Hypoxia Due to Cardiac Arrest Induces a Time-Dependent Increase in Serum Amyloid β Levels in Humans

Amyloid β (Aβ) peptides are proteolytic products from amyloid precursor protein (APP) and are thought to play a role in Alzheimer disease (AD) pathogenesis. While much is known about molecular mechanisms underlying cerebral Aβ accumulation in familial AD, less is known about the cause(s) of brain amyloidosis in sporadic disease. Animal and postmortem studies suggest that Aβ secretion can be up-regulated in response to hypoxia. We employed a new technology (Single Molecule Arrays, SiMoA) capable of ultrasensitive protein measurements and developed a novel assay to look for changes in serum Aβ42 concentration in 25 resuscitated patients with severe hypoxia due to cardiac arrest. After a lag period of 10 or more hours, very clear serum Aβ42 elevations were observed in all patients. Elevations ranged from approximately 80% to over 70-fold, with most elevations in the range of 3–10-fold (average approximately 7-fold). The magnitude of the increase correlated with clinical outcome. These data provide the first direct evidence in living humans that ischemia acutely increases Aβ levels in blood. The results point to the possibility that hypoxia may play a role in the amyloidogenic process of AD

Features of Aβ42 elevation profiles were compared with 6-month overall cerebral outcome.

<p>(<b>A</b>) Magnitude of Aβ42 rise; (<b>B</b>) ratio of maximum to baseline Aβ42; (<b>C</b>) duration of major Aβ42 rise; (<b>D</b>) sum of <b>A</b> to <b>C</b> plus maximum slope of Aβ42 rise. Error bars are the standard error of the means.</p

Assay characteristics.

<p>Arrays of femtoliter-volume wells permit isolation of 2.7 mm capture beads from a standard bead-based ELISA (<b>A</b>), enabling exquisite sensitivity to enzyme label by preventing fluorescent product from diffusing away into a bulk solution. At very low concentrations of Aβ42, beads contain either a single labeled immunocomplex or no complexes, giving rise to digital signal output corresponding to single molecules (<b>B</b>). Simultaneous counting of active wells across an array statistically powers estimates of average enzymes/bead. (<b>C</b>) Dose-response of digital immunoassay for Aβ42 (n = 3). Y-axis refers to average number of enzyme labels per individual microbead captured in the array. The concentration of label is reduced relative to standard immunoassays, resulting in improved signal∶background at very low Aβ42 concentration. Assay calibrators were purified Aβ42 (Merck) in PBS/BSA. (C inset) Limit of quantification (LoQ) was estimated from total coefficients of variation (CV) from five low panel members (spiked PBS/BSA panels, grey circles, and immunodepleted plasma, black circles) assayed repeatedly across five days. The Aβ42 concentration at which total assay imprecision reached 20% (LoQ) was 0.032 pg/mL. (<b>D</b>) Recovery at extremely low Aβ42 concentrations was tested by diluting spiked immunodepleted plasma with PBS/BSA zero calibrator (n = 3). Because samples are pre-diluted 4-fold prior to assay to reduce matrix effects, dilutions include an initial 4-fold dilution.</p

Characteristics of serum Aβ42 elevation profiles from resuscitated survivors of cardiac arrest during the first 96–108 hours following admission to the intensive care unit.

<p>Parameters were sorted on the basis of good or poor 6-month outcome. Blood sampling was terminated early for patients 1, 16, 19 and 22 for medical reasons, and rise durations could not be estimated for these patients. The Aβ42 score equals the sum of magnitude of rise, duration of rise, rise ratio and max slope.</p

Serum Aβ42 following resuscitation from cardiac arrest.

<p>CPC scores depicted are after discharge from the ICU and 6 months later. Panels on left (<b>A–C</b>) are profiles from patients exhibiting good outcomes, panels on the right (<b>D–F</b>) are from patients with poor outcome. (<b>E</b>) Illustration of Aβ42 profile analysis. Baseline Aβ42 was defined as the mean of the two lowest values in the initial 12 hours. The time of initial elevation was defined as the intersection between the baseline Aβ42 and the line of maximum ascension of the major elevation peak. The duration of the Aβ42 increase was defined as the difference between time of initial elevation and time beyond which no significant further rise was observed. The magnitude and maximum slope of rise are also indicated. (<b>A</b>) Patient exhibiting smallest relative increase in Aβ42 among all patients. For confirmation, the sample set was re-assayed on a different day. (<b>D</b>) Patient with poor outcome exhibiting the largest relative increase from baseline. (<b>C</b> and <b>F</b>) Patients with similar baseline Aβ42 and poor cerebral outcome upon discharge from the ICU. Six months later, patient BE had recovered good cerebral function, while patient LP had not. Error bars: standard deviation of triplicate measurements.</p