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Environmental
Health Perspectives Supplements Volume 110, Number 5, October 2002
Brain Uptake, Retention, and Efflux of Aluminum and Manganese
Robert A. Yokel
College of Pharmacy and Graduate Center for Toxicology, University
of Kentucky Medical Center, Lexington, Kentucky, USA
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Full Article in PDF
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Abstract
My colleagues and I investigated the sites and mechanisms of aluminum
(Al) and manganese (Mn) distribution through the blood-brain barrier
(BBB). Microdialysis was used to sample non-protein-bound Al in the
extracellular fluid (ECF) of blood (plasma) and brain. Brain ECF Al appearance
after intravenous Al citrate injection was too rapid to attribute to diffusion
or to transferrin-receptor-mediated endocytosis, suggesting another
carrier-mediated process. The brain:blood ECF Al concentration ratio was
0.15 at constant blood and brain ECF Al concentrations, suggesting carrier-mediated
brain Al efflux. Pharmacological manipulations suggested the efflux carrier
might be a monocarboxylate transporter (MCT). However, the lack of Al
14C-citrate uptake into rat erythrocytes suggested it is not
a good substrate for isoform MCT1 or for the band 3 anion exchanger. Al
14C-citrate uptake into murine-derived brain endothelial cells
appeared to be carrier mediated, Na independent, pH independent, and energy
dependent. Uptake was inhibited by substrate/inhibitors of the MCT and
organic anion transporter families. Determination of 26Al in
rat brain at various times after intravenous 26Al suggested
a prolonged brain 26Al half-life. It appears that Al transferrin
and Al citrate cross the BBB by different mechanisms, that much of the
Al entering brain ECF is rapidly effluxed, probably as Al citrate, but
that some Al is retained for quite some time. Brain influx of the Mn2+
ion and Mn citrate, determined with the in situ brain perfusion
technique, was greater than that attributable to diffusion, suggesting
carrier-mediated uptake. Mn citrate uptake was approximately 3-fold greater
than the Mn2+ ion, suggesting it is a primary Mn species entering
the brain. After Mn2+ ion, Mn citrate, or Mn transferrin injection
into the brain, brain Mn efflux was not more rapid than that predicted
from diffusion. The BBB permeation of Al and Mn is mediated by carriers
that may help regulate their brain concentrations. Key words: aluminum,
b.End5 cells, blood-brain barrier, brain efflux, brain endothelial
cells, brain influx, in situ brain perfusion, manganese, microdialysis,
rat. Environ Health Perspect 110(suppl 5):699-704 (2002).
http://ehpnet1.niehs.nih.gov/docs/2002/suppl-5/699-704yokel/abstract.html
This article is part of the monograph Molecular Mechanisms
of Metal Toxicity and Carcinogenicity.
Address correspondence to R.A. Yokel, College of Pharmacy
and Graduate Center for Toxicology, Pharmacy Bldg., Rose St., University
of Kentucky Medical Center, Lexington, KY 40536-0082 USA. Telephone:
(859) 257-4855. Fax: (859) 257-7585. E-mail: ryokel@uky.edu
I thank the following who conducted much of the work
presented herein: D.C. Ackley, D.D. Allen, B.L. Bukaveckas, J.S. Crossgrove,
and S.S. Rhineheimer. This work was made possible in part by financial
support from the National Institutes of Health (ES K04 174, R01 4640,
and F06 TW2343), the U.S. Environmental Protection Agency (R 825357),
the Health Effects Institute (Agreement 99-10), and a Burroughs Wellcome
Fund Research Travel grant.
Received 23 January 2002; accepted 29 May 2002.
