current strategies in the discovery of small-molecule biomarkers for alzheimers disease - 2011 -...

8/4/2019 Current Strategies in the Discovery of Small-molecule Biomarkers for Alzheimers Disease - 2011 - Whiley

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Review

At present, there is no denitive diagnosis avail-able or Alzheimers disease (AD). Current g-ures list those aected by the condition at over400,000 people in the UK alone [201], with ur-ther estimates suggesting there are as many as5.1 million suerers in the USA [1], meaningthe demand or an accurate indicative method

is high. This demand is also set to increase overthe coming years as currently it is understoodthat the main risk actor o sporadic AD is age,with the incidence o the disease doubling every5 years ater 65 years o age [2]. As the averageliespan o the population increases due toimproved worldwide healthcare, gures o ADincidence are expected to rise signicantly overthe coming decades (Figure 1)[3,4].

Current diagnosis and monitoring o dis-ease progression involves the completion o aselection o mental tests, or example a mini-

mental state examination (MMSE) [5].However, these tests have been shown to givehighly variable results [6], lacking accuracyand consistency between patients and, hence,predictive capabilities.

The identication o an indicative biomarkerwould be a major progressive step rom a diag-nostic viewpoint, but perhaps more impor-tantly it would have the potential to improveour knowledge o the disease pathways andpathology, hopeully leading to novel treatmentstrategies, improving the lives o those aected.

The elevation or reduction o small-molecule

concentrations can indicate abnormalities inbiological pathways, which can be ampliedat metabolite level [7,8], making small-moleculemetabolites ideal markers o disease. A urtheradvantage o using metabolites as biomarkers isthe ability to track metabolic changes over time,or instance ater the addition o a drug [9,10],

enabling a refection o their therapeutic eect,improving the ability to track disease progressionand treatment response.

This review sets out to discuss recent strate-gies employed or the discovery o small-mole-cule biomarkers in AD, providing examples omolecules o interest and their importance tothe knowledge and understanding o the dis-ease. As the review was ocused on describinganalytical methods that have been applied inAD biomarker discovery, selection or inclusiono articles related to specic analytical tech-

niques as well as ocus upon small molecules.Search terms were generally technique-specic,or example Alzheimers and LCMS, with theresults then sorted manually or publicationsrelevant or the review.

This review is not designed to be a compre-hensive review on all the AD small-moleculebiomarker candidates published, rather it isdesigned to be a reading source providing back-ground inormation and considerations to betaken regarding the potential approaches thatcan be taken when developing a method or

their identication.

Current strategies in the discovery of

small-molecule biomarkers forAlzheimers 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 ndings. A wide range of

techniques are discussed, including separation approaches (LC, GC and CE), magnetic resonance technologies

(NMR and magnetic resonance spectroscopy), and immunoassays.

Luke Whiley1,2 & Cristina

Legido-Quigley1

1Pharmaceutical Sciences Research

Division, Franklin-Wilkins Building,150 Stamford Street, Kings CollegeLondon, London, UK2Medical Research Council Centre

for Neurodegeneration Research,Institute of Psychiatry, KingsCollege London, London, UKAuthor for correspondence:

Tel.: +44 207 848 4722E-mail: [email protected]

1121ISSN 1757-618010.4155/BIO.11.62 2011 Future Science Ltd Bioanalysis(2011) 3(10), 11211142


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Sample considerations for AD

biomarker identifcation

When planning a strategy or small-moleculebiomarker investigations, a vital consideration tomake is the choice o biofuid analyzed. Studiesare oten designed to ocus on the disease site, andpost-mortem brain samples are regularly used orthis purpose [1113]. Although brain samples havean important use in initially identiying novelmarkers, there are obvious disadvantages and theyare o no practical use or diagnosis in a clinicalscenario, as taking a portion o brain or analysis isjust not easible in live patients. In some cases it ispossible to quantiy biomarkers in vivo within thebrain using noninvasive imaging techniques such

as magnetic resonance spectroscopy (MRS) [1416],removing the need or a brain homogenate; how-ever, due to the loud noise and conned space asso-ciated with a MRS scan this can be a distressingand conusing experience or suerers o AD.

Cerebrospinal fuid (CSF) is a very useulbiofuid due to its close proximity to the diseasesite and it has been used in many AD biomarkerstudies [1722]; however, the use o CSF is ar romideal due to the diculties involved in obtain-ing such samples via an uncomortable lumbarpuncture procedure, particularly rom patients

suering rom the disease. Thereore, biomarkerstudies that identiy molecules o interest in rela-tively noninvasive samples such as plasma [2325]or urine [2628] have signicant benet.

When analyzing plasma and urine, the abil-ity o hypothetical biomarkers to cross thebloodbrain barrier (BBB) should be consid-ered. A known pathological consequence o ADis the deterioration and subsequent damage othe BBB, resulting in an increased permeabilityand leakiness [2931], theoretically improving thechance that any prospective biomarkers or drugs

will be able to cross the BBB.

Analytical techniques used in

small-molecule biomarker discovery

for AD

A wide range o techniques are used to identiysmall-molecule biomarkers, with vast improve-

ments in recent years in the sensitivity andselectivity o analytical technologies includingNMR, UHPLC and MS. Not one technique isperect and oten a compromise has to be takendepending upon a range o criteria includingspeed, cost and properties o the analytes, sen-sitivity and the biofuid employed. These meth-ods are discussed below with a brie summaryo each technique, as well as providing exampleso the roles in biomarker identication in AD.

Targeted versus nontargeted approachesMethod development or biomarker discoverytends to take one o two options, rst, a tar-geted approach can be made, where the studyis designed using a hypothesis based upon a spe-cic molecule or group o molecules. Second,a nontargeted or top-down strategy can beused, which sets out to analyze a biofuid non-discriminately, identiying any dierences inthe concentrations o molecules detected. Thetwo routes are discussed here with methods pro-vided to show examples o how both options aresuccessully implemented.

Targeted approaches

NMR & MRS

NMR is a highly useul analytical technique thatis capable o the identication o atoms basedon their ability to absorb a specic requencyo electromagnetic radiation when a magneticeld is applied. The simplied theory behindthe technique is based upon certain commonatomic nuclei such as 1H, 13C, 15N, 19F 29Si and31P, which have properties that enable them tospin and thereore they align with a eld o amagnetic source. Radiorequency radiation is

then applied to the sample, which is used toexcite the atoms, switching the nuclei betweenaligned and nonaligned states. The requencyrequired or exciting each nuclei is highly specicor certain chemical structures, thereore struc-tural elucidation and chemical identication omolecules is possible.

NMR is extremely useul in biomarker dis-covery as it is capable o analyses with a non-destructive nature due to its ability to analyze whole biological samples without the require-ment o metabolite extraction and its ability

to provide important structural inormation

0

30

60

90

120

2000 2010 2020 2030 2040 2050

Year

A

Daffected

p

rediction

(m

illions)

Figure 1. Current and projected incidence o AD in the coming decades.Figure adapted rom [3] using data presented by [4].AD: Alzheimers disease.

Key Terms

Mini-mental state

examination:A brief 30

question approach currentlyemployed to assist Alzheimersdisease diagnosis.

Biomarker: A biologicalcomponent that is objectivelymeasured and used as anindicator of a biological state.

Targeted approach:Biomarker identication using a

hypothesis based approachdesigned to investigate a specic

molecule or group of molecules.

Nontargeted approach:

Biomarker identication usingan unknown hypothesisapproach designed to analyze alarge number of molecules in anunbiased manner.

Review | Whiley & Legido-Quigley

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o analytes o interest. However, the techniquehas some disadvantages that currently restrictNMR research. The rst and most important owhich is its relative insensitivity, especially whencompared with MS methods, which can heavily

restrict the molecules that can be analyzed bythe technique.

A second disadvantage is that although thetechnique is capable o whole sample analysis, insome instances processing o the samples is stillrequired; or example, those samples that con-tain high concentrations o water and are beinganalyzed by1H NMR require water removal asotherwise the high number o hydrogen atomspresent in water molecules interere with thespectra [32]. This problem also highlights anotherNMR weakness: within biological samples

concentrations o molecular components varygreatly, and as NMR generally analyses wholesamples, the molecules ound at high concen-trations can overlap those present at muchsmaller concentrations, meaning many potentialbiomarker candidates may be overlooked.

