Title: Diagnostic for early stage Alzheimer's disease
Abstract: A method for diagnosing Alzheimer's disease even at early stages by assessing the levels of sulfatides or its metabolites in biological fluids is disclosed.
Patent Number: 7,015,044 Issued on 03/21/2006 to Han, et al.
| Inventors:
|
Han; Xianlin (Clayton, MO);
Holtzman; David M. (St. Louis, MO)
|
| Assignee:
|
Washington University (St. Louis, MO)
|
| Appl. No.:
|
317031 |
| Filed:
|
December 10, 2002 |
| Current U.S. Class: |
436/71; 436/173; 436/811 |
| Current Intern'l Class: |
G01N 33/92 (20060101); G01N 24/00 (20060101) |
| Field of Search: |
436/71,173,811
|
References Cited [Referenced By]
U.S. Patent Documents
| 2001/0044126 | Nov., 2001 | Holtzman et al.
| |
| Foreign Patent Documents |
| WO 01/4987/5 | Jul., 2001 | WO.
| |
Other References
Bosio, A., et al., "Functional Breakdown of the Lipid Bilayer of the Myelin Membrane
in Central and Perioheral Nervous System by Disrupted Galactocerebroside Synthesis"
Proc. Natl. Acad. Sci. USA (1996) 93:13280-13285.
Coetzee, T., et al., "Myelination in the Absence of Galactocerebroside and Sulfatide:
Normal Structure with Abnormal Function and Regional Instability" Cell (1996) 86:209-219.
Fredman, P., et al., "Sulfatide as a Biochemical Marker in Cerebrospinal Fluid
of Patients with Vascular Dementia" Acta Neurol. Scand. (1992) 85:103-106.
Galasko, D., et al., "High Cerebrospinal Fluid Tau and Low Amyloid β42
Levels in the Clinical Diagnosis of Alzheimer Disease and Relation to Apolipoprotein
E Genotype" Arc. Neurol. (1998) 55:937-945.
Gottfries, C-G., et al., "Membrane Components Separate Early-Onset Alzheimer's
Disease from Senile Dementia of the Alzheimer Type" Int. Psycho-Geriatrics (1996) 8:365-372.
Han, X. et al., "Substantial Sulfatide Deficiency and Ceramide Elevation in Very
Early Alzheimer's Disease: Potential Role in Disease Pathogenesis" J. Neurochem.
(2002) 82:809-818.
Hannun, Y.A. et al., "Functions of Ceramide in Coordinating Cellular Reponses
to Stress" Science (1996) 274:1855-1859.
Hess, B., et al., Proc. "Phenotype of Arylsulfatase A-Deficient Mice: Relationship
to Human Metachromatic Leukodystrophy" Natl. Acad. Sci. USA (1996) 93:14821-14826.
Kolesnick, R.N., et al., "Regulation of Ceramide Production and Apoptosis" Ann.
Rev. Physiol. (1998) 60:643-665.
Morris, J.C., "The Clinical Dementia Rating (CDR): Current Version and Scoring
Rules" Neurology (1993) 43:2412-2414.
Paola, M.D., et al., "Ceramide Interaction with the Respiratory Chain of Heart
Mitochondria" Biochemistry (2000) 39:6660-6668.
Svennerholm, L., "Krabbe Disease: A Galactosylsphingosine (Psychosine) Lipidosis"
J. Lipid Res. (1980) 21:53-64.
International Preliminary Examination Report, mailed on Oct. 29, 2003, for PCT
patent application No. PCT/US02/39567, filed on Dec. 10, 2002, 5 pages.
Majocha et al., J. Neurochem. (1989) 53(3):953-961 (abstract).
Sorbi et al., Gerontology (1987) 33:197-202.
Supplementary European Search Report for EP 02 80 4772, mailed on Dec. 10, 2004,
1. page.
Tarvonen-Schröder et al., Dement. Geriatr. Cogn. Disord. (1997) 8:174-179.
