Abstract
A large body of experimental and postmortem findings indicate that Alzheimer's disease (AD) is associated with increased oxidative stress (OxS) levels in the brain. Despite the current limitations of OxS assessment in living subjects, recent data suggest that oxidative challenge might increase early both in the central nervous system and peripheral fluids. The aim of this review was to provide an overview of the existing literature linking systemic OxS to brain OxS in AD. We firmly believe that continued research aimed at overcoming the methodological and design issues affecting the body of studies in this field is mandatory for successful development of an effective antioxidant-based treatment of AD.
Background
Alzheimer's disease (AD) is the most frequent form of neurodegenerative disease associated with dementia in the older population.1 ,2 Recent estimates on the prevalence of AD in the USA indicate that about five million individuals suffer from this disease.1
A corollary of evidence suggests that oxidative stress (OxS), occurring in the presence of an imbalanced cellular redox state,3 might be an early feature of AD.4–7 However, the definitive in vivo confirmation of an interaction between OxS-induced biological damage and AD development has not yet been obtained, mainly as a result of unsolved methodological issues.8–10 Likewise, the few clinical trials conducted to assess the possible effect of antioxidant supplementation in slowing or preventing AD have yielded contradictory results.11 ,12
The purpose of this review is to describe and discuss the main findings of the current literature supporting the hypothesis that OxS might be a precocious central nervous system (CNS) as well as a systemic manifestation in patients with AD. Moreover, the importance of identifying reliable biomarkers for in vivo OxS assessment and the implications of marker development on the design of future human intervention and epidemiology AD studies will be discussed.
AD: diagnosis and neuropathological features
Two major forms of AD have been described: the early-onset familial form (occurring before 65 years), and the late-onset form, which accounts for more than 95% of all AD cases.1 AD can be diagnosed with >90% confidence, based on clinical criteria, including medical history, physical examination, laboratory tests, neuroimaging and neuropsychological evaluation. At present, the conclusive diagnosis of this disease requires both clinical assessment of the disease and histopathological brain examination.13 Postmortem brain tissue is examined for the presence of two neuropathological hallmarks of AD, extracellular amyloid-β (Aβ) peptide (Aβ1-40 and Aβ1-42) accumulated in extracellular senile plaques and intracellular neurofibrillary tangles (NFT) primarily comprising abnormal and hyperphosphorylated τ protein.13
The deposition of amyloid plaques and NFTs is estimated to start approximately 10–15 years before the earliest signs of cognitive impairment, a stage referred to as presymptomatic AD14 ,15 (in this potential stage of AD, the patient presents as a fully functional individual in cognitive examinations16). The recent advances in imaging techniques, in primis structural MR, have also revealed a reduction in volumes in specific brain regions prior to the first signs of cognitive impairment.17 Notably, the progress of this disease is chiefly associated with atrophy, reflecting neuronal shrinkage and death and synaptic and axonal loss.18 These volumetric changes (along with neuropathological lesions) initially affect the hippocampus and entorhinal cortex, which have been widely observed in the brains of individuals with early-stage AD and mild cognitive impairment (MCI).19 The latter is regarded as an intermediate state between normal aging and early AD20 and is primarily characterized by short-term/long-term memory impairment, which at variance of dementia is not associated with disability in activities of daily living.20 ,21 Patients with MCI represent a high-risk population; indeed, almost half of these individuals evolve to AD at a rate of approximately 10–15% per year, which is much greater than in control subjects.20 As the brain area affected by atrophy expands, neurodegeneration progresses from the limbic area to cortical regions, particularly the parietal, prefrontal, and orbitofrontal regions,17 where additional cognitive symptoms emerge and the full dementia syndrome becomes apparent.22
Main hypotheses on AD pathogenesis
Despite years of intense research, a complete understanding of the pathogenic mechanisms underlying AD has not been achieved. Experimental and epidemiological data have resulted in the generation of several hypotheses regarding AD pathophysiology.
