Current Strategies in The Discovery of Small-Molecule Biomarkers For Alzheimers Disease

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Current strategies in the discovery of small-molecule biomarkers for Alzheimers disease

With the number of patients suffering from Alzheimers disease rapidly increasing, there is a major requirement for an accurate biomarker capable of diagnosing the disease early. Much of the research is focused on protein and genetic approaches; however, small molecules may provide viable marker molecules. Examples that support this approach include known abnormalities in lipid metabolism, glucose utilization and oxidative stress, which have been demonstrated in patients suffering from the disease. Therefore, by-products of this irregular metabolism may provide accurate biomarkers. In this review we present the current approaches previously published in the literature used to investigate potential small-molecule and metabolite markers, and report their findings. A wide range of techniques are discussed, including separation approaches (LC, GC and CE), magnetic resonance technologies (NMR and magnetic resonance spectroscopy), and immunoassays.
At present, there is no definitive diagnosis available for Alzheimers disease (AD). Current figures list those affected by the condition at over 400,000 people in the UK alone [201] , with further estimates suggesting there are as many as 5.1 million sufferers in the USA [1] , meaning the demand for an accurate indicative method is high. This demand is also set to increase over the coming years as currently it is understood that the main risk factor of sporadic AD is age, with the incidence of the disease doubling every 5 years after 65 years of age [2] . As the average lifespan of the population increases due to improved worldwide healthcare, figures of AD incidence are expected to rise significantly over the coming decades (Figure 1) [3,4] . Current diagnosis and monitoring of disease progression involves the completion of a selection of mental tests, for example a minimental state examination (MMSE) [5] . However, these tests have been shown to give highly variable results [6] , lacking accuracy and consistency between patients and, hence, predictive capabilities. The identification of an indicative biomarker would be a major progressive step from a diagnostic viewpoint, but perhaps more importantly it would have the potential to improve our knowledge of the disease pathways and pathology, hopefully leading to novel treatment strategies, improving the lives of those affected. The elevation or reduction of small-molecule
10.4155/BIO.11.62 2011 Future Science Ltd
concentrations can indicate abnormalities in biological pathways, which can be amplified at metabolite level [7,8] , making small-molecule metabolites ideal markers of disease. A further advantage of using metabolites as biomarkers is the ability to track metabolic changes over time, for instance after the addition of a drug [9,10] , enabling a reflection of their therapeutic effect, improving the ability to track disease progression and treatment response. This review sets out to discuss recent strategies employed for the discovery of small-molecule biomarkers in AD, providing examples of molecules of interest and their importance to the knowledge and understanding of the disease. As the review was focused on describing analytical methods that have been applied in AD biomarker discovery, selection for inclusion of articles related to specific analytical techniques as well as focus upon small molecules. Search terms were generally technique-specific, for example Alzheimers and LCMS, with the results then sorted manually for publications relevant for the review. This review is not designed to be a comprehensive review on all the AD small-molecule biomarker candidates published, rather it is designed to be a reading source providing background information and considerations to be taken regarding the potential approaches that can be taken when developing a method for their identification.
Bioanalysis (2011) 3(10), 11211142
Luke Whiley1,2 & Cristina Legido-Quigley 1

Pharmaceutical Sciences Research Division, Franklin-Wilkins Building, 150 Stamford Street, Kings College London, London, UK 2 Medical Research Council Centre for Neurodegeneration Research, Institute of Psychiatry, Kings College London, London, UK Author for correspondence: Tel.: +44 207 848 4722 E-mail: cristina.legido_quigley@ kcl.ac.uk
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ISSN 1757-6180
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120 AD affected prediction (millions) 90 60 30
Whiley & Legido-Quigley