Gottfries et al. (1986) Prog Neuropsychopharmacol Biol Psychiatry 10(3-5):405-413 (Abstract).
|
Primary Examiner: Gakh; Yelena G.
Attorney, Agent or Firm: Morrison & Foerster LLP
Goverment Interests
ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT
At least a portion of this work was supported by National Institutes of Health
(NIH) contract NIH-AG05681. The U.S. government has certain rights in this invention.
Parent Case Text
RELATED APPLICATION
This application claims priority under 35, U.S.C. §119(e) to U.S. provisional
application No. 60/339,188 filed 10 Dec. 2001. The contents of this application
are incorporated herein by reference.
Claims
The invention claimed is:
1. A method to identify a subject who is at high risk for, or who has developed,
Alzheimer's disease, which method comprises
(a) assessing the level of sulfatides in a biological fluid of said subject;
(b) providing the level of sulfatides in the corresponding biological fluid in
normal subjects;
(c) comparing the level of sulfatides assessed in (a) with that of normal subjects
as provided in (b);
wherein when the comparison in (c) shows the level of sulfatides in said subject
in (a) is statistically significantly decreased from those in normal subjects as
provided in (b),
said subject is identified as at high risk for, or as a subject who has developed,
Alzheimer's disease.
2. The method of claim 1, wherein the biological fluid is plasma, urine, or cerebrospinal fluid.
3. The method of claim 2, wherein the biological fluid is cerebrospinal fluid.
4. The method of claim 1, wherein the level of sulfatides is measured as total sulfatides.
5. The method of claim 1, within the level of sulfatides is measured as the level
of a single species of sulfatide.
6. The method of claim 1, wherein the level of sulfatides is normalized to the
level of one or more non-variant lipids.
7. The method of claim 6, which the comparing in (c) is of the ratio of sulfatides
to one or more non-variant lipids.
8. The method of claim 6, wherein the non-variant lipid is phosphatidyl inositol.
9. The method of claim 1, wherein the biological fluid is cerebrospinal fluid
and wherein the sulfatide level is measured as the ratio of total sulfatides to
phosphatidylinositol (ST/PI).
10. The method of claim 1, wherein said level of sulfatides is measured using
electrospray ionization mass spectrometry.
11. The method of claim 9, wherein said level of sulfatides and of phosphatidyl
inositol is measured using electrospray ionization mass spectrometry.
12. A method to identify a subject who is at high risk for, or who has developed
Alzheimer's disease, which method comprises
(a) assessing the level of ceramide in a biological fluid of said subject;
(b) providing the level of ceramide in the corresponding fluid in normal subjects;
(c) comparing the level of ceramide assessed in (a) with that of normal subjects
as provided in (b);
wherein when the comparison in (c) shows the level of at least one sulfatide
metabolite in said subject in (a) is statistically significantly increased from
those of normal subjects provided in (b),
said subject is identified as at high risk for, or as a subject who has developed,
Alzheimer's disease.
13. The method of claim 12, wherein the biological fluid is plasma, urine, or
cerebrospinal fluid.
14. The method of claim 13, wherein the biological fluid is cerebrospinal fluid.
15. The method of claim 12, wherein the level of sulfatide metabolites is measured
using electrospray ionization mass spectrometry.
Description
TECHNICAL FIELD
The invention relates to methods and materials useful in diagnosing very early
stage Alzheimer's disease. More specifically, it concerns assessing sulfatides
in the cerebrospinal fluid, or other body fluid, whereby a decreased level of sulfatides
or an increase in its metabolites indicates the presence of Alzheimer's disease.
BACKGROUND ART
The diagnosis of Alzheimer's disease, especially in the early stages, is notoriously
difficult. Postmortem analysis of the brain shows the presence of neuritic plaques
and neurofibulary tangles. Studies of brain tissue in the past have focused on
gray matter pathology, but white matter has also been implicated in more recent
studies. White matter in Alzheimer's subjects shows a dramatic loss of myelin and
axons, DNA fragmentation, and plasmalogen deficiency in early stages of the disease.