Amyloid cascade hypothesis: the accumulation of Aβ in the brain is the primary pathogenic event, which then triggers the entire array of subsequent brain lesions.23
τ Hypothesis: τ hyperphosphorylation is the first event in AD.24 ,25
Mitochondrial cascade hypothesis: the impairment of brain mitochondria is the pathogenic trigger of AD neurodegeneration.26
Vascular hypothesis: AD develops first as a vascular disorder characterized by an abnormal reduction of cerebral blood flow.27 Reduction in oxygen and nutrient supply leads to a neuronal energy crisis that, in turn, promotes the cellular and molecular neuropathology that defines AD.27–30
At present, the amyloid cascade is the most commonly held hypothesis, although concerns about the centrality of Aβ aggregation in senile plaques in AD pathophysiology have been repeatedly raised.25–27 The difficulty of developing a unifying hypothesis about the pathogenesis of AD primarily reflects the multifactorial nature of this disease. Nevertheless, OxS is a factor that, regardless of the pathogenic trigger proposed, plays a role in AD pathophysiology.24 ,31–33
OxS: an imbalance leading to biological damage
Helmut Sies formulated the most inspired definition of OxS nearly three decades ago, describing this disturbance as “an imbalance between oxidants and antioxidants in favor of the oxidants, potentially leading to damage.”3 This definition implies a pivotal concept: oxidants become harmful for cellular components only when these compounds overcome the antioxidant cellular defense system. Oxidants are primarily represented by reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive aldehyde species (RAS).31 These species, although commonly described as ‘dangerous’, typically act in defense against pathogens31 ,34 and as regulators of cellular metabolism.35 However, most oxidants are free radicals forced (because of intrinsic instability) to react with and transform all biochemical constituents of living organisms.31 Thus, even a slight modification of the cellular redox state, through dysfunctional defensive enzymes or depleted dietary micronutrients, has the potential to result in biomolecular damage.3 ,31 Cumulative oxidative damage is a progressive and inevitable process associated with aging, and this progression is accelerated in several aging-related diseases, particularly cardiovascular diseases (CVD)36 and neurodegenerative disorders.37 ,38 Therefore, maintaining cellular ‘redox homeostasis’ might prevent the development of pathological status.31
Oxidants and antioxidants: the main sources, routes of formation, and the reaction of the major members of the two families
Free radicals are defined as an atom or a molecule containing at least one unpaired electron.31 Superoxide anion (
) is regarded as the ‘primary’ ROS that further interacts with other molecular species, either directly or through the mediation of metal or enzyme catalyzed reaction, to form other ‘secondary’ ROS.39 The intracellular increase in the superoxide radical primarily reflects mitochondrial dysfunction (with an increase in electron leak from complexes I and III of the electron transport chain) and the abnormal solicitation of both NADPH oxidase (NOX, present in both phagocytic and non-phagocytic cells) and xanthine oxidase (enzyme involved in purine catabolism).31 ,34 ,40 ,41 Extremely high concentrations of superoxide and nitric oxide (NO˙) are obtained during respiratory burst triggered through inflammatory responses.42 ,43 Under these conditions, these two species react to form a highly reactive peroxynitrite anion (ONOO−), which in turn induces lipid and protein oxidation and DNA fragmentation.42 This deleterious process can be prevented through the highly efficient scavenging activity of both cytosolic and mitochondrial isoforms of superoxide dismutase (SOD) that dismutate 
to a ‘less’ reactive ROS, hydrogen peroxide (H2O2).39 ,44 This is a critical step of the cellular redox pathway, since H2O2 can either be neutralized through other enzymes, such as catalase (CAT) or glutathione peroxidase (Gpx), or converted in the ‘chief instigator’ of OxS damage, that is, hydroxyl radical (˙OH), through the metal-catalyzed Fenton reaction.45 ,46 Hydroxyl radical is by far the most reactive ROS (half-life of 10−9 seconds), and this molecule indiscriminately reacts with all biomacromolecules.46 Indeed, hydroxyl radicals trigger the lipid peroxidation cascade (leading to alterations in the biological properties of membranes, such as degree of fluidity, permeability, inactivation of receptors, etc), oxidative damage to proteins (alterations in folding and either the alteration or loss of enzymatic functions, etc), nucleic acids (damage to DNA and RNA), and carbohydrates.31 ,47
The accumulation of ROS, particularly superoxide, results in biological damage when the responses from enzymes and non-enzymatic molecules with antioxidant properties is inappropriate.3 ,31 Defense mechanisms against reactive species essentially involve: (1) preventive mechanisms; (2) repair mechanisms; and (3) scavenging actions.31 ,37 The most effective strategies for preventing oxidative damage are dismutation (through SOD) and the reduction of hydrogen peroxide or lipid hydroperoxides (ROOH) through enzymes that use endogenous nucleophiles, such as glutathione and thioredoxin.48 The repair of damaged biomolecules is afforded through various proteinases, lipases, and DNA enzymes. Finally, according to Forman et al,48 only SOD, α-tocopherol, and most likely glutathione reach a suitable concentration in vivo and/or react with a sufficiently high-rate constant as effective radical scavengers. Indeed, most free radicals react, at nearly the same rate constant, with several low-molecular weight antioxidants and the biomolecules to be protected. To overcome these kinetic constraints, these antioxidants should be at intracellular concentrations that are difficult to achieve in vivo.48 However, the beneficial effects of some dietary micronutrients are undeniable. Indeed, there are several types of polyphenols that potently augment endogenous antioxidant mechanisms via alterations in gene expression.35 ,48
However, when the concentrations of endogenous antioxidants are insufficient to directly quench radicals, reflecting the kinetic limitations described above, low-molecular antioxidants, such as ascorbic acid, β-carotene, uric acid, and protein thiols, represent a diverse array of important antioxidants that act in synergy. These molecules are also essential for recycling α-tocopherol from tocopheryl radical in membranes and lipoproteins, chelating iron and other pro-oxidant metals.31 ,48
Biomarkers of OxS: a longstanding issue
