposing flow of newly-formed CSF, i.e., a negative solvent drag effect. However, the data do not allow us to exclude a component of transport (e.g., via carrier) from CSF to LVP (see Discussion).
Compartmentation Studies In order to characterize the intra-tissue distribution of the drugs studied, it was necessary to quantitate the volume of the various water compartments in the tissues of interest: choroid plexus and skeletal muscle. The water in the LVP, as determined by a technique involving electrobalance weighing and graphical analysis, comprises 0.79-0.80 the weight of wet tissue (Table 2). The amount of extracellular fluid in the plexus, as estimated by the steady-state distribution of [3H]inulin, is slightly greater than 0.20 of the wet weight of the tissue (Table 2). The relatively large amount of residual erythrocytes in the samples of LVP (i.e., an average of 0.066 mg erythrocytes/mg wet tissue) is not unexpected due to the vascularity of this secretory tissue. Data obtained for analogous compartments in skeletal muscle are also summarized in Table 2. The arterial pH of the etherized rats was generally in the range of 7.49-7.54. The effect of ether on respiration, together with the altitude of ca. 1350 m to
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C.E. Johanson and D.M. Woodbury
which the animals were acclimatized, are factors responsible for the relatively high pH.
Distribution of Tracers Between Intracellular and Extracellular Compartments The space data for [14C]antipyrine and [14C]barbital (Table 1), together with the steady-state spaces for the tissue water compartments (Table 2), have been used to analyse the partitioning of these radiolabelled drugs between intracellular and extracellular fluids, both in choroid plexus and skeletal muscle (Table 3). The data in Table 3 are relative concentrations of the radiolabelled drugs in various compartments based upon a plasma concentration of 100. At the steady state, [a4C]antipyrine distributed more or less evenly throughout the various water compartments in the tissues studied (Table 3). For example, the concentrations of radioantipyrine in the epithelial cell water of choroid plexus is approximately equal to that both in the plasma water and in the CSF water; moreover, within the limits of experimental error, the concentration of [14C] antipyrine in cellular water of skeletal muscle is the same as that in plasma water. However, [14C]barbital does not accumulate in the cellular compartment as extensively as [14C]antipyrine, even if 2 h are allowed for this compound to distribute. Thus, the concentration of [14C]barbital in the epithelial cells of choroid plexus as well as the CSF is considerably less (i.e., 20-30%) than that of [14C]antipyrine. Similarly, in skeletal muscle the concentration of [14C]barbital in the cellular compartment is less than that of [14C]antipyrine. The predicted values for the concentrations of radiobarbital in parenchymal cells (see figures in parentheses in Table 3) are in reasonably close agreement to those values determined experimentally; to obtain the theoretically-expected concentrations, values of 6.99 and 7.03 were utilized for the cell pH of LVP and muscle, respectively (Johanson, in preparation). Discussion
Rate of Uptake of [14C]Antipyrine and [a4C]Barbital by the Choroid Plexus, Cerebrospinal Fluid and Other Tissues The rate of penetration of [14C]barbital into the choroid plexus was considerably slower than that of [14C]antipyrine. At 9 min post injection, a time at which radioantipyrine had penetrated into more or less 100% of water in the LVP, [14C]barbital had distributed into only 0.44 of the total water in this tissue. The lipid solubility of these drugs, together with the extent of their ionization, are important factors to consider in the explanation of differences in permeation rates. Partition coefficients (n-heptane/water of less than 0.002 and 0.005, respectively, have been reported for the non-ionized forms of barbital and antipyrine (Hogben et al., 1959). On the basis of these oil/water coefficients, one could predict the rate of penetration of barbital into choroid plexus to be less than half that of antipyrine. At a pH of 7.4, barbital with a pKa of 7.8 and antipyrine with a pK~ of 1.4 (Shore et al., 1957) are 72% and > 99.9%
Penetration of Drugs into In Vivo Choroid Plexus
71
non-ionized, respectively; since the degree of ionization of a drug also influences its rate of movement into cells, it is useful in the analysis of penetration to consider the "effective partition coefficient" (i.e. the partition coefficient multiplied by the fraction of the drug which is non-ionized at the pH of interest.) The ratio of "effective partition coefficients" for barbital/antipyrine is 1.1/5 (Goldstein et al., 1969). On the basis of these "effective" coefficients it might be expected that barbital, if it were to distribute passively via uncomplicated diffusion, would take about four times longer than antipyrine to attain a steady-state distribution in the choroid plexus. The uptake data do not indicate the exact time at which radiobarbital reaches a steady-state distribution in the LVP; however, it is interesting to note that whereas 3 rain are required for [14C]antipyrine to achieve a space which is 80% that of the steady-state space, approximately 12 min are necessary for [14C]barbital to penetrate the LVP to the same extent. The respective time courses of uptake of radioisotopic antipyrine and barbital by CSF (and brain) in this study are similar to comparable determinations in other mammalian species. It has previously been established that 2 h of distribution time is sufficient for radiobarbital to achieve a steadystate distribution in various compartments of the central nervous system (Mayer et al., 1959). Mayer et al. (1959) and Brodie et al. (1960) both reported that antipyrine penetrates into the mammalian CSF system at a rate approximately four times faster than that of barbital. Such a ratio for the rates of penetration of antipyrine and barbital into the CSF system is similar to the ratio of rates at which these two compounds penetrate the choroid plexus (present study) during the attainment of steady-state distribution. Thus, there may be a fixed relationship between the respective rates of uptake of a given organic compound by choroid plexus and CSF. The uptake of [14C]barbital by the LVP lags behind that of skeletal muscle, despite the fact that blood flow to the choroid plexus is two orders of magnitude greater than that to skeletal muscle (Welch, 1963; Goldstein et al., 1969). Apparently, the rate-limiting step for the penetration of barbital into the LVP is a factor other than blood flow (e.g., parenchymal cell permeability; see discussion below). On the other hand, the uptake of [~4C]antipyrine by LVP leads that by muscle; thus, the penetration of antipyrine (in contrast to that of barbital) into choroid plexus may be limited primarily by blood flow.
