Preanalytical sample handling recommendations for Alzheimer's disease plasma biomarkers.

INTRODUCTION: We examined the influence of common preanalytical factors on the measurement of Alzheimer's disease-specific biomarkers in human plasma. METHODS: Amyloid ß peptides (Aß[1-40], Aß[1-42]) and total Tau plasma concentrations were quantified using fully automated Roche Elecsys assays. RESULTS: Aß(1-40), Aß(1-42), and total Tau plasma concentrations were not affected by up to three freeze/thaw cycles, up to five tube transfers, the collection tube material, or the size; circadian rhythm had a minor effect. All three biomarkers were influenced by the anticoagulant used, particularly total Tau. Aß concentrations began decreasing 1 hour after blood draw/before centrifugation and decreased by up to 5% and 10% at 2 and 6 hours, respectively. For separated plasma, time to measurement influenced Aß levels by up to 7% after 6 hours and 10% after 24 hours. DISCUSSION: Our findings provide guidance for standardizing blood sample collection, handling, and storage to ensure reliable analysis of Alzheimer's disease plasma biomarkers in routine practice and clinical trials.

Human cerebrospinal fluid 6E10-immunoreactive protein species contain amyloid precursor protein fragments.

In a previous study, we reported that levels of two types of protein species-a type of ~55-kDa species and a type of ~15-kDa species-are elevated in the lumbar cerebrospinal fluid (CSF) of cognitively intact elderly individuals who are at risk for Alzheimer's disease (AD). These species are immunoreactive to the monoclonal antibody 6E10, which is directed against amino acids 6-10 of amyloid-ß (Aß), and their levels correlate with levels of total tau and tau phosphorylated at Thr181. In this study, we investigated the molecular composition of these AD-related proteins using immunoprecipitation (IP)/Western blotting coupled with IP/mass spectrometry. We show that canonical Aß1-40/42 peptides, together with amyloid-ß precursor protein (APP) fragments located N-terminally of Aß, are present in the ~55-kDa, 6E10-immunoreactive species. We demonstrate that APP fragments located N-terminally of Aß, plus the N-terminal region of Aß, are present in the ~15-kDa, 6E10-immunoreactive species. These findings add to the catalog of AD-related Aß/APP species found in CSF and should motivate further study to determine whether these species may serve as biomarkers of disease progression.

Accuracy of cerebrospinal fluid Aß1-42 measurements: evaluation of pre-analytical factors using a novel Elecsys immunosassay.

BACKGROUND: A decreased level of Aß1-42 in cerebrospinal fluid (CSF) is characteristic of Alzheimer disease and often used to support clinical diagnosis. The measured concentration of CSF Aß1-42, however, depends strongly on several pre-analytical and analytical "confounding" factors such as sample collection, material of testing tube, CSF handling and storage procedures (e.g. transfer to new tubes after centrifugation, freeze-thaw effects). As a consequence, substantial variations in the measured levels of this biomarker are observed even for the same sample. This study investigates whether the accuracy of quantitative analysis of CSF Aß1-42 can be improved by pre-analytical treatment of CSF with agents that could potentially reduce a freeze-thaw and adhesion-related depletion of Aß1-42 from CSF, including modulators of Aß aggregation and cryoprotecting or anti-adhesion agents. METHODS: The concentration of CSF Aß1-42 was assessed with a novel Elecsys immunoassay developed for quantification of Aß1-42 in human CSF. RESULTS: Low-molecular weight Aß oligomerization inhibitors, ß-sheet breaker peptides, or the mid domain 4G8 antibody do not improve the stability of CSF Aß1-42 during a repeated freeze-thaw treatment. Cryoprotecting agents reduce a freeze-thaw dependent loss of Aß1-42 only when spiked to CSF to final concentration of 300 mM or higher. Adhesion of Aß1-42 can be prevented by pre-treating CSF with Tween or by using tubes with a siliconized surface. CONCLUSIONS: Between-center variability in measured level of CSF Aß1-42 can be reduced only by standardized CSF collection into one specific tube that, without centrifugation, transfer or other types of pre-analytical processing, is directly analyzed after sample collection.

Cu(II) complex formation by ACES buffer.

ACES (N-(2-Acetamido)-2-aminoethanesulfonic acid), a popular Good's buffer, binds Cu(II) ions with a moderate affinity. Although this interaction was the subject of previous studies, no consensus in the literature was found. We used potentiometry to establish binding constants, and controlled the potentiometric model selection and binding constant calculations by UV-vis spectroscopy. As a result, we obtained a consistent set of complex stoichiometries and binding constants in this system, which contains Cu(2+), CuL(+), CuL2, CuH-1L2(-1) and CuH(-)2L2(-2) complexes. The negative indexes at H atoms in these formulae denote the Cu(II) assisted deprotonation of the amide nitrogen present in the ACES molecule. The affinity of ACES for Cu(II) strongly depends on the concentration and ACES:Cu(II) ratio, reaching submicromolar apparent affinities at ratios higher than 100. These results will enable more accurate determinations of biologically relevant stability constants of Cu(II) complexes using ACES buffer.

Cu(II) affinity for the Alzheimer's peptide: tyrosine fluorescence studies revisited.

Copper(II) binding to the amyloid-ß peptide has been proposed to be a key event in the cascade leading to Alzheimer's disease. As a direct consequence, the strength of the Cu(II) to Aß interaction, that is, the Cu(II) affinity of Aß, is a very important parameter to determine. Because Aß peptide contain one Tyr fluorophore in its sequence and because Cu(II) does quench Tyr fluorescence, fluorescence measurements appear to be a straightforward way to obtain this parameter. However, this proved to be wrong, mainly because of data misinterpretation in some previous studies that leads to a conflicting situation. In the present paper, we have investigated in details a large set of fluorescence data that were analyzed with a new method taking into account the presence of two Cu(II) sites and the inner-filter effect. This leads to reinterpretation of the published data and to the determination of a unified affinity value in the 10(10) M(-1) range.