The measurement of OxS in living individuals is challenging, mainly because the direct assessment of reactive species requires time-consuming and expensive approaches that are not feasible for clinical applications.49 The alternative to these methods is the indirect assessment of ROS through fingerprinting, that is, the quantification of the byproducts of oxidative biomolecular damage.8 ,31 ,50 Among these potential indicators, a gold-standard biomarker for the quantification of OxS in the periphery (serum/plasma or urine) has not yet been validated. Indeed, none of the currently available indices of OxS can fulfill the widely accepted definition of biomarker as “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention.”9
The development and application of various potential markers for measuring OxS in biological fluids are a currently evolving research area. A multitude of indicators of OxS have been employed in population-based studies thus far, although there has been little direct comparison between different methods using identical samples.8 In addition, the available results appear inconsistent and variable, most likely reflecting the lack of accuracy and comparability of different analytical methods; hence, it is extremely difficult to provide absolute reference values for the specific markers in humans.8 ,51 A crucial advance in this framework is the application of multilaboratory validation studies to determine the reliability of markers used for non-invasive measurement of OxS in humans, which has previously been undertaken in animals.51 ,52
An additional concern regarding OxS assessment must be highlighted. Indeed, the detection of high levels of OxS in the blood/urine of patients with a given disease (eg, AD) may not be the definitive evidence of the implication of reactive species in the development of such a pathology.8 Increased OxS might reflect potential concomitant pathologies (eg, CVD, diabetes, and liver diseases) and subclinical pathological conditions that induce low-grade inflammation (eg, atherosclerosis and arthritis).8 ,53 Moreover, some large epidemiological studies have identified a number of demographic and physical lifestyles and dietary factors that markedly affect the systemic redox balance, even in the absence of a full-blown disease.54–57 The most common examples of these factors are older age, gender, race, as well as overall and central obesity (the latter being more associated with OxS58 ,59), smoking, alcohol abuse, and nutrition (particularly the intake of dietary antioxidants42). The failure to identify these factors (through anamnesis, detection of disease markers, assessment of anthropometrics, etc) or an underestimation of the impact of these factors in data analysis could considerably affect the reliability of the conclusions drawn from these data, as exhaustively outlined in two previous reviews.60 ,61
Fortunately, although conclusive analytical and pathophysiological validations have not as yet been achieved, there are several markers that possess the characteristics of chemical stability, easy accessibility, and the reproducibility required for a ‘reliable’ index of OxS status.8 ,51 ,62 In this review, we will describe the use of these markers in studies of AD.
Classification of the most assessed peripheral markers of oxidative stress
In general, markers of OxS can be classified as molecules modified through interactions with reactive species in a defined microenvironment and molecules of the antioxidant system that change in response to increased pro-oxidative burden.8 ,51 Traditionally, most markers of oxidative damage reflect ROS/RAS/RNS attack on polyunsaturated fatty acids DNA and protein.8
Markers of lipid peroxidation
The most widely employed index of lipid peroxidation in blood and urine is that of thiobarbituric acid-reactive substances (TBARS), extensively used because of the simplicity of their assessment.8 However, this simple assay lacks specificity and precision.51 ,63 The TBARS method has been frequently employed to measure malondialdehyde (MDA), a secondary product of lipid peroxidation. However, this RAS can only be reliably measured through mass spectrometry (MS) and ancillary techniques of chromatographic separation.63 ,64 In addition to MDA, one of the main peroxidation products studied as an oxidative biomarker with distinct biological effects, α-β unsaturated aldehyde 4-hydroxynonenal (4-HNE), derived from the oxidation of ω-6 fatty acid, has also been used as a biomarker. This RAS efficiently binds covalently to proteins, peptides, nucleic acids, etc.65
At present, gas chromatography (GC)-MS methods to analyze 4-HNE adducts show the highest sensitivity compared with other quantification techniques, such as western blotting and high-pressure liquid chromatography (HPLC).8 ,65 Recently, an ELISA test was proposed as a potential alternative to these time-consuming and labor-consuming methods, but the appropriateness of this assay has not been validated in blood samples.66
Similar considerations apply for the assessment of F2-isoprostanes (F2-iso), a family of prostaglandin F2α compounds generated in vivo through the non-enzymatic ROS-catalyzed peroxidation of esterified arachidonic acid.8 ,67 F2-iso and other isomers (derived from different PUFAs) are widely considered the most reliable serum and urinary markers of lipid peroxidation.68 The specificity and sensitivity of F2-iso as an OxS marker, however, strictly depend on the method used to assess this prostaglandin-like compound.67 ,68 In this regard, the most reliable analytical method (but also the least applicable in clinical routine) is GC/MS, while the easiest and most accessible method (although less accurate and occasionally misleading) is an ELISA approach.8 ,51
Hydroperoxides (ROOH) are prominent non-radical intermediates of the oxidative modification of all unsaturated phospholipids and glycolipids, cholesterol, and (to a minor extent) amino acids and carbohydrates.8 ROOH are primarily formed during the propagation phase in the lipid peroxidation cascade through the reaction of the highly reactive peroxyl radical (
) with a lipid molecule (figure 1).31 Reflecting diverse sources of origin, hydroperoxides reach high concentrations in the blood, and monitoring them does not require highly sensitive analytical procedures. The most commonly used assays for these determinations are based on the spectrophotometric assessment of the products formed through reactions between a chromogenic substrate and hydroperoxides. However, previous studies and reviews8 ,51 have concluded that, although presenting some positive features (low cost, simplicity, etc), none of the currently available methods afford an accurate and specific in vivo assessment of these lipid peroxidation byproducts.
