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INTRODUCTION |
Alzheimer's disease
(AD)1 is associated with the
accumulation of a specific form of amyloid in the brain parenchyma and
in meningocerebral blood vessels (1-3). The primary components of the
amyloid are polymers of a short peptide derived through proteolytic processing of a ubiquitous transmembrane protein (4, 5) termed the
-amyloid precursor protein. The amyloid
-peptide is usually
referred to as the A
. It is present in two principal variants (2,
6), one that contains 40 amino acid residues (A
1-40),
and one C-terminally extended variant that contains 42 amino acid
residues (A
1-42). The longer variant has been suggested
to be of major importance in the pathogenesis of AD because it has a
greater tendency to form amyloid fibrils in vitro and
possibly also in vivo (7-9). Certain mutations associated with familial AD lead to an increased secretion of the 42-amino acid
form (10) and an enhanced accumulation of amyloid.
-Amyloid displays several important features that distinguish it
from other types of amyloid. (i) The peptide forming the amyloid
deposits is present at very low concentrations in the circulation. This
is in contrast to peripheral amyloid disorders in which the amyloid
proteins are present at high concentrations. Examples of such
non-central nervous system amyloid proteins include serum amyloid A,
myeloma protein, and transthyretin (11). (ii) The levels of A
are
not higher and the peptide is not structurally different (except in
extremely rare cases of familial AD) in individuals with the disease
than in healthy controls (for a review, see Ref. 3). (iii) It is well
known that most and possibly all nucleated cells in the body produce
the A
(12, 13); however, for unknown reasons,
-amyloid is only
deposited in the central nervous system.
In the present study, we aimed at investigating why
-amyloid
exclusively is formed in the central nervous system. Previous work has
demonstrated that some plasma proteins and lipoproteins bind A
and
serve as carrier proteins (14, 15). Protein binding is a general
mechanisms for the transport of endogenous substances such as hormones
and lipids as well as clinically used drugs (16). Generally, it is only
the non-protein-bound fraction of the substances that is biologically
active. We therefore hypothesized that only the free fraction of A
can take part in the polymerization process generating amyloid fibrils.
Hence, A
-carrier proteins may have an important role in preventing
amyloid formation by increasing the bound fraction.
The bulk of large proteins do not penetrate the blood-brain barrier
efficiently. Thus, the levels of soluble proteins in the central
nervous system are much lower than those in peripheral tissues. It has
been estimated that the ratio between protein content in the CSF and
plasma is approximately 0.004 (17). However, in contrast to large
proteins, the A
levels are higher in CSF than in plasma (18, 19),
which probably reflects a higher rate of secretion from neuronal cells
than from other cell types. Overall, this suggests that a smaller
fraction of the A
is protein-bound in the central nervous system
than in the periphery.
With this background, we decided to investigate whether plasma and CSF
proteins can indeed inhibit
-amyloidogenesis. We established in vitro assays allowing quantitative and qualitative
studies of amyloid formation in the presence of several different
proteins. The proteins studied here represent more than 90% of the
protein content in plasma and CSF (20-25). Many drugs bind to
plasma proteins, which can lead to interactions with severe
consequences (16). If some drugs bind to the same site on plasma
protein molecules as A
, it may lead to increased levels of free A
and enhanced amyloid formation. Therefore, we decided to also address
this possibility experimentally.
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EXPERIMENTAL PROCEDURES |
Materials--
Synthetic A
1-40,
A
1-40, and PrP106-126 biotinylated at the
N terminus were obtained from ANAWA (Wangen, Switzerland). Nonlabeled
A
1-40, A
1-42, and
PrP106-126 were obtained from Bachem (Bubendorff,
Switzerland). The peptides were stored in Me2SO at
20 °C. Human serum albumin, (fatty acid-free; 99% purity) was
from Sigma. All other proteins were from Calbiochem. Streptavidin-peroxidase was bought from Roche Molecular Biochemicals. All other reagents were from Sigma. Iodinated A
1-42 was obtained from Amersham Pharmacia Biotech.
