U.S. patent application number 14/944311 was filed with the patent office on 2016-05-19 for methods for measuring the metabolism of neurally derived biomolecules in vivo.
The applicant listed for this patent is The Washington University. Invention is credited to Randall John Bateman, David Michael Holtzman.
Application Number | 20160139142 14/944311 |
Document ID | / |
Family ID | 37073989 |
Filed Date | 2016-05-19 |
United States Patent
Application |
20160139142 |
Kind Code |
A1 |
Bateman; Randall John ; et
al. |
May 19, 2016 |
METHODS FOR MEASURING THE METABOLISM OF NEURALLY DERIVED
BIOMOLECULES IN VIVO
Abstract
The present invention relates to methods of diagnosing,
monitoring, and assessing treatment effects for neurological and
neurodegenerative diseases and disorders, such as Alzheimer's
Disease, early in the course of clinical disease or prior to the
onset of brain damage and clinical symptoms. Methods of measuring
the in vivo metabolism of biomolecules produced in the CNS in a
subject are provided.
Inventors: |
Bateman; Randall John;
(Grover, MO) ; Holtzman; David Michael; (St.
Louis, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Washington University |
St. Louis |
MO |
US |
|
|
Family ID: |
37073989 |
Appl. No.: |
14/944311 |
Filed: |
November 18, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13534704 |
Jun 27, 2012 |
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14944311 |
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13005233 |
Jan 12, 2011 |
8232107 |
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13534704 |
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11910463 |
Oct 19, 2007 |
7892845 |
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PCT/US06/12200 |
Apr 4, 2006 |
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13005233 |
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60668634 |
Apr 6, 2005 |
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Current U.S.
Class: |
435/29 |
Current CPC
Class: |
A61K 49/0004 20130101;
G01N 2458/15 20130101; G01N 33/6896 20130101; Y02P 20/582 20151101;
G01N 33/6848 20130101; Y10T 436/143333 20150115; G01N 33/6875
20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Goverment Interests
ACKNOWLEDGEMENT OF FEDERAL RESEARCH SUPPORT
[0002] The present invention was made, at least in part, with
government support under grants P50-AG05681, M01 RR00036, RR000954,
and DK056341 awarded by the National Institutes of Health.
Accordingly, the United States Government has certain rights in
this invention.
Claims
1. A method for measuring the in vivo metabolism of a protein in a
subject, the protein being synthesized in the central nervous
system, the method comprising: (a) administering a labeled moiety
to the subject, the labeled moiety being capable of crossing the
blood brain barrier and incorporating into the protein as the
protein is synthesized in the central nervous system of the
subject; (b) obtaining a biological sample from the subject, the
biological sample comprising a protein fraction labeled with the
moiety and a protein fraction not labeled with the moiety; and (c)
detecting the amount of labeled protein and the amount of unlabeled
protein, wherein the ratio of labeled protein to unlabeled protein
is directly proportional to the metabolism of the protein in the
subject.
2. The method of claim 1, wherein the protein synthesized is from a
neuronal cell, glial cell, or other cell in the central nervous
system.
3. The method of claim 1, wherein the labeled moiety is an atom, or
a molecule with a labeled atom.
4. The method of claim 3, wherein the atom is a non-radioactive
isotope.
5. The method of claim 4, wherein the non-radioactive isotope is
selected from the group consisting of .sup.2H, .sup.13C, .sup.15N,
.sup.17O, .sup.18O, .sup.33S, .sup.34S, and .sup.36S.
6. The method of claim 5, wherein the non-radioactive isotope is a
component of or attached to an amino acid.
7. The method of claim 6, wherein the amino acid is leucine, the
nonradioactive isotope is .sup.13C, and the protein is
amyloid-beta.
8. The method of claim 1, wherein the labeled moiety is
administered to the subject intravenously, intra-arterially,
subcutaneously, intraperitoneally, intramuscularly, or orally.
9. The method of claim 1, further comprising separating the labeled
protein fraction and the unlabeled protein fraction from the
biological sample.
10. The method of claim 9, wherein the protein is separated by
immunoprecipitation.
11. The method of claim 1, wherein the amount of labeled
biomolecule and the amount of unlabeled biomolecule is detected by
mass spectrometry.
12. A kit for detecting the in vivo metabolism of a protein in a
subject, the kit comprising: (a) a labeled amino acid; (b) means
for administering the labeled amino acid to the subject, whereby
the labeled amino acid is capable of crossing the blood brain
barrier and incorporating into and labeling a protein as the
protein is being synthesized in the central nervous system of the
subject; (c) means for obtaining a biological sample at regular
time intervals from the subject, the biological sample comprising a
labeled protein fraction and an unlabeled protein fraction; and (d)
instructions for detecting and determining the ratio of labeled to
unlabeled protein over time so that the in vivo metabolism may be
calculated.
13. The kit of claim 12, wherein the protein synthesized is from a
neuronal cell, glial cell, or other cell in the central nervous
system.
14. The kit of claim 12, wherein the protein is selected from the
group consisting of amyloid-beta, apolipoprotein E, apolipoprotein
J, synuclein, soluble amyloid precursor protein, Tau, alpha-2
macroglobulin, S100B, myelin basic protein, an interleukin, and
TNF.
15. The kit of claim 12, wherein the labeled amino acid has a
radioactive or a non-radioactive atom.
16. The kit of claim 15, wherein the non-radioactive isotope is
selected from the group consisting of .sup.2H, .sup.13C, .sup.15N,
.sup.17O, .sup.18O, .sup.33S, .sup.34S, and .sup.36S.
17. The kit of claim 15, wherein the amino acid is leucine, the
non-radioactive isotope is .sup.13C, and the protein is
amyloid-beta.
18. The kit of claim 12, wherein the labeled amino acid is
administered to the subject intravenously, intra-arterially,
subcutaneously, intraperitoneally, intramuscularly, or orally.
19. The kit of claim 12, wherein the ratio of labeled protein to
unlabeled protein is determined from the amounts of labeled and
unlabeled protein detected by mass spectrometry.
20. The kit of claim 12, wherein the metabolic index comprises the
fractional synthesis rate (FSR) and the fractional clearance rate
(FCR).
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/224,933, filed Mar. 25, 2014, which is a is a continuation
of U.S. application Ser. No. 13/534,704, filed Jun. 27, 2012, which
is a divisional of U.S. application Ser. No. 13/005,233, filed Jan.
12, 2011, which issued as U.S. Pat. No. 8,232,107 on Jul. 31, 2012,
which is itself a divisional of U.S. application Ser. No.
11/910,463, filed Oct. 19, 2007, which issued as U.S. Pat. No.
7,892,845 on Feb. 22, 2011, which is a US National of PCT
Application PCT/US2006/012200, filed Apr. 4, 2006, which claims the
priority of U.S. provisional Application No. 60/668,634, filed Apr.
6, 2005, each of which is hereby incorporated by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The invention relates to methods for the diagnosis and
treatment of neurological and neurodegenerative diseases,
disorders, and associated processes. The invention also relates to
a method for measuring the metabolism of central nervous system
derived biomolecules in a subject in vivo.
BACKGROUND OF INVENTION
Alzheimer's Disease
[0004] Alzheimer's Disease (AD) is the most common cause of
dementia and is an increasing public health problem. It is
currently estimated to afflict 5 million people in the United
States, with an expected increase to 13 million by the year 2050
(Herbert et al 2001, Alzheimer Dis. Assoc. Disord. 15(4): 169-173).
AD, like other central nervous system (CNS) degenerative diseases,
is characterized by disturbances in protein production,
accumulation, and clearance. In AD, dysregulation in the metabolism
of the protein, amyloid-beta (A.beta.), is indicated by a massive
buildup of this protein in the brains of those with the disease. AD
leads to loss of memory, cognitive function, and ultimately
independence. It takes a heavy personal and financial toll on the
patient and the family. Because of the severity and increasing
prevalence of this disease in the population, it is urgent that
better treatments be developed.
[0005] Currently, there are some medications that modify symptoms,
however, there are no disease-modifying treatments.
Disease-modifying treatments will likely be most effective when
given before the onset of permanent brain damage. However, by the
time clinical diagnosis of AD is made, extensive neuronal loss has
already occurred (Price et al. 2001, Arch. Neurol. 58(9):
1395-1402). Therefore, a way to identify those at risk of
developing AD would be most helpful in preventing or delaying the
onset of AD. Currently, there are no means of identifying the
pathophysiologic changes that occur in AD before the onset of
clinical symptoms or of effectively measuring the effects of
treatments that may prevent the onset or slow the progression of
the disease.
[0006] A need exists, therefore, for a sensitive, accurate, and
reproducible method for measuring the in vivo metabolism of
biomolecules in the CNS. In particular, a method is needed for
measuring the in vivo fractional synthesis rate and clearance rate
of proteins associated with a neurodegenerative disease, e.g., the
metabolism of A.beta. in AD.
SUMMARY OF INVENTION
[0007] An aspect of the current invention is the provision of means
for diagnosing and monitoring the advent and progress of
neurological and neurodegenerative diseases, such as AD, prior to
the onset of brain damage and clinical symptoms. Another aspect of
the invention provides means for monitoring the effects of the
treatment of neurological and neurodegenerative diseases, such as
AD.
[0008] A further aspect of the invention provides methods for
measuring the in vivo metabolism (e.g., the rate of synthesis, the
rate of clearance) of neurally derived biomolecules.
[0009] An additional aspect of the invention encompasses kits for
measuring the in vivo metabolism of neurally derived proteins in a
subject, whereby the metabolism of the protein may be used as a
predictor of a neurological or a neurodegenerative disease, an
monitor of the progression of the disease, or an indicator in the
effectiveness of a treatment for the disease.
