Send Orders of Reprints at
[email protected] Current Alzheimer Research, 2013, 10, 191-198
191
Metal-Score as a Potential Non-Invasive Diagnostic Test for Alzheimer’s Disease Rosanna Squitti1,2,*, Patrizio Pasqualetti1, Renato Polimanti3, Carlo Salustri4, Filomena Moffa1, Emanuele Cassetta1, Domenico Lupoi5, Mariacarla Ventriglia2, Maurizio Cortesi1, Mariacristina Siotto6, Fabrizio Vernieri2 and Paolo Maria Rossini7,8 1
Department of Neuroscience, AFaR - Fatebenefratelli Hospital, Rome, Italy; 2Departement of Neurology, “Campus Biomedico” University, Rome, Italy; 3Department of Biology, University of Rome “Tor Vergata”, Rome, Italy; 4Institute of Cognitive Sciences and Technologies (CNR) - Fatebenefratelli Hospital, Rome, Italy; 5Department of Radiology, Fatebenefratelli Hospital, Rome, Italy; 6Don Carlo Gnocchi Foundation ONLUS, Milan, Italy; 7Casa di Cura San Raffaele, Cassino , Italy; 8Institute of Neurology, Catholic University, Rome, Italy Abstract: 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.
Keywords: Alzheimer’s disease, metal disarrangements, diagnostic test, statistical score, copper, transferrin, antioxidant capacity, vascular dementia. INTRODUCTION Alzheimer’s disease (AD) is a heterogeneous and progressive neurodegenerative disorder representing the most common cause of dementia in the elderly [1]. The disease results from a complex interaction of predisposing genes and biochemical variables with the cooperation of environmental factors. Recently, researchers have uncovered an important role played by iron and copper metabolisms in the pathogenesis of the disease [2]. In fact, although both iron and copper are essential for human life, they become dangerous when regulatory mechanisms fail, as they become available to participate in Fenton’s like reactions which generate highly noxious free radicals. So far, the two metals have been mostly studied separately, as researchers have mainly focused their attention on local brain accumulations in the case of iron and on assessment of general circulation levels in the case of copper. Sys*Address correspondence to this authors at the Department of Neuroscience, AFaR - Ospedale Fatebenefratelli, 00186, Rome, Italy; Tel: +39 06 6837 385; Fax: +39 06 6837 300; E-mail:
[email protected] 1875-5828/13 $58.00+.00
temic iron dyshomeostasis has been hardly investigated in AD, apart from a single recent study demonstrating the activation of the ceruloplasmin-transferrin antioxidant system in response to elevated oxidative stress and associated with both worsen Mini-Mental State Examination (MMSE) scores and isolated medial temporal lobe atrophy (MTA) [3]. Conversely, systemic copper dyshomeostasis has been quite extensively investigated and a recent meta-analysis of the data published since 1983 has concluded that circulating copper levels are slightly but significantly increased in AD [4]. The two pathological hallmarks of AD are amyloid plaques and neurofibrillary tangles. Although it has been clearly established that these hallmarks are associated with the peptide amyloid- and the tau protein respectively, the pathways linking them are not yet completely understood, and several hypotheses have been proposed. Among them, the notion that an abnormality in the balance of body metal levels may be one of the altered pathways promoting amyloid- toxicity and its precipitation in AD plaques [5] is sustained by solid clinical [6, 7], epidemiological [8] and experimental data [9-11]. Consequently, an analysis of sys© 2013 Bentham Science Publishers
192 Current Alzheimer Research, 2013, Vol. 10, No. 2
temic metal level changes promises not only to expose the role of biometals in AD pathogenesis but also to assess the potential of the analysis itself to provide non-invasive and inexpensive AD biomarkers. Currently, although there is no definitive biological marker of the disease, some researchers have proposed new diagnostic tests to help diagnosis [12, 13], such as those from cerebrospinal fluid which indicate the presence of AD pathological changes [14]. In fact, the diagnosis of AD during life is still mostly based on clinical examination using the criteria of the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association (NINCDS–ADRDA), as we used in this study [15]. Metaanalyses have shown that only 70–90% of participants fulfilling the NINCDS–ADRDA criteria of probable