Biological Marker Candidates of Alzheimer's Disease in Blood, Plasma, and Serum

Abstract

At the earliest clinical stages of Alzheimer's disease (AD), when first symptoms are mild, making a reliable and accurate diagnosis is difficult. AD related brain pathology and underlying molecular mechanisms precede symptoms. Biological markers can serve as supportive early screening and diagnostic tools as well as indicators of presymptomatic biochemical change. Moreover, biomarkers cover a variety of roles and functions such as disease prediction, indicating disease acuity and progression, and may ensure biological mapping of treatment outcome. Early screening, detection, and diagnosis of AD would permit earlier disease modifying intervention at potentially reversible stages. To date, most established biological markers from both cerebrospinal fluid neurochemistry and structural and functional neuroimaging have not reached widespread clinical application. Crucial remaining problems, such as easy acceptance and application of a test, cost‐effectiveness, and noninvasiveness, need to be resolved. The development and validation of precise, reliable, and robust tests and biomarkers in blood, plasma, or serum has therefore been for a long time the ultimate focus of many research groups worldwide. Blood‐based testing will most likely be the prerequisite to future sensitive screening of large populations at risk of AD and the baseline in a diagnostic flow approach to AD. The status and emerging perspectives on hypothesis and exploratory‐based candidate biomarkers derived from blood, plasma, and serum are reviewed and discussed.

Introduction

Although clinical manifestations of cognitive dysfunction and impairment of activities of daily living are the current standard for the diagnosis of Alzheimer's Disease (AD) [1], neurochemical and neuroimaging biomarker candidates are receiving increasing attention in research centers as objectively measurable diagnostic tools. Especially at very early stages when episodic memory impairment is still subtle and can go unrecognized by patients, informants, and even experienced physicians, underlying AD is very difficult to diagnose. In order to treat AD early before neurodegeneration has progressed to a widespread and basically irreversible stage of the disease process, there is need for a biomarker or combination of biomarkers that enable early presymptomatic and predementia diagnosis, at least at the symptomatic stage of mild cognitive impairment (MCI), and differentiation from other forms of dementia. Innovative, potentially disease‐modifying therapeutic strategies are of increasing importance and require biomarkers for risk enrichment in study populations, detection of disease progression, and verification of treatment effects (Fig. 1).

Figure 1.

Time course of AD – stage‐specific diagnostic and therapeutic windows (adapted and modified from [151]).

Biochemical biomarkers in cerebrospinal fluid (CSF) have been extensively studied and are already a well‐established diagnostic entity at last stages of diagnostic validation [2]. Measurement of amyloid β peptide 42 (Aβ42), tau protein, and hyperphosphorylated tau protein (p‐tau) in CSF of AD patients, as well as patients with other dementias and normal elderly controls revealed sensitivity and specificity levels between 80% and 90% for detecting AD versus normal elderly [3]. CSF concentration of Aβ42, tau, and p‐tau were also reported to be able to distinguish subjects with MCI who are likely to progress to AD from nonconverters [3]. However, lumbar puncture is considered as a relatively invasive procedure in many countries and obtaining CSF, especially repeatedly, on large numbers of elderly individuals in the community is challenging and currently far from realistic. Blood samples, on the other hand, are easy to obtain and offer a rich source of disease biomarkers, as approximately 500 mL of CSF are absorbed daily into the circulating blood. Furthermore, damage to the blood–brain barrier (BBB) which occurs in the course of AD may enhance exchange of proteins between CSF and blood in either direction [4].

The Food and Drug Administration (FDA) defines a biological marker as an objectively measured feature that is evaluated as an indicator of normal biologic or pathogenic processes or pharmacological responses to a therapeutic intervention. According to the 1998 Consensus Report of the Working Group on Molecular and Biochemical Markers of Alzheimer Disease [5], an ideal biomarker for AD should be able to detect a fundamental feature of Alzheimer's neuropathology, validated in neuropathologically confirmed AD cases, precise (able to detect AD early in its course and distinguish it from other dementias), reliable, noninvasive, simple to perform, and inexpensive. Blood‐based testing would be widely available, noninvasive, easy and rapid to perform, and economic.

In the context of the United States Alzheimer's Disease Neuroimaging Initiative (US‐ADNI) project, Frank et al. considered a wide range of biological measures with possible relevance to AD, and classified them into categories of “feasible, core”“feasible, con‐core” and “uncertain feasibility”[6]. Feasibility was determined by the availability of a validated assay for the biological measure in question, with properties that included high precision and reliability of measurement, where reagents and standards were well described. Core analytes were those judged by the group to have reasonable evidence for association with key mechanisms of pathology implicated in AD, while noncore were felt to be less clearly connected with mechanisms of pathogenesis and neurodegeneration in AD. In a recently completed pilot European Alzheimer's Disease Neuroimaging Initiative (E‐ADNI) biological marker program of CSF and plasma candidate biomarkers, feasibility and reliability of a European multicentre AD biomarker program have been confirmed [7].

