Clearance of specific substances
There are certain species that are critical for normal brain function and that must be transported into or out of the brain rapidly and in large quantities. The most prominent of these are O2, CO2, water and glucose. Influx and efflux of these species are so rapid that they entail movements of a large fraction of the amounts flowing through the brain vasculature, much more than could be delivered by the blood flow to just the choroid plexuses.

Water
Water permeability of the blood–brain barrier can be calculated in two very different ways. In the first tritiated water is introduced into the blood and the permeability, Pw,tracer calculated from the ratio of the undirectional influx of tracer, Jinf, to the concentration of the tracer, cTHO,7 Pw,tracer=Jinf/cTHO.
It is assumed that this permeability also applies to efflux and to unlabelled water. This permeability is often called the diffusional water permeability, Pd. The major difficulty with this method is that the influx is so great that 70–90% of the tracer arriving in the blood enters the parenchyma in a single pass (see chapter 4 in Bradbury [55]  and [283–289]). Thus along much of the length of the microvessels the concentration gradient of the tracer across the microvessel walls driving its influx is much less than the concentration that was added to the blood. The permeability calculated from Eq. 7 using the arterial concentration of the tracer thus seriously underestimates the true water permeability of the blood–brain barrier. Mathematical expressions to correct for this effect have been derived relating the fraction of the tracer extracted from the flow through the blood vessel to the PS product (reviewed in [159]). However, even after correction the calculated values are inaccurate when the extraction fraction is large. Paulson et al. [290] found values about 1/5th of the PS values calculated from osmotic flow as described below and similar values have been determined by others (see [159]).
The second method for measuring water permeability uses an osmotic gradient to generate a net flux, Jnet, of water across the barrier. In effect a water concentration gradient is produced by “diluting” or “concentrating” the water on one side by adding or removing solutes and the permeability is then calculated as8 Pw,osmotic=Jnet/Δcwith results close to 1.1 × 10−3 cm s−1 for both rats [291, 292] and humans [290]. (The original references and a recent review [4] can be consulted for the actual equations used which are based on arguments that avoid the rather woolly concepts of “diluting” and “concentrating” the water). Using S = 100 cm2 g−1, the value of the surface area of the microvessels employed in [290, 292], the permeability-area product, PS, i.e. the clearance, is ~ 0.11 mL g−1 s−1 = 6.7 mL g−1 min−1. Patlak and Paulson [293] have argued that for the blood–brain barrier the tracer value is likely to be a better estimate of the true water permeability because the measurement of osmotic permeability using a brief exposure to raised osmolality reflects partly water extraction from the endothelium rather than from the parenchyma. It is adequate for the present purpose to use the two estimates as brackets of the correct value.
Water influx and efflux across the human blood–brain barrier each amount to roughly 40,000 mol day−1. The difference between the influx and efflux is very much less. Not even the normal direction of the net flux of water across the blood–brain barrier is known with any certainty, partly because it is so small. The available evidence suggests that scaled for a human there is a net movement from blood to brain amounting perhaps to ~ 10 mol day−1 (see [4]). For comparison metabolic production of water within the brain is ~ 3.3 mol day−1 and the amount of water in the CSF produced by the choroid plexuses is ~ 28 mol day−1.

Carbon dioxide
It has long been known that CO2 crosses the blood–brain barrier sufficiently rapidly that its removal from the parenchyma is largely blood-flow limited (see Sect. 6.1), i.e. pCO2 in the venous effluent is closer to that within the parenchyma than to that in arterial blood. Rapid transfer between blood and brain has been confirmed directly by the observation that when CO2 labelled with the short-lived isotope 11C is added to arterial blood more than 70% is extracted from the cerebral blood flow in a single pass [294] (see Section 6.4.2 in [4] for further discussion).
A crude underestimate of the clearance for CO2 in humans can be calculated from the rate of CO2 production (in turn calculated from glucose and oxygen consumption) [295, 296], ~ 3.3 mol day−1, and the average difference in pCO2 between ISF and plasma along the length of the microvessels which must be less than the difference between the values in the parenchyma and arterial blood, ~ 8 mmHg [297]. 8 mmHg corresponds to a difference in free concentration of 0.24 mM [298] and thus the underestimate of the clearance for a 1400 g brain becomes9 CL>2200μmolmin-1/1400g/0.24μmolmL-1=6.5mLg-1min-1.
This is more than 5000 times larger than would be possible by perivascular clearance, which simply restates that the clearance of CO2 must be across the blood–brain barrier.

Glucose
Glucose and O2 are the most important substrates for brain energy metabolism. Glucose enters ISF across the blood–brain barrier via the more glycosylated form of a passive, selective carrier, GLUT1 (SLC2A1), that is present in membranes located on both surfaces of the endothelial cells. From ISF it rapidly enters both astrocytes by the less glycosylated form of GLUT1 and neurons via GLUT3 (see Fig. 11). The rate-limiting step in glucose metabolism is the effectively irreversible phosphorylation by hexokinase. Normally glucose influx into the parenchyma is higher than the rate of phosphorylation, and thus there must be some efflux corresponding to the difference. This efflux is also primarily across the blood–brain barrier via GLUT1. Because both influx and efflux of glucose take place by passive transport there is no additional metabolic cost caused by having influx greater than the metabolic rate.
Fig. 11 GLUT and MCT transporters at or near the blood–brain barrier. GLUT1 and MCT1 are present on endothelial cells; GLUT1 and MCT4 on astrocytes
(Figure simplified and redrawn from Simpson et al. [315])
It has long been known that glucose is able to cross the blood–brain barrier rapidly [189, 299–302]. Crone [299] found that at low concentrations as much as 50% of the glucose arriving in the arterial blood could be extracted in a single pass, but that this percentage decreased with concentration, falling to 28% at 5 mM and ~ 14% at 14 mM. This extensive but saturable transport implies the presence of a specific transporter, which as stated above is GLUT1 (SLC2A1) [303–305].
The expression of GLUT1 in the endothelial cell membranes has been measured in several different ways: by cytochalasin-B binding, by specific antibody binding, and by proteomic methods (see Table 2 for references). In the proteomic studies from the group of Terasaki, Uchida, Ohtsuki and colleagues, GLUT1 was found to be the most highly expressed of all the transporters that are present in the membranes of the endothelial cells [306].
Table 2 Expression of GLUT1 at the blood–brain barrier
Source Methoda Species Expression/pmol mg−1
Luminal Totalb Abluminal
Relative to microvessel protein
Dick et al. [303] Cytochalasin B binding Rat, pig 69–80
Kalaria et al. [571] Cytochalasin B binding Human 42
Farrell and Pardridge [572] Immunogold e.m Rat 12%c 100%c 48%c
Cornford et al. [573] Immunogold e.m Human 48% 100% 18%
Vannucci et al. [574] Cytochalasin B binding Rat 40–125
Kamiie et al. [182] Proteomics Mouse 90
Uchida et al. [184] Proteomics Human 139
Shawahna et al. [176] Proteomics Human 78.5
Hoshi et al. [185] Lysate digestion proteomics Rat 84–98
Relative to membrane protein
Simpson et al. [575] Fractionation, cytochalasin B binding Bovine 620 280
Kubo et al. [58] Fractionation, proteomics Porcine 79% 21%
Zhang et al. [188] Proteomics Porcine 300
aFractionation = fractionation of isolated plasma membranes
bFor immunogold detection, values are percentages of the immunogold particles where the total includes cytoplasmic
cAntigen for the antibody used by Farrell and Pardridge appears to be partially masked for GLUT1 most markedly in the luminal membrane in bovine endothelial cells [575]
A rough estimate of the glucose clearance in man can be calculated from the rate of consumption, about 0.55 mol day−1 = 380 µmol min−1 [295, 296] or, for a 1400 g brain, 270 nmol g−1 min−1. For a difference between the concentrations in plasma and ISF of 5 mM this corresponds to CL ~ 54 µL g−1 min−1. In isolated perfused dog brains Betz et al. [302] measured the loss of glucose from the blood flow through the brain and found about 0.6 µmol g−1 min−1 at 6 mM from which at this concentration CL = 100 µL g−1 min−1. Hawkins [307] lists values ranging from 158 to 352 µL g−1 min−1 (at 6 mM glucose) depending on brain region (inferior colliculus the highest). Note that the first two of the estimates above are based on the net flux of glucose while the values listed by Hawkins are based on the unidirectional influx. Because all of these estimates far exceed the clearance expected for perivascular efflux, ~ 1 µL g−1 min−1 (see Sect. 3 and Table 1), the perivascular route is likely to be of minor importance.
Cutler and Sipe [301] using anaesthetized cats, Bachelard et al. [308, 309] using anaesthetized rats and Betz et al. [302] using isolated perfused dog brains all found that the influx of glucose measured using tracers could exceed the net flux by two to threefold. This is a direct, experimental demonstration that there is efflux across the blood–brain barrier that can be as large as two-thirds of the influx. This would of course be less under conditions of increased metabolic demand.
Glucose distributes rapidly between intracellular and extracellular water within the parenchyma and thus its volume of distribution is close to the total aqueous volume, which is VD = 0. 77 mL g−1 [310–315].14 Pfeuffer et al. [316] used diffusion weighted NMR to distinguish between intracellular and extracellular glucose and found that only 19% of the glucose in the parenchyma was extracellular which is in agreement with the fraction of water that is extracellular. These observations imply that glucose transport across the membranes of astrocytes and neurons is rapid compared to the rate of metabolism.
When glucose concentrations in plasma are near 6 mM, the average concentration of glucose in brain water is roughly 1.3 mM (see Sect. 5.3.2). Even two fold changes in the concentration in brain water have little effect on the cerebral metabolic rate of glucose, CMRglc, because these concentrations are substantially greater than the Km for phosphorylation of glucose by hexokinase (0.04–0.05 mM [317–319]) and hence hexokinase, the first step in glucose metabolism, remains nearly saturated (compare e.g. [313]).
It is unclear why the passive glucose transport at the blood–brain barrier is mediated by a carrier rather than by a pore. Pores have the advantage that they do not undergo any large conformation changes during transport of each substrate. Hence they are capable of high turnover numbers, which would seem to be an advantage. On the other hand carriers allow more complicated coupling of transport between different solutes and it is possible that during transport of a relative large solute like glucose, it is easier for a carrier than the “open hole” of a pore to prevent unwanted transfer of other solutes. (Water can probably get through both carriers and pores. The possibility that water permeability of GLUT1 may or may not be important at the blood–brain barrier [320] was considered in Section 4.3.6, footnote 17 of [4]). While arguments for “why a carrier” are speculative, the structural and kinetic evidence, reviewed in the following subsections, leave little doubt but that glucose transport across the membranes of the endothelial cells of the blood–brain barrier is mediated by a carrier.