[0010] Other aspects and iterations of the invention are described
in more detail herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 depicts a schematic illustrating the processing of
amyloid precursor protein (APP) into amyloid-.beta. (A.beta.)
within a cell. Leucines (L), one of the possible labeling sites,
are indicated in black. The amino acid sequence of A.beta. (SEQ ID
NO:1) is shown at the bottom, with the trypsin digest sites
indicated to demonstrate the fragments that were analyzed by mass
spectrometry.
[0012] FIG. 2 depicts a mass spectrometer plot showing the
separation of the amyloid-.beta. peptides. A.beta. peptides were
immunoprecipitated from human CSF with the central domain
anti-A.beta. antibody, m266, and the eluted A.beta. was subjected
to mass spectrometry. Mass spectral peaks are labeled with their
corresponding peptide variants; A.beta..sub.38, A.beta..sub.39,
A.beta..sub.40, and A.beta..sub.42.
[0013] FIG. 3A-B presents mass spectrometer plots illustrating the
shift in molecular weight of the .sup.13C-labeled A.beta..sub.17-28
fragment. In (A), unlabeled media from a human neuroglioma cell
line producing A.beta. in vitro was collected and
immunoprecipitated. Amyloid-beta peptides were then cleaved with
trypsin at sites 5, 16, and 28 (see FIG. 1) producing the two
fragment envelopes shown at masses 1325 and 1336. Note the two mass
envelopes of A.beta. fragments A.beta..sub.17-28 (1325) and
A.beta..sub.6-16 (1336) showing the statistical distribution of
natural isotopes in unlabeled A.beta.. Also, note there is no
signal at mass of 1331, where the labeled signal would be. In (B),
media from human neuroglioma cells cultured for 24 hours in the
presence of .sup.13C.sub.6-leucine was collected and A.beta. was
immunoprecipitated and cleaved with trypsin to produce the fragment
envelopes shown at masses 1325, 1331, and 1336. Note the shift of
mass (arrow) of A.beta..sub.17-28 from 1325 to 1331 that
demonstrates the .sup.13C.sub.6-leucine labeled A.beta. fragment
(A.beta.*.sub.17-28). A.beta..sub.6-16 does not contain a leucine,
and so is not labeled or mass shifted. A minor amount of
A.beta..sub.17-28 remains unlabeled.
[0014] FIG. 4 depicts a graph showing a standard curve of the
labeling of A.beta. in vitro. A sample of labeled cultured media
was serially diluted to generate a standard curve to test the
linearity and variability of the measurement technique. The A.beta.
was precipitated from the media, trypsin digested, and the
fragments were analyzed on a Liquid Chromatography Electro-Spray
Injection (LC-ESI) mass spectrometer and the tandem mass spectra
ions were quantitated using custom written software. The software
summed both the labeled and the unlabeled tandem ions and
calculated the ratio of labeled to total A.beta.. The percent
labeled A.beta. versus the predicted value is shown with a linear
regression line. Note the good linear fit, in addition to the low
deviation.
[0015] FIG. 5 depicts a schematic illustrating the in vivo labeling
protocol. Shown is a diagram of participant with an intravenous
catheter in either antecubital vein and a lumbar catheter in the
L3-4 intrathecal space. In one IV, .sup.13C.sub.6-labeled leucine
was infused at a rate of 1.8 to 2.5 mg/kg/hr for 9 or 12 hours,
after an initial bolus of 2 mg/kg. Twelve ml of plasma was obtained
through the other IV every hour for the first 16 hours and every
other hour thereafter as depicted. Six ml of CSF was obtained
through the lumbar catheter every hour. Each sample was then
processed by immunoprecipitation of A.beta., trypsin digestion, and
LC-ESI-MS analysis to determine the percent of labeled A.beta. at
each time point.
[0016] FIG. 6A-B depicts mass spectrometer plots demonstrating the
MS/MS ions of labeled and unlabeled amyloid-.beta.. Human CSF was
collected after intravenous infusion of .sup.13C.sub.6-leucine.
Representative spectra of unlabeled and labeled A.beta..sub.17-28
(LVFFAEDVGSNK (SEQ ID NO:2)) are shown in (A) and (B),
respectively. The spectra were obtained using MS/MS analysis of
unlabeled parent ion A.beta..sub.17-28 at m/z 663.3 or labeled
parent ion A.beta..sub.17-28 at m/z 666.3. Note the MS/MS ions
containing .sup.13C-leucine (A.beta..sub.17) are mass shifted by 6
Daltons demonstrating the labeled leucine. The A.beta. ions without
leucine at position 17 are not labeled and are not mass shifted by
6 Daltons.
[0017] FIG. 7 depicts a graph showing a standard curve of the
labeling of A.beta. in vivo. A sample of labeled human CSF was
serially diluted with unlabeled human CSF to generate a standard
curve to quantify the accuracy and precision of the measurement
technique for in vivo labeled A.beta. in human CSF. The A.beta. was
precipitated from the CSF, trypsin digested, and the A.beta.
fragments were analyzed on a LC-ESI mass spectrometer and the
tandem mass spectra ions were quantitated using custom written
software. The software summed both the labeled and the unlabeled
tandem ions and calculated the ratio of labeled to total A.beta..
The predicted percent labeled A.beta. versus the measured value is
shown with a linear regression line. Note the good linear fit.
[0018] FIG. 8 depicts a graph illustrating the A.beta. metabolism
curves of three participants. Each participant started the labeled
leucine infusion at time zero and continued for 9 (squares) or 12
hours (triangles and circles). Hourly samples of CSF were obtained
through a lumbar catheter. A.beta. was immunoprecipitated and
trypsin digested. The percent of labeled A.beta. was determined by
measuring labeled and unlabeled tandem mass spectra ions on a
LC-ESI mass spectrometer as described above.
[0019] FIG. 9 depicts a graph illustrating the ratio of labeled
leucine in the CSF and blood from a participant during a 36-hour
study. The CSF and plasma labeled leucine levels reached near
steady state within an hour of the initial bolus of 2 mg/kg. After
the infusion of labeled leucine into the bloodstream was stopped at
9 hours, there was an exponential decay in labeled leucine levels.
The plasma level of labeled leucine was about 4% higher than the
CSF labeled leucine levels during the infusion period.
[0020] FIG. 10 depicts a graph illustrating the mean ratio of
labeled to unlabeled A.beta. in CSF from 6 participants over 36
hours. The labeled to unlabeled A.beta. metabolism curves were
averaged and the mean for each time point is shown +/-SEM. Each
participant was labeled for 9 or 12 hours, while sampling occurred
hourly from 0 to 12, 24, or 36 hours. There was no detectable
incorporation of label during the first 4 hours, followed by an
increase in percent labeled A.beta. that plateaued to near steady
state labeled leucine levels (.about.10%) before decreasing over
the last 12 hours of the study.
[0021] FIG. 11A-F depicts graphs showing the A.beta. metabolism
curves from 3 participants with 9-hour label infusion and 36 hour
sampling. (A, B, C) depict calculation of the fractional synthesis
rate (FSR), which was calculated by the slope of increasing labeled
A.beta. divided by the predicted steady state value. The predicted
steady state value was estimated as the average CSF labeled leucine
measured during labeling. The slope was defined to start after the
4 hours lag time when there was no increase in labeled A.beta. and
ending 9 hours later (solid diamonds). (D, E, F) show the
calculation of the fractional clearance rate (FCR), which was
calculated by the slope of the natural logarithm of percent labeled
A.beta. from hours 24 to 36 (solid diamonds). (A) and (D)
Participant 8; (B) and (E) Participant 9; (C) and (F) Participant
10.
[0022] FIG. 12 depicts a graph illustrating the average FSR and
FCR. The average A.beta. FSR of 6 participants and the average
A.beta. FCR of 3 participants is shown with standard deviation.
[0023] FIG. 13 depicts an illustration of the protocol for
quantifying ApoE from human CSF. ApoE from human CSF or astrocyte
media is bound to an anti-ApoE antibody (WUE4) coupled to sepharose
beads, and digested with trypsin. The supernatant containing ApoE
peptides is analyzed by LC-MS.
[0024] FIG. 14 depicts a graph showing the standard curve of
.sup.13C-Leu-labeled ApoE peptide SWFEPLVEDMQR (SEQ ID NO:3).
Stably-transfected mouse astrocytes expressing human ApoE were
labeled with .sup.13C.sub.6-Leu. ApoE was affinity purified from
cell media, digested with trypsin, and analyzed by nanoLC tandem
MS.
[0025] FIG. 15 depicts a graph showing the incorporation of
.sup.13C-Leu into CNS-derived ApoE. ApoE was affinity purified from
CSF of a young normal control participant infused with
.sup.13C.sub.6-Leu, and analyzed by nanoLC tandem MS. Fractional
synthesis rate (FSR) was calculated using the slope of the linear
regression divided by the .sup.13C-Leu precursor enrichment in CSF
(.sup.13C-Leu=9.86%).
[0026] FIG. 16 depicts a graph showing the incorporation of
.sup.13C-Leu and .sup.13C-Phe into CNS-derived ApoE. ApoE was
affinity purified from CSF of a young normal control participant
infused with .sup.13C.sub.6-Leu and .sup.13C.sub.6-Phe, and
analyzed by nanoLC tandem MS. Fractional synthesis rate (FSR) was
calculated using the slope of the linear regression divided by the
.sup.13C-Leu or .sup.13C-Phe precursor enrichment in CSF
(.sup.13C.sub.6-Leu=11.5% or .sup.13C.sub.6-Phe=22.3%).