Analysis of A
Polymerization--
96-well plates (Maxisorp;
Nunc) were coated with peptide by incubating them with a solution of
A
1-42 or A
1-40 (2.5 µM)
in Tris-buffered saline (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and NaN3). Solution (100 µl) was
added to each well, and the plates were incubated at 37 °C with
shaking for 48 h. The peptide solution was then flicked off.
Staining with a solution of Congo red (20 µM) in
Tris-buffered saline showed that the polymeric peptide had bound to the
wells (data not shown). After removal of the peptide solution, the
plates were placed upside down on absorbing paper and allowed to dry.
Coated plates were stored at
20 °C in a desiccator. On the day of
experiment, the plates were blocked by the addition of 300 µl of PBS
containing 0.05% Tween 20 (PBS-T) and 1% bovine serum albumin/well
for 2 h at room temperature. The plates were then washed with
PBS-Tween (0.05% Tween 20), and the fluid was flicked off.
Biotin-A
1-40 or biotin-A
1-42 was
dissolved in Me2SO and diluted with Tris-buffered saline
with NaN3 (0.05%). Unless stated otherwise, the final
concentration of the labeled peptide was 20 nM. The plates
were incubated overnight at 37 °C with agitation. Nonbound peptide
was removed by washing the plates three times with PBS-T (300 µl/well). Streptavidin-peroxidase was diluted with PBS-T and 1% BSA
and added to the plates (150 µl/well). After incubation (2 h at room
temperature), the solution was flicked off, and the plates were washed
four times with PBS-T. Tetramethyl-benzidin was used as chromogenic
substrate for the peroxidase. After termination of the reaction with
sulfuric acid (0.33 M, final concentration), absorbance was
measured at 455 nm with a SpectraMAX 250 96-well plate reader.
Nonspecific binding was defined as the binding of biotin-A
to wells
that had not been coated with A
. There was a linear relationship
between peroxidase activity and the amount of peptide bound (data not
shown). Nonspecific binding was, on average, approximately 15% of
total binding (data not shown). We also studied the incorporation of
125I-A
into tissue sections of human AD brain using the
method of Maggio et al. (29). In experiments with the prion
protein-derived peptide PrP106-126 (26), similar
methodology was used, but with two exceptions. First, the Maxisorp
plates were coated with a solution of 10 µM peptide for
14 days. Second, incubation with N-terminally biotinylated
PrP106-126 was performed for 4 h. The method used was
validated by several means. (i) biotinylated-A
1-42 or
A
1-40 was incubated at a high concentration (10 µM) for 72 h at 37 °C and then examined by
electron microscopy. Both peptides were capable of forming fibrils that
were indistinguishable from nonbiotinylated controls. (ii) Both
biotinylated peptides required the Maxisorp plates to be coated with
amyloid fibrils in order for them to bind. When the plates were coated
with truncated variants of A
(A
12-28,
A
35-42, A
10-20, A
1-16, or A
25-35), no significant binding over that obtained
with noncoated control wells was observed. (iii) We also studied the incorporation of biotin-A
1-40 into preformed
A
1-42 fibrils. A low concentration of fibrils
(corresponding to 20 nM monomeric peptide) was incubated
with equimolar amounts of biotin A
1-40. After overnight
incubation and centrifugation, the material was stained with
anti-biotin Ig labeled with colloidal gold and negatively stained with
uranyl acetate. Using this protocol, gold-labeled amyloid fibrils were
observed, demonstrating that biotin-A
1-40 could bind to
the preformed fibers. The controls used were
biotin-A
1-40 or preformed A
1-42 fibrils alone. In these control experiments, biotin-A
1-40 did not produce any detectable fibrils, whereas the A
1-42 fibrils were not labeled by gold.
Electron Microscopy--
Negative staining was performed by
adsorption of a 5-µl aliquot of the sample to a carbon-coated
200-mesh copper grid for 60 s. Staining was done by adding 10 µl
of 2% uranyl acetate directly to the adsorbed sample droplet for 2 min
and air drying after the removal of excess liquid with filter paper.