("Plasmalogen" refers to a class of phospholipid enriched in myelin.) Most Alzheimer's
patients show evidence of white matter degeneration and loss of oligodendrocytes
(Morris, J. C.,
Neurology (1993) 43:2412-2414).
Sulfatides are a class of sulfated galactocerebrosides. A general structure
for sulfatides is shown in FIG. 1. The various sulfatides differ only in the fatty
acid represented by RCO in the structure. Sulfatides are synthesized through the
mediation of ceramidase galactosyl transferase with subsequent sulfation mediated
by galactocerebroside sulfotransferase. They are degraded, ultimately to ceramides,
by the action of sulfatidase and galactosyl ceramidase. Sulfatides are important
in the regulation of cell growth, protein trafficking, signal transduction, cell
adhesion, neuronal plasticity and cell morphogenesis and are synthesized exclusively
by oligodendrocytes in the central nervous system. They are present predominantly
in the myelin sheath surrounding the axons and are present both in gray matter
and white matter in the brain.
It has previously been shown that a deficiency in sulfatidase leads to an accumulation
of sulfatides and to metachromatic leukodystrophy (Hess, B., et al.,
Proc. Natl.
Acad. Sci. USA (1996) 93:14821-14826). A galactosyl ceramidase deficiency also
leads to the accumulation of sulfatides as well as galactocebrosides in Krabbe's
disease (Svennerholm, L.,
J. Lipid Res. (1980) 21:53-64). Further, knockout
mice lacking ceramide galactosyl transferase show deficiencies in sulfatides and
galactocerebrosides and generally die by three months and demonstrate abnormal
axonal function. (Bosio, A., et al.,
Proc. Natl. Acad. Sci. USA (1996) 93:13280-13285;
Coetzee, T., et al.,
Cell (1996) 86:209-219.)
It is also known that the ceramides, which are sulfatide degradation products
are lipid second messengers and mediate inflammatory cytokines and growth factors
so that the accumulation of ceramides results in up-regulation of cytokines, generation
of reactive oxygen species, interruption of the mitochondrial respiratory chain
and apoptosis (Hannun, Y. A., et al.,
Science (1996) 274:1855-1859; Kolesnick,
R. N., et al.,
Ann. Rev. Physiol. (1998) 60:643-665; Paola, M. D., et al,
Biochemistry (2000) 39:6660-6668).
Gottfries, C-G., et al.,
Int. Psycho-
Geriatrics (1996) 8:365-372
describe studies which purport to distinguish the membrane components in brain
of subjects with Alzheimer's disease from those with senile dementia of the Alzheimer's
type (SDAT). SDAT and Alzheimer's disease are presently classified together since
they appear to differ only in the age of onset of dementia; the onset in "pure
Alzheimer's disease" is at an earlier age than that of SDAT. Nevertheless, this
study showed that phospholipids, cholesterol, cerebroside and sulfatides were reduced
in the frontal lobe white matter in the SDAT group as compared to age-matched controls
and Alzheimer's patients. Levels of sulfatides in body fluids such as cerebrospinal
fluid, blood, or urine in Alzheimer's patients or controls were not measured.
In a more recent publication, Han, X., et al.,
J. Neurochem. (2002) 82:809-818
further examined the status of sulfatide both in gray matter and white matter.
This study, by the group of which the present inventors are members, showed that
sulfatides were depleted both in gray matter and white matter in Alzheimer's subjects
with very mild dementia, mild, and severe dementia, whereas other major classes
of lipid, except plasmalogen were not altered. It was also established that there
was no deficiency in the biosynthesis of sulfatides in these individuals and that
the content of ceramides was elevated in the white matter. The authors concluded
that the decrease in sulfatides was associated with Alzheimer's pathology even
in very early stages in the gray and white matter of the brain. No measurements
were taken of body fluids of any kind.