Structure of GLUT1 (SLC2A1) and the kinetics of the glucose transport it mediates in red blood cells
A crystal structure for GLUT1 has been obtained using a GLUT1 construct purified from an expression system (see Fig. 12) [321]. In this structure a bundle of α-helices spans the membrane surrounding an inner cavity open at the cytoplasmic end. This structure and those for related transporters (for references see [322]) strongly support the widely held view that the transport kinetics should be described using a carrier model (see Appendix D). A binding site in the central cavity of the carrier can be exposed to either side of the membrane, but only one side at a time. While the site is exposed a substrate molecule can associate with or dissociate from the site. The side of exposure can be altered by a conformation change in the carrier and the substrate can then associate or dissociate on the other side of the membrane.
Fig. 12 Structure of the human glucose transporter GLUT1. The structure of full-length human GLUT1 containing two point mutations (N45T, E329Q) was determined in an inward-open conformation. The side and cytoplasmic views are shown. The corresponding transmembrane segments in the four 3-helix repeats are coloured the same. The extracellular and intracellular helices are coloured blue and orange, respectively. A slab of cut- open view of the surface electrostatic potential, which was calculated with PyMol50, is shown on the right to facilitate visualization of the inward-facing cavity. IC indicates intracellular helix. Reprinted by permission from Springer Nature from Nature 510, 121–126, Crystal structure of the human glucose transporter GLUT1 by Deng et al. [321]
Since GLUT1 is highly expressed in red blood cells, they have been used as the most convenient system in which to study the kinetics of its transport. There are two prominent features revealed by these studies that must be accommodated in any model. On the one hand the normal net transport of glucose occurs without input of energy from any source other than the concentration gradient, on the other hand downhill movement of one type of sugar can be coupled to uphill movement of another (see Fig. 13), a phenomenon called counter-flow or counter-transport [322–325]. A closely related phenomenon is trans-stimulation, an increase in influx when internal concentration is increased or an increase in efflux when external concentration is increased (see Fig. 13 and, for a quantitative example, Appendix D). In terms of a simple carrier model, the observation of net glucose transport when it is the only substrate implies that both the loaded and unloaded forms of the carrier can change conformation thus altering exposure of the binding site. This allows transport of solute in one direction to occur without transport in the opposite direction, i.e. the transport is not an obligatory exchange. Similarly counter-transport or trans-stimulation imply that the rate constants for the conformation changes when the carrier is loaded are at least comparable to those for the unloaded carrier so that solute on the trans side can assist transport from the cis side by increasing the rate of return of the carrier.
Fig. 13 Interpretation of net flux of a single solute, obligatory exchange, and trans-stimulation in terms of a simple carrier model. In each case the concentration of the first solute (filled black circle) is higher on the cis side (left) than on the trans side (right). a Net flux of solute from cis to trans is supported by return of the free carrier. b If return of the carrier is only possible with a solute bound, there is obligatory exchange, either self-exchange or counter-transport of another solute (circle). c Trans-stimulation is a combination of these two effects. Flux of the first solute from cis to trans can be increased if there is more solute (either sort) on the trans side (here the right) provided that increases the rate of return of the carrier—i.e. it increases the rate of conformation changes of the carrier from trans-facing to cis-facing
Trans-stimulation can markedly increase influx and efflux of glucose at high glucose concentrations (see Appendix D) and it is therefore very important in studies of the mechanism of transport. However, it has little if any effect on the net flux and it is the net flux that is important for the delivery of glucose for metabolism. The exchanges underlying trans-stimulation are likely to be much more important for large neutral amino acids where several compete for transport by the same carrier (see Sect. 5.5).
The kinetics of the simple carrier model are complex even in the steady-state [325–329]. GLUT1 (SLC2A1) kinetics are complicated further by the added twist that the GLUT1 protein may exist in the membranes as part of a homo-tetramer, each capable of transport, but in a coupled manner such that transport through one affects the transport through the others [322, 330]. Given these and further complexities considered in the next section, it should not be surprising that definitive characterization of glucose transport at the blood–brain barrier remains elusive (see Appendix D).

Glucose transport kinetics at the blood–brain barrier
Transport of glucose into and out of the brain is clearly more complex than that into and out of red blood cells. Firstly GLUT1 is needed in both membranes of the endothelial cells of the blood–brain barrier to allow the glucose to enter on one side and leave on the other. However, because the endothelial cells are very thin and correspondingly contain very little glucose, provided that the properties of the transport in the two membranes are similar, it is thought that the transport can still be described, at least qualitatively, as transport across a single barrier [331–333]. Secondly once across the blood–brain barrier, glucose is metabolized at a rate comparable to the rates of influx and efflux across the barrier while in red blood cells transport is much faster than metabolism. Thirdly there is also the technical difficulty that, with the important exception of the study in 1975 by Betz et al. [327], it has not proved possible to manipulate interstitial fluid glucose concentrations during the experiments. In most studies all that has been done is either to measure the extraction of glucose (total or labelled) from blood as described above or to measure the variation in the total amount of glucose present in the parenchyma with time as a function of glucose concentration in plasma. Mason et al. [334] compare the results obtained in many studies performed prior to 1992 but with the surprising omission of reference to studies from Betz’s group. Also in 1992, Gjedde [335] reviewed results obtained for glucose transport in rat and man. Glucose transport into and within the brain has been analyzed and reviewed by Simpson et al. [315], Barros et al. [314] and, more recently, by Patching [336].
In one of the first attempts to establish the mechanism of glucose transport at the blood–brain barrier, Buschiazzo et al. [319] found that 3-O-methyl-d-glucose, a non-metabolizable derivative of glucose, competes with glucose for transport, and furthermore that an inward gradient of glucose could drive 3-O-methyl-d-glucose uphill out of the brain, i.e. there is counter-transport for GLUT1 at the blood–brain barrier just as in red blood cells. Further evidence that GLUT1 behaves in a similar manner in the two environments was obtained by Betz et al. [327] who found that the rate of glucose influx was increased by increasing the concentration of glucose within the brain, i.e. there is trans-stimulation (see Appendix D).
Buschiazzo et al. [319] and Betz et al. [327] determined the total glucose in the parenchyma for different glucose concentrations in plasma (see Fig. 14). Subsequently NMR has been used to measure glucose content in conscious humans and lightly anaesthetized rats [334, 337–341]. The NMR results for humans and rats confirm under nearly physiological conditions (see Fig. 14) that brain glucose content continues to increase with plasma concentration for plasma concentrations up to at least 30 mM well above a typical resting value, 6 mM. They also confirm that the rates of glucose influx and efflux are respectively larger than and not much smaller than the rate of metabolism. Because influx and efflux substantially exceed the expected efflux via the perivascular route, the net flux across the blood–brain barrier is normally taken to be equal to CMRglc at steady-state.
Fig. 14 Four studies of brain glucose content versus glucose concentration in blood. In two studies glucose content was measured by chemical assay, a in anaesthetized rats by Buschiazzo et al. [319] and b in isolated perfused brains from dogs by Betz et al. [327]. In the latter it was assumed that brain water was 0.75 mL g−1. In the other two studies glucose content was determined by magnetic resonance spectroscopy, c in conscious humans by Gruetter et al. [337] and d in lightly anaesthetized rats by Choi et al. [338]. In all studies the glucose content continues to increase with plasma concentration even though it is known that the influx of glucose shows saturation. The explanation is that efflux also saturates and the increase in content must parallel the increase in plasma concentration in order for efflux to increase so that it is equal to influx minus the constant rate of glucose metabolism (see Appendix D)
In the results reported by Duarte et al. (see Figure 3 in [341]) using rats, following a step change in cplasma from 4 to 20 mM the brain content of glucose increased from about 0.5 to 4.5 µmol g−1 with a half life of about 16 min which indicates a net rate of accumulation of 0.122 µmol g−1 min−1, i.e. using their value of CMRglc, 0.52 µmol g−1 min−1, there is an influx of 0.64 µmol g−1 min−1 which is similar to that reported by Betz et al. in 1974 [302] for the dog.
It has so far not proved possible to analyse glucose efflux directly after injection of glucose into the brain. Any such measurements face major challenges including separating efflux from metabolism and avoiding disturbance of the efflux processes by the injection or infusion. The study by Ball et al. [85] established that during a 5 min, 0.1 µL min−1 infusion into the inferior colliculus glucose can move, presumably by a perivascular route, to the adjacent meninges strongly suggesting that as expected there is perivascular efflux of glucose. However, estimating the normal rate of this process to see if the perivascular clearance notably exceeds the 1 µL g−1 min−1 found in other regions would require measurement of the time course of the appearance of glucose in the meninges after the end of the infusion.15
The glucose efflux across the blood–brain barrier can be calculated if the influx and net flux are both known as indicated earlier in this discussion of glucose. Furthermore if it can be assumed that the fluxes are described by the expressions of the form derived from the carrier model, the rate of efflux can be calculated from the measured rates of influx versus the concentrations in plasma and ISF. An example of this using the data from Betz et al. [327] for the isolated perfused dog brain is given in Appendix D and Additional file 1. These data remain the only measurements of glucose influx versus plasma concentration for a range of known concentrations within the brain. Hence the calculated results in Appendix D are the only available results for efflux as a function of both plasma and ISF concentrations.
The fits to the data of Betz et al. [327] (see Additional file 1) indicate that the net flux = CMRglc for cplasma = 6 mM is 0.65 µmol g−1 min−1 with cisf = 1.2 mM. This value of CMRglc is close to those expected for rats but about twice that for humans. The fits also predict that glucose consumption, CMRglc, could increase to about 0.9 µmol g−1 min−1 with cisf approaching 0 without any change in transport capacity. However, larger increases in glucose consumption are required in order to support nervous activity. Changes in transport capacity are considered in Sect. 6.2.
Both neurons and astrocytes have transporters that will allow uptake of glucose and both can use it as a substrate for energy production. The proportions of glucose metabolism that occur in astrocytes and neurons remain controversial [315, 342–346] (see next section).