Analytical techniques used in small-molecule biomarker discovery for AD A wide range of techniques are used to identify small-molecule biomarkers, with vast improvements in recent years in the sensitivity and selectivity of analytical technologies including NMR, UHPLC and MS. Not one technique is perfect and often a compromise has to be taken depending upon a range of criteria including speed, cost and properties of the analytes, sensitivity and the biofluid employed. These methods are discussed below with a brief summary of each technique, as well as providing examples of the roles in biomarker identification in AD. Targeted versus nontargeted approaches Method development for biomarker discovery tends to take one of two options, first, a targeted approach can be made, where the study is designed using a hypothesis based upon a specific molecule or group of molecules. Second, a nontargeted or top-down strategy can be used, which sets out to analyze a biofluid nondiscriminately, identifying any differences in the concentrations of molecules detected. The two routes are discussed here with methods provided to show examples of how both options are successfully implemented.
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2020 Year
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Figure 1. Current and projected incidence of AD in the coming decades. Figure adapted from [3] using data presented by [4] . AD: Alzheimers disease.
Key Terms Mini-mental state examination: A brief 30
question approach currently employed to assist Alzheimers disease diagnosis.
Biomarker: A biological
component that is objectively measured and used as an indicator of a biological state.
Targeted approach:
Biomarker identification using a hypothesis based approach designed to investigate a specific molecule or group of molecules.
Nontargeted approach:
Biomarker identification using an unknown hypothesis approach designed to analyze a large number of molecules in an unbiased manner.
Sample considerations for AD biomarker identification When planning a strategy for small-molecule biomarker investigations, a vital consideration to make is the choice of biofluid analyzed. Studies are often designed to focus on the disease site, and post-mortem brain samples are regularly used for this purpose [1113] . Although brain samples have an important use in initially identifying novel markers, there are obvious disadvantages and they are of no practical use for diagnosis in a clinical scenario, as taking a portion of brain for analysis is just not feasible in live patients. In some cases it is possible to quantify biomarkers in vivo within the brain using noninvasive imaging techniques such as magnetic resonance spectroscopy (MRS) [1416], removing the need for a brain homogenate; however, due to the loud noise and confined space associated with a MRS scan this can be a distressing and confusing experience for sufferers of AD. Cerebrospinal fluid (CSF) is a very useful biofluid due to its close proximity to the disease site and it has been used in many AD biomarker studies [1722] ; however, the use of CSF is far from ideal due to the difficulties involved in obtaining such samples via an uncomfortable lumbar puncture procedure, particularly from patients suffering from the disease. Therefore, biomarker studies that identify molecules of interest in relatively noninvasive samples such as plasma [2325] or urine [2628] have significant benefit. When analyzing plasma and urine, the ability of hypothetical biomarkers to cross the bloodbrain barrier (BBB) should be considered. A known pathological consequence of AD is the deterioration and subsequent damage of the BBB, resulting in an increased permeability and leakiness [2931] , theoretically improving the chance that any prospective biomarkers or drugs will be able to cross the BBB.
Bioanalysis (2011) 3(10)
Targeted approaches NMR & MRS
NMR is a highly useful analytical technique that is capable of the identification of atoms based on their ability to absorb a specific frequency of electromagnetic radiation when a magnetic field is applied. The simplified theory behind the technique is based upon certain common atomic nuclei such as 1H, 13C, 15N, 19F 29Si and 31 P, which have properties that enable them to spin and therefore they align with a field of a magnetic source. Radiofrequency radiation is then applied to the sample, which is used to excite the atoms, switching the nuclei between aligned and nonaligned states. The frequency required for exciting each nuclei is highly specific for certain chemical structures, therefore structural elucidation and chemical identification of molecules is possible. NMR is extremely useful in biomarker discovery as it is capable of analyses with a nondestructive nature due to its ability to analyze whole biological samples without the requirement of metabolite extraction and its ability to provide important structural information
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of analytes of interest. However, the technique has some disadvantages that currently restrict NMR research. The first and most important of which is its relative insensitivity, especially when compared with MS methods, which can heavily restrict the molecules that can be analyzed by the technique. A second disadvantage is that although the technique is capable of whole sample analysis, in some instances processing of the samples is still required; for example, those samples that contain high concentrations of water and are being analyzed by 1H NMR require water removal as otherwise the high number of hydrogen atoms present in water molecules interfere with the spectra [32] . This problem also highlights another NMR weakness: within biological samples concentrations of molecular components vary greatly, and as NMR generally analyses whole samples, the molecules found at high concentrations can overlap those present at much smaller concentrations, meaning many potential biomarker candidates may be overlooked. Further to these disadvantages is the high cost of purchasing and maintaining an NMR, which makes NMR analysis an expensive option. Couple this to the fact that it can be difficult to interpret NMR spectra, often requiring specialized training and background knowledge, many researchers find the use of NMR daunting. However, despite these issues the technique is still a valuable biomarker tool, providing fast, nondestructive ana lysis, which can lead to reliable structural elucidation of marker molecules. MRS is a similar technique utilizing the theory of NMR combined with MRI, enabling quantification of metabolites in vivo via a scan of the live patient (usually the brain). An interesting review of MRS technology with a view to small-molecule and biomarker discovery has been compiled by Gujar et al. and describes the approach in further detail [33] . It is a useful technique for the identification of marker molecules as it is 100% noninvasive and no biofluids have to be collected; however, there are limitations to the technique. Interference is a major problem, particularly when scanning adjacent tissues that have major differences in magnetic susceptibility, for example, brain tissue and bone, therefore scans of areas such as the base of the skull are very difficult to obtain [33] . The technique also has difficulties in producing spectra from mobile tissues such as peripheral blood or cardiac muscle, and thus is of no use in identifying peripheral biomarkers for AD [33] . However, as
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the brain has a mainly homogenous construction and lacks any real movement, a spectrum is achievable [33] . Within the literature that describes NMRand MRS-targeted ana lysis, the brain is the main tissue of study, with NMR tending to be used to analyze post-mortem brain homogenates and MRS used to scan the brains of live patients.
NMR & MRS in AD-targeted molecular biomarkeridentification
Considering that the brain contains the second highest concentration of lipids in the body (after adipose tissue), it is of no surprise that a constant reoccurring factor throughout this review is the irregularities in lipids and their metabolite concentrations found in samples of AD patients. The first example of this reviewed here was determined using 31P-NMR, with differences observed in membrane phospholipids in postautopsy brain homogenate, including significant reductions in phosphatidylethanolamine and phosphatidylinositol, as well as increases in sphingomyelin and phosphotidylethanolamine-plasmalogen [34] . Neuronal acids and their metabolites also regularly occur in AD biomarker literature. The aspartic acid metabolite N-acetyl-aspartate is interesting as it is considered a neuronal marker thought to reflect condition and integrity of neurons [35] . Studies using NMR and MRS have demonstrated a significant decrease in the levels of N-acetyl-aspartate in a number of brain regions [3638] , suggesting neuronal damage. Metabolite concentrations such as N-acetyl-aspartate are difficult to comprehensively quantify in vivo using NMR/MRS as there is no access to an internal standard, therefore, biomarker studies often focus on ratios with creatine [35] (worryingly for the accuracy of studies, creatine has also been identified at increased levels in AD patients [38]), and many cases within the literature demonstrate reductions in the metabolite/creatine ratio when comparing AD to control subjects in different brain regions [3943] . This methodology has also been used to investigate the effects of rivastigmine treatment in AD, with results showing a significant increase in the N-acetyl-aspartate/creatine ratio in patients taking the drug, concluding that MRS may be suitable for future monitoring of response of patients undergoing treatment programs [9] . The carbohydrate myo-inositol is abundant within the brain and by developing NMR/MRS methodologies it has been shown to exist at
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significantly increased levels in the brains of AD patients [38,44] . As with N-acetyl-aspartate, when myo-inositol is analyzed via MRS, ratios with other small molecules are often used to enable quantification. The ratio between myo-inositol and creatine has been shown to increase in those who suffer from AD [40,43,45] and ratios between myo-inositol and N-acetyl-aspartate have shown a decrease, and this ratio was found to be the strongest predictor of the pathogenic likelihood of AD [43,45] . Targeted studies could not be identified in the literature, which used plasma or urine as the source biofluids, possibly because of the relative insensitivity of NMR as an analytical technique and the relatively low concentrations of small-molecule biomarkers found in these biofluids; however, this may change as NMR technology improves.
MS
MS is a widely used analytical technique that detects molecules from a sample by measuring their atomic mass. The simplified theory behind the technique relies on the mass spectrometer providing the analyte with a charge, via a process known as ionization. The charged molecule then enters the detector, which manipulates the molecule with electric or magnetic fields, resulting in the determination of the molecular weight. This can then be compared with standards resulting in an identification and biomarker ana lysis. MS is a highly useful technique due to its high specificity, sensitivity and ability to confirm biomarker candidates with comparison to standards. Furthermore, it is possible to apply techniques to fragment the analytes of interest, enabling information about structure to be determined, making it a highly powerful technique. A major disadvantage of the technique is known as ion-suppression, which occurs in the ionization procedure of the detector. When a high number of molecules are within a sample they compete for charge and ionization, meaning a percentage of analytes do not undergo ionization and therefore go to waste without being analyzed. This could be a major disadvantage as molecules of interest can be lost. This can be overcome by the introduction of a separation technique prior to MS ana lysis and this is discussed later. Further to the problem of ion-suppression in comparison to NMR, the ability of MS to elucidate structural information is relatively weak, and because many biological molecules share
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the same molecular mass and sometimes even chemical formula, often a mass figure alone is not enough data for confirmation, with a comparison to an internal standard required. With regard to targeted ana lysis, this is not a difficult problem to overcome as the study is designed with a hypothesis molecule or group of molecules in mind, meaning it is easier to select internal standards. A further disadvantage when compared with NMR is that whole samples cannot be analyzed without extensive pretreatment and it is a requirement of the technique that analytes must be extracted from the biofluids prior to ana lysis and analytical variation is higher. Within the literature, MS is widely used in targeted small-molecule and metabolite biomarker identification, due to its sensitivity, relative ease of use for quantitation and ability to detect a wide range of analytes. The technique can be utilized as a standalone (particularly when analyzing lipid extracts) [46] , or coupled to a chromatographic system such as GC [47,48] or LC [4951] .
Direct infusion-MS & AD-targeted molecular biomarkeridentification
Abnormalities in the products of lipid metabolism in AD are again highlighted in the results of a direct infusion (DI)-MS ana lysis on a lipid extract of post-mortem brain material. A significant decrease in the subclass of glycerophospholipids known as plasmalogens was observed. This was particularly apparent in extracts of white brain matter where the total plasmalogen level was approximately 40% lower in AD than in control patients. A decrease was also witnessed in gray matter, although not as extreme with a decrease of approximately 10%. Interestingly, correlation between disease severity and plasmalogen level was significantly apparent in the gray matter, with levels decreasing in accordance with severity. In contrast, in white matter levels quickly decreased and remained relatively constant regardless of AD severity [52] . These findings are of particular note as plasmalogens are a major component of nerve tissue, with the ethanolamine plasmalogens subgroup responsible for approximately 32% of total phospholipids in myelin [53] , suggesting loss within the brain may play a role in AD pathology. Continuing the lipid imbalance theme, DI-MS ana lysis was completed on a lipid extract from brain tissue to analyze sulfatide levels (sulfated metabolites of the glycosphingolipid,