Further to these disadvantages is the highcost o purchasing and maintaining an NMR,which makes NMR ana lysis an expensive option.Couple this to the act that it can be dicult tointerpret NMR spectra, oten requiring special-ized training and background knowledge, manyresearchers ind the use o NMR daunting.

However, despite these issues the technique is stilla valuable biomarker tool, providing ast, non-destructive analysis, which can lead to reliablestructural elucidation o marker molecules.

MRS is a similar technique utilizing thetheory o NMR combined with MRI, enablingquantication o metabolites in vivo via a scano the live patient (usually the brain). An inter-esting review o MRS technology with a viewto small-molecule and biomarker discovery hasbeen compiled byGujar et al. and describes theapproach in urther detail [33]. It is a useul tech-

nique or the identication o marker moleculesas it is 100% noninvasive and no biofuids haveto be collected; however, there are limitations tothe technique. Intererence is a major problem,particularly when scanning adjacent tissues thathave major dierences in magnetic susceptibil-ity, or example, brain tissue and bone, there-ore scans o areas such as the base o the skullare very dicult to obtain [33]. The techniquealso has diculties in producing spectra rommobile tissues such as peripheral blood or car-diac muscle, and thus is o no use in identiying

peripheral biomarkers or AD [33]. However, as

the brain has a mainly homogenous construc-tion and lacks any real movement, a spectrumis achievable [33].

Within the literature that describes NMR-and MRS-targeted analysis, the brain is the

main tissue o study, with NMR tending to beused to analyze post-mortem brain homogenatesand MRS used to scan the brains o live patients.

NMR & MRS in AD-targeted molecular


Considering that the brain contains the secondhighest concentration o lipids in the body (ateradipose tissue), it is o no surprise that a con-stant reoccurring actor throughout this reviewis the irregularities in lipids and their metaboliteconcentrations ound in samples o AD patients.

The rst example o this reviewed here was deter-mined using 31P-NMR, with dierences observedin membrane phospholipids in postautopsy brainhomogenate, including signicant reductions inphosphatidylethanolamine and phosphatidyl-inositol, as well as increases in sphingomyelinand phosphotidylethanolamine-plasmalogen [34].

Neuronal acids and their metabolites alsoregularly occur in AD biomarker literature.The aspartic acid metabolite N-acetyl-aspartateis interesting as it is considered a neuronalmarker thought to refect condition and integ-rity o neurons [35]. Studies using NMR and

MRS have demonstrated a signicant decreasein the levels oN-acetyl-aspartate in a numbero brain regions [3638], suggesting neuronaldamage. Metabolite concentrations such asN-acetyl-aspartate are dicult to comprehen-sively quantiy in vivo using NMR/MRS asthere is no access to an internal standard, there-ore, biomarker studies oten ocus on ratios with creatine [35] (worryingly or the accuracyo studies, creatine has also been identied atincreased levels in AD patients [38]), and manycases within the literature demonstrate reduc-

tions in the metabolite/creatine ratio when com-paring AD to control subjects in dierent brainregions [3943]. This methodology has also beenused to investigate the eects o rivastigminetreatment in AD, with results showing a signi-cant increase in the N-acetyl-aspartate/creatineratio in patients taking the drug, concludingthat MRS may be suitable or uture monitor-ing o response o patients undergoing treatmentprograms [9].

The carbohydrate myo-inositol is abundantwithin the brain and by developing NMR/MRS

methodologies it has been shown to exist at

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signicantly increased levels in the brains o ADpatients [38,44]. As with N-acetyl-aspartate, whenmyo-inositol is analyzed via MRS, ratios withother small molecules are oten used to enablequantication. The ratio between myo-inositol

and creatine has been shown to increase in thosewho suer rom AD [40,43,45] and ratios betweenmyo-inositol and N-acetyl-aspartate have showna decrease, and this ratio was ound to be thestrongest predictor o the pathogenic likelihoodo AD [43,45].

Targeted studies could not be identied inthe literature, which used plasma or urine asthe source biofuids, possibly because o therelative insensitivity o NMR as an analyticaltechnique and the relatively low concentrationso small-molecule biomarkers ound in these

biofuids; however, this may change as NMRtechnology improves.

MS

MS is a widely used analytical technique thatdetects molecules rom a sample by measuringtheir atomic mass. The simplied theory behindthe technique relies on the mass spectrometerproviding the analyte with a charge, via a processknown as ionization. The charged molecule thenenters the detector, which manipulates the mol-ecule with electric or magnetic elds, resulting inthe determination o the molecular weight. This

can then be compared with standards resultingin an identication and biomarker analysis.

MS is a highly useul technique due to its highspecicity, sensitivity and ability to conrm bio-marker candidates with comparison to standards.Furthermore, it is possible to apply techniques toragment the analytes o interest, enabling inor-mation about structure to be determined, makingit a highly powerul technique.

A major disadvantage o the technique isknown as ion-suppression, which occurs in theionization procedure o the detector. When a

high number o molecules are within a samplethey compete or charge and ionization, mean-ing a percentage o analytes do not undergo ion-ization and thereore go to waste without beinganalyzed. This could be a major disadvantageas molecules o interest can be lost. This canbe overcome by the introduction o a separa-tion technique prior to MS analysis and this isdiscussed later.

Further to the problem o ion-suppression incomparison to NMR, the ability o MS to elu-cidate structural inormation is relatively weak,

and because many biological molecules share

the same molecular mass and sometimes evenchemical ormula, oten a mass gure alone isnot enough data or conrmation, with a com-parison to an internal standard required. Withregard to targeted analysis, this is not a dicult

problem to overcome as the study is designedwith a hypothesis molecule or group o moleculesin mind, meaning it is easier to select internalstandards. A urther disadvantage when com-pared with NMR is that whole samples cannotbe analyzed without extensive pretreatment andit is a requirement o the technique that analytesmust be extracted rom the biofuids prior toanalysis and analytical variation is higher.

Within the literature, MS is widely used intargeted small-molecule and metabolite bio-marker identication, due to its sensitivity, rela-

tive ease o use or quantitation and ability todetect a wide range o analytes. The techniquecan be utilized as a standalone (particularlywhen analyzing lipid extracts) [46], or coupledto a chromatographic system such as GC [47,48]or LC [4951].

Direct infusion-MS & AD-targeted molecular


Abnormalities in the products o lipid metabo-lism in AD are again highlighted in the resultso a direct inusion (DI)-MS analysis on a lipidextract o post-mortem brain material. A sig-

nicant decrease in the subclass o glycerophos-pholipids known as plasmalogens was observed.This was particularly apparent in extracts owhite brain matter where the total plasmalo-gen level was approximately 40% lower inAD than in control patients. A decrease wasalso witnessed in gray matter, although notas extreme with a decrease o approximately10%. Interestingly, correlation between diseaseseverity and plasmalogen level was signicantlyapparent in the gray matter, with levels decreas-ing in accordance with severity. In contrast,

in white matter levels quickly decreased andremained relatively constant regardless o ADseverity[52]. These ndings are o particularnote as plasmalogens are a major component onerve tissue, with the ethanolamine plasmalo-gens subgroup responsible or approximately32% o total phospholipids in myelin [53], sug-gesting loss within the brain may play a role inAD pathology.

Continuing the lipid imbalance theme,DI-MS analysis was completed on a lipid extractrom brain tissue to analyze sulatide levels

(sulated metabolites o the glycosphingolipid,




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galactocerebroside). Sulatides are again oundin the CNS, predominantly as a component othe myelin sheath [54], and were depleted up to93% in the gray matter and 53% in the whitematter o autopsy-conrmed AD patients com-

pared with controls [55]. In the same study, asecond potential galactocerebroside metaboliteproduct, ceramide, was ound to be elevatedthreeold in AD patients, urther highlightingirregularities in the metabolism o neuronalglycosphingolipids in the disease pathology[55].

Chromatographic separation techniques & MS

MS detectors can also be used in combinationwith a separation technique, or example LCor GC. LC works on the theory o injectingextracted metabolites in a solvent into a small

diameter stainless steel column lled with solidparticles, normally silica-based bound to a vari-ation o carbon chains, or example C

18or C

8.