Lactate
When at rest and even more during nervous activity, there is net production of lactate within the brain parenchyma and thus there must be means for its efflux. Clearance of lactate from the brain has recently been reviewed in some detail [146] (see also footnote 26 in [4]). In brief lactic acid is transported across the blood–brain barrier by passive transport mediated by MCT1 (SLC16A1) present in both luminal and abluminal membranes. Lactate both enters and leaves the brain by this route. Lactate is generated within the brain by partial metabolism of glucose and by metabolism of glutamate [347, 348]). Under resting conditions when lactate concentrations are low, the clearance, CL = PS ~ 60–100 µL g−1 min−1 [349–352], far exceeds the expected clearance, ~ 1 µL g−1 min−1, by a strictly perivascular route.
It is often said that transport of lactate across the blood–brain barrier is slow (see e.g. Pardridge’s account [189]). But these statements refer to the amounts transported not the permeability. The lactate clearance (= PS product) calculated for low concentrations from the kinetic constants that Pardridge presents, Kt = 1.8 mM and Tmax = 91 nmol g−1 min−1, is 50 µL g−1 min−1, close to that stated above. Quistorff et al. [353] and Boumezbeur et al. [352] have emphasized that lactate from the periphery can be an important source of energy in the brain during heavy exercise.
There is clear evidence that during periods of increased neural activity the blood–brain barrier is not the only route of lactate removal from the sites of activity [354–357]. This may be particularly important in circumstances where the lactate concentration is also increased in the rest of the body, e.g. as a result of physical exercise. Under these circumstances the net transport across the blood–brain barrier is likely to be inwards [352, 353]. Other routes for efflux cannot be just perivascular transport as seen with inulin because that isn’t fast enough. One suggested explanation is perivascular transport augmented by transfer between astrocyte endfeet via gap junctions. This can lead to movement of lactate from sites of activity either to inactive regions or to perivascular spaces of larger blood vessels [356–358] (see Fig. 15). Much of the lactate removed from the parenchyma via perivascular transport is likely to be removed from the brain along with CSF, though a proportion reaches lymph, possibly via the meninges, without first mixing with CSF. Lactate in CSF that leaves via the cribriform plate is delivered to the nasal mucosa from which it may return to blood either indirectly via lymph or directly by crossing peripheral capillary walls [85, 120, 125].16
Fig. 15 Lactate removal from the brain. Lactate produced within the brain can be effluxed via the blood–brain barrier or via perivascular routes. It may reach the latter locally near the site of its production or at more distant sites having been transferred between astrocytes via gap junctions
(Diagram modified from Figure 7c in Gandhi et al. [358])
It remains puzzling why so much of the lactate produced within the brain during nerve activity appears to be removed rather than serving as fuel for oxidation in neurons as proposed in the astrocyte neuron lactate shuttle (ANLS) hypothesis (G. A. Dienel, personal communication). However, at least according to Dienel [345] the available evidence is that the oxygen consumption does not increase sufficiently during nerve activity for shuttling of lactate from astrocytes to neurons and further oxidative metabolism of lactate in neurons to be an important mechanism. Furthermore using expression of a genetically encoded NAD sensor that can be monitored in real time with cellular resolution, Diaz-Garcia et al. [346] have found in mice that nervous activity induces neural production rather than consumption of lactate. For an alternative view see e.g. [344].

Amino acids
In order to put the importance of efflux of amino acids from brain parenchyma into context, it is necessary to consider not just the fluxes and transporters but also the need for fluxes.
Amino acids are required within the brain for protein synthesis (see Fig. 16) and for maintenance of pools of neurotransmitters, in particular glutamate and GABA (see Fig. 17). Amino acids are also needed for synthesis of many other substances, e.g. nucleosides, but when considering overall balance this demand has usually been ignored as being relatively minor and it will not be considered further here (compare [359]). The required amino acids must either be synthesized inside the brain or enter from outside primarily across the blood–brain barrier.
Fig. 16 Simplified overview of fates of amino acids in the brain parenchyma. Essential amino acids enter and leave the parenchyma across the blood–brain barrier. Non-essential amino-acids, e.g. glutamate (Glu), glutamine (Gln), and GABA can be synthesized within the brain. The amino groups for the synthesis are supplied either by transamination as shown for glutamate or to some extent [359] by incorporation of NH4+ by glutamate dehydrogenase. The latter route is believed to be minor [359, 641]. NH4+ is added to form the amide group of glutamine by glutamine synthetase (see Fig. 17). Within the parenchyma amino acids are used for synthesis of proteins and (not shown) formation of other nitrogen containing compounds, e.g. nucleotides. New amino acids must be supplied to replace those lost by metabolism. In the brain, input of amino acids is also required to provide amino groups to replace glutamate lost from the pool of amino acids involved in glutaminergic (and GABAergic, not shown) neurotransmission (see Fig. 17). α-KG α-ketoglutarate, e.a.a essential amino acids, t.a transaminase
Fig. 17 The glutamate/glutamine cycle shown in bold with indication of some of the losses and of replenishment of glutamate by denovo synthesis. Glutamate (Glu) in the presynaptic neuron is packaged into vesicles and released into the ISF during neurotransmission. Most of the glutamate is taken up into the astrocytes by the transporter Eaat1 (glast, Slc1a3) where it is converted to glutamine (Gln) by addition of an NH4+ by the enzyme glutamine synthase (g.s) [642, 643]. The glutamine is transported into the ISF by Snat3 and/or Snat5 (Slc38a3 and Slc38a5) from which it is taken up into the presynaptic terminals again by a transporter that may be a Snat. The glutamate is then regenerated by glutaminase (g.a). This cycle represents a large turnover of the amide group at the end of the side chain in glutamine, estimated to be 55% of the CMRglc (cerebral metabolic rate of glucose, see Sect. 5.3) for the entire brain in rats amounting to 490 nmol g−1 min−1 (estimated value in humans 280 nmol g−1 min−1) [644] (G. A. Dienel, personal communication). However the requirement for NH4+ consumed in the conversion of glutamate to glutamine within the astrocytes is balanced by an equal release of NH4+ in the reverse conversion in neurons. Whether diffusion of NH4+ itself is adequate to transfer the nitrogen from neurons to astrocytes as shown or some other form of N carrier is required remains controversial [385, 641, 645]. Regardless, if there were no losses of glutamine or glutamate from the cycle, there would be no need for any fluxes of amino acids into or out of the parenchyma to support glutaminergic neurotransmission. However, there are losses of glutamate and glutamine from the cycle [347, 646, 647]. At least in rodents, such losses are made good by de novo synthesis of glutamate in the astrocytes. Estimates of the total rate of loss and of de novo synthesis are around 11% of CMRglc ([648, 649] (G. A. Dienel, personal communication), i.e. about 0.11 × 0.9 µmol g−1 min−1 ≅ 100 nmol g−1 min−1. The carbon skeletons for the de novo synthesis are derived ultimately from glucose. Glucose is metabolized to two molecules of pyruvate one of which is carboxylated by pyruvate carboxylase (p.c) (thought to be present within the brain only in astrocytes) to form oxaloacetic acid (OAA) a component of the citric acid cycle. Addition of acetyl-CoA from the second pyruvate then forms citrate, which is decarboxylated to form α-ketoglutarate (α-KG). Glutamate is then formed either a by transamination (t.a) of α-ketoglutarate using leucine or other amino acids as source (see e.g. [383, 641, 645, 650], or b by addition of NH4+ [366] catalyzed by glutamate dehydrogenase (g.d). The latter is believed to be a minor pathway [359, 366, 641]. The source of the amino groups for transamination is considered further in Sect. 5.5.3 and Fig. 18. Data for the pathways involved in glutamate synthesis are much less extensive for human than for rat. Rothman and colleagues [651, 652] have argued that the α-ketoglutarate is synthesized in astrocytes based on measurements of incorporation of 13C (see [653, 654]). However, the failure to find a key transaminase in human astrocytes by immunohistochemistry [655, 656] has cast some doubt on astrocytes being the major site for the conversion from α-ketoglutarate to glutamate. For recent reviews of glutamate synthesis see [386, 641, 645]
The need for amino acid input is different from the need for glucose input. Glucose is the basic fuel consumed in metabolism and must be supplied continually in large quantities. Amino acids are needed to allow the maintenance of cell structure and composition. But, the N containing constituents of the cells either are not consumed during metabolism or if they are they are partly replaced internally. The balance between influx and efflux across the blood–brain barrier need only provide sufficient amounts of amino acids to top up losses. Any metabolic losses that do occur will either be by efflux from the brain or by generation of NH4+ and carbon compounds. The latter become part of the carbon metabolism of cells. Possible fates of the NH4+ include: diffusion across the blood–brain barrier; reaction with glutamate to form glutamine, which is then exported from the parenchyma; and use in amino acid synthesis [359, 360]. Glutamate synthesis is considered further in Sect. 5.5.5.
For each amino acid at steady-state, its net fluxes across the blood–brain barrier and via perivascular routes and its net rate of synthesis must add to zero so that the concentrations in the brain parenchyma can remain constant. However, there are major complications in applying this principle to the interpretation of data: there are more than 20 different amino-acids, inter-conversions between them by transamination are common, and they compete with each other for the many amino acid transporters. Indeed the major application of this principle comes when considering overall N balance.
Allowing the fluxes that are required (see Sect. 5.5.1) while maintaining ISF concentrations of all amino acids except glutamine well below those in plasma (see Sect. 5.5.2) is a major challenge and it is not yet certain how the available transporters (see Sect. 5.5.4) achieve these objectives.

