Probing Phospholipid Structure in Alzheimer’s Disease for Possible Biomarkers of Disease
Daniel Willems | Montana State University - Billings
Alzheimer’s disease (AD) is a terminal neurodegenerative disorder affecting the brain. The human brain is nearly 60 percent lipid; thus, it is reasonable to assume that the loss of brain volume observed in AD is, in significant part, phospholipid. Phospholipids that make-up neuronal brain tissues are catabolized in a highly regulated manner into metabolic intermediates, such as DHA and other fatty acids, choline, and other small molecule metabolites. These metabolites are involved in neuronal protection, signal transduction, and many other brain functions. Extensive loss of these metabolites results in the brain physiology observed in AD. Many researchers and treatment professionals agree that early detection and intervention of AD is a crucial requirement for effective treatment. However, a low-cost, effective, and non-invasive option for such early and often testing is currently not available. Previous studies targeting DHA and other metabolic intermediates for this purpose have failed to establish such tests.
This study seeks to examine the bioavailability of precursors to important metabolic intermediates in AD as a first step to develop robust diagnostic tests for rapid and cost-effective deployment into rural communities.
This study seeks to measure levels of LFA’s esterified at the sn2 position of PCs in AD brain tissue, ultimately to determine the bioavailability of LFAs coupled to choline in AD brain. Using stereospecific enzyme-mediated phospholipid digestion and tandem mass spectrometry (MSMS) both LFA and choline levels will be measured. Additionally, a new technology, ion mobility mass spectrometry (IMMS) will be evaluated for its utility in quantitative analysis of phospholipids generally and phosphocholines more specifically for this purpose.
Decreased levels of free LFAs and choline in the brains of AD patients is hypothesized to be due to the lack of LFAs esterified at the sn2 position of PCs specifically. Resolving the stereospecific structure of PC in AD may significantly improve our understanding of the role bioavailability of choline and LFAs play in the brain of AD. Specific aims include:
- Determine the bioavailability levels of fatty acids and choline in the AD brain and CSF from individuals diagnosed with AD at death and compare those levels to that of NC individuals at death. Fatty acid bioavailability is likely to play a crucial role in AD but is poorly understood due in part to a lack of robust analytic tools. Carefully chosen PLA2 enzymes will specifically hydrolyze fatty acid ester linkages at the sn2 position of phospholipids. Fatty acids released by this method will subsequently be derivatized for detection by GCMS to assess the global bioavailability of fatty acids in the brain of AD.
- Quantify specific phospholipids dysregulated in AD versus NC brain tissue and CSF samples with an emphasis on phosphatidylcholine species using high resolution MSMS data. Of critical importance to this hypothesis is the native structure of the phospholipid species. After digestion with PLA2, some crucial inter-phospholipid structure data becomes blurred as LFAs are detected by GCMS in ‘bulk’.
- Determine the applicability of Ion Mobility Mass Spectrometry (IMMS) to elucidating phospholipid structure in biological samples for use in future work. IMMS exploits differences in cross-sectional profiles of small molecules to differentiate structural isomers from each other. This methodology would allow absolute identification of phospholipids. Initially developed in 2016, these methodologies continue to be expanded and are likely to become the gold standard in mass spectrometry-based lipid identification.