galactocerebroside). Sulfatides are again found in the CNS, predominantly as a component of the myelin sheath [54] , and were depleted up to 93% in the gray matter and 53% in the white matter of autopsy-confirmed AD patients compared with controls [55] . In the same study, a second potential galactocerebroside metabolite product, ceramide, was found to be elevated threefold in AD patients, further highlighting irregularities in the metabolism of neuronal glycosphingolipids in the disease pathology [55] .
Chromatographic separation techniques & MS
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MS detectors can also be used in combination with a separation technique, for example LC or GC. LC works on the theory of injecting extracted metabolites in a solvent into a small diameter stainless steel column filled with solid particles, normally silica-based bound to a variation of carbon chains, for example C18or C8. Liquid solvent is then passed through the column at a high pressure, with components interacting with the stationary phase or mobile phase at different rates depending on their chemical properties. GC works in a similar way, however the liquid mobile phase is replaced with an inert gas and the stationary phase column is replaced with a stainless steel or glass column, which contains either a stationary solid or a stationary liquid phase. Separation occurs based upon the analyte interaction with the stationary phase. The result of the two techniques is a separation of analytes, and therefore the introduction of molecules into the mass spectrometer detector at separate time points. This is advantageous as separation of analytes greatly reduces competition for ionization within the source of the mass spectrometer, and therefore reduces ion-suppression. This means more information about the molecular contents is collected for ana lysis enabling a greater range of molecular properties and concentrations to be analyzed. The more complex mixtures require greater separation and this can be achieved by increasing the analysis time or by using solvent gradient in LC or a temperature gradient in GC, which introduces variables enabling greater control of component elution. However, the introduction of a separation technique does have the disadvantage of adding time to sample ana lysis. A further addition to the time of the sample ana lysis is that when preparing samples for LC, the removal of large molecules and the denaturing of proteins is required in order to free the small molecules (e.g., small-molecule co-factors
bound to enzymes), preventing overlapping of peaks, blockages of the chromatographic system and obstructions in the column. With GC, a further step of analysis is required when compared with LCMS as an extra derivatization step is required to increase analyte volatility, enabling successful separation and detection. This is a disadvantage to the technique as it introduces a further step in sample preparation, which not only increases analysis time, but increases the chance of analytical variation. However, the increased sensitivity of the techniques makes separation a necessity in smallmolecule biomarker ana lysis, especially due to the low trace levels and varied concentration range involved in the nature of the work. The technology is continually advancing, in particular recent advances in LC have greatly improved speed, resolution and sensitivity [56] and the option is now there for laboratories to use HPLC and UHPLC in their methods, which is capable of greatly reducing run times and increasing productivity, especially in high-throughput studies [5255] .
GCMS & AD-targeted molecular biomarkeridentification
As well as abnormalities in lipid metabolism, disruptions in glucose utilization [57,58] and deficiencies in mitochondrial function [59] are thought to occur in AD, subsequently leading to an energy deficit within the brain. GCMS ana lysis of patients CSF investigated the concentration of a range of molecules that require mitochondrial metabolism, with results demonstrating that AD patients have an increase in CSF lactate as well as a decrease in CSF succinate and fumarate [60] . Oxidative stress, particularly of lipids, is also thought to play a major role in AD pathology [61,62] ; therefore, GCMS has been used with this in mind to identify biomarkers formed as a result of this free radical oxidation process. An ideal group of molecules for this purpose are isoprostanes, which are chemically stable metabolites formed via the lipid peroxidation of various fatty acids during oxidative stress. GCMS has been used to demonstrate an elevation of a number of isoprostanes in many biofluids, including various brain region homogenates [12,48] , CSF [47,48,6366] , plasma [47] and urine [26,47] . Isoprostanes have also been used as markers in a GCMS study designed to monitor the therapeutic effect of antioxidant vitamin supplements on oxidative stress in AD. Those patients who were dosed with increased vitamin E and
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C showed less signs of oxidative damage than those taking no treatment [67] . Not only does this highlight the role that oxidative stress plays in AD, it also demonstrates the potential for the use of biomarkers in monitoring treatment regimes and overall disease progression. Oxidation of fatty acids in AD is also the cause of a significant rise in the levels of hydroxyoctadecadienoic acid, found via a GCMS analysis of both plasma and erythrocyte extracts. Interestingly, the level of hydroxyoctadecadienoic acid also correlated with patient clinical dementia ratings [68] , again suggesting a possible role in tracking disease progression using this biomarker. Previously in this review, irregular oxidation in AD has been discussed with regard to lipids and proteins; however, evidence also exists suggesting DNA is affected too. Wang et al. applied a GCMS approach to AD brain samples in a targeted attempt aimed at investigating oxidized DNA bases in AD [69] . The study identified a significant increase in 8-hydroxyguanine as well as an increase in multiple oxidized DNA bases. This suggests products of DNA oxidation may be able to play a role in AD diagnosis. The neurosteroid dehydroepiamdrosterone (DHEA) is also thought to be susceptible to oxidative stress [70] . This theory was examined by applying GCMS analytical methods to serum samples, which resulted in the identification of a significant increase in 7-hydroxylated metabolites of DHEA in AD patients [71] . DHEA and its metabolites regularly appear in the literature regarding AD biomarkers with conflicting results, particularly within the LC methods described below.
LCMS&ADmolecularbiomarkeridentification
Although it is a widely used analytical technique, to date only a couple of examples of LCMS use in AD small-molecule biomarker research exist in the literature. The first of these developed methods is capable of identifying and quantifying glutathione conjugates of trans-4-hydroxy-2nonenal, a product of lipid peroxidation in oxidative stress, in a selection of post-mortem brain regions. The study found a significant increase of the conjugate in hippocampus and substantia innomina of AD patients [50] , again providing yet further evidence of oxidative stress, particularly of lipids, playing a major pathological role within AD. A method has also been published identifying F2-isoprostanes (previously mentioned) in urine, however it found no significant differences when
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comparing between AD and control [51] , again conflicting the results identified in GCMS studies described earlier. A recently developed method described in the literature has demonstrated the ability of LCMS techniques to quantify cholesterol metabolites found within CSF [22] . Some of the higher concentration metabolites identified were intermediates of bile acid synthesis, for example, 3b-hydroxycholest-5-en-26-oic acid and 7a-hydroxy-3-oxocholest-4-en-26-oic acid. The study also tested the metabolites ability to activate liver X receptors (LXRs), finding that 3b-hydroxycholest-5-en-26-oic acid was an LXR ligand, and although 7a-hydroxy-3-oxocholest4-en-26-oic acid was not a direct ligand to the receptor, it is formed from metabolism of 3b,7adihydroxycholest-5-en-26-oic acid, which is an active ligand. This relates to AD as it has recently been demonstrated in mice models that LXR activation reduces AD symptoms, including amyloid load, improving cognitive response [72] . It has also been shown that a deficiency in LXR in vivo has been shown to increase AD symptoms [72] . This evidence suggests a possible role of bile acids in neurodegenerative disorders such as AD, and this published LCMS method capable of quantifying intermediates of bile acid synthesis may play a role in AD biomarker identification. LCMS approaches have also been employed to investigate small-molecule products of protein glycation, oxidation and nitration by Ahmed et al. [73] . The method analyzed the CSF of AD patients in a targeted manner before comparing the results to those of controls. The study identified a number of small molecules at increased levels including 3-nitrotyrosine, Ne-carboxymethyl-lysine, 3-deoxyglucosonederived hydroimidazolone, N-formylkynurenine, methylglyoxal-derived hydroimidazolone and glyoxal-derived hydroimidazolone, suggesting extensive oxidation, nitration and glycation of protein products in AD pathology.
LC & non-MS detection & AD molecular biomarkeridentification
Despite reducing costs, MS is still generally considered to be an expensive approach; however, there are alternative detectors available that can be coupled to separation-based instruments, which are often more affordable and still remain valuable biomarker discovery tools. A suggested reason for the increase of oxidative stress in AD is that a significant reduction of antioxidant species exists within diseased