[0027] FIG. 17 depicts a graph showing the percent labeled protein
over time. Soluble APP has a much slower production and clearance
rate compared to A.beta., as indicated by the slower rise in
plateau and clearance of labeled sAPP. Open circles=A.beta., Black
triangles=soluble amyloid precursor protein (sAPP).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0028] The present invention relates to methods for the early
diagnosis, prognosis, and assessment of treatment effectiveness of
neurological and neurodegenerative diseases, disorders, and
processes in a subject. Specifically, the invention provides a
method for diagnosis prior to and after the onset of clinical
symptoms associated with neural damage by determining the synthesis
and clearance rates of CNS derived biomolecules. It also provides a
method to assess whether a treatment is affecting the production or
clearance rate of biomolecules in the CNS relevant to neurological
and neurodegenerative diseases. The usefulness of this invention
will be evident to those of skill in the art in that early
diagnosis provides the opportunity for early treatment and,
possibly, the prevention of significant neural damage in those
afflicted. This invention provides a method for monitoring the
effectiveness of disease-modifying therapies or for the screening
of therapies likely to have a significant effect directly in
humans. For example, one may determine if a treatment alters the
synthesis or clearance rate of a biomolecule derived from the CNS.
Ultimately, this method may provide a predictive test for the
advent of neurological and neurodegenerative diseases, provide a
method for more accurate diagnosis, and a means to monitor the
progression of such diseases.
I. Methods for Monitoring the In Vivo Metabolism of Neurally
Derived Biomolecules
[0029] The current invention provides methods for measuring the in
vivo metabolism of neurally derived biomolecules. By using this
method, one skilled in the art may be able to study possible
changes in the metabolism (synthesis and clearance) of a relevant
neurally derived biomolecule implicated in a particular disease
state. In addition, the invention permits the measurement of the
pharmacodynamic effects of disease-modifying therapeutics in a
subject.
[0030] In particular, this invention provides a method to label a
biomolecule as it is synthesized in the central nervous system in
vivo; to collect a biological sample containing labeled and
unlabeled biomolecules; and a means to measure the labeling of the
biomolecule over time. These measurements may be used to calculate
metabolic parameters, such as the synthesis and clearance rates
within the CNS, as well as others.
(a) Degenerative Diseases
[0031] Alzheimer's Disease (AD) is a debilitating disease
characterized by accumulation of amyloid plaques in the central
nervous system (CNS) resulting from increased production, decreased
clearance, or both of amyloid-.beta. (A.beta.) protein. The
inventors have developed a method for measuring the in vivo
metabolism of A.beta. in a human by measuring the in vivo synthesis
and clearance rates of A.beta. in the cerebrospinal fluid (CSF) or
plasma. The in vivo synthesis and clearance rates of A.beta. then
may be used to assess whether a subject has an alteration in
A.beta. synthesis or clearance as compared to a control group. Such
a comparison may allow for the diagnosis of AD early in its course,
i.e., prior to the onset of clinical symptoms and significant
neural damage. In addition, the present invention provides means to
determine whether apolipoprotein E (ApoE) causes a change in
A.beta. metabolism. This determination may provide new insights
about why particular ApoE genotypes are a risk factor for AD.
[0032] Those of skill in the art will appreciate that, while AD is
the exemplary disease that may be diagnosed or monitored by the
invention, the invention is not limited to AD. It is envisioned
that the method of the invention may be used in the diagnosis and
assessment of treatment efficacy of several neurological and
neurodegenerative diseases, disorders, or processes including, but
not limited to, Parkinson's Disease, stroke, frontal temporal
dementias (FTDs), Huntington's Disease, progressive supranuclear
palsy (PSP), corticobasal degeneration (CBD), aging-related
disorders and dementias, Multiple Sclerosis, Prion Diseases (e.g.
Creutzfeldt-Jakob Disease, bovine spongiform encephalopathy or Mad
Cow Disease, and scrapie), Lewy Body Disease, and Amyotrophic
Lateral Sclerosis (ALS or Lou Gehrig's Disease). It is also
envisioned that the method of the invention may be used to study
the normal physiology, metabolism, and function of the CNS.
[0033] Neurological and neurodegenerative diseases are most common
in subjects of advanced age. For example, 10% of people over the
age of 65 have AD, while about 50% of people over age 85 are
afflicted with AD. Because of the prevalence of neurological and
neurodegenerative diseases among the aging human population and the
health care costs associated with these diseases, it is envisioned
that the in vivo metabolism of biomolecules will be measured in a
human subject, and in particular, in a human subject with advanced
age. Alternatively, the in vivo metabolism of biomolecules may be
measured in other mammalian subjects. In another embodiment, the
subject is a companion animal such as a dog or cat. In another
alternative embodiment, the subject is a livestock animal such as a
cow, pig, horse, sheep or goat. In yet another alternative
embodiment, the subject is a zoo animal. In another embodiment, the
subject is a research animal such as a non-human primate or a
rodent.
(b) Biomolecule
[0034] The present invention provides a method for measuring the
metabolism of a biomolecule derived from the CNS in vivo. The
biomolecule may be a protein, a lipid, a nucleic acid, or a
carbohydrate. The possible biomolecules are only limited by the
ability to label them during in vivo synthesis and collect a sample
from which their metabolism may be measured. In a preferred
embodiment, the biomolecule is a protein synthesized in the CNS.
For example, the protein to be measured may be, but is not limited
to, amyloid-.beta. (A.beta.) and its variants, soluble amyloid
precursor protein (APP), apolipoprotein E (isoforms 2, 3, or 4),
apolipoprotein J (also called clusterin), Tau (another protein
associated with AD), glial fibrillary acidic protein, alpha-2
macroglobulin, synuclein, S100B, Myelin Basic Protein (implicated
in multiple sclerosis), prions, interleukins, TDP-43, superoxide
dismutase-1, huntingtin, and tumor necrosis factor (TNF).
Additional biomolecules that may be targeted include products of,
or proteins or peptides that interact with GABAergic neurons,
noradrenergic neurons, histaminergic neurons, seratonergic neurons,
dopaminergic neurons, cholinergic neurons, and glutaminergic
neurons.
[0035] In an exemplary embodiment, the protein whose in vivo
metabolism is measured may be amyloid-beta (A.beta.) protein. In a
further embodiment, other variants of A.beta. (e.g., 40, 42, 38 or
others) may be measured. In yet a further embodiment, digestion
products of A.beta. (e.g., A.beta..sub.6-16, A.beta..sub.17-28) may
be measured.
(c) Labeled Moiety
[0036] Several different moieties may be used to label the
biomolecule of interest. Generally speaking, the two types of
labeling moieties typically utilized in the method of the invention
are radioactive isotopes and non-radioactive (stable) isotopes. In
a preferred embodiment, non-radioactive isotopes may be used and
measured by mass spectrometry. Preferred stable isotopes include
deuterium .sup.2H, .sup.13C, .sup.15N, .sup.17 or 18O, .sup.33, 34,
or 36S, but it is recognized that a number of other stable isotope
that change the mass of an atom by more or less neutrons than is
seen in the prevalent native form would also be effective. A
suitable label generally will change the mass of the biomolecule
under study such that it can be detected in a mass spectrometer. In
one embodiment, the biomolecule to be measured is a protein, and
the labeled moiety is an amino acid comprising a non-radioactive
isotope (e.g., .sup.13C). In another embodiment, the biomolecule to
be measured is a nucleic acid, and the labeled moiety is a
nucleoside triphosphate comprising a non-radioactive isotope (e.g.,
.sup.15N). Alternatively, a radioactive isotope may be used, and
the labeled biomolecules may be measured with a scintillation
counter rather than a mass spectrometer. One or more labeled
moieties may be used simultaneously or in sequence.
[0037] In a preferred embodiment, when the method is employed to
measure the metabolism of a protein, the labeled moiety typically
will be an amino acid. Those of skill in the art will appreciate
that several amino acids may be used to provide the label of a
biomolecule. Generally, the choice of amino acid is based on a
variety of factors such as: (1) The amino acid generally is present
in at least one residue of the protein or peptide of interest. (2)
The amino acid is generally able to quickly reach the site of
protein synthesis and rapidly equilibrate across the blood-brain
barrier. Leucine is a preferred amino acid to label proteins that
are synthesized in the CNS, as demonstrated in Examples 1 and 2.
(3) The amino acid ideally may be an essential amino acid (not
produced by the body), so that a higher percent of labeling may be
achieved. Non-essential amino acids may also be used; however,
measurements will likely be less accurate. (4) The amino acid label
generally does not influence the metabolism of the protein of
interest (e.g., very large doses of leucine may affect muscle
metabolism). And (5) availability of the desired amino acid (i.e.,
some amino acids are much more expensive or harder to manufacture
than others). In one embodiment, .sup.13C.sub.6-phenylalanine,
which contains six .sup.13C atoms, is used to label a neurally
derived protein. In a preferred embodiment, .sup.13C.sub.6-leucine
is used to label a neurally derived protein. In an exemplary
embodiment, .sup.13C.sub.6-leucine is used to label
amyloid-.beta..
[0038] There are numerous commercial sources of labeled amino
acids, both non-radioactive isotopes and radioactive isotopes.
Generally, the labeled amino acids may be produced either
biologically or synthetically. Biologically produced amino acids
may be obtained from an organism (e.g., kelp/seaweed) grown in an
enriched mixture of .sup.13C, .sup.15N, or another isotope that is
incorporated into amino acids as the organism produces proteins.
