- Email: [email protected]
POTENTIAL NON-CLASSICAL BIOMARKERS IN THE CEREBROSPINAL FLUID OF SPORADIC ALZHEIMER’S DISEASE
P702
Poster Presentations: Monday, July 17, 2017
Abhinav Saxena1, Wessam Sourour1, Bishoy Elgallab1, Vivek Nuguri1, Ian V. J. Murray1,2, 1St George’s University School of Medicine, True Blue, Grenada; 2The University of Texas at San Antonio, San Antonio, TX, USA. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is currently incurable and
the majority of investigational drugs have failed clinical trials. Thus, there is need to identify novel AD therapeutic targets. Metabolomic analysis of AD CSF has quantified several metabolites. We used meta-analysis to identify the metabolites sginificanlty altered in AD. These metabolites were then correlated to novel or underrepresented AD mechanisms in the literature. Methods: Pubmed and Google Scholar were searched for published English articles, without date restrictions, using the keywords “AD”, “CSF”, and “human” plus 41 markers for cardiovascular, metabolic, glial, neuronal, and repair functions. Inclusion criteria were: CSF biomarkers measured in both AD and control groups, with means, standard errors/deviations, and subject numbers reported. Data was extracted by 13 authors and checked by two for accuracy. Data were transformed to log ratio of the means (AD/Control) and analyzed by random effects model in meta-analysis software (Cochrane Review Manager). Results: Of 4,309 publications identified, after exclusion and removal of duplicates, 92 articles were included comprising of 3,492 AD patients and 3,622 control subjects. Markers for neuronal cardiovascular, and glial functions, metabolism, and repair were significantly altered in AD are listed below. Neuronal markers significantly changed in AD/Controls: acetylcholine (average ratio 0.36, 95% CI 0.24-0.53, p<0.00001), GABA (0.68, 0.54-0.85, p<0.0008), and taurine (0.88, 0.77-0.99, p¼0.04). Cardiovascular markers: homocysteine (1.19, 1.11-1.28, p< 0.00001), folate/vitamin B9 (0.82, 0.68-0.99, p¼0.03), s-adenylmethionine (0.62, 0.40-0.98, p¼0.04), cobalamin/B12 (0.90, 0.81-1.0, p¼0.04), and 5-methyltetrahydrofolate (0.91, 0.82-1.00, p¼0.05). Glial marker: YKL-40 (1.27, 1.22-1.31, p< 0.00001). Metabolic markers: pyruvate (0.48, 0.24-0.94, p¼0.03), glutathione (1.11, 1.01-1.21, p¼0.03), and lactate (1.17, 0.98-1.39, p¼0.09). Markers related to neuronal or wound repair: 24(s)hydroxycholesterol (1.27, 1.03-1.57, p¼0.02), and serum amyloid P (SAP: 1.3, 1.01-1.67, p¼0.05). Conclusions: Meta-analysis of markers associated with AD identified well known pathways (cholinergic, homocysteine, vitamin B), as well as under-appreciated mechanisms. Examples include a hypoxic-induced response in anaerobic glycolysis (lactate, pyruvate), reduced neuronal inhibition (GABA, taurine decreases), and wound/vascular/glial repair mechanisms (SAP, 24(s)hydroxycholesterol, YKL-40). This meta-analysis of metabolomic data holds promise for in silico evaluation and generation of novel hypotheses in the AD field.