patients. LC methods have been developed using UV detection, which uses the property of certain molecular structures known as chromophores, which can absorb certain wavelengths of UV light, enabling their detection. Generally, UV detectors are very cost effective and easy to obtain, however they lack sensitivity, especially for molecules lacking chromophore regions. This can be overcome by the completion of a derivatization reaction adding a chromophore to molecules of interest; however, as with GC, this adds an extra sample preparation step increasing time and the risk of technical variation. Publications that have employed LCUV in AD biomarker discovery have demonstrated decreases in uric acid [74] , and vitamins C [74] , A [75] and E [75] in plasma, as well as a decrease in the plasma carotenoid antioxidants including luetin, zeaxanthin, a-carotene and b-cryptoxanthin [75] . Luetin levels were also found at decreased levels in AD plasma in a second study along with another carotinoid, b-carotene [76] . Interestingly, the decrease of these two antioxidants appeared to correlate with patient MMSE score and disease severity, suggesting a possible use in monitoring disease progression. The antioxidant vitamin A has also been demonstrated to exist at lower concentrations in AD plasma by using a technique similar to UV [74] ; however, instead of relying on UV absorbance it relies on the emission of fluorescence after excitation of a florescent region of the molecule. It is more sensitive than UV, but it is less universal as few molecules contain fluorescence regions, although again this can be solved with derivitization [74] . A reduction in concentration of antioxidant species (vitamin C and uric acid) has also been demonstrated with LC coupled to electrochemical detection [75] . This form of detector employs two electrodes (working and reference) set at a fixed voltage. The working electrode oxidizes or reduces analytes resulting in a flow of electrons that can be measured. Electrochemical detection was also used to identify a decrease in the CSF concentration of two monoamine metabolites: 5-hydroxyindoleacetic acid and homovanillic acid [77] . Homovanillic acid is of particular interest as it can be used as a marker of metabolic stress in relation to irregularities in glucose metabolism, which as discussed previously is pathologically linked to AD. Furthermore, homovanillic acid has been used to demonstrate metabolic stress in the neurodegenerative disease schizophrenia [78] , therefore, in theory could be applied to AD.
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LCelectrochemical detection methods have determined the existence of metabolites of the membrane phospholipid phosphatidylcholine at significantly increased levels in AD patient CSF. Glycerophosphocholine was demonstrated to be significantly increased by 76%, phosphocholine by 52% and free choline by 39% [79] . This again suggests irregularities in membrane phospholipid metabolism potentially playing a role in AD pathology. LC has also been implemented as a useful isolation tool prior to further sample ana lysis. For example, an LC separation was applied to patient CSF where fractions containing metabolites of interest were collected. These fractions were then analyzed using radioimmunoassays and GCMS methods to quantify metabolites [49] . The study identified a significant increase in both the previously mentioned DHEA and its sulfated metabolite DHEAS, although there were no differences in the levels of DHEA hydroxyl metabolites. These results complement work discussed previously in the GCMS section where 7-hydroxylated metabolites were found at significantly increased levels in AD patient serum [71] . However, significant differences were observed in the ratios between DHEA and hydroxyl metabolites, including 7a-hydroxy-dehydroepiamdrosterone, 7b-hydroxyl-dehydroepiamdrosterone and 16a-hydroxyl-dehydroepiamdrosterone, indicating irregularities in DHEA metabolism associated with the disease.
CE
CE is a widely used analytical technique that can be applied to targeted biomarker identification from biological samples. Separation occurs by applying a high voltage potential across a mobile phase housed in a fused-silica capillary. Analytes then migrate through the mobile phase based upon their charge and ionic radius. Neutral analytes undergo migration due to the overall electro-osmotic flow of the mobile phase under charged conditions and several techniques are also available to separate neutral compounds, such as using charged micelles. CE has advantages of small-injection volume of sample, low solvent consumption, short run times and high separation efficiency. The technique is not without drawbacks; currently it struggles for robustness and reproducibility, with peak drift from run-to-run being a major issue. Sensitivity is also a problem with CE as generally it is connected to a UV
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Key Term

or electrochemical detector, this is improving and it is now possible to purchase a CE coupled to a TOF mass spectrometer. CE has been commonly used for protein, peptide and larger molecule ana lysis with regard to AD [80,81] and to test small molecules inhibiting formation of fibrils from b-amyloid oligomerization [82] . Although there are currently no specific CE methods published focused on AD biomarkers in human samples, there are some examples of interest in the literature where CE methods have been developed relating to small molecules that are thought to play a role in AD pathogenesis [8387] . CE methods have been developed that are capable of separating l- and d- form amino acids from biofluid samples, and a range of amino acids have been extracted from both human CSF and urine [83] , this is relevant as the d-amino acid, d-asp, has been found at increased levels in AD patients white matter, and d-ala has been found at increased concentrations in gray matter using a spectrophotometric enzyme assay [84] . The use of CE chiral methodologies to analyze amino acid enantiomers could potentially enable high-throughput biomarker identification without the need to continuously invest in expensive enzyme assays. The use of an optimized CE method may also improve the quantitative sensitivity of the ana lysis. CE has also been used to quantify 5-hydroxyindoleacetic acid and homovanillic acid in CSF from a range of neuropsychiatric disorders including AD [85] (although the sample size was very small). As discussed previously, these two acids have been identified at decreased levels in AD CSF using LCelectrochemical detection techniques; however, this methodology shows that CE methods may be a viable alternative. The potential of the technique has also been demonstrated in targeted methods developed for animal models. These are briefly discussed below, and are only included as a demonstration that CE has the ability to perform AD smallmolecule biomarker ana lysis. For this reason, and to avoid confusion, the results of this section are not included in Table 1. The first example of these is a method developed using capillary zone electrophoresis (CZE), that was capable of quantifying quinolinic acid in rat brain and plasma [86] . The method was designed with neurological diseases such as AD in mind as quinolinic acid is a potent neurotoxin that has been demonstrated to cause neuronal death via increased lipid peroxidation
Immunoassays: Measure the

concentration of molecules by using the interaction of labeled antibodies to its antigen.
in vivo [87] and in vitro [88] , as well as inducing lipid peroxidation resulting in increases in neurotoxic products [89] . Quinolinic acid is a product of the kynurenine pathway and it has been suggested that irregularities in this pathway may lead to an increase in quinolinic acid, and therefore increased neurotoxicity [90] . Thus, the CZE method described here may be suitable to investigate the merits of quinolinic acid as a biomarker for the disease.
Immunoassays
Immunoassays come in a variation of formats,
however the theory behind them is similar. They employ an antibody labeled by the addition of a fluorescent or radioactive tag. The antibody is specific for the molecule of interest and binds to any molecules that are present. The sample is then washed to remove excess material and viewed under the appropriate detector, with the intensity of detection directly related to the concentration of target molecule present. The highly specific nature of immunoassays enables their use as a suitable technique for targeted metabolite biomarker identification; furthermore, the assays are capable of quantification analysis when coupled to the labeled tags. Despite incurring some disadvantages, including high costs and relatively low throughput of ana lysis, there are instances within the literature where imunnoassays have been used to quantify small molecules in biological samples from AD patients. One such developed method purified the steroid, dehydroepiandrosterone, using HPLC then quantified the isolated sample using a specific radiolabeled immunoassay. The results found increased levels in various AD brain region samples and CSF, with the findings thought to indicate the presence of an alternative metabolism pathway linked to oxidative stress and therefore occurring at greater regularity in AD patient brains [70] . An ELISA method was published that is capable of quantifying the level of F2 isoprostanes in human plasma [91] . As discussed earlier, different studies have produced conflicting reports about the levels of F2 isoprostanes in AD, with GCMS results suggesting a significant elevation in a range of biofluids [6366] , whilst LCMS results suggested no significant change in urine [51] . The immunoassay results identified no significant change in the plasma concentrations, complementing the LCMS results. The same study also analyzed uric acid by a similar immunoassay and found this to be at
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Table 1. Summary of the small molecules that are discussed in this review and the methods associated with their identification.
Molecule or group of Tissue used for molecules identification
5-hydroxyindoleacetic Cerebrospinal fluid acid 7-hydroxylated Serum metabolites of dehydroepiamdrosterone 8-hydroxyguanine Brain Bile acids (GCA, GDA, GCDCA) Ceramide Citrate Creatine Creatinine
d -aspartic d -alanine
Trend in Alzheimers Targeted or disease compared nontargeted with controls analysis