The objective of this article is to review the literature on proposed “feasible, core” hypothesis‐based biomarker candidates in blood, plasma, and serum that are believed to detect a constituent part of the presumed AD pathophysiology. Additionally, we consider new data about novel exploratory proteome‐based biomarker candidates in plasma. We comprehensively searched PubMed Library of Medicine by entering the following search terms: Alzheimer's disease, biological marker, blood, plasma, serum, diagnosis, screening, early detection, prediction, and proteomics. In addition, we entered the single marker candidates’ names referring, with few exceptions, to core biological marker candidates as proposed by Frank et al. [6]. Since we did not make out suitable and relevant data before 1986, we refer to data that were published after 1986. We performed a survey of past and recent research, focusing on the most relevant and convincing data of the most promising biomarker candidates, not applying any specific exclusion criteria. Therefore, this article is to be considered as an opinion piece and not as an evidence‐based review of all available literature. The reviewed articles were published between February 1986 and May 2009 (Table 1).

Table 1.

List of biological measures

Category	Biological marker	Core references
Markers related to the amyloidogenic pathway	Amyloid beta peptides (Aβ40, Aβ42, Aβ42/40 ratio)	5–27
	Aβ autoantibodies	28–37
	APP isoforms in platelet membranes	38–47
Markers of tau	Tau protein	48, 3
	Phosphorylated tau protein	49, 3
Markers related to cholesterol metabolism	Cholesterol	50–70
	Oxysterols/24S‐hydroxycholesterol	71–78
	Apolipoprotein E, apolipoprotein E genotype	79–98
Markers of oxidation	Antioxidants (vitamins C, E and A, lycopene, beta‐carotene, urate, bilirubin)	99–107
	Isoprostanes/8,12‐iso‐iPF2α‐VI	108–116
Markers of immunological mechanisms and inflammation	α1‐antichymotrypsin	121–129
	Interleukin‐6, soluble interleukin‐6‐receptor‐complex	130–143
Markers of microvascular changes	C‐terminal endothelin‐1 precursor fragment, midregional pro‐adreno‐ medullin, midregional pro‐atrial natriuretic peptide	144–146
Exploratory proteome‐based plasma biomarkers	Numerous	147–150

Markers Related to the Amyloidogenic Pathway

Amyloid β Peptides

Amyloid plaques are a pathologically defining feature of AD neuropathology. Therefore, amyloid beta (Aβ) measures in biological fluids such as CSF and blood plasma are natural candidate biomarkers for AD [5]. The Aβ peptide in vivo occurs in several forms. Two forms, consisting of 40 (Aβ40) or 42 (Aβ42) amino acids, have been widely investigated. As involved in the amyloidogenic cascade, Aβ42 which is more toxic and fibrillogenic than Aβ40 in vitro is initially deposited in the brain [8, 9, 10]. The development of sensitive and specific ELISAs enabled the detection and quantitation of Aβ40 and Aβ42 in biological fluids. The circulating pool of Aβ is composed of Aβ produced by peripheral tissues and organs, as well as of Aβ produced by brain tissue and transported across the blood–brain barrier by means of predominantly two receptors responsible for Aβ transport [receptor for advanced glycation end products = RAGE and low‐density lipoprotein (LDL) receptor‐related protein‐1 = LRP‐1][11].

Plasma total Aβ[12] and Aβ42 [13] were increased in subjects with familial AD with presenilin 1 and 2 and amyloid precursor protein (APP) mutations, and Aβ42 was increased in Down syndrome with APP triplication [14]. These as well as other results were strengthening the hypothesis that sporadic cases of AD might be detectable by altered plasma levels of Aβ. However, most groups investigating Aβ plasma levels found no significant differences between AD and control cases [12, 13, 15, 16, 17]. Several longitudinal studies suggested that high plasma Aβ42 (but not Aβ40) levels in nondemented elderly people were a risk factor for developing cognitive decline or AD [18, 19, 20, 21]. In contrast, van Oijen et al. reported an association between high Aβ40, low Aβ42, and risk of dementia [22], a result that is in agreement with the findings of Graff‐Radford et al. [23], who observed a weak association between low plasma Aβ42/Aβ40 ratio and risk of future MCI or AD in a healthy, elderly population. In a recently published longitudinal cohort study [21] including 1125 elderly subjects, conversion to AD was correlated with a significant decline in plasma Aβ42, a decreased Aβ42/Aβ40 ratio and, with the onset of cognitive impairment, decreased protofibrillar Aβ42 levels. In cross‐sectional studies, however, plasma Aβ40 or Aβ42 levels did not correlate with cognitive impairment or dementia severity [17, 24, 25], but strongly with age [17, 18, 19], and with serum creatinine levels [26].

Measurement of Aβ in plasma was also thought to be of use in clinical treatment trials as outcomes of pharmacological effects of compounds influencing APP processing. For instance, reduction of Aβ plasma levels were observed under treatment with compounds that inhibit the enzymes β‐secretase or γ‐secretase which produce Aβ. In a phase‐II trial targeting Aβ production with the γ‐secretase inhibitor LY450139, oral administration of the compound was associated with reduced Aβ plasma levels [27].

Despite this pharmacologic effect, treatment‐related changes in plasma Aβ levels would not necessarily implicate clinical benefit, because plasma Aβ levels correlate poorly with severity of dementia. The overall contradictory findings could be partly explained by different measuring techniques, including the employment of different antibodies for Aβ detection. Furthermore, it is unclear which conformational state(s) of Aβ is/are measured by ELISA technique. The tendency of Aβ to adhere to many plasma proteins may also mask the epitopes required for Aβ detection. Lastly, little is known about the contribution of peripherally produced Aβ to cerebral Aβ deposition, and whether there is any correlation between plasma and CSF Aβ levels. Further investigation with high methodological standardization will be necessary in order to clarify the role of plasma Aβ peptides as biological markers for AD. The previous data give, however, rise to the speculation that with the onset of disease, the decline of plasma Aβ42 may reflect deposition of Aβ42 peptides in the brain.

Amyloid β Autoantibodies

Active and passive immunization against Aβ42 reduced cerebral amyloid deposition in APP transgenic mice [28, 29]. Several studies evaluated the effect of active immunotherapy with Aβ42 peptide which induced the production of antibodies with a high degree of selectivity for the pathogenic target structures [30, 31]. These observations led to the question whether individuals in the general population may harbor anti‐Aβ antibodies, either as a response to having AD or as an incidental phenomenon that may have a protective role against AD. In fact, naturally occurring autoantibodies directed against Aβ were traceable in human CSF and blood plasma [32, 33]. In CSF, significantly lower titers of anti‐Aβ antibodies have been observed in AD patients compared to nondemented controls (NDCs) using an ELISA [32]. Anti‐Aβ42 autoantibody levels were also significantly (P= 0.001) decreased in the serum of AD patients compared to healthy controls (HC) by use of an immuno‐precipitation assay with radiolabeled Aβ42 peptide [34]. In another study, however, plasma autoantibody titers did not differ between AD and healthy control subjects [33]. Yet, these studies concentrated on autoantibodies against monomeric Aβ (Aβ_mon). Hence, it was hypothesized that autoantibodies against oligomeric or aggregated Aβ were the clinically more relevant target [35]. Indeed, Moir et al. were testing blood plasma of AD patients and nondemented controls for auto‐antibodies specific for soluble low molecular weight oligomeric cross‐linked β‐amyloid protein species (CAPS) [36]. Plasma of nondemented controls and AD patients demonstrated similar immunoreactivity to synthetic unmodified Aβ_mon. In contrast, anti‐CAPS antibodies in AD plasma were found to be significantly reduced compared with nondemented controls (P= 0.018). In addition, age at onset for AD correlated significantly (P= 0.041) with plasma immunoreactivity to CAPS. These data suggest that autoantibodies to CAPS could be a future candidate plasma biomarker for AD and furthermore raise the prospect that immunization with anti‐CAPS antibodies might provide therapeutic benefit for AD.

Aβ antibody titers were also evaluated as a marker of treatment effectiveness in a placebo‐controlled randomized clinical trial which investigated active immunization with Aβ42 (AN1792) in 30 AD patients [35]. Intramuscular administration of the antigen AN1792 was followed by production of antibodies reactive against amyloid deposits in AD brain tissue. In this clinical trial, patients who generated such antibodies indicated reduced rates of decline of cognitive functions and activities of daily living, as compared to patients without antibodies. In another randomized placebo‐controlled trial investigating active immunization with AN1792 in AD patients [37], mean anti‐Aβ antibody titers were also raised in the treatment versus the placebo group (P= 0.02). In this trial, however, no correlation was found between anti‐Aβ antibody titers and rate of cognitive decline measured by Alzheimer's Disease Assessment Scale‐Cognitive Subscale (ADAS‐Cog), Mini‐Mental State Examination (MMSE) and Disability Assessment for Dementia (DAD) after a follow‐up‐period of 80 months.

To what extent human antibodies against Aβ‐related proteins may serve as a diagnostic marker for prediction, progression, or therapeutic monitoring of AD remains to be clarified in future studies.

APP Isoforms in Platelet Membranes

An altered pattern of APP isoforms in platelet membranes consisting of a reduced ratio between the higher (130 kDa) and the lower (106–110 kDa) molecular weight in the immunoreactivity bands has been described in patients with AD [38]. Soluble forms of cleaved APP are released into the extracellular space, analogous to processing in neurons, after platelets have been activated [39]. In platelets, 150 kDa intact APP is processed into 120–130 kDa and 110 kDa carboxy‐truncated forms. These two isoforms can be detected by means of a Western blot. Alterations in the APP isoform ratio in platelet membranes of AD patients were reported by several research groups [38, 40, 41, 42, 43]. The ratio of truncated forms of higher molecular weight (120–130 kDa) to truncated forms of lower molecular weight (110 kDa), defined as the platelet APP isoform ratio, was decreased in AD and MCI [38, 42, 43], but not in other dementias [38], and the extent of decrease correlated with indices of dementia severity [40, 43, 44]. Sensitivities and specificities for AD diagnosis lied in between the 80% and 95% range [38, 43]. APP isoform ratios were found to be independent of gender or apolipoprotein E (ApoE) genotype [42], and correlated inversely with age in one study [42], but not another [38]. Simvastatin [45] and cholinesterase inhibitors [46] interestingly corrected abnormally low APP isoform ratios in AD cases. Limitations of this candidate biomarker include technical factors (use of tourniquet, use of anticoagulants, platelet aggregation), and the Western blot technique itself which precludes high throughput and consistent standardization [47]. To interpret the value of this potential blood biomarker more clearly, we are in need of a better understanding of the biochemical nature of the platelet APP isoforms and their relationship to pathophysiological processes in AD.

Markers of Tau

The main component relating to intraneuronal changes in AD is the microtubule‐associated tau protein. It is increasingly evident that the disengagement of tau from microtubules is likely to comprise a cardinal step that sets the stage for tau‐mediated neurodegeneration. The link between this process and Aβ‐mediated toxicity and oxidative stress remains less clear. Although tau‐mediated neurodegeneration probably results from the combination of losses of function and toxic gains of function, the specific roles played by the various forms of misfolded and aggregated tau are not fully understood [48]. Hyperphosphorylation of the tau protein can result in the self‐assembly of tangles or paired helical or straight filaments which are involved in the pathogenesis of AD and other tauopathies [49].

Total tau protein and tau protein hyperphosphorylated at threonine 231 (p‐tau_231P) or at threonine 181 (p‐tau_181P) are already established CSF biomarkers for AD and reach diagnostic sensitivity and specificity levels between 80% and 90%[3].

Only recently, novel serum‐based diagnostic tests have been developed, for example, a sandwich ELISA tracing p‐tau_231P (Applied NeuroSolutions, Vernon Hills, IL, USA). Since at present there are no data available about the diagnostic usability of tau and p‐tau measures in serum or plasma, we excitedly await first results of these novel assay prototypes.

Markers Related to Cholesterol Metabolism

Cholesterol

A series of reports has been published that establish a connection between cholesterol, Aβ, and AD. Recent evidence suggests that amyloidogenic APP processing may preferentially occur in the cholesterol‐rich regions of membranes known as lipid rafts, and that changes in cholesterol levels could exert their effects by altering the distribution of APP‐cleaving enzymes within the membrane [50]. Experimental studies suggest that high total cholesterol (TC) accelerates the production of Aβ in AD, by shifting APP metabolism from alpha to beta cleavage products [51, 52]. Wirths et al. [53] reported the results of a neuropathologic study on brain Aβ and plasma TC levels in transgenic mouse models of AD and age‐matched control mice. The authors found a significant decrease of plasma TC in the APP transgenic animals, whereas plasma TC in young and aged control mice remained almost unchanged. In the APP transgenic mice, levels of plasma TC correlated inversely with brain Aβ42 levels. These results correspond with epidemiological studies in humans who have found decreased TC levels in AD patients [52, 54, 55]. In another study, cholesteryl ester levels in the brain of transgenic mice showed a direct correlation with Aβ production [56]. The use of cholesterol‐lowering drugs decreased neuronal production of Aβ in cultured cells and in transgenic mice [56, 57, 58], while diets rich in cholesterol increased amyloid deposition in rabbits [59]. Statins not only inhibit cellular cholesterol synthesis, they also affect cellular signaling by several different mechanisms. Among others, reduced cholesterol levels may reduce the cholesterol‐rich membrane domains, which in turn are believed to affect cellular signaling [60]. Animal experiments as well as human trials have shown that the lipophilic statins simvastatin and lovastatin are capable of crossing the blood–brain barrier (BBB), while the more hydrophilic pravastatin cannot [60].

High serum TC levels were associated with an increased risk of developing AD or cognitive impairment in humans in several cross‐sectional and longitudinal studies [61, 62, 63, 64, 65]. Furthermore, the use of cholesterol‐lowering drugs (HMG‐CoA reductase inhibitors, statins) was associated with a decreased risk of AD in several observational studies [66, 67, 68]. However, two large randomized controlled trials failed to show that use of statins reduced the incidence of dementia. One trial compared pravastatin (40 mg/day) with placebo in 5804 men and women aged 70–80 years who had a baseline TC level of 4.0–9.0 mmol/L [69]. The mean duration of follow‐up was 3.2 years. By the end of the trial, there were no significant differences in the MMSE scores or in the instrumental activities of daily living between the pravastatin and the placebo group. The other study compared simvastatin (40 mg/day) with placebo in 20536 men and women aged 40–80 years who had a baseline TC level greater than 3.5 mmol/L [70]. The duration of follow‐up was 5 years. By the end of the trial, the incidence of dementia was 0.3% in each of the two groups, despite a prominent lipid‐lowering benefit.

The lack of consensus regarding the association between TC and AD might be explained by the hypothesis that only midlife elevation in TC may be a risk factor for AD, and that the TC may be lowered by some aspect of the incipient dementing process years before onset of symptoms. Cholesterol lowering could be an effect rather than a cause of dementia. Large‐scale randomized controlled trials evaluating the capability of cholesterol‐lowering drugs to decrease the incidence of AD in amnestic MCI subjects could help clarify this issue.

Oxysterols/24S‐Hydroxycholesterol

Within the brain, cholesterol from damaged or dying neurons is converted to 24S‐hydroxycholesterol by cholesterol 24‐hydroxylase (CYP46) [71]. 24S‐hydroxycholesterol is subsequently transferred across the blood–brain barrier, transported to the liver by LDLs, and excreted as bile acids. Most of plasma 24S‐hydroxycholesterol is derived from brain cholesterol. Consequently, plasma levels of this oxysterol are believed to reflect brain cholesterol catabolism. Changes in the cholesterol equilibrium across the whole body may, to some extent, cause alterations in sterol recycling and ApoE expression within the central nervous system (CNS), which, in turn, may affect neuronal and myelin integrity. In two trials, 24S‐hydroxycholesterol was elevated in AD CSF [72, 73], but was only inconsistently increased in AD plasma [72, 73, 74]. Plasma levels showed only a weak, if any, correlation with CSF levels [72, 75]. In another trial cholesterol‐corrected concentrations of plasma 24S‐hydroxycholesterol were significantly reduced in patients with dementing disorders compared to nondemented subjects and depressed patients [76]. In addition, there is evidence that 24S‐hydroxycholesterol in CSF and plasma is reduced by statin treatment [71, 77, 78]. Since 24S‐hydroxycholesterol is a parameter which reflects brain cholesterol catabolism, it could be of use as a marker of treatment effectiveness in future trials evaluating cholesterol‐lowering drugs as a therapeutic or preventive strategy for AD.

Apolipoprotein E and Apolipoprotein E Genotype

The ApoE ɛ4 allele is of special interest in AD research since the presence of this allele is associated with a two‐ to four‐fold increased risk for late‐onset alzheimer's disease (LOAD), increased risk of sporadic and familial LOAD, earlier age of AD onset, increased amyloid plaque load, and elevated levels of Aβ40 in the AD brain [79, 80, 81, 82]. The ApoE genotype influences ApoE protein levels in plasma, with the ApoE ɛ4 allele being associated with less ApoE protein in plasma [83]. ApoE is the major apolipoprotein in the CNS where it is involved in the mobilization and redistribution of cholesterol, necessary for the maintenance of myelin and neuronal membranes during development and following injury [84].

Present data suggest that ApoE genotyping has low sensitivity and specificity with regard to AD diagnosis [85]. Possession of an ApoE ɛ4 allele increased the risk of AD conversion from MCI [86]. However, its predictive values (positive predictive value 0.48; negative predictive value 0.65) did not support its utility as a diagnostic test for predicting progression from MCI to AD. Therefore, ApoE genotyping is neither recommended for use in routine clinical diagnosis nor should it be used for predictive testing [87]. It may be useful, though, in the context of research studies in order to enrich or stratify study samples of MCI subjects which may have a higher and faster mean rate of progression to AD. Since the number of ApoE ɛ4 alleles has shown to influence CSF levels of the core AD biomarker candidates Aβ, tau, p‐tau, and beta‐site amyloid precursor protein‐cleaving enzyme‐1 (BACE1) [88, 89, 90, 91], ApoE genotyping is generally considered as part of investigatory biomarker studies.

Reports on the concentrations of serum or plasma ApoE protein levels in AD are conflicting. Studies documented elevated plasma or serum ApoE protein levels in AD [92], no difference [93, 94, 95] or reduced protein levels in AD relative to controls [96, 97]. The ratio of ApoE4 protein to ApoE3 protein in the plasma of heterozygous ApoE ɛ3/ɛ4 individuals did not correlate with AD diagnosis [98]. Because of the heterogeneity of the results, ApoE protein levels currently cannot be recommended as a diagnostic test.

Markers of Oxidation

Antioxidants

Oxidative stress is believed to play a major role in the pathogenesis of AD. Increased markers of protein, lipid, and nucleic acid oxidation and reduced activities of antioxidant enzymes in the AD brain support the contribution of oxidative stress to neurodegeneration and AD [99, 100]. A causative role of Aβ42‐induced oxidative stress in AD neurodegeneration is supported by findings from in vitro studies of lipid peroxidation, as well as by postmortem studies of lipid peroxidation in the AD brain [100]. Antioxidants in plasma include the vitamins C, E, and A, lycopene, beta‐carotene, urate, and bilirubin. Both vascular dementia and AD were variably associated with reduced serum or plasma levels of vitamins A, C, and E [101, 102, 103]. To what extent malnutrition, as a consequence of advanced dementia, might have contributed to these results remains unclear. Although vitamin E (oral administration of 2000 IU/day) delayed progression of AD in a double‐blind, placebo‐controlled, randomized, multicenter trial including 341 patients with a follow‐up period of 2 years [104], antioxidant vitamin supplementation did not affect incident cognitive impairment over 5 years as a secondary outcome of a large placebo‐controlled cardiological study involving 20536 individuals [105]. Recent systematic reviews have also concluded that, in the absence of evidence from randomized controlled trials, there is no justification for consuming antioxidant vitamins C or E with the intention of reducing the risk of subsequent AD [106]. Furthermore, doses of vitamin E greater than 400 IU/day have been shown to have negative cardiovascular effects [107].

Isoprostanes/8,12‐iso‐iPF2α‐VI

Growing evidence implicates oxidative and nitrative damage in the pathogenesis of AD and other neurodegenerative disorders [108, 109]. Free radical damage of proteins and polyunsaturated fatty acids results in modified forms that can be measured in body fluids as markers of oxidation state. Isoprostanes arise from free‐radical‐mediated peroxidation of polyunsaturated fatty acids. The isoprostane 8,12‐iso‐iPF2α‐VI is a sensitive and specific marker of in vivo lipid peroxidation. Isoprostanes were markedly elevated in both frontal and temporal poles of AD brains compared to the corresponding cerebella, as well as compared to corresponding brain areas from patients who had died with schizophrenia or Parkinson's disease or from non‐neuropsychiatric disorders [110]. In a transgenic mouse model of AD amyloidosis, levels of 8,12‐iso‐iPF2α‐VI in urine, plasma, and brain correlated with increasing brain Aβ levels and deposits [111]. Diseases implicating oxidative stress (ischemia, reperfusion, atherosclerosis, and inflammation) are associated with elevated plasma F(2)‐isoprostane levels, which in turn are modifiable by antioxidant treatment [112]. Elevated plasma levels of 8,12‐iso‐iPF2α‐VI were found in AD patients, with the levels correlating with measures of cognitive and functional impairment, established biomarkers of AD pathology (CSF tau and Aβ), and the number of ApoE ɛ4 alleles [109, 113, 114]. These results, however, were not confirmed by other groups [115, 116]. Because of the inconsistency of results, plasma isoprostane levels currently cannot be recommended for diagnostic purposes.

Markers of Immunological Mechanisms and Inflammation

Degenerating tissue and the deposition of highly insoluble abnormal materials are classical stimulants of inflammation. Likewise, in the AD brain damaged neurons and neurites, and highly insoluble Aβ peptide deposits and neurofibrillary tangles, provide obvious stimuli for inflammation [117]. The amyloid deposition in the AD brain is thought to elicit a range of reactive inflammatory responses including astrocytosis, microgliosis, upregulation of proinflammatory cytokines, complement activation, and acute phase reactions, with the proteins associated with inflammatory responses being found in tight association with the senile plaques [118]. Evidence now suggests that AD pathophysiology has important inflammatory and immune components and may respond to treatment by anti‐inflammatory and immunotherapeutic agents [118]. Whether the accumulation of cytokines and acute phase reactants within the brain is reflected in serum or plasma is not straightforward, because many of these proteins do not easily cross the blood–brain barrier [119]. Interpretation of immune mediator levels in AD serum and plasma has been limited so far by inconsistent results. Inflammatory molecules that were variably increased in AD include C‐reactive protein, interleukin (IL)‐1β, tumor necrosis factor‐α, IL‐6, IL‐6‐receptor‐complex (IL‐6RC), α1‐antichymotrypsin (ACT), and transforming growth factor‐β[119]. These inflammatory molecules were unchanged in other studies, as were other cytokines including IL‐12, interferon‐α, and interferon‐γ[120]. One longitudinal study of inflammatory factors in serum, CSF, and brain tissue in AD [121] did not find any significant differences between AD subjects and controls in the mean serum levels of the following mediators: IL‐1β, IL‐6, IL‐1 receptor antagonist, tumor necrosis factor α, the soluble tumor necrosis factor receptors I and II, and alpha1‐antichymotrypsin. There was also no correlation in either subject group between the levels of these inflammatory mediators in serum and the change in cognitive status or the progression of the atrophy of the medial temporal lobe measured by computed tomography. In this study, though, the sample size was very small (only eight patients and nine controls). To date, it has not yet been clarified if inflammation is an important molecular mechanism or driving force of AD, or if it is simply a byproduct of the disease process and does not substantially alter its course, or if the components of the inflammatory response are even beneficial and slow the progression of disease. Peripheral markers of inflammation could in any case serve as safety markers in therapeutic monitoring during trials evaluating anti‐inflammatory, immunotherapeutic agents, i.e. the immunization trials. Here, we focused on two peripheral inflammatory markers that were regarded as core analytes with reasonable evidence for association with key mechanisms of the assumed AD pathology, and whose measurement was associated with high feasibility determined by availability of a validated assay, high precision, and reliability [6].

α1‐Antichymotrypsin

The serum concentration of the serine protease inhibitor α1‐Anti‐chymotrypsin (ACT) seems to be one of the most convincing markers for CNS inflammation. Concentrations of ACT are elevated in the AD brain, and ACT is one of the components of the senile plaque [121, 122]. In addition to being produced in the liver and released into the serum, ACT is expressed in the AD brain, particularly in areas that develop amyloid lesions. ACT has been shown to promote Aβ polymerization in vitro and in vivo. Transgenic mice expressing human ACT alone or ACT along with mutant human APP showed a significant increase in tau phosphorylation, suggesting that ACT can induce tau hyperphosphorylation [123]. This result was further confirmed by the finding that addition of purified ACT induced AD‐related tau hyperphosphorylation in cortical neurons cultured in vitro[123]. The ACT‐treated neurons showed neurite loss and subsequently underwent apoptosis. Various studies reporting ACT levels in serum or plasma of AD patients have been published, but the findings are controversial. Some studies showed no difference in ACT serum concentrations between AD and controls [124], whereas others showed higher serum concentrations in AD than in controls [125, 126]. Two studies showed that plasma ACT levels correlated with severity of dementia in AD patients [127, 128]. In a large population‐based prospective cohort study comprising 6713 subjects, high plasma levels of ACT were associated with an increased risk of dementia [129].

The above findings indicate that inappropriate inflammatory responses are a potential threat to the brain and that the intervention directed at inhibiting the expression or function of ACT and possibly other immune modulating molecules could be of therapeutic value in neurodegenerative diseases such as AD. Therefore, ACT may be helpful as a within subject biomarker in interventions (particularly with anti‐inflammatory agents) directed at slowing or halting disease progression.

Interleukin‐6 and Soluble Interleukin‐6‐Receptor‐Complex (IL‐6RC)

IL‐6 participates in a variety of biological processes, such as immune responses, acute phase reactions, and differentiation of immune cells, and furthermore has been reported to serve as a relevant mediator of neuroregulatory and inflammatory processes in the CNS [130]. IL‐6, which is expressed by a variety of cells including microglia, astroglia, neurons, and endothelial cells, exerts its biological actions by complex interactions with specific soluble or membrane bound receptors, forming the biologically active IL‐6RC. IL‐6 has been consistently detected in the frontal, parietal and occipital cortex, and hippocampus of AD patients [131], where IL‐6 has been demonstrated in early plaques without neuritic pathology of isocortical and hippocampal brain samples of AD patients, whereas IL‐6 immunoreactivity was rare in advanced and solid plaques [117]. Based on these findings, it has been suggested that IL‐6 expression may appear before neuritic changes rather than follow neuritic degeneration. CSF levels of IL‐6 and the IL‐6R were inconsistently altered in AD patients compared to healthy controls (HC) [132, 133]. Several groups reported increased IL‐6 plasma or serum levels in AD patients [127, 134, 135, 136, 137, 138], although this was not the case in other cohorts [139, 140, 141, 142]. In one study, plasma levels of the soluble IL‐6RC were higher in AD patients than in control subjects [143]. In a large population‐based prospective cohort study, high plasma levels of IL‐6 at baseline were associated with an increased risk of subsequent development of dementia [129]. Since cytokines are degraded rapidly in blood and CSF, and rapid freezing as well as multiple freeze‐thaw cycles are likely to influence blood concentrations, homogenous processing methods are required in order to compare study results. Due to the numerous possible confounding factors in these studies (differences in plasma collection protocols, assay methodology, assay sensitivity, small sample sizes, heterogeneous patient populations, effects of disease severity, age, comorbid inflammatory illness), a definitive statement about the usefulness of IL‐6 or the IL‐6RC as diagnostic markers of AD cannot be made. Yet they could serve as markers of therapeutic monitoring in clinical trials evaluating substances directed at altering inflammatory or immunomodulatory processes in AD.

Markers of Microvascular Changes

Growing evidence suggests that vascular factors and cardiovascular dysfunction may contribute to the pathogenesis of AD [144]. Vascular lesions may unmask or worsen the clinical expression of AD [145]. Several vascular factors are presently under investigation in a variety of diseases associated with vascular dysfunction, such as adrenomedullin (ADM), endothelin‐1 (ET‐1), and atrial natriuretic peptide (ANP).

ADM, a peptide with 52 aminoacids, has immune modulating, metabolic, and vascular properties and has potent vasodilating activity. ET‐1, a 21 amino acid peptide, acts in an autocrine or paracrine manner as a vasoconstrictor on vasculature. ANP, consisting of 28 amino acids, promotes natriuresis and diuresis, inhibits the renin‐angiotensin‐aldosterone axis, and acts as a vasodilator. There are reliable assay systems available to detect precursor fragments of these circulation and microcirculation regulating factors C‐terminal endothelin‐1 precursor fragment (CT‐proET‐1), midregional pro‐adrenomedullin (MR‐proADM), and midregional pro‐atrial natriuretic peptide (MR‐proANP). Only recently, these markers were found to be altered in AD patients when compared to HC, showing a consistent pattern of elevated vasodilators (MR‐proANP and MR‐proADM) and a decreased vasoconstrictor (CT‐proET‐1) in AD patients [146]. The highest diagnostic accuracy was found for the MR‐proANP/CT‐proET‐1 ratio (specificity and sensitivity approximately 80%). These results indicate an altered expression of microcirculation parameters and support the hypothesis of a disturbed microvascular homeostasis in AD.