P2-238
ANALYTICAL PERFORMANCE CHARACTERISTICS OF A PROTOTYPE IMMUNOASSAY FOR QUANTIFICATION OF ALPHA-SYNUCLEIN IN HUMAN CEREBROSPINAL FLUID
Emeric Chassaing1, Philippe Vergnaud1, Tanja Schubert1, Hugo Marcel Vanderstichele2, Erik Stoops2, Melanie Bodnar-Wachtel1, 1 Bioclinica Lab, Lyon, France; 2ADx NeuroSciences, Gent, Belgium. Contact e-mail: [email protected] Background: a-Synuclein
is a pre-synaptic protein whose abnormal aggregation is the main characteristic of a sub-group
of neurodegenerative disorders called a-synucleinopathies, including but not limited to Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). CFS a-Synuclein levels reflect the presence of Lewy bodies in the brain and have been shown to be decreased in PD and DLB patients as compared to healthy aging controls. Robust assays with good lot-to-lot consistency are required for integration into clinical trials. This study documents the analytical performance characteristics of a novel colorimetric ELISA targeting a-Synuclein in biological fluids, with a focus on cerebrospinal fluid (CSF). Methods: a-Synuclein concentrations have been determined using a colorimetric ELISA designed and developed by ADx NeuroSciences using two monoclonal antibodies. The assay was qualified for CSF, but can be modified for application for EDTA-plasma samples. After having performed familiarization runs and some adaptation of the test procedures to the needs of a service provider, the assay was challenged internally for its analytical performance characteristics such as precision, parallelism, spike-recovery, working range, sample stability, and interference using commercially available CSF samples (n¼40). Results: a-Synuclein is measured in undiluted CSF and 10-50-fold diluted EDTA-plasma using a 3-hour incubation period for the samples. The new assay format specifically quantifies a-Synuclein in the absence of matrix interference as verified after dilution in a-Synuclein low CSF. Spike/recovery testing revealed good recovery rates (93 % 6 7 %). Our internal precision experiments with CSF resulted in an intra- and inter-assay variability (% CV) below 12 % and 11 %, respectively. The working range is confirmed between 90 pg/mL (Lower Limit of Quantitation) to 6142 pg/mL (Upper Limit of Quantitation). The stability of CSF a-Synuclein was assessed with respect to freeze/thawing and short-term stability at room temperature. No major difference with the reference condition was observed. Conclusions: The newly developed a-Synuclein colorimetric ELISA assay meets all of our internal acceptance criteria for clinical trial sample testing and represents an additional tool for patient enrichment in clinical trials or treatment follow-up.
P2-239
POTENTIAL NON-CLASSICAL BIOMARKERS IN THE CEREBROSPINAL FLUID OF SPORADIC ALZHEIMER’S DISEASE
Fatima Mesa-Herrera1, Raquel Marin1, David Quinto-Alemany2, Jose Rojo3, Eduardo Torrealba4, Luis Galindo1, Mario Diaz5, 1University of La Laguna, La Laguna, Tenerife, Spain; 2Univerity of La Laguna, La Laguna, Tenerife, Spain; 3University Hospital of Canarias, La Laguna, Tenerife, Spain; 4Dr Negrın Hospital, Las Palmas de Gran Canaria, Spain; 5 University of La Laguna, Santa Cruz de Tenerife, Spain. Contact e-mail: [email protected] Background: Sporadic Alzheimer disease (AD) is the most common form of AD, yet its causes are currently unknown. Several studies have indicated that abnormal levels of electrolytes and transition metals may play a role in the pathogenesis of AD. It has been shown that altered homeostasis of brain copper, zinc, and particularly, iron, can generate oxidative stress, a process that has been linked to the pathogenesis of sporadic AD. Methods: We have obtained samples of cerebrospinal fluids (CSF) from well-defined AD patients (n¼8) and control patients (n¼7). Atomic elements (Na, K, Ca, Mg, Cu, Fe, Mn, Zn and
Poster Presentations: Monday, July 17, 2017
Al) were measured by flame or graphite furnace atomic absorption spectrometry. SOD activity was measured spectrophotometrically. Results: We have observed that, compared to controls, K and Fe levels were significantly increased in the CSF of AD individuals, while the concentration of Zn and Al were decreased. Further, we observed that total SOD activity was elevated CSF from AD individuals. Conclusions: We have demonstrated the existence of disturbances in the concentrations of K, Fe and Zn, total protein and SOD activity in AD subjects, compared to controls. Therefore ionic markers and their relationship with classical and oxidative stress biomarkers could provide new diagnostic tools for AD.