Liquid solvent is then passed through the col-umn at a high pressure, with components inter-acting with the stationary phase or mobile phaseat dierent rates depending on their chemicalproperties. GC works in a similar way, howeverthe liquid mobile phase is replaced with an inertgas and the stationary phase column is replaced with a stainless steel or glass column, whichcontains either a stationary solid or a stationaryliquid phase. Separation occurs based upon the

analyte interaction with the stationary phase.The result o the two techniques is a separa-

tion o analytes, and thereore the introductiono molecules into the mass spectrometer detectorat separate time points. This is advantageous asseparation o analytes greatly reduces competi-tion or ionization within the source o the massspectrometer, and thereore reduces ion-sup-pression. This means more inormation aboutthe molecular contents is collected or analysisenabling a greater range o molecular propertiesand concentrations to be analyzed. The more

complex mixtures require greater separationand this can be achieved by increasing the ana-lysis time or by using solvent gradient in LC ora temperature gradient in GC, which introducesvariables enabling greater control o componentelution. However, the introduction o a sepa-ration technique does have the disadvantage oadding time to sample analysis.

A urther addition to the time o the sampleanalysis is that when preparing samples or LC,the removal o large molecules and the denatur-ing o proteins is required in order to ree the

small molecules (e.g., small-molecule co-actors

bound to enzymes), preventing overlapping opeaks, blockages o the chromatographic systemand obstructions in the column.

With GC, a urther step o analysis is required when compared with LCMS as an extra

derivatization step is required to increase ana-lyte volatility, enabling successul separation anddetection. This is a disadvantage to the techniqueas it introduces a urther step in sample prepara-tion, which not only increases analysis time, butincreases the chance o analytical variation.

However, the increased sensitivity o the tech-niques makes separation a necessity in small-molecule biomarker analysis, especially due tothe low trace levels and varied concentrationrange involved in the nature o the work. Thetechnology is continually advancing, in particu-

lar recent advances in LC have greatly improvedspeed, resolution and sensitivity [56] and theoption is now there or laboratories to use HPLCand UHPLC in their methods, which is capa-ble o greatly reducing run times and increas-ing productivity, especially in high-throughputstudies [5255].

GCMS & AD-targeted molecular


As well as abnormalities in lipid metabolism, dis-ruptions in glucose utilization [57,58] and decien-cies in mitochondrial unction [59] are thought to

occur in AD, subsequently leading to an energydecit within the brain. GCMS analysis opatients CSF investigated the concentration oa range o molecules that require mitochondrialmetabolism, with results demonstrating that ADpatients have an increase in CSF lactate as wellas a decrease in CSF succinate and umarate [60].

Oxidative stress, particularly o lipids, isalso thought to play a major role in AD pathol-ogy [61,62]; thereore, GCMS has been usedwith this in mind to identiy biomarkers ormedas a result o this ree radical oxidation process.

An ideal group o molecules or this purpose areisoprostanes, which are chemically stable metab-olites ormed via the lipid peroxidation o variousatty acids during oxidative stress. GCMS hasbeen used to demonstrate an elevation o a num-ber o isoprostanes in many biofuids, includ-ing various brain region homogenates [12,48],CSF [47,48,6366] , plasma [47] and urine [26,47].Isoprostanes have also been used as markersin a GCMS study designed to monitor thetherapeutic eect o antioxidant vitamin supple-ments on oxidative stress in AD. Those patients

who were dosed with increased vitamin E and




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C showed less signs o oxidative damage thanthose taking no treatment [67]. Not only does thishighlight the role that oxidative stress plays inAD, it also demonstrates the potential or the useo biomarkers in monitoring treatment regimes

and overall disease progression.Oxidation o atty acids in AD is also the

cause o a signicant rise in the levels o hydroxy-octadecadienoic acid, ound via a GCMS ana-lysis o both plasma and erythrocyte extracts.Interestingly, the level o hydroxyoctadecadienoicacid also correlated with patient clinical demen-tia ratings [68], again suggesting a possible role intracking disease progression using this biomarker.

Previously in this review, irregular oxidationin AD has been discussed with regard to lipidsand proteins; however, evidence also exists sug-

gesting DNA is aected too. Wang et al. applieda GCMS approach to AD brain samples in atargeted attempt aimed at investigating oxidizedDNA bases in AD [69]. The study identied asignicant increase in 8-hydroxyguanine as wellas an increase in multiple oxidized DNA bases.This suggests products o DNA oxidation maybe able to play a role in AD diagnosis.

The neurosteroid dehydroepiamdrosterone(DHEA) is also thought to be susceptible tooxidative stress [70]. This theory was examinedby applying GCMS analytical methods toserum samples, which resulted in the identi-

cation o a signicant increase in 7-hydroxyl-ated metabolites o DHEA in AD patients [71].DHEA and its metabolites regularly appear inthe literature regarding AD biomarkers withconficting results, particularly within the LCmethods described below.

LCMS & AD molecular biomarker identifcation

Although it is a widely used analytical technique,to date only a couple o examples o LCMS usein AD small-molecule biomarker research existin the literature. The rst o these developed

methods is capable o identiying and quantiy-ing glutathione conjugates otrans-4-hydroxy-2-nonenal, a product o lipid peroxidation in oxi-dative stress, in a selection o post-mortem brainregions. The study ound a signicant increaseo the conjugate in hippocampus and substantiainnomina o AD patients [50], again providingyet urther evidence o oxidative stress, particu-larly o lipids, playing a major pathological rolewithin AD.

A method has also been published identiyingF

2-isoprostanes (previously mentioned) in urine,

however it ound no signicant dierences when

comparing between AD and control [51], againconficting the results identied in GCMSstudies described earlier.

A recently developed method described inthe literature has demonstrated the ability o

LCMS techniques to quantiy cholesterolmetabolites ound within CSF [22]. Some othe higher concentration metabolites identiedwere intermediates o bile acid synthesis, orexample, 3b-hydroxycholest-5-en-26-oic acidand 7a-hydroxy-3-oxocholest-4-en-26-oic acid.The study also tested the metabolites ability toactivate liver X receptors (LXRs), nding that3b-hydroxycholest-5-en-26-oic acid was an LXRligand, and although 7a-hydroxy-3-oxocholest-4-en-26-oic acid was not a direct ligand to thereceptor, it is ormed rom metabolism o 3b,7a-

dihydroxycholest-5-en-26-oic acid, which is anactive ligand. This relates to AD as it has recentlybeen demonstrated in mice models that LXR acti-vation reduces AD symptoms, including amyloidload, improving cognitive response [72]. It has alsobeen shown that a deciency in LXRin vivo hasbeen shown to increase AD symptoms [72]. Thisevidence suggests a possible role o bile acids inneurodegenerative disorders such as AD, and thispublished LCMS method capable o quantiy-ing intermediates o bile acid synthesis may playa role in AD biomarker identication.

LCMS approaches have also been employed

to investigate small-molecule products o pro-tein glycation, oxidation and nitration byAhmed et al. [73]. The method analyzed theCSF o AD patients in a targeted manner beorecomparing the results to those o controls. Thestudy identied a number o small moleculesat increased levels including 3-nitrotyrosine,Ne-carboxymethyl-lysine, 3-deoxyglucosone-derived hydroimidazolone, N-ormylkynurenine,methylglyoxal-derived hydroimidazolone andglyoxal-derived hydroimidazolone, suggestingextensive oxidation, nitration and glycation o

protein products in AD pathology.

LC & non-MS detection & AD molecular


Despite reducing costs, MS is stil l generally con-sidered to be an expensive approach; however,there are alternative detectors available that canbe coupled to separation-based instruments,which are oten more aordable and still remainvaluable biomarker discovery tools.

A suggested reason or the increase o oxida-tive stress in AD is that a signicant reduction

o antioxidant species exists within diseased




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patients. LC methods have been developedusing UV detection, which uses the property ocertain molecular structures known as chromo-phores, which can absorb certain wavelengthso UV light, enabling their detection. Generally,

UV detectors are very cost eective and easy toobtain, however they lack sensitivity, especiallyor molecules lacking chromophore regions.This can be overcome by the completion o aderivatization reaction adding a chromophore tomolecules o interest; however, as with GC, thisadds an extra sample preparation step increasingtime and the risk o technical variation.