Requirements for amino acid fluxes (and NH4+)
While it is clear that there are losses of essential amino acids from brain parenchyma and thus that some influx of amino acids must occur, it is difficult to obtain a quantitative estimate of the influx required. Using radiolabelled amino acids in rats, Dunlop et al. [361–363] found a turnover rate for the protein content of rat brains to be about 0.6% h−1. Using a protein content of about 100 mg for each gram of brain and the molecular weight of an average amino acid, perhaps 125 Da, that corresponds to a rate of incorporation of amino acids of about 80 nmol g−1 min−1. Similarly amino groups required for de novo synthesis of glutamate amount to about 100 nmol g−1 min−1 (see legend to Fig. 17).
Many of the amino acids needed for protein synthesis are supplied either by de novo synthesis (which, however, still requires some source of amino groups, see Fig. 16) or by recycling those released during protein breakdown, which averaged over enough time must be occurring at the same rate as synthesis. Furthermore it may be possible to reuse some of the NH4+ lost from the glutamate/glutamine cycle in the de novo synthesis of glutamate. Thus the sum of the estimates above, 180 nmol g−1 min−1, is likely to exceed the actual requirement for amino-acid input.
Because the brain parenchyma must be in N balance and there must be net inputs of essential amino acids, there must also be a route or routes for N removal. As the brain normally doesn’t produce urea as a means of disposing of NH4+ [364–366], the two main routes for exit to be considered are efflux of NH4+ and efflux of glutamine. Fluxes of NH4+ are easily demonstrated to occur in both directions across the blood–brain barrier and are almost certainly by diffusion across the membranes of NH3 combined with transport either of H+ in the same direction or, more likely, of HCO3− in the opposite direction [4, 359, 367]. Because concentrations of NH4+ in brain, 150–300 µM, and CSF, 100–300 µM, normally exceed those in arterial plasma, 50–250 µM [359], it is likely that there is some net NH4+ efflux. However, an arterio-venous difference in NH4+ concentration and thus its net transport have only been demonstrated in the brain when plasma NH4+ concentration is raised as in hepatic insufficiency [359, 368]. There is then net NH4+ entry, rapid incorporation of the NH4+ into glutamine by reaction with glutamate [359], and efflux of the resultant glutamine. Glutamine efflux is considered further in Sect. 5.5.4.
Lee et al. [360] made the interesting suggestion that much of the NH4+ that moves from brain microvascular endothelial cells to plasma is produced within the endothelial cells by glutaminase acting on glutamine. However, that taken alone would suggest that there should also be a substantial efflux of glutamate, which has not been observed. Alternatively the NH4+ effluxed may derive from metabolism of both glutamine and glutamate. This is considered further in Sect. 5.5.4.

Concentrations of amino acids in CSF and ISF
Values of amino acid concentrations measured in blood plasma, CSF and ISF are summarized in Table 3. There is agreement in all studies that, with the exception of glutamine, the concentrations of all other amino acids in CSF and ISF are substantially less than those in plasma. This could arise if the rates of consumption were to reduce the concentrations greatly or if there were active transport of amino acids from brain fluids to blood. Whether or not there is a substantial difference in amino acid concentrations between CSF and ISF is less clear.17
Table 3 Amino acid concentrations in plasma, CSF and ISF
Plasma concentration/µM CSF concentration/µM ISF concentration/µM
Human1 Rat2 Human3 Rabbit4 Rat5 Mice6 Human1 Human7 Rat2, c Rat1, 2 Human3 Rabbit4 Rat5 Mice6 Rabbit4 Rabbit8 Rabbit9 Rat5 Mice6
Gln 619 641 868 834 598 863 780 552 583 524 517 250 547 159 193 80
Asn 112 55 Trace 14 16 3 7.8 4.3 1.4
Ala 330 489a 382 430 408 27 18.6 34a 39 85.4 57 15 19 15 7.7 9.3
Ser 149 196 140 263 247 109 28.9 30.4 79 80 30 116 62.3 37 34 24.9 9.8
Gly 249 221 283 246 1.3 6.4 20 15 6 34 7.8
Pro 212 60 Trace 0.6 4.2 1.8
Thr 142 166 113 28.5 27.7 36 22 3.5
Val 222 430 309 191 173 14 16.1 78 47 20 14 9.4 13 12.5 7.4 2.9
Leu 109 432 155 81 127 13.1 11.9 66 42 15 7 6.6 9 1.5
Ile 61 77 101 5.2 4.7 6 7 9
Tyr 70 73 43 54 7.3 8.1 10 7 7.2 5 0.7
His 85 80 59 10.8 12.0 12 8.9 1.2
Lys 158 290 171 137 321 19.7 22.0 120 87 21 8 46 19 10
Arg 80 94 81 227 103 17 18.6 55 46 22 32.5 18 6.9 3.2
Glu 83 61 56 159.6 37 7.2 8.7 26 10 11.4 21 4 4.3 3.4 2.9 4.1
Asp 7 33.8 2.4 1.5 5.8 0.6 0.5 1.7
Phe 71 64 45 8.3 8.2 10 7 6
Met 41 28 30 2.8 2.6 3 3
Trp 62
Concentrations measured in CSF are, with the exception of glutamine always substantially less, than the concentrations in plasma. Concentrations in ISF are measured by microdialysis with extrapolation to zero flow (see text). If these are correct, ISF concentrations are substantially lower than those in CSF. c cisternal, l lumbar
1Plum et al. [576]
2Franklin et al. [577]
3McGale et al. [578]
4Hamberger et al. [579]
5Lerma et al. [580]
6Dolgodilina et al. [581]
7Table 8.15 in Davson and Segal [56]
8Jacobson et al. [582] microdialysis by concentration profile
9Jacobson et al. [582] microdialysis by recovery of samples

The relative importance of perivascular supply and removal for amino acid turnover in ISF
Excluding glutamine, concentrations of each of the amino acids in CSF and ISF are usually < 1/5th of those in plasma (see below) and in total < 1 mM. With a perivascular clearance of 1 µL g−1 min−1, and an amino acid concentration at the high end of the observed range, 100 µM, the rate of loss or gain of any particular amino acid by the perivascular route is expected to be of the order of 0.1 nmol g−1 min−1 or less, which is likely to be negligible. Amino acid loss from the brain by outflow of CSF at 0.25 µL g−1 min−1 (500 mL day−1 for a 1400 g brain) at 100 µM would be 0.025 nmol g−1 min−1 which again is likely to be negligible.