CompartmentalAnalys& An accurate assessment of the partitioning of antipyrine and barbital among the various compartments in choroid plexus and skeletal muscle is dependent upon values obtained for the magnitude of the various water compartments (or spaces) in these tissues. The volume of extracellular fluid in LVP, as estimated from the distribution of [3H]inulin, was 0.202 mg (or ~tl) water/mg wet tissue; this finding agrees well with the value of 0.206 previously reported for the same parameter determined with [14C]inulin (Johanson et al., 1974). Because of the relatively large volume of residual erythrocytes in the plexus, it is important to
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correct for this cellular compartment in any transport or distribution study which is focused on the epithelial cells of this tissue. Compartmentation data obtained for rat muscle (Table 2) are similar to those previously reported. Metabolism and binding are important factors to consider in the calculation of cellular concentrations of tracers. Previous investigators have established that barbital is not chemically modified by CSF, brain or plasma (Lal et al., 1964); moreover, barbital is not metabolized by the liver (Rubin et al., 1964). There is only a negligible degree of binding of barbital to the proteins of plasma and tissue (Goldbaum and Smith, 1954; Brodie et al., 1960). [a4C]antipyrine has a metabolic half life of 90 rain in the rat (Bakke et al., 1974); thus, in the short-term experiments (i.e., 3-15 rain) of the present study, the level of metabolites of antipyrine in tissue should not be appreciable. Since radioantipyrine is an indicator which accurately measures total water content (Soberman et al., 1949) of most (if not all) tissues, it is not surprising that the binding of this tracer to macromolecules is negligible (Shore et al., 1957). Therefore, since both barbital and antipyrine are neither bound nor metabolized appreciably (under the conditions of the experiment), it is likely that the data in Table 3 are reliable estimates of the concentrations of unbound, unmetabolized tracers.
Analysis of the Intra-Tissue Distribution of [14C]Antipyrine and [14C]Barbital Antipyrine is almost completely unionized at a pH of 7.4; thus, the predominance, as well as the extreme lipid solubility, of the non-ionized form of this weak base is the main reason for its rapid penetration even into the CNS. The steady-state distribution of [14C] antipyrine is characterized by its penetration into virtually the entire water of all tissue compartments investigated (Table 3). Because of the rapid attainment of equilibrium distribution of radioantipyrine between plasma water and CSF water, there is no sin~:~effect,exerted by the CSF to this compound. Barbital, because it is less lipid soluble than antipyrine (and more ionized at the existing pH) penetrates tissues both within and outside the CNS less rapidly and extensively than antipyrine. The steady-state concentration of barbital in the parenchymal cells of both choroid plexus and skeletal muscle is 20-25 % less than that in plasma water. The hydrogen ion gradient between extracellular and intracellular fluids, which is equivalent to approximately 0.5 pH unit, is probably the best explanation for the observed asymmetrical distribution of [14C]barbital between cell water and plasma water. Moreover, the concentration of barbital in cell water tends to be lower in the LVP than in muscle; if, during the CSF secretory process, barbital is less able than antipyrine to keep up with the volume flow of water across the choroid plexus (Welch et al., 1966), the resultant sieving of barbital would tend to lower its concentration in the choroid cell. The CSF data support this postulated sieving of barbital; at the steady state, 14C-barbital concentration in the CSF water is only 67 % that in plasma water. Moreover, we have recently obtained evidence that 14C-urea is substantially sieved by the choroidal membrane (unpublished data).
Penetration of Drugs into In Vivo Choroid Plexus
73
Is active transport also a mechanism for keeping barbital in the CSF at a lower concentration than that in plasma? The finding that 14C-barbital space in the choroid plexus was not affected significantly by increasing the dose of non-radioactive barbital by 33 fold (i.e., from 0.6-20 mg/kg) might be explained by one of the following ways: 1) barbital is not actively transported by the LVP, 2) barbital is actively transported by a carrier mechanism of extremely low capacity which is saturated by relatively low IP doses of barbital, i.e., < 1 mg/kg or 3) barbital is actively transported by the LVP via a carrier system of relatively high capacity which is not saturated even at an IP dose of barbital of 20 mg/kg. We favour explanation 2 for several reasons: carrier transport systems which translocate several different organic anions have been demonstrated in in vitro preparations of choroid plexus (Forn, 1972; Spector and Lorenzo, 1974; Sampath and Neff, 1976); using an in vivo approach, Rollins and Reed (1970) showed that the weak organic acid, DMO, ([14C]5,5-dimethyloxazolidine-2,4-dione) is actively removed from CSF by a carrier system which is saturated at relatively low IP doses of carrier, i.e., from 1-5 mg/kg (a comparison of D M O with barbital in this context would not be valid if these two anions have substantially different affinities for an anion carrier); in the present study, the calculated concentration of radiobarbital in the choroid cell is approximately that expected if barbital were to distribute passively across the choroid cell membrane according to the p H gradient (if a high-capacity anion transport system were present in the LVP, one might expect the steady-state concentration of radiobarbital to be considerably greater than that observed). Since a substantial part of the overall exchange of materials between blood and ventricular CSF occurs across the epithelial membrane of the choroid plexus, it seems important to gain more insight on the nature of transport at this important interface. The compartmentation aspect of the present study represents a new approach to the problem of delineating transport and permeability phenomena associated with the choroid plexus. Additional studies inthis area are indicated since a better understanding of the basic physiology' of transport at this interface should contribute to the fulfilment of the therapeutic goal of being able to manipulate the transport of materials across the blood-CSF barrier.
