Evidence of altered phosphatidylcholine metabolism in Alzheimer's disease

Abstract

Abberant lipid metabolism is implicated in Alzheimer's disease (AD) pathophysiology, but the connections between AD and lipid metabolic pathways are not fully understood. To investigate plasma lipids in AD, a multiplatform screen (n = 35 by liquid chromatography-mass spectrometry and n = 35 by nuclear magnetic resonance) was developed, which enabled the comprehensive analysis of plasma from 3 groups (individuals with AD, individuals with mild cognitive impairment (MCI), and age-matched controls). This screen identified 3 phosphatidylcholine (PC) molecules that were significantly diminished in AD cases. In a subsequent validation study (n = 141), PC variation in a bigger sample set was investigated, and the same 3 PCs were found to be significantly lower in AD patients: PC 16:0/20:5 (p < 0.001), 16:0/22:6 (p < 0.05), and 18:0/22:6 (p < 0.01). A receiver operating characteristic (ROC) analysis of the PCs, combined with apolipoprotein E (ApoE) data, produced an area under the curve predictive value of 0.828. Confirmatory investigations into the background biochemistry indiciated no significant change in plasma levels of 3 additional PCs of similar structure, total choline containing compounds or total plasma omega fatty acids, adding to the evidence that specific PCs play a role in AD pathology.

Keywords: Addneuromed; Alzheimer's disease; ApoE; Lipid; Mild cognitive impairment; Phosphatidylcholine; Plasma.

Fig. 1

Experimental pipeline overview. Initially a typical liquid chromatography–mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) metabolite screen led to the identification of 3 phosphatidylcholine (PC) molecules that significantly decrease in individuals with Alzheimer’s disease (AD) compared to controls. This led to a comprehensive multiplatform lipidomic analysis consisting of 3 major components. During NMR analysis of patient plasma, particular attention was focused on total choline-containing molecules, a vital component of the phosphatidylcholine molecules identified in the screen experimental section. LC-MS analysis of plasma fatty acids (arachidonic acid, docosahexaenoic acid, and eicosapentaenoic acid) was also conducted. Again, as with NMR analysis of choline species, these fatty acid species are components of phosphatidylcholine structures indentified in the screen experimental section. Finally, comprehensive lipidomic validation was conducted with a large sample cohort. Here a specially developed lipidomic LC-MS method was applied to increased sample numbers. The method is able to detect >3000 lipid markers from a single plasma extraction.

Fig. 2

Results of the multivariate data analysis completed with SIMCA-P+ 12.0 software, (Umetrics, Umeå, Sweden). (A) Unsupervised principal components analysis (PCA) with 41 samples (15 from controls, 10 from individuals with mild cognitive impairment [MCI], and 10 from individuals with Alzheimer’s disease [AD]). Orange inverted triangle signifies a repeat extraction of a plasma pool used as a quality control (QC) (n = 6). The QCs cluster in same area of the PCA, suggesting across-run reproducibility. (B) Supervised orthogonal partial least-squares discriminate analysis (OPLS-DA) of all 3 groups (n = 35). Red square and green triangles relate to control (n = 15) and MCI (n = 10), individuals, respectively. Blue diamonds relate to individuals with AD (n = 10). A clear intergroup separation is achieved when observing AD samples. (C) S-Plot corresponding to the OPLS-DA in B. Highlighted in red boxes are masses with the p[1] value of >0.15, which underwent further investigation. The 3 features in the bottom left of the S-Plot correspond to the 3 phosphatidylcholine species shown to significantly decrease in AD during the screen experimental phase.

Fig. 3

An overview of the plasma phosphatidylcholine (PC) analysis and resultant predictive properties. (A) Box plots for the 3 significant PC species identified in the comprehensive lipidomic analysis. * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Receiver operating characteristic (ROC) analysis results of the 3 peak area ratios for the 3 PC masses of interest (n = 141). Mass 780 (PC16:0/20:5) area under the curve (AUC) of 0.722, mass 806 (PC16:0/22:6) AUC of 0.662, and mass 834 (PC18:0/22:6) AUC of 0.680. Combining the 3 PC features into an ROC analysis returned an AUC of 0.788. Inclusion of the ApoE genotyping data within disease classes provided an AUC of 0.828. As a comparison, apolipoprotein E (ApoE) data used on its own yielded an AUC of 0.667.

Fig. 4

Phosphatidylcholine (PC) molecules reduced in plasma from individuals with Alzheimer’s disease (AD) plasma versus control. This overview presents background information on the identified PC species, including their source side chains.

Fig. 5

Phosphatidylcholine (PC) metabolism and Alzheimer’s disease (AD). This overview illustrates the major routes to PC biosynthesis, with an emphasis on steps involving choline-containing compounds and those that have been previously associated with the pathology of AD. Phosphatidate (phosphatidic acid [PA]) is synthesized endogenously from glycerol-3-phosphate in most tissues, and is a key signaling molecule and a precursor for other 1,2-diacylglycerophospholipids. PA is converted to 1,2-diacylglycerol (DAG), which is then fed into the CDP-choline Kennedy pathway to form PC (PC can also be formed in the liver from phosphatidylethanolamine [PE] in a multi-step pathway catalyzed by PE-methyl transferase). PC is subject to the action of a variety of phospholipases, 2 main families of which are the phospholipase D (PLD) and phospholipase A2 (PLA2): PLD enzymes hydrolyze PC to yield free choline (Ch) and PA, whereas PLA2 enzymes cleave acyl groups from the sn-2 position of PC to yield lysoPC.