Increased levels of plasma p3-alcα35, a major fragment of Alcadeinα by γ-secretase cleavage, in Alzheimer's disease.

p3-Alcα is a metabolic fragment of Alcadeinα (Alcα). Similar to the generation of the p3 fragment from amyloid-ß protein precursor (AßPP) processing, Alcα is cleaved by α- and γ-secretases, leading to the secretion of p3-Alcα peptides into cerebrospinal fluid (CSF). p3-Alcα is also detected in the plasma, similar to amyloid-ß (Aß), which is a metabolic fragment of AßPP cleaved by amyloidogenic ß- and γ-secretases. Because p3-Alcα is a non-aggregatable and stable peptide, unlike aggregatable Aß and metabolically labile p3 of AßPP, the changes of p3-Alcα in quality and/or quantity in CSF and plasma are expected to be a marker for assessing alteration of substrate cleavage by γ-secretase, such as Aß generation from AßPP. The present study describes a sandwich enzyme-linked immunosorbent assay for quantifying levels of p3-Alcα35, the major form of the p3-Alcα species, and examines levels of p3-Alcα35 in the plasma of three independent Japanese cohorts. In two of the three cohorts, the p3-Alcα35 levels were significantly increased with a concomitant decrease in the Mini-Mental State Examination score, or in clinically diagnosed Alzheimer's disease (AD) patients, when compared with age-matched non-demented subjects. The values were significantly lower in AD subjects who were administered donepezil, when compared to AD subjects without donepezil treatment. The increase in plasma p3-Alcα35 levels may indicate an endophenotype in subjects in whom AD is due to a progressing cognitive impairment in subjects with a γ-secretase malfunction, or a disorder of the clearance of peptides.

Simple and novel screening assay of natural antioxidants for Cu(II) ion/adrenaline-mediated oxidation of N-terminal amyloid beta by liquid chromatography/mass spectrometry.

In the present study, a novel assay for screening inhibitors of Cu(II) ion/adrenaline-mediated oxidative modification of N-terminal amyloid beta (Abeta) peptides was developed using liquid chromatography with mass spectrometry (LC/MS). The physiological condition of Cu(II) ion/adrenaline in buffer (pH 7.4) at 37 degrees C for 90 min revealed a specific modification of N-terminal Abeta peptides, such as Abeta1-6, Abeta1-40, and Abeta1-42, using trypsin digestion and LC/MS detection of the modified Abeta peptide. When this oxidative modification of the shorter N-terminal Abeta1-6 was subjected to LC/MS, single charged ions from native peptide ([M+H]+, m/z 774) were observed at m/z 729 and 685, corresponding to a decrease in mass of 45 and 89 Da, respectively, as compared with the original peptide. To determine the effect of specific antioxidants, a screening assay to find inhibitors of Cu(II) ion/adrenaline-mediated oxidation was developed based on the response ratio of m/z 685 to 774. LC/MS detection of the modified peptides allowed us to identify antioxidants that inhibit oxidative modification of Abeta1-6 model peptide. The oxidative modification of Abeta1-6 was inhibited by curcumin but not an isoflavone or catechin mixture or saponin or capsaicin, revealing a clear difference between antioxidants that inhibit oxidative modification and other antioxidants. This novel assay may allow for the identification of antioxidants that protect against oxidative modification of Abeta and other proteins related to oxidative stress by adrenaline and Cu(II) ions under normal physiologic conditions.