Initiation and propagation phases of lipid peroxidation cascade reaction. Hydroperoxides are relatively stable intermediates of lipid peroxidation, but in the presence of iron or copper in reduced form can form a highly reactive radical (alcoxyl radical), which is able to damage lipids and other biomolecules.
Markers of protein and nucleic acid oxidation
Proteins are highly sensitive targets for ROS/RAS/RNS because of the overall abundance of these molecules in biological systems.8 ,62 The primary markers derived from the oxidation of these macromolecules are carbonyls, nitrotyrosine and the least specific advanced oxidized protein products (AOPP).62 ,69 ,70 Among these, carbonyls are now considered the most reliable markers, but only when they are measured through MS or HPLC.3 ,5 ,53 Simpler, but less reliable, spectrophotometric or ELISA-based assays are also now available for large-scale determination.71 Conversely, 3-nitrotyrosine (formed from a reaction with peroxynitrite or nitrogen dioxide) is widely considered as a promising early marker of protein oxidation in AD-related neurodegenerative processes and is extremely difficult to measure in serum or urine because of the high instability of this compound in these fluids.8 ,70
Studies have shown that cellular DNA and RNA damage results from radical species generated under different conditions, and a number of assays have been developed to oxidatively measure modified bases in several biological specimens.72 The most used markers are derived from the ROS-induced modification of guanosine of DNA (hydroxy-2′-deoxyguanosine, acronym: 8-OHdG) and RNA (hydroxy-2′-guanosine, acronym: 8-OHG), which can be accurately measured through ELISA (particularly in urine, where these molecules are more stable compared with other fluids), although MS technology is considered the gold standard.73
Involvement of OxS in AD pathogenesis
Several lines of evidence from in vitro and animal studies suggest that OxS might be an early event in AD pathogenesis.5–7 ,74 ,75 According to these experimental observations, oxidative burst could precede and/or contribute to the formation of Aβ deposits and NFT and plays a role in AD-related neurodegeneration. Evidence of a primary role for OxS in disease development has also been provided through postmortem examination, revealing fingerprints of oxidative challenge in MCI and AD.76–79 In contrast, clinicoepidemiological studies on peripheral markers of OxS have generated inconsistent findings.10 ,80–84
Evidence of the early involvement of OxS in AD and potential mechanisms linking OxS with disease pathogenesis
Extensive oxidative damage has been observed in vulnerable neurons that do not yet show any pathological hallmarks of AD.85 As stressed in the next paragraph, oxidatively modified biomolecules have been observed in MCI and AD postmortem brain tissue.86 ,87 In vivo experiments on transgenic mouse models support the concept that oxidative burden temporally precedes the deposition of Aβ and other typical neuropathological traits of AD.5
In an exhaustive review, Cai et al88 suggested that OxS might play a role in the generation and deposition of Aβ in brain parenchyma by diverse mechanisms. Briefly, the formation of Aβ occurs through two sequential cleavages of amyloid precursor protein (APP), elicited through β-secretase and γ-secretase. Accumulating experimental evidence from transgenic animal models4 ,75 suggests that OxS significantly increases the catalytic activity of these two enzymes, which in turn augments Aβ production. The deposition of Aβ in the brain could reflect a compromised blood–brain barrier (BBB),89 and OxS could contribute to damaging BBB either directly32 or through the activation of metalloproteinases.88 ,90 As a consequence of an alteration in BBB permeability, Aβ clearance from the brain might be delayed, and Aβ influx from cerebrospinal fluid (CSF) might be increased.88
The factors that contribute to elevated ROS levels most likely include aging, genetic factors, environmental exposure to contaminants (eg, pesticides), metal dyshomeostasis, etc.39 ,91–93 This deleterious increase in reactive species most likely involves impaired neuronal mitochondria, which have been consistently observed as an early and prominent feature of AD.24 ,26 Mitochondrial dysfunction could reflect reduced energetic substrates or oxygen delivery to the highly active neurons following chronic brain hypoperfusion (from stroke or heart failure)27 ,32 or the defective expression of glucose receptors in the BBB.29 The ensuing reduction of ATP synthesis is the precursor of a biochemical cascade (altered protein and lipid synthesis, signal transduction breakdown, etc), eventually leading to OxS30 and cellular hypometabolism, with the latter preceding any clinical manifestations of AD.27 Moreover, according to Morris et al94 and Kaminsky et al,95 this form of dementia might be associated with several systemic manifestations (ie, obesity, diabetes, chronic inflammation, insulin resistance, etc), which are well-recognized factors with the potential to negatively influence peripheral redox balance.8 ,31 ,36 ,54 ,55 ,57