Observed fluxes of amino acids
Quantitative measurements of fluxes of amino acids have been either of influx or net flux. Influx is measured by adding a tracer to the blood perfusing the brain and measuring the amount that enters the brain over a short period. Net flux of an amino acid is calculated as10 net flux=A-V difference×blood flowby using measurements of the blood flow and the A − V difference equal to the difference between the concentrations in arterial blood entering and venous blood leaving the brain. Direct measurements of efflux have proved difficult. In practice efflux into the blood has been calculated as the difference between influx and net flux from the blood.
Influx of amino acids into brain parenchyma across the blood–brain barrier has been studied in rats. In a highly influential early study, rates were compared to that for water using 14C-labeled amino acids and 3HOH added together as a single bolus arterial injection. The results were reported as the brain uptake index (BUI), defined as a ratio of ratios ((uptake of 14C-aa)/[14C-aa])/((uptake of 3HOH)/[3HOH]) [300]. When added one at a time, the influxes of the amino acids varied greatly, with BUI for phenylalanine or leucine found to be more than 50% (i.e. each enters about half as easily as water) while at the other extreme influxes of proline, glutamate, asparagine and glycine were below the background limit of detection by the technique, BUI < ~ 3%. Influx of each of the essential amino acids (those not able to be formed within the brain) was easily measurable.
All of the influxes that were clearly above baseline were inhibited when the radiolabeled amino acids were added using serum rather than a simple buffer suggesting competition for transport with the amino acids present in serum. Competition was investigated further and confirmed by measuring uptake of tracer in the presence of an excess of individual unlabelled amino acids [300].18 Quantitative estimates of the influxes of various amino acids in rats when plasma concentrations of tracers were held constant by controlled infusions [369] or during perfusion of isolated brains [43, 370, 371] have confirmed the pattern seen using BUI measurements [300, 372] (see Table 4).
Table 4 Influx and net flux of amino acids in the indicated species
Rat1 Rat2 Rat3 Rat4 Rat5 Rat6 Dog7 Dog7 Cat8 Sheep9 Human10 Human11 Human12 Human13 Human14
Influx* Influx Influx Influx Influx Net Net* Net* Net Net Net Net Net Net Net
Phenylalanine 6.5 7 8 13.2 − 0.6 0.12 0.01 2.2 0.06 0.3 − 0.5
Leucine 6.2 15 14.5  9.7 1.56 1.28 1.9 6 5.2 3.2 2.2 0.6
Isoleucine 1.8 4  5.7 0.78 0.46 1.5 3.1 1.2 1.4 0.7 0.3
Valine 1.3 1.8  2.9 1.26 0.37 2.7 11.0 1.7 0.8 − 1.7
Tyrosine 5.3 7 4.1 − 1.1 0 0.06 1.6 0.1 − 0.1 − 0.1
Methionine 1.6 1.7 − 1.1 0 − 0.12 1.0 0.6 0.2 0.2
Tryptophan − 1.1
Histidine 2.5 2.5 − 1.7 − 0.23 − 0.7 0.7 − 0.3 − 1.3
threonine 1.2 0.8 − 3.4 0.62 − 0.1 5.0 2.6 0.1
Arginine 1 − 7.4 − 0.2 − 0.05 2 0.7 − 0.5 − 0.1
Lysine 6.2 9 − 1.1 0.58 − 1.6 2.5 − 0.3 − 0.9
Glutamate  1.7 − 0.24 − 0.18 − 0.05 1.4 − 0.5 − 6
Glutaminea 11.6 1 − 15.4 − 6.6 − 4.3 − 1.94 − 7.4 11.0 − 20 − 20.4
nerv. stim.b glutamate − 1.26
nerv. stim.b glutamine − 6.69
Values are stated in nmol g−1 min−1. Influxes have been measured only in rats. The only available measurements of net fluxes in rats, shown in the italicized column, failed to reach statistical significance
* Data used by Pardridge [379] in his comparison of influxes and net fluxes
aIn some columns the values are for glutamine + asparagine as the assay used detected both
bRelease measured during nervous stimulation
1Banos et al. [369]
2Hawkins et al. [583]
3Mans et al. [584]
4Ennis et al. [415]
5Smith and Stoll [43]
6Brosnan et al. [378]
7Betz and Gilboe [365]
8Abdul-Ghani et al. [394]
9Pell and Bergman [585]
10Felig et al. [586]
11Lying-Tunell et al. [587]
12Eriksson et al. [588]
13Grill et al. [589]
14Strauss et al. [590]
From the patterns of competition between amino acids for influx across the blood–brain barrier it appeared that there were four separate systems of transport (see e.g. [43, 44]).System L primarily for neutral amino acids, which can be inhibited by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH);
System ASC primarily for neutral amino acids, which is not inhibited by BCH;
System y+ (sometimes called system Lys+) primarily for basic amino acids;
System N primarily for the nitrogen-rich amino acids glutamine, histidine and asparagine.
A number of amino acids fit into more than one of these groups. Most of the amino acids with large BUI values are substrates for system L.
Studies with isolated brain microvessels, which provide access to the abluminal membranes of the endothelial cells, identified two more systems.A Na+-linked transport system for small neutral amino acids (system A, with identifying substrate N-methyl-a-aminoisobutyric acid, MeAIB) [373].
Another system for glutamate [374].
The ability to prepare vesicles enriched in membranes from either the luminal or abluminal membranes of the endothelial cells [375] allowed localization of transport activities to the separate membranes with the generalization (since revised, see Sect. 5.5.6) that transporters in the luminal membrane are not Na+-linked and hence bidirectional while those in the abluminal membrane are Na+-linked favouring transport from ISF into the endothelial cells [44]. There are now known to be many more types of transporter present at the blood–brain barrier than initially suggested by identification of these systems (see Sect. 5.5.6).
Large rates of efflux of amino acids from CSF to blood were detected in cats [376] and rabbits [377] undergoing ventriculo-cisternal or ventriculo-cortical subarachnoid space perfusions. However, it was not possible in these studies to determine how much of the efflux was going via the choroid plexuses and how much via the parenchyma and the blood–brain barrier. Evidence that the latter route is important derives from the observation that transfer was much more rapid in ventriculo-subarachnoid infusion than in ventriculo-cisternal infusion. Both types of perfusion expose the infused fluid to the choroid plexuses, but in the former a much larger surface area of parenchyma is exposed to the fluid [376].
The net flux into a region can be calculated if the blood flow to that region and the concentrations of the solute in arterial blood and the venous outflow can be measured (see Sect. 5.5.6). (Equating net flux out of blood with net flux into the brain ignores possible metabolism within the endothelial cells, see the end of Sect. 5.5.6). Net flux measurements have been attempted using rats [378], but all except one of the A − V differences were not statistically significant. The rest of the net flux data in Table 4 are for larger species.
Pardridge [379] compared the influx data for rats obtained by Banos et al. [369] with the net flux data for dogs obtained by Betz et al. [365] (see Table 4) and noted that the net fluxes are much smaller than the unidirectional influxes. With the assumption that the fluxes are similar in various species, this comparison implies that there must be large effluxes, comparable in size to the influxes. Measurements of net fluxes in dogs, sheep, and humans have produced data broadly comparable with each other (see Table 4) favouring the assumption that when expressed per gram of tissue the fluxes are the same in all species.19
At present there are strong indications that the net flux of glutamine is outwards. This was seen in five out of six studies. There is also indication that the combined net flux of the branched chain amino acids, leucine + isoleucine + valine, is inwards. This was seen in six out of seven studies. But as described in the next section there is no evidence for a sufficiently large inwards net flux of neutral amino acids to provide for all of the transamination invoked in the explanations of glutamate turnover, at least in rats.

Observed fluxes of neutral amino acids compared with their requirement in glutamate synthesis
A major difficulty is revealed by comparison of the small net fluxes for the large, essential neutral amino acids and the large provision of these amino acids required for transamination to convert α-ketoglutarate into glutamate (see Figs. 16 and 17). For this requirement to be satisfied by influx across the blood–brain barrier of leucine, isoleucine and valine, their combined net influx would need to be > 100 nmol min−1 g−1 (see Sect. 5.5.1). For a cerebral blood flow of 0.57 mL min−1 g−1 (see e.g. Sect. 5.3) that would correspond to an A − V difference > 175 µM. Given that the total of the arterial plasma concentrations for these amino acids is only 392 µM (see Table 3), this A − V difference and hence net rate of transport should have been well above the “noise” in all of the studies, even that in rats (see Table 4).
If, as indicated by all available studies, sufficient net inward flux of amino acids does not in fact exist, the amino groups for synthesis of glutamate in the astrocytes must be obtained from sources within the brain. Independent evidence that such a source is available comes from studies comparing isotope dilution in the brain when plasma leucine was labeled with 13C or 15N. 62% of the N in brain leucine was derived from reverse transamination [380–383].
One detailed suggestion (see Fig. 18) is that loss of the branched chain α-ketoacids (BCKA), e.g. α-ketoisocaproate, generated in the transamination in the astrocytes is prevented by using a branched chain amino acid (BCAA) shuttle ([382, 384], reviewed in [385]). In this scheme instead of being further metabolized within the astrocytes as shown in Fig. 17, the BCKA are transferred to neurons where the branched chain amino acids (BCAA), e.g. leucine, can be regenerated by transamination from glutamate producing α-ketoglutarate. The leucine is then exported back to the astrocytes while the glutamate within the neuron is regenerated by glutamate dehydrogenase from NH4+ and the α-ketoglutarate [384, 386]. In this scheme NH4+ is taken from the neuron where it is released from glutamine and will be at relatively high concentration. This is shifted to the astrocyte by the BCAA shuttle where it can be combined with new α-ketoglutarate to complete the de novo synthesis of glutamate. This scheme greatly reduces the need for net flux of BCAA across the blood–brain barrier.
Fig. 18 The branched chain amino acid shuttle for provision of branched chain amino acids (BCAA) in the astrocytes to allow de novo synthesis of glutamate. Leucine (Leu) is used as example of a BCAA. α-KG α-ketoglutarate, α-KIC α-ketoisocaproic acid, Gln glutamine, Glu glutamate, g.a glutaminase, g.d glutamate dehydrogenase, g.s glutamine synthetase, t.a transaminase. Losses of Gln, primarily by efflux, and of Glc, primarily by catabolism are replaced by de novo synthesis of α-KG in astrocytes and transamination using Leu producing α-KIC. Leu is regenerated from α-KIC in the neuron by transamination from Glu producing α-KG. The Glu is in turn regenerated from the α-KG and NH4+ by gdh. Loss of N via efflux of Gln, Glu, and Leu is made good by net inward flux of Leu and NH4+. The BCAA shuttle greatly reduces the need for net inward flux of Leu as this is only required to make good the metabolic loss of α-KIC
(Based on Figure 1 in Hutson [384])