P2-240
SALIVARY BIOMARKERS FOR THE DETECTION OF ALZHEIMER’S DISEASE
Gustavo AA. Santos1, Niraldo Paulino2, Paulo Celso Pardi3, 1 SENAC, Sao Paulo, Brazil; 2Medical Lex, Florianopolis, Brazil; 3 FAMA College, Mineiros, Brazil. Contact e-mail: gusfarma@ hotmail.com Background: The clinical features of Alzheimer’s disease coincide with a number of other dementia pathologies, and conclusive diagnosis is only achieved at autopsy. In the case of AD, early detection of disease-related symptoms is critically important for the design of the treatment; in addition, saliva is easy to obtain, simple transport and can be a repeated test for days at a time, allowing efficient monitoring of the evolution of the disease. Stages of the disease. Methods: 120 elderly people were invited to participate in this experiment: Group without AD: 60 cognitively healthy individuals with no diagnosis of DA, aged 60 or over. Group with AD:60 patients with probable AD diagnosis. All the research volunteers, healthy or with a probable diagnosis
P703
of AD, were Selected from the inclusion and exclusion criteria, and in a randomized fashion. After being withdrawn from the freezing temperature, the saliva was centrifuged for five minutes at 3,000 rpm. At this stage, called the sample processing, SalivetteÒ cotton was discarded, and the saliva separated into Aliquots of 200 mL in EppendorfÒ tubes. For the quantification of Ab40 by the ELISA method in the saliva of the research participants, we used the Amyloid beta 40 ELISA kit human. For the quantification of t-Tau by the saliva ELISA method, the TAU (total) human kit ELISA. Results: All data from this experiment were statistically treated with the aid of Software GRAPH PAD PRISM 5.0. We used the non-parametric Mann-Whitney test to compare the different groups (concentrations). We can observe that the expression of t-Tau in patients with AD is significantly higher when compared to patients without AD, confirming the hypothesis that T-Tau can be used as a biomarker for AD. Through the concentration of Ab42 in the saliva of patients without AD and AD, it is possible to observe that the variation, although small, points to a higher concentration of Ab42 in patients with AD. Conclusions: Experimental results and their validation by the literature search demonstrated that the reduction of t-Tau expression and the increase of salivary p-Tau occurred in the same group of patients with probable AD, demonstrating the feasibility of using these substances as biomarkers.
P2-241
CSF NEUROGRANIN IS INCREASED IN FAMILIAL ALZHEIMER’S DISEASE
Henrietta Wellington1, Philip SJ. Weston2, Teresa Poole3, Ross W. Paterson4, Ulrika Tornqvist5, Erik Portelius6,7,8, Nick C. Fox4, Kaj Blennow6,8, Henrik Zetterberg1,7,9,10, Jonathan M. Schott4, 1University College London, London, United Kingdom; 2UCL Institute of Neurology, London, United Kingdom; 3 London School of Hygiene and Tropical Medicine, London, United Kingdom; 4Dementia Research Centre, Institute of Neurology, University College London, London, United Kingdom; 5 University of Gothenberg, Gothenberg, Sweden; 6Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg, M€olndal, Sweden; 7Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, M€olndal, Sweden; 8Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, M€olndal, Sweden; 9Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden; 10University of Gothenburg, Gothenburg, Sweden. Contact e-mail: [email protected] Background: CSF neurogranin (Ng), a neuron specific post-synap-
tic protein, is a promising biomarker for synapse degeneration in pre-dementia (mild cognitive impairment) as well as dementia stages of Alzheimer’s disease (AD). CSF Ng concentration is higher in patients with AD than in other neurodegenerative diseases or controls, with promising specificity and clinical utility. To date, it is unknown how early CSF Ng concentrations increase in the AD trajectory. Familial AD, where individuals at risk of dementia can be reliably determined provides an ideal paradigm for exploring biomarkers at very early disease stages. We therefore performed a pilot study exploring CSF Ng levels in both presymptomatic and symptomatic mutation carriers. Methods: We measured CSF Ng by ELISA in 23 subjects, including controls (n¼8), presymptomatic (n¼8) and symptomatic carriers (n¼7).