Excess ROS generation might also influence the clinical progression of AD. Indeed, it has been suggested that these noxious species mediate the neurotoxicity of the Aβ soluble oligomers (ie, the precursors of insoluble fibrils of amyloid plaques)74 and hyperphosphorylated τ.96 Specifically, experimental data have shown that Aβ oligomers, mainly those formed from the Aβ1-42 isoform, directly generate H2O2 through copper-dependent SOD-like activity.6 ,31 ,74 Copper present in high levels in amyloid plaques reacts with hydrogen peroxide to form highly reactive hydroxyl free radicals.7 The excessive generation of ROS reflects the interaction between Aβ oligomers and neuronal and microglial and NOX,34 ,97 with the latter presumably activated through increased intracellular free calcium.98 Furthermore, these aberrant oligomers could also induce the release of pro-inflammatory and pro-oxidant cytokines from astrocytes and microglia97 and alter ROS production in mitochondria through modulating the activity of enzymes, such as Aβ-binding alcohol dehydrogenase, or impairing the activity of respiratory chain complexes (eg, cytochrome oxidase, figure 2).99 ,100 Moreover, increasing evidence suggests that OxS might also be involved in the hyperphosphorylation and polymerization of the τ protein.101 Indeed, the neurotoxic action of neurofibrillary deposits in the brain is depressed or induced through the downregulation or upregulation, respectively, of mitochondrial SOD.96
Sources of reactive oxygen species (ROS) in the Alzheimer's disease (AD) brain. Soluble amyloid-β (Aβ) oligomers and mitochondrial dysfunction are the main sources of ROS and related biomolecular damage in the brain tissue of patients affected by AD. Once formed, the Aβ oligomers are able to generate oxygen peroxide (H2O2), which acts with the reduced transition metals (Fe2+ or Cu+) and produces hydroxyl radicals (˙OH). In turn, this noxious radical is able to: directly damage biomolecules (1); cause damage to neuronal mitochondria (2) and, consequently, a large leak of superoxide anion (
) and oxygen peroxide from the respiratory chain (3); directly activate microglial NOX (NADPH oxidase) (4) (and promote production of superoxide anion (5)) and/or induce the release of pro-inflammatory cytokines from astrocytes and microglia (6), which in turn can promote the release of ROS from other glial cells (7). Mitochondrial dysfunction, and consequent emanation of ROS from the organelle to cellular constituents (8), may also occur prior to Aβ oligomer accumulation (and be one of the causes of their formation (9)) (IL, interleukin; TNF, tumour necrosis factor).
The concept of a primary role for OxS as a mediator of NFT and Aβ neurotoxicity is further supported by the potential high vulnerability of neurons to reactive species insults. Indeed, neurons have a long life, relatively low levels of endogenous antioxidants (particularly glutathione), high levels of polyunsaturated fatty acids (PUFA, the primary target of reactive species), high levels of pro-oxidant metals, and the conspicuous use of oxygen, which enhance the ‘physiological’ extent of ROS derived by mitochondria.91 ,102 In this scenario, even a moderate increase in the normal rate of ROS formation could lead to detrimental consequences, as demonstrated through the extensive accumulation of oxidative damage.
Markers of OxS in human studies: brain (postmortem tissue and CSF) versus peripheral fluids (blood and urine)
Higher concentrations of carbonyls and 3-nitrotyrosine have been repeatedly detected in neocortical/hippocampal postmortem tissue samples from patients with AD compared with controls.77 ,103 Butterfield et al78 and other groups have shown that protein oxidation could be a precocious event in AD pathogenesis, as the quantity of carbonyls is significantly increased in the hippocampi or in the superior and middle temporal gyri103 of MCI subjects compared with age-matched and sex-matched controls. In contrast with the results from postmortem analyses, a recent meta-analysis did not report any significant evidence of higher protein oxidation levels in the serum of patients with AD compared with healthy individuals.80 Indeed, with a few exceptions,104 ,105 no observational studies have reported significant changes in the serum concentration of AOPP, carbonyls, or 3-nitrotyrosine in relation to the occurrence of AD.80 ,81 ,106 ,107
High levels of biomarkers of oxidative DNA damage in circulating lymphocytes from patients affected with AD108–110 have been reported in a small number of studies. In contrast, Abe et al111 reported that the higher (fivefold compared with controls) levels of 8-OHG observed in the CSF of patients with AD were not observed in the serum of these individuals. The results from postmortem studies are much more consistent than those conducted in living individuals. Specifically, Mecocci et al112 observed an increase of 8-OHdG in mitochondrial and nuclear DNA in three regions of the cortex of patients with AD compared with controls. Moreover, Wang et al113 showed higher levels of this marker in nuclear DNA from frontal and temporal lobe tissues and mitochondrial DNA from temporal lobe tissues in MCI compared with age-matched control subjects.