Amino acid transporters at the blood–brain barrier
The transporters currently thought to be involved in amino acid transport across the blood–brain barrier are indicated in Fig. 19. These will be discussed below according to the categories of amino acids transported.
Fig. 19 Amino acid transporters thought to exist at the blood–brain barrier. Based on Nalecz [200]; Broer [393]; Mann et al. [520]; O’Kane et al. [657]; and Hawkins et al. [44]. #See [44, 398] but contrast [399, 400]
Anionic amino acids, in particular glutamate, are transported by EAATs 1, 2 and/or 3 (coded by SLC1A3, 2, 1 respectively) which are found only in the abluminal membrane of the endothelial cells [387]. These EAATs mediate co-transport of the anionic amino acid together with 3 Na+ ions and 1 H+ ion followed by return transport of 1 K+ ion [388–390]. Because the electrochemical gradient for Na+ is directed from ISF into the endothelial cells and 3 Na+ ions are transported, this coupling renders the amino acid transport effectively unidirectional into the cells. Glutamate is also produced within the endothelial cells from breakdown of glutamine mediated by glutaminase [360]. Glutamate in the endothelial cells can then either be metabolized releasing NH4+, as argued by Helms and colleagues [391, 392], or be transported to blood plasma by a transporter other than an EAAT. Glutamate metabolism within endothelial cells is analogous to the extensive metabolism known to occur within gut epithelial cells (see e.g. [393]). Glutamate transport from brain endothelial cells to plasma has been demonstrated after sensory stimulation in vivo, which increases glutamate production [394]. This transport is likely to be via the glutamate/cystine exchanger, Xc− (SLC7A11 + SLC3A2), [200, 395]), though there is also evidence for a transporter, yet to be identified, that functions in the absence of cystine [396].
Cationic amino acids such as arginine and lysine are transported by CAT-1 (SLC7A1), which is known to exist in the abluminal membrane of the endothelial cells. Transport of these amino acids across the luminal membrane is less well-characterized but may be also via CAT-1 or possibly ATB0,+ (SLCA14). Transport of cationic amino acids by CAT-1 can involve exchange of one amino acid for another (trans-stimulation see Sect. 5.3.1), but this is not essential [397]. There may be at least one more transporter for cationic amino acids at the abluminal membrane (but see [398]). Hawkins et al. [44] reported that cationic amino-acid transport across both membranes can be inhibited by a number of neutral amino acids in the presence of Na+. CAT-1 is thought not to be so affected [397, 399, 400]. The additional transporter may be y+L [4F2hc (SLC3A2) + either y+LAT2 (SLC7A6) or y+LAT1 (SLC7A7)] [399, 400].
Neutral amino acids are transported by several systems as indicated in Fig. 19.System L, primarily the heterodimer 4F2hc/Lat1 (Slc3a2 + Slc7a5) which is present in both membranes and functions independently of Na+;
System A, primarily ATA2 (Slc38a2) in the abluminal membrane which because it is a Na+-linked transporter is biased towards transport from ISF into the endothelial cells;
ASC, primarily ASCT2 (Slc1a5), an obligatory exchanger that requires the presence of Na+-but is not driven by the Na+ gradient;
System Na+-LNAA a Na+-linked system whose molecular basis is still unknown;
ATB0,+ (SLC6A14) which allows net fluxes without exchange;
And possibly the y+L transporter [4F2hc (SLC3A2) + either y+LAT2 (SLC7A6) or y+LAT1 (SLC7A7)].
The large influxes of neutral amino acids from blood-to-brain seen in the early work and ascribed to system L have subsequently been shown to be mediated by 4F2hc/Lat1 [401–403]. The discovery that not only can this system mediate exchanges of amino acids [404, 405] but the exchange is obligatory [406–408] has far reaching consequences for amino acid transport at the blood–brain barrier [409]. It provides an important part of the explanation for how it is that there are large unidirectional fluxes (influx and efflux) but only small net fluxes. In order for system L to mediate a net inward flux of one amino acid, it must have net outward flux of another. An exchanger of neutral solutes, like system L, tends to equilibrate the concentration ratios for all of its substrates. Thus predicting the flux of any one of the amino acids across a membrane requires knowledge of the concentrations of all of the substrates on both sides of the membrane.20 Consumption of any system L substrate within the parenchyma will by reducing its ISF concentration tend to lead to net inward flux of that substrate and net outward flux of others. Similarly production of any system L substrate will tend to lead to its net outward flux together with net inward flux of others.
The function of 4F2hc/Lat1 (Slc3a2/Slc7a5), the principal component of system L, was explored in mice by Tarlungeanu et al. [410]. They compared the concentrations of amino acids in brain (amount per unit weight of brain) between a conditional Slc7a5 knockout [411] and normal controls. In adult mice they found that the levels of methionine, leucine and isoleucine in the knockouts were about 0.66 times the levels in normals, i.e. a reduction of about 35%. This suggests that there is normally a net inward flux of these amino acids via 4F2hc/Lat1 but that there are other routes at least as important. By contrast levels of phenylalanine, proline, glycine, threonine, and serine in the knockouts were about 1.3 times higher than in normals, i.e. an increase of about 30%. This suggests that for these amino acids there is normally a net outward flux via Lat1 but that there are other important routes for their elimination. More dramatically with histidine the level in knockouts was sevenfold higher, a 600% increase compared to normals. This suggests that 4F2hc/Lat1 is normally the main route for eliminating histidine from the parenchyma and that a net inward flux of histidine occurs by some route other than 4F2hc/Lat1. However, it is important to note that while these results show that 4F2hc/Lat1 is very important for the fluxes of histidine, they do not in themselves show that histidine efflux is a large fraction of the total efflux carried by 4F2hc/Lat1. Further evidence for exchanges involving histidine have been obtained using 4F2hc/Lat1 expressed in proteoliposomes. High concentrations of cysteine inside the vesicles can allow or drive influx of histidine and high concentrations of many amino acids outside of the vesicles can allow or drive efflux of histidine [412].
It has been tempting to propose that the combined net flux of neutral amino acids, inward or outward, is determined by their fluxes via systems other than system L and by their synthesis and breakdown in the parenchyma. System L is, however, still important, because it is the combined action of system L with the other transporters that determines which of the neutral amino acids move inwards and which outwards. A coherent overall account of the transport of neutral amino acids across the blood–brain barrier is still awaited.
With regard to glutamine, which is synthesized within the parenchyma, it has been tempting to propose that a substantial part of its efflux occurs via a system L mediated exchange for the essential large neutral amino acids such as leucine, isoleucine, valine and phenylalanine entering the parenchyma. Indeed such exchanges can be observed with isolated microvessels under experimental conditions [413, 414]. However, there is no evidence for this effect under conditions that exist in vivo.
The observation that there is a net efflux of glutamine is especially important because it is present at high concentration in plasma and ISF and it is the obvious sink for excess NH4+ in the brain. Glutamine is a substrate not only of 4F2hc/LAT1 (system L) as outlined above but also of Snat3 (SLC38A3) (system N), ATA2 (SLC38A2) (system A), and CAT (SLC7A1) (system y+) [200]. Of these the principal transport that has been observed is mediated by system N. Localization of system N has been controversial. Lee et al. [360] (see also [44]) found that vesicles prepared from abluminal membranes displayed a Na+-linked transport for glutamine while vesicles prepared from luminal membranes had only Na+-independent transport. This combination would explain net outward flux of glutamine from the brain. However, Ennis et al. [415] found marked Na+-dependent tracer influx of glutamine. While there are alternatives (see footnote 3 on p. 9 in [4]) the simplest interpretation is that there are Na+-linked transporters in both membranes. More recently immunohistochemical localization studies [416] have shown Snat3 primarily on the abluminal membrane but also on the luminal membrane of brain capillaries. It should be noted that while linking transport of glutamine to that of a single Na+ confers a bias towards transport into the endothelial cells, it does not preclude flux in the opposite direction via the same transporter and thus it is possible that Snat3 is responsible for the transport across both membranes.
As already mentioned, Lee et al. [360] found that brain endothelial cells have glutaminase activity, and thus following glutamine transport from ISF into the cells, at least some of the glutamine will be broken down to glutamate and NH4+. Helms et al. [391, 392] have suggested that some of the glutamate can be metabolized further releasing more NH4+. As a consequence of metabolism within the endothelial cells, glutamine removal from the parenchyma and glutamine appearance in plasma need not be the same. Glutamine net flux cannot be assessed in isolation.

Na+ and Cl−
It has been known for almost 50 years [152, 417] that influx and efflux of Na+ and Cl− across the blood–brain barrier are much larger than the net flux [4]. It was proposed by Crone [151, 418] that these apparently passive fluxes might well be paracellular, a suggestion that is still in agreement with all available data [4]. (The partial inhibitions seen in some studies with amiloride derivatives are discussed in Sections 4.3.3 and 4.3.4 of [4]).
The permeability of the blood–brain barrier to Na+ was measured by Davson and Welch in 1971 [417] and subsequently using a different experimental and analytical approach by Smith and Rapoport in 1986 [419] (see Appendix E). Because the fluxes in and out across the barrier are nearly in balance and the potential difference across the barrier is small, the PS product measured for influx, ~ 1 µL min−1 g−1 for each ion, can be used as an estimate for that for efflux, i.e. for the clearance via the barrier (see Appendix A). This cannot be exactly true, because there is a component of active transport, but inhibition of the Na+-pump has little effect on the tracer fluxes. This is consistent with both passive influx and passive efflux being much larger than both active transport and the net flux, and with active transport making a major contribution to the net flux (see Section 4.3.5 in [4]).
Perivascular transport does make a contribution to the clearances of Na+ and Cl−. Perhaps more importantly the net perivascular transport of each, the difference between influx and efflux, will be closely similar in size to the net transport across the blood–brain barrier, so that the volume, Na+ content and Cl− content of the parenchyma can be constant. The net transport of each across the blood–brain barrier is close to its concentration times the rate of fluid secretion across the blood–brain barrier in the steady-state. The controversy over whether perivascular influx and efflux occur along the same vessels or instead there is a glymphatic circulation with influx primarily by periarterial routes and efflux primarily by perivenular routes was considered in Sect. 4.2.
The net transports across the blood–brain barrier and via perivascular routes need not be exactly equal because there will be some component of diffusion between ISF and CSF at the brain surfaces, e.g. across the ependyma lining the ventricles and across the pia/glial layers. As indicated in Fig. 2 (see also [420], blood vessels enter and leave the parenchyma from subarachnoid spaces and cisterns and not from the ventricles. Thus transport from parenchyma to the ventricles will be primarily by diffusion probably with a component of flow in white matter (see Footnote 2) but it cannot be perivascular.
The possibility that there can be a small but significant net perivascular outflow from the parenchyma of Na+, Cl− and accompanying water may be the resolution of a long-standing difficulty. In non-communicating hydrocephalus, CSF production by the choroid plexuses continues at a nearly normal rate, but the normal route for CSF outflow from the IIIrd ventricle is blocked. Because after an initial period the ventricles do not continue to enlarge at a rate sufficient to accommodate the CSF production, CSF must be escaping via an alternative route (see Sections 4.2.2.1–4.2.2.2 in [41] and Section 4.1 in [4] for discussion and references). The periventricular parenchyma is oedematous which may allow flow of fluid from the ventricles, but the oedema only extends a small distance. In cats with kaolin induced hydrocephalus, Sahar et al. [421] observed penetration of serum albumin only up to about 2.5 mm which they took to mean that the albumin was being absorbed into the blood. There is no known mechanism by which this absorption could have occurred. It would be very interesting to know whether this distance corresponds instead to the distance from the ventricular surface to perivascular pathways that would allow sufficiently rapid removal of albumin to CSF in the subarachnoid spaces and/or to lymph that the concentrations observed deeper in the parenchyma would be small. The importance of fluid escape from the ventricles across the ependyma into the parenchyma in hydrocephalus has recently been given further support by observations of gadobutrol movements in normal pressure hydrocephalus in humans [15].

