Alterations in cerebrospinal fluid glycerophospholipids and phospholipase A2 activity in Alzheimer's disease

Abstract

Our aim is to study selected cerebrospinal fluid (CSF) glycerophospholipids (GP) that are important in brain pathophysiology. We recruited cognitively healthy (CH), minimally cognitively impaired (MCI), and late onset Alzheimer's disease (LOAD) study participants and collected their CSF. After fractionation into nanometer particles (NP) and supernatant fluids (SF), we studied the lipid composition of these compartments. LC-MS/MS studies reveal that both CSF fractions from CH subjects have N-acyl phosphatidylethanolamine, 1-radyl-2-acyl-sn-glycerophosphoethanolamine (PE), 1-radyl-2-acyl-sn-glycerophosphocholine (PC), 1,2-diacyl-sn-glycerophosphoserine (PS), platelet-activating factor-like lipids, and lysophosphatidylcholine (LPC). In the NP fraction, GPs are enriched with a mixture of saturated, monounsaturated, and polyunsaturated fatty acid species, while PE and PS in the SF fractions are enriched with PUFA-containing molecular species. PC, PE, and PS levels in CSF fractions decrease progressively in participants from CH to MCI, and then to LOAD. Whereas most PC species decrease equally in LOAD, plasmalogen species account for most of the decrease in PE. A significant increase in the LPC-to-PC ratio and PLA2 activity accompanies the GP decrease in LOAD. These studies reveal that CSF supernatant fluid and nanometer particles have different GP composition, and that PLA2 activity accounts for altered GPs in these fractions as neurodegeneration progresses.

Keywords: lipid metabolism; liquid chromatography; mass spectrometry; minimal cognitive impairment; nanoparticles; neurodegeneration.

Fig. 1.

LC-MS/MS of SF and NP GPs. GPs extracted from CSF SF or NP fractions were separated using HILIC and detected using tandem MS. The total ion current (TIC) and scan-specific chromatographs for NAPE, xPE and PE, PS, PC, PAF_LL, and LPC for SF (A) and NP (B). These data are representative of injections from 70 and 67 SF and NP, respectively.

Fig. 2.

Spectra of major GPs in SF and NP. The major GPs from LC-MS/MS were processed using the spectral feature of Qual Browser software. Shown are the spectra for PE, PS, and PC for SF (A) and NP (B). These data are representative of 70 SF and 67 NP injections.

Fig. 3.

PC spectra showing the major clusters in SF and NP. PC from LC-MS/MS was processed using the spectral feature of Qual Browser software. Shown are the PC spectra for SF (A) and NP (B). These data are representative of 70 SF and 67 NP injections and show major clusters of 1-acyl-linked, 1-ether-linked, and 1-plasmalogen-linked species in both SF and NP.

Fig. 4.

EICs of PC molecular species. The major PC molecular species were identified using a Lipid Maps algorithm. The most intense molecular species not influenced by isobars were extracted from LC-MS/MS chromatograph of SF and NP. Shown are the TIC, SRM of PC(11:0/11:0) internal standard, and EIC of PC species in SF (A) and NP (B). These data are representative of injections from 70 and 67 SF and NP, respectively.

Fig. 5.

Distribution of selected PC species in SF and NP. After LC-MS/MS of SF and NP, selected PC species not encumbered by isobaric interference were identified using a Lipid Maps algorithm. These PC species were extracted and their intensities normalized to PC(11:0/11:0) as an internal standard. Amounts of each molecular species were determined and expressed as a percentage of total in each CSF fraction. These data are the mean ± SEM for SF (A) (n = 70), and NP (B) (n = 67). The P values were obtained using a paired t-test. (A) *P < 0.05 for three PC species whose proportion is higher in SF than in NP. (B) *P < 0.05 for nine PC species whose proportion is higher in NP than in SF, while the proportion of the four unmarked species are similar in both fractions (P > 0.05).

Fig. 6.

Distribution of the top PE and PS molecular species in SF. After LC-MS/MS of SF, the four most intense PE and 3 PS molecular species were identified using a Lipid Maps algorithm (A). PE and PS molecular species were extracted, their intensities were normalized to PC(11:0/11:0) as an internal standard, and the levels were determined from standards curves (B). Data are the mean ± SEM (n = 70).

Fig. 7.

GPC lipids change in LOAD. The levels of GPC lipids (PC, LPC, PAF_LL) in SF or NP were determined using LC-MS/MS. (A–C) Levels of PC, LPC, and LPC/PC, respectively, in SF for CH, MCI, and LOAD subjects. (D–F) Levels of PC, LPC, and LPC/PC, respectively, in NP for CH, MCI, and LOAD subjects. The number of samples for each group (n) is indicated for each group, and the bar is the mean ± SEM. Group comparisons were performed using Kruskal-Wallis test and comparisons using Mann-Whitney test. *P < 0.05.

Fig. 8.

PC species change in LOAD. Levels of selected PC species in SF or NP were determined using LC-MS/MS. (A) Levels of PC species in SF for CH, MCI, and LOAD subjects. (B) Levels of PC species in NP for CH, MCI, and LOAD subjects (mean ± SEM). Group comparisons were performed using Kruskal-Wallis test and comparisons using Mann-Whitney test. *P < 0.05 compared with CH.

Fig. 9.

PE and PS decrease in LOAD. Levels of PE and PS in SF and PE and xPE in NP were determined using LC-MS/MS. (A, B) Levels of PE and PS, respectively, in SF for CH, MCI, and LOAD subjects. (C, D) Levels of PE and xPE, respectively, in NP for CH, MCI, and LOAD subjects. The number of samples for each group (n) is indicated for each group, and the bar is the mean ± SEM. Group comparisons were performed using Kruskal-Wallis test and comparisons using Mann-Whitney. *P < 0.05 compared with CH.

Fig. 10.

PE and PS species change in LOAD. Levels of PE (A) and PS (B) species in SF were determined using LC-MS/MS. Data are the mean ± SEM. Group comparisons were performed using Kruskal-Wallis test and comparisons using Mann-Whitney test. *P < 0.05 compared with CH.

Fig. 11.

PLA₂ activity increases in LOAD. PLA₂ activity in CSF (10 μg protein per assay) was determined using a fluorescent assay. Data are expressed as the RFU per min for different clinical groups. Data are the mean ± SEM for CH, MCI, and LOAD. *P < 0.05 compared with CH.