Ed with the progressive accumulation of amyloid b-peptide (Ab) in the human brain, a pathogenic role of Ab in the brain has been widely recognized [1,2]. Over the last decade or so, the presence of Amyloid b-peptide (Ab) in peripheral blood plasma has received increasing attention [3?], and plasma Ab is hypothesized to readily contact red blood cells (RBCs) and impair the capacity of RBCs in circulating human blood [7,8]. Our group and other researchers have investigated the hypothesis, and found that Ab induces oxidative injury to RBC by binding to them, and causing accumulation of phospholipid hydroperoxides (PLOOH), a specific marker for RBC membrane oxidative injury [9,10]. Ab also induces the binding of erythrocytes to endothelial cells and decreases endothelial viability, perhaps by the generation of oxidative and inflammatory stress [11]. These studies [9?1] provide a possibility that Ab plays a key role in blood and oxidatively impairs RBC Bexagliflozin function (e.g., oxygen delivery to the brain), thereby potentially facilitating AD. However, to the best of our knowledge, no extensive study of the presence and distribution of Ab in human RBC has been undertaken. The aim of this study was to ascertain the distribution of Ab in the RBCs of young and Fexinidazole senior subjects by applying a commercial ELISA assay. The RBC Ab concentrations were comparedbetween young and senior subjects and also compared to plasma Ab levels. In addition, we previously conducted a randomized, double-blind, placebo-controlled human study to evaluate whether nutritional supplementation with the antioxidant astaxanthin (a polar carotenoid) affected 1480666 RBC PLOOH [12]. Thus, RBCs that had been obtained from the human study [12] were subjected to Ab determination in order to evaluate the relationship between RBC Ab and the antioxidant/oxidant profile.Materials and Methods Ethics StatementThe study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Anti-Aging Science (Tokyo, Japan; ethics No. I030807). All of the subjects provided written informed consent to the experimental protocol before participating in the study.Blood Samples from Young and Senior VolunteersTwenty-four young healthy human volunteers [12 men and 12 women, between 22 and 29 years of age (mean 6 SE, 24.260.6)] and 38 senior healthy volunteers [20 men and 18 women, between 48 and 69 years of age (mean 6 SE, 56.261.0)] participated in this study. Blood was collected into a tube containing EDTA-2Na as an anticoagulant and was subjected to centrifugation at 1,000 g for ten min at 1407003 4uC. After the plasma and buffy coat were removed,Amyloid b Determination in Human ErythrocytesRBCs were washed three times with phosphate buffered saline (PBS, pH 7.4) to prepare packed cells.Measurement of Ab40 and Ab42 in RBCs and PlasmaFor determination of Ab40 and Ab42 in RBCs, human b Amyloid (1?0) ELISA kits (WAKO, Osaka, Japan) and human b Amyloid (1?2) ELISA kits (WAKO) were used, respectively. These kits are commercially available and used worldwide. We tested conditions for measurement of RBC Ab, and the optimized protocol is as follows. Packed cells (200 mL) were mixed with 200 mL of water and one mL of 70 formic acid. A 40 mL aliquot was collected and mixed with 760 mL of 1 mol/L Tris-HCl with protease inhibitors, and the mixture was diluted two-fold with the standard diluent present in each Ab40 and Ab42 ELISA kit. A 100 mL aliquot was subjected to either the Ab40 or the Ab4.Ed with the progressive accumulation of amyloid b-peptide (Ab) in the human brain, a pathogenic role of Ab in the brain has been widely recognized [1,2]. Over the last decade or so, the presence of Amyloid b-peptide (Ab) in peripheral blood plasma has received increasing attention [3?], and plasma Ab is hypothesized to readily contact red blood cells (RBCs) and impair the capacity of RBCs in circulating human blood [7,8]. Our group and other researchers have investigated the hypothesis, and found that Ab induces oxidative injury to RBC by binding to them, and causing accumulation of phospholipid hydroperoxides (PLOOH), a specific marker for RBC membrane oxidative injury [9,10]. Ab also induces the binding of erythrocytes to endothelial cells and decreases endothelial viability, perhaps by the generation of oxidative and inflammatory stress [11]. These studies [9?1] provide a possibility that Ab plays a key role in blood and oxidatively impairs RBC function (e.g., oxygen delivery to the brain), thereby potentially facilitating AD. However, to the best of our knowledge, no extensive study of the presence and distribution of Ab in human RBC has been undertaken. The aim of this study was to ascertain the distribution of Ab in the RBCs of young and senior subjects by applying a commercial ELISA assay. The RBC Ab concentrations were comparedbetween young and senior subjects and also compared to plasma Ab levels. In addition, we previously conducted a randomized, double-blind, placebo-controlled human study to evaluate whether nutritional supplementation with the antioxidant astaxanthin (a polar carotenoid) affected 1480666 RBC PLOOH [12]. Thus, RBCs that had been obtained from the human study [12] were subjected to Ab determination in order to evaluate the relationship between RBC Ab and the antioxidant/oxidant profile.Materials and Methods Ethics StatementThe study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Anti-Aging Science (Tokyo, Japan; ethics No. I030807). All of the subjects provided written informed consent to the experimental protocol before participating in the study.Blood Samples from Young and Senior VolunteersTwenty-four young healthy human volunteers [12 men and 12 women, between 22 and 29 years of age (mean 6 SE, 24.260.6)] and 38 senior healthy volunteers [20 men and 18 women, between 48 and 69 years of age (mean 6 SE, 56.261.0)] participated in this study. Blood was collected into a tube containing EDTA-2Na as an anticoagulant and was subjected to centrifugation at 1,000 g for ten min at 1407003 4uC. After the plasma and buffy coat were removed,Amyloid b Determination in Human ErythrocytesRBCs were washed three times with phosphate buffered saline (PBS, pH 7.4) to prepare packed cells.Measurement of Ab40 and Ab42 in RBCs and PlasmaFor determination of Ab40 and Ab42 in RBCs, human b Amyloid (1?0) ELISA kits (WAKO, Osaka, Japan) and human b Amyloid (1?2) ELISA kits (WAKO) were used, respectively. These kits are commercially available and used worldwide. We tested conditions for measurement of RBC Ab, and the optimized protocol is as follows. Packed cells (200 mL) were mixed with 200 mL of water and one mL of 70 formic acid. A 40 mL aliquot was collected and mixed with 760 mL of 1 mol/L Tris-HCl with protease inhibitors, and the mixture was diluted two-fold with the standard diluent present in each Ab40 and Ab42 ELISA kit. A 100 mL aliquot was subjected to either the Ab40 or the Ab4.
