Measurements of serum non-ceruloplasmin copper by a direct fluorescent method specific to Cu(II).

BACKGROUND: Meta-analyses indicated the breakdown of copper homeostasis in the sporadic form of Alzheimer's disease (AD), comprising copper decreases within the brain and copper increases in the blood and the pool not bound to ceruloplasmin (non-Cp Cu, also known in the literature as "free" copper). The calculated non-Cp Cu (Walshe's) index has many limitations. METHODS: A direct fluorescent method for non-Cp Cu detection has been developed and data are presented herein. The study included samples from 147 healthy subjects, 36 stable mild cognitive impairment (MCI) and 89 AD patients, who were tested for non-Cp Cu through the direct method, total serum copper, ceruloplasmin concentration and o-dianisidine ceruloplasmin activity. The indirect non-Cp Cu Walshe's index was also calculated. RESULTS: The direct method was linear (0.9-5.9 µM), precise (within-laboratory coefficient variation of 9.7% for low and 7.1% for high measurements), and had a good recovery. A reference interval (0-1.9 µM) was determined parametrically in 147 healthy controls (27-84 years old). The variation of non-Cp Cu was evaluated according to age and sex. Non-Cp Cu was 1.5 times higher in AD patients (regarding the upper value of the reference interval) than in healthy controls. Healthy, MCI and AD subjects were differentiated through the direct non-Cp Cu method [areas under the curve (AUC)=0.755]. Considering a 95% specificity and a 1.91 µmol/L cut-off, the sensitivity was 48.3% (confidence interval 95%: 38%-58%). The likelihood ratio (LR) was 9.94 for positive test results (LR+) and 0.54 for negative test result (LR-). CONCLUSIONS: The direct fluorescent test reliably and accurately measures non-Cp Cu, thereby determining the probability of having AD.

Value of serum nonceruloplasmin copper for prediction of mild cognitive impairment conversion to Alzheimer disease.

OBJECTIVE: Meta-analyses show that nonbound ceruloplasmin (non-Cp) copper (also known as free or labile copper) in serum is higher in patients with Alzheimer disease (AD). It differentiates subjects with mild cognitive impairment (MCI) from healthy controls. However, a longitudinal study on an MCI cohort has not yet been performed to assess the accuracy of non-Cp copper for the prediction of conversion from MCI to AD during a long-term follow-up. METHODS: The study included 42 MCI converters and 99 stable MCI subjects. We assessed levels of copper, ceruloplasmin, non-Cp copper, iron, transferrin, ferritin, and APOE genotype. A multiple Cox regression analysis-with age, sex, baseline Mini-Mental State Examination, APOE4, iron, non-Cp copper, transferrin, ferritin, hypercholesterolemia, and hypertension as covariates-was applied to predict the conversion from MCI to AD. RESULTS: Among the evaluated parameters, the only significant predictor of conversion to AD was non-Cp copper (hazard ratio = 1.23, 95% confidence interval = 1.03-1.47, p = 0.022); for each additional micromole per liter unit (µmol/l) of non-Cp copper, the hazard increased by ~20%. Subjects with non-Cp copper levels >1.6 µmol/l had a hazard conversion rate (50% of conversion in 4 years) that was ~3× higher than those with values ≤1.6 µmol/l (<20% in 4 years). The rate of conversion was similar between APOE4 carriers and noncarriers (p = 0.321), indicating that the non-Cp copper association was independent of APOE4. INTERPRETATION: Non-Cp copper appears to predict conversion from MCI to AD. These results encourage healthy life style choices and dietary intervention to modify this risk.

The influence of sex on non-motor wearing-off in Parkinson's disease: A WORK-PD post-hoc study.