Poster Presentations: Monday, July 17, 2017
Abhinav Saxena1, Wessam Sourour1, Bishoy Elgallab1, Vivek Nuguri1, Ian V. J. Murray1,2, 1St George’s University School of Medicine, True Blue, Grenada; 2The University of Texas at San Antonio, San Antonio, TX, USA. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is currently incurable and
the majority of investigational drugs have failed clinical trials. Thus, there is need to identify novel AD therapeutic targets. Metabolomic analysis of AD CSF has quantified several metabolites. We used meta-analysis to identify the metabolites sginificanlty altered in AD. These metabolites were then correlated to novel or underrepresented AD mechanisms in the literature. Methods: Pubmed and Google Scholar were searched for published English articles, without date restrictions, using the keywords “AD”, “CSF”, and “human” plus 41 markers for cardiovascular, metabolic, glial, neuronal, and repair functions. Inclusion criteria were: CSF biomarkers measured in both AD and control groups, with means, standard errors/deviations, and subject numbers reported. Data was extracted by 13 authors and checked by two for accuracy. Data were transformed to log ratio of the means (AD/Control) and analyzed by random effects model in meta-analysis software (Cochrane Review Manager). Results: Of 4,309 publications identified, after exclusion and removal of duplicates, 92 articles were included comprising of 3,492 AD patients and 3,622 control subjects. Markers for neuronal cardiovascular, and glial functions, metabolism, and repair were significantly altered in AD are listed below. Neuronal markers significantly changed in AD/Controls: acetylcholine (average ratio 0.36, 95% CI 0.24-0.53, p<0.00001), GABA (0.68, 0.54-0.85, p<0.0008), and taurine (0.88, 0.77-0.99, p¼0.04). Cardiovascular markers: homocysteine (1.19, 1.11-1.28, p< 0.00001), folate/vitamin B9 (0.82, 0.68-0.99, p¼0.03), s-adenylmethionine (0.62, 0.40-0.98, p¼0.04), cobalamin/B12 (0.90, 0.81-1.0, p¼0.04), and 5-methyltetrahydrofolate (0.91, 0.82-1.00, p¼0.05). Glial marker: YKL-40 (1.27, 1.22-1.31, p< 0.00001). Metabolic markers: pyruvate (0.48, 0.24-0.94, p¼0.03), glutathione (1.11, 1.01-1.21, p¼0.03), and lactate (1.17, 0.98-1.39, p¼0.09). Markers related to neuronal or wound repair: 24(s)hydroxycholesterol (1.27, 1.03-1.57, p¼0.02), and serum amyloid P (SAP: 1.3, 1.01-1.67, p¼0.05). Conclusions: Meta-analysis of markers associated with AD identified well known pathways (cholinergic, homocysteine, vitamin B), as well as under-appreciated mechanisms. Examples include a hypoxic-induced response in anaerobic glycolysis (lactate, pyruvate), reduced neuronal inhibition (GABA, taurine decreases), and wound/vascular/glial repair mechanisms (SAP, 24(s)hydroxycholesterol, YKL-40). This meta-analysis of metabolomic data holds promise for in silico evaluation and generation of novel hypotheses in the AD field.