Decrease Increase Targeted Targeted
Identifying technique
LCelectrochemical GCMS
Refs
[77] [71]
Increase Increase Increase Decrease Increase Increase Increase Increase Increase
Targeted Nontargeted Targeted Nontargeted Targeted Nontargeted Targeted Targeted Targeted
GCMS LCMS Direct infusion-MS

1 1 1
[69] [95] [55] [104] [38] [32] [84] [84] [49]
Plasma Brain Cerebrospinal fluid Brain Cerebrospinal fluid
H-NMR H-MRS H-NMR
acid
Brain Brain
Dehydroepiamdrosterone Cerebrospinal fluid
Dehydroepiandrosterone Brain and cerebrospinal fluid Fumarate Homovanillic acid Cerebrospinal fluid Cerebrospinal fluid
Increase
Targeted
Decrease Decrease Increase Increase Increase Increase Increase No significant change Increase No significant change Increase Decrease Increase Increase in ratio between the two molecules Reduction in ratio between the two molecules Decrease
Targeted Targeted Targeted Targeted Targeted Targeted Targeted Targeted Targeted Targeted Targeted Targeted Targeted Targeted
Spectrophotometric enzyme assay Spectrophotometric enzyme assay LC purification followed by immunoassay and GCMS Purification using HPLC followed by immunoassay GCMS LCelectrochemical GCMS LCMS GCMS GCMS GCMS ELISA GCMS LCMS GCMS LCUV
1 1
[70]
[60] [77] [68] [73] [12,48] [47,48,6366] [47] [91] [26,47] [51] [60] [75,76] [38,44] [40,43,45]
Hydroxyoxtadecadienoic Plasma and erythrocytes acid Hydroimidazolone Cerebrospinal fluid Isoprostanes Isoprostanes Isoprostanes Isoprostanes Isoprostanes Isoprostanes Lactate Luetin Myo-inositol Myo-inositol combined with creatine Myo-inositol combined with N-acetyl-aspartate N-acetyl-aspartate Brain Cerebrospinal fluid Plasma Plamsa Urine Urine Cerebrospinal fluid Plasma Brain Brain
H-MRS H-MRS
Brain
Targeted
H-MRS
[43,45]
Brain
Targeted
NMR/MRS
[3638]
It is interesting to note the sheer number of different molecules encountered and the wide variety of methods used. Particular interest should be paid to molecules such as creatine, which was identified at increased concentrations in human Alzheimers disease brains, however, in decreased concentrations in mouse model brains. Another particular class of molecules of interest are the isoprostanes, which have been found to increase in many studies using GCMS, however, they displayed no significant change using LCMS and ELISA methodologies. MRS: Magnetic resonance spectroscopy.
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Table 1. Summary of the small molecules that are discussed in this review and the methods associated with their identification (cont.).
Molecule or group of molecules
N-acetyl-aspartate combined with creatine Ne-carboxymethyl-lysine N-formylkynurenine Nitrotyrosine Nitrotyrosine Phosphatidylcholine Phosphatidylethanolamine Phosphatidylethanolamineplasmalogen Phosphatidylinositol Phospholipid plasmalogens Sphingomyelin Succinate Sulfated metabolites of dehydroepiamdrosterone Sulfatides Uric acid Uric acid Uric acid Vitamin A Vitamin A Vitamin C Vitamin C Vitamin E Zeaxanthin a-carotene b-carotene b-cryptoxanthin
Tissue used for identification

Brain
Trend in Alzheimers disease compared with controls

Reduction in ratio between the two molecules Increase Increase Increase Increase Increase Decrease Increase Decrease Decrease Increase Decrease Increase
Targeted or Identifying nontargeted technique analysis

Targeted
1
Refs
H-MRS
[3943]
Cerebrospinal fluid Cerebrospinal fluid Brain homogenate Cerebrospinal fluid Cerebrospinal fluid Brain Brain Brain Brain Brain Cerebrospinal fluid Cerebrospinal fluid
Targeted Targeted Targeted Targeted Targeted Targeted Targeted Targeted Targeted Targeted Targeted Targeted
LCMS LCMS Immunoassay LCMS LCelectrochemical

31 31
[73] [73] [93] [73] [79] [34] [34] [34] [52] [34] [60] [49]
P-NMR
P-NMR P-NMR
31
Direct infusion-MS P-NMR GCMS

31
Brain Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plamsa Plasma
Decrease Decrease Decrease Decrease Decrease Decrease Decrease Decrease Decrease Decrease Decrease Decrease Decrease
Targeted Targeted Targeted Targeted Targeted Targeted Targeted Targeted Targeted Targeted Targeted Targeted Targeted
LC purification followed by immunoassay and GCMS Direct infusion-MS LCUV LCelectrochemical ELISA LCUV LCfluorescence LCUV LCelectrochemical LCUV LCUV LCUV LCUV LCUV
[55] [74] [74,75] [91] [75] [74] [74] [75] [75] [75] [75] [76] [75]
It is interesting to note the sheer number of different molecules encountered and the wide variety of methods used. Particular interest should be paid to molecules such as creatine, which was identified at increased concentrations in human Alzheimers disease brains, however, in decreased concentrations in mouse model brains. Another particular class of molecules of interest are the isoprostanes, which have been found to increase in many studies using GCMS, however, they displayed no significant change using LCMS and ELISA methodologies. MRS: Magnetic resonance spectroscopy.
a significantly lower concentration in AD plasma than in control plasma [91] , again complementing the results found via LC methodologies discussed previously [74,75] . A further published study set out to analyze a hypothesis that within AD there is a dysregulation of the adrenal pituitary hormonal axis by using an immunoassay to quantify the levels of the hormone cortisol in the plasma of AD patients [92] . The levels of the hormone showed
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a significant increase, supporting the initial hypothesis. These results present evidence that suggests further investigation into this hypothesis may yield biomarker results, possibly leading to a novel therapeutic target through hormone treatment. Smith et al. developed an immunoassay that quantifies levels of nitrotyrosine, a small-molecule product of tyrosine nitration that can act as an indicator of the presence of peroxynitrite,

