Peptides and Proteins in Plasma and CerebrospinalFluid as Biomarkersfor the
Prediction, Diagnosis, and Monitoring of Therapeutic Efficacy of Alzheimer’s
Christopher D. Aluise, Ren˜ a A. Sowell, D. Allan Butterfield
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Please cite this article as: Christopher D. Aluise, Ren˜ a A. Sowell, D. Allan Butterfield,
Peptides and Proteins in Plasma and Cerebrospinal Fluid as Biomarkers for the Predic-
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Peptides and Proteins in Plasma and Cerebrospinal Fluid as
Biomarkers for the Prediction, Diagnosis, and Monitoring of
Therapeutic Efficacy of Alzheimer’s Disease
Christopher D. Aluise,1 Renã A. Sowell,1 and D. Allan Butterfield 1
1Department of Chemistry, Center of Membrane Sciences, and
Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY 40506-0055,
*Address correspondence to:
Professor D. Allan Butterfield
Department of Chemistry, Center of Membrane Sciences,
and Sanders-Brown Center on Aging,
University of Kentucky
Lexington, KY 40506-0055
Fax: (859) 257-5876
identified from CSF, plasma, and urine that may serve as biomarkers in AD.
Alzheimer’s disease (AD) affects millions of persons worldwide. Earlier detection
and/or diagnosis of AD would permit earlier intervention, which conceivably could delay
progression of this dementing disorder. In order to accomplish this goal, reliable and
specific biomarkers are needed. Biomarkers are multidimensional and have the potential
to aid in various facets of AD such as diagnostic prediction, assessment of disease stage,
discrimination from normally cognitive controls as well as other forms of dementia, and
therapeutic efficacy of AD drugs. To date, biomarker research has focused on plasma and
cerebrospinal fluid (CSF), two bodily fluids believed to contain the richest source of
biomarkers for AD. CSF is the fluid surrounding the central nervous system (CNS), and
is the most indicative obtainable fluid of brain pathology. Blood plasma contains proteins
that affect brain processes from the periphery, as well as proteins/peptides exported from
the brain; this fluid would be ideal for biomarker discovery due to the ease and non-
invasive process of sample collection. However, it seems reasonable that biomarker
discovery will result in combinations of CSF, plasma, and other fluids such as urine, to
serve the aforementioned purposes. This review focuses on proteins and peptides
Keywords: biomarker, plasma, CSF, Alzheimer’s disease
biomarker or set of biomarkers to diagnose AD earlier, distinguish AD from other forms
Alzheimer’s disease (AD) is a neurodegenerative disorder that currently affects
five million Americans. AD is the most common form of dementia among the elderly, as
approximately 50% of adults aged 85 years develop this condition. Diagnosis of AD is
based on National Institute of Neurological and Communicative Disorders and
Alzheimer’s Disease and Related Disorders (NINCDS-ADRDA) Reagan criteria,
neuropsychiatric testing, neuroimaging such as PET scanning/MRI, and an exclusion of
other forms of dementia. AD is inherently difficult to diagnose, especially in very early
stages when memory impairment is mild and may go unrecognized. Despite intense
investigation into the mechanisms of neurodegeneration and possible causes of AD,
currently no cure or definitive underlying cause exists. Therapeutics such as memantine,
an NMDA receptor antagonist, and Aricept®, an acetlycholinesterase inhibitor, slow the
rate of degeneration and are among the most widely prescribed medications to treat AD.
These drugs are effective only briefly in the course of this disease and are usually
administered at a time when the patient has exhibited a noticeable decline in cognition,
and neurodegeneration has progressed to an irreversible state. Therefore, the need for a
of dementia, and monitor therapeutic efficacy is of critical need to aid and better protect
the elderly from this devastating dementing disorder.
A biomarker is an abnormal signal from a bodily fluid or tissue that can provide
distinguishable pathological information for a patient. According to the 1998 Consensus
Report of the Working Group on Molecular and Biochemical Markers of Alzheimer
Disease , ideal biomarkers for AD should be: 1) reflective or indicative of AD
(therefore, no anticoagulant necessary) prior to centrifugation. During the coagulation
pathology; 2) reliable; 3) easy to perform/analyze; and 4) relatively inexpensive. Because
biomarkers have the potential to assist in the diagnosis, delineate the disease stage, and
monitor drug efficacy in AD, the notion of a single biomarker serving all of these
functions is somewhat unrealistic. Rather, a combination of markers for diagnosing and
managing AD and pre-clinical stages of AD, such as amnestic mild cognitive impairment
(MCI), are continually being investigated. For AD and other neurodegenerative diseases,
blood plasma and cerebrospinal fluid (CSF) have evolved as prime targets for biomarker
investigation. This review focuses mainly on proteins and peptides in plasma and CSF as
candidate biomarkers for AD, and briefly discusses advances in potential urinary
biomarkers for AD.