Publications that have employed LCUV in AD biomarker discovery have demonstrateddecreases 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 includ-ing luetin, zeaxanthin,a-carotene and b-crypto-xanthin [75]. Luetin levels were also ound atdecreased levels in AD plasma in a second studyalong with another carotinoid, b-carotene [76].Interestingly, the decrease o these two anti-oxidants appeared to correlate with patientMMSE score and disease severity, suggesting apossible use in monitoring disease progression.

The antioxidant vitamin A has also been dem-onstrated to exist at lower concentrations in ADplasma by using a technique similar to UV[74];however, instead o relying on UV absorbance

it relies on the emission o fuorescence aterexcitation o a forescent region o the molecule.It is more sensitive than UV, but it is less uni-versal as ew molecules contain fuorescenceregions, although again this can be solved withderivitization [74]. A reduction in concentrationo antioxidant species (vitamin C and uric acid)has also been demonstrated with LC coupledto electrochemical detection [75]. This orm odetector employs two electrodes (working andreerence) set at a xed voltage. The workingelectrode oxidizes or reduces analytes resulting

in a fow o electrons that can be measured.Electrochemical detection was also used to

identiy a decrease in the CSF concentrationo two monoamine metabolites: 5-hydroxy-indoleacetic acid and homovanillic acid [77].Homovanillic acid is o particular interest as itcan be used as a marker o metabolic stress inrelation to irregularities in glucose metabolism,which as discussed previously is pathologicallylinked to AD. Furthermore, homovanillic acidhas been used to demonstrate metabolic stress inthe neurodegenerative disease schizophrenia [78],

thereore, in theory could be applied to AD.

LCelectrochemical detection methods havedetermined the existence o metabolites o themembrane phospholipid phosphatidylcholine atsignicantly increased levels in AD patient CSF.Glycerophosphocholine was demonstrated to be

signicantly increased by 76%, phosphocholineby 52% and ree choline by 39% [79]. This againsuggests irregularities in membrane phospho-lipid metabolism potentially playing a role inAD pathology.

LC has also been implemented as a useulisolation tool prior to urther sample analysis.For example, an LC separation was applied topatient CSF where ractions containing metabo-lites o interest were collected. These ractionswere then analyzed using radioimmunoassaysand GCMS methods to quantiy metabo-

lites[49]

. The study identiied a signiicantincrease in both the previously mentionedDHEA and its sulated metabolite DHEAS,although there were no dierences in the levelso DHEA hydroxyl metabolites. These resultscomplement work discussed previously in theGCMS section where 7-hydroxylated metabo-lites were ound at signicantly increased levelsin AD patient serum [71]. However, signicantdierences were observed in the ratios betweenDHEA and hydroxyl metabolites, includ-ing 7a-hydroxy-dehydroepiamdrosterone,7b-hydroxyl-dehydroepiamdrosterone and

16a-hydroxyl-dehydroepiamdrosterone, indi-cating irregularities in DHEA metabolismassociated with the disease.

CE

CE is a widely used analytical technique thatcan be applied to targeted biomarker identi-cation rom biological samples. Separationoccurs by applying a high voltage potentialacross a mobile phase housed in a used-silicacapillary. Analytes then migrate through themobile phase based upon their charge and

ionic radius. Neutral analytes undergo migra-tion due to the overall electro-osmotic fow othe mobile phase under charged conditions andseveral techniques are also available to sepa-rate neutral compounds, such as using chargedmicelles. CE has advantages o smal l-injectionvolume o sample, low solvent consumption,short run times and high separation eciency.The technique is not without drawbacks; cur-rently it struggles or robustness and reproduc-ibility, with peak drit rom run-to-run beinga major issue. Sensitivity is also a problem

with CE as generally it is connected to a UV




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or electrochemical detector, this is improvingand it is now possible to purchase a CE coupledto a TOF mass spectrometer.

CE has been commonly used or protein, pep-tide and larger molecule analysis with regard to

AD [80,81] and to test small molecules inhibit-ing ormation o brils rom b-amyloid oligo-oligo-merization [82]. Although there are currently nospecic CE methods published ocused on ADbiomarkers in human samples, there are someexamples o interest in the literature where CEmethods have been developed relating to smallmolecules that are thought to play a role in ADpathogenesis [8387].

CE methods have been developed that arecapable o separating l- and d- orm amino acidsrom biofuid samples, and a range o amino

acids have been extracted rom both human CSFand urine [83], this is relevant as the d-aminoacid, d-asp, has been ound at increased levelsin AD patients white matter, and d-ala has beenound at increased concentrations in gray matterusing a spectrophotometric enzyme assay [84].The use o CE chiral methodologies to analyzeamino acid enantiomers could potentially enablehigh-throughput biomarker identication with-out the need to continuously invest in expen-sive enzyme assays. The use o an optimizedCE method may also improve the quantitativesensitivity o the analysis.

CE has also been used to quantiy 5-hydroxy-indoleacetic acid and homovanillic acid in CSFrom a range o neuropsychiatric disordersincluding AD [85] (although the sample size wasvery small). As discussed previously, these twoacids have been identied at decreased levels inAD CSF using LCelectrochemical detectiontechniques; however, this methodology showsthat CE methods may be a viable alternative.

The potential o the technique has also beendemonstrated in targeted methods developedor animal models. These are briefy discussed

below, and are only included as a demonstrationthat CE has the ability to perorm AD small-molecule biomarker analysis. For this reason,and to avoid conusion, the results o this sectionare not included in Table 1.

The rst example o these is a method devel-oped using capillary zone electrophoresis (CZE),that was capable o quantiying quinolinic acidin rat brain and plasma [86]. The method wasdesigned with neurological diseases such asAD in mind as quinolinic acid is a potent neu-rotoxin that has been demonstrated to cause

neuronal death via increased lipid peroxidation

in vivo [87] and in vitro [88], as well as induc-ing lipid peroxidation resulting in increases inneurotoxic products [89]. Quinolinic acid is aproduct o the kynurenine pathway and it hasbeen suggested that irregularities in this path-

way may lead to an increase in quinolinic acid,and thereore increased neurotoxicity[90]. Thus,the CZE method described here may be suitableto investigate the merits o quinolinic acid as abiomarker or the disease.

Immunoassays

Immunoassays come in a variation o ormats,however the theory behind them is similar. Theyemploy an antibody labeled by the addition oa fuorescent or radioactive tag. The antibody isspecic or the molecule o interest and binds

to any molecules that are present. The sampleis then washed to remove excess material andviewed under the appropriate detector, withthe intensity o detection directly related to theconcentration o target molecule present.

The highly specic nature o immunoassaysenables their use as a suitable technique or tar-geted metabolite biomarker identication; ur-thermore, the assays are capable o quanticationanalysis when coupled to the labeled tags. Despiteincurring some disadvantages, including highcosts and relatively low throughput o analysis,there are instances within the literature where

imunnoassays have been used to quantiy smallmolecules in biological samples rom AD patients.

One such developed method puried the ste-roid, dehydroepiandrosterone, using HPLC thenquantied the isolated sample using a specicradiolabeled immunoassay. The results oundincreased levels in various AD brain region sam-ples and CSF, with the ndings thought to indi-cate the presence o an alternative metabolismpathway linked to oxidative stress and thereoreoccurring at greater regularity in AD patientbrains [70].

An ELISA method was published that iscapable o quantiying the level o F

2isopros-

tanes in human plasma [91]. As discussed ear-lier, dierent studies have produced confict-ing reports about the levels o F

2isoprostanes

in AD, with GCMS results suggesting a sig-nicant elevation in a range o biofuids [6366],whilst LCMS results suggested no signicantchange in urine [51]. The immunoassay resultsidentied no signicant change in the plasmaconcentrations, complementing the LCMSresults. The same study also analyzed uric acid

by a similar immunoassay and ound this to be at

Key Term

Immunoassays: Measure theconcentration of molecules byusing the interaction of labeledantibodies to its antigen.




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Table 1. Summary o the small molecules that are discussed in this review and the methods associated with

their identication.

