Abstract 1796 A C30 RP-LC-MS/MS Method for Analyzing Cholesteryl Esters from Human Plasma

Older adults are at an increased risk for injury and illness resulting in extended periods of physical inactivity or hospitalization. Previous research has found that prolonged periods of physical inactivity / bed rest alters the blood lipid profile. Particularly, high-density lipoprotein particles (HDL), also known as "good cholesterol", decrease while low-density lipoprotein particles (LDL; "bad cholesterol") increase. Lipoprotein particles are classified by their molecular cargo which includes cholesterol and cholesteryl esters (CE). Prior studies investigating the effects of bed rest on blood lipids were conducted with young adult subjects and did not determine changes in specific molecular lipid species. Our objective in this study was to investigate the changes in CE molecular species in middle-aged and older adults from two separate bed rest studies. In the middle-aged bed rest study, subjects underwent a 14-d bed rest with plasma samples taken before, during, and after bed rest, as well as after a 7-d recovery period following bed rest. The subjects of the elderly bed rest study underwent a 7-d bed rest with plasma samples taken before and after bed rest, and after a 7-d recovery period following bed rest. We developed a liquid chromatography (LC) tandem mass spectrometry (MS / MS) method that uses C30 reverse phase (RP) technology to separate and analyze the molecular species of CE from plasma. The C30 RP-LC-MS / MS method showed good separation of CE molecular species by their acyl carbon chain lengths and degrees of unsaturation. Limits of quantitation were as low as 1 ng / mL with extraction recoveries of > 90% using a methyl tert-butyl ether-based extraction. When assessing the intraday and interday precision, we found %CV were less than 20% and 25%, respectively, for CE detected in SRM 1950 plasma. Using this LC-MS / MS method, we found CE(18:2) and CE(20:4) were the most abundant in both middle-aged and elderly subjects. None of the CE molecular species showed statistically significant changes in the middle-aged subjects when compared to pre-bed rest CE content. However, the elderly subjects showed significant decreases in 12 CE molecular species that ranged in acyl carbon chain lengths from 16 to 24 and degrees of unsaturation from 0 to 4. Overall, most CE molecular species tended to decrease in older adults following bed rest whereas several CE increased in middle-aged adults. However, following a recovery period the changes in the CE profiles between middle-aged and older adults nearly mirrored each other for each CE molecular species. Further investigation is warranted to determine the biological effects of altered CE content and associated impacts on metabolic health to provide appropriate interventions for older patients during periods of physical inactivity. Additionally, we show this LC-MS / MS method is well-suited for the analysis of CE molecular species. We would like to acknowledge funding from the Cancer Prevention and Research Institute of Texas awarded to WKR (RP 190682), and from the National Space Biomedical Research Institute (NNJ08ZSA002N) and from the National Institutes of Health (R01NR012973) awarded to Douglas Paddon-Jones who supervised EJA-L.