Specimens were examined in a JEOL 1210 electron microscope operated at
100 kV. Digitized micrographs were recorded with a slow scan
charge-coupled device camera (Gatan; model 679). Data acquisition with
the slow scan charge-coupled device camera and processing of the
digitized images were controlled by a Macintosh PowerPC 8500 using
DigitalMicrograph software from Gatan. Images were printed on a
Thermoprinter Phaser 440 (Tektronix). Magnification calibration was
performed as described previously (27) using negatively stained
catalase crystals.
Surface Plasmon Resonance Spectroscopy--
Interactions between
albumin and monomeric/polymeric A
were measured using a BiaCore 2000 instrument (BiaCore AB, Uppsala, Sweden) essentially as described
previously (28). Briefly, monomeric biotin-A
1-40 at a
concentration of 115 nM was attached to a sensor cell to
which streptavidin had been coupled. The peptide was stored in
Me2SO and diluted in running buffer (see below) immediately
before it was coupled to the chip. Under these conditions, no evidence
suggesting that the peptide polymerized could be obtained (data not
shown). Polymeric peptide was attached via a monoclonal antibody
against amino acid residues 2-8 of A
(BAP-1A). Unless otherwise
stated, the running buffer used contained 10 mM Hepes, pH
7.5, 150 mM NaCl, and 0.05% P20 detergent. After each
experiment, the cells were washed with ethanolamine (1 M,
pH 8.5) until the sensor signal remained stable in contact with running buffer.
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RESULTS |
Albumin-mediated Inhibition of A
Polymerization--
In the
first set of experiments, we investigated whether albumin, which is
quantitatively the most important A
-binding protein (14) and also
the most abundant protein in plasma and CSF, could interfere with the
incorporation of biotin-A
1-40 into amyloid fibrils. The
test system used is based on the finding that A
monomers bind with
high affinity to preformed polymers of A
(29). We immobilized
A
1-42 polymers as seeds (7) in 96-well plates and
measured the incorporation of soluble biotin-A
1-40 in
the presence of human serum albumin (HSA) or BSA. In Fig.
1A, the effects of different
concentrations of HSA and BSA on biotin-A
1-40 incorporation are shown. The highest concentrations of albumin used
corresponded approximately to the plasma levels of a healthy human
adult (21). Both HSA and BSA had the capacity to completely inhibit the
incorporation of biotin-A
1-40 into immobilized A
polymers with apparent IC50 values of 10 and 12 µM, respectively. The effects of HSA on the
polymerization of A
1-42 in this system were also
studied. Here, nonlabeled A
1-40 or
A
1-42 was immobilized in the Maxisorp plates as
described under "Experimental Procedures." Biotinylated
A
1-40 or A
1-42 was then allowed to bind
the immobilized peptide in the presence of various concentrations of
HSA. The IC50 values of HSA on the inhibition of
A
1-40 or A
1-42 binding were essentially
identical (data not shown), suggesting that HSA is indeed capable of
inhibiting polymerization of the two major forms of the A
.

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Fig. 1.
Albumin inhibits the incorporation of
biotin-A 1-40 into immobilized
amyloid polymers. Biotin-A 1-40 was incubated at a
concentration 20 nM in 96-well plates coated with
A 1-42 polymers in the presence of the indicated
concentrations of HSA ( ) or BSA ( ) (A). After an
overnight incubation at 37 °C with agitation, the reaction was
stopped and processed as described under "Experimental Procedures."
The biotin-A 1-40 incorporation in the absence of
albumin is equal to 100%. The experiments were performed in
quadruplicate. The experiment was repeated three times with essentially
identical results. Data are indicated as the mean ± S.E. The
effect HSA on the incorporation of
125I-A 1-42 into genuine amyloid deposits in
human AD brain tissue was also studied. In B and
C, the effect of buffer alone or buffer containing HSA (227 µM), respectively, is demonstrated.
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These findings were confirmed in a second set of experiments in which
the incorporation of 125I-A
1-42 into brain
tissue sections from an individual with AD was measured. As seen in
Fig. 1B, the radiolabeled peptide bound to the amyloid
deposits in the tissue, as demonstrated previously (29). In the
presence of 227 µM HSA, binding was heavily reduced (Fig.