A number of studies have shown increased levels of F2-iso in frontal and temporal cortical areas or in the CSF of patients with AD compared with healthy individuals or patients affected with other neuropsychiatric diseases114–117 (table 1). Markesbery et al79 observed precocious lipid peroxidation in AD pathogenesis and reported higher F2-iso levels in the frontal, parietal, and occipital lobes in MCI and AD compared with controls, whereas no significant differences were present between MCI and late AD. Postmortem CSF levels of F2-iso in patients with AD were positively associated with the severity of neurodegeneration, as assessed through histopathological examination.118 However, alterations in the blood/urine concentration of F2-iso in living patients with AD are inconsistent.82–84 Other isoprostane-like compounds, such as D4-isoprostanes, E4-isoprostanes, and F4-isoprostanes (or neuroprostanes), might be promising indicators of brain damage, given that these isoforms are derived from the oxidation of the major PUFA in the CNS docosahexaenoic acid (DHA).117 Interestingly, neuroprostanes levels were more elevated in the temporal and occipital lobes of patients with MCI and AD 79 and in the CSF119 of patients with AD compared with controls. Unfortunately, data concerning this marker in the blood/urine from patients with AD remain scarce.
OxS markers in peripheral fluids and brain of patients with MCI or AD
A large body of data have been gathered through measurements of peripheral MDA, 4-HNE, and hydroperoxides.80 ,81 ,107 ,120–125 A meta-analysis of 29 studies revealed that MDA was significantly increased in the plasma/serum of patients with AD and MCI compared with controls,80 thereby reflecting the results obtained in brain tissue.103 ,126 A similar trend has also been described for 4-HNE, which was increased both in the serum127 and brain128–130 of patients with AD and MCI129. Moreover, an association between higher serum levels of hydroperoxides and AD was observed in a previous study involving one of the largest population samples (422 participants) examined in studies on this topic.131 To the best of our knowledge, there are no published studies that evaluate hydroperoxide levels in autopsied brains or CSF samples.
Table 1 summarizes the results of some of the largest studies evaluating OxS markers in brain tissue and CSF or peripheral fluid samples from patients with MCI or AD.
Some, but not all, lipid peroxidation markers are elevated in the peripheral fluids of patients with AD: potential explanatory hypotheses
Considering the data discussed in the previous paragraph, the markers most consistently altered in the peripheral fluids of patients with AD are those derived from lipid peroxidation. These byproducts, at variance with oxidized proteins, are small lipophilic molecules that freely cross the BBB, even when this barrier is not damaged, and subsequently diffuse into the blood. Indeed, the level of these compounds should be considerably higher in the brain and CSF of patients with AD to become detectable in the systemic circulation. In a pioneering study, Montine reported that the difference in the mean concentration of CSF F2-iso between the AD and controls was 28 pg/mL, which could only reflect the oxidation of arachidonic acid (10% of the brain lipid content)132; this surplus is then diluted 20–80 times in the blood, becoming undetectable even by the most sensitive methods. These considerations might account for the mixed results obtained with this superior marker of OxS.82–84
The most reasonable explanation for the increase in lipid oxidation markers, particularly lipid hydroperoxides, may be the induction of peripheral lipid peroxidative cascades through ROS. While the most noxious free radicals (hydroxyl and superoxide) are too unstable to translocate from the brain without reacting within neuronal tissue, H2O2 and hydroperoxides are stable enough to travel from the brain to the blood.31 In the presence of even small concentrations of free iron or copper ions in reduced form, both species generate two highly reactive species, hydroxyl and alcoxyl radicals (figure 1), which in turn initiate and/or accelerate the peroxidative modification of lipids, such as the plasmatic cholesterols and phospholipids of circulating cells.31 ,48 Some recent observations are consistent with these considerations: (1) circulating copper levels are increased in patients with AD133 ,134; (2) ceruloplasmin enzymatic activity, promoting the incorporation of the free pro-oxidant Fe2+ into transferrin, is decreased in AD80 ,134; (3) AD has often been associated with an increase in oxidized-low density lipoprotein (LDL) level106 and a loss of erythrocyte membrane stability, reflecting the oxidative modification of structural lipids.135
Abnormal levels of lipid peroxidation byproducts in the serum of patients with AD could also result from systemic processes and manifestations that often accompany (and mutually influence67) CNS dysfunctions, such as impaired glucose and lipid metabolism (type 2 diabetes and insulin resistance are risk factors for AD) and chronic inflammation. Increases in inflammatory cytokines136 and altered energy metabolism are also associated with OxS.94
Alterations in the peripheral levels of antioxidants: co-factors for systemic OxS in AD
The propagation of oxidative damage occurs rapidly when the antioxidant system affords an inadequate buffer.3 ,31 Previous studies80 ,81 ,124 ,131 ,137 have shown low serum concentrations of dietary-derived and endogenous low-molecular non-enzymatic antioxidants, such as uric acid, vitamin E, and vitamin C, in patients with AD or MCI, compared with healthy controls. Although they rarely reach extracellular and intracellular concentrations sufficient to scavenge radicals,48 these compounds act synergistically to prevent, or slow down, the perpetuation of oxidative damage to biological molecules.3 ,31 However, it is difficult to ascertain whether the low levels of exogenous antioxidants might be secondary to excessive consumption (reactions with radicals) or low dietary intake (or malabsorption syndromes).