INTRODUCTION: The wearing-off phenomenon is characterized by the recurrence of motor and non-motor symptoms of Parkinsonism during a period free from levodopa. It is a pivotal aspect marking the end of the pharmacological "honeymoon" period in Parkinson's disease (PD). A growing body of literature is connecting sex with the likelihood of developing fluctuations. We investigated such an association in a post-hoc analysis of the large WORK-PD study. METHODS: WORK-PD analyzed the usability of the wearing-off questionnaire 19 (WOQ19) in clinical practice and included cross-sectional data on age, disease duration, time on levodopa, Hoehn and Yahr stage, and WOQ19 scores of 532 PD patients. In the present study, we selected patients with an exposure time to levodopa of at least 1 year. RESULTS: A total of 380 patients were included. Women reported a higher number of wearing-off symptoms than men (6.09 ± 3.39 vs 4.96 ± 3.11, p = 0.0006). Sex groups also differed in non-motor symptoms (2 ± 1.9 vs 1.5 ± 1.5, p = 0.021), particularly behavioral wearing-off scores being higher in women (p < 0.001). The latter were primarily featured by anxiety-related phenomena. Finally, there was a significant interaction between behavioral symptoms, sex, and age at onset (df = 2, F = 9.79, p < 0.0001), whereas no such interaction was observed with levodopa exposure and motor impairment, unlike motor symptoms. DISCUSSION: Women showed a greater propensity than men to experience wearing-off, particularly non-motor fluctuations on the anxiety spectrum. The latter may demonstrate a lesser reliance on dopamine compared to motor symptoms. This observation could be underpinned by biological variances between genders at the neurotransmitter level.

Evaluating the Safety and Efficacy of Crenezumab vs Placebo in Adults With Early Alzheimer Disease: Two Phase 3 Randomized Placebo-Controlled Trials.

Importance: Alzheimer disease (AD), a neurodegenerative disease characterized by ß-amyloid plaques and τ tangles in the brain, represents an unmet medical need with no fully approved therapeutics to modify disease progression. Objective: To investigate the safety and efficacy of crenezumab, a humanized monoclonal immunoglobulin G4 antibody targeting ß-amyloid oligomers, in participants with prodromal to mild (early) AD. Design, Setting, and Participants: Two phase 3 multicenter randomized double-blind placebo-controlled parallel-group efficacy and safety studies of crenezumab in participants with early AD, CREAD and CREAD2, were initiated in 2016 and 2017, respectively, and were designed to evaluate the efficacy and safety of crenezumab in participants with early AD. CREAD (194 sites in 30 countries) and CREAD2 (209 sites in 27 countries) were global multicenter studies. A total of 3736 and 3664 participants were screened in CREAD and CREAD2, respectively. A total of 3736 and 3664 participants were screened in CREAD and CREAD2, respectively. Both trials enrolled individuals aged 50 to 85 years with early AD. Participants with some comorbidities and evidence of cerebral infarction or more than 4 microbleeds or areas of leptomeningeal hemosiderosis on magnetic resonance imaging were excluded. After 2923 and 2858 were excluded, respectively, 813 participants in CREAD and 806 in CREAD2 were randomly assigned in a 1:1 ratio to either placebo or crenezumab. In the final analysis, there were 409 participants in the placebo group and 404 in the crenezumab group in CREAD and 399 in the placebo group and 407 in the crenezumab group in CREAD2. Data were analyzed up until January 2019 and August 2019, respectively. Interventions: Participants received placebo or 60 mg/kg crenezumab intravenously every 4 weeks for up to 100 weeks. Main Outcomes and Measures: The primary outcome was change from baseline to week 105 in Clinical Dementia Rating-Sum of Boxes (CDR-SB) score. Results: There were 813 participants in CREAD (mean [SD] age, 70.7 [8.2] years; 483 female and 330 male) and 806 in CREAD2 (mean [SD] age, 70.9 [7.7] years; 456 female and 350 male). Baseline characteristics were balanced between both groups. The between-group difference in mean change from baseline in CDR-SB score (placebo minus crenezumab) was -0.17 (95% CI, -0.86 to 0.53; P = .63) at week 105 in the CREAD study (88 placebo; 86 crenezumab). Compared with previous trials, no new safety signals were identified, and amyloid-related imaging abnormalities with edema were rare, mild, and transient. No meaningful changes in AD biomarkers were observed. Both studies were discontinued following a preplanned interim analysis indicating that CREAD was unlikely to meet the primary end point. Conclusions and Relevance: Crenezumab was well tolerated but did not reduce clinical decline in participants with early AD. Trial Registration: ClinicalTrials.gov Identifiers: CREAD, NCT02670083; CREAD2, NCT03114657.

