Supplemental Material
Supplemental Methods
Measurements of LCAT protein, apolipoproteins, lipids, and additional parameters in whole plasma
LCAT protein was measured using a commercially available ELISA kit (ALPCO Diagnostics). Plasma apoAI was determined using an internally developed ELISA on the DELFIA platform (Perkin Elmer). Briefly, goat anti mouse apoAI antibody (Rockland) was used as both coating and detection antibody, and purified mouse HDL was used as standard. Sample dilution was 1:2000 and assays were carried out in the standard procedure for DELFIA-ELISA on 96-well plates, which were then read on EnVision 2103 Multi-label reader (Perkin Elmer). Plasma apoB was quantitated via an LC/MS assay as described previously [1].
Total cholesterol (TC), HDL cholesterol, and triglycerides (TG) were measured using commercially available kits (Wako Diagnostics), and free cholesterol (FC) was measured using a kit from BioVision. Cholesteryl ester (CE) levels were derived by subtracting FC from TC. Total phospholipids (PL), including phosphatidylcholine (PC), lysophosphatidylcholine (LysoPC), and sphingomyelin, was determined by a commercially available kit (Wako Diagnostics).
FPLC separation of lipoproteins was carried out using previously described procedures [2].
Plasma LCAT activity assay
LCAT activity was measured using a modified LC/MS assay based on the originally described proteoliposome substrate-thin layer chromatography method[3], with the incorporation of stable isotope-labeled FC in the substrate, to allow LC/MS detection of newly synthesized CE species. Briefly, proteoliposome substrate that contained egg-yolk PC (Sigma), deuterium-labeled FC (cholesterol-2,2,3,4,4,6-d6, Isotec, referred to as d6-FC hereafter), and purified human apoAI (Academy Bio-medical Co.) at a molar ratio of 250:12.5:0.8 was prepared by the cholate dialysis technique. Plasma LCAT activity assay reaction was carried out in a 50ul system on a 96-well plate that included the following components: 1.5 ul mouse plasma, 10 ul proteoliposome substrate (250 nmol PC, 12.5 nmol d6-chol, 0.8 nmol apoAI), 0.5% bovine serum albumin, 5 mM b-mercaptoethanol. Reaction was allowed to proceed at 37°C for 2 hour, then mixed with 50 ul stop solution (5 uM internal standard (CE 17:0, MP Biomedicals, Solon, OH) and 2% formic acid in methanol). 50 ul pentanol was then added to the reaction mixture to extract lipids and prepare the samples for LC/MS analysis. When combining the reaction sample, stop solution and pentanol as described, a two-phase system resulted. A 5 uL aliquot of the upper, pentanol phase containing the extracted lipids was injected on a Hypersil Gold C18 LC column (2.1 x 50 mm; 1.9 mm) using a Waters 2777 autosampler. Lipids were eluted using a binary gradient of Solvent A (10 mM Ammonium Formate in 40% H2O : 60% Acetonitrile) and Solvent B (10% Acetonitrile : 90% Isopropanol). The LC column was initially equilibrated with 40% Solvent B at a flow rate of 600 mL/min and held for 0.2 minutes after injection. The proportion of Solvent B was then increased to 70% at 0.5 minutes and 80% at 3.3 minutes. All changes in solvent composition followed a linear ramping scheme. The flow rate was then increased to 800 uL/min and 95% B at 3.32 minutes and held for 0.1 minutes to flush the column. The composition was returned to 40% solvent B at a flow rate of 600 mL/min before the next injection. Cholesteryl esters were analyzed using positive electrospray ionization and multiple reaction monitoring on a Waters Acquity UPLC and Xevo-TQMS mass spectrometer (Waters). The NH4+ adducts were selected as the parent ions in the first quadrupole. Esters formed from the d6-cholesterol were fragmented to a common daughter ion of m/z = 375.2; a daughter ion of m/z/ = 369.2 was obtained for the CE 17:0 internal standard without d6 labeling. The peak areas of the major, newly formed CE species (d6-CE 18:1, d6-CE 18:2, d6-CE 20:4, d6-CE 22:6) were normalized to the peak area of the CE 17:0 internal standard to provide relative quantitation of newly formed CE species, and this measure was used to represent LCAT activity of the corresponding plasma sample.
LC/MS lipid analyses for lipoproteins from polyacylamide gel electrophoresis
Lipid composition characterization in lipoprotein particles was conducted by gradient gel electrophoresis (GGE) followed by high resolution LC/MS. The lipoprotein separation from 25 ml plasma samples was carried out on the Lipoprint GGE system (Quantimetrix, Redondo Beach, CA) according to the manufacturer's instructions. Following electrophoresis, the gel bands that contained different lipoprotein fractions (VLDL, LDL, and HDL) were excised and homogenized in phosphate-buffered saline. Lipids in each fraction were then extracted using the Bligh and Dyer method [4]. Major lipid classes (CE, TG, PC, LysoPC) in each lipoprotein fraction were then analyzed via an Acquity Ultra Performance Liquid Chromatography (UPLC) system coupled to a high resolution LC/MS system (Synapt G2 HDMS QTof, hybrid quadrupole orthogonal time of flight mass spectrometer (Waters, Milford, MA)) operated in positive ion electrospray mode. Detailed methods are described in previous publications [2, 5].
Supplemental References
1. Chen, Z., Strack, A. M., Stefanni, A. C., Chen, Y., Wu, W., Pan, Y., Urosevic-Price, O., Wang, L., McLaughlin, T., Geoghagen, N., Lassman, M. E., Roddy, T. P., Wong, K. K., Hubbard, B. K. & Flattery, A. M. (2011) Validation of Human ApoB and ApoAI Immunoturbidity Assays for Non-human Primate Dyslipidemia and Atherosclerosis Research, J Cardiovasc Transl Res.
2. Tadin-Strapps, M., Peterson, L. B., Cumiskey, A. M., Rosa, R. L., Mendoza, V. H., Castro-Perez, J., Puig, O., Zhang, L., Strapps, W. R., Yendluri, S., Andrews, L., Pickering, V., Rice, J., Luo, L., Chen, Z., Tep, S., Ason, B., Sommers, E. P., Sachs, A. B., Bartz, S. R., Tian, J., Chin, J., Hubbard, B. K., Wong, K. K. & Mitnaul, L. J. (2011) siRNA induced liver ApoB knockdown lowers serum LDL-cholesterol in a mouse model with human-like serum lipids, J Lipid Res.
3. Chen, C. H. & Albers, J. J. (1982) Characterization of proteoliposomes containing apoprotein A-I: a new substrate for the measurement of lecithin: cholesterol acyltransferase activity, J Lipid Res. 23, 680-91.
4. Bligh, E. G. & Dyer, W. J. (1959) A rapid method of total lipid extraction and purification, Can J Biochem Physiol. 37, 911-7.
5. Castro-Perez, J. M., Kamphorst, J., DeGroot, J., Lafeber, F., Goshawk, J., Yu, K., Shockcor, J. P., Vreeken, R. J. & Hankemeier, T. (2010) Comprehensive LC-MS E lipidomic analysis using a shotgun approach and its application to biomarker detection and identification in osteoarthritis patients, J Proteome Res. 9, 2377-89.