Enrichment of cholesterol in microdissected Alzheimer's disease senile plaques as assessed by mass spectrometry

Abstract

Extensive knowledge of the protein components of the senile plaques, one of the hallmark lesions of Alzheimer's disease, has been acquired over the years, but their lipid composition remains poorly known. Evidence suggests that cholesterol contributes to the pathogenesis of Alzheimer's disease. However, its presence within senile plaques has never been ascertained with analytic methods. Senile plaques were microdissected from sections of the isocortex in three Braak VI Alzheimer's disease cases and compared with a similar number of samples from the adjoining neuropil, free of amyloid-beta peptide (A beta) deposit. Two cases were apo epsilon 4/apo epsilon 3, and one case was apo epsilon 3/apoepsilon3. A known quantity of (13)C-labeled cholesterol was added to the samples as a standard. After hexane extraction, cholesterol content was analyzed by liquid chromatography coupled with electrospray ionization mass spectrometry. The mean concentration of free cholesterol was 4.25 +/- 0.1 attomoles/microm(3) in the senile plaques and 2.2 +/- 0.49 attomoles/microm(3) in the neuropil (t = 4.41, P < 0.0009). The quantity of free cholesterol per senile plaque (67 +/- 16 femtomol) is similar to the published quantity of A beta peptide. The highly significant increase in the cholesterol concentration, associated with the increased risk of Alzheimer's disease linked to the apo epsilon 4 allele, suggests new pathogenetic mechanisms.

Fig. 1.

Analytical procedure. The tissue sections were immunostained with the 6F/3D anti-Aβ antibody. SPs were microdissected and catapulted in a cap by laser pressure catapulting (LPC). The samples were spiked with 20 ng of [3,4-¹³C]cholesterol. After sterol extraction with hexane, the preparations were evaporated and dissolved in acetonitrile. MS analysis was performed and quantification was achieved by comparing the ratio of the peak area of natural isotope of free cholesterol with the added [3,4-¹³C]cholesterol internal standard.

Fig. 2.

Calibration curves: quantity of cholesterol as a function of the peak areas. Increasing and known amounts of natural cholesterol and of internal [3,4-¹³C]cholesterol standards were extracted and analyzed by LC-MS. The curves obtained with natural and [3,4-¹³C₂]cholesterol are indicated. Since they were not statistically different, a pooled curve was obtained and used for the subsequent assessment. The parameters of the curve were as follows: quantity of cholesterol = 1.50·10⁻⁵·peak area − 5.68. The linear regression explained 98% of the variance. The curve is drawn only in the segment where the measurements were made (see text). LLOQ, lower limit of quantification.

Fig. 3.

Precision and accuracy. The quantities of assessed cholesterol are plotted against the known quantities of cholesterol injected into the column. A line of slope 1, representing the theoretical line for which estimated quantity = injected quantity, is drawn to show deviation from the theoretical value. The bars are 1 SD above and below the mean estimates.

Fig. 4.

Example of a chromatogram single ion recording (SIR) chromatogram of 20 ng of extracted cholesterol (at m/z 369.3) and [3,4-¹³C₂]cholesterol standards (at m/z 371.3).

Fig. 5.

Quantification of free cholesterol content in the neuropil and in SPs. Samples from three different human AD brains were used, and three measurements were performed on each case. Cholesterol concentration per microdissected volume is given in attomol/µm³. Bars = 1 SE of the mean for three measurements. The ANOVA indicated no difference between the cases and a mean difference of 2.04 attmol/µm³ between SP and Aβ free neuropil (ddl = 1, F = 19.45, t = 4.41, and P < 0.0009).