PINK1 heterozygous rare variants: prevalence, significance and phenotypic spectrum.

Heterozygous rare variants in the PINK1 gene, as well as in other genes causing autosomal recessive parkinsonism, have been reported both in patients and healthy controls. Their pathogenic significance is uncertain, but they have been suggested to represent risk factors to develop Parkinson disease (PD). The few large studies that assessed the frequency of PINK1 heterozygotes in cases and controls yielded controversial results, and the phenotypic spectrum is largely unknown. We retrospectively analyzed the occurrence of PINK1 heterozygous rare variants in over 1100 sporadic and familial patients of all onset ages and in 400 controls. Twenty patients and 6 controls were heterozygous, with frequencies (1.8% vs. 1.5%) not significantly different in the two groups. Clinical features of heterozygotes were indistinguishable to those of wild-type patients, with mean disease onset 10 years later than in carriers of two mutations but worse disease progression. A meta-analysis indicated that, in PINK1 heterozygotes, the PD risk is only slightly increased with a non significant odds ratio of 1.62. These findings suggest that PINK1 heterozygous rare variants play only a minor susceptibility role in the context of a multifactorial model of PD. Hence, their significance should be kept distinct from that of homozygous/compound heterozygous mutations, that cause parkinsonism inherited in a mendelian fashion.

An observational study on the influence of the APOE-epsilon4 allele on the correlation between 'free' copper toxicosis and EEG activity in Alzheimer disease.

Since many years the apolipoprotein E epsilon4 allele (APOE-epsilon4) is known to be associated with Alzheimer disease (AD) but the mechanisms of these associations remained unclear. In the last years, the potential pathogenetic role of 'free' copper (i.e. non-ceruloplasmin bound copper) has been evidenced in AD. Recently, elevated 'free' copper was found to be correlated with slowing of cortical electroencephalographic (EEG) rhythms. The present work aimed to check the hypothesis that the strength of the correlations between free-copper and alterations of cortical rhythms might be different in carriers and non-carriers of the APOE-epsilon4 allele. Fifty-four AD patients and 20 healthy controls were included in the study. In all of them 1) APOE genotyping was performed; 2) total serum copper and ceruloplasmin was determined in order to calculate the serum 'free' copper; and 3) resting eyes-closed EEG rhythms were recorded and spectral brain activity was estimated via LORETA. A 'two correlation coefficients comparison' test was used to test the strength of the correlation in APOE-epsilon4 carriers and non-carriers. 'Free' copper levels were higher in patients than in controls and correlated positively with parietal-temporal delta and negatively with parieto-temporal alpha-1 activities. The correlation between 'free' copper and temporal alpha-1 activity was stronger in APOE-epsilon4 carriers than in non-carriers. Peroxide levels correlated with higher temporal delta in the AD group. APOE-epsilon4 appears to modulate the effect of copper on the altered AD brain activities, suggesting that modulation of oxidative stress related to copper dysfunction may be one of the mechanisms that make APOE-epsilon4 a risk factor for AD.

Copper dyshomeostasis in Wilson disease and Alzheimer's disease as shown by serum and urine copper indicators.

