**3. Oxidized proteins: Potential candidate biomarkers in AD**

Although the pathogenesis of AD is not yet fully known, it is clear that the disease is caused by a combination of risk factors. Among several hypotheses, oxidative stress is considered to play a significant role (Butterfield, 2007). Although CSF represents the most suitable biological fluid to study neurodegenerative diseases since it can reflect the biochemical changes occurring in brain, its analysis is not always easily feasible for a large scale screening, because the costs involved are enormous and procedures are invasive, uncomfortable and not without risk. For a full screening and early diagnosis, biomarkers easily detectable in biological samples, such as plasma, are needed. Up to now, the search for reliable biomarkers for AD in peripheral blood is very challenging because of difficulties with the standardization of the methods of analysis and the low reproducibility of the results. Although a set of plasma markers that differentiated AD from controls have been shown to be useful in predicting conversion from MCI to AD (Song., 2009), the study has not been yet verified by other researchers and the application of these candidate biomarkers have yet to achieve the diagnostic power, sensitivity, and reproducibility necessary for widespread use in a clinical setting. Oxidized proteins may represent potential candidate biomarkers for "oxidative stress diseases", such as AD.

The first report on protein oxidation in CSF samples was from Tohgi et al. (1999) who dem‐ onstrated that 3-nitrotyrosine moderately but significantly increased with advancing age, and showed a remarkable increase in patients with AD. As the free tyrosine concentration did not decrease, the increase in 3-nitrotyrosine with age or associated with AD did not appear to be directly related to an increase in free-nitrated tyrosines. Rather, the increased 3-nitrotyrosine was likely due to an increase in nitrated tyrosines in proteins or increased degradation of 3 nitrotyrosin containing proteins, which are highly vulnerable to degradation.The most reliable CSF markers in AD are Aβ42 and tau. Low CSF Aβ 42 is associated with amyloid pathology in the brain and high Tau is linked with neurofibrillary pathology (Frey etal. 2005). Most subjects with decreased CSF Aβ42 and high tau develop AD during the follow-up (Herukka et al., 2007). Therefore, these CSF markers may reflect brain pathology and identify preclinical AD. Interestingly, the levels of CSF Aβ42 showed a tendency to correlate positively with serum oxidative markers in the whole study population and with plasma nitrotyrosines in AD patients. Moreover, a negative correlation between CSF tau and serum nitrotyrosine levels was evidenced in controls (Korolainen et al., 2009). The correlation between CSF AD markers and blood oxidative markers may suggest that oxidative metabolism is changed in AD. This hypothesis is further supported by the finding of decreased CSF protein carbonylation in APOE ε4 carriers, which is considered an important risk factor for developing AD (Raber et al., 2004) and correlates with redox proteomics studies that identified metabolic proteins as oxidatively modified and dysfunctional (Choi et al., 2004).

2006). Newman et al (2007) also reported that a number of proteins modified by glutathiony‐

Candidate Bio-Markers of Alzheimer's Disease

http://dx.doi.org/10.5772/55069

205

Previous studies on CSF nitrite and nitrate levels in patients with AD have provided contra‐ dictory results, with some showing decreased nitrate levels (Kuiper.,1994), others showing unaltered nitrite/nitrate levels (Ikeda.,1995), and still others increased nitrate levels (Tohgi., 1998). However, another study from the same group showed that nitrite/nitrate levels in AD were stage-dependent, being elevated only in the early phase of AD and decreasing to control levels with disease progression (Tohgi., 1998). This finding was interpreted to reflect progres‐ sive reduction of neurons. In contrast, free 3-nitrotyrosine levels increased significantly in parallel with the severity of AD, suggesting that protein degradation increases with disease progression, resulting in increased release of free 3-nitrotyrosine from tyrosine residues that have been nitrated. 3-nitrotyrosine and the 3-nitrotyrosine/tyrosine ratios in the CSF, both of which are believed to reflect degradation of nitrated tyrosine-containing proteins, increased significantly with age and were remarkably higher in patients with AD than in controls.