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Aluminum and Manganese As Neurotoxicants
Aluminum (Al) and manganese (Mn) are neurotoxicants that have the potential
to contribute to neurodegenerative disorders (1-5). The contributory
role of Al in the dialysis encephalopathy syndrome has been shown. Avoidance
of the major Al sources contributing to the syndrome has greatly reduced this
problem, although occasional outbreaks still occur (6). There has been
concern about the suggested role of Al in Alzheimer disease (AD) since the initial
report of elevated brain Al in victims of this condition (7). Some studies
have shown a small positive correlation between drinking-water Al concentration
and dementias, including AD [reviewed by Yokel (2)]. However, the role
of Al in AD etiology is highly controversial because of the conflicting results
of studies of brain Al of AD victims, the association of drinking-water Al and
the risk for AD, and other reported end points of Al toxicity. As a result of
continued concern about the neurotoxicity of Al, the U.S. Environmental Protection
Agency has put Al on its contaminant candidate list, the U.S. Food and Drug
Administration recently implemented labeling requirements for Al in large- and
small-volume parenterals, and Canada established operational guidance limits
for drinking-water Al on the basis of the precautionary principle (8-10).
Manganese can produce a parkinsonism-like syndrome (11,12). This has
occurred in miners after prolonged exposed to manganese dioxide by inhalation
(13), families drinking well water contaminated by buried dry-cell batteries
[cited by Hudnell (11)], workers in dry-cell battery factories, and perhaps
from environmental exposure (14). There is currently concern about airborne
exposure from the use of Mn in fungicides such as ethylenebis(dithiocarbamato)manganese
(Maneb; Cerexagri, Inc., King of Prussia, PA, USA) and in the fuel-additive
octane-enhancer methylcyclopentadienyl manganese tricarbonyl (MMT) (11).
The use of MMT in the United States has been permitted by a court decision (Ethyl
Corporation v. Carol M. Browner, Administrator of the United States Environmental
Protection Agency, and the United States Environmental Protection Agency. 1995.
Case No. 94-1516, U.S. Court of Appeals for District of Columbia Circuit, Washington,
DC), although MMT has apparently not been extensively used as a fuel additive
in the United States since the decision. In contrast to Al, for which no mammalian
essentiality has been shown, Mn is essential, required for brain development
and function (15).
There was very little published information on the sites and mechanisms of
brain Al entry prior to our initiation of such studies, and nothing on efflux
from the brain. My colleagues and I have elucidated the predominant site and
have investigated the rates and mechanisms of blood-brain barrier (BBB)
permeation of Al, as well as the duration of Al retention in the brain. Brain
Mn uptake has been attributed to a carrier-mediated process [reviewed by Aschner
and colleagues (16,17)]. However, the studies were not conducted under
conditions that controlled the Mn species, leaving uncertainty about the Mn
species that enter the brain. There have been no reports of studies of Mn efflux
from the brain.
The Sites and Mechanisms of Metal Distribution into
the Brain
The potential routes that substances may distribute from the nasal cavity
into the brain include absorption into systemic circulation followed by permeation
through the BBB or choroid plexuses, and absorption from the nasal cavity into
olfactory nerves followed by neuronal distribution directly into the brain.
The ability of Al to enter the brain via this latter route has been addressed
in two preliminary studies (18,19). In contrast, Mn has been shown to
slowly enter the brain via this route, although quantitation (bioavailability)
has not been reported (20,21).
Al and Mn may enter the brain from blood, either through the choroid plexuses
or the BBB. These routes of distribution between blood and brain are shown diagrammatically
in Figure 1. There is a choroid plexus in each of the four cerebral ventricles
of the brain. They synthesize most of the cerebrospinal fluid (CSF) that fills
the brain ventricles and the subarachnoid space that surrounds the brain and
spinal cord. The total surface area of the choroid plexuses is approximately
10 cm2. Brain atlases of the rat and human show brain regions as
far as 1 mm away from the nearest component of the CSF compartment.
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Figure 1. The
routes of distribution between plasma and brain: through the BBB and the
choroid plexus (CP). Arrows show membranes through which distribution might
occur by carrier-mediated transport in addition to diffusion. The dashed
line depicts the absence of a membrane barrier to distribution between the
CSF and brain ECF. |
The BBB provides the other route of distribution from blood to brain. The
anatomical basis of the BBB is primarily attributed to the tight junctions between
the cerebral microvascular endothelial cells that line the microvessels that
perfuse the brain. Additional impediments to permeation through the BBB come
from the absence of fenestrations and the low transcytotic activity of the endothelial
cells, the pericytes that surround 30% of their surface, and the astrocyte foot
processes that cover 99% of the surface of the endothelial and pericyte cells.
