Assay Services

Blood Lipoprotein Subspecies Assay

This assay measures subspecies defined by apolipoproteins within each broad category of blood lipids, including the very-low density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) particles. The assay, depending on the target lipoprotein categories, may involve a separate ultracentrifugation step. 

To measure subspecies within the categories of VLDL and LDL particles, there are three steps involved to separate lipoprotein subspecies by their density and apolipoprotein profiles and then measure the concentration of apolipoproteins. Briefly, we first use immunoaffinity chromatography to bind apolipoproteins with their specific antibody resin (polyclonal goat anti–human apolipoprotein antibody bound to Sepharose 4B Resin; Academy Biomedical Company Inc, Houston, TX). Plasma samples are filtered, and 700 μL filtered plasma is loaded into 20 mL Econo-Pac columns (Bio-Rad Laboratories, Hercules, CA) packed with the anti-apo resin. The unbound fraction is eluted from the column by gravity followed by washes with phosphate-buffered saline. The bound fraction is then eluted from the columns with 3 mol/L sodium thiocyanate in phosphate-buffered saline and was immediately desalted with the use of PD-10 columns (GE Healthcare, Little Chalfont, United Kingdom). The immunoaffinity columns consisted of 2.5 mL anti–apo resin prepared with the use of polyclonal goat anti–human apo antibody bound to Sepharose 4B Resin at a minimum concentration of 5 mg antibody/mL resin. Ultracentrifugation is then used to separate the lipoprotein particles (within the bound and unbound fractions) by density into the VLDL (d<1.006 g/mL), LDL (1.006<d<1.063g/mL), and higher-density (d>1.063 g/mL) lipoprotein particles. Lastly, sandwich enzyme-linked immunoabsorbent assay (ELISA) procedures with the use of affinity-purified antibodies (Academy Biomedical Company Inc) are performed to determine the concentrations of apoB, apoCIII, apoE, and other lipoproteins in whole plasma and the lipoprotein fractions. Liquid transfer for 96-well plate loading and ELISA dilutions are handled robotically with a Multiprobe II (Perkin-Elmer, Waltham, MA) to minimize pipetting error. Both ELISA and lipid plates are read with a BioTek ELx808iu 96-well plate reader controlled by KCJUNIOR software (BioTek, Winooski, VT). 

To measure the subspecies within the HDL category, the ultracentrifugation step is not needed. Instead, the HDL fraction will be isolated using the immunoaffinity chromatography through anti-apoAI resin binding and then the ELISA assay will be applied to measure the concentrations of apolipoproteins in the HDL fration. All assays are completed in triplicate, and any sample with an intraassay CV > 15% is repeated.

References

1. Furtado JD, Yamamoto R, Melchior JT, Andraski AB, Gamez-Guerrero M, Mulcahy P, He Z, Cai T, Davidson WS and Sacks FM. Distinct Proteomic Signatures in 16 HDL (High-Density Lipoprotein) Subspecies. Arterioscler Thromb Vasc Biol. 2018;38:2827-2842.
2. Furtado JD, Campos H, Appel LJ, Miller ER, Laranjo N, Carey VJ and Sacks FM. Effect of protein, unsaturated fat, and carbohydrate intakes on plasma apolipoprotein B and VLDL and LDL containing apolipoprotein C-III: results from the OmniHeart Trial. Am J Clin Nutr. 2008;87:1623-30.

Blood Fatty Acid Assay

This assay measures a full profile of fatty acids (n≥40) in blood, red blood cells, adipose tissue, or food samples. Fatty acids are first extracted from biospecimens and analyzed by gas-liquid chromatography (GLC). Briefly, the fatty acids are extracted using a hexane/isopropanol (3:2) mixture and esterified with methanol and acetyl chloride. After esterification, the methanol and acetyl chloride are evaporated, and the fatty acid methyl esters are redissolved in isooctane. The methyl esters are quantitated by GLC. Peak retention times and area percentages of total fatty acids were identified by injecting known standards (Nu-Chek-Prep, Ely- sium, MN), and analyzed with the Agilent Technologies ChemSta- tion A.08.03 software (Agilent Technologies, Inc., Palo Alto, CA). Two internal QC samples are inserted into each batch for monitoring lab variabilities.

References

1. Baylin A, Kabagambe EK, Ascherio A, Spiegelman D, Campos H. High 18:2 trans-fatty acids in adipose tissue are associated with increased risk of nonfatal acute myocardial infarction in costa rican adults. J Nutr. 2003 Apr;133(4):1186-91. doi: 10.1093/jn/133.4.1186. PMID: 12672941.

Blood Antioxidant Panel Assay

This assay measures several antioxidants of dietary origin in blood and food samples. The antioxidants include lutein/zeaxanthin, b-cryptoxanthin, trans-lycopene, cis-lycopene, a-carotene, trans-b-carotene, cis b-carotene, retinol, and d-, g-, and a-tocopherol.

Samples (250 mL) are mixed with 250 mL ethanol containing 10 mg ractocopherol/mL (Tocol; Matreya Inc, Pleasant Gap, PA) as an internal standard, extracted with 4 mL hexane, evaporated to dryness under nitrogen, and reconstituted in 100 mL ethanol:dioxane (1:1, by vol) and 150 mL acetonitrile. The extracted samples are saponified with potassium hydroxide for 15 min at 50°C. Before saponification, 500 mL of 20% (wt:vol) ascorbic acid in aqueous solution and 1000 mL of 0.167 mg trans-b-apo-8’-carotenal/mL in ethanolic solution are added to all samples. Samples are reconstituted with 100 mL of a dioxane:ethanol solution (1:1, by vol) and 150 mL acetonitrile. Concentrations of the antioxidants are then quantitated by HPLC on a Restek Ultra C18 150 mm × 4.6 mm column with a 3-mm particle size (Restek Corp, Bellefonte, PA). The column was encased in a water bath to prevent temperature fluctuations and is equipped with a trident guard cartridge system. A mixture of acetonitrile, tetrahydrofuran, methanol, and a 1% ammonium acetate solution (68:22:7:3) is used as the mobile phase with a flow rate of 1.1 mL/min. The system includes a Hitachi L-7100 pump in isocratic mode, an L-4250 ultraviolet-visible light (445 nm) detector, and a programmable AS-4000 autosampler with water-chilled tray interfaced with a D-6000 interface module (Hitachi, San Jose, CA). The system manager software (D-7000, version 3.0; Hitachi) is used for peak integration and data acquisition. The minimum detection limits in plasma are 7.74 mg/L for a-carotene, 7.31 mg/L for b-carotene, 5.51 mg/L for b-cryptoxanthin, 8.49 mg/L for lycopene, and 6.31 mg/L for lutein+zeaxanthin.

References

1. El-Sohemy A, Baylin A, Kabagambe E, Ascherio A, Spiegelman D, Campos H. Individual carotenoid concentrations in adipose tissue and plasma as biomarkers of dietary intake. Am J Clin Nutr. 2002 Jul;76(1):172-9. doi: 10.1093/ajcn/76.1.172. PMID: 12081831.