Lipidomic alterations in lipoproteins of patients with mild cognitive impairment and Alzheimer’s disease by asymmetrical flow field-flow fractionation and nanoflow ultrahigh performance liquid chromatography-tandem mass spectrometry
Introduction
Alzheimer’s disease (AD) is an irreversible neurodegenerative disorder that is the most common cause of dementia in adults older than 65 years of age; notably, it presents a challenging task for health care in the developed countries, since it will dramatically increase in the future [[1], [2], [3]]. The clinical symptoms of AD comprise a progressive loss of cognitive function, typically memory; AD is pathologically distinguished by an extensive loss of synapses and neurons, as well as by the presence of neuritic plaques enriched with amyloid β (Aβ) peptides in brain [4] and of neurofibrillary tangles composed of hyperphosphorylated tau proteins [5]. Mild cognitive impairment (MCI) refers to a transitional state between the cognitive changes of normal aging and AD. Because 80% of MCI has been reported to progress to AD over a period of 6 years [6], and changes in the brain caused by AD pathogenesis may occur earlier than the onset of MCI symptoms [7], the identification of the earliest signs of disease progression is important in establishing preventive treatments for potential AD patients [8,9]. Therefore, reliable and convenient blood biomarkers, which clinicians could use to predict the potential of AD development, are crucial to enable the commencement of preventive intervention at the earliest stages.
Although several AD biomarkers, such as Aβ peptides and tau protein have been developed using cerebrospinal fluid (CSF) [[10], [11], [12], [13]], lumbar puncture to obtain CSF is rather invasive. Few plasma proteins, based on proteomic approaches have been reported to predict the progression of MCI to AD [9,14,15]; however, most of them require further clinical validation. Alterations in lipid levels are known to modulate the generation of Aβ [3,16,17]. Studies show decreased levels of phosphatidylethanolamine plasmalogen and sulfatide, but increases in ceramide and diacylglycerol, in the AD-brain or CSF [3,[18], [19], [20]], as well as few putative lipid signatures from AD-plasma [21]. Also reported are higher levels of low-density lipoprotein (LDL) cholesterol, which is closely linked with AD [22]. While the roles and consequences of lipids in AD have been described, detailed profiles of blood lipids with AD, especially those of different lipoproteins, have not yet been thoroughly investigated.
Lipid analysis at the molecular level is often complicated, due to the diversity in lipid molecular structures. Rapid growth in mass spectrometry (MS) has facilitated lipidomic analysis. Direct analysis of lipids using electrospray ionization-tandem MS (ESI-MS/MS) provides the high throughput analysis and the accurate determination of lipid molecular structures [23,24], however, it can not avoid the ion suppression effect caused by high abundance lipid species and the difficulty in identifying isobaric lipids. Lipid analysis has been empowered by hyphenating chromatographic methods with ESI-MS/MS such as reversed phase liquid chromatography (RPLC) in most cases, hydrophilic-interaction liquid chromatography (HILIC), supercritical fluid chromatography (SFC), two-dimensional LC, and etc. [[25], [26], [27], [28], [29], [30], [31]]. Lately, we have demonstrated that nanoflow ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry (nUHPLC-ESI-MS/MS) can facilitate the identification of lipid structures at low fmol levels with a high speed quantitation: lipid profiles in the plasma of patients with Gaucher diseases upon enzyme replacement therapy, in the muscle tissues of diabetic rats upon physical exercise, and in urinary exosomes from patients with prostate cancer [[32], [33], [34]].
In this study, a comprehensive targeted profiling of lipids has been performed with human plasma lipoproteins of patients who were diagnosed with AD and MCI in comparison to age-matched healthy controls. Lipoproteins were size-sorted into high density lipoprotein (HDL) and LDL including very low-density lipoprotein (VLDL) using semi-preparative scale asymmetrical flow field-flow fractionation (AF4), an elution-based size-separation technique [35]. Then, lipids in HDL and LDL/VLDL were analyzed for non-targeted identification of molecular structures, followed by targeted quantitation using nUHPLC-ESI-MS/MS based on selective reaction monitoring method and statistical evaluation of the dependence of lipids with brain damage. This study aimed to elucidate the lipoprotein-dependent lipids in the progression of MCI and AD, which can be utilized in the future as candidate molecules to enhance the predictability of disease by engaging with the mini-mental state examination (MMSE) results.
Section snippets
Materials & reagents
A total of 25 lipid standards were used to optimize nUHPLC-ESI-MS/MS run conditions, together with 14 internal standards having odd-numbered acyl chains. These were lysophosphatidylcholine (LPC) 12:0, phosphatidylcholine (PC) 13:0/13:0, PC 16:0/14:0, PC 16:0/16:0, PC 18:1/18:0, PC 20:0/20:0, lysophophatidylethanolamine (LPE) 14:0, phophatidylethanolamine (PE) 12:0/12:0, phophatidylethanolamine plasmalogen (PEp) 18:0p/18:1, lysophosphatidic acid (LPA) 14:0, phosphatidic acid (PA) 12:0/12:0,
Size sorting of lipoproteins and lipid identification
Size separation of plasma lipoproteins by preparative scale AF4 channel is shown with the fractograms of the control, MCI, and AD patient plasma samples (50 μL each) in Fig. 1 obtained at 0.4 mL/min of outflow rate and 3.6 mL/min of crossflow rate. It appears that LDL and VLDL peak intensities of cognitively impaired groups (MCI and AD groups) are larger than those of age-matched healthy controls, supporting that the LDL and VLDL levels are elevated in the patient groups. While HDL and LDL were
Conclusions
The present study shows that lipid analysis of entire blood samples without lipoprotein separation may not reveal the perturbed changes in the development of AD, as is also reported by the evidence of cholesterols in HDL-C and LDL-C associating with Aβ levels [52]. Although the mechanisms of alterations in individual lipid levels related to AD development are not yet known, the observed changes in lipid levels reveal that not all lipids in the same class show a same trend of changes and a
Disclosure statement
The authors report no actual or potential conflict of interest.
Acknowledgements
This study was supported by a grant (NRF-2015R1A2A1A01004677) through the National Research Foundation (NRF) of Korea, funded by the Ministry of Science, ICT & Future Planning.
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