The amino acids are then separated and purified. Alternatively,
amino acids may be made with known synthetic chemical
processes.
(d) Administration of the Labeled Moiety
[0039] The labeled moiety may be administered to a subject by
several methods. Suitable methods of administration include
intravenously, intra-arterially, subcutaneously, intraperitoneally,
intramuscularly, or orally. In a preferred embodiment, the labeled
moiety is a labeled amino acid, and the labeled amino acid is
administered by intravenous infusion. In another embodiment,
labeled amino acids may be orally ingested.
[0040] The labeled moiety may be administered slowly over a period
of time or as a large single dose depending upon the type of
analysis chosen (e.g., steady state or bolus/chase). To achieve
steady-state levels of the labeled biomolecule, the labeling time
generally should be of sufficient duration so that the labeled
biomolecule may be reliably quantified. In one embodiment, the
labeled moiety is labeled leucine and the labeled leucine is
administered intravenously for nine hours. In another embodiment,
the labeled leucine is administered intravenously for 12 hours.
[0041] Those of skill in the art will appreciate that the amount
(or dose) of the labeled moiety can and will vary. Generally, the
amount is dependent on (and estimated by) the following factors.
(1) The type of analysis desired. For example, to achieve a steady
state of about 15% labeled leucine in plasma requires about 2
mg/kg/hr over 9 hr after an initial bolus of 2 mg/kg over 10 min.
In contrast, if no steady state is required, a large bolus of
labeled leucine (e.g., 1 or 5 grams of labeled leucine) may be
given initially. (2) The protein under analysis. For example, if
the protein is being produced rapidly, then less labeling time may
be needed and less label may be needed--perhaps as little as 0.5
mg/kg over 1 hour. However, most proteins have half-lives of hours
to days and, so more likely, a continuous infusion for 4, 9 or 12
hours may be used at 0.5 mg/kg to 4 mg/kg. And (3) the sensitivity
of detection of the label. For example, as the sensitivity of label
detection increases, the amount of label that is needed may
decrease.
[0042] Those of skill in the art will appreciate that more than one
label may be used in a single subject. This would allow multiple
labeling of the same biomolecule and may provide information on the
production or clearance of that biomolecule at different times. For
example, a first label may be given to subject over an initial time
period, followed by a pharmacologic agent (drug), and then a second
label may be administered. In general, analysis of the samples
obtained from this subject would provide a measurement of
metabolism before AND after drug administration, directly measuring
the pharmacodynamic effect of the drug in the same subject.
[0043] Alternatively, multiple labels may be used at the same time
to increase labeling of the biomolecule, as well as obtain labeling
of a broader range of biomolecules.
(e) Biological Sample
[0044] The method of the invention provides that a biological
sample be obtained from a subject so that the in vivo metabolism of
the labeled biomolecule may be determined. Suitable biological
samples include, but are not limited to, cerebral spinal fluid
(CSF), blood plasma, blood serum, urine, saliva, perspiration, and
tears. In one embodiment of the invention, biological samples are
taken from the CSF. In an alternate embodiment, biological samples
are collected from the urine. In a preferred embodiment, biological
samples are collected from the blood.
[0045] Cerebrospinal fluid may be obtained by lumbar puncture with
or without an indwelling CSF catheter (a catheter is preferred if
multiple collections are made over time). Blood may be collected by
veni-puncture with or without an intravenous catheter. Urine may be
collected by simple urine collection or more accurately with a
catheter. Saliva and tears may be collected by direct collection
using standard good manufacturing practice (GMP) methods.
[0046] In general when the biomolecule under study is a protein,
the invention provides that a first biological sample be taken from
the subject prior to administration of the label to provide a
baseline for the subject. After administration of the labeled amino
acid or protein, one or more samples generally would be taken from
the subject. As will be appreciated by those of skill in the art,
the number of samples and when they would be taken generally will
depend upon a number of factors such as: the type of analysis, type
of administration, the protein of interest, the rate of metabolism,
the type of detection, etc.
[0047] In one embodiment, the biomolecule is a protein and samples
of blood and CSF are taken hourly for 36 hours. Alternatively,
samples may be taken every other hour or even less frequently. In
general, biological samples obtained during the first 12 hours of
sampling (i.e., 12 hrs after the start of labeling) may be used to
determine the rate of synthesis of the protein, and biological
samples taken during the final 12 hours of sampling (i.e., 24-36
hrs after the start of labeling) may be used to determine the
clearance rate of the protein. In another alternative, one sample
may be taken after labeling for a period of time, such as 12 hours,
to estimate the synthesis rate, but this may be less accurate than
multiple samples. In yet a further alternative, samples may be
taken from an hour to days or even weeks apart depending upon the
protein's synthesis and clearance rate.
(f) Detection
[0048] The present invention provides that detection of the amount
of labeled biomolecule and the amount of unlabeled biomolecule in
the biological samples may be used to determine the ratio of
labeled biomolecule to unlabeled biomolecule. Generally, the ratio
of labeled to unlabeled biomolecule is directly proportional to the
metabolism of the biomolecule. Suitable methods for the detection
of labeled and unlabeled biomolecules can and will vary according
to the biomolecule under study and the type of labeled moiety used
to label it. If the biomolecule of interest is a protein and the
labeled moiety is a non-radioactively labeled amino acid, then the
method of detection typically should be sensitive enough to detect
changes in mass of the labeled protein with respect to the
unlabeled protein. In a preferred embodiment, mass spectrometry is
used to detect differences in mass between the labeled and
unlabeled proteins. In one embodiment, gas chromatography mass
spectrometry is used. In an alternate embodiment, MALDI-TOF mass
spectrometry is used. In a preferred embodiment, high-resolution
tandem mass spectrometry is used.
[0049] Additional techniques may be utilized to separate the
protein of interest from other proteins and biomolecules in the
biological sample. As an example, immunoprecipitation may be used
to isolate and purify the protein of interest before it is analyzed
by mass spectrometry. Alternatively, mass spectrometers having
chromatography setups may be used to isolate proteins without
immunoprecipitation, and then the protein of interest may be
measured directly. In an exemplary embodiment, the protein of
interest is immunoprecipitated and then analyzed by a liquid
chromatography system interfaced with a tandem MS unit equipped
with an electrospray ionization source (LC-ESI-tandem MS).
[0050] The invention also provides that multiple proteins or
peptides in the same biological sample may be measured
simultaneously. That is, both the amount of unlabeled and labeled
protein (and/or peptide) may be detected and measured separately or
at the same time for multiple proteins. As such, the invention
provides a useful method for screening changes in synthesis and
clearance of proteins on a large scale (i.e.
proteomics/metabolomics) and provides a sensitive means to detect
and measure proteins involved in the underlying pathophysiology.
Alternatively, the invention also provides a means to measure
multiple types of biomolecules. In this context, for example, a
protein and a carbohydrate may be measured simultaneously or
sequentially.
(g) Metabolism Analysis
[0051] Once the amount of labeled and unlabeled biomolecule has
been detected in a biological sample, the ratio or percent of
labeled biomolecule may be determined. If the biomolecule of
interest is a protein and the amount of labeled and unlabeled
protein has been measured in a biological sample, then the ratio of
labeled to unlabeled protein may be calculated. Protein metabolism
(synthesis rate, clearance rate, lag time, half-life, etc.) may be
calculated from the ratio of labeled to unlabeled protein over
time. There are many suitable ways to calculate these parameters.
The invention allows measurement of the labeled and unlabeled
protein (or peptide) at the same time, so that the ratio of labeled
to unlabeled protein, as well as other calculations, may be made.
Those of skill in the art will be familiar with the first order
kinetic models of labeling that may be used with the method of the
invention. For example, the fractional synthesis rate (FSR) may be
calculated. The FSR equals the initial rate of increase of labeled
to unlabeled protein divided by the precursor enrichment. Likewise,
the fractional clearance rate (FCR) may be calculated. In addition,
other parameters, such as lag time and isotopic tracer steady
state, may be determined and used as measurements of the protein's
metabolism and physiology. Also, modeling may be performed on the
data to fit multiple compartment models to estimate transfer
between compartments. Of course, the type of mathematical modeling
chosen will depend on the individual protein synthetic and
clearance parameters (e.g., one-pool, multiple pools, steady state,
non-steady-state, compartmental modeling, etc.).
[0052] The invention provides that the synthesis of protein is
typically based upon the rate of increase of the labeled/unlabeled
protein ratio over time (i.e., the slope, the exponential fit
curve, or a compartmental model fit defines the rate of protein
synthesis). For these calculations, a minimum of one sample is
typically required (one could estimate the baseline label), two are
preferred, and multiple samples are more preferred to calculate an
accurate curve of the uptake of the label into the protein (i.e.,
the synthesis rate).
[0053] Conversely, after the administration of labeled amino acid
is terminated, the rate of decrease of the ratio of labeled to
unlabeled protein typically reflects the clearance rate of that
protein. For these calculations, a minimum of one sample is
typically required (one could estimate the baseline label), two are
preferred, and multiple samples are more preferred to calculate an
accurate curve of the decrease of the label from the protein over
time (i.e., the clearance rate). The amount of labeled protein in a
biological sample at a given time reflects the synthesis rate
(i.e., production) or the clearance rate (i.e., removal or
destruction) and is usually expressed as percent per hour or the
mass/time (e.g., mg/hr) of the protein in the subject.
[0054] In an exemplary embodiment, as illustrated in the examples,
the in vivo metabolism of amyloid-.beta. (A.beta.) is measured by
administering labeled leucine to a subject over 9 hours and
collecting biological samples at regular intervals over 36 hours.