Fredman, P., et al.,
Acta Neurol. Scand. (1992) 85:103-106 measured
sulfatides in the cerebrospinal fluid of patients with vascular dementia (i.e.,
dementia caused by stroke) and showed that the sulfatide concentrations in the
patients with vascular dementia were at a significantly higher level than those
in controls and in the Alzheimer's disease group. The levels in Alzheimer's patients
and in controls appeared to be similar. The sulfatide levels in the group with
vascular dementia were 307±118 nm/l while that in controls was 145±86
nm/l and in Alzheimer's patients 178±79 nm/l. The authors suggest that the
elevation in sulfatide concentration reflects demyelination.
Alternative diagnoses for Alzheimer's disease and early stages thereof
which measure components of biological fluids have been described in PCT publication
WO 01/49875 and U.S. publication No. 2001/0044126A1 incorporated herein by reference.
These documents describe assays in cerebrospinal fluid and blood which are predictive
of Alzheimer's disease. These assays involve the levels of circulating amyloid-beta
(Aβ) peptide in the blood and in the cerebrospinal fluid (CSF). The Aβ
peptide in circulating form is composed of 39-43 amino acids (mostly 40 or 42 amino
acids) and results from the cleavage of a common precursor, amyloid precursor protein,
designated APP. It was found that the ratio of Aβ
40 to Aβ
42
is elevated in individuals with Alzheimer's disease or a propensity therefor.
The total circulating levels of Aβ peptide, however, do not differ in Alzheimer's
patients and in normal individuals. While statistically this method is helpful,
there is some overlap in the values of this ratio between normal and Alzheimer's subjects.
It has also been shown that Aβ
42 decreases and tau protein increases
in patients with moderate to severe clinical Alzheimer's disease (Galasko, D.,
et al.,
Arc. Neurol. (1998) 55:937-945).
A potential dramatic improvement in this assay is described in U.S. Provisional
Applications 60/313,221 filed 17 Aug. 2001 and 60/334,987 filed 23 Oct. 2001, and
incorporated herein by reference. In this method, antibodies that sequester Aβ
peptide from its bound circulating form in blood alter the clearance of soluble
and bound forms of Aβ in the central nervous system and alter the levels
of circulating Aβ peptides in the blood when the subject is in clinical or
preclinical stage of Alzheimer's. Thus, the level of Aβ
40, Aβ
42
or the ratio of Aβ
40/Aβ
42 in the blood of the
subject can be measured after injecting these antibodies and an elevation in any
of these is indicative of Alzheimer's disease. This method, however, has not yet
been assessed in human patients.
The present invention offers a method which clearly separates patients in even
the very early stages of Alzheimer's disease from normal subjects. Virtually no
overlap occurs between values obtained in subjects who are normal as compared to
those with early stage Alzheimer's disease.
DISCLOSURE OF THE INVENTION
The invention is directed to a method to diagnose Alzheimer's disease at a very
early or later stage using sulfatides in body fluids such as cerebrospinal fluid
(CSF), blood, including plasma, or urine as an index. A decrease in the level of
sulfatides in these fluids is positively correlated with the presence of Alzheimer's disease.
Thus, in one aspect, the invention is directed to a method to identify a subject
with the condition of Alzheimer's disease or a propensity therefor, including early
stage Alzheimer's disease, which method comprises measuring the level of sulfatides
in the body fluid of said subject, whereby a subject whose level of sulfatides
in said fluids is statistically lower than the level in normal subjects is identified
as an individual with Alzheimer's disease. In addition, levels of ceramides and
other sulfatide metabolites can be measured instead of or in addition to the sulfatides.
Generally, an increased level of at least one metabolite is indicative of an individual
with Alzheimer's propensity or disease.
If desired, the level of sulfatides as measured can be normalized with regard
to other lipid components whose levels are not altered by the disease or conditions,
such as the levels of phosphatidyl inositol. Total sulfatides, or a selected individual
sulfatide can be measured.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the structure of sulfatides in general and the mechanisms for synthesis
and breakdown of this class of compounds.
