(a) the novel Aβ1-40 and Aβ1-42 ELISA assays characterize with very good analytical performance; (b) we reconfirm that the CSF Aβ42/40 concentration ratio shows significantly better diagnostic performance compared to the CSF Aβ1-42 concentration alone.
IntroductionSynaptic dysfunction and degeneration are central events in Alzheimer’s disease (AD) pathophysiology that are thought to occur early in disease progression. Synaptic pathology may be studied by examining protein biomarkers specific for different synaptic elements. We recently showed that the dendritic protein neurogranin (Ng), including the endogenous Ng peptide 48 to 76 (Ng48–76), is markedly increased in cerebrospinal fluid (CSF) in AD and that Ng48–76 is the dominant peptide in human brain tissue. The aim of this study was to characterize Ng in plasma and CSF using mass spectrometry and to investigate the performance of plasma Ng as an AD biomarker.MethodsPaired plasma and CSF samples from patients with AD (n = 25) and healthy controls (n = 20) were analyzed in parallel using an immunoassay developed in-house on the Meso Scale Discovery platform and hybrid immunoaffinity-mass spectrometry (HI-MS). A second plasma material from patients with AD (n = 13) and healthy controls (n = 17) was also analyzed with HI-MS. High-resolution mass spectrometry was used for identification of endogenous plasma Ng peptides.ResultsNg in human plasma is present as several endogenous peptides. Of the 16 endogenous Ng peptides identified, seven were unique for plasma and not detectable in CSF. However, Ng48–76 was not present in plasma. CSF Ng was significantly increased in AD compared with controls (P < 0.0001), whereas the plasma Ng levels were similar between the groups in both studies. Plasma and CSF Ng levels showed no correlation. CSF Ng was stable during storage at −20°C for up to 2 days, and no de novo generation of peptides were detected.ConclusionsFor the first time, to our knowledge, we have identified several endogenous Ng peptides in human plasma. In agreement with previous studies, we show that CSF Ng is significantly increased in AD as compared with healthy controls. The origin of Ng in plasma and its possible use as a biomarker need to be further investigated. The results suggest that CSF Ng, in particular Ng48–76, might reflect the neurodegenerative processes within the brain, indicating a role for Ng as a potential novel clinical biomarker for synaptic function in AD.Electronic supplementary materialThe online version of this article (doi:10.1186/s13195-015-0124-3) contains supplementary material, which is available to authorized users.
Preanalytical sample handling and storage procedures play an extremely important role in reliably measuring neurochemical dementia diagnostics (NDD) biomarkers: Aβ(1-40), Aβ(1-42), Tau, and pTau181. To test different handling and storage conditions, the following protocols were applied: (a) storage at room temperature for one week, (b) deep-freezing and thawing up to three cycles, (c) deep-freezing, thawing and keeping under +4°C for two days before the analysis, and (d) long-term stability of a deeply frozen sample. Between the first and the seventh day of the storage at room temperature, the percentage of the concentrations (compared to the starting concentrations) fluctuated: 104.3-105.3, 97.6-93.2, 100.6-96.8, and 97.9-90.2 for Aβ(1-40), Aβ(1-42), Tau, and pTau181, respectively. Re-freezing cycles resulted in the percentage fluctuations of the concentrations: 101.1-105.5, 95.4-99.7, 98.3-100.0, and 100.5-101.4 for Aβ(1-40), Aβ(1-42), Tau, and pTau181, respectively. Keeping previously frozen/thawed samples under +4°C for two days resulted in the percentage differences of the concentrations: +15.9, +2.2, -1.1, and -0.1 for Aβ(1-40), Aβ(1-42), Tau, and pTau181, respectively. During long-term stability, the coefficients of linear correlation (R(2)) were: Aβ(1-40), 0.007; Aβ(1-42), 0.02; Tau, 0.011; and pTau181, 0.02, and the corresponding inter-assay coefficients of variation: 13.9%, 13.9%, 11.0%, and 10.7% for Aβ(1-40), Aβ(1-42), Tau, and pTau181, respectively. We conclude that the NDD biomarkers are relatively stable when the cerebrospinal fluid sample is kept at room temperature for about four days; one or two thawing/refreezing cycles do not profoundly affect the biomarkers concentrations, however three cycles result in increased unsystematic variation. The four biomarkers seem to be stable in a sample stored deeply frozen for more than two years.
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