References Bakke, O.M., Bending, M., Aarbakke, J., Davies, D.S.: 14C-antipyrineas a model compound in the study of drug oxidation and enzyme induction in individual surviving rats. Acta pharmaeol. (Kbh.) 35, 91-97 (1974) Bradbury, M.W.B., Stubbs, J., Hughes, I.E., Parker, P.: The distribution of potassium, sodium, chloride and urea between lumbar cerebrospinal fluid and blood serum in human subjects. Clin. Sci. 25, 97-105 (1963) Brodie, B. B., Kurz, H., Schanker, L.S.: The importance of dissociationconstant and lipid-solubility in influencing the passage of drugs into the cerebrospinal fluid. J. Pharmacol. exp. Ther. 130, 20-25 (1960) Cserr, H.: Physiologyof the choroid plexus. Physiol. Rev. 51, 273-311 (1971) Dohrmann, G.J.: The choroid plexus: A historical review. Brain Res. 18, 197-218 (1970)
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Forn, J.: Active transport of 5-hydroxyindoleacetic acid by the rabbit choroid plexus in vitro. Biochem. Pharmacol. 21, 619--624 (1972) Goldbaum, L.R., Smith, P.K.: The interaction of barbiturates with serum albumin and its possible relation to their disposition and pharmacological actions. J. Pharmacol. exp. Ther. 111, 197-209 (1954) Goldstein, A., Aronow, L., Kalman, S.M.: Principles of drug action: The basis of pharmacology, pp. 884. New York: Harper and Row 1969 Hogben, C.A.M., Tocco, D.J., Brodie, B.B., Schanker, LS.: On the mechanism of intestinal absorption of drugs. J. Pharmacol. exp. Ther. 125, 275-282 (1959) Johanson, C.E., Reed, D.J., Woodbury, D.M.: Active transport of sodium and potassium by the choroid plexus of the rat. J. Physiol. (Lond.) 241, 359-372 (1974) Johanson, C.E., Reed, D. J., Woodbury, D.M.: Developmental studies of the compartmentalization of water and electrolytes in the choroid plexus of the neonatal rat brain. Brain Res. 116, 35-48 (1976) Kleeman, C.R., Davson, H., Levin, E.: Urea transport in the central nervous system. Amer. J. Physiol. 203,739-747 (1962) Lal, H., Barlow, C.F., Roth. L.J.: Barbital-C 14 in cat and mouse. Arch. int. Pharmacodyn. 149, 25-36 (1964) Mayer, S., Maickel, R.P., Brodie, B.B.: Kinetics of penetration of drugs and other foreign compounds into cerebrospinal fluid and brain. J. Pharmacol. exp. Ther. 127, 205-211 (1959) Rollins, D.E., Reed, D.J.: Transport of DMO out of cerebrospinal fluid of rats. Amer. J. Physiol. 219, 1200--1204 (1970) Rubin, A., Tephly, T.R., Mannering, G.J.: Kinetics of drug metabolism by hepatic microsomes. Biochem. Pharmacol. 13, 1007-1016 (1964) Sampath, S. S., Neff, N.H.: Transport of 3-methoxy-4-hydroxymandelic acid by isolated rat choroid plexus. Neurochem. Res. 1, 47-52 (1976) Shore, P.A., Brodie, B.B., Hogben, C.A.M.: The gastric secretion of drugs: a pH partition hypothesis. J. Pharmacol. exp. Ther. 119, 361-369 (1957) Soberman, R., Brodie, B.B., Levy, B.B., Axelrod, J., Hollander, V., Steele, J.M.: The use of antipyrine in the measurement of total body water in man. J. biol. Chem. 179, 31-42 (1949) Spector, R., Lorenzo, A.V.: Specificity of ascorbic acid transport system of the central nervous system. Amer. J. Physiol. 226, 1468-1473 (1974) Welch, K.: Secretion of cerebrospinal fluid by choroid plexus of the rabbit. Amer. J. Physiol. 205, 617-624 (1963) Welch, K., Sadler, K., Gold, G.: Volume flow across choroidal ependyma of the rabbit. Amer. J. Physiol. 210, 232-236 (1966) Received January 17, 1977
Experimental Brain Resea"ch 9
Springer-u
1977
Penetration of 14C-Antipyrine and 14C-Barbital into the Choroid Plexus and Cerebrospinal Fluid of t h e Rat in Vivo C.E. Johanson and D.M. Woodbury Department of Pharmacology,Universityof Utah Collegeof Medicine, Salt Lake City, Utah 84132, USA