The biological sample may be collected from blood plasma or CSF.
The amount of labeled and unlabeled A.beta. in the biological
samples is typically determined by immunopreciptitation followed by
LC-ESI-tandem MS. From these measurements, the ratio of labeled to
unlabeled A.beta. may be determined, and this ratio permits the
determination of metabolism parameters, such as rate of synthesis
and rate of clearance of A.beta..
II. Kits for Diagnosing or Monitoring the Progression or Treatment
of Neurological and Neurodegenerative Diseases
[0055] The current invention provides kits for diagnosing or
monitoring the progression or treatment of a neurological or
neurodegenerative disease by measuring the in vivo metabolism of a
central nervous system-derived protein in a subject. Generally, a
kit comprises a labeled amino acid, means for administering the
labeled amino acid, means for collecting biological samples over
time, and instructions for detecting and determining the ratio of
labeled to unlabeled protein so that a metabolic index may be
calculated. The metabolic index then may be compared to a metabolic
index of a normal, healthy individual or compared to a metabolic
index from the same subject generated at an earlier time. These
comparisons may enable a practitioner to predict the advent of a
neurological or neurodegenerative disease, diagnose the onset of a
neurological or neurodegenerative disease, monitor the progression
of a neurological or neurodegenerative disease, or verify the
effectiveness of a treatment for a neurological or
neurodegenerative disease. In a preferred embodiment, the kit
comprises .sup.13C.sub.6-leucine or .sup.13C.sub.6-phenylalanine,
the protein to be labeled is A.beta., and the disease to be
assessed is AD.
Definitions
[0056] Unless defined otherwise, all technical and scientific terms
used herein have the meaning commonly understood by a person
skilled in the art to which this invention belongs. The following
references provide one of skill with a general definition of many
of the terms used in this invention: Singleton et al., Dictionary
of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge
Dictionary of Science and Technology (Walker ed., 1988); The
Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer
Verlag (1991); and Hale & Marham, The Harper Collins Dictionary
of Biology (1991). As used herein, the following terms have the
meanings ascribed to them unless specified otherwise.
[0057] "Clearance rate" refers to the rate at which the biomolecule
of interest is removed.
[0058] "Fractional clearance rate" or FCR is calculated as the
natural log of the ratio of labeled biomolecule over a specified
period of time.
[0059] "Fractional synthesis rate" or FSR is calculated as the
slope of the increasing ratio of labeled biomolecule over a
specified period of time divided by the predicted steady state
value of the labeled precursor.
[0060] "Isotope" refers to all forms of a given element whose
nuclei have the same atomic number but have different mass numbers
because they contain different numbers of neutrons. By way of a
non-limiting example, .sup.12C and .sup.13C are both stable
isotopes of carbon.
[0061] "Lag time" generally refers to the delay of time from when
the biomolecule is first labeled until the labeled biomolecule is
detected.
[0062] "Metabolism" refers to any combination of the synthesis,
transport, breakdown, modification, or clearance rate of a
biomolecule.
[0063] "Metabolic index" refers to a measurement comprising the
fractional synthesis rate (FSR) and the fractional clearance rate
(FCR) of the biomolecule of interest. Comparison of metabolic
indices from normal and diseased individuals may aid in the
diagnosis or monitoring of neurological or neurodegenerative
diseases.
[0064] "Neurally derived cells" includes all cells within the
blood-brain-barrier including neurons, astrocytes, microglia,
choroid plexus cells, ependymal cells, other glial cells, etc.
[0065] "Steady state" refers to a state during which there is
insignificant change in the measured parameter over a specified
period of time.
[0066] "Synthesis rate" refers to the rate at which the biomolecule
of interest is synthesized.
[0067] In metabolic tracer studies, a "stable isotope" is a
nonradioactive isotope that is less abundant than the most abundant
naturally occurring isotope.
[0068] "Subject" as used herein means a living organism having a
central nervous system. In particular, the subject is a mammal.
Suitable subjects include research animals, companion animals, farm
animals, and zoo animals. The preferred subject is a human.
EXAMPLES
[0069] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples that
follow represent techniques discovered by the inventors to function
well in the practice of the invention, and thus can be considered
to constitute preferred modes for its practice. However, those of
skill in the art should, in light of the present disclosure,
appreciate that many changes can be made in the specific
embodiments which are disclosed and still obtain a like or similar
result without departing from the spirit and scope of the
invention.
Example 1
Measurement of Amyloid-.beta. Metabolism In Vitro Rationale
[0070] Biochemical, genetic, and animal model evidence implicates
A.beta. (FIG. 1) as a pathogenic peptide in AD. In order to develop
a method to measure A.beta. in vivo labeling, an in vitro system
was designed using four basic steps: 1) label A.beta. in vitro in
culture, 2) isolate A.beta. from other labeled proteins, 3)
specifically cleave A.beta. into fragments that could be analyzed
for the label, and 4) quantitate the labeled and unlabeled
fragments.
Amyloid-.beta. Immunoprecipitation and Cleavage
[0071] First, a method was developed for isolating and measuring
unlabeled A.beta. from biologic fluids. A.beta. was
immunoprecipitated from samples of CSF or cell culture media using
a highly specific monoclonal antibody (m266), which recognizes the
central domain (residues 13-28) of the molecules. Antibody beads
were prepared by covalently binding m266 antibody to CNBr sepharose
beads per the manufacturers protocol at a concentration of 10 mg/ml
of m266 antibody. The antibody beads were stored at 4.degree. C. in
a slurry of 50% PBS and 0.02% azide. The immunoprecipitation
mixture was 250 .mu.l of 5.times. RIPA, 12.5 .mu.l of 100.times.
protease inhibitors, and 30 .mu.l of antibody-bead slurry in an
Eppendorf tube. To this, 1 ml of the biological sample was added
and the tube was rotated overnight at 4.degree. C. The beads were
rinsed once with 1.times. RIPA and twice with 25 mM ammonium
bicarbonate. They were aspirated dry after the final rinse and
A.beta. was eluted off the antibody-bead complex using 30 .mu.l of
pure formic acid. A.beta. was directly characterized (molecular
weight and amino acid sequence) using mass spectrometry. Results
were similar to previously published findings (Wang et al. 1996, J
Biol. Chem. 271(50): 31894-31902), as shown in FIG. 2.
[0072] Amyloid-.beta. may be cleaved into smaller fragments by
enzymatic digestion using trypsin. Cleavage of A.beta. by trypsin
produces the A.beta..sub.1-5, A.beta..sub.6-16, A.beta..sub.17-28,
and A.beta..sub.29-40/42 fragments, as depicted in FIG. 1.
Labeling of Amyloid-.beta.
[0073] Second, a method was developed to label newly synthesized
A.beta.. .sup.13C.sub.6-leucine was used as a metabolic label
because leucine equilibrates across the blood brain barrier quickly
via active transport (Smith et al. 1987, J Neurochem 49(5):
1651-1658), is an essential amino acid, does not change the
properties of A.beta., and is safe and nonradioactive. .sup.13C
stable isotopes do not change the chemical or biologic properties
of amino acids or proteins; only the mass weight is increased by
one Dalton for each .sup.13C label. In fact, entire organisms have
been grown on pure .sup.13C without any deleterious effect. The
labeled leucine is incorporated into the amino acid sequence of
A.beta. at positions 17 and 34 (see FIG. 1).
[0074] The naturally occurring isotopes .sup.13C (1.1% of all
carbon) and .sup.15N cause a natural distribution of mass of larger
molecules, including proteins. Due to the size of A.beta. and the
presence of these naturally occurring isotopes, the peptide may be
broken into smaller peptides for direct measurement of the label.
Alternatively, separation may be made using whole undigested
A.beta..
Liquid Chromatography/Mass Spectrometry
[0075] Third, a method to accurately quantitate labeled and
unlabeled A.beta. was developed. For this, a Waters (Milford,
Mass.) capillary liquid chromatography system with auto injector
was interfaced to a Thermo-Finnigan (San Jose, Calif.) LCQ-DECA
equipped with an electrospray ionization source (LC-ESI-tandem MS).
A 5 .mu.l aliquot of each sample was injected onto a Vydac C-18
capillary column (0.3.times.150 mm MS 5 .mu.m column). The
A.beta..sub.17-28 fragment contains one leucine residue and
incorporation of .sup.13C.sub.6-leucine shifts the molecular weight
of the fragment by 6 Daltons. In positive-ion scanning mode,
LC-ESI-MS analysis of trypsin-digested synthetic and
immunoprecipitated A.beta. yielded the expected parent ions at
masses 1325.2 for A.beta..sub.17-28 and 1331.2 for
.sup.13C.sub.6-leucine labeled A.beta..sub.17-28 (FIGS. 3A and 3B).
The percent of labeled A.beta. (A.beta.*) was calculated as the
ratio of all labeled MS/MS ions from labeled A.beta..sub.17-28
divided by all unlabeled MS/MS ions from unlabeled
A.beta..sub.17-28. A custom Microsoft Excel spreadsheet with macros
was used to calculate the ratio as the tracer to tracee ratio (TTR)
of A.beta..sub.17-28 by the following formula:
TTR A .beta. = MS / MS ions A .beta. 17 - 28 * MS / MS ions A
.beta. 17 - 28 ##EQU00001##
[0076] It was concluded that this method provided a highly specific
"fingerprint" of the A.beta. in both labeled and unlabeled forms,
as it quantitated the amounts of each form and determined the amino
acid sequence at the same time. In this way, excellent separation
and specificity of the labeled to unlabeled A.beta. peptide was
achieved. Accuracy and precision were tested by generating a
standard curve from serial dilutions of labeled and unlabeled
culture media (FIG. 4). The linear fit from a range of 0% to 80%
labeled A.beta. serial dilution standard curve gave an R.sup.2 of
0.98 and slope of 0.92. Alternative measuring techniques that were
evaluated included measuring parent ions directly in selective ion
mode only and also using a MALDI-TOF mass spectrometer. However,
these methods were unable to offer the sensitivity and specificity
that was achieved by the LC-ESI using quantitative tandem mass
spectrum analysis.