Substances must either diffuse through the membranes of these cells or be transported
by cell membrane carriers to penetrate the BBB. The surface area of the 400
miles of brain capillaries that are the site of the BBB is roughly 12 m2,
about 1,000-fold that of the choroid plexuses. Visual examination of electron
micrographs suggests that no cell in the brain is >30-40 µm from
the nearest microvessel (BBB site) (22). There is much greater opportunity
for rapid exchange between blood and brain through the BBB than through the
choroid plexuses and CSF compartment.
The potential mechanisms of distribution of substances across the BBB are
the same as those across any cell membrane: diffusion, carrier- and receptor-mediated
transport by facilitated diffusion, and active transport. The roles of lipophilicity
(hydrogen bonding potential, polar surface area) and molecular weight (size)
as predictors for diffusion of small molecules across the BBB have been well
described (23,24), providing the opportunity to estimate the permeability
rates of Al, Mn, and their complexes across the rat BBB from their octanol/buffer
partitioning coefficient and molecular weight (23). Studies have identified
carrier-mediated transport of some metals through the BBB, including copper
as an l-histidine complex, iron by transferrin-receptor-mediated endocytosis
(TfR-ME), mercury as a cysteine complex, and zinc as an amino acid complex (25).
These suggest the possibility that Al, Mn, and/or their complexes may cross
the BBB by carrier-mediated transport. We conducted studies in rats and in cells
derived from the BBB to elucidate the distribution of Al and Mn into and out
of the brain and the mechanisms mediating these processes. Animal work was approved
by the University of Kentucky Institutional Animal Care and Use Committee and
was conducted following the Guiding Principles in the Use of Animals in Toxicology
of the Society of Toxicology (26).
The Predominant Site of Aluminum Distribution into and out
of the Brain
We initially studied Al distribution across the rat BBB using microdialysis
probes implanted in the frontal cortex, lateral ventricle, and blood (jugular
vein). This provided the ability to sample the extracellular fluid (ECF) in
those compartments and therefore in the fluids on both sides of the BBB and
a choroid plexus. Microdialysis provides a reflection of unbound substances
in the ECF. It was used because it enables repeated sampling and therefore determination
of distribution across the BBB and choroid plexus at multiple time points within
the same subject. The substance concentrations in dialysates exiting the brain
or CSF compared with blood were used to generate a brain:blood ratio (BBr).
It has been pointed out that consideration of the chemical species of a metal
in a biofluid indicates the species available to distribute across membranes,
such as the BBB, and that might serve as substrates for carriers (25).
Speciation calculations conducted by Wesley Harris [cited by Yokel and McNamara
(27)] indicated that 91% of plasma Al is Al transferrin and 7-8% is Al
citrate, whereas 90% of Al in brain ECF was predicted to be Al citrate and only
4% Al transferrin. Al citrate was administered in the microdialysis studies.
The Al concentration in the dialysate exiting the frontal cortex microdialysis
probe reached a maximal steady value in the first 5-min sample after Al citrate
was given as an intravenous bolus, indicating quite rapid distribution of Al
from blood to brain ECF (Figure 2A). The microdialysis probe in the frontal
cortex was some distance from the cerebral ventricles where Al might enter the
CSF from a choroid plexus. Maximal penetration of substances through brain parenchyma
is thought to be <1 mm (28), perhaps in part due to a bulk flow of
ECF from brain parenchyma toward the cerebral ventricles (29). The appearance
of the maximal value within 5 min in the frontal cortex suggests entry through
the BBB. However, there is evidence of rapid distribution of solutes from CSF
to the brain ECF surrounding the microvasculature. This distribution is believed
to occur through the paravascular space (30). Comparison of the Al concentration
in the dialysates obtained from the frontal cortex versus the CSF and comparison
of the Al BBr calculated from brain/blood and CSF:blood ratio for each sample
interval (Figure 2B) revealed a higher Al concentration, and higher BBr, in
the frontal cortex than in CSF. This supports the suggestion that Al enters
the brain from blood through the BBB rather than through the choroid plexuses
because entry through the choroid plexus and diffusion through the brain would
not be expected to produce a higher Al concentration in the frontal cortex than
in the CSF. The frontal cortex BBr was consistently <1, suggesting a process
other than diffusion was mediating Al transfer across the BBB.