Plasma is the liquid portion of blood that suspends cells such as red blood cells,
white blood cells, and platelets. Plasma is pale-yellow in color, rather viscous, and can be
isolated from whole blood by centrifugation at low speeds in the presence of an
anticoagulant. Serum, which is oftentimes confused with plasma, is plasma with clotting
factors, such as fibrinogen, removed. Serum is isolated by allowing blood to clot
process for serum preparation, the concentrations of some proteins in the sample are
altered, and also, protein fragments are released by platelets and other cells . The
plasma proteome, consequently, is altered and the opportunity for artifactual results is
increased. Also, high fibrinogen levels have been shown to be associated with cognitive
decline in patients with MCI  and increased risk of dementia . With this protein
removed from the sample in serum preparation, a potential biomarker for AD may be
Plasma Biomarkers for Differentiating AD from Controls
overlooked. Therefore, depending on the investigation, plasma may be preferable for
proteomic analysis/biomarker research instead of serum.
The ease with which blood, and therefore plasma, can be obtained from a human
makes this fluid ideal for biomarker investigation. Human plasma is estimated to contain
thousands of proteins and peptides that span a dynamic range in concentration of >1015
. Although an extremely rich source for disease-related biomarkers, the concentrations
of the most abundant proteins, the “classical proteins,” make plasma a difficult sample to
analyze. Albumin, the most abundant protein in plasma (albumin comprises greater than
half of the total plasma protein concentration), is capable of masking lower abundance
proteins that may be important as biomarkers. Depletion methods for high abundance
proteins have spawned a plethora of commercially available dyes, spin cartridges, and
high performance liquid chromatography (HPLC) columns that are capable of depleting
approximately 20 of the most highly abundant proteins in plasma. Thus, depletion of high
abundance proteins will increase the accessible dynamic range (Fig 1) and uncover lower
abundance proteins that would otherwise be masked in a non-depleted sample (for
example, see the 2D electrophoretogram in Fig 2).
In the last decade, numerous possible candidate biomarkers for AD have been
reported. A considerable number of AD studies of plasma have exploited differences in
the expression of individual proteins or peptides in AD patients compared to healthy
controls. Some of these proteins have direct effects on brain/cognitive processes or are
localized in brain, and therefore fulfill the requirement that an ideal biomarker be
reflective of the pathology it represents. Also, because of the multidimensionality of a
activity, and as a result, contribute to inflammatory processes observed in conditions such
biomarker, peptides or proteins identified as having a characteristic difference in AD
plasma may be useful in other ways. For example, proteins indicative of pathological
conditions of AD may lead to new therapeutic strategies.
Numerous studies have identified plasma proteins whose expression levels in AD
patients differ from controls. One proteomic study found alpha-2-macroglobulin (α2M)
and complement factor H (CFH) to have increased expression in AD plasma compared to
controls, and the increase for CFH was observed only for AD when compared with other
forms of dementia . Both α2M and CFH have been shown to be present in senile
plaques [7, 8], a hallmark of AD pathology. α2M is associated with damage to the blood
brain barrier (BBB) , which may be evident in AD . Also, α2M has been proposed
as a genetic polymorphism in AD [11, 12]
Alpha-1-antitrypsin (A1AT), a serine protease inhibitor, was found to be
increased in AD plasma [13, 14] and serum  compared to controls and oxidized in its
precursor form in AD . A1AT is localized in both senile plaques and neurofibrillary
tangles (NFT) . The physiological role of A1AT is to suppress overexpressed
proteases during inflammation; oxidation of this protein can lead to an alteration of its
as AD .
Alpha-1-antichymotrypsin (A1ACT) has been reproducibly found to be increased
in plasma/serum of AD patients compared to controls [19-25], although some groups
report otherwise [26, 27]. A1ACT is a serine protease inhibitor synthesized by the liver.
Elevated levels of this protein in the periphery may indicate the presence of systemic
inflammation . Plasma and CSF A1ACT levels have been shown to correlate with
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