Summary. The nature of the transport and distribution of two organic compounds, antipyrine and barbital, in the in vivo choroid plexus was studied in the adult rat. The rate of penetration of *4C-barbital into the lateral ventricular plexus (LVP) was approximately one fourth that of 14C-antipyrine; :a similar quantitative relationship was found with respect to the penetration of these two drugs into the cerebrospinal fluid (CSF). The 14C-antipyrine and 14C-barbital spaces for the choroid plexus (as well as for skeletal~muscle for comparison), together with radioisotope data for the extracellular fluid volume (3H-inulin) and residual erythrocyte volume ( ~ r , e r y t h r o c y t e space~, were used to analyze the partitioning of these drugs between the extracellular and intracellular fluids. The steady-state distribution o f ~4C-antipyrine was characterized by its penetration into virtually the entire water of all the tissue compartments investigated; there was no concentration gradient for radioantipyrine from plasma to choroidal epithelium to CSF. On the other hand, the steady-state concentration of [~4C]barbital in the epithelial cells of the LVP was about 20% less than that in plasma H20, but about 10% greater than that in the CSF. Additional data suggest that the steady-state level of radiobarbital in the epithelium of the LVP is determined primarily by the H § gradient across the cell membrane and perhaps also by sieving of barbital by the CSF secretory process. Although carrier barbital (20 mg/kg) did not significantly change the [~4C]barbital space in LVP, it is not possible to rule out carrier-mediated transport of barbital in this tissue. Key words: Cerebrospinal fluid transport - Rat choroid plexus - Drug distribution- Antipyrine - Barbital
Introduction The penetration of organic compounds into the cerebrospinal fluid (CSF) has been explained on the basis that the barrier between blood and CSF behaves as
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C.E. Johanson and D.M. Woodbury
a l i p o i d l a y e r ( M a y e r et al., 1959; B r o d i e et al., 1960). H o w e v e r , t h e r e has b e e n a p a u c i t y o f in vivo studies d e s c r i b i n g t h e u p t a k e o f o r g a n i c c o m p o u n d s b y t h e c h o r o i d plexus, i.e., t h e m e m b r a n o u s i n t e r f a c e s e p a r a t i n g b l o o d f r o m C S F . A c o n s i d e r a b l e b o d y o f e v i d e n c e i n d i c a t e s t h a t t h e c h o r o i d p l e x u s is an i n t e g r a l p a r t o f t h e b l o o d - C S F b a r r i e r (see r e v i e w b y Cserr, 1971); thus, with r e s p e c t to those organic compounds which are neither transported by a carrier mechanism n o r t r a p p e d i n t r a c e l l u l a r l y , it is c o n c e i v a b l e t h a t t h e r e l a t i v e r a t e o f u p t a k e o f such c o m p o u n d s b y t h e c h o r o i d p l e x u s a r e similar to t h e i r r e l a t i v e r a t e s o f p e n e t r a t i o n into t h e CSF. Thus, o n e a s p e c t o f this i n v e s t i g a t i o n c o n c e r n s a c o m p a r a t i v e analysis o f t h e r a t e s o f u p t a k e o f two r a d i o l a b e l l e d drugs o f d i f f e r e n t lipophilicity, a n t i p y r i n e a n d b a r b i t a l , b y c h o r o i d p l e x u s a n d CSF. It is also o f i n t e r e s t to a n a l y s e t h e e x t e n t o f the s t e a d y - s t a t e u p t a k e o f p h a r m a c o l o g i c a l agents b y t h e c h o r o i d p l e x u s in o r d e r to a s c e r t a i n m e c h a n i s m s o f t r a n s p o r t in this r e g u l a t o r y tissue. Thus, a n o t h e r a s p e c t o f this i n v e s t i g a t i o n has i n v o l v e d t h e c o m p a r t m e n t a l analysis o f t h e i n t r a - t i s s u e d i s t r i b u t i o n o f 14C-antipyrine a n d 14C-barbital b o t h in t h e c h o r o i d p l e x u s ( a n e p i t h e l i a l tissue) and, for c o m p a r i s o n , in s k e l e t a l m u s c l e (a n o n - e p i t h e l i a l tissue). S u c h a n analysis o f t h e p a r t i t i o n i n g o f t h e s e drugs b e t w e e n e x t r a c e l l u l a r a n d i n t r a c e l l u l a r fluids at t h e s t e a d y s t a t e p r o v i d e s d e d u c t i v e i n f o r m a t i o n c o n c e r n i n g m e c h a n i s m s o f t r a n s p o r t in t h e tissues of i n t e r e s t .