1C). Measurement of the incorporated radioactivity using a
phosphorimager showed that overall binding (binding to amyloid deposits
in the tissue and background together) had been reduced with 55% by
the addition of HSA.
A
incubated at high concentrations also rapidly polymerizes in the
absence of preformed polymers, but through primary nucleation (30, 31).
Therefore, in other experiments, we studied the effects of HSA on
soluble A
1-40 and A
1-42 in the absence
of seeds. As seen in Fig. 2, A
and C, both peptides formed fibrils when incubated for
24 h at 37 °C at a concentration of 20 µM. This
concentration is approximately 6,000 times higher than that in CSF (17,
29, 39). When incubated in the presence of 227 µM HSA,
the polymerization of A
1-40 into amyloid fibrils was
completely inhibited (Fig. 2B). Under the same conditions, A
1-42 only formed occasional fibrils (Fig.
2D). Moreover, spherical structures of 10-30 nm in diameter
were also detected frequently.

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Fig. 2.
Effects of HSA on
A 1-40 and
A 1-42 polymerization studied with
electron microscopy. A and B,
A 1-40 (20 µM) incubated for 24 h in
the absence (A) and presence (B) of 227 µM HSA. C and D,
A 1-42 (20 µM) incubated in the absence
(C) and presence (D) of 227 µM HSA.
Scale bars, 500 nm. The area in each panel that is enclosed
by a square is shown at a higher magnification in the
inset (upper left).
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HSA Inhibits the Polymerization of a Prion-derived
Peptide--
PrP106-126 represents the central core of
the prion protein (for a recent review, see Ref. 26) and spontaneously
forms amyloid-like fibrils. Similar to the A
, prion protein can form amyloid deposits in the CNS and cause neurodegeneration. We therefore decided to study whether albumin can also prevent polymerization of
this peptide. As seen in Fig. 3, HSA
dose-dependently inhibited the binding of biotinylated
PrP106-126 to immobilized homologous peptide. The
IC50 value for HSA in this system was approximately 100 µM, 10 times higher than that for A
. This
concentration represents less then one-sixth of the albumin
concentration in blood but is more than 30 times higher than the
albumin concentration in CSF. Hence, these findings demonstrate that
HSA displays a certain degree of specificity for
-amyloid.

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Fig. 3.
Effects of HSA on
PrP106-126 polymerization.
Biotin-PrP106-126 was incubated at a concentration of 20 nM in 96-well plates coated with PrP106-126
polymers in the presence of the indicated concentrations of HSA. The
incorporation of biotin-PrP106-126 was defined as the
total binding minus the binding obtained in wells that had not been
coated with Biotin-PrP106-126.
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Characterization of the Mode of Action for Albumin on A
Polymerization--
Surface plasmon resonance spectroscopy allows
protein-protein interaction studies in real time (28), and this
methodology was therefore used to study how BSA and HSA interact with
monomeric and polymeric A
. Preformed A
1-42 fibrils
were immobilized to the sensor chip as described under "Experimental
Procedures." A solution (25 µM) of BSA (Fig.
4A) or HSA (Fig.
4B) was then allowed to flow through the cell. The protein
bound avidly to the polymers, indicating that albumin indeed has an
affinity for the polymeric peptide. In parallel flow cells, monomeric
biotin-A
1-40 was immobilized using streptavidin. In
this case, no binding was observed with either BSA (Fig. 4A)
or HSA (Fig. 4B). When the experiment was repeated using
nonbiotinylated A
1-40 that was immobilized with a
monoclonal antibody, essentially identical results were obtained (data
not shown).

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Fig. 4.
Albumin binding to HSA studied using surface
plasmon resonance spectroscopy. Preformed A 1-42
polymers or biotin-A 1-40 were immobilized on the sensor
chip using the monoclonal antibody MAP-1A (A 1-42
polymers) or streptavidin (biotin-A 1-40). A 25 µM solution of BSA (A) or HSA (B)
was injected into the flow cells and allowed to bind to the peptides
between 0 and 300 s.