A study by Choi et al. (2002) identified uniquely oxidized proteins in AD plasma. These authors applied two-dimensional gel electrophoresis (2DE) coupled with immunological staining of protein carbonyl and the oxidized proteins observed in the plasma of both AD subjects and non-AD controls were determined. However, the level of oxidation of these protein spots was markedly higher in the AD samples. They also found that the increased oxidation was not a generalized phenomenon. In the total protein stain profile, more than 300 spots were detected, but less than 20 spots were positive by immunostain‐ ing with anti-DNP antibody. Furthermore, of the seven proteins that were most intensive‐ ly oxidized, their relative levels of oxidation differed. These studies found that fibrinogen gamma chain precursor and alpha 1 antitrypsinprecursor showed increased levels of

**4. Identification of a new plasma biomarker of AD using metabolomics**

Current metabolomics research involves the identification and quantification of hundreds to thousands of small-molecular-mass metabolites (<1,500 Daltons) in cells, tissues, or biological fluids. The aims of such studies are typically to understand new diagnosis biomarkers, to understand the mechanism of action of therapeutic compounds, and to uncover the pharma‐ codynamics and kinetic markers of drugs in patients and in preclinical in vivo and in vitro models (Wilcoxen et al., 2010). Lipidomics is one of the metabolomics approaches used to analyze lipid species in biological systems (Hu et al., 2009; Han et al., 2005; Han and Gross, 2003). Investigating lipid biochemistry using a lipidomics approach will not only provide insights into the specific roles of lipid molecular species in healthy individuals and patients but will also assist in identifying potential biomarkers for establishing preventive or thera‐ peutic approaches for human health (Hu et al., 2009,Wenk.,2005; Rosenson.,2010 ). Lipidomics has recently captured attention, owing to the well-recognized roles of lipids in numerous

carbonyl groups in AD comparedwith controls (Stief et al., 1989).

lation in AD IPL.

**technology**

Subsequently, Ahmed et al. (2005) measured in CSF the levels of protein glycation, oxidation and nitration. The authors found that the concentrations of 3-nitrotyrosine,Nε-carboxymethyllysine,3-deoxyglucosone-derived hydroimidazolone and N-formylkynurenine (as markers of protein glycation) were increased in subjects with AD. The Mini-Mental State Examination (MMSE) score correlated negatively with 3-nitrotyrosine residue concentration. These findings indicated that protein glycation, oxidation and nitration were increased in the CSF of subjects with AD. A combination of nitration and glycation adduct estimates of CSF may conceivably provide an indicator for the diagnosis of AD. Increased levels of protein aggregates in the form of fibrils together with increased lipid peroxidation have been shown, both in AD andMCI brain (Butterfield et al., 2010).

Advanced oxidation end products (AOEs,) during AD, colocalize with neurofibrillary tangles, senile plaques, microglia, and astrocytes and have been also measured in plasma. Advanced oxidation protein products (AOPPs), a relatively novel marker of oxidative damage, are considered as reliable markers to estimate the degree of oxidant-mediated protein damage. A significant increase in protein carbonyls in hippocampus (HP) and inferior parietal lobule (IPL) of AD subjects compared with age-matched controls was observed. Dityrosine and 3-NT total levels were reported to be elevated in the hippocampus, IPL, and neocortical regions of AD brain. Alterations in brain phospholipids pattern, a more specific assessment of lipid peroxi‐ dation, have been reported for AD brain (Lovell et al., 1995; Nitsch etal.,1992; Prasad etal., 1998). The levels of phosphatidylinositol (PI) and phosphatidylethanolamine (PE), rich in easily oxidizable PUFA, are decreased in AD brain. The levels of F(2)-isoprostanes [F(2)-IsoP], F(4)-neuroprostane[F(4)-NP], and isoprostane 8,12-iso-iPF2(α)-VI were also found to be increased in AD brain compared to controls (Montine et al.,2002; Mark et al,1999). An increase in free HNE has been demonstrated in amygdala, hippocampus, and parahippocampal gyrus of the AD brain compared with age matched controls (Markesbery.,1998). Several proteins mainly involved in energy metabolism pathways, pH regulation, and mitochondrial func‐ tionsamong others, were found carbonylated, HNE-bound or nitrated in AD brain (Sultana, 2006). Newman et al (2007) also reported that a number of proteins modified by glutathiony‐ lation in AD IPL.

was likely due to an increase in nitrated tyrosines in proteins or increased degradation of 3 nitrotyrosin containing proteins, which are highly vulnerable to degradation.The most reliable CSF markers in AD are Aβ42 and tau. Low CSF Aβ 42 is associated with amyloid pathology in the brain and high Tau is linked with neurofibrillary pathology (Frey etal. 2005). Most subjects with decreased CSF Aβ42 and high tau develop AD during the follow-up (Herukka et al., 2007). Therefore, these CSF markers may reflect brain pathology and identify preclinical AD. Interestingly, the levels of CSF Aβ42 showed a tendency to correlate positively with serum oxidative markers in the whole study population and with plasma nitrotyrosines in AD patients. Moreover, a negative correlation between CSF tau and serum nitrotyrosine levels was evidenced in controls (Korolainen et al., 2009). The correlation between CSF AD markers and blood oxidative markers may suggest that oxidative metabolism is changed in AD. This hypothesis is further supported by the finding of decreased CSF protein carbonylation in APOE ε4 carriers, which is considered an important risk factor for developing AD (Raber et al., 2004) and correlates with redox proteomics studies that identified metabolic proteins as