P2-238
ANALYTICAL PERFORMANCE CHARACTERISTICS OF A PROTOTYPE IMMUNOASSAY FOR QUANTIFICATION OF ALPHA-SYNUCLEIN IN HUMAN CEREBROSPINAL FLUID
Emeric Chassaing1, Philippe Vergnaud1, Tanja Schubert1, Hugo Marcel Vanderstichele2, Erik Stoops2, Melanie Bodnar-Wachtel1, 1 Bioclinica Lab, Lyon, France; 2ADx NeuroSciences, Gent, Belgium. Contact e-mail: [email protected] Background: a-Synuclein
is a pre-synaptic protein whose abnormal aggregation is the main characteristic of a sub-group
of neurodegenerative disorders called a-synucleinopathies, including but not limited to Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). CFS a-Synuclein levels reflect the presence of Lewy bodies in the brain and have been shown to be decreased in PD and DLB patients as compared to healthy aging controls. Robust assays with good lot-to-lot consistency are required for integration into clinical trials. This study documents the analytical performance characteristics of a novel colorimetric ELISA targeting a-Synuclein in biological fluids, with a focus on cerebrospinal fluid (CSF). Methods: a-Synuclein concentrations have been determined using a colorimetric ELISA designed and developed by ADx NeuroSciences using two monoclonal antibodies. The assay was qualified for CSF, but can be modified for application for EDTA-plasma samples. After having performed familiarization runs and some adaptation of the test procedures to the needs of a service provider, the assay was challenged internally for its analytical performance characteristics such as precision, parallelism, spike-recovery, working range, sample stability, and interference using commercially available CSF samples (n¼40). Results: a-Synuclein is measured in undiluted CSF and 10-50-fold diluted EDTA-plasma using a 3-hour incubation period for the samples. The new assay format specifically quantifies a-Synuclein in the absence of matrix interference as verified after dilution in a-Synuclein low CSF. Spike/recovery testing revealed good recovery rates (93 % 6 7 %). Our internal precision experiments with CSF resulted in an intra- and inter-assay variability (% CV) below 12 % and 11 %, respectively. The working range is confirmed between 90 pg/mL (Lower Limit of Quantitation) to 6142 pg/mL (Upper Limit of Quantitation). The stability of CSF a-Synuclein was assessed with respect to freeze/thawing and short-term stability at room temperature. No major difference with the reference condition was observed. Conclusions: The newly developed a-Synuclein colorimetric ELISA assay meets all of our internal acceptance criteria for clinical trial sample testing and represents an additional tool for patient enrichment in clinical trials or treatment follow-up.
P2-239
POTENTIAL NON-CLASSICAL BIOMARKERS IN THE CEREBROSPINAL FLUID OF SPORADIC ALZHEIMER’S DISEASE
Fatima Mesa-Herrera1, Raquel Marin1, David Quinto-Alemany2, Jose Rojo3, Eduardo Torrealba4, Luis Galindo1, Mario Diaz5, 1University of La Laguna, La Laguna, Tenerife, Spain; 2Univerity of La Laguna, La Laguna, Tenerife, Spain; 3University Hospital of Canarias, La Laguna, Tenerife, Spain; 4Dr Negrın Hospital, Las Palmas de Gran Canaria, Spain; 5 University of La Laguna, Santa Cruz de Tenerife, Spain. Contact e-mail: [email protected] Background: Sporadic Alzheimer disease (AD) is the most common form of AD, yet its causes are currently unknown. Several studies have indicated that abnormal levels of electrolytes and transition metals may play a role in the pathogenesis of AD. It has been shown that altered homeostasis of brain copper, zinc, and particularly, iron, can generate oxidative stress, a process that has been linked to the pathogenesis of sporadic AD. Methods: We have obtained samples of cerebrospinal fluids (CSF) from well-defined AD patients (n¼8) and control patients (n¼7). Atomic elements (Na, K, Ca, Mg, Cu, Fe, Mn, Zn and
Poster Presentations: Monday, July 17, 2017
Al) were measured by flame or graphite furnace atomic absorption spectrometry. SOD activity was measured spectrophotometrically. Results: We have observed that, compared to controls, K and Fe levels were significantly increased in the CSF of AD individuals, while the concentration of Zn and Al were decreased. Further, we observed that total SOD activity was elevated CSF from AD individuals. Conclusions: We have demonstrated the existence of disturbances in the concentrations of K, Fe and Zn, total protein and SOD activity in AD subjects, compared to controls. Therefore ionic markers and their relationship with classical and oxidative stress biomarkers could provide new diagnostic tools for AD.