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Drug-enhanced A
Polymerization--
It is well known that
several clinically important drugs bind to albumin with various
affinities. We speculated that some of these substances may bind to the
same site(s) on the albumin molecule as A
and may therefore be able
to displace the peptide from its binding site(s). This may lead to
increased levels of free A
and the enhancement of amyloid formation.
We therefore screened a number of albumin ligands with regard to their
effects on biotin-A
1-40 incorporation into preformed
amyloid polymers in the presence or absence of 100 µM
HSA. It was found that tolbutamide, at concentrations corresponding to
therapeutic levels (17), enhanced biotin-A
1-40
incorporation in the presence but not in the absence of HSA (Fig.
5). This strongly suggests that tolbutamide is capable of interfering with A
-albumin binding and
indirectly stimulating amyloid fibril formation.

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Fig. 5.
Tolbutamide increases
biotin-A 1-40 incorporation in the
presence but not in the absence of HSA.
Biotin-A 1-40 (20 nM) was incubated in the
absence ( ) or presence ( ) of HSA (100 µM) with the
indicated concentrations of tolbutamide at 37 °C overnight. Binding
in the absence and presence of HSA was 374 ± 6 and 279 ± 6 milli-optical density units, respectively. Data are indicated as
mean ± S.E. (n = 5).
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Endogenous Regulators of
-Amyloidogenesis--
In these
experiments, we investigated the effects of various plasma/CSF proteins
on A
polymerization (Table I). The
proteins listed in Table I represent more than 90% of the protein
content in plasma and CSF. The concentrations tested covered the levels in plasma and CSF for all but two proteins, IgM and
1-antichymotrypsin. The highest concentrations used were
0.55 and 1.0 µM, respectively, which were lower than
their plasma concentrations but higher than their CSF concentrations
(see Table I). Seven of the 13 tested proteins had very little or no
effect (i.e. the IC50 was higher than the plasma
concentrations). Of the remaining six proteins, three had
IC50 values in the range of 10-30 µM, and
three had IC50 values below 10 µM. Albumin,
1-antitrypsin, IgG, and IgA had IC50 values
that were substantially below their plasma concentrations, which
strongly suggests that these proteins may be potent inhibitors of
-amyloidogenesis in vivo.
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Table I
IC50 values for the inhibition of biotin-A 1-40
incorporation into immobilized A 1-42 polymers
The two columns to the right indicate plasma and CSF concentrations of
the studied proteins in healthy adults (21-24, 41).
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1 unit of inhibitory activity was defined as the number of µmol/liter
of protein required to inhibit polymerization by 50% under the
conditions specified under "Experimental Procedures." When taking
the plasma concentration of the studied proteins into consideration, it
is possible to estimate how much inhibitory activity each protein
contributes (Fig. 6). Albumin is probably the most important regulator of
-amyloidogenesis in plasma. Although
1-antitrypsin has an IC50 value eight times
lower than that of albumin (1.25 and 10 µM,
respectively), the concentration of the former is substantially lower
(25.3 and 644 µM, respectively). Therefore, despite its
higher efficacy, it probably plays a less important role in the
regulation of A
polymerization.

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Fig. 6.
A polymerization
inhibitory activity of individual proteins and the total content of
inhibitory activity in plasma and CSF of these proteins. A,
1 inhibitory unit is defined as the number of µmol/liter of the
indicated protein required for a 50% inhibition of A polymerization
under the conditions defined under "Experimental Procedures" and
the Fig. 1 legend. B, the total inhibitory
activity in plasma and CSF contributed by the studied proteins
specified in Table I.
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Cerebrospinal fluid contains essentially the same proteins as plasma,
but the concentrations are considerably lower (see Table I). None of
the tested proteins are present in the CSF in a concentration equal to
or higher than its IC50 value, which was obtained in the
amyloid formation assay (see Table I). When comparing the total amount
of inhibitory activity in plasma and CSF, we found that CSF contains
only about 0.3% of that seen in plasma (Fig. 6).