Methods Drug Uptake Studies Forty-five male rats (Sprague-Dawley, 250-4 I0 g) were utilized in this aspect of the investigation. Each animal was anaesthetized with ether via cone, bilaterally nephrectomized (to minimize fluctuations in plasma drug concentration), and injected intraperitoneaUy with 10 ~Ci of either [a4C]antipyrine (11 mCi/mM, New England Nuclear) or [14C]barbital (8 mCi/mM, International Chemical & Nuclear). Rats injected with [14C]antipyrine were killed at 3, 6, 9, 12 and 15 rain post injection. Those animals which received radiobarbital were killed at 9, 12, 15 and 120 min after injection; in the study of uptake of 14C-barbital at 120 min, one group of rats received barbital (0.6 mg/kg) in the injection of t4C-barbital (10 ~tCi)while another group of rats received a dose of carrier barbital (20 mg/kg) administered concurrently with the injection of radioisotope (10 ~Ci). To terminate a given experiment, each rat was reanaesthetized with ether; immediately therafter, a 5 ml sample of blood was withdrawn from the abdominal aorta prior to exsanguination via this artery. (Each animal was etherized for a brief period, i.e., for a few minutes, at the start of an experiment when the renal pedicles were ligated and again at the termination of that same experiment when the rat was killed; this procedure was adopted so as not to introduce into the long-term (2 h) experiments complicating variables arising from continuous exposure to ether.) The pH of anaerobically-collectedblood was analysed on an Instrumentation Laboratory blood-gas apparatus (Model 213). CSF and brain were sampled from the cisterna magna and cerebral cortex, respectively. (Fluid withdrawn from the cisterna magna undoubtedly represents subarachnoid as well as ventricular fluid; however, that the concentration of solutes (e.g., urea) in cisternal fluid is similar to that in ventricular fluid is indicated from data by Kleeman et al., 1962 and by Bradbury et al., 1963.) With the aid of a microscope and fine forceps, the entire plexus from each lateral ventricle (LVP) was removed without fragmentation. Finally, a piece of skeletal muscle was excised from the thigh. A thorough description of the sampling techniques has been given previously (Johanson et al., 1974). Plasma samples, as well as those of various tissues, were digested in 1 M piperidine and added to scintillation cocktail (PPO, ethoxyethanol and toluene) for counting of a4C activity in a programmed Isocap 300. The tissue uptake of a given radioisotope was expressed as a space (Table 1) according to the formula:
Penetration of Drugs into In Vivo Choroid Plexus
space =
67
dpm/mg tissue water (or ~tl CSF water) dpm/~l plasma water
Thus, the finding of space equal to unity would be consistent with the interpretation that the radioisotope distributes into the entire water of a tissue.
Table 1. Distribution of 14C-antipyrine and 14C-barbital between plasma and various tissues in the rat a Time (min) after intraperitoneal injection of radioisotope
A. a4C-antipyrine spaces Choroid plexus Cerebral cortex Cerebrospinal fluid Skeletal muscle
B. a4C-barbital spaces Choroid plexus Cerebral cortex Cerebrospinal fluid Skeletal muscle
3 0.82 0.87 0.57 0.25
6 0.95 0.97 0.77 0.58
9 1.02 0.99 0.90 0.81
12 1.00 0.97 0.95 0.93
15 0.99 0.98 0.96 0.95
9 0.44 0.52 0.28 0.68
12 0.67 0.59 0.35 0.70
15 0.65 0.68 0.44 0.69
120 0.80 0.82 0.65 0.84
120 b 0.82 0.78 0.68 0.87
a Each entry in the table is a mean value for 3-5 animals. Spaces for both radioisotopes were determined by the formula given in Methods. Standard errors are generally 2-8 % of the respective means. For each tissue (and CSF) listed in part A there are no statistically significant differences between a4C-antipyrine spaces at 12 rain and those at 15 rain (P > 0.05, multiple range test); with respect to part B, the 14C-barbital spaces at 120 rain (without carrier barbital) are not significantly different from those corresponding spaces at 120 rain (with carrier barbital) b Results for rats receiving carrier barbital (20 mg/kg) in addition to radiobarbital
Compartmentation Studies Eighteen rats were used in the compartmentation studies, the purpose of which was to quantify the volume of the various water compartments in the tissues of interest (Table 2) for use in the calculation of intracellular and extracellular concentrations of [14C]antipyrine and [14C]barbital. For both LVP and muscle, water content was determined by the difference between wet and dry tissue weight. Because of the problem of evaporation associated with the small size of the LVP (i.e., < 1 rag), it was necessary to determine the true wet weight indirectly by a technique involving electrobalance weighing and graphical analysis (Johanson et al., 1976). With technique previously described (Johanson et al., 1974), measurements of extracellular fluid volume (by the steady-state distribution of [all]inulin, i.e., 60 rain uptake) and residual erythrocyte volume (with 5~Cr-tagged erythrocytes) were carried out in separate animals because of the limited amount of plexus tissue for analysis. In general, the materials and procedures (i.e. tissue sampling, analytical techniques and calculations) utilized in the compartmentation studies were similar to those used in the drug uptake studies. To calculate the concentration of a drug in a given tissue compartment, we applied the general equation: concentration = amount/volume (1). For example, the concentration of radiolabeled drug in parenchymal cell water is calculated by the formula: dpm(wt) - dpm(~0 - dpm(r~) V(wt) - V(e 0 - V(re)
(2)
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C.E. Johanson and D.M. Woodbury
where dpm = disintegrations/min of radioactive drug, wt = wet tissue, ef = extracellular fluid, re = residual erythrocytes in sampled tissue, and V = fractional volume of water in a given compartment. The experimental values substituted into the terms of formula 2 were pooled means of steady-state values. Values of V for the various compartments are listed in Table 2; the dpm data for the respective compartments, which are expressed on a unit weight (of tissue) basis, were derived from the steady-state spaces listed in Table 1 and from untabulated data for erythrocytes. The calculation of drug concentration in the parenchymal cell compartment is based on observations that the volume comprised by endothelial ceils and connective tissue is negligible compared with parenchymal cell volume; see diagram drawn to scale of a choroidal villus by Dohrmann (1970). The pH gradient across the cell membrane may be the main factor affecting the distribution of a weak organic acid (such as barbital) between the intracellular fluid (icf) and extracellular fluid (ecf) of a given tissue. To test for this possibility, a modified form of the Henderson-Hasselbalch equation [base]/[acid] = antilog (pH-pKa) (3) was used to calculate the relative concentrations of ionized and unionized forms of the drug both in the icf and ecf; see footnote a in Table 3.