FIGS. 2A and 2B show a comparison of ESI-MS results of lipid extracts from
temporal gray matter from a normal subject with results from a subject with very
mild Alzheimer's dementia.
FIGS. 3A and 3B show graphical representations of sulfatide content in cerebral
and cerebellar gray and white matter correlated with clinical dementia ratings.
FIGS. 4A and 4B show comparison of negative ion ESI mass spectra of lipid extracts
from temporal white matter from a normal and an Alzheimer's disease subject to
show ceramide levels.
FIGS. 5A and 5B show correlation of ceramide content in gray and white matter
with clinical dementia ratings.
FIGS. 6A and 6B show representative positive ion ESI mass spectra on lipid
extracts from temporal white matter of a cognitively normal and a mild Alzheimer's
disease subject.
FIG. 7 shows comparisons of galactocerebroside sulfotransferase activity in
microsomal preparations of white and gray matter from normal and mild Alzheimer's
disease subjects.
FIGS. 8A and 8B show a comparison of negative ion ESI-mass spectrum of a lipid
extract of cerebrospinal fluid from a normal and a mild Alzheimer s disease subject.
FIGS. 9A-9E show the results obtained from CDR 0 and CDR 0.5 patients using
various parameters measured in the cerebrospinal fluid as well as the results of
psychological tests. FIG. 9A shows a comparison of the sulfatide/phosphatidylinositol
ratio in the cerebrospinal fluid of normal individuals as compared to individuals
with very mild dementia. FIGS. 9B-9D show similar comparisons when the levels of
Aβ
42, tau protein, and pTau
231 were measured. FIG.
9E compares the results of the two groups using a test for cognitive ability.
MODES OF CARRYING OUT THE INVENTION
The invention employs biological fluids as the biological samples for assessing
sulfatide or ceramide levels as measures of the presence of Alzheimer's disease
in the subject. Preferably, the biological fluid is CSF, but blood, plasma, or
urine could also be used. Desirably, the biological fluid is first extracted to
obtain a lipid fraction for evaluation of the level of sulfatides (or ceramides).
However, depending on the method employed for assessing the level of sulfatides
(and/or ceramides), such extraction may not be necessary. The sample ultimately
used for the assessment may be subjected to fractionation procedures to obtain
the most convenient ultimate sample for measurement
The invention resides in the discovery that the level of sulfatides, including
individual sulfatides, is significantly decreased in body fluids including CSF
in a reliable manner in subjects who have Alzheimer's disease, even at very early
stages. The stage of the disease can be measured by trained health care providers,
with a clinical dementia rating (CDR). This measurement (CDR) shows a score of
0 where there is no dementia, 0.5 when the individual has very mild dementia, 1
for mild dementia, 2 for moderate dementia and 3 for severe dementia. (Morris,
J. C.,
Neurology (1993) 43:2412-2414.) By correlating these ratings with
neuropathological findings, the diagnostic accuracy for Alzheimer's disease appears
to be 93% (Morris, J. C., et al.,
Neurology (1996) 46:707-719; Berg, L.,
et al.,
Arch. Neurol. (1998) 55:326-335). The present invention allows accurate
diagnosis of the presence of, or propensity for, Alzheimer's disease even at the
very mild dementia stage of CDR 0.5. This stage of abnormality is also called mild
cognitive impairment (MCI).
The methods described by the invention are particularly advantageous since, in
addition to showing statistically significant differences in mean values between
groups of tested individuals, the degree of overlap in the results derived from
populations with normal cognition and those with mild cognitive impairment is extremely
low. Thus, the measurement of the appropriate sulfatide level in the biological
fluids of a single individual can effectively be used to evaluate the state of
that individual. In the case of other prior art measurements, the measurement of
the value for a particular individual becomes almost meaningless in view of the
high degree of overlap among the results from the two populations.