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| Figure 2. Aluminum concentration
determined by atomic absorption spectrometry in three compartments of rats
simultaneously and repeatedly using microdialysis after a bolus intravenous
Al injection. (A) Al concentration in dialysates exiting from microdialysis
probes implanted in blood ECF (plasma; circles), brain ECF (squares), and
the lateral ventricle CSF (triangles), after an intravenous bolus injection
of 0.5 mmol Al citrate/kg. (B) BBr of Al for the results shown in
A: brain ECF:blood (diamonds) and CSF:blood (inverted triangles).
Values shown are mean ± SEM of at least three animals at each time.
Lines show two-term nonlinear regressions in A and one-term nonlinear
regressions in B. From Allen and Yokel (53). Published with
permission of Blackwell Publishing. |
Further evidence of carrier-mediated distribution of Al across the BBB was
obtained based on the calculated plasma Al concentration in the microdialysis
studies (1 mM), the rate of Al citrate flux through membranes (4 
10-16 mol/cm2/sec) (31), the brain capillary surface
area (240 cm2/g brain) (32), and brain ECF volume (0.15 mL/g
brain). Calculating the amount of Al that would diffuse across the BBB in 5
min from 1 mL of blood into 0.15 mL of ECF provided an estimate of the resultant
Al concentration in the brain ECF. When compared with the blood Al concentration,
this produced a BBr of 0.00003. This is much less than the observed BBr of approximately
0.15.
To further assess Al distribution across the BBB from the relationship between
brain and blood ECF Al concentrations, Al citrate was bolus dosed and infused
at various rates. This achieved quite constant Al concentrations in the sampled
compartments, shown by the consistent Al concentration over time in the dialysates
exiting microdialysis probes in the brain and blood. Plasma and brain ECF Al
concentration increased proportionally to the increase in the Al infusion rate
(Figure 3A), suggesting linear (concentration independent) Al distribution and
elimination. However, the BBr was consistently approximately 0.15, significantly
less than 1 (Figure 3B). These results support the hypothesis that Al BBB permeation
is carrier mediated because at steady state the concentration of Al in blood
ECF (plasma)
the brain influx rate must equal the concentration of Al in brain ECF
the brain efflux rate. Because the concentration of Al in blood ECF was greater
than the concentration of Al in brain ECF, the brain influx rate must be less
than the efflux rate. Therefore, BBB permeation of Al cannot be attributed solely
to diffusion.
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| Figure 3. Steady-state Al
concentrations determined by atomic absorption spectrometric analysis of
microdialysis samples from the brain and blood of rats during intravenous
infusion of four Al citrate concentrations. (A) Steady-state Al concentration
in dialysate exiting microdialysis probes implanted in blood ECF (plasma;
circles) and brain ECF (squares) at various infusion rates of Al citrate.
(B) Steady-state BBr of Al for the results shown in A. Values
shown are means ± SEM of five animals at each infusion rate. Lines
show linear regressions. From Allen et al. (54). |
Efflux of Al from the brain also likely occurs across the BBB rather than through
CSF. In these studies it was necessary to administer doses of Al citrate that
exceeded physiological Al and citrate concentrations and exceeded the metal-binding
capacity of transferrin. These high concentrations were necessary to reliably
quantitate increases in Al above its background concentration, considering the
low relative recovery of Al citrate (~3.25%) by microdialysis (33). The
calculated plasma Al concentration at the highest Al citrate infusion rate was
approximately 1 mM. Because the Al was introduced as Al citrate, and it is estimated
transferrin could not have complexed more than 15% of the Al in plasma at the
lowest Al blood level and only 2% at the highest, it is likely that most Al
in plasma and essentially all in brain ECF, where the transferrin concentration
is <1% of that in plasma, was associated with citrate. The Al recovered by
microdialysis could not have been Al transferrin because transferrin is too
large to penetrate the membrane of the microdialysis probes we used. The marker
of BBB permeability included in most of these studies, 4-trimethylammonium antipyrine
(34), did not indicate that BBB integrity was significantly compromised.
These results suggest Al citrate can enter the brain from blood through the
BBB by a mechanism other than diffusion.