Table 2. Compartmentalization data for choroid plexus and skeletal musclea. Each entry represents a mean for 6 rats. Limits are standard errors Fractional volume (or "space") of water (mg/mg wet wt. of tissue) Choroid plexus Skeletal muscle
Compartment Total tissue ExtraceUular fluid Erythrocytesb (residual) Parenchymal cells
0.793-+0.010 a 0.202 + 0.007 0.045+-0.006 r 0.546 a
0.760+-_0.003 ~ 0.111 + 0.005 0.004-+0.001 r 0.645 a
" Determined by difference between wet and dry weights b Residual erythrocytes in sampled tissue constitute 6.6 and 0.6% of the wet weight of choroid plexus and skeletal muscle, respectively c Calculated from data for tissue content of erythrocytes (see footnote b) and an erythrocyte water content of 67.4 % d Calculated by difference (using pooled mean results for other compartments)
Table 3. Steady-state partitioning of tracers between intracellular and extracellular fluidsa. Each entry represents a mean concentration of radioisotope which has been calculated from average steady-state values in Tables 1 and 2; because of data pooling for the calculations, statistical parameters are not available Relative concentration (as % of plasma) of tracers in Cerebrospinal Choroid cell Skeletal muscle fluid H20 HzO cell H20 ~4C-antipyrine 14C-barbital
96 67 (90)"
99 78 (77) ~
94 81 (78) a
a The numbers in parentheses represent those concentrations expected if the unionized (but not the ionized) form of barbital penetrates the cell membrane to distribute between intracellular and extracellular fluids according to the pH gradient. A value of 7.8 was used for the pKa of barbital (Hogben et al., 1959); if a pK~ of 7.5 (Brodie et al., 1960) is used, the expected concentrations of barbital are approximately 10 % less than those values in parentheses
Penetration of Drugs into In Vivo Choroid Plexus
69
Results
Uptake of [14C]Antipyrine and [a4C]Barbital by Choroid Plexus and Other Tissues The uptake of [14C]antipyrine by both choroid plexus and cerebral cortex was extremely rapid; at 6 min post injection, this tracer had distributed into virtually all of the water of these richly-perfused tissues (Table 1, part A). The CSF, a large-cavity fluid, required several minutes longer to reach a steady state; thus, at 12-15 rain radioantipyrine had penetrated into virtually all of the water of the sampled CSF. The uptake of [14C]antipyrine by skeletal muscle trailed that of the various compartments in the central nervous system (CNS). The rate of uptake of [a4C]barbital by tissues within the CNS was markedly slower than the rate of uptake of [a4C]antipyrine (Table 1, part B). At 15 min post injection, a time at which [*4C]antipyrine had nearly achieved an equilibrium distribution in all tissues investigated, [~4C]barbital had distributed into only 0.65 of the water in choroid plexus and into 0.68 of that in cerebral cortex; with respect to the build-up in concentration of radiobarbital, the sampled CSF lagged considerably behind that of the plexus and cortex. The steady-state distribution of radiobarbital in all tissues (and CSF) was not significantly affected by carrier barbital (20 mg/kg IP); see 2 h data. In contrast to the findings for [~4C]antipyrine, the uptake of [~4C]barbital by the LVP was slower than that of muscle. That the influx of radiolabeled antipyrine and barbital into LVP is mainly from the blood, rather than from the CSF, is suggested by the finding that the CSF concentration of both drugs is always lower than that in the LVP (Table 1). Moreover, the movement of solute by diffusion from CSF to blood via the choroid plexus would be thwarted by an opposing flow of newly-formed CSF, i.e., a negative solvent drag effect. However, the data do not allow us to exclude a component of transport (e.g., via carrier) from CSF to LVP (see Discussion).
Compartmentation Studies In order to characterize the intra-tissue distribution of the drugs studied, it was necessary to quantitate the volume of the various water compartments in the tissues of interest: choroid plexus and skeletal muscle. The water in the LVP, as determined by a technique involving electrobalance weighing and graphical analysis, comprises 0.79-0.80 the weight of wet tissue (Table 2). The amount of extracellular fluid in the plexus, as estimated by the steady-state distribution of [3H]inulin, is slightly greater than 0.20 of the wet weight of the tissue (Table 2). The relatively large amount of residual erythrocytes in the samples of LVP (i.e., an average of 0.066 mg erythrocytes/mg wet tissue) is not unexpected due to the vascularity of this secretory tissue. Data obtained for analogous compartments in skeletal muscle are also summarized in Table 2. The arterial pH of the etherized rats was generally in the range of 7.49-7.54. The effect of ether on respiration, together with the altitude of ca. 1350 m to
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C.E. Johanson and D.M. Woodbury
which the animals were acclimatized, are factors responsible for the relatively high pH.