The level of sulfatides or of an individual sulfatide in a subject may be assessed
as a total concentration, but is preferably normalized to the levels of a component
which does not vary with the presence of the disease. The levels of phospholipids
in general, cholesterol, and cerebrosides are similar in Alzheimer's patients and
normal individuals, but levels of these lipids may vary among particular individuals
or with the conditions of the assay. Thus, a more accurate assessment for the levels
of sulfatides may be obtained by normalizing the sulfatide levels measured to one
of these indicators. A particularly preferred and convenient substance for such
normalization is the level of phosphatidyl inositol. Factors independent of Alzheimer's
disease which cause similar variations in levels of phosphatidyl inositol are expected
also to cause variations in the levels of sulfatides. Thus, by taking this ratio,
this source for possible error is eliminated.
The levels of sulfatides and, if desired, normalizing components may be measured
by any convenient method. Such measurements may include immunoassay methods using
specific antibodies or fragments for detection of the various components, chromatographic
separation techniques and measurements of individual fractions, or any other suitable
method. A particularly preferred and convenient method is electrospray ionization
mass spectrometry (ESI-MS) using the lipid extracts per se. This method includes
identifying individual species by ESI tandem mass spectrometry. This method is
described in detail by Han, X., et al.,
Proc. Natl. Acad Sci. USA (1994)
91:10635-10639; Han, X., et al.,
Biochemistry (1996) 35:5822-5832; and Han,
X., et al.,
J. Neurochem. (2001) 77:1168-1180. The contents of these documents
are incorporated herein by reference.
Thus, to identify an individual as an Alzheimer's subject, a biological fluid
sample is obtained from the subject, subjected, if desired, to fractionation procedures,
and assessed for the level of sulfatides, or for an individual sulfatide. The sulfatide
level may, if desired, be normalized with regard to a different lipid component,
known not to vary with Alzheimer's disease presence, preferably phosphatidyl inositol.
The level of sulfatides thus obtained is compared with the values associated with
normal individuals; comparison with a single normal individual is possible but
it is preferred that a statistical value for the normal population be obtained
using an appropriately sized sample. If normalized levels are used, one suitable
comparison is based on the ratio of sulfatide to the normalizing substance. For
example, the CSF of normal individuals shows a ratio of sulfatides/phosphatidyl
inositol of approximately 0.88 based on a sample of 19 normal subjects.
Regardless of the method used for comparison, a decrease in the level
of sulfatides in the biological fluid of an individual, relative to control subjects
of the same age, identifies that individual as someone with Alzheimer's disease,
even in very early clinical stages.
Alternatively, using similar techniques, the levels of ceramide and/or
other sulfatide metabolites can be used as an index for the presence of Alzheimer's disease.
The following examples are intended to illustrate but not to limit the invention.
EXAMPLE 1
Levels of Sulfatides and Ceramides in Brain Tissue
Brain tissue samples were taken from subjects who, at the time of death, had
various CDR scores and were matched for age of death and postmortem interval. Lipids
from individual samples were prepared in the presence of 14:1-14:1 phosphatidyl
choline (29 nm/mg protein); 14:0-16:0 phosphatidyl glycerol (PG) (10 nm/mg protein),
perdeuterated N-18:0 galactocerebrosides (30 nm/mg protein) and N-17:0 ceramides
(5 nm/mg protein) which were used as internal standards for quantitating choline-containing
phospholipids, sulfatides, galactocerebrosides, and ceramide respectively. This
method was described previously by Han, X., et al.,
J. Neurochem. (2001) 77:1168-1180.
The lipid extracts were analyzed by ESI-MS as described hereinabove.
More specifically, sulfatides in the lipid extracts were prepared by the method
of Bligh, E. G., et al.,
Can. J. Biochem. Physiol. (1959) 37:911-917. Sulfatides
in the lipid extracts were determined by negative ion ESI-MS and identified by
tandem mass spectrometry as described by Hsu, F. F., et al.,
Biochim. Biophys.