The Cu(II)/Abeta/human serum albumin model of control mechanism for copper-related amyloid neurotoxicity.

Alzheimer's disease (AD) is the most common neurodegenerative disorder in the elderly population, above 65 years of age. Multiple lines of evidence confirm the central role of 40-42 residue Abeta peptides in the pathogenesis of AD, but exact mechanisms of Abeta toxicity remain unclear. Recently, evidence has accumulated in favor of small oligomers of the Abeta42 peptide as major toxic species. Metal ions, copper(II) in particular, have been implicated in molecular mechanisms of Abeta neurotoxicity, including oxidative damage of lipid membranes. While monomeric Abeta peptides are not neurotoxic, the deep understanding of their chemical properties is prerequisite for significant progress in Alzheimer research. Monomeric Abeta40 and Abeta42 form a specific mononuclear complex with Cu(II), recruiting donor atoms within their common 16 amino acid N-terminal sequence. The formation of this complex, the exact structure of which is debated, correlates with increased Abeta toxicity. Human serum albumin (HSA) is a versatile carrier protein present, among others, in blood and cerebrospinal fluid. It binds one Cu(II) ion with a high, picomolar affinity and one Abeta molecule with a moderate, micromolar affinity. In this perspective, we present a model of interactions, which make HSA a likely guardian against Cu/Abeta toxicity in extracellular brain compartments.

A direct determination of the dissociation constant for the Cu(II) complex of amyloid beta 1-40 peptide.

Interactions of amyloid beta (Abeta) peptides with Cu(II) are believed to play a crucial role in the molecular mechanisms of neurotoxicity of Alzheimer's disease. There is, however, a serious disagreement regarding the strength of Cu(II) binding to these peptides. We used recombinant amyloid beta peptide 1-40 (Abeta40) to determine the stoichiometry and dissociation constants of Cu(II)-Abeta40 complexes using fluorescence spectroscopy. A single Cu(Abeta40) complex, characterized with the conditional dissociation constant K(d)(cond) = 57 +/- 5 nM was identified. This complex does not bind Hepes buffer molecules, as indicated by the total lack of relationship between K(d)(cond) values and Hepes concentration. The differences between this and other determinations of this constant and its relevance for the understanding of Cu(II) interaction with Abeta peptides are discussed.

The Cu(II) complex of Abeta40 peptide in ammonium acetate solutions. Evidence for ternary species formation.

Fluorimetric Cu(ii) titrations of Alzheimer's disease (AD) Abeta40 peptide at various ammonium acetate concentrations demonstrated that ternary Cu(Abeta40)L complexes are formed with buffer components L, thereby providing a novel perspective on Cu(ii)-Abeta interactions.

An integrated LC-ESI-MS platform for quantitation of serum peptide ladders. Application for colon carcinoma study.

Mounting evidence indicates that MS analysis of the human blood peptidome allows to distinguish between cancer and non-cancer samples, giving promise for a new MS-based diagnostic tool. However, several aspects of already published work have been criticized and demand for more methodical approach has been formulated. Motivated by this we undertook a systematic study of the plasma and serum peptidome using an integrated ESI-LC-MS-based platform, equipped with new data analysis tools for relative and absolute peptide quantitation. We used a high resolution LC-ESI-MS to analyze well-separated MS signals corresponding to peptides, and measured the variability of >1000 peptide signal amplitudes across a set of plasma and serum samples from healthy individuals. By spiking serum samples with known amounts of isotopically labeled versions of a selected set of peptides we measured the variability of their absolute concentration in this sample set and demonstrated a strong influence of clotting time on the concentration of these peptides in serum. Finally, we used this new LC-ESI-MS analytical platform for the differential analysis of healthy versus colon cancer serum samples and found that it was possible to distinguish the two groups with 89.8% sensitivity and 94.6% specificity.

The binding constant for amyloid Abeta40 peptide interaction with human serum albumin.

Human serum albumin (HSA) is the major carrier of Abeta peptides in blood plasma. 1:1 interaction stoichiometries were established in previous indirect antibody-based studies for both Abeta40 and Abeta42, but corresponding binding constants were not provided. In this study we applied direct titrations of HSA with Abeta40 monitored using circular dichroism spectroscopy and obtained a dissociation constant (K(d)) of 5+/-1 microM for a HSA complex with Abeta40. The interaction resulted in an increase of the alpha-helical contents in the complex, compared to its components, which is quantitatively consistent with the known ability of Abeta40 to adopt a partially alpha-helical conformation in a hydrophobic environment. The relevance of these findings for the role of HSA in Abeta physiology is discussed.

Human serum albumin coordinates Cu(II) at its N-terminal binding site with 1 pM affinity.

The conditional stability constant at pH 7.4 for Cu(II) binding at the N-terminal site (NTS) of human serum albumin (HSA) was determined directly by competitive UV-vis spectroscopy titrations using nitrilotriacetic acid (NTA) as the competitor in 100 mM NaCl and 100 mM N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid (Hepes). The log Kc (NTS) value of 12.0 +/- 0.1 was determined for HSA dissolved in 100 mM NaCl. A false log log Kc (NTS) (c) value of 11.4 +/- 0.1 was obtained in the 100 mM Hepes buffer, owing to the formation of a ternary Cu(NTA)(Hepes) complex. The impact of the picomolar affinity of HSA for Cu(II) on the availability of these ions in neurodegenerative disorders is briefly discussed.