Distribution of Tracers Between Intracellular and Extracellular Compartments The space data for [14C]antipyrine and [14C]barbital (Table 1), together with the steady-state spaces for the tissue water compartments (Table 2), have been used to analyse the partitioning of these radiolabelled drugs between intracellular and extracellular fluids, both in choroid plexus and skeletal muscle (Table 3). The data in Table 3 are relative concentrations of the radiolabelled drugs in various compartments based upon a plasma concentration of 100. At the steady state, [a4C]antipyrine distributed more or less evenly throughout the various water compartments in the tissues studied (Table 3). For example, the concentrations of radioantipyrine in the epithelial cell water of choroid plexus is approximately equal to that both in the plasma water and in the CSF water; moreover, within the limits of experimental error, the concentration of [14C] antipyrine in cellular water of skeletal muscle is the same as that in plasma water. However, [14C]barbital does not accumulate in the cellular compartment as extensively as [14C]antipyrine, even if 2 h are allowed for this compound to distribute. Thus, the concentration of [14C]barbital in the epithelial cells of choroid plexus as well as the CSF is considerably less (i.e., 20-30%) than that of [14C]antipyrine. Similarly, in skeletal muscle the concentration of [14C]barbital in the cellular compartment is less than that of [14C]antipyrine. The predicted values for the concentrations of radiobarbital in parenchymal cells (see figures in parentheses in Table 3) are in reasonably close agreement to those values determined experimentally; to obtain the theoretically-expected concentrations, values of 6.99 and 7.03 were utilized for the cell pH of LVP and muscle, respectively (Johanson, in preparation). Discussion
Rate of Uptake of [14C]Antipyrine and [a4C]Barbital by the Choroid Plexus, Cerebrospinal Fluid and Other Tissues The rate of penetration of [14C]barbital into the choroid plexus was considerably slower than that of [14C]antipyrine. At 9 min post injection, a time at which radioantipyrine had penetrated into more or less 100% of water in the LVP, [14C]barbital had distributed into only 0.44 of the total water in this tissue. The lipid solubility of these drugs, together with the extent of their ionization, are important factors to consider in the explanation of differences in permeation rates. Partition coefficients (n-heptane/water of less than 0.002 and 0.005, respectively, have been reported for the non-ionized forms of barbital and antipyrine (Hogben et al., 1959). On the basis of these oil/water coefficients, one could predict the rate of penetration of barbital into choroid plexus to be less than half that of antipyrine. At a pH of 7.4, barbital with a pKa of 7.8 and antipyrine with a pK~ of 1.4 (Shore et al., 1957) are 72% and > 99.9%
Penetration of Drugs into In Vivo Choroid Plexus
71
non-ionized, respectively; since the degree of ionization of a drug also influences its rate of movement into cells, it is useful in the analysis of penetration to consider the "effective partition coefficient" (i.e. the partition coefficient multiplied by the fraction of the drug which is non-ionized at the pH of interest.) The ratio of "effective partition coefficients" for barbital/antipyrine is 1.1/5 (Goldstein et al., 1969). On the basis of these "effective" coefficients it might be expected that barbital, if it were to distribute passively via uncomplicated diffusion, would take about four times longer than antipyrine to attain a steady-state distribution in the choroid plexus. The uptake data do not indicate the exact time at which radiobarbital reaches a steady-state distribution in the LVP; however, it is interesting to note that whereas 3 rain are required for [14C]antipyrine to achieve a space which is 80% that of the steady-state space, approximately 12 min are necessary for [14C]barbital to penetrate the LVP to the same extent. The respective time courses of uptake of radioisotopic antipyrine and barbital by CSF (and brain) in this study are similar to comparable determinations in other mammalian species. It has previously been established that 2 h of distribution time is sufficient for radiobarbital to achieve a steadystate distribution in various compartments of the central nervous system (Mayer et al., 1959). Mayer et al. (1959) and Brodie et al. (1960) both reported that antipyrine penetrates into the mammalian CSF system at a rate approximately four times faster than that of barbital. Such a ratio for the rates of penetration of antipyrine and barbital into the CSF system is similar to the ratio of rates at which these two compounds penetrate the choroid plexus (present study) during the attainment of steady-state distribution. Thus, there may be a fixed relationship between the respective rates of uptake of a given organic compound by choroid plexus and CSF. The uptake of [14C]barbital by the LVP lags behind that of skeletal muscle, despite the fact that blood flow to the choroid plexus is two orders of magnitude greater than that to skeletal muscle (Welch, 1963; Goldstein et al., 1969). Apparently, the rate-limiting step for the penetration of barbital into the LVP is a factor other than blood flow (e.g., parenchymal cell permeability; see discussion below). On the other hand, the uptake of [~4C]antipyrine by LVP leads that by muscle; thus, the penetration of antipyrine (in contrast to that of barbital) into choroid plexus may be limited primarily by blood flow.