a powerful oxidant molecule that can not only nitrate tyrosine, but is also capable of directly oxidizing proteins and other macromolecules [93] . Levels of nitrotyrosine were shown to significantly increase in brain homogenates of AD patients when compared with controls, suggesting its potential to act as a biological marker for the disease.
Conclusions regarding a targeted approach
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There are clearly many different approaches that can be taken when planning a biomarker investigation with a molecular target in mind. Each technique has advantages over any other. For example, approaches involving MS are certainly more sensitive, but do not allow the ana lysis of whole samples in a way that NMR ana lysis can achieve. When planning, consideration of what each method can provide should be taken. However, it should be noted that the discrepancies that ana lysis between publications and instruments produces is often of great concern. For example, isoprostane ana lysis by GCMS found a significant increase in many biofluids, including various brain region homogenates [12,48] , CSF [47,48,6366] , plasma [47] and urine [26,47] , but there was no significant change in LCMS ana lysis of urine [51] and an immunoassay ana lysis of plasma [91] . This is of particular importance as inter-technique variability and conflicting results will not result in the development of a reliable marker molecule. Whilst not without problems, the technology required for targeted ana lysis is constantly improving as time progresses. Combine this with the falling costs of accurate instrumentation, then it is certain that targeted approaches will play a role in the future of AD small-molecule biomarker ana lysis. Nontargeted approaches: metabonomics/metabolomics There is an increasing trend for biological samples to be analyzed without a specific target molecule in mind and the fields of metabonomics and metabolomics are forever expanding. Confusingly, there is great overlap between the two expressions and often it depends on which laboratory publishes the study as to which terminology they use. Recently however, a definition structure is appearing to take shape, with metabolomics now commonly defined as the comprehensive detection and quantitation of all the low-molecular-weight molecules and
metabolites present in cells, tissues or organisms under a set of given conditions, and metabo, nomics described as the quantitative ana lysis of the metabolic response of living systems to pathophysiological stimuli or genetic modification over time [94] . Further to these terminologies and adding to the confusion is the possibility of completing nontargeted analysis of specific biological extracts; for example, lipidomics aims to nondiscriminately analyze the differences in concentrations of lipids extracts. This style of ana lysis becomes increasingly important, especially as over recent years our traditional view of metabolism has altered from a simple linear structure to a more complex network, with many intracellular outcomes still not fully understood. As nontargeted metabonomics/metabolomic studies improve and the discipline becomes commonly practiced, data will be combined with similar studies designed with genetics and proteins to give us an overall picture of systems biology and the whole networked process (Figure 2) . In order for a nontargeted approach to be interpreted in a reliable and useful manner, the ana lysis of the raw data is heavily reliant on rigorous data processing methods, including peak retention time alignment between samples and normalization between samples by use of an internal standard. This treatment of the raw data is then processed using a selection of mathematical models, for example partial least squares [95] or constrained total-line-shape [32] , which are capable of grouping samples dependant on the differences in small molecules identified. This approach highlights metabolite differences, which can then be identified leading to individual components being linked to disease states. An extremely informative table describing the various forms of data treatment and chemometric approaches available in metabolomic studies can be found in the review compiled by Madsen et al. [96] . Furthermore, a general workflow outlining the stages and processes involved in a typical metabolomic study can be viewed in Figure 3. Such approaches are integrative and commonly correlate analytical methods or other -omics in an effort to achieve a systems biology outcome. In order to understand neurodiseases and in particular AD and its complex effects in brain biochemistry, novel experimental and computational approaches are needed. Fundamental to such understanding is the contribution of systems biology, where data from
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Genome
e om ct ra te In
Transcriptome
Proteome
Metabolome
Figure 2. Simple overview of systems biology and the different disciplines required for study. The analysis of these will hopefully lead to complete knowledge of the overall system network, enabling the determination of novel disease pathways in Alzheimers disease. The upper right segment of the figure demonstrates the complex nature of biological pathways with multiple interactions possible across the entire network.
genomics, transcriptomics, proteomics and the mentioned metabonomics/metabolomics are integrated [95] . Nontargeted approaches hold a lot of potential, and many publications are now constantly produced that cover many different conditions and disorders. However, it is still a relatively new technique and disadvantages exist. First, and the greatest issue, is the problem of interference. The concept of the approach is to extract and analyze the greatest variation and quantity of molecules as possible and this understandably leads to overlap of molecules, leading to shielding and masking those presenting at lower concentrations. To counter this, separation techniques can be employed, however, due to the sheer number of molecular features extracted separation run times can be in excess of an hour. This adds to the overall study time frame, increasing costs and solvent use. Further adding to the time of the study is confirmation of molecules of interest. During the study, samples are analyzed and compared by data manipulation and modeling providing a possible feature. This has to be confirmed by comparison with standards, which can be expensive or difficult-to-impossible to obtain if it is not a common biological molecule. Again, this adds time and cost to the study. Sample treatment is also
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extremely important with consistent and reproducible methods a necessity. This disadvantage is countered by the implementation of pooled quality control (QC) samples placed intermittently throughout the analysis. In any data model of successful studies, the QCs should all group together with minimal variation. If this is achieved the method is suitable for nontargeted ana lysis, although results will be always semi-quantitative. Last, a major disadvantage in AD human studies, and not one that is only metabolomics related but is a major issue, is the amount of drugs that old patients and controls might be having, with the inherent variability and complexity of knowing for certain all the drug-related changes that the biofluid is showing. This problem becomes even greater when the diagnosis power is seen on the whole fingerprint, not only will many metabolites in that case be unidentified, but the specificity of a method is certainly not proven by comparing AD and controls. Yet again, AD mice, or other animal AD models, are not the perfect choice either as they might have the drawback of disease diagnosis inaccuracy or nontransferability of results. In general, nontargeted approaches such as this involve the profiling of metabolic content in a selection of biofluids comparing different groups (i.e., healthy and diseased), therefore

the number of samples is of utmost importance. Typically, a pilot study would utilize 1520 samples per group, providing a unique and comprehensive source of information on the biochemical state of the system. Metabonomics has been employed in many applications, such as disease diagnosis and pathway interpretation [96] , as well as the beneficial and adverse effects of pharmaceuticals [9799] . Regarding the study of mental illness, NMR-based metabonomic strategies have been effectively applied in the past to schizophrenia [100,101] and bipolar disorder [102] . To date, very few groups have attempted nontargeted techniques in AD biomarker discovery, however, a few instances do appear in the literature and they are discussed below.
Nontargeted NMR techniques for AD biomaker identification
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The earliest example in the literature of AD nontargeted biomarker analysis is a preliminary study comparing patient and control CSF samples by
NMR. The method was capable of investigating a wide variety of components, the results of which were processed by principle component ana lysis, and was able to partially separate the classes (control and AD) based on the concentrations of metabolites found. Further ana lysis of the data resulted in the identification of one of the metabolites responsible for the class grouping, and it was demonstrated that citrate existed at significantly different concentrations between the classes, therefore, this was recommended for further investigation as a biomarker [103] . Jukarainen et al. also completed a nontargeted CSF metabolite ana lysis via NMR methods, evaluating the data using a constrained totalline-shape model [32] . Varying differences in metabolite concentrations were observed in the study, although the only significant difference between AD and control samples was in the level of creatinine, which was found at higher concentrations in the AD group. These results complement a targeted study discussed earlier
MS Study design Extraction Derivatization Analysis Alignment Baseline connection Peak-picking or deconvolution Peak identification Normalization Scaling Problem formulation Study design Sample collection
NMR
Analysis
(Extraction) Analysis
Data processing
Phasing and baseline correction Alignment Normalization Bucketing/peak-picking/deconvolution Scaling
Statistical analysis
Data overview (e.g., principle component analysis) Model building (OPLS, PLS, NN or other) Model optimization Model validation Predictions and identification of biomarkers Identification of perturbed metabloic pathways Mechanistic explanation Follow-up studies
Biological interpretation
Figure 3. General overview of a typical study workflow when undertaking a nontargeted approach to biomarker identification. Here, both MS and NMR approaches are compared. NN: Neural network; OPLS: Orthogonal partial least square; PLS: Partial least square. Reprinted from [96] with permission from Elsevier.
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in this review where creatine (the parent molecule of creatinine) was identified at increased concentrations in the brain, suggesting possible irregularities in this metabolism pathway in AD [38] . A nontargeted study has been published analyzing serum samples via a 1H-NMR method [104] . In this instance the patient population did not contain any AD patients, but consisted of those suffering from mild cognitive impairment (MCI), a neurodegenerative condition associated with a significant chance of further developing into AD [105] . The study used a self-organizing map ana lysis to process the serum 1H-NMR data, identifying a decrease in the relative amount of w-3 fatty acids present in MCI, concluding that this irregularity may possibly indicate an increased risk of AD [104] . F igure 4 demonstrates the NMR spectrum windows used in this study, highlighting the
(-CH2-)n A Lipoproteins and albumin -N(CH3)3 =CH-CH2-CH= Albumin B Low-molecular-weight metabolites Unsaturated lipids Urea 5.5 Lipid extract Glucose Lactate Water 4.0
=CH-CH2-CH2CH2 CH2 Albumin
-CH3 -C(18)H3
Glucose
Free choline Creatinine 3.0
Glycoprotein Acetoacetate 2.0 Proline
Lactate Alanine Valine
18:2 FA -CH2-CO FA -CH2-CO TC 2.2
16:1, 18:1 FA =CH-CH2-CH2TC
22:6 FA -CH=CH-CH2-CH2-CO TC
20:4 FA -CH2-CH2-CO
FA -CH2-CH2-CO TC
1.0 ppm TC -C(26)H3 -C(27)H3 FA -9 (-CH2-)n sat Fa -CH -C(19)H3 -6 3 EC, FC -CH3 TC -C(21)H3
2.4
2.0
1.8
1.6
ppm
PGLY backbone -CH2TG backbone -CH2PC -PO-CH2