Abnormal handling of copper is the cause of Wilson disease (WD), a rare disorder typified by increased levels in plasma copper not-bound to ceruloplasmin (nCp-Cu, also known as 'free' copper). In Alzheimer's disease (AD), meta-analyses show that copper decreases in brain but increases in serum, due to the nCp Cu component increase. Despite the similarities, a direct comparison of copper biological status in the two diseases has never been carried out. To fill this gap, we evaluated serum copper, ceruloplasmin, nCp-Cu and Cu:Cp in 385 CE and 336 healthy controls previously investigated that were compared with 9 newly diagnosed WD patients. We then assessed 24h copper urinary excretion in 24 WD patients under D-penicillamine (D-pen) treatment and in 35 healthy controls, and compared results with those of AD patients participating to a D-pen phase II clinical trial previously published. After adjusting for sex and age, serum nCp-Cu and Cu:Cp resulted higher in AD and in WD than in healthy controls (both p<0.001). While nCp-Cu was similar between AD and WD, Cu:Cp was higher in WD (p<0.016). 24h urinary copper excretion in AD patients (12.05µg/day) was higher than in healthy controls (4.82µg/day; p<0.001). 77.8% of the AD patients under D-pen treatment had a 24h urinary excretion higher than 200µg/day, suggestive of a failure of copper control. This study provides new insight into the pathophysiology of copper homeostasis in AD, showing a failure of copper control and the Cu:Cp ratio as an eligible marker.

Free copper and resting temporal EEG rhythms correlate across healthy, mild cognitive impairment, and Alzheimer's disease subjects.

OBJECTIVE: The present study tested the hypothesis that the serum copper abnormalities were correlated with alterations of resting electroencephalographic (EEG) rhythms across the continuum of healthy elderly (Hold), mild cognitive impairment (MCI), and AD subjects. METHODS: Resting eyes-closed EEG rhythms delta (2-4Hz), theta (4-8Hz), alpha 1 (8-10.5Hz), alpha 2 (10.5-13Hz), beta 1 (13-20Hz), beta 2 (20-30Hz), and gamma (30-40Hz), estimated by LORETA, were recorded in 17 Hold, 19 MCI, 27 AD- (MMSE< or =20), and 27 AD+ (MMSE20) individuals and correlated with copper biological variables. RESULTS: Across the continuum of Hold, MCI and AD subjects, alpha sources in parietal, occipital, and temporal areas were decreased, while the magnitude of the delta and theta EEG sources in parietal, occipital, and temporal areas was increased. The fraction of serum copper unbound to ceruloplasmin positively correlated with temporal and frontal delta sources, regardless of the effects of age, gender, and education. CONCLUSIONS: These results sustain the hypothesis of a toxic component of serum copper that is correlated with functional loss of AD, as revealed by EEG indexes. SIGNIFICANCE: The present study represents the first demonstration that the fraction of serum copper unbound to ceruloplasmin is correlated with cortical delta rhythms across Hold, MCI, and AD subjects, thus unveiling possible relationships among the biological parameter, advanced neurodegenerative processes, and synchronization mechanisms regulating the relative amplitude of selective EEG rhythms.

Patients with Increased Non-Ceruloplasmin Copper Appear a Distinct Sub-Group of Alzheimer's Disease: A Neuroimaging Study.

BACKGROUND: Meta-analyses show that copper non-bound to ceruloplasmin (non-Cp Cu, also known as 'free' copper) in serum is higher in a percentage of Alzheimer's disease (AD) patients. Genetic heterogeneity in AD patients stratified on the basis of non-Cp Cu cut-off sustains the existence of a copper AD metabolic subtype. OBJECTIVE: In order to find evidence of the existence of a detectable metabolic subtype of AD associated to copper abnormalities, we explore the hypothesis of a neuroimaging pattern heterogeneity in an homogenous and well characterized AD population classified in two groups by the stratification of patients on the basis non-Cp Cu cut-off. METHOD: We assessed levels of copper, ceruloplasmin, non-Cp Cu, cerebrospinal levels of total Tau protein (h-tau), Thr 181 phosphorylated tau protein (P-tau) and ß-amyloid 1-42, and APOE4 genotype in 66 AD patients and compared neuroimaging indices of a visual rating scale of cerebral atrophy and neurovascular burden in AD patients stratified in 'Normal' and 'High' non-Cp Cu groups. RESULTS: The stratification for non-Cp Cu originated AD groups which did not differ for medial temporal lobe atrophy, periventricular hyperintensities, deeper hyperintensities (including frontal, parietooccipital and temporal white matter hyperintensities), infratentorial hyperintensities indices, while they differed for global atrophy. More specifically, AD patients within the high non-Cp Cu group had a less severe burden of global atrophy (p=0.042). CONCLUSION: This neuroimaging heterogeneity between AD groups is suggestive of the existence of a copper metabolic subtype of AD; non-Cp Cu appears a good marker of this copper AD.