Amyloid-.beta. In Vitro Labeling
[0077] Human neuroglioma cells that produce A.beta. (Murphy et al.
2000, J Biol. Chem. 275(34): 26277-26284) were grown in the
presence of .sup.13C.sub.6-labeled leucine (Cambridge Isotope
Laboratories, Cambridge, Mass.) or unlabeled leucine. A.beta. was
isolated from the media by immunoprecipitation with m266 antibody
(see above). The eluted A.beta. was digested with trypsin for 4
hours at 37.degree. C., and the fragments were analyzed by LC-ESI
MS. As expected, the A.beta..sub.17-28 fragment of A.beta. isolated
from cells incubated in the presence of unlabeled leucine had a
molecular weight of 1325.2 and the A.beta..sub.17-28 fragment of
A.beta. isolated from .sup.13C.sub.6-labeled leucine incubated
cells had a molecular weight of 1331.2 (FIG. 3B). These findings
indicate that the cells incorporated .sup.13C.sub.6-leucine into
A.beta., confirming that A.beta. synthesized in the presence of
.sup.13C.sub.6-leucine incorporates the labeled amino acid and that
the shift of 6 Daltons in the molecular weight of the
leucine-containing peptide can be distinguished using mass
spectrometry.
[0078] Cell culture media at 4 hours and 24 hours of
.sup.13C.sub.6-leucine labeling were analyzed to determine the
relative amount of labeling that occurs as a function of time. The
4-hour labeling experiment revealed approximately 70% labeling,
while the 24-hour labeling experiment revealed more than 95%
labeling. These findings indicate that within hours after exposure
to the label, amyloid precursor protein (APP) incorporated the
labeled amino acid, and the labeled A.beta. was cleaved from
labeled APP and released into the extracellular space.
Example 2
Measurement of Amyloid-.beta. Metabolism In Vivo Rationale
[0079] Protein production and clearance are important parameters
that are tightly regulated and reflect normal physiology as well as
disease states. Previous studies of protein metabolism in humans
have focused on whole body or peripheral body proteins, but not on
proteins produced in the central nervous system (CNS). No methods
were previously available to quantify protein synthesis or
clearance rates in the CNS of humans. Such a method would be
valuable to assess not only A.beta. synthesis or clearance rates in
humans but also the metabolism of a variety other proteins relevant
to diseases of the CNS. In order to address critical questions
about underlying AD pathogenesis and A.beta. metabolism, a method
for quantifying A.beta. fractional synthesis rate (FSR) and
fractional clearance rate (FCR) in vivo in the CNS of humans was
developed.
Participants and Sampling
[0080] All human studies were approved by the Washington University
Human Studies Committee and the General Clinical Research Center
(GCRC) Advisory Committee. Informed consent was obtained from all
participants. All participants were screened to be in good general
health and without neurologic disease. Seven men and three women
(23-45 yrs old) participated. Each research participant was
admitted to the GCRC at 7:00 AM after an overnight fast from 8 PM
the preceding evening. The GCRC Research Kitchen provided meals
(60% carbohydrate, 20% fat, 20% protein, low leucine diet during
labeled leucine infusion) at 9 AM, 1 PM, and 6 PM and the
participant had free access to water. All food and water
consumption was recorded during the admission by nursing staff and
the GCRC kitchen. One intravenous catheter was placed in an
antecubital vein and used to administer the stable isotope labeled
leucine solution. A second intravenous catheter was placed in the
contra-lateral antecubital vein to obtain blood samples. A
subarachnoid catheter was inserted at the L3-L4 interspace via a
Touhy needle, so that CSF could be sampled without performing
multiple lumbar punctures (Williams, 2002, Neurology 58:
1859-1860). The intravenous catheters were placed by trained
registered nurses and the lumbar catheter was placed by trained
physicians with extensive experience in inserting catheters into
the lumbar subarachnoid space. Blood samples were obtained hourly,
unless the study was 36 hours, in which case, blood was obtained
hourly for the first 16 hours and every other hour thereafter. CSF
samples were obtained hourly throughout the study. See the
schematic of the in vivo experimental protocol presented in FIG. 5.
The participants were encouraged to stay in bed except to use the
restroom.
[0081] .sup.13C.sub.6-leucine (Cambridge Isotope Laboratories) was
dissolved in medical grade normal saline and then filtered through
a 0.22 micron filter the day before each study. The labeled leucine
was infused intravenously using a medical IV pump at a rate of 1.8
to 2.5 mg/kg/hr.
In Vivo Labeling and Quantitation of A.beta. in One Participant
[0082] To determine if labeled A.beta. could be produced and
detected in vivo in a human, a single participant underwent a
24-hour infusion of labeled leucine followed by a lumbar puncture
to obtain CSF. .sup.13C.sub.6-labeled leucine was infused at a rate
of 1.8 mg/kg/hr in one IV. Every hour, 10 ml of plasma was obtained
through the other IV. After 24 hours of continuous infusion, a
single lumbar puncture was performed and 30 ml of CSF was obtained.
A.beta. was immunoprecipitated from the CSF sample, digested with
trypsin, and a 5 .mu.l aliquot of each sample was injected into a
Vydac C-18 capillary column (0.3.times.150 mm MS 5 .mu.m). In
positive-ion scanning mode, LC-ESI-MS analysis of trypsin-digested
synthetic and immunoprecipitated A.beta. yielded the expected
parent ions at masses 1325.2 for A.beta..sub.17-28 and 1331.2 for
.sup.13C.sub.6-leucine labeled A.beta..sub.17-28. To obtain amino
acid sequence and abundance data, these parent ions were subjected
to collision induced dissociation (CID 28%), and tandem MS analysis
of their doubly-charged species ([M+2H].sup.+2; m/z 663.6 and
666.6) were scanned in selected reaction monitoring mode (SRM), so
that the y- and b-series ions generated were used for isotope ratio
quantitation (FIG. 6). In addition, plasma and CSF
.sup.13C.sub.6-leucine were measured to determine the maximum
amount of .sup.13C.sub.6-leucine A.beta. to be expected. The
results demonstrated that unlabeled A.beta..sub.17-28 and
.sup.13C-labeled A.beta..sub.17-28 could be detected and measured
in human CSF.
[0083] The results of the first human participant demonstrated
three significant findings: 1) plasma .sup.13C.sub.6-leucine
averaged 12% of total plasma leucine at an IV infusion rate of 1.8
mg/kg/hour; 2) CSF .sup.13C.sub.6-leucine was measured in CSF
demonstrating a similar level (11.9%) as plasma at 24 hours; and 3)
A.beta. was labeled with .sup.13C.sub.6-leucine in human CSF at
approximately 8% of total A.beta. levels at 24 hours.
[0084] In order to quantify the accuracy and precision of the
measurement technique of in vivo labeled A.beta. in human CSF, a
standard curve was generated from serial dilutions of labeled and
unlabeled human CSF (FIG. 7). The measurement technique was the
same as for the in vitro standard curve (FIG. 4). LC-ESI MS was
used to quantitate the amounts of labeled and unlabeled
A.beta..sub.17-28 in selective ion monitoring mode with tandem mass
spectrometer recording MS-2 ions. The linear fit from a range of 0%
to 8% labeled A.beta. serial dilution standard curve gave an
R.sup.2 of 0.98 and slope of 0.81. From these data, it was
predicted that in vivo samples from human participants will likely
range from 1 to 10% labeling. Note, the 1% measurement in unlabeled
CSF at the Y-axis, whereas 0% was predicted. Due to the baseline
noise of the detection system, it was not possible to measure less
than 1% labeling with this system. It was concluded that A.beta.
can be labeled in vivo in humans and measured with good accuracy
and precision using LC-ESI mass spectrometry.
Pharmacokinetics of In Vivo Labeling
[0085] To ensure that detectable .sup.13C.sub.6-leucine labeling of
A.beta. was achieved and maintained for an adequate period of time
so that steady-state equations could be used to calculate A.beta.
synthesis and clearance rates, the optimal labeling and sampling
times were determined. A range of .sup.13C.sub.6-leucine
intravenous infusion dosages (1.8 to 2.5 mg/kg/hr), durations (6,
9, or 12 hours) and CSF/blood sampling times (from 12 to 36 hours
duration) were tested (see Table 1).
TABLE-US-00001 TABLE 1 Participant Labeling and Sampling Parameters
Participant Infusion Dosage Infusion CSF/blood Number (mg/kg/hr)
Duration, hours sampling, hours 1 1.8 24 hours 1 time at 24 hours 2
1.9 12 hours 24 hours 3 2.5 12 hours 13 hours 4 2.5 9 hours 24
hours 5 2.4 6 hours 6 hours 6 2 6 hours 36 hours 7 2 6 hours 36
hours 8 2 9 hours 36 hours 9 2 9 hours 36 hours 10 2 9 hours 36
hours
[0086] A.beta. metabolic labeling curves of three participants are
presented in FIG. 8. .sup.13C.sub.6-labeled leucine was infused
through one IV at a rate of 1.9 (circles), 2.5 (triangles), or 2.5
(squares) mg/kg/hr for 12 (circles, triangles) or 9 (squares)
hours. Each hour, 10 ml of plasma and 6 ml of CSF were obtained
through the other IV and the lumbar catheter, respectively. There
was a 5-hour lag time before significant rises in labeled A.beta.
was detected. This was followed by a 9 (squares) or 12 (triangles
or circles) hour increase in labeled A.beta. until it plateaued for
another 5 hours. The 9 hours of labeling (squares) had decreasing
levels of labeled A.beta. for the last 3 hours, while 12 hours of
labeling (triangles or circles) did not show a decrease in labeled
A.beta..