PC -CH2-N -N(CH3)3 PC, SM
-6 -CH3
-C(18)H3 FC, EC 0.9 ppm
4.4
4.3
4.2
4.1
4.0 ppm
1.0 PUFA =CH-CH2-CH= 3.0 2.0
-CH=CHEC TG, PGLY -C(3)H backbone -CH 5.0 4.0
FC -C(3)H
TC
1.0
ppm
Figure 4. Three NMR spectrum windows used by Tukiainen et al. when analyzing serum samples. (A) Lipoproteins and albumin. (B) Low-molecular-weight metabolites. (C) Lipid extract. Different molecular components visible in 1H-NMR analysis are clear and some important metabolites are labeled. Interestingly, the sample preparation procedure results in lipoprotein breakdown providing valuable information regarding the individual lipid species that normally reside inside these particles. As can be seen in this diagram, the use of this technique in nontargeted analysis is clearly of great value due to the many components identified. EC: Esterified cholesterol; FA: Fatty acid; FC: Free cholesterol; PC: Phosphatidylcholine; PUFA: Polyunsaturated fatty acid; PGLY: Phosphoglyceride; SM: Sphingomyelin; TC: Total serum cholesterol; TG: Total serum triglyceride. Reprinted from [106] with permission from Elsevier.
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techniques ability to identify a large range of metabolite families as well as a high number of individual components. Currently, these are the only examples of nontargeted NMR approaches applied to human AD samples; however, to demonstrate the potential of the technique and to highlight its potential in AD small-molecule biomarker discovery, discussed here is an approach developed by Salek et al. [106] , which applies a nontarget NMR method to AD mouse model brain. This is included as a demonstration of the capabilities of the technique, the methods of which could be applied to human samples. As the majority of this review covers human AD biomarkers this approach should not be considered in parallel, but purely as a demonstrative piece. To further avoid confusion, the results from this have not been included in Table 2 . A nontargeted NMR study has been completed using an AD transgenic mouse model (TgCRND8) capable of developing amyloid deposits in brain associated with AD development within 23 months of birth. Extracts were examined from a variety of brain regions using 1 H-NMR methods, before data ana lysis was completed via a multivariate model, producing a principle component ana lysis and a partial least squared ana lysis. Findings from the comprehensive study indicated a decrease in many different metabolites, including N-acetyl-l-aspartate, glutamate, glutamine, taurine, g-amino butyric acid, choline and phosphocholine (co-resonance), creatine, phosphocreatine and succinate, as well as an increase in lactate, aspartate, glycine, alanine, leucine, iso-leucine, valine and finally a group of soluble free fatty acids [106] . These findings correlate with many of the human studies discussed previously within this review and suggest that irregularities in metabolism in AD patient brains are widespread and not a simple issue. This mouse study highlights the ability of metabonomics/metabolomics to identify a wide range of metabolite variations, enforcing the high potential for the technique to improve our understanding of the disease and supporting the call for nontargeted type ana lysis to be frequently used when analyzing human AD samples.
Nontargeted LCMS techniques for AD biomaker identification
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diagnostic model that could separate based on the whole fingerprint of the three groups could not be validated, the potential of metabonomics within AD was demonstrated by the identification of a number of molecules of interest recommended for further investigation. The first, glycerophosphocholine, as discussed previously in this review, is a metabolite of the membrane phospholipid phosphatidylcholine, and suggestions have been made about its role in relation to neuronal membrane degradation in AD. Unfortunately, to date no link has been determined between AD and the second molecule of interest identified in the study, d-glucosaminide, therefore more work on this molecule could lead to pathological clues about the mechanism of AD disease. Finally, a trio of bile acids (GCA, GDA and GCDCA) were identified at increasing concentrations, with controls containing the lowest concentration and AD patients containing the highest. The study commented that although a trend was noted, due to the small number of patient samples the data found could only be treated as a preliminary result and require more work before being statistically significant.
Nontargeted GCMS techniques for AD biomaker identification
With regard to small-molecule discovery in AD, there are few nontargeted GCMS data currently available in the literature, with only a single example available at present. This recently developed approach combines GCMS ana lysis with multivariate data treatment to analyze free fatty acids in a nontargeted approach [107] . Although the study is focusing upon fatty acids as an area of particular ana lysis, it has no particular target acid, and aims to analyze a wide selection in a nonbiased manner. Following intensive data treatment and the creation of analytical models, the paper identified a number of fatty acids that had significantly decreased in samples derived from AD, these included: myristic acid (C14:0), palmitic acid (C16:0), oleic acid (C18:1), linolenic acid (C18:3) and docosahexaenoic acid (C22:6). Conclusions regarding a nontargeted approach Nontargeted analysis is a relatively new approach to biomarker discovery, hence there is less data available when compared with those constructed with a molecular target in the design. However, it has been demonstrated here that this approach
A preliminary UPLCMS nontargeted study was developed to analyze plasma samples from AD, MCI and control patients [95] . Although a
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Table 2. Comparison of the various analytical techniques available to small-molecule biomarker discovery with regard to Alzheimers disease.
Analytical technique
NMR
Targeted or nontargeted analysis