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DISCUSSION |
Plasma and CSF proteins with affinity to A
serve as carriers
for the peptide (14, 15). This study and previous studies (32, 33) have
demonstrated that A
-binding proteins in plasma and CSF may also have
a function in regulating
-amyloid formation. The most abundant
plasma protein, albumin, is present in a concentration more than
60-fold higher than its IC50 in the A
polymerization assay used here (see Table I). Albumin is the most abundant protein in
CSF, but it is present at a concentration below its IC50
value at which only a partial inhibition of A
polymerization is
obtained (see Fig. 1). Plasma also contains significant levels of other proteins, such as
1-antitrypsin, IgG, and IgA, that are
capable of inhibiting A
polymerization. The other studied proteins
capable of inhibiting polymerization are also present in CSF in
concentrations substantially below their IC50 values (see
Table I). These results point to a dramatic difference between plasma
and CSF: the former contains large quantities of inhibitory proteins,
whereas the latter contains small quantities of inhibitory proteins.
For unknown reasons,
-amyloid deposits are not formed outside the
central nervous system (34). The present results suggest that the high concentrations of inhibitory proteins in plasma prevent the formation of
-amyloid in peripheral tissues, but the low levels in CSF do not
block
-amyloid formation in the central nervous system. This
conclusion is also supported by previous experimental data (35),
showing that CSF only partially inhibits the formation of
thioflavin-binding amyloid from synthetic A
1-40.
Pathologically reduced levels of albumin might promote
-amyloidosis
and possibly also AD. In clinical studies, it was observed that
anti-inflammatory drugs may have beneficial effects on AD (36). Levels
of albumin are often reduced in association with inflammation (25) and,
hence, the antiamyloidogenic activity in plasma and CSF is also
reduced. However, even heavily reduced plasma levels of albumin are
probably still sufficiently high to prevent amyloid formation in
peripheral tissues. It may be different in the central nervous system.
Because albumin (and other inhibitory proteins) is present in low
concentrations having limited effects on amyloid formation (35), even
small reductions in albumin levels in association with inflammation may
lead to increased amyloid formation.
The structural background as to why A
binds albumin and other
proteins is not known. However, it is reasonable to assume that
hydrophobic interactions are involved. It was surprising that monomeric
A
did not display binding to albumin when studied by surface plasmon
resonance spectroscopy, considering the findings of Biere et
al. (14) showing that soluble A
binds albumin and lipoproteins.
One explanation may be that A
molecules rapidly form small, soluble,
oligomers with an affinity to albumin (37, 38).
Tolbutamide is a drug used to regulate blood glucose levels in diabetes
mellitus. It also displays a high affinity for albumin. As a result,
its clinical use is often associated with interactions with other drugs
when the compounds compete for the same binding site on the albumin
molecule (17). Here, we found that tolbutamide, at concentrations
corresponding to therapeutic levels, enhanced amyloid formation in the
presence but not in the absence of HSA. A reasonable explanation is
that tolbutamide and A
bind to the same site on albumin. Tolbutamide
may therefore displace A
from albumin and generate higher free A
fractions that can participate in amyloid formation. Drugs that can
penetrate into the central nervous system, bind to the A
site(s) on
albumin, and increase the free fraction of the peptide may thus be
capable of enhancing amyloid formation in vivo.
Mutations affecting proteins capable of binding A
may promote the
development of AD (39, 40). It is therefore possible that mutations
affecting the proteins studied here may also have an impact on the
development of AD through a similar mechanism.
In conclusion, the present data suggest a novel and possibly important
physiological role for albumin and other plasma/CSF proteins in
controlling amyloidogenesis in the central nervous system and possibly
also in peripheral tissues. The data also suggest that drugs with
certain pharmacokinetic properties may be capable of enhancing
amyloidogenesis. Moreover, the reduced levels of albumin seen in
association with inflammatory reactions may provide an opportunity for
the A
to polymerize and thereby more easily form amyloid in the
central nervous system.