[0087] Additional studies revealed that labeled A.beta. could be
reliably quantified after 9 or 12 hours of label infusion, but not
after 6 hours of label infusion. The synthesis portion of a
labeling curve could be determined in the first 12 hours of
sampling; however, the clearance portion of the labeling curve
could only be determined with 36 hours of sampling. Based on these
results, optimal labeling parameters for A.beta. were defined to be
9 hours of IV infusion of the label and 36 hours of sample
collection. These parameters allowed for assessment of both the
fractional synthesis rate (FSR) and fractional clearance rate (FCR)
portions of a labeling curve.
In Vivo Labeling Protocol
[0088] In the last three participants, .sup.13C.sub.6-labeled
leucine was administered with an initial bolus of 2 mg/kg over 10
minutes to reach a steady state of labeled leucine, followed by 9
hours of continuous intravenous infusion at a rate of 2 mg/kg/hr.
Blood and CSF were sampled for 36 hours in the last 3 participants.
Serial samples of 12 ml blood and 6 ml CSF were taken at one or two
hour time intervals. CSF has a production rate of .about.20 ml per
hour (Fishman R A, 1992, Cerebrospinal fluid in diseases of the
nervous system, Saunders, Pa.) in a normal sized adult and
replenishes itself throughout the procedure. Over a 36-hour study,
the total amount of blood collected was 312 ml and the total amount
of CSF collected was 216 ml.
[0089] There were a total of 10 participants enrolled in the study,
with 8 completing the predefined protocols and 2 studies stopped
before completion due to post-lumbar puncture headache associated
with the study (see Table 1). Two of the 8 completed studies had a
6 hour labeled leucine infusion, and labeled A.beta. levels in
these 2 participants were too low to accurately measure and were
not used for analysis. Thus, the findings from the remaining 6
studies are reported below.
Labeled Leucine Quantitation
[0090] Plasma and CSF samples were analyzed to determine the amount
of labeled leucine present in each fluid (FIG. 9). The labeled to
unlabeled leucine ratios for plasma and CSF .sup.13C.sub.6-leucine
were quantified using capillary gas chromatography-mass
spectrometry (GC-MS) (Yarasheski et al. 2005, Am J Physiol.
Endocrinol. Metab. 288: E278-284; Yarasheski et al. 1998 Am J
Physiol. 275: E577-583), which is more appropriate than LC-ESI-MS
for low mass amino acid analysis. The .sup.13C.sub.6-leucine
reached steady state levels of 14% and 10% in both plasma and CSF,
respectively, within an hour. This confirmed that leucine was
rapidly transported across the blood-brain-barrier via known
neutral amino acid transporter systems (Smith et al. 1987 J
Neurochem. 49(5): 1651-1658).
Labeled A.beta. Dynamics
[0091] For each hourly sample of CSF collected, the ratio of
labeled to unlabeled A.beta. was determined by
immunoprecipitation-MS/MS, as described above. The MS/MS ions from
.sup.13C.sub.6-labeled A.beta..sub.17-28 were divided by the MS/MS
ions from unlabeled A.beta..sub.17-28 to produce a ratio of labeled
A.beta. to unlabeled A.beta. (see TTR formula, above). The mean
labeled A.beta. ratio and standard error (n=6) of each time point
are shown in FIG. 10. There was no measurable labeled A.beta. for
the first 4 hours, followed by an increase from 5 to 13 hours.
There was no significant change from 13 to 24 hours. The labeled
A.beta. decreased from 24 to 36 hours.
Calculation of FSR and FCR:
[0092] The fractional synthesis rate (FSR) was calculated using the
standard formula, presented below:
FSR = ( E t 2 - E t 1 ) A .beta. ( t 2 - t 1 ) / Precursor E
##EQU00002##
Where (E.sub.t2-E.sub.t1).sub.A.beta./(t.sub.2-t.sub.1) is defined
as the slope of labeled A.beta. during labeling and the Precursor E
is the ratio of labeled leucine. FSR, in percent per hour, was
operationally defined as the slope of the linear regression from 6
to 15 hours divided by the average of CSF .sup.13C.sub.6-labeled
leucine level during infusion (see FIG. 11A-C). For example, a FSR
of 7.6% per hour means that 7.6% of total A.beta. was produced each
hour.
[0093] The fractional clearance rate (FCR) was calculated by
fitting the slope of the natural logarithm of the clearance portion
of the labeled A.beta. curve, according to the following
formula:
FCR = ln ( .DELTA. TTR A .beta. .DELTA. time ( hours ) 24 - 36 )
##EQU00003##
The FCR was operationally defined as the natural log of the labeled
A.beta. from 24 to 36 hours (FIG. 11D-F). For example, a FCR of
8.3% per hour means that 8.3% of total A.beta. was cleared each
hour. The average FSR of A.beta. for these 6 healthy young
participants was 7.6%/hr and the average FCR was 8.3%/hr (FIG. 12).
These values were not statistically different from each other.
Example 3
A Technique to Measure Percent Labeled A.beta. in Plasma
Rationale
[0094] Plasma A.beta. metabolism likely occurs in a separate
compartment with different metabolism rates compared to CSF.
However, a significant amount of plasma A.beta. is probably derived
from the CNS. In a mouse model of AD, the amount of A.beta.
captured by an antibody in the plasma, predicted CNS pathology of
neurally derived A.beta.. Therefore, the metabolism rate of A.beta.
in plasma may be a defining feature of pathology in AD. In
addition, plasma A.beta. metabolism may be an equally effective
method of measuring A.beta. metabolism in humans compared to CSF.
If it proves to be diagnostic or predictive of dementia, this
method may be more viable as a diagnostic test of pre-clinical or
clinical AD.
Experimental Design
[0095] As was done for CSF in the prior examples, a method can be
developed to measure labeled and unlabeled A.beta. in plasma. There
are two major differences in obtaining A.beta. from plasma compared
to CSF: 1) there is -100.times. less A.beta. in plasma and 2) there
is approximately a 200.times. increase in non-A.beta. protein
concentration. The efficiency and specificity of the
immunoprecipitation may have to be optimized using methods known to
one of skill in the art. The immunoprecipitations can be tested by
analysis with a Linear Trap Quadrapole (LTQ) mass spectrometer to
identify numbers and relative amounts of contaminating proteins.
The LTQ provides up to a 200-fold increase in sensitivity over
LC-ESI. Preliminary results indicate that it has excellent signal
to noise ratio at 50 fold dilution of A.beta. fragments from 1 ml
of human CSF.
[0096] Testing of the optimized methods can be done with 5-10 ml of
plasma. Labeled and unlabeled A.beta. can by immunoprecipitated,
digested with trypsin, and analyzed by mass spectrometry. Labeled
plasma samples from subjects can be used to detect and generate
plasma labeling standard curves. The sample with the most labeling
can be used to create 5 samples by serial dilution. The labeled
A.beta. can be quantitated by the LTQ in parent ions and tandem
mass spectrometry ions, and the results can generate a standard
curve. From this curve, the linearity and variability can be
determined by a linear fit model. This standard curve can be
compared to the standard curve generated for the human CSF A.beta.
labeling. Labeled plasma A.beta. curves can be compared to labeled
CSF curves from control versus AD individuals to determine if
plasma levels of A.beta. can detect or predict AD.
Results
[0097] As in data for CSF (see Examples 1 and 2), it is expected
that a technique can be developed that can provide reproducible and
quantitative measurements of labeled and unlabeled A.beta. from
human plasma. The standard curves are expected to be near linear
and with low variability. It is expected that the plasma labeled
A.beta. curves from human in vivo studies should closely reflect
the CNS/CSF labeled A.beta. curves. FSR and FCR of plasma A.beta.
from participants can also be generated. It is expected that the
clearance rates of A.beta. in plasma will be much quicker than in
the CSF, as has been shown in animal models after A.beta. infusion
into plasma.
Alternative Approaches
[0098] If labeled and unlabeled plasma A.beta. cannot be accurately
measured as detailed above, then increased sample per time point
with fewer time points may be used (20 ml every other hour as
opposed to 10 ml every hour). This would decrease temporal
resolution of the measurements, but may be still sufficient to
generate FSR and FCR. If protein contamination is still be a
problem with plasma, then purification by HPLC, protein 2D gel, or
even more stringent rinse steps familiar to those of skill in the
art may be necessary.
[0099] The LTQ is a sensitive mass spectrometer commercially
available and provides a very good opportunity to generate
measurements based on attomole amounts. There are no better
alternatives to this mass spectrometer at present; however, mass
spectrometry sensitivity is constantly improving with technology
improvements. Those of skill in the art will recognize that the use
of such improvements in mass spectrometry is within the scope and
spirit of the current invention.
Example 4
Determination of the Effect of ApoE Genotype on CSF A.beta.
Metabolism
Rationale
[0100] ApoE genotype is a well-validated genetic risk factor for
AD. Immunohistochemical studies revealed that ApoE co-localized to
extracellular amyloid deposits in AD. Furthermore, ApoE .epsilon.4
genotype was found to be a risk factor for AD in human populations.
