**3. Plasma markers**

Compared with CSF, blood sampling is a less invasive procedure, more easily accessible, and cost reductive, thus the finding of reliable blood biomarkers for AD is being given the highest priority. There has been an increasing research effort to examine the potential biomarkers of AD in blood plasma. However, for blood-based biomarkers, it has to be noted that blood plasma contains several tens of thousands of different proteins. In addition, the range of protein concentrations are extremely varied (attaining to 12 orders of magnitude), and the lower the concentration, the greater the diversity of proteins [24, 25]. Moreover, none of the current methods allows us to directly detect components in the low concentration region [25]. These conditions make it extremely challenging or almost impossible to directly analyze blood, even though possible biomarker candidates are more likely to be present in the areas of low concentration. The change in concentration of the blood components may often be on a very small scale and cover a wide range of both peripheral and central processes. Additionally, the less abundant proteins may be masked by highly abundant plasma proteins such as albumin and immunoglobulin. Therefore, focusing on concentration change of a particular AD-specific marker, which may be in low concentration, can be the most challenging to discover [26, 27].

It was reported that the BBB is disrupted resulting in increased permeability with aging and in AD [28, 29]. It is also thought that this event occurs in the relatively early stage of the aging brain, which is related to increased cognitive impairment. Although the relationship between an analyte found as a biomarker candidate in blood plasma and the behavioral changes in the brain is not easily demonstrated, there is the possibility of a connection due to BBB disruption during the early stage. This might lead one to expect the possible appearance of a brain component in the peripheral blood stream.

The widely accepted CSF biomarker, Aβ peptides, have also been examined in blood, but its concentration in blood plasma is considerably lower than reported in CSF by about 100-fold [30]. Elevated plasma level of either Aβ<sup>40</sup> [31, 32] or Aβ<sup>42</sup> [33, 34] levels was used as an indicator for the development of AD, while the opposite results [35] or no association at all between plasma Aβ level and AD development [36, 37] were reported. Thus, results based on plasma Aβ as a biomarker have been inconsistent. A low plasma Aβ42 to Aβ40 ratio was utilized for the prediction of future AD [32, 38, 39], while contrary results, reporting a higher ratio [31, 33] in the nascent AD stage patients than the subjects who did not develop AD, and no significant differences were also shown [36].

Another promising candidate of a plasma protein biomarker was identified by means of the proteomic approach. The protein clusterin/apolipoprotein J, which is involved in the clearance of cellular debris and apoptosis, was associated with both hippocampal atrophy and clinical progression [40]. Increased plasma concentration of clusterin indicated the prediction of greater fibrillary amyloid-β burden in the medial temporal lobe and AD patients had increased clusterin messenger RNA in blood. Moreover, in the transgenic AD mouse model (APP/PS1), increased plasma clusterin level, age-dependent increase in brain clusterin, as well as amyloid and clusterin colocalization in plaques were shown [40, 41]. The recent finding is that increased plasma clusterin levels have been associated with increased risk of conversion to AD and the rate of cognitive decline [42]. Clusterin may have a role in Aβ aggregation and clearance [43, 44], and at high concentrations, clusterin may prevent Aβ aggregation through its binding to Aβ. Furthermore, clusterin possesses neurotoxic properties by involvement in noncanonical wnt signaling, which mediates Aβ toxicity [45]. Therefore, clusterin might fulfill different roles. Other plasma biomarker candidates have been reported, such as desmosterol [46], transthyr‐ etin [47], chitinase 3-like 1 protein [48], and matrix metalloproteinase 2 [49], which may be associated with AD. Using protein array technology, Ray et al. found 18 signaling proteins in blood plasma that can discriminate AD samples from control subjects with approximately 90% accuracy [50].