CompartmentalAnalys& An accurate assessment of the partitioning of antipyrine and barbital among the various compartments in choroid plexus and skeletal muscle is dependent upon values obtained for the magnitude of the various water compartments (or spaces) in these tissues. The volume of extracellular fluid in LVP, as estimated from the distribution of [3H]inulin, was 0.202 mg (or ~tl) water/mg wet tissue; this finding agrees well with the value of 0.206 previously reported for the same parameter determined with [14C]inulin (Johanson et al., 1974). Because of the relatively large volume of residual erythrocytes in the plexus, it is important to
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C.E. Johanson and D.M. Woodbury
correct for this cellular compartment in any transport or distribution study which is focused on the epithelial cells of this tissue. Compartmentation data obtained for rat muscle (Table 2) are similar to those previously reported. Metabolism and binding are important factors to consider in the calculation of cellular concentrations of tracers. Previous investigators have established that barbital is not chemically modified by CSF, brain or plasma (Lal et al., 1964); moreover, barbital is not metabolized by the liver (Rubin et al., 1964). There is only a negligible degree of binding of barbital to the proteins of plasma and tissue (Goldbaum and Smith, 1954; Brodie et al., 1960). [a4C]antipyrine has a metabolic half life of 90 rain in the rat (Bakke et al., 1974); thus, in the short-term experiments (i.e., 3-15 rain) of the present study, the level of metabolites of antipyrine in tissue should not be appreciable. Since radioantipyrine is an indicator which accurately measures total water content (Soberman et al., 1949) of most (if not all) tissues, it is not surprising that the binding of this tracer to macromolecules is negligible (Shore et al., 1957). Therefore, since both barbital and antipyrine are neither bound nor metabolized appreciably (under the conditions of the experiment), it is likely that the data in Table 3 are reliable estimates of the concentrations of unbound, unmetabolized tracers.
Analysis of the Intra-Tissue Distribution of [14C]Antipyrine and [14C]Barbital Antipyrine is almost completely unionized at a pH of 7.4; thus, the predominance, as well as the extreme lipid solubility, of the non-ionized form of this weak base is the main reason for its rapid penetration even into the CNS. The steady-state distribution of [14C] antipyrine is characterized by its penetration into virtually the entire water of all tissue compartments investigated (Table 3). Because of the rapid attainment of equilibrium distribution of radioantipyrine between plasma water and CSF water, there is no sin~:~effect,exerted by the CSF to this compound. Barbital, because it is less lipid soluble than antipyrine (and more ionized at the existing pH) penetrates tissues both within and outside the CNS less rapidly and extensively than antipyrine. The steady-state concentration of barbital in the parenchymal cells of both choroid plexus and skeletal muscle is 20-25 % less than that in plasma water. The hydrogen ion gradient between extracellular and intracellular fluids, which is equivalent to approximately 0.5 pH unit, is probably the best explanation for the observed asymmetrical distribution of [14C]barbital between cell water and plasma water. Moreover, the concentration of barbital in cell water tends to be lower in the LVP than in muscle; if, during the CSF secretory process, barbital is less able than antipyrine to keep up with the volume flow of water across the choroid plexus (Welch et al., 1966), the resultant sieving of barbital would tend to lower its concentration in the choroid cell. The CSF data support this postulated sieving of barbital; at the steady state, 14C-barbital concentration in the CSF water is only 67 % that in plasma water. Moreover, we have recently obtained evidence that 14C-urea is substantially sieved by the choroidal membrane (unpublished data).
Penetration of Drugs into In Vivo Choroid Plexus
73
Is active transport also a mechanism for keeping barbital in the CSF at a lower concentration than that in plasma? The finding that 14C-barbital space in the choroid plexus was not affected significantly by increasing the dose of non-radioactive barbital by 33 fold (i.e., from 0.6-20 mg/kg) might be explained by one of the following ways: 1) barbital is not actively transported by the LVP, 2) barbital is actively transported by a carrier mechanism of extremely low capacity which is saturated by relatively low IP doses of barbital, i.e., < 1 mg/kg or 3) barbital is actively transported by the LVP via a carrier system of relatively high capacity which is not saturated even at an IP dose of barbital of 20 mg/kg. We favour explanation 2 for several reasons: carrier transport systems which translocate several different organic anions have been demonstrated in in vitro preparations of choroid plexus (Forn, 1972; Spector and Lorenzo, 1974; Sampath and Neff, 1976); using an in vivo approach, Rollins and Reed (1970) showed that the weak organic acid, DMO, ([14C]5,5-dimethyloxazolidine-2,4-dione) is actively removed from CSF by a carrier system which is saturated at relatively low IP doses of carrier, i.e., from 1-5 mg/kg (a comparison of D M O with barbital in this context would not be valid if these two anions have substantially different affinities for an anion carrier); in the present study, the calculated concentration of radiobarbital in the choroid cell is approximately that expected if barbital were to distribute passively across the choroid cell membrane according to the p H gradient (if a high-capacity anion transport system were present in the LVP, one might expect the steady-state concentration of radiobarbital to be considerably greater than that observed). Since a substantial part of the overall exchange of materials between blood and ventricular CSF occurs across the epithelial membrane of the choroid plexus, it seems important to gain more insight on the nature of transport at this important interface. The compartmentation aspect of the present study represents a new approach to the problem of delineating transport and permeability phenomena associated with the choroid plexus. Additional studies inthis area are indicated since a better understanding of the basic physiology' of transport at this interface should contribute to the fulfilment of the therapeutic goal of being able to manipulate the transport of materials across the blood-CSF barrier.
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