Acta. (1998) 1392:202-216, incorporated herein by reference. Sulfatides were
directly quantitated by comparison of individual peak intensities with an internal
standard (i.e., PG) after correction for
13C isotope effects and ionization
efficiency factor. The efficiency factor was obtained by measuring the slope of
a linear correlation (γ
2=0.994) of the concentration profile between
N-16:0 sulfatide and the internal standard. (A correction factor of 1.385 was then applied.)
The results of the ESI-MS of the chloroform extract of superior temporal cortex
gray matter from a CDR 0 subject as compared to a CDR 5 subject are shown in FIGS.
2A and 2B. FIG. 2A shows the CDR 0 subject has a total sulfatide mass of 42.1±8.8
nm/mg; N-24:1 sulfatide was the major species. The sulfatide content in the cerebral,
frontal and parietal gray matter gave essentially identical results, but the sulfatide
content in cerebellar gray matter was considerably less representing 17% of that
found in cerebral gray matter. The profiles obtained from white matter were substantially
similar to those shown in FIG. 2A; however, the total sulfatide in white matter
of all cerebral regions was almost three times that in cerebral gray matter.
FIG. 2B shows a comparable ESI-MS for a subject with a CDR of 0.5. It is immediately
apparent that the levels of sulfatides are greatly diminished. When a chloroform
extract of superior temporal cortex gray matter, similar to that in FIG. 2A was
employed, the level of sulfatides dropped to 3.2±0.8 nm/mg protein, a reduction
of 92%.
FIGS. 3A and 3B summarize the results obtained for gray matter and white matter
as a function of CDR. The total content of sulfatide in chloroform extracts of
cerebral frontal (diamonds), temporal (circles), parietal (triangles), and cerebellar
(squares) gray matter (FIG. 3A) and white matter (FIG. 3B) was determined using
negative ion ESI-MS as described above. The data were normalized to the protein
content. It is apparent that a dramatic decrease occurs between normals and very
early stage dementia and that the levels are similar at all stages of dementia.
It was also found that there was no alteration in the level of phosphatidylserine
(PS) or phosphatidylinositol (PI) between the samples (FIGS. 2A and 2B).
Similar procedures were used to determine ceramide content as shown in FIGS.
4A (CDR 0) and 4B (CDR 0.5). CDR 0 subjects had two major species of ceramide
corresponding to N-18:0 and N-24:1 ceramide; the total ceramide mass was 2.59±0.56
nm/mg protein in superior temporal white matter. The ceramide content of white
matter from all examined brain regions was essentially identical in the CDR 0 subjects.
However, as shown in FIG. 4B, the total mass of ceramides in the CDR 0.5
subject was 8.7 nm/mg protein, an increase of 3.3-fold; the majority of the increase
was of N-24:1 ceramide.
FIGS. 5A and 5B show ceramide content as a function of CDR in the temporal
cortex and the cerebellum in both gray matter and white matter. FIG. 5A shows variations
in the ceramide content of the temporal cortex white matter (circles) and gray
matter (squares); FIG. 5B shows the content of the cerebellar white matter (circles)
and gray matter (squares). As shown ceramide content of gray matter appears not
to change, with a change in CDR while the white matter content is highly elevated
at CDR 0.5 but diminishes as CDR score increases.
This study also measured sphingomyelin and galactocerebrosides, which are also
possible degradation products of sulfatides. Using positive ion ESI-MS, these results
are shown in FIGS. 6A (CDR 0) and 6B (CDR 0.5) for lipid extracts of the
white matter of the superior temporal cortex. As shown, the values for these compounds
as well as for phosphatidylcholine were substantially similar for CDR 0 and CDR
0.5 subjects, the values were approximately 410, 138, 330 nm/mg protein, respectively
for CDR 0. Similar comparative results were found with other examined brain tissue
samples including gray and white matter of cerebral and cerebellar regions, although
there were differences between these regions per se.
The level of galactocerebroside sulfotransferase activity was also measured as
a function of CDR in samples obtained from the brain bank of Washington University
ADRC Neuropathology/Tissue Research Core from three CDR 0 and three CDR 5 individuals.