Both
Strengths
Limitations
Provides detail on chemical structure, good for metabolite identification, can analyze whole biological samples
MRS
For AD only small molecules currently targeted, but there is potential for both
Direct infusion-MS For AD only small molecules currently targeted, but there is potential for both LCMS Both
GCMS
Both
LCUV detection
Targeted
LC electrochemical detection
Targeted
CE
For AD only small molecules currently targeted, but there is potential for both Targeted
Immunoassays
Relatively insensitive, high instrument and operation costs, specialist operator required, high-concentration metabolites can mask those with small concentration In vivo technique 100% noninvasive, Interference between adjacent tissues, provides detail on chemical structure, good cannot produce spectra in mobile tissues for metabolite identification (e.g., blood), instrumentation can be confusing/frightening for elderly (especially dementia patients ), relatively insensitive, highly expensive, specialist operator required Highly sensitive, fragmentation provides Samples must be pretreated to extract some structural data, confirmation metabolites, interference and possible with comparison to standards, ion-suppression issues, less structural high throughput information than NMR and MRS Separation prior to MS reduces interference Samples must be pretreated to extract and ion-suppression, easy to operate, highly metabolites, less structural information than sensitive, high selectivity, confirmation NMR and MRS, separation adds time to possible with comparison to standards, analysis, especially with complex nontargeted fragmentation provides some structural data approaches, generates high quantities of organic solvent waste can be expensive to dispose of Separation prior to MS reduces interference Samples must be pretreated to extract and ion-suppression, easy to operate, highly metabolites, extra time-consuming sensitive, high selectivity, confirmation dramatization step also a necessity to possible with comparison to standards, increase analyte volatility, less structural fragmentation provides some structural data information than NMR and MRS, due to high temperatures molecule stability issues can arise, aqueous solutions and salts cannot be injected into the instrument, separation time can be lengthy Considerably cheaper than LCMS, Poor sensitivity, molecules must contain high-throughput methods can be developed, chromophore region to be detected, samples easy to operate, high selectivity must be pretreated to extract metabolites, generates high quantities of organic solvent waste can be expensive to dispose of, no structural information must be compared with standards, no nontargeted approach possible Considerably cheaper than LCMS, Molecules must be able to undergo oxidation high-throughput methods can be developed, or reduction for detection, generates high easy to operate, high selectivity quantities of organic solvent waste can be expensive to dispose of, no structural information must be compared with standards, no nontargeted approach possible Potential for more efficient separation than Currently suffers from reproducibility and LC approaches, shorter analysis time than LC robustness issues, poor sensitivity (improving and GC, negligible solvent consumption, with new CEMS instruments), specialist high selectivity operation required due to difficulties in optimization Highly specific antibody approach, highly Highly expensive, only targeted analysis sensitive, relatively easy to complete, can possible, low throughput prebuy many kits for specific molecule identification
AD: Alzheimers disease; MRS: Magnetic resonance spectroscopy.
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can be successful, with a number or publications already available that can identify small molecules via data modeling approaches. Currently, investigations based upon a nontargeted approach are a lot more time consuming than those with a target. This is because of a combination of criteria including the know-how, longer ana lysis time, more intensive data treatment, identification involving database searches and finally study confirmation using standards. This extended time obviously has other implications too, including increased man hours and increased cost of ana lysis. Despite these disadvantages, the technologies, methods and statistical models are constantly improving, and it is likely that nontargeted approaches will become the start point for many future discovery studies and may lead the way to small-molecule biomarker discovery in AD. Small-molecule findings in comparison with protein marker molecules Presented in this review are a wide variety of analytical approaches aimed at small-molecule biomarker determination; however, as it currently stands, research into proteins is the major focus of AD biomarker discovery. This is in part because of the nature of the disease and the characterized nature of b-amyloid and tau proteins, and the known connection with the disease. Although these approaches are currently the major area of research, there is yet to be a reliable biomarker progressing to the clinic. Promising candidate markers are appearing in publications, for example, a major recent study comprising a number of centers internationally identified clusterin and apolipoprotein J, as molecules that could be used to identify the disease as well as determine disease severity [108] . As demonstrated within this review, small molecules such as lipids, carbohydrates and products of protein oxidation have the potential to act either individually, or in combination with currently established protein markers, ideally to result in an accurate diagnostic tool. Small molecules hold plenty of advantages as tools for diagnosis and disease progression monitoring. If an appropriate molecule is identified a routine ana lysis could then be developed. Generally, routine ana lysis of small molecules is relatively cost effective and can be completed with minimal technical knowledge. Further to this, the identification of a novel small molecule may direct research towards a new metabolic pathway, either unknown or not previously
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associated with the disease, opening up possibilities of new protein biomarker targets such as enzymes or receptors molecules. In addition, small molecules could be utilized in a combination approach, confirming disease presence and progression via the ana lysis of metabolites, proteins and genetics. For example, as mentioned previously the clusterin protein findings complement a similar international approach aimed at identifying genetic markers with the clusterin gene (CLU ) shown to be significantly associated with AD [109] , therefore, potentially any metabolites associated with the protein and its metabolic pathway may add sensitivity and selectivity to any test developed. Although small-molecule biomarkers are living in the shadow of their larger neighbors, they will play a role in AD research, particularly in biomarker approaches. With the development and improvement of nontargeted methods, more small molecules will be identified, hopefully providing biomarkers and opening up new areas of research. Conclusion There are many possible techniques available for the determination of biomarkers in AD, and advantages apply to the use of each method; however, it should be noted that each technique also has the disadvantages to ensure no one procedure is perfect for each and every scenario required for the role. Each mode of ana lysis is capable of providing important information about molecules of interest, and if the correct technique is applied it provides vital biomarker knowledge enabling the development of treatments and improved care. As this review has highlighted, there are several metabolic groups of particular interest that continuously reappear within the literature no matter which technology is used to identify them. It is these markers of oxidative stress, particularly of lipids, which appear most promising, and are the most frequented small-molecule biomarkers of AD mentioned in the literature. The sheer amount of citations that exist regarding lipid oxidation in AD, combined with the high concentrations of lipids that exist within the brain, suggest that irregularities in these pathways have a role to play in both AD mechanisms and the identification of a reliable biomarker. Therefore, it may potentially be worth a great deal of further investigation as this area may contain major clues and provide important biomarkers for the disease.
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However, it should also be noted that throughout the review numerous instances have occurred where there have been conflicting results between methodologies and their biomarker findings, particularly in the concentrations identified of F2 isoprostanes. These may be genuine results due to coincides in sample sources, but it is more likely that differences in sample treatment and experimental protocol are the culprits providing these irregularities [110] . Either way, this requires investigation to ensure progress in AD biomarker development as inter-study variability suggests problems with the approaches that investigations are taking. Furthermore, these irregularities highlight the importance of confirming results via repetition of studies, perhaps even in different laboratories, to ensure the reproducibility of results before bold claims regarding biomaker molecules can be made. Future perspective Alzheimers disease is going to be one of the greatest challenges to modern day research in the coming years. With an increasingly aging population, patient numbers are going to expand rapidly. The discovery of an accurate biomarker will be of vital importance and is of a pressing need. Irregularities in metabolism have already been demonstrated in many pathways associated with the disease, suggesting that small molecules and metabolites will have a role to play in the complexities associated with diagnosis and disease progression. The use of small-molecule biomarkers as a tool used in the fight against AD may be as a standalone biomarker/group of biomarkers; however, there is also the potential for the development of a combination approach that merges data from the genome, proteome and metabolome in a more complete systems biology approach. Whilst targeted studies will certainly continue to hold a role in AD biomarker research, particularly for biomarker candidate confirmation and further study, analytical technologies and the methods of raw data treatment will improve, enabling nontargeted analysis to come to the forefront of biomarker research. This will be especially apparent in complex disease such as AD, where the mechanism behind the disease is not fully understood. This will provide a further advantage in that it increases the chance of a novel biomarker, and therefore an unknown/misunderstood pathway of the disease, being found to investigate.
Executive summary
Introduction

The number of patients suffering from Alzheimers disease (AD) is rapidly increasing. Currently, AD diagnosis relies on subjective analysis. An objective noninvasive test for the disease is desperately required. Tissues closest to disease site (brain and cerebrospinal fluid) are hard to obtain from AD patients. Blood or urine are the ideal samples for an AD biomarker. Evidence suggesting bloodbrain barrier degradation in AD increases this possibility. Targeted approaches employ a hypothesis of a specific molecule biomarker presence. Nontargeted approaches aim to analyze a wide range of molecules in an unbiased approach. Currently widely used in AD biomarker investigations. These approaches employ many techniques including NMR, magnetic resonance spectroscopy, direct infusion-MS, LCMS, GCMS and immunoassays. Potential biomarkers include lipids, sugars and products of oxidative stress. Currently less common with regard to AD biomarker identification. Improving analytical equipment and data models make nontargeted approaches a valuable alternative. A wide array of techniques are available for small-molecule biomarker discovery in AD. Small molecules have serious potential for AD biomarker discovery. Small-molecule biomarkers for AD will be an important field of research over the next 510 years.
Sample considerations

Targeted versus nontargeted approaches

Targeted approaches

Nontargeted approaches

Conclusion

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Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
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Luke Whiley1,2 & Cristina Legido-Quigley 1

Whiley & Legido-Quigley

Key Terms Mini-mental state examination: A brief 30

question approach currently employed to assist Alzheimers disease diagnosis.

component that is objectively measured and used as an indicator of a biological state.

Targeted approaches NMR & MRS