MASS SPECTROMETRY-BASED METHOD TO MEASURE CIRCULATING OXYSTEROLS IN ALZHEIMER’S DISEASE

Abstract

Hypercholesterolemia is an established risk factor for Alzheimer's disease (AD). Unlike cholesterol, oxysterols; oxygenated derivatives of cholesterols are able to pass the blood brain barrier. Oxysterols are involved in many biological processes including the regulation of cholesterol homeostasis within the brain. However, the modifications to cholesterol are not fully explored due to challenges in detecting these entities at low physiological abundance. The aim of this project is to develop and validate a multiple reaction monitoring (MRM) based liquid chromatography-mass spectrometry (LC-MS/MS) method for the absolute quantification of oxysterols (24OHC, 25OHC, 27OHC, 7βOHC and 7-keto-OHC) in human plasma. A multiple reaction monitoring (MRM) based liquid chromatography-mass spectrometry (LC-MS/MS) method was developed and validated for the absolute quantification of five oxysterols (24OHC, 25OHC, 27OHC, 7βOHC and 7-keto-OHC) in human plasma. Blood from healthy control, age-, sex-matched AD and AD with vascular complications (AD plus) subjects (n=10/group) was collected. Total oxysterols were extracted from plasma (70μl) spiked with internal standards (24OHC-d7, 25OHC-d6, 27OHC-d6, 7βOHC-d7, and 7-keto-OHC-d5) and oxysterols were enriched using two-step solid phase extraction (SPE) using a polymeric SPE column (HLB PRiME, Waters). MRM assay has a linear dynamic range (R2 > 0.94) of 0.02ng/ml-20ng/ml for the five oxysterols tested from. High intra- and inter-day precision (CV < 9%) and high concentration accuracy (< 15% absolute error) were observed from the QC plasma samples. All five oxysterols measured in AD and AD plus patients were not significantly different from healthy control subjects (27±5pg/ml 24OHC, 104±9pg/ml 25OHC, 150±21pg/ml 27OHC, 310±39pg/ml 7βOHC, and 342±42pg/ml 7-keto-OHC). The method described has been validated for five oxysterol measurement in human plasma. These data suggest that patients with Alzheimer's disease have similar levels of oxysterols to healthy individuals.