Sources of cortical rhythms change as a function of cognitive impairment in pathological aging: a multicenter study.

OBJECTIVE: The present study tested the hypothesis that cortical electroencephalographic (EEG) rhythms. change across normal elderly (Nold), mild cognitive impairment (MCI), and Alzheimer's disease (AD) subjects as a function of the global cognitive level. METHODS: Resting eyes-closed EEG data were recorded in 155 MCI, 193 mild AD, and 126 age-matched Nold subjects. EEG rhythms of interest were delta (2-4 Hz), theta (4-8 Hz), alpha 1 (8-10.5 Hz), alpha 2 (10.5-13 Hz), beta 1 (13-20 Hz), and beta 2 (20-30 Hz). EEG cortical sources were estimated by LORETA. RESULTS: Occipital delta and alpha 1 sources in parietal, occipital, temporal, and 'limbic' areas had an intermediate magnitude in MCI subjects compared to mild AD and Nold subjects. These five EEG sources presented both linear and nonlinear (linear, exponential, logarithmic, and power) correlations with the global cognitive level (as revealed by mini mental state examination score) across all subjects. CONCLUSIONS: Cortical EEG rhythms change in pathological aging as a function of the global cognitive level. SIGNIFICANCE: The present functional data on large populations support the 'transitional hypothesis' of a shadow zone across normality, pre-clinical stage of dementia (MCI), and AD.

Alpha rhythms in mild dements during visual delayed choice reaction time tasks: a MEG study.

Can simple delayed response tasks affect latency and amplitude of magnetoencephalographic midline alpha rhythms (6-12 Hz) in early dementia? We recruited 15 mild Alzheimer's disease (AD) and 10 vascular dementia (VaD) patients (paired mini mental state exam of 17-24). The control groups comprised 18 young and 22 elderly normal subjects. In the first task, a simple "cue" stimulus (one bit) was memorized along a brief delay period (3.5-5.5s) up to a "go" stimulus triggering (right or left) button press. In the second task, the "cue" stimulus remained available along the delay period. Event-related reduction in power of the alpha rhythms indexed the cortical activation (event-related desynchronization, ERD) for the trials associated with correct behavioral responses. Behavioral performances to both tasks were lower in the AD and VaD patients than in the normal subjects. In particular, just four AD and five VaD patients executed a sufficient amount of correct responses for the alpha ERD analysis, so they were included in a unique group. In both tasks, the alpha ERD peak was later in latency in the demented and normal elderly subjects than in the normal young subjects. Furthermore, the alpha ERD peak was stronger in amplitude in the demented patients than in the normal subjects. These results suggest that simple delayed response tasks during physiological recordings are quite difficult for patients even at an early dementia stage. Such difficulty may induce the abnormal amount of the related cortical activation in dementia as revealed by the alpha ERD.

Copper perturbation in 2 monozygotic twins discordant for degree of cognitive impairment.

BACKGROUND: Recent evidence indicates that peripheral tissue markers can provide information regarding changes affecting cellular metabolism in Alzheimer disease (AD). We previously reported that serum copper levels can discriminate subjects with AD from normal control subjects (with 60% sensitivity and 95% specificity) and from patients with vascular dementia (with 63% sensitivity and 85% specificity). OBJECTIVE: To study the correlation between AD and serum levels of transition metals and markers of peripheral oxidative stress. DESIGN: Case study. SETTING: General hospital inpatient wards and outpatient clinics. Patients A pair of elderly monozygotic female twins discordant for AD. MAIN OUTCOME MEASURES: Biochemical analyses of peripheral-blood transition metals and indicators of oxidative stress and neurologic and neuropsychological assessments of clinical status for presence of cognitive impairment and AD. RESULTS: Serum copper and total peroxide levels were both 44% higher in the twin with greater cognitive impairment and a diagnosis of AD. CONCLUSIONS: The cases reported support the hypothesis of a major involvement of copper and oxidative abnormalities in AD.