Subsequently, Ahmed et al. (2005) measured in CSF the levels of protein glycation, oxidation and nitration. The authors found that the concentrations of 3-nitrotyrosine,Nε-carboxymethyllysine,3-deoxyglucosone-derived hydroimidazolone and N-formylkynurenine (as markers of protein glycation) were increased in subjects with AD. The Mini-Mental State Examination (MMSE) score correlated negatively with 3-nitrotyrosine residue concentration. These findings indicated that protein glycation, oxidation and nitration were increased in the CSF of subjects with AD. A combination of nitration and glycation adduct estimates of CSF may conceivably provide an indicator for the diagnosis of AD. Increased levels of protein aggregates in the form of fibrils together with increased lipid peroxidation have been shown, both in AD andMCI

Advanced oxidation end products (AOEs,) during AD, colocalize with neurofibrillary tangles, senile plaques, microglia, and astrocytes and have been also measured in plasma. Advanced oxidation protein products (AOPPs), a relatively novel marker of oxidative damage, are considered as reliable markers to estimate the degree of oxidant-mediated protein damage. A significant increase in protein carbonyls in hippocampus (HP) and inferior parietal lobule (IPL) of AD subjects compared with age-matched controls was observed. Dityrosine and 3-NT total levels were reported to be elevated in the hippocampus, IPL, and neocortical regions of AD brain. Alterations in brain phospholipids pattern, a more specific assessment of lipid peroxi‐ dation, have been reported for AD brain (Lovell et al., 1995; Nitsch etal.,1992; Prasad etal., 1998). The levels of phosphatidylinositol (PI) and phosphatidylethanolamine (PE), rich in easily oxidizable PUFA, are decreased in AD brain. The levels of F(2)-isoprostanes [F(2)-IsoP], F(4)-neuroprostane[F(4)-NP], and isoprostane 8,12-iso-iPF2(α)-VI were also found to be increased in AD brain compared to controls (Montine et al.,2002; Mark et al,1999). An increase in free HNE has been demonstrated in amygdala, hippocampus, and parahippocampal gyrus of the AD brain compared with age matched controls (Markesbery.,1998). Several proteins mainly involved in energy metabolism pathways, pH regulation, and mitochondrial func‐ tionsamong others, were found carbonylated, HNE-bound or nitrated in AD brain (Sultana,

oxidatively modified and dysfunctional (Choi et al., 2004).

brain (Butterfield et al., 2010).

204 Understanding Alzheimer's Disease

Previous studies on CSF nitrite and nitrate levels in patients with AD have provided contra‐ dictory results, with some showing decreased nitrate levels (Kuiper.,1994), others showing unaltered nitrite/nitrate levels (Ikeda.,1995), and still others increased nitrate levels (Tohgi., 1998). However, another study from the same group showed that nitrite/nitrate levels in AD were stage-dependent, being elevated only in the early phase of AD and decreasing to control levels with disease progression (Tohgi., 1998). This finding was interpreted to reflect progres‐ sive reduction of neurons. In contrast, free 3-nitrotyrosine levels increased significantly in parallel with the severity of AD, suggesting that protein degradation increases with disease progression, resulting in increased release of free 3-nitrotyrosine from tyrosine residues that have been nitrated. 3-nitrotyrosine and the 3-nitrotyrosine/tyrosine ratios in the CSF, both of which are believed to reflect degradation of nitrated tyrosine-containing proteins, increased significantly with age and were remarkably higher in patients with AD than in controls.

A study by Choi et al. (2002) identified uniquely oxidized proteins in AD plasma. These authors applied two-dimensional gel electrophoresis (2DE) coupled with immunological staining of protein carbonyl and the oxidized proteins observed in the plasma of both AD subjects and non-AD controls were determined. However, the level of oxidation of these protein spots was markedly higher in the AD samples. They also found that the increased oxidation was not a generalized phenomenon. In the total protein stain profile, more than 300 spots were detected, but less than 20 spots were positive by immunostain‐ ing with anti-DNP antibody. Furthermore, of the seven proteins that were most intensive‐ ly oxidized, their relative levels of oxidation differed. These studies found that fibrinogen gamma chain precursor and alpha 1 antitrypsinprecursor showed increased levels of carbonyl groups in AD comparedwith controls (Stief et al., 1989).