The ApoE .epsilon.2 allele has been shown to be protective in the
risk of AD. ApoE genotype has also been shown to dramatically
effect changes in AD pathology in several mouse models of AD
(Holtzman et al. 2000 PNAS 97(2892); Fagan et al. 2002 Neurobiol.
Dis 9 (305); Fryer et al. 2005 J Neurosci 25 (2803))
[0101] ApoE .epsilon.4 dose dependently increases the density of
A.beta. deposits in AD and in cerebral amyloid angiopathy (CAA).
ApoE is associated with soluble A.beta. in CSF, plasma and in
normal and AD brain. It is likely that ApoE4 is associated with AD
and CAA through the common mechanism of influencing A.beta.
metabolism, although ApoE4 has been shown to be involved in a
variety of other pathways.
[0102] ApoE isoform has been shown to cause dose and allele
dependent changes in time of onset of A.beta. deposition and
distribution of A.beta. deposition in mouse models of AD (Holtzman
et al., 2000, Proc. Natl. Acad. Sci. 97: 2892-2897; DeMattos et al.
2004, Neuron 41(2): 193-202). Human ApoE3 was shown to cause a dose
dependent decrease in A.beta. deposition. In addition, clearance
studies have shown that A.beta. transport from the CNS to plasma
has a t.sub.1/2 of <30 minutes, which is decreased without ApoE.
Together, this suggests that ApoE has A.beta. binding and clearance
effects on CNS A.beta..
Experimental Design and Analysis
[0103] ApoE genotype can be determined in each participant. The
Buffy coat (white blood cell layer) from centrifuged plasma can be
collected and immediately frozen at -80.degree. C. using standard
techniques known to those of skill in the art. The ApoE genotype of
the sample is determined by PCR analysis (Talbot et al. 1994,
Lancet 343(8910): 1432-1433). The effect of gene dose of ApoE2 (0,
1, or 2 copies) and ApoE4 (0, 1, or 2 copies) can be analyzed with
the continuous variable of CSF or plasma FSR or FCR of A.beta.
metabolism.
[0104] Methods for statistical analysis can be made using standard
techniques known to those of skill in the art. For example, for the
FSR and FCR of A.beta. a two-way or three-way ANOVA can be
performed with human ApoE isoform and age as factors in the control
group and also in the AD group. If the data are not normally
distributed, a transformation can be utilized to meet necessary
statistical assumptions regarding Gaussian distributions.
Results
[0105] It is expected that ApoE4 can decrease clearance of A.beta.
compared to ApoE3 in the CNS. Conversely, ApoE2 is expected to
increase clearance of A.beta. compared to ApoE3 in the CNS. A
change in synthesis rate of A.beta. based on ApoE genotype is not
expected. If changes in A.beta. metabolism are detected, this would
be evidence of the effect of ApoE status on in vivo A.beta.
metabolism in humans.
Example 5
Comparison of the ApoE Genotype to Human Plasma A.beta. FSR and FCR
Metabolism Rationale
[0106] Transport of A.beta. from the CNS to plasma may be affected
by ApoE genotype, as demonstrated in mouse models of AD.
Measurement of this effect in humans may reveal transport changes
via ApoE.
[0107] In vivo animal data has shown there are different clearance
rates of A.beta. in plasma vs. CSF vs. brain. The cause of these
differences and the relationship between them is not well
understood. It is likely that ApoE genotype expression plays a
significant role in the transport and clearance of A.beta. from the
CNS to the CSF and the plasma. ApoE in the CNS is mostly produced
by astrocytes and is sialylated and is structurally different
compared to plasma A.beta.. To better understand the relationship
between these compartments as a function of ApoE genotype, the
technique from Example 3 can be used to measure the metabolism of
A.beta. in plasma.
[0108] Experimental Design and Analysis
[0109] ApoE genotype can be determined in each participant. The
Buffy coat (white blood cell layer) from centrifuged plasma can be
collected and immediately frozen at -80.degree. C. using standard
techniques known to those of skill in the art. The sample can be
analyzed using the technique used in Example 4. The effect of gene
dose of ApoE2 (0, 1, or 2 copies) and ApoE4 (0, 1, or 2 copies) can
be analyzed to the continuous variable of FSR or FCR of plasma
A.beta. metabolism. Methods for statistical analysis can be made
using standard techniques known to those of skill in the art and as
described above in Example 4.
Results
[0110] It is expected that ApoE4 can decrease the clearance of
plasma A.beta. compared to ApoE3, and that ApoE2 can increase the
clearance of A.beta. from the plasma compared to ApoE3. A change in
synthesis rate of A.beta. based on ApoE genotype is not expected to
be observed. If changes in plasma A.beta. metabolism are detected,
however, this would be the first assessment of the effect of ApoE
status on A.beta. metabolism in humans.
Alternative Approaches
[0111] The relationship between plasma, CSF, and CNS compartments
for A.beta. metabolism are not well understood. There are changes
in the ratios of A.beta. in each compartment depending on presence
of AD. This indicates a differential effect in the disturbance of
A.beta. metabolism between compartments. The relationship of
peripheral plasma A.beta. metabolism compared to CSF A.beta.
metabolism may be more complex than just ApoE genotype dependent.
It is likely that not only AD status, but other factors may
interact to effect this relationship. Therefore, a clear pattern of
change in A.beta. metabolism may not be dependent on ApoE
genotype.
[0112] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the claims.
Example 6
Measurement of ApoE Metabolism In Vivo Rationale
[0113] For reasons analogous to those detailed for Example 2, a
method for quantifying ApoE FSR and FCR in vivo in the CNS of
humans was developed.
Experimental Design and Analysis
[0114] As depicted in FIG. 13, human ApoE was immunoprecipitated
from CSF or astrocyte cell culture media using WUE4, a specific
anti-human ApoE monoclonal (anti-ApoE) antibody. An immortalized
astrocyte cell line from transgenic mice expressing human ApoE was
used to produce .sup.13C.sub.6-labeled leucine ApoE standards.
Immunoprecipitated ApoE was then digested with sequencing-grade
trypsin. Released peptides were separated by nano liquid
chromatography and quantitated by tandem mass spectrometry
(nanoLC-MS). The efficiency and selectivity of the immuno-affinity
purification was confirmed by analyzing the samples following a
general proteomics LC-MS method. Results from Bioworks.TM. 3.2,
using the SEQUEST algorithm, confirmed ApoE was the predominant
protein present in samples, and identified the most abundant
peptides containing leucines, which could be used for quantitation.
Due to the 6 Da mass shift of a .sup.13C.sub.6-labeled leucine,
unlabeled peptides were distinguishable from labeled peptides.
Tandem MS was performed on the peptides of interest, and the
percent of .sup.13C-leucine incorporated into ApoE was determined
by calculating the ratio of labeled product ions to unlabeled
product ions. A standard curve of .sup.13C.sub.6-labeled leucine
ApoE from cell culture media is shown in FIG. 14. The excellent
linearity from 0-25% labeling (R.sup.2=0.99) indicates the method
developed to measure ApoE is sensitive, specific, and accurate.
[0115] In a preliminary experiment, a young normal control
participant was infused with .sup.13C.sub.6-Leucine (Leu) (2
mg/kg/h) from 0-9 hours. CSF was collected every hour for 36 hours.
ApoE was affinity purified from CSF, and analyzed by nanoLC-MS.
Percent label was calculated from the ratio of the labeled to
unlabeled product ions for ApoE tryptic peptide SWFEPLVEDMQR (FIG.
15). Similar results were observed for a young normal control
participant infused with .sup.13C.sub.6-Leu (2 mg/kg/h) from 0-9
hours, followed by infusion with .sup.13C.sub.6-phenylalanine (Phe)
(3 mg/kg/h) from 16-25 hours (FIG. 16). Fractional synthetic rate
(FSR) was calculated using the standard formula, dividing the slope
of the linear regression by the .sup.13C-Leu or .sup.13C-Phe
precursor enrichment in CSF. Precursor enrichment for these
participants was determined by gas chromatography mass spectrometry
(GCMS).
Results
[0116] FSR was shown to be approximately 2 percent per hour in
these young normal controls (FIG. 15 and FIG. 16). This indicates
that approximately half of the ApoE pool is synthesized in the
human brain in about 24 hours.
Example 7
Measurement of Soluble Amyloid Precursor Protein Metabolism In
Vivo
Rationale
[0117] Analogous to the rationale for Examples 2 and 6, a method
for quantifying soluble amyloid precursor protein (sAPP) FSR and
FCR in vivo in the CNS of humans was developed.
Experimental Design and Analysis
[0118] Similar to the method detailed in Examples 2 and 6, sAPP was
labeled in the CNS of a human subject. Tandem MS was performed on
the peptides of interest, and the percent of .sup.13C.sub.6-leucine
incorporated into sAPP was determined by calculating the ratio of
labeled product ions to unlabeled product ions.
Results
[0119] Soluble APP has a much slower production and clearance rate
compared to A.beta., as indicated by the slower rise to plateau and
clearance of labeled sAPP (FIG. 17). This indicates that individual
protein turnover rates are different in the same samples in the
same subject.
Sequence CWU 1
1
3142PRTHomo sapiens 1Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu
Val His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly
Ser Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val
Val Ile Ala 35 40 212PRTHomo sapiens 2Leu Val Phe Phe Ala Glu Asp
Val Gly Ser Asn Lys 1 5 10 312PRTHomo sapiens 3Ser Trp Phe Glu Pro
Leu Val Glu Asp Met Gln Arg 1 5 10
* * * * *