Zsuzsanna Kuklenyik (Presenter)
Centers for Disease Control
Bio: Earned BS/MS degree in Chemical Engineering at Technical University of Budapest, Hungary. Received PhD degree in Bioinorganic Chemistry at Emory University, Atlanta. Currently she is a senior research chemist in the Biological Mass Spectrometry Laboratory at the Centers for Disease Control and Prevention in Atlanta. She has published numerous hyphenated chromatographic and mass spectrometry applications for biomonitoring of wide range of environmental chemicals, analysis of pre-exposure prophylactic drugs against HIV, protein toxins, and more recently, quantitative analysis lipoprotein sub-classes.
Authorship: Zsuzsanna Kuklenyik, Christopher A Toth, Jeffrey I Jones, Bryan A Parks, Michael S Gardner, David M Schieltz, Jon C Rees, Michael L Andrews, Lisa G McWilliams, James L Pirkle and John R Barr
Centers for Disease Control, Division of laboratory Sciences
| Short Abstract Lipoproteins circulating in fasting blood are classified by density into high, low, intermediate and very low density lipoproteins (HDL, LDL, IDL and vLDL). In this work lipoprotein classes/sub-classes were size fractionated using <100 µL serum from 200 donors (normal, hyperglycemia, hypercholesterolemia, hypertriglyceridemia and hyperlipidemia), collecting size fractions of 6-40 nm. Each serum sample and their corresponding 40 size fractions were analyzed parallel by three different quantitative LC-MS/MS methods for measurements of non-polar lipid classes (free cholesterol, cholesterol esters, and triglycerides), five phospholipids classes, and 8 apolipoproteins. The data demonstrate how LC-MS/MS can provide highly effective means to measure lipoprotein sub-class composition in cohort studies, allowing system biology investigations of various dyslipidemias and cardiovascular diseases. |
Long Abstract
Introduction: Lipoproteins are dynamic lipid-protein assemblies that function as transporters of various biomolecules circulating in blood. Lipoproteins have unique lipid/protein composition which determine their ability of to scavenge other proteins and enzymes circulating in blood or interact with membrane receptor sites on cells of various tissues and organs. Lipoproteins circulating in fasting blood are classified by density into high, low, intermediate and very low density lipoproteins (HDL, LDL, IDL and vLDL). These main density classes consist sub-classes from small/dense to large/buoyant characteristics. Liporptein classes/sub-classes are recognized as important indicators that can be linked to cardiovascular disease (CVD).
Methods: Size fractions of HDL and LDL were separated using 50 µL serum drawn from 200 donors (normal, hyperglycemia, hypercholesterolemia, and hypertriglyceridemia and hyperlipidemia), collecting 40 size fractions in a size range of 5-40 nm with <2 nm resolution by asymmetric flow field flow fractionation (AF4) [1]. The average particle size in each fraction was directly measured by dynamic light scattering (DLS). Each serum sample and their corresponding 40 size fractions were analyzed parallel by three different quantitative isotope dilution MS/MS methods: 1) UPLC and in-source-CID coupled MS/MS for free cholesterol (FC), cholesterol esters (CE) and triglycerides (TG) content (without pre-analytical ester hydrolysis); 2) on-line trypsin digestion coupled LC-MS/MS [2] for apolipoproteins: A-I, A-II, A-IV, B, C-I, C-II, C-III, and E (monitoring others); and 3) UPLC-MS/MS for phospholipids [3] (PLs) classes, including phosphatidyl choline (PC) sphingomyelins (SM), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI), and lysophosphatidyl choline (LPC).
Results: From the size and multiplexed concentration measurements, the composition of HDL, LDL, IDL and vLDL sub-classes could be reconstructed, including calculation of hydrated particle volume, surface volume (PLs, proteins, and FC), core volume (FC, CE and TG), and lipoprotein particle count [4]. The average numbers of ApoA-I/HDL particle (1-6) and ApoB-100/LDL particle (~1) were consistent with current consensus models of HDL and LDL. Differences in lipoprotein size and lipid/protein composition indicated irregularities in specific lipid metabolism pathways. For example, all dyslipidemic donors showed lower small HDL and higher small LDL particle counts than normal donors. The small HDL and small LDL sub-classes from different type of dyslipidemia groups could be differentiated based on TG/TC ratio, SM/PC ratio, FC/surface area, average A-I/A-II ratio, and numbers of ApoC-I/C-II/C-III/E molecules per particle, indicating variations in degree of cholesterol efflux, lipase enzyme activity, and efficacy of uptake by HDL and LDL receptors.
Conclusions: Our findings demonstrate that this highly multiplexed, hyphenated, high throughput MS/MS workflow allows the generation of comprehensive quantitative data, and provide effective means to system biology investigations of CVD.
References & Acknowledgements:
[1] Z. Kuklenyik, , M.S. Gardner, B.A. Parks, D.M. Schieltz, J.C. Rees, L.G. McWilliams , Y. Williamson, J.L. Pirkle, J.R. Barr, Multivariate DoE optimization of asymmetric flow field flow fractionation coupled to quantitative LC-MS/MS for analysis of lipoprotein subclasses, Chromatography (Separations) 2 (2015) 96-117.
[2] F.E. Regnier, J.H. Kim, Accelerating trypsin digestion: The immobilized enzyme reactor, Bioanalysis 6 (2014) 2685-98.
[3] T. Cajka, O. Fiehn, Comprehensive analysis of lipids in biological systems by liquid chromatography-mass spectrometry, Trends Analyt. Chem. 61 (2014) 192–206.
[4] J.P. Segrest, M.C. Cheung, M.K. Jones, Volumetric determination of apolipoprotein stoichiometry of circulating HDL subspecies, J. Lipid Res. 54 (2013) 2733-44.
| Description | Y/N | Source |
| Grants | no | |
| Salary | no | |
| Board Member | yes | IFCC Working Group on Apolipoprotein Standardisation |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |