Biochemical biomarkers may be assessed in different matrices or compartments, including CSF, blood, and urine. Many of the same considerations given to imaging techniques also apply to these biochemical measures (eg, the validity and accuracy of the analytical method and the variability among multiple sites). More specific to biochemical measures are the need to standardize sample-handling techniques and to standardize methods for obtaining and storing CSF.
Lumbar punctures and CSF analyses have been used routinely in the practice of neurology for decades, although with the advent of other diagnostic modalities, this procedure is now performed most frequently in research settings in the United States. Nevertheless, two large studies of lumbar punctures performed as part of an evaluation of possible AD biomarkers have shown that the procedure can be applied broadly and that it is well tolerated.39,40
The only recorded complication was post-lumbar puncture headache. With the use of a small diameter needle (0.7 mm), the rate of mild headache (duration less than 1 day, not affecting daily life) was less than 4%, and the rate of moderate or severe headache (duration more than 1one day and/or affecting daily life) was less than 1%.
While the initial pathogenic events in AD are not known with certainty, biochemical markers of the disease can be considered as more proximal or upstream, compared with more distal or downstream events. As shown in , a number of potential biomarkers can be measured that may be proximal or distal in the pathogenic process.
FIGURE 3 Potential biochemical biomarkers in AD. Question marks indicate processes or anatomic areas that may be proximal in the disease process. Possible biomarkers that can be considered given these postulated disease processes include tau, phospho-tau, sulfatides, (more ...)
Each potential biomarker must have certain characteristics to be useful in multicenter trials. The assay must have excellent sensitivity and test/retest reliability. Sample handling requirements must be such that analyses have acceptable variability when samples are obtained at multiple sites. The biomarker analyte should reflect a key feature of AD pathology or a mechanism of disease. Finally, the pattern of change in the biomarker over time and variability of that change should be adequately described.
Aβ, and in particular Aβ1–42
, has been studied frequently as a biomarker for AD. CSF concentrations of Aβ1–42
are reduced by 40% to 50%, whereas concentrations of Aβ1–40
” (using an ELISA that does not distinguish C-terminal length) are similar to those of age-matched controls. CSFAβ1–42
does correlate to an extent with dementia severity; however, in most studies concentrations are stable over intervals as long as 12 months.41
Plasma concentrations of Aβ1–42
do not correlate with those in CSF.42
Longitudinal studies have not shown a consistent change in plasma Aβ over time in AD patients,43
and cross-sectional differences between AD patients and controls that would allow plasma Aβ concentrations to be used as a diagnostic measure have not been identified.
Cerebrospinal fluid tau has also been studied as a potential biomarker in AD.44
Elevations of 2- to 3-fold of CSF total tau (T-tau) levels in patients with AD have been demonstrated in cross-sectional studies. In longitudinal studies, weak correlations are present with changes in cognitive scores, and CSF T-tau levels remain stably elevated in AD over time intervals of 12 months or longer. Tau may be phosphorylated at various sites, and forms of CSF tau reflecting specific sites of phosphorylation (P-tau 181, 199, 231, 235, 396, and 404) have been studied.
Three species of p-tau (p-thr231, p-ser199, and p-thr181) have been examined in detail in cross-sectional studies.45–49
All three species are elevated in the CSF of patients with AD, and concentrations of all three species appear to be linearly related. When assessed as diagnostic measures, these three measures have similar sensitivity, although p-thr231 may have somewhat greater specificity for AD versus other forms of dementia.45
Interestingly, p-thr231 tau, as well as other forms, is elevated in MCI patients compared with control subjects, but longitudinal studies of AD patients show a progressive decline in concentration with disease progression.50
There are several studies in which the diagnostic performance of the combination of CSF T-tau and Aβ1–42
has been evaluated.44
In most but not all studies,51
the sensitivity and specificity for the combination of these two biomarkers have been slightly higher (89% and 90%, respectively) than for T-tau (81% and 91%, respectively) or Aβ1–42
(86% and 89%, respectively) alone. Other combinations of CSF biomarkers have also resulted in slightly better diagnostic performance than the use of single markers. In a study on the combination of CSF p-tau181 and Aβ1–42
, the sensitivity was 86% at a specificity of 97%,52
and in another study the combination of CSF T-tau and p-tau396/404 resulted in a sensitivity of 96% at a specificity of 100%.53
Further studies examining the value of combinations of biomarkers in larger series of patients and controls, and in particular in MCI, are needed.
As with imaging measures, sample size calculations for clinical trials can be made using Aβ and tau measures. As shown in , samples sizes using biochemical measures are similar to those achieved with imaging and are smaller than sample sizes based on clinical cognitive measures.
Besides the pathologic hallmarks of the disease, which include amyloid plaques and neurofibrillary tangles, AD pathology is characterized by evidence of reactive-oxygen species (ROS)–mediated damage.54
ROS are formed under normal conditions, and although they are chemically unstable and highly reactive, their levels are kept relatively low by efficient antioxidant systems including catalase, glutathione, uric acid, and vitamins E and C. However, in some situations their generation can exceed the endogenous capacity to destroy them. As a consequence, the oxidant versus the antioxidant balance is altered and oxidative damage is the final result.55
Depending on the substrate attacked by ROS, oxidative damage will manifest as protein oxidation, DNA oxidation, or lipid peroxidation products, all of which have been described in AD brain (). In general, oxidative damage in the central nervous system predominantly manifests as lipid peroxidation because of its high content of polyunsaturated fatty acids that are easily susceptible to oxidation.56
FIGURE 4 Products of reactive oxygen species–dependent attack of different substrates (nucleic acid, protein, lipid) and relative most employed analytical methods (GC/MS: gas chromatography/mass spectrometry; HPLC: high performance liquid chromatography; (more ...)
Isoprostanes are members of a complex family of lipid oxidation products derived from an ROS-mediated attack on free or esterified fatty acids. One group of them, called F2
-iPs) (), are present in detectable quantities in all normal biologic fluids and tissues. Assays for specific F2
-iPs isomers using gas chromatography/mass spectrometry have identified 8,12-iso
-VI (IPF2A) to be the most abundant F2
-iP in human as well as in animals.57
FIGURE 5 The four classes of F2-isoprostane deriving from the ROS-mediated oxidation of arachidonic acid.86
IPF2A concentrations are elevated in brain, CSF, and plasma of AD patients compared with controls.58,59
In cross-sectional studies, concentrations of IPF2A in CSF correlate directly with concentrations of total tau and inversely with Aβ1–42
In patients with MCI, CSF concentrations of IPF2A are intermediate between those of AD patients and those of control subjects; interestingly, patients with MCI who progress to AD have higher concentrations than those who do not.59
Recent investigations were conducted to determine whether the increase in this marker of lipid peroxidation is present in neurodegenerative diseases other than AD. For this reason, histopathologically confirmed AD was compared with frontotemporal dementia (FTD) subjects, a heterogeneous group of dementing disorders with neurodegeneration. Levels of IPF2A were found to be markedly elevated in postmortem AD brains compared with corresponding areas of FTD and control brain tissues.60
This observation was also confirmed in CSF from living patients with clinical diagnosis of FTD.61
Longitudinal studies in MCI patients showed that CSF F2-iPs levels were elevated at both baseline (P < 0.001) and follow-up (P < 0.01) compared with controls. This resulted in an overall classification accuracy of 88%, both at baseline and follow-up. Moreover, a significant longitudinal change was seen in the MCI patients relative to controls. The longitudinal change yielded an overall classification accuracy of 76%, and post hoc examination showed a significant isoprostane increase restricted to the MCI group (de Leon M, DeSanti S, Zinkowski R, et al. Biomarkers for Alzheimer’s disease improve early diagnosis. Neurobiol Aging. 2005 [in press]).
Many, including Alzheimer himself, have observed enlarged (more recently referred to as activated) microglia and astrocytes in brain of Alzheimer patients. A 1989 report provided the first evidence of a neuropathogenic role for two of the principal cytokines derived from activated microglia and astrocytes, viz., IL-1, a potent pro-inflammatory cytokine, and S100B, a potent neurotogenic cytokine: (1) overexpression was shown to be already dramatic in neonates, children, and young adults with Down’s syndrome (a virtually certain risk for precocious development of AD)62
and (2) a similar overexpression was demonstrated in end-stage AD.63–65
The neuro-pathogenic role of these two cytokines has been further supported by findings that both S100B66
regulate production of the β-amyloid precursor protein (βAPP) as well as reports that the number of activated astrocytes and microglia overexpressing these two cytokines are related to the progression of β-amyloid plaques.68,69
Overexpression of these cytokines has been implicated in the pathogenesis of AD by their demonstrated influences on the genesis and formation of the two principal features in AD, neuritic β-amyloid plaques and neurofibrillary tangles, as well as synaptic loss, and neuronal dysfunction and loss.70–79
This strong evidence for cytokine involvement in such neurodegenerative processes, underscores the importance of astrocyte-derived and microglia-derived cytokines in the brain’s innate immune system and its role pathogenesis. In addition to overexpression of IL-1 and S100B, expression of IL-6,80
are increased in AD brain. Moreover, there is evidence that expression of each is up-regulated by IL-1 and that expression of each is decreased by suppression of IL-1.83–85
Findings such as these suggest that neuroinflammation, powered by glia-derived cytokines, drives a self-sustaining, self-amplifying cycle that leads to a vicious circle of inflammation and neuronal dysfunction and death. The way in which neuronal insults are implicated in AD neuropathogenesis is summarized in .
FIGURE 6 Inflammatory changes implicated in Alzheimer disease (AD). This conceptual scheme of the cytokine cycle illustrates the relationship of glial activation and inflammatory cytokine overexpression to neurodegenerative events in AD, with the effect of overexpression (more ...)
An important goal is to discover markers of conversion from a situation in which the brain can cope with a given level of insults and when the insults are sufficient to mildly impair brain function. Most of the studies cited above used brain tissue, but the need for studies of more accessible tissue has resulted in studies using CSF, some of which show tantalizing, but inconclusive, changes in α1
-ACT and IL-6.2
Inflammatory markers have also been measured in serum, and early preliminary studies show that the relative levels of IL-1, IL-6, IL-8, IL-10, IL-12 and may be different in serum from patients who converted from control to MCI (Griffin et al, unpublished data). Measuring serum cytokine levels may serve as peripheral biomarkers of innate brain immune responses. At sufficient sensitivity, such biomarkers, although not likely to be specific for AD or other neurodegenerative condition, may be useful as peripheral indicators of progression of neural pathologies typical of AD. One way in which they might prove most useful is in testing the efficacy of therapeutic interventions. Not only the sensitivity but also the specificity of these putative biomarkers will need to be established in future trials.