P2-240
SALIVARY BIOMARKERS FOR THE DETECTION OF ALZHEIMER’S DISEASE
Gustavo AA. Santos1, Niraldo Paulino2, Paulo Celso Pardi3, 1 SENAC, Sao Paulo, Brazil; 2Medical Lex, Florianopolis, Brazil; 3 FAMA College, Mineiros, Brazil. Contact e-mail: gusfarma@ hotmail.com Background: The clinical features of Alzheimer’s disease coincide with a number of other dementia pathologies, and conclusive diagnosis is only achieved at autopsy. In the case of AD, early detection of disease-related symptoms is critically important for the design of the treatment; in addition, saliva is easy to obtain, simple transport and can be a repeated test for days at a time, allowing efficient monitoring of the evolution of the disease. Stages of the disease. Methods: 120 elderly people were invited to participate in this experiment: Group without AD: 60 cognitively healthy individuals with no diagnosis of DA, aged 60 or over. Group with AD:60 patients with probable AD diagnosis. All the research volunteers, healthy or with a probable diagnosis
P703
of AD, were Selected from the inclusion and exclusion criteria, and in a randomized fashion. After being withdrawn from the freezing temperature, the saliva was centrifuged for five minutes at 3,000 rpm. At this stage, called the sample processing, SalivetteÒ cotton was discarded, and the saliva separated into Aliquots of 200 mL in EppendorfÒ tubes. For the quantification of Ab40 by the ELISA method in the saliva of the research participants, we used the Amyloid beta 40 ELISA kit human. For the quantification of t-Tau by the saliva ELISA method, the TAU (total) human kit ELISA. Results: All data from this experiment were statistically treated with the aid of Software GRAPH PAD PRISM 5.0. We used the non-parametric Mann-Whitney test to compare the different groups (concentrations). We can observe that the expression of t-Tau in patients with AD is significantly higher when compared to patients without AD, confirming the hypothesis that T-Tau can be used as a biomarker for AD. Through the concentration of Ab42 in the saliva of patients without AD and AD, it is possible to observe that the variation, although small, points to a higher concentration of Ab42 in patients with AD. Conclusions: Experimental results and their validation by the literature search demonstrated that the reduction of t-Tau expression and the increase of salivary p-Tau occurred in the same group of patients with probable AD, demonstrating the feasibility of using these substances as biomarkers.
P2-241
CSF NEUROGRANIN IS INCREASED IN FAMILIAL ALZHEIMER’S DISEASE
Henrietta Wellington1, Philip SJ. Weston2, Teresa Poole3, Ross W. Paterson4, Ulrika Tornqvist5, Erik Portelius6,7,8, Nick C. Fox4, Kaj Blennow6,8, Henrik Zetterberg1,7,9,10, Jonathan M. Schott4, 1University College London, London, United Kingdom; 2UCL Institute of Neurology, London, United Kingdom; 3 London School of Hygiene and Tropical Medicine, London, United Kingdom; 4Dementia Research Centre, Institute of Neurology, University College London, London, United Kingdom; 5 University of Gothenberg, Gothenberg, Sweden; 6Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg, M€olndal, Sweden; 7Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, M€olndal, Sweden; 8Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, M€olndal, Sweden; 9Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden; 10University of Gothenburg, Gothenburg, Sweden. Contact e-mail: [email protected] Background: CSF neurogranin (Ng), a neuron specific post-synap-
tic protein, is a promising biomarker for synapse degeneration in pre-dementia (mild cognitive impairment) as well as dementia stages of Alzheimer’s disease (AD). CSF Ng concentration is higher in patients with AD than in other neurodegenerative diseases or controls, with promising specificity and clinical utility. To date, it is unknown how early CSF Ng concentrations increase in the AD trajectory. Familial AD, where individuals at risk of dementia can be reliably determined provides an ideal paradigm for exploring biomarkers at very early disease stages. We therefore performed a pilot study exploring CSF Ng levels in both presymptomatic and symptomatic mutation carriers. Methods: We measured CSF Ng by ELISA in 23 subjects, including controls (n¼8), presymptomatic (n¼8) and symptomatic carriers (n¼7).