Exploratory Proteome‐based Plasma Biomarkers

A relatively novel approach is classification and prediction of clinical AD diagnosis based on plasma signaling proteins. Since the brain is controlling many body functions via the release of signaling proteins, and since central and peripheral immune and inflammatory mechanisms are increasingly implicated in AD, it was hypothesized that the pathological processes leading to AD would cause characteristic changes in the concentrations of signaling proteins in the blood, generating a detectable disease‐specific molecular phenotype [147]. In CSF, comparative peptide profiling revealed significant differences in correlation with diagnosis of AD supporting the working hypothesis that the degenerative character of AD is mirrored in the CSF peptide pattern [148]. Ray et al. found 18 signaling proteins in blood plasma that could be used to classify blinded samples of AD and control subjects with close to 90% accuracy, and to identify patients who had MCI that progressed to AD 2–6 years later [147]. A total of 120 known signaling proteins were measured in 259 plasma samples from individuals with presymptomatic to late‐stage AD and from various controls by use of filter‐based, arrayed sandwich ELISAs. Statistical analysis was performed by use of an unsupervised clustering algorithm and a shrunken centroid algorithm, called predictive analysis of microarrays (PAM), both applied to a training set and a test set equally consisting of AD and NDC samples. PAM identified 18 predictors out of the 120 analyzed signaling proteins and classified blinded samples of AD and NDC with 90% positive agreement (for the AD samples) and 88% negative agreement (for the non‐AD samples) with the clinical diagnosis. Additionally, plasma samples of two previously published MCI cohorts were analyzed at baseline and after a follow‐up period of 2–6 years. After application of the 18 predictors to the MCI test set, PAM classified 20 of 22 MCI patients who developed AD 2–5 years later correctly as AD (91% positive agreement with the clinical diagnosis). All eight MCI patients who later developed other dementias were correctly classified as non‐AD. Biological analysis of the 18 proteins pointed to an overall reduction in the abundance of factors associated with hematopoesis and inflammation during AD, as well as to deficits in neuroprotection, neurotrophic activity, phagocytosis, and energy homeostasis. Marksteiner et al. examined 16 of these signaling proteins by quantitative Searchlight multiplex ELISA in order to determine their sensitivity and specificity in 201 plasma samples from AD, MCI, depression, and healthy subjects [149]. Five out of 16 signaling proteins were elevated in plasma of patients with MCI and AD. Receiver operating characteristic (ROC) analysis predicted a sensitivity of 65–75% and a specificity of 52–63% when comparing HC versus MCI or AD. Another research group identified a total of 11 proteins that differed between AD cases and controls in a case–control study including 511 individuals with probable AD, other neurodegenerative diseases and normal elderly controls [150]. Analysis of the protein distribution by use of two‐dimensional gel electrophoresis identified AD cases with 56% sensitivity and 80% specificity. The biological function of almost all of these proteins was related to immune regulation. A general problem of this exploratory approach is that its mechanistic relevance with respect to AD is uncertain as compared to the hypothesis‐generated biological marker candidates.

Conclusions

Since it is hypothesized that pathology in AD commences roughly 20–30 years prior to diagnosis, early diagnosis would shift the initiation of treatment from late decelerating neurodegeneration to early potential disease modification and prevention. The aim is to have early and easily accessible diagnostic markers ready in clinical practice for initial broad screening of at risk subjects when disease‐modifying treatments become available. Thus, patients who would benefit from these strategies could be identified and treated in time. Blood‐based markers for early detection would be very advantageous due to the ease of sample collection and cost effectiveness. Studies conducted so far on blood, plasma, and serum in search of a biomarker for AD have not yielded a single candidate marker which is reliable, sensitive, and specific for AD diagnosis, risk assessment, disease progression, or monitoring of treatment effects. Several markers related to APP and Aβ metabolism, cholesterol metabolism, oxidative stress and inflammation appear to be altered in AD relative to controls, but yield insufficient discriminatory power (Table 2). Aβ40, Aβ42, and the Aβ42/Aβ40 ratio in plasma have been most intensively studied candidates, but to date not (yet) proven to achieve sensitivity or specificity above 80%. Some measures respond to medications, for instance HMG‐CoA reductase inhibitors reduce cholesterol and 24S‐hydroxycholesterol levels, active immunization with Aβ peptides induces production of anti‐Aβ antibodies, anti‐inflammatory drugs (e.g. NSAID) decrease levels of inflammatory cytokines and molecules, and antioxidants presumably reduce isoprostane levels. The importance of safety markers in clinical trials is evident, for example, the measurement of peripheral markers of inflammation in the context of innovative immunization trials. Anyhow, a consistent correlation with clinical benefit should be demonstrated before any parameter can be recommended as a surrogate marker in clinical trials. A combination of several peripheral markers and the integration of proteomic and metabolomic profiles currently seems the most promising approach, as long as there has not been detected a single marker fulfilling the required criteria of high reliability, specificity, and sensitivity.

Table 2.

Overall direction of change of biological markers in serum or plasma of patients with AD or MCI

Biological marker	AD	MCI	HC at risk
Aβ40	↑↔	↔	↑
Aβ42	↑↓↔	↓↔	↑↓
Aβ42/Aβ40 Ratio	↓	↓	↓
Monomeric Aβ autoantibodies	↓↔
Oligomeric Aβ autoantibodies (CAPS)	↓
Platelet APP isoform ratio	↓	↓
Tau
Phospho‐tau
Cholesterol	↔		↑
Oxysterol 24S‐Hydroxycholesterol	↑↓↔
Apolipoprotein E	↑↓↔
Antioxidants vitamins C, E & A	↓
Antioxidants lycopene & beta‐carotene	↓
Isoprostane 8,12‐iso‐iPF2α‐VI	↑↔
α1‐Antichymotrypsin	↑↔		↑
Interleukin‐6	↑↔
Soluble interleukin‐6‐receptor‐complex	↑↓		↑
C‐terminal endothelin‐1 precursor fragment	↓	↓
Midregional pro‐adrenomedullin	↑
Midregional pro‐atrial natriuretic peptide	↑
Pro‐ANP/pro‐ET‐1 ratio	↑	↑

Regarding elderly or middle‐aged healthy controls, we listed changes of biological measures that indicate increased risk of developing AD or cognitive impairment. Blanks indicate that, to our knowledge, the issue has not been addressed so far. Please note that the database for this overview, i.e. the number of studies investigating a particular marker, varies considerably between markers. ↑=increased; ↓=decreased; ↔=not different in comparison to non‐demented or cognitively normal controls, respectively.

Conflict of Interest

The authors have no conflict of interest.