Individual analysis of EEG frequency and band power in mild Alzheimer's disease.

OBJECTIVE: This EEG study investigates the role of the cholinergic system, cortico-cortical connections, and sub-cortical white matter on the relationship between individual EEG frequencies and their relative power bands. METHODS: EEGs were recorded at rest in 30 normal elderly subjects (Nold), 60 mild Alzheimer disease (AD) and 20 vascular dementia (VaD) patients, comparable for Mini Mental State Evaluation scores (MMSE 17-24). Individual EEG frequencies were indexed by the theta/alpha transition frequency (TF) and by the individual alpha frequency (IAF) with power peak in the extended alpha range (5-14 Hz). Relative power was separately computed for delta, theta, alpha1, alpha2, and alpha3 bands, on the basis of the TF and IAF. RESULTS: Using normal subjects as a reference, VaD patients showed 'slowing' of alpha frequency (TF-IAF) and lower alpha2 power; Mild AD patients showed lower alpha2 and alpha3 power; delta power was higher in both AD and VaD patients; Theta power was higher only in VaD patients. CONCLUSIONS: Individual analysis of the alpha frequency and power can discriminate mild AD from VaD and normal elderly subjects. SIGNIFICANCE: This analysis may probe pathophysiological mechanisms causing AD and VaD.

Promoter haplotypes of interleukin-10 gene and sporadic Alzheimer's disease.

Clinical and immunopathological evidence support a potential role of the pro- and anti-inflammatory cytokine network in neurodegeneration of Alzheimer's disease (AD). Moreover, association studies suggest a possible involvement of cytokine-related genes in the susceptibility to sporadic AD. Since conflicting results are associated with the pro-inflammatory pathway, we investigated a putative effect of the anti-inflammatory counterpart focusing on the interleukin-10 (IL-10) gene. The 5' flanking region contains numerous polymorphisms; in particular, three single nucleotide polymorphisms (-1082 G/A, -819 T/C, -592 C/A) are in linkage disequilibrium resulting in three haplotypes GCC, ACC and ATA. We analyzed the IL-10 haplotype distributions in 215 Italian sporadic AD patients and 153 controls in an association case-control study. Haplotype frequencies did not reveal differences between the two samples, however the genotype GCC/ACC was more represented in AD patients (OR 1.91, 95% CI: 1.18-3.07). This putative risk factor could be independent of the presence of the ApoE epsilon 4 allele. Our results provide new insights on a possible involvement of the IL-10 gene in susceptibility to sporadic AD even though further functional and genetic investigations are necessary to clarify its role in AD.

Red blood cell copper, zinc superoxide dismutase activity is higher in Alzheimer's disease and is decreased by D-penicillamine.

Copper, zinc superoxide dismutase (Cu, Zn SOD) activity was measured in red blood cells (RBC) of 32 patients affected by Alzheimer's disease (AD), eight other AD patients treated with the copper-chelating agent D-penicillamine, 13 first-degree relatives and 22 controls. All AD patients enrolled in our study showed a higher level of Cu, Zn SOD activity early in the disease. No correlation between apolipoprotein E genotype and SOD activity was found in AD patients. D-penicillamine treatment of AD patients for 24 weeks lowered the enzyme activity even below the control value. These results support the hypothesis that a higher level of Cu, Zn SOD activity in RBC can be an early diagnostic peripheral marker of this disease and a sensor to monitor treatments with copper-chelating drugs.