Mechanism(s) of Aluminum Distribution across the BBB
Many carriers have been reported at the BBB (35). A review of these
carriers suggests some candidates that might mediate Al citrate transport across
the BBB, including TfR-ME. However, Al citrate can also enter the brain by one
or more other mechanisms. Assuming that TfR-ME clears Al from blood into brain
ECF across the BBB at the same rate as it clears iron [3
10-3 mL blood/gm brain/hr (36)], the Al that could enter brain
ECF from blood in 5 min would result in a BBr of 0.0004 based on estimated plasma
ECF Al concentrations up to 1 mM from our microdialysis results. The observed
BBr was much greater than this value. Calculation of Al speciation at pH 7.4
showed approximately 1.25 and 29% of the Al would be present as Al(H-1citrate)-
and Al(H-1citrate)(OH)2-, respectively, in the presence
of 1 mM citrate. The percentage of these species would increase at lower Al
citrate concentrations (37). In these complexes there is a single, noncomplexed,
ionized carboxylate group. The presence of this monocarboxylate suggested that
Al citrate may be a substrate for the monocarboxylate transporter (MCT) at the
BBB. At least one MCT isoform, MCT1, has been shown to be expressed at the BBB.
The reported rate of substrate transfer by the MCT across the BBB, 60 nmol/g
brain/min, could transport 40,000 ng Al, as the citrate, into 1 mL brain ECF
in 5 min after the intravenous bolus Al injection described above if Al citrate
serves as a comparably transported substrate. This is about 15 times the estimated
rate of appearance of Al in brain ECF that we observed. This calculation is
based on the observed 2.8 mM Al in frontal cortex dialysate within 5 min of
intravenous Al citrate injection and an estimated 3.25% relative recovery, yielding
an estimated 2,520 ng Al/mL ECF.
To test the hypothesis that MCT1 mediates Al citrate transport across the
BBB, substances were included in the dialysate of the microdialysis probe implanted
in the frontal cortex of rats that were receiving an intravenous infusion of
Al citrate. These substances were expected to diffuse from the dialysate into
the brain to achieve sufficient concentrations to produce local effects in the
same brain region where BBB function was being studied. Addition of 15 µM
CN- or 10 µM 2,4-dinitrophenol, as metabolic inhibitors,
1 M pyruvate as a substrate for the MCT, and a pH 10.2 dialysate or 1 mM carbonylcyanide-p-(trifluoromethoxy)phenylhydrazone
to reduce proton availability and proton gradients significantly increased the
BBr to approximately 1 (38,39). Similar increases in the BBr were not
observed in the contralateral frontal cortex nor was the integrity of the BBB
significantly compromised. These results suggest inhibition of an energy-dependent
process, competition for MCT transport, and reduction of protons inhibited carrier-mediated
Al citrate transport across the BBB, leaving only diffusion mediating its BBB
permeation. The results were consistent with the hypothesis that Al citrate
is a substrate for MCT-mediated transport across the BBB. To further test this
hypothesis, studies were conducted with the rat erythrocyte because it expresses
MCT1 and the band 3 anion exchange transporter. However, the uptake of a known
substrate, 14C-lactate, was not inhibited by Al citrate, nor was
there significant uptake of Al 14C-citrate. These results suggest
Al citrate is not an effective substrate for either MCT1 or the band 3 anion
exchange transporter.
To characterize, and perhaps identify, the carrier(s) mediating Al citrate
transport across the BBB, Al citrate uptake studies were conducted using b.End5
cells, an immortalized cell line derived from murine brain endothelial cells.
Al 14C-citrate uptake was slow and reasonably linear for 4 hr. Uptake
of Al 14C-citrate was approximately 70% greater than 14C-citrate
uptake. Uptake after 1 hr achieved an intracellular Al citrate concentration
of approximately 25% of the medium concentration. In comparison, diffusion was
calculated to produce an intracellular Al citrate concentration of approximately
1% of the medium concentration. These results suggested carrier-mediated uptake.