AD have their diagnosis later confirmed by brain histopathology [16]. Therefore, although the NINCDS–ADRDA criteria have good reliability and validity [17, 18], any new measure that increases diagnostic sensitivity and specificity is highly valuable for improving early detection and intervention. To address these issues, we performed a comprehensive study of iron- and copper-related biological variables in a group of 93 AD patients, 45 patients with vascular dementia (VaD) and 48 controls. Specifically, we measured the serum levels of iron, copper, ceruloplasmin, transferrin, and total antioxidant capacity (TAS). We also calculated nonceruloplasmin-bound copper (‘free copper’), transferrin saturation, total iron binding capacity, ceruloplasmin-transferrin ratio (Cp/Tf), which is a statistical index conceived to reflect the ratio between electron paramagnetic signals of the ceruloplasmin - bonding a copper in the oxidized state (Cu+2) and of the apotransferrin [3]. Moreover, we performed Apolipoprotein E (APOE) genotyping. We finally investigated the association of the results with the patient clinical status, as assessed by neuropsychological examinations and brain imaging, and computed a score expressing the evaluation of the analysis, which we propose as AD biomarker. METHODS Subjects The study was approved by the local IRB and all participants or legal guardians signed an informed consent. 93 AD patients (NINCDS-ADRDA criteria), 45 VaD patients (NINCDS-AIREN criteria), both groups with MMSE score 25, and 48 cognitively normal subjects with no clinical evidence of neurological and psychiatric disease were enrolled in the study. Partially overlapping patient cohorts had been investigated in a previous study [3]. VaD patients were selected on the basis of clinical history consistent with VaD as well as brain MRI criteria suggestive of microangiopathic (small vessel) pathology (Jellinger class two) [19]. Criteria for exclusion for all individuals participating to current study were conditions known to affect copper metabolism and biological variables of oxidative stress on the basis of the past medical history and screening laboratory tests, reported in details elsewhere [3]. Patients and controls with abnormal values of thyroid, liver, kidney and cardiac functions were also excluded from the study. We also excluded patients with a history of stroke, presence of focal neurological signs, severe subcortical leukoencephalo-
Squitti et al.
pathy, presence of hemodynamically significant neck and intracranial arteries stenosis or occlusion, or cardiopathy. Briefly, All AD and VaD patients underwent neurologic, neuroimaging (magnetic resonance imaging or computerized tomography) and extensive neuropsychological evaluations as well as routine laboratory tests to best characterize the cohorts under study. Mixed dementia were excluded. Brain MRI was performed using a 1.5 Tesla superconductor magnet. The imaging protocol consisted of axial T2 W double Spin Echo (SE) sequences and T1 W SE images in axial, coronal, and sagittal planes, with 5 mm slice thickness and intersection gap = 0.5 mm. MR images were evaluated by two experienced neuroradiologists, blind to the patients’ diagnoses and laboratory results, with an agreement of about 95%. Atrophy and white matter lesions were graded following standardized visual rating scales on plain MRI [20-23]. The degree of MTA was evaluated with a ranking procedure and validated by linear measurements of the medial temporal lobe including the hippocampal formation and surrounding spaces occupied by CSF, following standardized criteria (five point rating scale) [23]. Generalized brain atrophy (ventricular and sulcal atrophy) was rated as present (=1) or absent (=0; global atrophy). The visual rating scale of white matter changes included both anatomical distribution and severity of the lesions. Based on the anatomical distribution, a distinction was made between areas of periventricular hyperintensities (PVH, caps and rims), deep white matter hyperintensities (DWMH, including frontal, parieto-occipital and temporal hyperintensities), basal ganglia hyperintensities (BGH), and infratentorial hyperintensities (ITH). Large vessel cortical infarcts in the anterior, posterior and medial cerebral artery were also evaluated. Exclusion criteria were presence of mass lesions and lobar hemorrhages [23]. Average