Linkage disequilibrium and haplotype analysis of the ATP7B gene in Alzheimer's disease.

Copper dyshomeostasis leading to a labile Cu(2+) not bound to ceruloplasmin ("free" copper) may influence Alzheimer's disease (AD) onset or progression. To investigate this hypothesis, we investigated ATP7B, the gene that controls copper excretion through the bile and concentrations of free copper in systemic circulation. Our study analyzed informative ATP7B single-nucleotide polymorphisms (SNPs) in a case-control population (n=515). In particular, we evaluated the genetic structure of the ATP7B gene using the HapMap database and carried out a genetic association investigation. Linkage disequilibrium (LD) analysis highlighted that our informative SNPs and their LD SNPs covered 96% of the ATP7B gene sequence, distinguishing two "strong LD" blocks. The first LD block contains the gene region encoding for transmembrane and copper-binding, whereas the second LD block encodes for copper-binding domains. The genetic association analysis showed significant results after multiple testing correction for all investigated variants (rs1801243, odds ratio [OR]=1.52, 95% confidence interval [CI]=1.10-2.09, p=0.010; rs2147363, OR=1.58, 95% CI=1.11-2.25, p=0.010; rs1061472, OR=1.73, 95% CI=1.23-2.43, p=0.002; rs732774, OR=2.31, 95% CI=1.41-3.77, p<0.001), indicating that SNPs in transmembrane domains may have a stronger association with AD risk than variants in copper-binding domains. Our study provides novel insights that confirm the role of ATP7B as a potential genetic risk factor for AD. The analysis of ATP7B informative SNPs confirms our previous hypothesis about the absence of ATP7B in the significant loci of genome-wide association studies of AD and the genetic association study suggests that transmembrane and adenosine triphosphate (ATP) domains in the ATP7B gene may harbor variants/haplotypes associated with AD risk.

Metal-score as a potential non-invasive diagnostic test for Alzheimer's disease.

The link between biometals and Alzheimer's disease (AD) has been investigated with a focus on local metal accumulations. In this work, we have looked at systemic metal changes and computed a score (M-score) based on metal disarrangements to discriminate patients with AD from patients with vascular dementia (VaD) and from controls. We measured serum levels of iron, copper, ceruloplasmin, transferrin, and total antioxidant capacity (TAS), performed Apolipoprotein E (APOE) genotyping and calculated non-ceruloplasmin copper ('free' copper') levels, transferrin saturation, total iron binding capacity, and ceruloplasmin-transferrin ratio (Cp/Tf) in 93 patients with AD, 45 patients with VaD, and 48 controls. All subjects underwent biochemical, neuroimaging and cognitive evaluations. Significant differences were observed among the tested groups for the levels of copper, free copper, peroxides, and TAS and for the Cp/Tf with disparity in couple comparison. On this basis we created the M-score as linear combination of biometal variables and APOE genotype. Besides its ability to discriminate AD patients vs. controls (ROC AUC=90%), M-score was able to distinguish AD vs. VaD (ROC AUC=79%). Moreover, we calculated the sensitivity and the specificity for M-score and for the other significant variables: M-score reached the highest sensitivity without a relevant loss in terms of specificity. When we compared M-score with APOE genotype and Medial Temporal Atrophy score, it resulted statistically better than these diagnostic markers. In conclusion, we confirm the link between biometals and AD and suggest its potential as diagnostic tool. Further studies may elucidate its potential role as reliable diagnostic test.

Antioxidant status and APOE genotype as susceptibility factors for neurodegeneration in Alzheimer's disease and vascular dementia.