Molecule or group omolecules

Tissue used oridentication

Trend in Alzheimersdisease compared

with controls

Targeted ornontargeted

analysis

Identiyingtechnique

Res

5-hydroxyindoleacetic

acid

Cerebrospinal uid Decrease Targeted LCelectrochemical [77]

7-hydroxylated

metabolites o

dehydroepiamdrosterone

Serum Increase Targeted GCMS [71]

8-hydroxyguanine Brain Increase Targeted GCMS [69]

Bile acids (GCA, GDA,

GCDCA)

Plasma Increase Nontargeted LCMS [95]

Ceramide Brain Increase Targeted Direct inusion-MS [55]

Citrate Cerebrospinal uid Decrease Nontargeted 1H-NMR [104]

Creatine Brain Increase Targeted 1H-MRS [38]

Creatinine Cerebrospinal uid Increase Nontargeted 1H-NMR [32]

d-aspartic acid Brain Increase Targeted Spectrophotometric

enzyme assay

[84]

d-alanine Brain Increase Targeted Spectrophotometric

enzyme assay

[84]

Dehydroepiamdrosterone Cerebrospinal uid Increase Targeted LC purifcation ollowed

by immunoassay and

GCMS

[49]

Dehydroepiandrosterone Brain and

cerebrospinal uid

Increase Targeted Purifcation using HPLC

ollowed by

immunoassay

[70]

Fumarate Cerebrospinal uid Decrease Targeted GCMS [60]

Homovanillic acid Cerebrospinal uid Decrease Targeted LCelectrochemical [77]Hydroxyoxtadecadienoic

acid

Plasma and erythrocytes Increase Targeted GCMS [68]

Hydroimidazolone Cerebrospinal uid Increase Targeted LCMS [73]

Isoprostanes Brain Increase Targeted GCMS [12,48]

Isoprostanes Cerebrospinal uid Increase Targeted GCMS [47,48,6366]

Isoprostanes Plasma Increase Targeted GCMS [47]

Isoprostanes Plamsa No signifcant change Targeted ELISA [91]

Isoprostanes Urine Increase Targeted GCMS [26,47]

Isoprostanes Urine No signifcant change Targeted LCMS [51]

Lactate Cerebrospinal uid Increase Targeted GCMS [60]

Luetin Plasma Decrease Targeted LCUV [75,76]

Myo-inositol Brain Increase Targeted 1H-MRS [38,44]

Myo-inositol combined

with creatine

Brain Increase in ratio

between the

two molecules

Targeted 1H-MRS [40,43,45]

Myo-inositol combined

with N-acetyl-aspartateBrain Reduction in ratio

between the

two molecules

Targeted 1H-MRS [43,45]

N-acetyl-aspartate Brain Decrease Targeted NMR/MRS [3638]

It is interesting to note the sheer number o dierent molecules encountered and the wide variety o methods used. Particular interest should be paid to moleculessuch as creatine, which was identifed at increased concentrations in human Alzheimers disease brains, however, in decreased concentrations in mouse model brains.Another particular class o molecules o interest are the isoprostanes, which have been ound to increase in many studies using GCMS, however, they displayed nosignifcant change using LCMS and ELISA methodologies.MRS: Magnetic resonance spectroscopy.




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a signicantly lower concentration in AD plasmathan in control plasma [91], again complement-ing the results ound via LC methodologiesdiscussed previously[74,75].

A urther published study set out to analyze ahypothesis that within AD there is a dysregula-tion o the adrenal pituitary hormonal axis byusing an immunoassay to quantiy the levelso the hormone cortisol in the plasma o AD

patients [92]. The levels o the hormone showed

a signicant increase, supporting the initialhypothesis. These results present evidencethat suggests urther investigation into thishypothesis may yield biomarker results, possi-bly leading to a novel therapeutic target throughhormone treatment.

Smith et al. developed an immunoassay thatquanties levels o nitrotyrosine, a small-mole-cule product o tyrosine nitration that can act

as an indicator o the presence o peroxynitrite,

Table 1. Summary o the small molecules that are discussed in this review and the methods associated with

their identication (cont.).

Molecule or group omolecules

Tissue used oridentication

Trend in Alzheimersdisease comparedwith controls

Targeted ornontargetedanalysis

Identiyingtechnique

Res

N-acetyl-aspartate combined withcreatine

Brain Reduction in ratio

between the

two molecules

Targeted 1H-MRS [3943]

Ne-carboxymethyl-lysine Cerebrospinal uid Increase Targeted LCMS [73]

N-ormylkynurenine Cerebrospinal uid Increase Targeted LCMS [73]

Nitrotyrosine Brain homogenate Increase Targeted Immunoassay [93]

Nitrotyrosine Cerebrospinal uid Increase Targeted LCMS [73]

Phosphatidylcholine Cerebrospinal uid Increase Targeted LCelectrochemical [79]

Phosphatidylethanolamine Brain Decrease Targeted 31P-NMR [34]

Phosphatidylethanolamine-

plasmalogen

Brain Increase Targeted 31P-NMR [34]

Phosphatidylinositol Brain Decrease Targeted 31P-NMR [34]

Phospholipid plasmalogens Brain Decrease Targeted Direct inusion-MS [52]

Sphingomyelin Brain Increase Targeted 31P-NMR [34]

Succinate Cerebrospinal uid Decrease Targeted GCMS [60]

Sulated metabolites o

dehydroepiamdrosterone

Cerebrospinal uid Increase Targeted LC purifcation ollowed

by immunoassay and

GCMS

[49]

Sulatides Brain Decrease Targeted Direct inusion-MS [55]

Uric acid Plasma Decrease Targeted LCUV [74]

Uric acid Plasma Decrease Targeted LCelectrochemical [74,75]

Uric acid Plasma Decrease Targeted ELISA [91]

Vitamin A Plasma Decrease Targeted LCUV [75]Vitamin A Plasma Decrease Targeted LCuorescence [74]

Vitamin C Plasma Decrease Targeted LCUV [74]

Vitamin C Plasma Decrease Targeted LCelectrochemical [75]

Vitamin E Plasma Decrease Targeted LCUV [75]

Zeaxanthin Plasma Decrease Targeted LCUV [75]

a-carotene Plasma Decrease Targeted LCUV [75]

b-carotene Plamsa Decrease Targeted LCUV [76]

b-cryptoxanthin Plasma Decrease Targeted LCUV [75]

It is interesting to note the sheer number o dierent molecules encountered and the wide variety o methods used. Particular interest should be paid to moleculessuch as creatine, which was identifed at increased concentrations in human Alzheimers disease brains, however, in decreased concentrations in mouse model brains.Another particular class o molecules o interest are the isoprostanes, which have been ound to increase in many studies using GCMS, however, they displayed nosignifcant change using LCMS and ELISA methodologies.MRS: Magnetic resonance spectroscopy.




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a powerul oxidant molecule that can not onlynitrate tyrosine, but is also capable o directlyoxidizing proteins and other macromole-cules [93]. Levels o nitrotyrosine were shown tosignicantly increase in brain homogenates o

AD patients when compared with controls, sug-gesting its potential to act as a biological markeror the disease.

Conclusions regarding a targeted approach

There are clearly many dierent approachesthat can be taken when planning a biomarkerinvestigation with a molecular target in mind.Each technique has advantages over any other.For example, approaches involving MS arecertainly more sensitive, but do not allow theanalysis o whole samples in a way that NMR

analysis can achieve. When planning, consider-ation o what each method can provide shouldbe taken.

However, it should be noted that the dis-crepancies that analysis between publicationsand instruments produces is oten o greatconcern. For example, isoprostane analysisby GCMS ound a signiicant increase inmany biofuids, including various brain regionhomogenates [12,48], CSF [47,48,6366], plasma [47]and urine [26,47], but there was no signicantchange in LCMS analysis o urine [51] and animmunoassay analysis o plasma [91]. This is o

particular importance as inter-technique vari-ability and conficting results will not result inthe development o a reliable marker molecule.

Whilst not without problems, the technol-ogy required or targeted analysis is constantlyimproving as time progresses. Combine this withthe alling costs o accurate instrumentation,then it is certain that targeted approaches willplay a role in the uture o AD small-moleculebiomarker analysis.