A more concrete contribution to abrogate the systemic propagation of ROS damage is obtained through enzymatic activities; these proteins, by definition, are much faster and more efficient ROS scavengers than low-molecular weight antioxidants.48 Most importantly, the modulation of the activity of these enzymes depends on both genetic factors and interactions with micronutrients procured through the diet (eg, polyphenols).48 ,138 ,139 Nonetheless, the most effective scavengers of ROS (CAT, Gpx and SOD) have a predominantly intracellular localization, and thus they cannot be used as serum indicators of systemic antioxidant status. On the contrary, there is increasing evidence that the measurement of some antioxidant enzymes in erythrocytes, which are considered as passive ‘reporter cells’ for the oxidative status of the whole organism, could be a useful marker in this field.140 In the milieu of plasma enzymes with antioxidant capacity, one of the most thoroughly investigated is paraoxonase 1 (PON-1). The next paragraph will focus on this enzyme and recent findings suggesting the potential implication of this enzyme in AD pathogenesis.
PON-1: potential relevance in AD pathogenesis
PON-1 is synthesized in the mammalian liver and circulates in the blood, bound to the surface of high-density lipoprotein (HDL), apolipoprotein (apo) A-1, and apo J. PON-1 exerts a number of enzymatic activities, including paraoxonase, arylesterase, and lactonase, which are spectrophotometrically detected using three different substrates, organophosphates, aromatic esters, and lactones, respectively.141 ,142 There are simple and fast spectrophotometric assays available for the measurement of the activities of serum PON-1.141 However, despite the wide use and adequate standardization of these methods, normal reference intervals have not been established to date.
Although the natural physiological substrate and catalytic mechanisms of this enzyme remain unknown, it has been suggested that PON-1 endows HDL particles with antioxidant properties. In vitro, PON-1 scavenges hydrogen peroxide and lipid hydroperoxides either free or present in atherosclerotic lesions or in minimally oxidized LDL.142 Further validation of the antioxidant properties of PON-1 has been generated through in vivo experiments in PON-1/apo E double knockout mice.143 Moreover, data from large population-based studies have consistently demonstrated a role for PON-1 in protecting the human body from CVD through inhibiting oxidative and pro-atherogenic modifications in lipoproteins and decreasing systemic lipoperoxidative damage.144
The finding that PON-1 single nucleotide polymorphisms (particularly Q192R and L55M) affect enzymatic activity and influence the risk of OxS-related diseases suggests the potential involvement of this enzyme in AD pathogenesis.145 ,146 Unfortunately, a recent meta-analysis showed inconsistent results regarding the association between PON-1 phenotypes (Q192R) and AD prevalence.146 These results do not rule out the potential relationship between PON-1 and this disease, as multiple polymorphisms in the PON-1 gene influence the catalytic function of this enzyme.147 Moreover, recent studies148–150 have shown that subjects affected with AD have lower levels of serum PON-1 enzymatic activity compared with healthy controls.
While the mechanism(s) for PON-1-mediated systemic antioxidant effects remain elusive, there is clear evidence that this HDL-associated protein might impact systemic OxS in humans.151 Thus, in the light of the prominent role of OxS as a ‘primary progenitor of AD’,85 it is reasonable to speculate that the high level of PON-1 might confer protection from AD development, or at least might contrast the clinical progression of this disease. In support of this hypothesis, it has been demonstrated that high paraoxonase activity provides protection from CVD,152 which in turn is a well-recognized risk factor for vascular dementia and AD development.21 ,24 A recent study conducted with 596 older participants revealed a deficit in PON-1 activity in MCI150; consequently, this deficit might reflect an early feature of dementia.