Different factors interact to develop neurodegeneration in patients with dementia and other neurodegenerative disorders. Oxidative stress and the ε4 allele of apolipoprotein E (ApoE) are associated with significant alteration in lipid metabolism, in turn connected to a variety of neurodegenerative diseases and aging. Thus, a better understanding of the pathogenetic pathways associated with lipid dyshomeostasis may elucidate the causes of neurodegenerative processes. To address this issue, we evaluated the effects of antioxidant status and APOE genotype on neurodegeneration in patients with dementia of the Alzheimer type (AD), with vascular dementia (VaD), and in elderly healthy controls. Eighty-two AD, 42 VaD patients, and 26 healthy controls were recruited and underwent medial temporal lobe atrophy (MTA) assessment, white matter hyperintensities rating (WMH), serum total antioxidant status assaying (TAS), and APOE genotyping. A logistic regression algorithm applied to our data revealed that a 0.01 mmol/L decrease of TAS concentration increased the probability of MTA by 24% (p=0.038) and that carriers of the APOE ε4 allele showed higher WMH scores (p=0.018), confirming that small variations in antioxidant systems homeostasis are associated with relevant modifications of disease risk. Furthermore, in individuals with analogous TAS values, the presence of the ε4 allele increased the predicted probability of having MTA. These outcomes further sustain the interaction of oxidative stress and APOE genotype to neurodegeneration.

GSTM1 null genotype as risk factor for late-onset Alzheimer's disease in Italian patients.

Alzheimer's disease (AD) is the most common form of dementia in the elderly. The causes of AD are very complex but there is general agreement about the existence of a link between Alzheimer's disease and oxidative stress. The Glutathione S-transferases (GSTs) act to detoxify products of oxidation that cause damage to macromolecules. Particular attention has been focused on GST genes because polymorphisms are important determinants of disease risk. To evaluate if GSTA1, GSTM1, GSTP1, and GSTT1 genes are associated with LOAD we screened a case-control population (n=311). Differences in genotype distributions between AD patients and controls were found only for the GSTM1 null genotype (P<0.001). In addition, a logistic regression analysis also conferred a positive association between the GSTM1 null genotype and LOAD after adjustment for age and gender (OR=2.09; 95%CI=1.31-3.35). The GSTM1 enzyme detoxifies substances such as exogenous and endogenous metabolites and plays a regulatory role in cellular signaling. Previous studies have highlighted that GSTM1 has a role in neurodegenerative disorders, but no data have associated the GSTM1 gene with AD risk. Our outcome suggests that the GSTM1 null genotype is a risk factor for AD in Italian patients.

Effects of hemochromatosis and transferrin gene mutations on iron dyshomeostasis, liver dysfunction and on the risk of Alzheimer's disease.

It is now accepted that transition metals, such as iron and copper, are involved in the pathogenesis of the Alzheimer's disease (AD) through their participation in toxic oxidative phenomena. In this context, hemochromatosis (Hfe) and transferrin (Tf) genes are of particular importance, since they play a key role in iron homeostasis. Also, signs of liver distress which accompany metal dysmetabolisms have been shown to be linked to AD. In order to investigate whether and how all these factors are interconnected, in this study we have explored the relationship of the gene variants of Hfe H63D and C282Y and of Tf C2 with serum markers of iron status (iron, ferritin, TF, TF-saturation, ceruloplasmin -CP-, CP and TF serum concentrations (CP/TF) ratio), and of liver function (albumin, transaminases, prothrombin time-prothrombin time (PT)) in a sample of 160 AD patients and 79 healthy elderly controls. Albumin resulted in lower, PT longer and AST/ALT higher ratios in AD patients than in controls, indicating a distress of the liver. Also TF was lower and ferritin higher in AD. Multiple logistic regression backward analyses, performed to evaluate the effects of our biochemical variables upon the probability of developing AD, revealed that a one-unit TF serum-decrease increases the probability of AD by 80%, a one-unit albumin serum-decrease reduces this probability by 20%, and a one-unit increase of AST/ALT ratio generates a 4-fold probability increase. Patients who were carriers of the H63D mutation showed higher levels of iron, lower levels of TF and CP and higher CP/TF ratios, a panel resembling hemochromatosis. This picture was found neither in H63D non-carrier patients, nor in healthy controls. Our results suggest the existence of a link between Hfe mutations and iron abnormalities that increases the probability of developing AD when accompanied by a distress of the liver.