Amyloid-β
Accumulation of amyloid-β (Aβ) in plaques within the parenchyma and deposition in the walls of arteries are both closely associated with the development of Alzheimer’s disease. Because the rate of production of Aβ appears not to be altered in the more common, late onset form of Alzheimer’s [422] attention has focused on the possible defects in clearance of Aβ that may lead to its accumulation. Aβ may be removed from the brain via metabolism within the parenchyma, via efflux across the blood–brain barrier or or via perivascular efflux [52]. Attempts to estimate the relative importance of each of these routes were reviewed by Hladky and Barrand [146]. For low nanomolar ISF concentrations, which are in or above the normal or clinical range (see [423]), Aβ is eliminated by all three routes, but efflux via the blood–brain barrier is likely to be the most important (see also [424]). However, as emphasized in a key early study, efflux across the blood–brain barrier is saturable with a half-maximal concentration of only 15 nM [62]. Many studies of Aβ metabolism have used much higher concentrations, e.g. > 1 µM, and at these concentrations metabolism is dominant. A recent study on appearance of Aβ in lymph nodes may also reflect the behaviour at higher concentrations as it was performed in mice with mutant APP and high Aβ production rate [425].
Differences in Aβ clearance between sleep and wakefulness have been reviewed by Hladky and Barrand [146] and by Boespflug et al. [426] who emphasized the role of ISF-CSF exchange. The effects of sleep were found to be more complicated than a simple increase in perivascular clearance. Both reviews [146, 426] should be consulted for more detail and discussion (see also Sect. 3.3).
Aβ polypeptides are produced by neurons (and to some extent by other cell types) by cleavage of the membrane bound amyloid precursor protein (APP) [427]. While there is still uncertainty, the final cleavage step is thought to release Aβ directly into ISF.
Most work has focussed on Aβ1-40 and Aβ1-42, these being the predominant forms of the Aβ polypeptides present in the parenchyma. In solution at or below low nanomolar concentrations they exist as monomers and, particularly for Aβ1-42, also as oligomers [423, 428, 429]. Only soluble forms of Aβ are detectable in young animals. However, in older animals and older people deposits mainly of Aβ1-40 accumulate along cerebral arteries (cerebral amyloid angiopathy or CAA) and large aggregates or plaques mainly of Aβ1-42 form in the brain parenchyma. Small changes in soluble Aβ concentrations may over time lead to large changes in the formation of Aβ aggregates [430–436]. While it is not known which forms of Aβ are toxic, current evidence appears to suggest that within the parenchyma the main culprits are the oligomers [437–441].
There is evidence that plaques in the brain can be removed by reducing the ISF concentration of Aβ [428, 442]. However, it is likely that this only occurs if the Aβ concentration can be reduced to levels below those present before aggregate formation began [431]. This has been shown experimentally but it may not be achievable in practice without both inhibition of Aβ production (see e.g. [443]) and enhancement of Aβ clearance.

Clearance of Aβ from ISF
In the young, Aβ is present in soluble form and is eliminated as rapidly as it is produced with about 7–8% of the total soluble Aβ being replaced each hour [422, 444]. Monomeric and small oligomeric forms of soluble Aβ are cleared from ISF by at least four routes: incorporation into plaques, metabolism [445–451], efflux across the blood–brain barrier [62, 429, 452–454] and efflux via perivascular routes [25, 85, 128, 455]. The relative importance of each of these routes remains controversial [52, 146, 456–458].

Evidence for transport of soluble Aβ across the blood–brain barrier
The ways in which soluble Aβ can be transported across the blood–brain barrier have been investigated by several different groups. Shibata et al. [62] were the first to propose that Aβ could cross the blood–brain barrier by transcytosis mediated by low density lipoprotein receptor related protein (LRP1). This they said could account for the loss of 125I-Aβ1-40 from the brain that they observed. In support of their proposal they found that the loss of total 125I from the brain was reduced by antibodies against LRP1, by receptor (LRP1) associated protein (RAP), which interferes with binding of all known substrates to LRP1, and by absence of apoE seen in knockout mice. (ApoE affects the interaction of Aβ with LRP1). In addition the elimination process appeared to be saturable with Km of 15 nM. All of these observations are consistent with the idea that the elimination of soluble 125I-Aβ1-40 is primarily efflux across the blood–brain barrier and is via an LRP1-dependent process. However it should be kept in mind that demonstrating the importance of LRP1 is not the same as demonstrating elimination via the blood–brain barrier because LRP1 is also present on neurons, astrocytes and vascular smooth muscle cells where it can mediate endocytosis of Aβ leading to its metabolism inside the cells [448, 456, 459] (see [146] for further discussion). Further results supporting the involvement of efflux have been reported by Bell et al. [429], who found that the rate constant for elimination of Aβ1-42 was about half that for Aβ1-40 and also in other papers by Deane, Zhao, Nelson, Zlokovic and coworkers [452, 454, 460].
Results from several other groups also support the idea that efflux of soluble Aβ does occur at the blood–brain barrier and that LRP1 is involved in this elimination.Jaeger et al. [461] showed that antisense oligonucleotides against LRP-1 substantially decreased the loss of Aβ1-42 after intraparenchymal injection.
Pflanzner et al. [462] demonstrated LRP1-dependent Aβ1-40 transport across monolayers of primary mouse brain capillary endothelial cells, a transport not observed in monolayers of cells with genetically modified LRP1.
Roberts et al. [457] confirmed that efflux of Aβ from brain to blood occurs in vivo by finding that the concentration in venous blood leaving the brain was 7.5% higher than that in arterial blood.
Qosa et al. [424] using the brain efflux index method found that 62% of added 125I-Aβ1-40 appeared in the blood.
Storck et al. [453] developed a mouse model in which LRP1 could be knocked out selectively in endothelial cells and showed that the knockout reduced the initial rate of loss of 125I-Aβ1-42 by 48%.
Collectively the studies discussed above leave little doubt that LRP1 dependent transport across the blood–brain barrier plays a substantial role in Aβ elimination. However, the actual mechanisms governing the net inward or outward flux of Aβ across the blood–brain barrier are considerably more complicated and involve complexing Aβ with soluble factors including clusterin (also called apoJ), apoE and a soluble, truncated form of LRP1 (sLRP1). In addition there are at least four endocytotic/transcytotic systems. Figure 20, based mainly on the views of Zlokovic and colleagues [429, 452, 454, 460, 463–466], is a simplified diagram indicating the mechanisms of Aβ transport across the blood–brain barrier. Notable in this scheme is the involvement of apoE, clusterin and the phosphatidylinositol-binding clathrin assembly protein, PICALM (also called CALM). Genetic variations for each of these have been shown to be associated with increased risk of Alzheimer’s disease [467, 468].
Fig. 20 Simplified outline of Aβ transport across the blood–brain barrier. Possible movements of Aβ are shown by solid or dashed lines with arrowheads indicating the principal direction. Endocytotic and exocytotic vesicles are shown as invaginations of the membranes. There is intracellular processing once the vesicles have been endocytosed. Aβ from ISF can bind directly to LRP1 on the abluminal membrane with the complex then being incorporated into a clathrin coated pit which can be endocytosed. The Aβ-LRP1 complex is stabilized by binding of the phosphatidylinositol-binding clathrin assembly protein (PICALM). Aβ in ISF can also be complexed with any of the forms of apoE, 2, 3 or 4 or with clusterin. Aβ-apoE2 and Aβ-apoE3 are substrates for interaction with LRP1 and endocytosis. By contrast Aβ-apoE4 inhibits LRP1 mediated endocytosis (dotted line), but can be endocytosed slowly after binding with the very low density lipoprotein receptor (VLDLR). Aβ-clusterin is a substrate for LRP2 mediated endocytois with transport across the blood–brain barrier to plasma. As Aβ-clusterin can also be transported in the opposite direction by LRP2-mediated endocytosis this is almost certainly by transcytosis of vesicles with LRP2 in the membrane. Vesicles with LRP1 in the membrane are also thought to discharge their contents on the far side of the barrier—i.e. this is transcytosis [465]. Some of the intracellular processing steps for the LRP1 vesicles are now known [452, 658]. Aβ is also transported from plasma to ISF. Aβ clusterin can be transported by LRP2 vesicles, but on the plasma side almost all of the LRP2 receptors are occupied by clusterin (dotted double headed arrow) rather than Aβ-clusterin which greatly reduces blood-to-brain transport by this route. Aβ is however, endocytosed after binding to the receptor for advanced glycation products, RAGE, and somehow transported to the brain side. Pgp may, in a manner which has not been well defined, assist transfer of Aβ from the endothelial cells to plasma whether it has entered the cells from ISF, via the LRP1 system, or from plasma, via the RAGE system. Figure based on [452, 464, 465]
Much of the soluble Aβ in ISF may be in the form of complexes with apoE or clusterin while in plasma most Aβ is complexed with clusterin or sLRP1, a truncated, soluble form of LRP1 [469]. The apoE gene has three alleles called apoE2, apoE3 and apoE4. Expression of the apoE4 allele is the greatest genetic risk factor known for developing the late-onset form of Alzheimer’s disease [467, 468].
LRP1 mediated transport of Aβ occurs via clathrin pits, with the LRP1, Aβ, clathrin system stabilized by interaction with PICALM. In addition to this transport of Aβ there is LRP1-mediated transport from brain-to-blood of Aβ complexes with apoE2 or apoE3 and LRP2-mediated transport of Aβ complexes with clusterin. Complexes with apoE4 inhibit LRP1-mediated transport but are transported at a much lower rate by very low-density lipoprotein receptor (VLDLR) mediated transport. This inhibition and slow transport with the resulting tendency to accumulate Aβ in the brain may account for the increased risk of Alzheimer’s disease.
The receptor for advanced glycation end products (RAGE) mediates transport of Aβ from blood-to-brain. Aβ-clusterin blood-to-brain transport by LRP2 can also be demonstrated under experimental conditions, however, in vivo it is likely that the Aβ-clusterin complexes are out-competed by clusterin for inwards transport [470–472]. The net flux of complexes via LRP2 is thus brain-to-blood [429, 464]. sLRP1 is released from LRP1 at the luminal membrane by removal of the membrane binding domain. Aβ complexes with sLRP1 are apparently not transported across the blood–brain barrier but can be delivered to the liver. Thus these serve as a sink reducing backflux of Aβ that has emerged from the brain [469].
The role of p-glycoprotein (Pgp) has been considered in many studies [243, 473–490] that indicate that it does play a role, but there have also been studies suggesting that it does not [491–494]. P-glycoprotein is present in the luminal membranes of the endothelial cells (see Sect. 4.2.1). With LRP1 mediating entry of Aβ into the endothelial cells from ISF, an obvious role to suggest for p-glycoprotein is Aβ efflux to plasma. Another function of p-glycoprotein may be to return to plasma some of the Aβ brought into the cells by RAGE [423, 495, 496]. However the intervening steps between endocytosis mediated by either LRP1 or RAGE and efflux by p-glycoprotein remain to be established.
The overall net flux of Aβ across the blood–brain barrier is thus seen to be the resultant of a number of transport mechanisms mediating both inward and outward fluxes. The use of complexing agents in plasma to reduce Aβ flux from blood-to-brain is one strategy being tried to reduce Aβ accumulation in the brain.