disease duration (from symptom onset) was 27 (range 696) months. Descriptive characteristics and MRI evaluations of the subjects are listed in Table 1. Biochemical and Molecular Investigations Serum from fasting blood was collected in the morning and rapidly stored at -80°C. Serum copper concentrations were measured both following the method of Abe [24] and by utilizing an A Aanalyst 300 Perkin Elmer atomic absorption spectrophotometer equipped with a graphite furnace with platform HGA 800. For each serum copper and ceruloplasmin pair we computed the amount of copper bound to ceruloplasmin (CB) and the amount of copper bound to lowmolecular-weight compounds (‘free’ copper) as follows: CB = n * ceruloplasmin (mg/L); n=0.0472 (mol/mg); free copper = absolute serum copper – CB [25]. We also computed the ratio between ceruloplasmin and transferrin serum concentrations (Cp/Tf) reflecting the activity of ceruloplasmintransferrin antioxidant system [3]. Hydro-peroxide content was assessed by d-ROMs test (Diacron, Italy) and expressed in arbitrary units (U.CARR), 1 U.CARR corresponding to 0.08 mg/100 ml of hydrogen peroxide. Normal range was between 230 and 310 U.CARR [26]. TAS was assayed by the TAS kit (Randox Laboratories, Crumlin, UK), based on published methods [27]. The serum reference range is 1.30 – 1.77 mmol/l [27]. Iron was measured by photometric test
Metal-Score as a Potential Non-Invasive Diagnostic Test
Current Alzheimer Research, 2013, Vol. 10, No. 2
using Ferene in the following way: iron bound to transferrin is released in an acidic medium as ferric iron and is then reduced to ferrous iron in the presence of ascorbic acid; ferrous iron forms a blue complex with Ferene [28]. Transferrin [29] and ceruloplasmin [30] levels were measured by immunoturbidimetric assays (Horiba ABX, Montpellier, France): serum was mixed with the purified immunoglobulin fraction of respectively a rabbit anti-human Tf antibody solution and a rabbit anti-human ceruloplasmin antiserum, containing 15 mM NaN3 as stabiliser. The resulting immune complexes are measured by turbidimetry. More details on these methods can be found in Hussain and collaborators [31]. Transferrin saturation (%Tf-sat) was calculated by dividing serum iron by the total iron-binding capacity (TBC= transferrin in mg/dL *1.25) multiplied by 100. All biochemical measures were automated on a Hitachi 912 analyser (Roche Diagnostics) and performed in duplicate. APOE genotyping was performed according to established methods [32]. For details on methods for measuring biological variables of metals and oxidative stress, see the work of Squitti and colleagues [33].
Table 1.
Statistical Analyses AD patients, VaD patients and healthy controls were described in terms of their main demographic, genetic and clinical characteristics (Table 1). The effects of both age and sex were considered in all statistical analyses. ANOVA was followed by pairwise comparisons with Bonferroni adjustment for multiple comparisons. A Multinomial (more than two groups in the dependent categorical variable) Multiple (more than one independent covariates/factors) Logistic Regression model was applied, considering the AD condition as the reference category. In particular, we calculated Relative Risk Ratios (RRR) and corresponding 95% Confidential Intervals (CI) to assess the effects of the biological variable alterations on the probability of developing AD. On the basis of a linear discriminant analysis, we defined an M-score (where ‘M’ stands for metal) as the linear combination of all the above defined metal-related biological variables. Such combination was the function able to maximize the discrimination between AD
Descriptive Characteristics and MRI Evaluations of the Subjects Participating in the Study. AD patients
VaD patients
Controls
Age
Mean (SD)
75.14 (8.69)
76.27 (8.04)
70.29 (8.98)
Sex
% female
77%
53%
48%
Median
6
8
12
Min
1
3
5
Max
18
17
17
MMSE
Mean (SD)
18.98 (5.18)
21.49 (4.49)
28.43 (1.30)
APOE
% 4 carriers
37%
29%
9%
Median
0
4
0
Min
0
0
0
Max
12
12
3
Median
0.5
6.5
0
Min
0
0
0
Max
36
36
12
Median
0
2
0
Min
0
0
0
Max
13
36
6
Median
0
0
0
Min
0
0
0
Max
3
7
2
Median
1
0
0
Min
0
0
0
Max
4
4
1
%
16%
21%
4%
Education (years)
PVH
DWMH
BGH
ITH
MTA
Global atrophy
193
194 Current Alzheimer Research, 2013, Vol. 10, No. 2
Squitti et al.
-2log likelihoods (age p=0.006; sex p=0.022; free copper p
