Combination of metabolomic and phospholipid-profiling approaches for the study of Alzheimer's disease

Highlights

• Combined metabolomic-profiling approach allows broader study of phospholipids.

• Different phospholipid species are altered in Alzheimer's disease.

• Alterations depend on the phospholipid class and fatty acid composition.

• Abnormal phospholipid metabolism in Alzheimer has a multifactorial origin.

Abstract

Alzheimer's disease is closely related to abnormal metabolism of phospholipids from neural membranes, so that the study of their dyshomeostasis could be of great interest for the discovery of potential biomarkers for diagnosis and disease monitoring. In this work, it has been developed a metabolomic multi-platform for the characterization of phospholipid alterations occurring in serum from Alzheimer's disease patients. For this purpose, we performed a metabolomic screening by direct infusion mass spectrometry and profiling analysis by reversed phase ultra-high performance liquid chromatography with complementary detection by molecular and atomic mass spectrometry, which allowed combining the high-throughput capability of shotgun metabolomics and the targeted character of profiling approaches. Thus significant changes were detected in the levels of several molecular species of phosphatidylcholines, phosphatidylethanolamines, plasmenylcholines, plasmenylethanolamines and different classes of lysophospholipids, which provided a global vision of the possible factors triggering membrane breakdown. In this sense, alterations of phospholipids metabolism appears to have a multifactorial origin involving overactivation of phospholipases, increased anabolism of lysophospholipids, peroxisomal dysfunction, imbalances in the levels of saturated/unsaturated fatty acids contained in the structure of phospholipids and oxidative stress.

Biological significance

This work represents the first comprehensive characterization of serum phospholipids alterations in relation to Alzheimer's disease, by combining shotgun metabolomics and phospholipids profiling through different analytical approaches.This article is part of a Special Issue entitled: Environmental and structural proteomics.

Introduction

Phospholipids play a central role in the biochemistry of all living organisms, constituting the lipid bilayer that serves as a structural barrier to protect cells and subcellular components from external conditions, and being required for the proper function of integral membrane proteins, receptors, and ion channels [1]. Moreover, phospholipids also act as storage depot of a complex meshwork of lipid mediators, such as eicosanoids, lysophospholipids, platelet activating factors or diacylglycerides, which exert a diverse array of effects on cellular functional activities including neural cell homeostasis, immune responsiveness, oxidative stress, and neuroinflammation [2]. Thus, defects in lipid metabolism are involved in numerous human diseases, particularly from the central nervous system because of its high lipid content, so changes in brain phospholipids levels may lead to many neurological disorders, including bipolar disorders, schizophrenia and neurodegenerative diseases, as for example Alzheimer's and Parkinson's disease [3]. Alzheimer's disease (AD) is the most common neurodegenerative disorder among older people, with a complex etiology in which multiple pathologic processes are involved [4]. Thereby, although major hallmarks of AD are the formation of senile plaques and neurofibrillary tangles, related to aberrant processing of amyloid precursor protein (APP) by secretases leading to the deposition of amyloid β, and hyperphosphorylation of tau protein [5], lipids also may be linked to pathogenesis of Alzheimer. In this sense, both APP and secretases are integral membrane proteins, so lipid surroundings play an important regulatory role in APP processing and formation of Aβ peptides [6]. In addition, genomic studies have demonstrated that the ε4 allele of apolipoprotein E (apoE), a lipid transport protein, is a major risk factor for late-onset AD, so apoE-mediated lipid alterations appear to be an important trigger of AD [7].

For these reasons, numerous studies have attempted to characterize the changes in the lipid profile occurring in the brain of AD individuals. These alterations have been traditionally associated with abnormal metabolism of brain phospholipids leading to breakdown of cellular membranes, so that post mortem studies of human AD brain usually showed decreased total levels of phospholipids [8] and accumulation of their degradation products [9]. However, confusing results are observed when specific changes in individual phospholipids are considered. Decreased levels of total phosphatidylethanolamine (PE) [10], [11], [12], [13] and phosphatidylinositol (PI) [12], [13], [14] have been previously reported, while phosphatidylserine (PS) was found significantly increased [11], [15]. In addition, considerably reductions have been described for plasmalogens, in both ethanolamine (PPE) [15], [16] and choline (PPC) species [17]. In contrast, findings about phosphatidylcholine (PC) levels are less clear since different studies found its content unchanged [11], [12], increased [18] or decreased [10] in specific brain regions. On the other hand, only a few studies attempted to monitor phospholipid alterations in cerebrospinal fluid (CSF) or blood samples, accessible in vivo to investigate possible diagnostic markers of neurodegeneration. Mulder et al. found a significant reduction of total CSF phospholipids [19], but recently PI, PE and PC levels were found unchanged in the CSF from AD patients representing different stages of the disease [20], [21]. In other study, unchanged PC levels and decreased lysoPC/PC ratio have been reported in the CSF from individuals with AD [22], while different water-soluble PC metabolites increased, suggesting that AD development is accompanied with increased phospholipid hydrolysis [23]. In case of blood samples, circulating plasmenylethanolamine (PPE) levels were observed to be significantly decreased in serum from clinically and pathologically diagnosed AD subjects [24], while by using a 2D-phospholipidomics method was possible to distinguish plasma samples of AD patients from those of elderly controls [25]. Therefore, it can be concluded that analysis of phospholipids offers a great potential for biomarker discovery in AD, but a comprehensive characterization of the phospholipid profile of AD individuals versus controls needs to be addressed.

Metabolomics presents a high potential in health survey and biomarker discovery, because changes in specific groups of metabolites may be sensitive to pathogenically relevant factors. Thus, metabolomics is emerging as a powerful tool for characterization of complex phenotypes affected by both genetic and environmental factors [26]. Nevertheless, metabolomic fingerprinting often lacks of robustness, so targeted or profiling approaches may be useful techniques for validation purposes, with the necessary specificity, precision, accuracy, linearity, sensitivity, recovery and stability in the presence of potentially interfering compounds [27]. In this study, shotgun metabolomics of serum samples was performed by direct infusion mass spectrometry (DIMS) for the screening of phospholipids involved in neurodegenerative processes associated with AD. Furthermore, a targeted analytical approach focused on phospholipids was optimized, based on separation of different species by reversed phase ultra-high performance liquid chromatography (RP-UPLC) and complementary detection by molecular and atomic mass spectrometry. Liquid chromatography coupled to mass spectrometry is widely employed for metabolomic and profiling analyses due to its high resolution and sensitivity, fast analysis and good potential for biomarker identification [28]. However, the high complexity of samples makes necessary to develop selective methods by targeting particular classes of compounds. In this sense, the hyphenation of inductively coupled plasma mass spectrometry (ICP-MS) to LC has been previously described for the sensitive and selective detection of phospholipids, enabling their quantification by phosphorous-tagging without the use of structurally matched standards [29], [30]. Thus, the combination of different mass spectrometry-based metabolomics strategies, from untargeted to targeted, allows us to shed light on the dyshomeostasis of different classes of phospholipids in AD.