Evidence for Aβ elimination via perivascular routes
The perivascular route has also been considered as a likely pathway for elimination of Aβ peptides from the brain. In initial studies, following exogenous Aβ introduction into the brain, aggregates were first found along the external boundaries of arterial walls [497, 498] (see also [499, 500]) but at later times were seen throughout the smooth muscle layer of the arteries ([497], see also [501]). The results from these initial studies are consistent with the idea that growth of the deposits starts occurring adjacent to an efflux route for Aβ along the outside of the arteries, i.e. an extramural periarterial route.
Subsequent studies followed the routes of exit from the parenchyma of fluorescent dextran. This was used as a non-metabolizable marker for substances of the size of Aβ. Within minutes of its injection fluorescence could be visualized throughout the smooth muscle layer of the arterial walls [70]. From this observation it was proposed that both the fluorescent dextran and the Aβ enter the smooth muscle layer near its end closest to the capillaries and move along the vessel wall towards the subarachnoid space with little further exchange between the smooth muscle layer and the surrounding parenchyma. However, it remains difficult to see how there could be sufficient driving force for movement through the extracellular matrix along the entire length, perhaps a millimeter, of the vessel (compare the discussion in Sect. 3.2.1) while at the same time movement over a 10- to 20-fold shorter distance perpendicular to the vessel wall is prevented. For a different viewpoint see [88, 95, 502–504]).
There may be an alternative explanation. The higher observed density of dextran or Aβ within the extracellular spaces of the smooth muscle layer than in the interstitial spaces of the parenchyma [102] might suggest that it binds, reversibly, to some component of the extracellular matrix in the layer. There is in fact good evidence for interaction of the Aβ peptides with some components [505, 506]. If the high concentrations within the basement membranes of the layer reflect binding rather than some form of impermeant sheath, then it is not clear whether Aβ and the dextrans reach the sites of the binding by moving parallel to the vessel wall or by traversing it (see Fig. 21). If the latter, movements parallel to the vessel would be occurring via an extramural route that might have a much lower resistance to flow. Transverse movement has been observed for both horseradish peroxidase and 3H-leucine with large cerebral arteries [507], and no additional impermeant layer is known to exist around smaller arteries inside the parenchyma [98]. There is at present no compelling evidence to decide between the intramural and extramural routes for movement parallel to the vessels.
Fig. 21 Putative routes for periarterial efflux. In the intramural proposal solutes move parallel to the vessel wall along the basement membranes of the smooth muscle layer, shown as blue trajectories. In the extramural proposal movements of solutes parallel to the vessel occur primarily in a perivascular space with lower resistance to flow. They also move in and out of the wall by a combination of diffusion and convection, shown as the red trajectories. As discussed in Sect. 3.1 the nature of the extramural pathway is still controversial including whether it is a space one side or the other of the pial cells or alternatively the pial and glial basement membranes themselves. endo endothelium, s.m smooth muscle, BM basement membrane. Pial cells and pial basement membrane(s) are shown together because they are very thin
The importance of the perivascular route for Aβ elimination may be not so much that it removes Aβ from the parenchyma but rather that it delivers Aβ into the vessel walls of arterioles and arteries. Cerebral amyloid angiopathy is often seen before formation of senile plaques within the parenchyma (see e.g. [508]) and the damage to the arterioles and arteries may have secondary consequences for the well-being of parenchymal cells, either by effects on blood flow or via local inflammation [509–511].

Relative importance of metabolism, blood–brain barrier transport and the perivascular route for elimination of soluble Aβ
Attempts have been made to estimate the proportions of soluble Aβ removed from the brain by metabolism, by transport across the blood–brain barrier, and by perivascular efflux. It is possible to get an estimate of perivascular elimination alone using inulin. When this was done in mice, Shibata et al. [62] found that the half-time for the elimination of 125I-Aβ1-40 was much shorter than could be explained by elimination by the perivascular route, with calculated rate constants of 0.027 min−1 and 0.0029 min−1 respectively (see Table 5). As they had concluded that metabolism played little part, the faster, non-perivascular elimination was held to be transfer across the blood–brain barrier. Bell et al. [429] (see Appendix 2 in [146] for corrections to their calculations) extended these observations to 125I-Aβ1-42.
It is interesting to note that Xie et al. [128] found the half-lives for both Aβ and inulin to be different when the mice were asleep as compared to when they were awake. In both conditions the rate constant was larger for Aβ than for inulin (see Table 5). The interpretation of these differences in rate constants between wakefulness and sleep has been considered in some detail in [146] and will not be considered further here.
Table 5 Rate constants for elimination of 125I-Aβ1-40, 125I-Aβ1-42 and inulin in mice
Rate constant/min−1
Shibtata et al. Bell et al. Xie et al. awake Xie et al. asleep
Inulin 0.0029 0.0024 0.006 0.016
Aβ1-40 0.027 0.0184 0.024 0.053
Aβ1-42 0.011
Data from Shibata et al. [62], Bell et al. [429] (see Appendix 2 of [146]) and Xie et al. [128]
The results of Shibata et al. [62], Iliff et al. [25] and Xie et al. [128] all imply that the rate constant of perivascular elimination, as estimated by the constant for inulin efflux, is considerably less than the rate constant of elimination by other means.21
Roberts et al. [457] sought to compare rates of metabolism of Aβ with those of Aβ efflux. To do this they used values for: the turnover rate for Aβ [512]; the pool size for Aβ; the difference between Aβ concentrations in arterial blood and in venous blood leaving the brain; the cerebral blood flow and the rate of return of CSF to the general circulation. From these values they calculated that 25% of Aβ elimination was via efflux across the blood–brain barrier, 25% was via CSF and the remaining 50% was via metabolism. As discussed in [146] while the results of Roberts et al. do suggest that all of these mechanisms are involved, the fraction of Aβ leaving the brain across the blood–brain barrier may have been underestimated and could be as high as 50%. By contrast the fraction accounted for by metabolism may have been smaller than estimated.
On balance the available data suggests a significant involvement in elimination of Aβ from the brain for all three routes of elimination: metabolism, net outward flux across the blood–brain barrier and net perivascular outward flux.

Estimating the value of the total clearance of soluble Aβ from ISF
Calculating a clearance value for the elimination of Aβ from ISF is not straightforward as much of the Aβ in ISF is complexed with other solutes, e.g. apoE and clusterin. However, an estimate can be made if it is assumed that all the forms that are accessible to be eliminated are dissolved in the ISF and eliminated with the same rate constant. The volume of distribution for the total soluble Aβ, whether or not as part of complexes, will be that of ISF and thus the clearance can be calculated as rate constant × volume of distribution = 0.05 min−1 × 0.2 mL g−1 = 10 µL g−1 min−1. On this basis perivascular clearance, expected using the same assumptions to be about 1 µL g−1 min−1, may be about 1/10th as large, a small but still significant fraction of the total.
In all of the preceding, the rates of elimination by various routes have been considered almost as if they are constant. However, reduction in the overall clearance and thus in the rates of elimination by some of the routes are likely to be very important in the development of Alzheimer’s disease [422]. In this regard LRP1 expression has been found to be reduced and RAGE expression increased with age [478, 513]. Similarly perivascular elimination has been found to decrease with age possibly as a result of decreased variations in the size of arteries and arterioles during the cardiac cycle [514] (see Sect. 3.2). All of these changes will tend to increase Aβ ISF concentration and hence lead to increased formation of plaques and vascular Aβ deposits.