Nontargeted approaches:metabonomics/metabolomicsThere is an increasing trend or biologicalsamples to be analyzed without a specic targetmolecule in mind and the elds o metabonom-ics and metabolomics are orever expanding.Conusingly, there is great overlap between thetwo expressions and oten it depends on whichlaboratory publishes the study as to which ter-minology they use. Recently however, a deni-tion structure is appearing to take shape, withmetabolomics now commonly dened as thecomprehensive detection and quantitation o

all the low-molecular-weight molecules and

metabolites present in cells, tissues or organismsunder a set o given conditions, and metabo-a set o given conditions, and metabo-, and metabo-nomics described as the quantitative analysiso the metabolic response o living systems topathophysiological stimuli or genetic modica-

tion over time [94]. Further to these terminolo-gies and adding to the conusion is the possibil-ity o completing nontargeted analysis o specicbiological extracts; or example, lipidomics aimsto nondiscriminately analyze the dierences inconcentrations o lipids extracts.

This style o analysis becomes increasinglyimportant, especially as over recent years ourtraditional view o metabolism has altered roma simple linear structure to a more complex net-work, with many intracellular outcomes still notully understood. As nontargeted metabonom-

ics/metabolomic studies improve and the disci-pline becomes commonly practiced, data willbe combined with similar studies designed withgenetics and proteins to give us an overall pic-ture o systems biology and the whole networkedprocess (Figure 2).

In order or a nontargeted approach to beinterpreted in a reliable and useul manner, theanalysis o the raw data is heavily reliant on rig-orous data processing methods, including peakretention time alignment between samples andnormalization between samples by use o aninternal standard. This treatment o the raw data

is then processed using a selection o mathemati-cal models, or example partial least squares [95]or constrained total-line-shape [32], which arecapable o grouping samples dependant on thedierences in small molecules identied. Thisapproach highlights metabolite dierences,which can then be identied leading to indi-vidual components being linked to disease states.An extremely inormative table describing thevarious orms o data treatment and chemo-metric approaches available in metabolomicstudies can be ound in the review compiled by

Madsen et al.[96]. Furthermore, a general work-fow outlining the stages and processes involvedin a typical metabolomic study can be viewedin Figure 3.

Such approaches are integrative and com-monly correlate analytical methods or other-omics in an eort to achieve a systems biol-ogy outcome. In order to understand neuro-diseases and in particular AD and its complexeects in brain biochemistry, novel experimen-tal and computational approaches are needed.Fundamental to such understanding is the

contribution o systems biology, where data rom




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genomics, transcriptomics, proteomics and thementioned metabonomics/metabolomics areintegrated [95].

Nontargeted approaches hold a lot o poten-

tial, and many publications are now constantlyproduced that cover many dierent conditionsand disorders. However, it is still a relatively newtechnique and disadvantages exist. First, and thegreatest issue, is the problem o intererence. Theconcept o the approach is to extract and analyzethe greatest variation and quantity o moleculesas possible and this understandably leads tooverlap o molecules, leading to shielding andmasking those presenting at lower concentra-tions. To counter this, separation techniques canbe employed, however, due to the sheer number

o molecular eatures extracted separation runtimes can be in excess o an hour. This adds tothe overall study time rame, increasing costsand solvent use.

Further adding to the time o the study isconrmation o molecules o interest. Duringthe study, samples are analyzed and comparedby data manipulation and modeling providing apossible eature. This has to be conrmed by com-parison with standards, which can be expensive ordicult-to-impossible to obtain i it is not a com-mon biological molecule. Again, this adds time

and cost to the study. Sample treatment is also

extremely important with consistent and repro-ducible methods a necessity. This disadvantage iscountered by the implementation o pooled qual-ity control (QC) samples placed intermittently

throughout the analysis. In any data model o suc-cessul studies, the QCs should all group togetherwith minimal variation. I this is achieved themethod is suitable or nontargeted analysis,although results will be always semi-quantitative.

Last, a major disadvantage in AD human stud-ies, and not one that is only metabolomics relatedbut is a major issue, is the amount o drugs thatold patients and controls might be having, withthe inherent variability and complexity o know-ing or certain all the drug-related changes thatthe biofuid is showing. This problem becomes

even greater when the diagnosis power is seenon the whole ngerprint, not only will manymetabolites in that case be unidentied, but thespecicity o a method is certainly not proven bycomparing AD and controls.

Yet again, AD mice, or other animal ADmodels, are not the perect choice either as theymight have the drawback o disease diagnosisinaccuracy or nontranserability o results.

In general, nontargeted approaches such asthis involve the proling o metabolic contentin a selection o biofuids comparing dierent

groups (i.e., healthy and diseased), thereore

Figure 2. Simple overview o systems biology and the dierent disciplines required orstudy. The analysis o these will hopeully lead to complete knowledge o the overall systemnetwork, enabling the determination o novel disease pathways in Alzheimers disease. The upperright segment o the fgure demonstrates the complex nature o biological pathways with multipleinteractions possible across the entire network.

Genome

Transcriptome

Proteome

Metabolome

In

teractome




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the number o samples is o utmost importance.Typically, a pilot study would utilize 1520samples per group, providing a unique andcomprehensive source o inormation on the bio-chemical state o the system. Metabonomics has

been employed in many applications, such as dis-ease diagnosis and pathway interpretation [96],as well as the benecial and adverse eects opharmaceuticals [9799]. Regarding the study omental illness, NMR-based metabonomic strate-gies have been eectively applied in the past toschizophrenia [100,101] and bipolar disorder [102].

To date, very ew groups have attempted non-targeted techniques in AD biomarker discov-ery, however, a ew instances do appear in theliterature and they are discussed below.

Nontargeted NMR techniques for ADbiomaker identication

The earliest example in the literature o AD non-targeted biomarker analysis is a preliminary studycomparing patient and control CSF samples by

NMR. The method was capable o investigat-ing a wide variety o components, the results owhich were processed by principle componentanalysis, and was able to partially separate theclasses (control and AD) based on the concentra-

tions o metabolites ound. Further analysis othe data resulted in the identication o one othe metabolites responsible or the class group-ing, and it was demonstrated that citrate existedat signicantly dierent concentrations betweenthe classes, thereore, this was recommended orurther investigation as a biomarker [103].

Jukarainen et al. also completed a nontargetedCSF metabolite analysis via NMR methods,evaluating the data using a constrained total-line-shape model [32]. Varying dierences inmetabolite concentrations were observed in the

study, although the only signicant dierencebetween AD and control samples was in thelevel o creatinine, which was ound at higherconcentrations in the AD group. These resultscomplement a targeted study discussed earlier

MS NMR

Problem formulation

Study designSample collection

Studydesign

ExtractionDerivatization

Analysis

(Extraction)AnalysisAnalysis

Biologicalinterpretation

Dataprocessing

AlignmentBaseline connection

Peak-picking or deconvolutionPeak identification

Normalization

Scaling

Phasing and baseline correction

AlignmentNormalization

Bucketing/peak-picking/deconvolutionScaling

Statistical

analysis

Data overview (e.g., principle component analysis)

Model building (OPLS, PLS, NN or other)Model optimization

Model validationPredictions and identification of biomarkers

Identification of perturbed metabloic pathways

Mechanistic explanationFollow-up studies

Figure 3. General overview o a typical study workfow when undertaking a nontargetedapproach to biomarker identication. Here, both MS and NMR approaches are compared.NN: Neural network; OPLS: Orthogonal partial least square; PLS: Partial least square.Reprinted rom [96] with permission rom Elsevier.




14/22

in this review where creatine (the parent mol-ecule o creatinine) was identied at increasedconcentrations in the brain, suggesting possibleirregularities in this metabolism pathway inAD [38].