Al 14C-citrate uptake into b.End5 cells was pH independent, sodium
independent, and energy dependent and was inhibited by numerous nonspecific
substrates and inhibitors of MCT and organic anion transporters. Uptake was
concentration dependently inhibited by two relatively specific organic anion
transporter substrates (37). Comparison of the characteristics of the
carrier(s) mediating Al citrate uptake to those of carriers described at the
BBB suggests Al citrate transport may be mediated by an organic anion transporter
such as an MCT isoform and/or an organic anion-transporting polypeptide
(oatp). Further work elucidating which of the MCT and oatp isoforms are expressed
at the BBB and the identification of isoform-selective inhibitors that could
be used in future studies of Al citrate uptake into brain endothelial cells
or the brain would advance the ability to identify the BBB Al citrate transporter(s).
Some Aluminum Persists in the Brain for a Long Time
We conducted a study to ascertain the residence time of Al in the brain. Administration
of the stable, ubiquitous isotope of Al, 27Al, is not well suited
to determine the brain half-life of Al because brain Al concentrations are relatively
small and do not greatly increase in response to Al exposure. Therefore, an
elevation above, and subsequent decrease toward, the endogenous Al concentration
is difficult to reliably determine. The natural abundance of 26Al
is extremely small. 26Al can be quantified with exquisite sensitivity
(~1
106 atoms) as the 26Al:27Al isotopic ratio
by accelerator mass spectrometry (AMS), enabling the study of Al toxicokinetics
at physiologically relevant Al exposures (40). A disadvantage is the
cost of AMS analysis of 26Al (~$200/sample). We gave rats intravenous
26Al transferrin or 26Al citrate and terminated them from
4 hr to 256 days later to determine the percentage of Al in blood that enters
the brain, the time course of Al efflux from the brain and whether brain Al
clearance is enhanced by repeated chelation therapy with desferrioxamine, a
clinically-useful Al chelator. The peak brain 26Al concentration,
approximately 0.005% of the 26Al dose per gram of brain, was similar
after 26Al transferrin and 26Al citrate dosing and was
similar to some previous, smaller, short-term studies (41). The comparable
results from Al citrate and Al transferrin are probably due to the change in
the Al ligand from citrate to transferrrin, the preferred ligand, within minutes
because the administered Al citrate dose was only about 1% of the plasma transferrin
metal-binding capacity. The half-life of brain 26Al could not be
accurately calculated but was estimated to be about 150 days. The brain half-life
was roughly 55 days in rats that received desferrioxamine injections three-times
weekly, demonstrating brain Al retention in compartments from which it can be
mobilized (Figure 4). It is difficult to extrapolate these results to the human
because of the lack of comparable metal half-life determinations in rat versus
human brain or sufficient insight into allometric scaling from rat to human
brain for metals. The residence of Al in the brain and other soft tissue organs
may reflect the residence of Al in bone, which contains approximately 50-70%
of the Al body burden. The bone 26Al half-life is prolonged in the
rat (42).
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Figure 4. Brain
26Al determined by AMS in rats terminated at various times after
intravenous administration of 26Al transferrin. The rats received
an injection of saline 3 times weekly (squares) or 0.15 mmol/kg desferrioxamine
(circles). Values are the means ± SEM of results from four to six rats
and are shown as the percentage of the injected 26Al per gram
of brain. From Yokel et al. (41) with permission of Oxford Press.
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Manganese Citrate Enters the Brain by a Carrier-Mediated Process
We recently began studies of Mn distribution across the BBB. Brain uptake
of small-molecular-weight Mn species was previously attributed to a carrier-mediated
process, but the work was not conducted under conditions that controlled the
Mn species (43,44). The experimental approach we employed was to compare
brain influx and efflux rates determined in the rat to BBB permeation rates
predicted for capillary diffusion to ascertain if there was evidence for carrier-mediated
influx or efflux of Mn. To study brain Mn uptake under conditions where better
control of the Mn species could be maintained, we used 54Mn species
and the in situ brain perfusion technique (45), as modified by
Allen and Smith (46). Brain influx of the Mn2+ ion and Mn
citrate, which were calculated to represent 40 and 12%, respectively, of non-protein-bound
Mn in plasma (47), were studied, as well as Mn transferrin. The brain
uptake rates determined in the intact rat were compared with the estimated brain
capillary diffusion rates of these Mn species, which were calculated from the
their molecular weight and lipophilicity (Figure 5). Lipophilicity was determined
as the partitioning coefficient between octanol and an aqueous phase. The estimated
brain capillary diffusion rates were then calculated from the relationship between
molecular weight and lipophilicity for substances that diffuse through the BBB
(23) times the brain capillary surface area (240 cm2/g brain).