Enzymatic activity was assayed by quantitation of the reaction product
35S-labeled
sulfatide, and normalized to the protein content. In FIG. 7, STG represents superior
temporal gray matter; STW is superior temporal white matter, CBG represents cerebellar
gray matter and CBW represents cerebellar white matter. Although there were large
differences depending on the source of the sample, no differences were observed
between CDR 0 and CDR 5 subjects.
EXAMPLE 2
Determination of Sulfatides in Cerebrospinal Fluid
Spinal taps (after overnight fast) were performed on longitudinally followed
subjects at the Washington University Memory and Aging Project. Nineteen subjects
with a CDR of 0 and 20 subjects with a CDR score of 0.5 were used in the study.
None of the subjects had prior history of stroke or other neurological disease.
The mean age was 71 for CDR 0 and 72 for CDR 0.5. Sulfatide (ST) and phosphatidyl
inositol (PI) levels were measured by ESI-MS, and the ratio of sulfatides to phosphatidyl
inositol (ST/PI) was determined. The results are shown for individual samples in
FIGS. 8A (CDR 0) and 8B (CDR 0.5). As shown, the level of sulfatides is
greatly decreased in the individual with a CDR 0.5 score, while the level of PI
is essentially the same for both the CDR 0 and CDR 5 subjects.
The reliability of the CSF ST/PI ratio in 19 CDR 0 and 20 CDR 0.5 subjects was
verified in our study using ESI-MS for measurement of the ST/PI ratio. These results
are shown in FIGS. 9A-E. Subjects who are cognitively normal (CDR 0) and those
with very mild dementia (CDR 0.5) were compared. The lack of severity of the dementia
itself is illustrated in FIG. 9E which compares the Mini-Mental State Exam scores
(MMSE scores) between the two groups. The mean score is lower in the CDR 0.5 group,
but there is a great deal of overlap between individual subjects: CDR 0=29.42±0.18;
CDR 0.5=26.35±0.72; p=0.002.
FIG. 9A shows a dramatic difference between the two groups in terms of the sulfatide/phosphatidylinositol
ratio with very little overlap. There were 20 subjects in the CDR 0.5 group and
19 in the CDR 0 group. Only 2 subjects in the CDR 0.5 group overlap with the CDR
0 group: ST/PI ratio (mean±SEM): CDR 0=0.86±0.019; CDR 0.5=0.499±0.38; p<0.0001.
The superior ability of this method to differentiate the two groups is further
illustrated by comparing the results in FIG. 9A with those in FIG. 9B. FIG. 9B
shows the results of comparison of levels of Aβ
42 according to
one measure practiced in the prior art. CSF Aβ
42 is significantly
lower in CDR 0.5 than CDR 0 subjects but there is marked overlap between the groups.
Aβ
2 (mean±SEM, pg/ml): CDR 0=636.6±48.4; CDR 0.5=498.9±45.8; p=0.04.
Similarly, FIGS. 9C and 9D show that other commonly used biomarkers of
Alzheimer's disease, while providing different mean values, show significant overlaps.
As shown in FIG. 9C, CSF tau is significantly increased in CDR 0.5 subjects (p<0.05)
but there is a also a large amount of overlap between CDR 0 and 0.5 subjects. Total
tau (mean±SEM, pg/ml): CDR 0=350.5±53.1; CDR 0.5=706.6±126.7; p=0.0153.
Further, as shown in FIG. 9D, CSF pTau
231 is significantly increased
in CDR 0.5 subjects but as for total tau, there is a large amount of overlap between
CDR 0 and 0.5 subjects. pTau
231 (mean±SEM, pg/ml): CDR 0=57.98±15.32;
CDR 0.5=85.69±9.61; p=0.0175.
Thus, the invention method provides not only a statistical difference between
individuals who are normal and those who have mild dementia but also provides an
assay which is applicable to individuals in view of the fact that there is very
little overlap between the cognitively normal and very mildly demented groups.
*