Notably, since longitudinal studies employing PON-1 are lacking, conclusions concerning the cause-effect link between the levels of this enzyme and AD remain elusive. Furthermore, PON-1 might not have a high clinical specificity for AD, being a multifactorial enzyme widely associated with many different disorders.150 ,152 ,153
OxS and AD: from theory to practical applications
Research needs and perspectives
Considering the published data discussed thus far, it is conceivable to ascribe OxS as a member of the proposed pathogenic mediators of AD. In addition, increasing evidence suggests that this condition might precociously affect both CNS and non-CNS compartments. Notably, the definitive proof of this ‘simultaneity’ is still lacking, reflecting the underscored methodological shortcomings concerning the in vivo assessment of OxS.
The association between OxS and AD represents the rationale of many cross-sectional and prospective population-based studies examining whether the intake of antioxidants in the diet or in nutritional supplements might be beneficial to disease prevention and/or course.154–156 The results of these observational studies are mixed. Two prospective studies showed lower risks of dementia or AD in patients who consumed higher amounts of dietary antioxidants.154 ,156 In contrast, a recent study has shown a lack of association between AD and antioxidant vitamin consumption (either in the diet or in supplements).155 Farina et al11 described similar disappointing results, obtained from double-blind randomized trials in which vitamin E treatment was compared with placebo in patients with AD or MCI. In particular, Petersen et al found that vitamin E supplementation failed to prevent the progression from MCI to AD during the 3 years of study.12
The reported failure of vitamin E supplementation in AD does not preclude that antioxidants might be beneficial for this disease. It has been suggested that combinations of antioxidants or combinations of antioxidants with aldehyde-trapping agents157 might be worthy of clinical investigation. For example, it has been observed that the combined intake of a mixture of various tocopherols might be more neuroprotective than α-tocopherol alone.158 Moreover, the use of other antioxidants in combination with vitamin E could improve the effects of therapy because antioxidants work synergistically against ROS.48 Relevant to this notion, a study of a large cohort of elderly individuals demonstrated an association between the self-reported use of vitamin E supplement and the reduced incidence of AD but only in combination with ascorbic acid.12 Unfortunately, to the best of our knowledge, large and well-designed double-blind studies on the use of a mixture of different antioxidant compounds in patients with AD and/or MCI are lacking. Although we propose that this approach might potentially be the most effective, we are also aware that it would be mandatory to identify the components of this ‘cocktail’ and determine the dosage that would decrease systemic OxS. Although the available experimental and epidemiological evidence is too sparse and disconnected to obtain a definitive solution to this issue, there are some interesting indications. Bonda et al85 suggested that the most effective therapeutic antioxidants for AD should target the mitochondrial components because these organelles are the primary generators of ROS, and in AD, oxidative injury to mitochondrial structures occurs prior to that in any other cell components.26 ,85 Thus, coenzyme Q10 and R-α-lipoic acid could be promising compounds, based on the results of preclinical studies.85 Other antioxidant candidates for AD might be the variegated family of plant-derived polyphenols. Cumulating evidence in transgenic mice has shown that the administration of a single polyphenol (eg, quercetin or resveratrol) or a mixture of polyphenols increased the serum activity of PON-1,159 which is depressed at the preclinical stage of AD.150 In addition, phytochemicals are not good ROS-scavengers per se but rather positively modulate the expression of key cellular antioxidants and phase 2 enzymes involved in ROS-scavenging activity, such as SOD, CAT, glutathione, and Gpx.160 ,161 Thus, the postulated modulation of PON-1 activity might be ascribed as part of the ROS-detoxifying mechanisms triggered by polyphenols.
The identification of the most suitable population sample that could be treated with antioxidants is a further important issue to consider. Since the herein reported converging evidence indicates that redox balance is already compromised in the preclinical stage of AD, it should be recommended to target cognitively healthy individuals (by using the most commonly used cognitive test), rather than MCI or AD, for possible antioxidant interventions. Indeed, it must be borne in mind that the antioxidants are effective in preventing OxS-related injuries while being unable to repair them.24 In agreement with other authors,74 we believe that,74 a possible new research avenue for studying the response to antioxidants in AD might consist in including the assessments of antioxidants (such as PON-1, low-molecular non-enzymatic antioxidants) and markers of oxidative damage. To accomplish this aim, it becomes mandatory to overcome the challenging methodological shortcomings affecting the in vivo assessment of OxS.
Conclusions
AD is regarded as a heterogeneous and multifactorial CNS disease, with no single etiopathogenetic pathway leading to the typical dementia syndrome. OxS might be ascribed as one of the ‘smoking guns’ that accounts for the pathogenesis of the disease, in the light of the multiple roles of OxS in systemic and neurological abnormalities characterizing AD.
Footnotes
Contributors CC and GZ developed the idea of the work and contributed equally to the writing. GV, PLW, AR, JMS, MS and BO contributed to the writing and editing at various extent.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
References