A nontargeted study has been publishedanalyzing serum samples via a 1H-NMRmethod [104]. In this instance the patient popu-lation did not contain any AD patients, butconsisted o those suering rom mild cognitive

impairment (MCI), a neurodegenerative con-dition associated with a signicant chance ourther developing into AD [105]. The studyused a sel-organizing map analysis to processthe serum 1H-NMR data, identiying a decrease

in the relative amount ow-3 atty acids presentin MCI, concluding that this irregularity maypossibly indicate an increased risk o AD [104].Figure 4 demonstrates the NMR spectrumwindows used in this study, highlighting the

Urea

Unsaturatedlipids

Glucose

Water

Glucose

LactateCreatinine

Freecholine

ProlineAceto-acetate

Glycoprotein Lactate

Alanine Valine

5.5 4.0 3.0 2.0 1.0 ppm

ppm

ppm

Low-molecular-weight metabolites

Lipoproteins and albumin -N(CH3)3=CH-CH

2-CH=

=CH-CH2-CH

2-

(-CH2-)

n

-CH3

CH2

CH2

-C(18)H3

Albumin

Albumin

18:2 FA

-CH2-CO

PC

-PO-CH2

-CH=CH-

TG, PGLYbackbone -CH

EC-C(3)H

FC-C(3)H

PC

-CH2-N

-N(CH3)

3

PC, SM

PGLY backbone -CH2-

TG backbone -CH2-

FA

-CH2-CO

16:1, 18:1 FA

=CH-CH2-CH

2-

22:6 FA

-CH=CH-CH2-CH

2-CO

FA

-CH2-CH

2-CO

FA

(-CH2-)

n

TC

-C(26)H3

-C(27)H3

TC

-C(21)H3

-C(19)H3

EC, FC

-9

sat Fa

-CH3

-6

-CH3

-6

-CH3

20:4 FA-CH2-CH

2-CO

PUFA

=CH-CH2-CH=

TC

TC TC TC

Lipid extract

2.4 2.2 2.0 1.8 1.6

1.0 0.9 ppm

ppm

-C(18)H3

FC, EC

TC

1.02.03.04.05.0

4.4 4.3 4.2 4.1 4.0

A

B

C

Figure 4. Three NMR spectrum windows used by Tukiainen et al. when analyzing serum samples. (A) Lipoproteins andalbumin. (B) Low-molecular-weight metabolites. (C) Lipid extract. Dierent molecular components visible in 1H-NMR analysis are clearand some important metabolites are labeled. Interestingly, the sample preparation procedure results in lipoprotein breakdownproviding valuable inormation regarding the individual lipid species that normally reside inside these particles. As can be seen in thisdiagram, the use o this technique in nontargeted analysis is clearly o great value due to the many components identifed.EC: Esterifed cholesterol; FA: Fatty acid; FC: Free cholesterol; PC: Phosphatidylcholine; PUFA: Polyunsaturated atty acid;PGLY: Phosphoglyceride; SM: Sphingomyelin; TC: Total serum cholesterol; TG: Total serum triglyceride.Reprinted rom [106] with permission rom Elsevier.




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techniques ability to identiy a large range ometabolite amilies as well as a high number oindividual components.

Currently, these are the only examples o non-targeted NMR approaches applied to human

AD samples; however, to demonstrate thepotential o the technique and to highlight itspotential in AD small-molecule biomarker dis-covery, discussed here is an approach developedby Saleket al. [106], which applies a nontargetNMR method to AD mouse model brain. Thisis included as a demonstration o the capabilitieso the technique, the methods o which could beapplied to human samples. As the majority othis review covers human AD biomarkers thisapproach should not be considered in parallel,but purely as a demonstrative piece. To urther

avoid conusion, the results rom this have notbeen included in Table 2.A nontargeted NMR study has been com-

pleted using an AD transgenic mouse model(TgCRND8) capable o developing amyloiddeposits in brain associated with AD develop-ment within 23 months o birth. Extracts wereexamined rom a variety o brain regions using1H-NMR methods, beore data analysis wascompleted via a multivariate model, producinga principle component analysis and a partial leastsquared analysis. Findings rom the comprehen-sive study indicated a decrease in many dier-

ent metabolites, including N-acetyl-l-aspartate,glutamate, glutamine, taurine, g-amino butyricacid, choline and phosphocholine (co-reso-nance), creatine, phosphocreatine and succi-nate, as well as an increase in lactate, aspartate,glycine, alanine, leucine, iso-leucine, valine andnally a group o soluble ree atty acids [106].These indings correlate with many o thehuman studies discussed previously within thisreview and suggest that irregularities in metabo-lism in AD patient brains are widespread andnot a simple issue. This mouse study highlights

the ability o metabonomics/metabolomics toidentiy a wide range o metabolite variations,enorcing the high potential or the techniqueto improve our understanding o the disease andsupporting the call or nontargeted type analysisto be requently used when analyzing humanAD samples.

Nontargeted LCMS techniques for AD

biomaker identication

A preliminary UPLCMS nontargeted studywas developed to analyze plasma samples rom

AD, MCI and control patients [95]. Although a

diagnostic model that could separate based onthe whole ngerprint o the three groups couldnot be validated, the potential o metabonomicswithin AD was demonstrated by the identica-tion o a number o molecules o interest rec-

ommended or urther investigation. The rst,glycerophosphocholine, as discussed previouslyin this review, is a metabolite o the membranephospholipid phosphatidylcholine, and sugges-tions have been made about its role in relationto neuronal membrane degradation in AD.Unortunately, to date no link has been deter-mined between AD and the second molecule ointerest identied in the study,d-glucosaminide,thereore more work on this molecule could leadto pathological clues about the mechanism oAD disease.

Finally, a trio o bile acids (GCA, GDA andGCDCA) were identied at increasing concen-trations, with controls containing the lowestconcentration and AD patients containing thehighest. The study commented that althougha trend was noted, due to the small number opatient samples the data ound could only betreated as a preliminary result and require morework beore being statistically signicant.

Nontargeted GCMS techniques for AD

biomaker identication

With regard to small-molecule discovery in

AD, there are ew nontargeted GCMS datacurrently available in the literature, with only asingle example available at present. This recentlydeveloped approach combines GCMS analysis with multivariate data treatment to analyzeree atty acids in a nontargeted approach [107].Although the study is ocusing upon atty acidsas an area o particular analysis, it has no par-ticular target acid, and aims to analyze a wideselection in a nonbiased manner. Followingintensive data treatment and the creation oanalytical models, the paper identied a num-

ber o atty acids that had signicantly decreasedin samples derived rom AD, these included:myristic acid (C14:0), palmitic acid (C16:0),oleic acid (C18:1), linolenic acid (C18:3) anddocosahexaenoic acid (C22:6).

Conclusions regarding anontargeted approachNontargeted analysis is a relatively new approachto biomarker discovery, hence there is less dataavailable when compared with those constructedwith a molecular target in the design. However,

it has been demonstrated here that this approach




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Table 2. Comparison o the various analytical techniques available to small-molecule biomarker discovery with

regard to Alzheimers disease.

Analyticaltechnique

Targeted ornontargeted

analysis

Strengths Limitations

NMR Both Provides detail on chemical structure, good

or metabolite identifcation, can analyze

whole biological samples

Relatively insensitive, high instrument and

operation costs, specialist operator required,

high-concentration metabolites can mask

those with small concentration

MRS For AD only small

molecules currently

targeted, but there is

potential or both

In vivo technique 100% noninvasive,provides detail on chemical structure, good

or metabolite identifcation

Intererence between adjacent tissues,

cannot produce spectra in mobile tissues

(e.g., blood), instrumentation can be

conusing/rightening or elderly (especially

dementia patients ), relatively insensitive,

highly expensive, specialist operator required

Direct inusion-MS For AD only small

molecules currently


potential or both

Highly sensitive, ragmentation provides

some structural data, confrmation

possible with comparison to standards,

high throughput

Samples must be pretreated to extract

metabolites, intererence and

ion-suppression issues, less structural

inormation than NMR and MRS

LCMS Both Separation prior to MS reduces intererence

and ion-suppression, easy to operate, highly

sensitive, high selectivity, confrmation


ragmentation provides some structural data


metabolites, less structural inormation than

NMR and MRS, separation adds time to

analysis, especially with complex nontargeted

approaches, generates high quantities o

organic solvent waste can be expensive to

dispose o

GCMS Both Separation prior to MS reduces intererence

and ion-suppression, easy to operate, highly

sensitive, high selectivity, confrmation


ragmentation provides some structural data


metabolites, extra time-consuming

dramatization step also a necessity to

increase analyte volatility, less structural

inormation than NMR and MRS, due to high

temperatures molecule stability issues canarise, aqueous solutions and salts cannot be

injected into the instrument, separation time

can be lengthy

LCUV detection Targeted Considerably cheaper than LCMS,

high-throughput methods can be developed,

easy to operate, high selectivity

Poor sensitivity, molecules must contain

chromophore region to be detected, samples

must be pretreated to extract metabolites,

generates high quantities o organic

solvent waste can be expensive to dispose

o, no structural inormation must be

compared with standards, no nontargeted

approach possible

LC

electrochemical

detection

Targeted Considerably cheaper than LCMS,

high-throughput methods can be developed,

easy to operate, high selectivity

Molecules must be able to undergo oxidation

or reduction or detection, generates high

quantities o organic solvent waste can beexpensive to dispose o, no structural

inormation must be compared with

standards, no nontargeted approach possible

CE For AD only small

molecules currently


potential or both

Potential or more efcient separation than

LC approaches, shorter analysis time than LC

and GC, negligible solvent consumption,

high selectivity

Currently suers rom reproducibility and

robustness issues, poor sensitivity (improving

with new CEMS instruments), specialist

operation required due to difculties

in optimization

Immunoassays Targeted Highly specifc antibody approach, highly

sensitive, relatively easy to complete, can

prebuy many kits or specifc molecule

identifcation

Highly expensive, only targeted analysis

possible, low throughput

AD: Alzheimers disease; MRS: Magnetic resonance spectroscopy.