The estimated brain capillary diffusion rates of the Mn ion, Mn citrate, and
Mn transferrin ranged from 1.5 to 2.8
10-5 mL/sec/g (48).
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Figure 5. The brain (parietal
cortex) uptake rates for three Mn species determined after their intravenous
infusion as 54Mn in the rat, using the in situ brain
perfusion method. The rat received an intra-arterial perfusion for a variable
duration (shown) of one of the three Mn species: Mn citrate (diamonds),
Mn transferrin (triangles), or Mn2+ ion (circles). Values are
mean from 7-11 rats per data point except for Mn citrate (diamonds), n
= 4 for 90 sec. The symbols for Mn transferrin and Mn citrate at 30 and
60 min are shown slightly to the left and right, respectively, of their
true values to enable their visualization. Squares show upper and lower
range of brain uptake predicted for these three Mn species for capillary
diffusion. Lines are best-fitted linear regressions of the means.
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The average observed brain uptake rates of the Mn citrate, Mn ion, and Mn transferrin
into the nine brain regions sampled were 25, 8.6, and 5.7
10-5 mL/sec/g, respectively, suggesting carrier-mediated uptake of
each Mn species. To verify that Mn was entering the brain, compared to being
adsorbed onto or taken into but not through brain endothelial cells, the capillary
depletion method was used to separate brain capillary cells from the rest of
the brain (49). Only 8-25% of the Mn was associated with capillary cells,
suggesting the in situ brain perfusion studies were showing Mn distribution
across the BBB (47).
Brain Manganese Efflux Does Not Appear to Be Carrier Mediated
The efflux of Mn out of the brain across the BBB was determined using an established
method (50). Efflux through brain capillaries (the BBB) was calculated
from the product of the volume of distribution of the Mn within the brain, from
which it can efflux to blood, times the brain elimination rate constant. The
volume of Mn distribution in the brain was determined from uptake of 54Mn
as the ion, citrate, and transferrin into rat parietal brain slices versus time.
In contrast to brain slice uptake of para-aminohippurate, which reached
a plateau at approximately 60 min (51), uptake of these Mn species continued
to increase for up to 180 min, suggesting continued brain cell uptake. The brain
elimination rate constant of these three Mn species was determined from the
percentage of 54Mn remaining in the brain at various times after
injection as 54Mn ion, citrate, or transferrin into the parietal
cortex, compared with the percentage of 14C-sucrose or 14C-dextran,
which are expected to very slowly efflux from the brain by diffusion across
the BBB. The Mn species did not efflux from the brain more rapidly than sucrose
or dextran. Taken with the parietal slice uptake results, the lack of brain
efflux suggests Mn continues to be taken up into brain cells over time and is
not transported out of the brain by carrier-mediated processes (52).
Carriers Mediate the Permeation of Aluminum and Manganese
across the BBB
The results of the studies reviewed herein suggest that there are carriers
at the BBB that mediate the uptake of Al and Mn into the brain. This may be
a beneficial process for Mn, an essential element for brain metabolism. However,
Al is not essential and, like Mn, is neurotoxic when sufficient brain concentrations
are achieved. The results suggest that there is a carrier-mediated mechanism
to protect the brain from Al, by effluxing it across the BBB into blood. It
does not appear that a similar protective mechanism is present for Mn. Further
work is necessary to identify the carriers mediating transport of these metals
across the BBB.
References and Notes
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3. Spencer PS. Aluminum and its compounds. In: Experimental
and Clinical Neurotoxicology (Spencer PS, Schaumburg HH, eds). New York:Oxford
University Press, 2000;142-151.
4. Chu N-S, Huang C-C, Calne DB. Manganese. In: Experimental
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University Press, 2000;752-755.
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Int 59:746-753 (2001).
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8. U.S. Environmental Protection Agency. Announcement
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Last Updated: October 4, 2002