17/22

can be successul, with a number or publica-tions already available that can identiy smallmolecules via data modeling approaches.

Currently, investigations based upon a non-targeted approach are a lot more time consuming

than those with a target. This is because o acombination o criteria including the know-how,longer analysis time, more intensive data treat-ment, identication involving database searchesand nally study conrmation using standards.This extended time obviously has other impli-cations too, including increased man hours andincreased cost o analysis.

Despite these disadvantages, the technologies,methods and statistical models are constantlyimproving, and it is likely that nontargetedapproaches will become the start point or many

uture discovery studies and may lead the wayto small-molecule biomarker discovery in AD.

Small-molecule fndings in comparison

with protein marker molecules

Presented in this review are a wide variety oanalytical approaches aimed at small-moleculebiomarker determination; however, as it cur-rently stands, research into proteins is themajor ocus o AD biomarker discovery. Thisis in part because o the nature o the diseaseand the characterized nature ob-amyloid andtau proteins, and the known connection with

the disease. Although these approaches are cur-rently the major area o research, there is yet tobe a reliable biomarker progressing to the clinic.Promising candidate markers are appearing inpublications, or example, a major recent studycomprising a number o centers internationallyidentied clusterin and apolipoprotein J, as mol-ecules that could be used to identiy the diseaseas well as determine disease severity[108].

As demonstrated within this review, smallmolecules such as lipids, carbohydrates andproducts o protein oxidation have the poten-

tial to act either individually, or in combina-tion with currently established protein markers,ideally to result in an accurate diagnostic tool.Small molecules hold plenty o advantages astools or diagnosis and disease progression mon-itoring. I an appropriate molecule is identieda routine analysis could then be developed.Generally, routine analysis o small moleculesis relatively cost eective and can be completedwith minimal technical knowledge. Further tothis, the identication o a novel small moleculemay direct research towards a new metabolic

pathway, either unknown or not previously

associated with the disease, opening up pos-sibilities o new protein biomarker targets suchas enzymes or receptors molecules. In addition,small molecules could be utilized in a combi-nation approach, conrming disease presence

and progression via the analysis o metabolites,proteins and genetics. For example, as men-tioned previously the clusterin protein ndingscomplement a similar international approachaimed at identiying genetic markers with theclusterin gene (CLU) shown to be signicantlyassociated with AD [109], thereore, potentiallyany metabolites associated with the protein andits metabolic pathway may add sensitivity andselectivity to any test developed.

Although small-molecule biomarkers are liv-ing in the shadow o their larger neighbors, they

will play a role in AD research, particularly inbiomarker approaches. With the developmentand improvement o nontargeted methods,more small molecules will be identied, hope-ully providing biomarkers and opening up newareas o research.

Conclusion

There are many possible techniques availableor the determination o biomarkers in AD, andadvantages apply to the use o each method;however, it should be noted that each techniquealso has the disadvantages to ensure no one

procedure is perect or each and every scenariorequired or the role. Each mode o ana lysis iscapable o providing important inormationabout molecules o interest, and i the cor-rect technique is applied it provides vital bio-marker knowledge enabling the development otreatments and improved care.

As this review has highlighted, there areseveral metabolic groups o particular interestthat continuously reappear within the literatureno matter which technology is used to identiythem. It is these markers o oxidative stress, par-

ticularly o lipids, which appear most promis-ing, and are the most requented small-moleculebiomarkers o AD mentioned in the literature.The sheer amount o citations that exist regard-ing lipid oxidation in AD, combined with thehigh concentrations o lipids that exist withinthe brain, suggest that irregularities in thesepathways have a role to play in both AD mech-anisms and the identication o a reliable bio-marker. Thereore, it may potentially be wortha great deal o urther investigation as this areamay contain major clues and provide important

biomarkers or the disease.




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However, it should also be noted that through-out the review numerous instances have occurredwhere there have been conficting results betweenmethodologies and their biomarker ndings, par-ticularly in the concentrations identied o F2

isoprostanes. These may be genuine results dueto coincides in sample sources, but it is morelikely that dierences in sample treatment andexperimental protocol are the culprits providingthese irregularities [110]. Either way, this requiresinvestigation to ensure progress in AD biomarkerdevelopment as inter-study variability suggestsproblems with the approaches that investigationsare taking. Furthermore, these irregularitieshighlight the importance o conrming resultsvia repetition o studies, perhaps even in dier-ent laboratories, to ensure the reproducibility o

results beore bold claims regarding biomakermolecules can be made.

Future perspective

Alzheimers disease is going to be one o the great-est challenges to modern day research in the com-ing years. With an increasingly aging population,patient numbers are going to expand rapidly. Thediscovery o an accurate biomarker will be o vital

importance and is o a pressing need. Irregularitiesin metabolism have already been demonstrated inmany pathways associated with the disease, sug-gesting that small molecules and metabolites willhave a role to play in the complexities associated

with diagnosis and disease progression.The use o small-molecule biomarkers as a tool

used in the ght against AD may be as a stand-alone biomarker/group o biomarkers; however,there is also the potential or the development oa combination approach that merges data romthe genome, proteome and metabolome in amore complete systems biology approach.

Whilst targeted studies will certainly continueto hold a role in AD biomarker research, par-ticularly or biomarker candidate conrmationand urther study, analytical technologies and

the methods o raw data treatment will improve,enabling nontargeted analysis to come to the ore-ront o biomarker research. This will be especiallyapparent in complex disease such as AD, where themechanism behind the disease is not ully under-stood. This will provide a urther advantage inthat it increases the chance o a novel biomarker,and thereore an unknown/misunderstoodpathway o the disease, being ound to investigate.

Executive summary

Introduction

The number o patients suering rom Alzheimers disease (AD) is rapidly increasing. Currently, AD diagnosis relies on subjective analysis.

An objective noninvasive test or the disease is desperately required.Sample considerations

Tissues closest to disease site (brain and cerebrospinal uid) are hard to obtain rom AD patients. Blood or urine are the ideal samples or an AD biomarker. Evidence suggesting bloodbrain barrier degradation in AD increases this possibility.

Targeted versus nontargeted approaches

Targeted approaches employ a hypothesis o a specifc molecule biomarker presence. Nontargeted approaches aim to analyze a wide range o molecules in an unbiased approach.

Targeted approaches

Currently widely used in AD biomarker investigations. These approaches employ many techniques including NMR, magnetic resonance spectroscopy, direct inusion-MS, LCMS, GCMS

and immunoassays.

Potential biomarkers include lipids, sugars and products o oxidative stress.Nontargeted approaches

Currently less common with regard to AD biomarker identifcation. Improving analytical equipment and data models make nontargeted approaches a valuable alternative.

Conclusion

A wide array o techniques are available or small-molecule biomarker discovery in AD. Small molecules have serious potential or AD biomarker discovery. Small-molecule biomarkers or AD will be an important feld o research over the next 510 years.




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Financial & competing interests

disclosure

The authors have no relevant aliations or nancial

involvement with any organization or entity with a

nancial interest in or nancial confict with the

subject matter or materials discussed in the manu-script. 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 o this manuscript.

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